University of Southern California Dissertations and Theses

The neural basis of estradiol conditioned taste aversion learning and estradiol anorexia

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The neural basis of estradiol conditioned taste aversion learning and estradiol anorexia

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Content THE NEURAL BASIS OF ESTRADIOL CONDITIONED TASTE AVERSION
LEARNING AND ESTRADIOL ANOREXIA
by
Houri Hintiryan
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PSYCHOLOGY)
May 2009
Copyright 2009 Houri Hintiryan
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Acknowledgments
I would like to express my deepest gratitude to my faculty advisor, Dr.
Kathleen Chambers, for all of her guidance and support throughout my graduate
career. I am forever grateful to my mentor and my friend.
I also would like to thank my teammate and dear friend Nicholas Foster,
who unselfishly devoted his time and efforts to helping me throughout the years.
Last, but not least, I would like to thank my husband and best friend Raffie,
who patiently and lovingly supported and encouraged me throughout this
challenging journey.
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Table of Contents
Acknowledgments
List of Tables
List of Figures
Abstract
Chapter 1 Neural Regulation of Eating Behavior
1.1 Introduction
1.2 The Dual Center Theory of Ingestive Behavior
1.3 Key Neural Substrates of Eating Behavior
1.3.1 Hypothalamus
Organization of the Hypothalamus
Connections of the Hypothalamus
Connections within Hindbrain Structures
Hypothalamic Nuclei and Neuropeptides Regulating Eating
1.3.1.1 Arcuate Nucleus (ARC)
Anabolic Peptides within the Arcuate
Neuropeptide Y (NPY)
Agouti Related Protein (AgRP)
Galanin
Catabolic Peptides within the Arcuate
Proopiomelanocortin (POMC) and Cocaine-
Amphetamine Related Transcript (CART)
Interactions between NPY/AgRP and POMC/CART Neurons
1.3.1.2 Lateral Hypothalamus (LH)
Anabolic Peptides within the Lateral Hypothalamus
Melanin-Concentrating Hormone (MCH)
Orexin
1.3.1.3 Paraventricular Nucleus (PVN)
1.3.1.4 Dorsomedial Hypothalamus (DMH)
1.3.1.5 Ventromedial Hypothalamus (VMH)
1.3.1.6 Tuberomammillary Nucleus (TM)
1.3.2 Hindbrain Structures
1.3.2.1 Area Postrema (AP)
1.3.2.2 Nucleus of the Solitary Tract (NST)
1.3.2.3 Parabrachial Nucleus (PBN)
The Lateral Parabrachial Nucleus
The Medial Parabrachial Nucleus
1.3.3 Forebrain Structures
1.3.3.1 Amygdala (Amg)
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1.3.3.2 Bed Nucleus of Stria Terminalis (BNST)
1.4 Short-term Regulation of Eating
1.4.1 Glucose and the Glucostat Hypothesis
1.4.2 Peripheral Feeding Signals
Ghrelin
1.4.3 Peripheral Satiety Signals
Cholecystokinin (CCK)
Peptide YY (PYY)
Glucagon-like Peptides-1and 2 (GLP-1/GLP-2)
1.5 Long-term Regulation of Eating
1.5.1 Set Point Theory of Body Weight
1.5.2 Peripheral Adiposity Signals
Leptin
Insulin
Amylin
1.6 Conclusion
Chapter 2 Estradiol and Eating Behavior
2.1 Estradiol: Overview
2.2 Estradiol and Eating Behavior
Tonic and Phasic Effects of Estradiol
Estradiol and Anorexia Nervosa
2.3 Neural Substrates of Estradiol Hypophagia
C-fos Like Immunoreactivity (c-FLI) Studies
Central Implant Studies
Lesion Studies
2.4 Estradiol and the Lateral Parabrachial Nucleus (PBN)
2.5 Estradiol, Neuropeptides and Eating
Cholecystokinin (CCK) and Estradiol
Neuropeptide Y (NPY) and Estradiol
Melanin-Concentrating Hormone (MCH) and Estradiol
Chapter 3 Conditioned Taste Aversion Learning
3.1 Conditioned Taste Aversion: Overview
Attributes of Conditioned Taste Aversion Learning
Evolutionary Significance of Conditioned Taste Aversion
3.2 Gustatory (CS) and Malaise (US) Information
Neural Gustatory Pathway (CS Pathway)
Neural Visceral Pathway (US Pathway)
3.3 Neural Substrates of Conditioned Taste Aversion Learning
3.3.1 The Vagus and Nucleus of the Solitary Tract (NST)
3.3.2 The Area Postrema (AP)
3.3.3 The Parabrachial Nucleus (PBN)
Lateral Parabrachial Nucleus (lateral (PBN)
Medial Parabrachial Nucleus (medial PBN)
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3.3.4 The Amygdala (Amg)
3.3.5 The Insular Cortex (IC)
3.3.6 Configuring the Conditioned Taste Aversion Neural Pathway
3.4 Neurotransmitters and Conditioned Taste Aversion Learning
Methodological Issues
3.4.1 Acetylcholine (ACh)
Synthesis of Acetylcholine
Cholinergic Receptors and Receptor Sites
Acetylcholine and Learning
Acetylcholine and Conditioned Taste Aversions
3.4.2 Histamine (HA)
Synthesis of Histamine
Histamine Receptors and Receptor Sites
Histamine and Learning
Histamine and Conditioned Taste Aversions
3.5 Estradiol and Conditioned Taste Aversion Learning
Conditioned Taste Aversions Revisited
Neural Substrates of Estradiol Conditioned Taste Avoidance
Neurochemical Mediation of Estradiol Conditioned Taste Avoidance
Chapter 4 Conducted Experiments
4.1 Outline of Experiments
4.2 Primary Aims
4.2.1 Primary Aim 1
Specific Aim 1.1
Specific Aim 1.2
Specific Aim 1.3
4.2.2 Primary Aim 2
Specific Aim 2.1
Specific Aim 2.2
4.3 Secondary Aims
4.3.1 Secondary Aim 1
4.3.2 Secondary Aim 2
4.4 General Methodology
4.4.1 Subjects and Husbandry
4.4.2 Chemical Agents
Conditioning Agents
Lesioning Agent
4.4.3 Surgical Procedures
Anesthetics and Surgical Techniques
Analgesics, Antibiotics, and Post-operative Care
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4.4.4 Behavior Testing
Conditioned Taste Avoidance/Aversion (CTA) Procedure
Measurements of Fluid Intake
Measurements of Food Intake
4.4.5 Histological Staining
Neuron-Specific Nuclear Protein
4.4.6 Statistical Analyses
Description of Analyses
Assessment of CTA Formation
Assessment of Extinction
Assessment of Fluid Intake
Assessment of Food Decrement
Additional Analyses
4.5 Presentation of Experiments
4.5.1 Experiment 1
Methods
Results
Discussion
4.5.2 Experiment 2
Methods
Results
Discussion
4.5.3 Experiment 3
Methods
Results
Discussion
4.5.4 Experiment 4
Methods
Results
Discussion
4.5.5 Experiment 5A
Methods
Results
Discussion
4.5.6 Experiment 5B
Methods
Results
Discussion

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4.5.7 Experiment 5 Discussion
CTA1: Estradiol: 6 Hour CS-US Interval
CTA1: Estradiol: 0 Hour CS-US Interval
CTA2: Lithium Chloride: 0 Hour CS-US Interval
Estradiol (CTA1) and Lithium Chloride (CTA2)
Unilateral Lesions and CTAs
Experiment 5b and CTAs: Some Important Issues
Summary of CTA Findings
Estradiol Hypophagia
Summary of Experiment 5 Findings
4.6 General Discussion
Dissociating Estradiol CTA from Estradiol Hypophagia
What is Estradiol CTA?
How is the Lateral PBN Involved in Estradiol CTA?
What is Meant by “Attenuated CTA”?
4.7 Future Direction
Excitotoxic Lesions
CTA Behavioral Procedure
Measurements of Food and Fluid Intake
Additional Considerations
References

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List of Tables
Table 1. Effects of Peripherally and Centrally Administered
Cholinergic Antagonists on Conditioned Taste Aversions
Table 2. Histamine and Estradiol Conditioned Taste Avoidance
Table 3. Criteria for Acquisition of a CTA: Changes in Sucrose
Consumption Across Acquisition and Either Post-Acquisition
Test 1 or Post-Acquisition Test 2
Table 4. Number of Animals that Acquired or Did Not Acquire an
Estradiol Conditioned Taste Avoidance Based on Criteria for
Conditioned Taste Avoidance Qualification
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List of Figures
Figure 1. Neural Connectivity Among Hypothalamic Nuclei, Hindbrain
Structures and Forebrain Structures
Figure 2. Neural Circuitry of Conditioned Aversions
Figure 3. Methodology for Determining Chemical Mediation of Estradiol
Conditioned Taste Avoidance
Figure 4. Effect of Two Different Time Intervals between Acquisition and
Post-acquisition Testing on Estradiol Conditioned Taste Avoidance
and Calorie Intake
Figure 5. Effect of Two Different Doses of Estradiol on Conditioned
Taste Avoidance and Calorie Intake
Figure 6. Effect of a 24 hour CS-US Interval on Estradiol Conditioned
Taste Avoidance
Figure 7. Effect of Various CS-US Intervals on Estradiol Conditioned
Taste Avoidance
Figure 8. Replication of the Effect of a 6 hour CS-US Interval on an
Estradiol Conditioned Taste Avoidance
Figure 9. Number of Animals in each Estradiol and Oil Group that
Acquired or Did Not Acquire Estradiol Conditioned Taste Avoidance
for Each CS-US Interval
Figure 10. Methodology for Determining Chemical Mediation of
Estradiol Conditioned Taste Avoidance
Figure 11. Neuron-Specific Nuclear Protein (NeuN) Stain of an Ibotenic
Acid Lesion of Lateral Amygdala
Figure 12. Neuron-Specific Nuclear Protein (NeuN) Stain of Ibotenic
Acid Lesions of Lateral Parabrachial Nucleus
Figure 13. Sucrose and Water Intake for Animals with Excitotoxic
Lesions of the Lateral Parabrachial Nucleus Across 7 Acquisition
Trials of an Estradiol Conditioned Taste Avoidance

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Figure 14. Sucrose and Water Intake for Animals with Excitotoxic
Lesions of the Lateral Parabrachial Nucleus Across Extinction Phases
of an Estradiol Conditioned Taste Avoidance
Figure 15. Chocolate Milk Intake in Animals with Excitotoxic Lesions
of the Lateral Parabrachial Nucleus During Acquisition and Extinction
of a Lithium Chloride Conditioned Taste Aversion
Figure 16. Size of Lateral Parabrachial Nucleus Lesion in Bilateral E
and Bilateral Oil Groups on Right and Left Sides of the Brain
Figure 17. Amount of Food Consumed During Twenty Four Hour
Periods that Followed Estradiol Injections in Animals with Excitotoxic
Lesions of the Lateral Parabrachial Nucleus
Figure 18. Amount of Food Consumed During Twelve Hour Dark Phase
Periods that Followed Estradiol Injections in Animals with Excitotoxic
Lesions of the Lateral Parabrachial Nucleus
Figure 19. Amount of Food Consumed During Twelve Hour Light Phase
Periods that Followed Estradiol Injections in Animals with Excitotoxic
Lesions of the Lateral Parabrachial Nucleus
Figure 20. Neuron Specific Nuclear Protein (NeuN) Stain of Electrolytic
Lesions of the Lateral Parabrachial Nucleus
Figure 21. Sucrose Intake in Animals with Electrolytic Lesions of the
Lateral Parabrachial Nucleus During Acquisition and the First Post-
Acquisition Test of a of a Lithium Chloride Conditioned Taste Aversion
Figure 22. Sucrose Intake in Animals with Electrolytic Lesions of the
Lateral Parabrachial Nucleus During Extinction of a Lithium Chloride
Conditioned Taste Aversion
Figure 23. Chocolate Milk Intake in Animals with Electrolytic Lesions
of the Lateral Parabrachial Nucleus During Acquisition and Post-
Acquisition Tests of an Estradiol Conditioned Taste Avoidance
Figure 24. Chocolate Milk Intake in Animals with Electrolytic Lesions
of the Lateral Parabrachial Nucleus During Extinction of an Estradiol
Conditioned Taste Avoidance
Figure 25. Amount of Food Consumed During Twenty-Four Hour Periods
that Followed Estradiol Injections in Animals with Electrolytic
Lesions of the Lateral Parabrachial Nucleus
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Abstract
When consumption of a novel tasting substance is followed by administration
of a chemical agent that produces physiological changes indicative of malaise,
animals will reduce their consumption of the substance during subsequent encounters.
This learned response is traditionally referred to as a conditioned taste aversion
(CTA). Studies have shown that the hormone estradiol is capable of producing this
learned gustatory aversion. In addition, estradiol produces reductions in food intake
and body weight, a phenomenon that is referred to as its anorectic effects. As a
consequence of this anorectic effect, we question whether estradiol truly can induce
CTA learning. Therefore, one of the purposes of the experiments presented in this
dissertation was to test the dissociability of estradiol CTA and estradiol anorexia. The
second purpose of this thesis was to examine the neural basis of estradiol CTA and
estradiol anorexia. Four approaches were adopted to test the ability of estradiol to
condition independent of its ability to produce reductions in eating. First, we show
that estradiol can produce a CTA in the absence of its anorectic effects. Second, we
demonstrate that a low dose of estradiol that produces reductions in eating does not
produce CTA. Next, we show that contingent pairing is necessary for a CTA since
non-contingent pairing does not result in the gustatory aversion. Finally, we
dissociate the conditioning effect of estradiol from its anorectic effect by showing that
both excitotoxic and electrolytic lesions of the lateral parabrachial nucleus (PBN)
either attenuated or blocked an estradiol CTA, while leaving estradiol anorexia
unaffected. Together, all of the data suggest that estradiol can condition a gustatory
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aversion. The data also suggest that estradiol elicits a CTA based on its aversion
inducing properties since lesions of the lateral PBN, an area that processes visceral
information, blocked the CTA. Third, the data show that the lateral PBN is necessary
for estradiol CTA. Finally, the studies show that although the lateral PBN is involved
in different types of anorexia, it is not involved in estradiol anorexia.
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CHAPTER 1
Neural Regulation of Eating Behavior
1.1 Introduction
Ingestion is a complex behavior that encompasses four different phases that
are distinguished from one another using behavioral and neurological criteria
(Watts, 2000). Briefly, the initiation phase consists of a change in the internal drive
state, which shifts the organism’s attention to food. The procurement phase
follows and involves the various aspects of foraging, while the consummatory
phase entails the stereotyped action patterns of eating. During this latter phase,
associations regarding the nutritional load and hedonic valence of the food also are
formed. Satiation or a competing motivated behavior, leads to the last stage,
namely the termination of a meal. This chapter will discuss the main neural areas
and neuropeptides involved in the initiation and termination of a meal, bypassing
the mechanisms involved in foraging and in the motor regulation of consummatory
behavior. The latter half of the chapter will be designated to the neural regulation
of body weight. Thus, the focus of the chapter will remain on the maintenance of
energy homeostasis, the process by which energy intake is matched to energy
expenditure. Although environmental and social stimuli, such as time of day,
financial circumstances, and the presence of food and people, should not be
discarded as important factors in motivating eating behavior, they too are beyond
the scope of this chapter.
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1.2 The Dual Center Theory of Ingestive Behavior
For an extended period of time, the predominant conception regarding the
neural control of eating revolved around the “dual center” theory. This hypothesis
gained solidity following the publication of the classic paper The Physiology of
Motivation, in which Stellar (1954) purported that most motivated behaviors,
including eating, were controlled by two specific brain structures: one that activated
the behavior and another that played an inhibitory role. Following this proposal,
the prevalent conception from the 1950’s to the 1970’s was that eating was under
the control of two functional centers, namely a feeding center and a satiety center.
Stellar reported evidence that was available at the time on the neural control of
eating that substantiated this binary theory. To that point, it had been demonstrated
that following lesions of the ventromedial nucleus of the hypothalamus (VMH),
animals would exhibit hyperphagia, which eventually would lead to severe obesity
(Hetherington & Ranson, 1940; Anand & Brobeck, 1951; Brobeck, Tepperman &
Long, 1943). Electrical stimulation of this area had the opposite effect, resulting in
decreased eating (Anand & Dua, 1955). On the contrary, bilateral lesions of the
lateral hypothalamus (LH) lead to hypophagia (Anand & Brobeck, 1951a), while
stimulation studies showed that activation of this area increased eating (Anand &
Brobeck, 1951b; Delgado & Anand, 1953). Predicated on these observations, the
VMH appropriately got labeled the satiety center, while the LH earned the title of
the feeding center.
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Due to recent advancements in the field of food intake and body weight
regulation, it is clear that not only does ingestive behavior extend beyond the VMH
and the LH within the diencephalic structure, it also involves regions of the
hindbrain and the forebrain. As such, the idea of functional centers has become
obsolete. Presently, the effects of the neuropeptides that are localized within a
neural structure are used in combination with lesion studies to assess the role of a
particular area in eating behavior. In addition, there are separate mechanisms that
work in conjunction to control the size of individual meals and to regulate body
weight. Although the neural machinery is highly conserved for these separate
processes (control of meal frequency, meal size, and body weight), the molecular
signals that initiate each process appear to be disparate.
1.3 Key Neural Substrates of Eating Behavior
Since the hypothalamus is the central player in eating behavior, the specific nuclei
of the diencephalic structure and their residing neuropeptides will be covered first.
Subsequently, discussion of hindbrain areas, like the area postrema (AP), nucleus
of the solitary tract (NST), and the parabrachial nucleus (PBN), and of forebrain
structures, like the amygdala (Amg) and the bed nucleus of the stria terminalis
(BNST), that work in concert with the hypothalamus to regulate eating and body
weight will follow.
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1.3.1 Hypothalamus
Organization of the Hypothalamus
The hypothalamus contains numerous nuclei, some of which are divided
further into subnuclei (Simerly, 1995). Generally, along the medial-lateral axis, the
hypothalamus is organized into three zones. Starting from the most lateral, they are
the (1) lateral, (2) medial, and (3) periventricular zones. Each of these contains
nuclei that have been shown to play an integral role in eating. The lateral zone
includes the lateral hypothalamus (LH), while the medial zone contains the
dorsomedial (DMH) and ventromedial (VMH) nuclei. The paraventricular nucleus
(PVN) resides in the periventricular zone, while the arcuate (ARC) extends across
the medial and periventricular zones. The mammillary nucleus (MM), also an
important regulator of eating behavior, lies in between the medial and
periventricular zones. Along the anterior-posterior axis, the hypothalamus is
divided into several regions as well. Starting with the most anterior, the
hypothalamic nuclei discussed in this chapter are organized into the following
regions: (1) anterior, (2) tuberal, and (3) mammillary regions. The LH extends
across all of these regions, while the PVN is restricted to the anterior region. The
DMH, VMH, and ARC nuclei are found in the tuberal region. The most posterior
of the nuclei are the mammillary bodies, which reside in the mammillary region.
Connections of the Hypothalamus
The nuclei of the hypothalamus are heavily interconnected, but they also
share neural connections with the AP, NST, and PBN in the hindbrain and with the
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Amg and BNST of the forebrain. The connectivity among these eating-related
structures gives insight into the potential circuitries underlying eating behavior. Of
course, fibers of the hypothalamus innervate many more regions of the brain;
however, since these are the eating-related structures covered in this chapter,
discussion regarding the connections of the hypothalamus will be limited to these
neural areas (see Figure 1 for neural connections discussed below).
Within the hypothalamus the ARC is a key regulator of eating. It receives
its densest afferent projections from other hypothalamic structures including the
PVN, while weaker inputs come from the LH (Simerly, 1995). It receives
projections from extra-hypothalamic structures implicated in eating like the lateral
subdivision of the PBN, NST, central nucleus of the Amg and the BNST (Ricardo
& Koh, 1978; Li, Chen & Smith, 1999).
Tracing studies have shown that there are reciprocal connections between
the LH and the ARC, PVN, DMH, and VMH (Simerly, 1995). The LH also shares
reciprocal connections with the PBN and NST where it receives viscero-sensory
information pertaining to food (Luiten, ter Horst & Steffens, 1987; Larsen, Moller
& Mikkelson, 1991; Simpson & Raubenheimer, 2000). Finally, the LH is the target
of afferent projections from the Amg (Simerly, 1995).
Like the LH, the PVN receives afferents from forebrain regions such as the
BNST and central Amg (Sawchenko & Swanson, 1983) and hindbrain structures
like the PBN (Thompson & Swanson, 1998). In turn, the PVN sends information
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to the NST (Hosoya, Sugiura, Okado, Loewy & Kohno, 1991) and to the PBN
(Moga, Saper & Gray, 1989; Moga, Herbert, Hurley, Yasui, Gray et al., 1990).
Most of the inputs received by the DMH are provided by other
hypothalamic nuclei such as the LH, ARC, VMH, and PVN (Simerly, 1995;
Thompson & Swanson, 1998). Much like the PVN and LH, although to a lesser
degree, it is the target of the PBN (Thompson & Swanson, 1998). The efferent
projections of the DMH also are mainly limited to the hypothalamus, with the
strongest recipient being the PVN (Simerly 1995).
Although the VMH sends weak inputs to eating-related areas (Canteras,
Simerly & Swanson, 1994), it receives strong inputs from the Amg, the LH, the
PBN, and the NST (Fulwiler & Saper, 1985).
Connections within Hindbrain Structures
Since the neural connectivity of the AP, NST, and PBN are going to be
important in the circuitry of peptides that regulate eating, the connections amongst
the structures are briefly introduced here. Neuroanatomical studies have
established that the AP receives information from the viscera via the afferent vagus
nerve (Leslie & Gwyn, 1984; Shapiro & Miselis, 1985). In addition, it sends and
receives information from the NST and the PBN, although predominantly from the
lateral subdivision of the PBN (van der Kooy & Koda, 1983; Shapiro & Miselis,
1985). The PBN and NST also share connections with one another (Lowey &
Burton, 1979; Saper & Lowey, 1980). Finally, the PBN has bi-directional
communication with the LH, VMH, and PVN (Moga et al., 1990) and the central
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Amg and BNST (Bernard, Peschanski & Besson, 1989; Moga et al., 1989; Moga et
al., 1990; Bernard, Carroue & Besson, 1991).
Anatomical studies have shown that the NST and central Amg share
reciprocal connections (Norgren, 1978; Ricardo & Koh, 1978; Gray & Magnuson,
1987). This connection has been confirmed by studies using electrical stimulation
of the central Amg and measurement of c-fos (the product of the immediate early
gene c-Fos and an indicator of neuronal activity). The results showed that
stimulation of the amygdaloid nucleus produced activation of c-fos in the NST
(Petrov, Jhamandas & Krukoff, 1996).
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Figure 1. Neural Connectivity Among Hypothalamic Nuclei, Hindbrain
Structures and Forebrain Structures.

DMH
VMH
ARC LH

BNST
PVN
Amg

Medial PBN Lateral PBN

NST

Area Postrema
Vagus Nerve
Figure 1. Neural connectivity among hypothalamic nuclei, hindbrain structures and forebrain
structures. Not all connections are depicted. Double arrows ( ) indicate reciprocal connections.
AP: Area Postrema; NST: Nucleus of the Solitary Tract; PBN: Parabrachial Nucleus; PVN:
Paraventricular Nucleus; ARC: Arcuate; VMH: Ventromedial Hypothalamus; DMH: Dorsomedial
Hypothalamus; LH: Lateral Hypothalamus; Amg: Amygdala; and BNST: Bed Nucleus of the
Solitary Tract
Hypothalamic Nuclei and Neuropeptides Regulating Eating
The role of each hypothalamic nucleus is largely defined by the
neuropeptides that are localized within each structure. These structures and their
regulatory peptides are discussed below.
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1.3.1.1 Arcuate Nucleus (ARC)
The different populations of neuropeptides that are localized within the
ARC largely define its role in eating behavior. This periventricular nucleus
contains peptides that are involved in both catabolic and anabolic effector systems
(Kaiyala et al., 1995; Schwartz, Woods, Porte, Seeley & Baskin, 2000). Briefly,
catabolic systems work to decrease food intake and increase energy expenditure
and are activated by positive energy states. Anabolic systems do the opposite by
increasing eating and decreasing energy expenditure and are activated by negative
energy states.
Numerous studies using various types of techniques ranging from
immunohistochemistry to in situ hybridization have identified two distinct
populations of neurons in the ARC. One of these populations contains the
orexigenic protein Neuropeptide Y (NPY), which is coexpressed with a second
feeding related agent, Agouti Related Protein (AgRP; Broberger, de Lecea,
Sutcliffe & Hokfelt, 1998; Hahn, Breininger, Baskin & Schwartz, 1998). Galanin
is a third orexigenic that is synthesized in the ARC, but is not colocalized with any
other peptide. A second population of neurons in the ARC co-expresses peptides
that have the opposite effect on eating. These anorexigenics are
proopiomelanocortin (POMC) and cocaine-amphetamine related transcript (CART;
Kristensen, Judge, Thim, Ribel, Christjansen et al., 1998). These different
neuropeptides with their various effects on eating define the diverse role the ARC
plays in eating behavior.
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Anabolic Peptides within the Arcuate Nucleus
Neuropeptide Y (NPY)
Neuropeptide Y is an orexigenic peptide that potently stimulates eating.
This is an effect that consistently has been demonstrated across various species of
animals (Clark, Karla, Crowley & Karla, 1984; Kuldosky, Glazner, Moore, Low &
Woods, 1988; Morley, Hernandez & Flood, 1987; Pau, Pau & Spies, 1985) and
across the life span of rats, ranging from the time of peri-natal development
(Capuano, Barr & Leibowitz, 1986) to late adulthood (Pinch, Messori, Zoli,
Ferraguti, Marrama et al., 1992). Infusions of the peptide produce hyperphagia
when infused into the lateral ventricles or directly into the PVN (Stanley &
Leibowitz, 1985; Stanley, Kyrkouli, Lampert & Leibowitz, 1986), the LH, and the
VMH (Stanley, Chin & Leibowitz, 1985). Evidence implicates the perifornical
area (PFA) as the most sensitive hypothalamic site for NPY infusions (Stanley,
Willet, Donias, Ha & Spears, 1993). The PFA is immediately adjacent to the LH
and stimulation of this area has been shown to produce increases in eating (Stanley
et al., 1993). Infusions of NPY into the PFA produce the strongest effects on
eating probably due to the substantial NPYergic connections it receives from the
ARC (Booth & Thibault, 2000). Extrahypothalamic infusions of NPY have no
effect on eating (Morley et al., 1987). Interestingly, NPY increases the motivation
to eat since following NPY infusions, animals will work extremely hard at a lever
press in order to get a food reward, they will eat food adulterated with an
unpalatable agent such as quinine, and they will drink milk that is paired with an
11
electric shock (Flood & Morley, 1991; Jewett, Cleary, Levine, Schaal &
Thompson, 1992). Further, intracerebroventricular infusions of NPY reduce
energy expenditure (Billington, Briggs, Grace & Levine, 1991; Billington, Briggs,
Harker, Grace & Levine, 1994), stimulate lipogenesis in the liver and in white
adipose tissue (Billington et al., 1991; Zarjevski, Cusin, Vettor, Rohner-Jeanrenaud
& Jeanrenaud, 1993), and increase circulating levels of insulin (Zarjevski et al.,
1993). These effects were independent of the peptide’s effects on eating since
since restricting the animals’ consumption following the infusions resulted in
elevated lipogenesis and circulating insulin. It is not surprising then that chronic
administration of the peptide readily leads to obesity in both mice and rats (Stanley
et al., 1986; Zarjevski et al., 1993).
Evidence implicates the involvement of NPY in a prominent anabolic
effector system that includes the ARC and PVN. Although NPYergic neurons in
the ARC heavily innervate other hypothalamic nuclei, they most densely project to
the PVN (Bai, Yamano, Shiotani, Emson, Smith, et al., 1985; Elias, Saper,
Maratos-Flier, Tritos, Lee et al., 1998b), another diencephalic structure shown to be
involved in the suppression of eating (Leibowitz, Hammer & Chang, 1981; Aravich
& Scalfani, 1983). This ARC-PVN connection has been implicated in the NPY
correctional mechanism during states of depleted energy. For instance, NPY
synthesis in the ARC and its subsequent release in the PVN are both elevated in
response to fasting (Sahu, Karla & Karla, 1988), lactation (Li, Chen & Smith,
1998; Smith, 1993), physical exercise (Lewis, Shellard, Koeslag, Boer, McCarthy
12
et al., 1993), and in the period of insulin deficiency during which cells are glucose
deprived (Sahu, Sninsky, Phelps, Dube, Karla, et al., 1992). These findings suggest
that NPY is released to increase eating in order to correct for the ensuing negative
energy state. Further, they demonstrate the important interaction between the ARC
and PVN in regulating energy homeostasis.
Despite the transparent importance of this neuropeptide in energy
homeostasis, NPY knock out mice continue to display normal patterns of eating
(Erickson, Clegg & Palmer, 1996; Hollopeter, Erickson, Seeley, Marsh & Palmiter,
1998). This finding may suggest that compensatory mechanisms exist which take
over the responsibilities of NPY during its absence. Agouti Related Protein
(AgRP), discussed in the next section, is a possible candidate that assumes this role.
Evidence for this hypothesis is provided by the finding that AgRP mRNA is
upregulated in NPY knock out mice (Marsh, Miura, Yagaloff, Schwartz, Barsh et
al., 1999).
Nevertheless, NPY appears to be a critical regulator of energy intake. Many
molecules that regulate energy intake interact with NPY to induce their effects on
eating whether it is for the cessation of an individual meal or the control of body
weight through glucose, leptin, or insulin. These interactions will be discussed in
later sections.
Agouti Related Protein (AgRP)
Agouti Related Protein is synthesized exclusively in the ARC and is
coexpressed with NPY. Messenger RNA for AgRP is found in all NPYergic
13
neurons of the ARC and thus far has not been localized in any other region of the
brain (Broberger et al., 1998). Similar to NPY, AgRP is an orexigenic compound
that increases eating when infused centrally into the lateral ventricles (Ollman,
Wilson, Yang, Kerns, Chen et al., 1997; Hagan, Rushing, Pritchard, Schwartz,
Strack et al., 2000). Its overexpression in transgenic mice produces increased
eating and culminates in obesity (Graham, Shutter, Sarmiento, Sarosi & Stark,
1997). Although intracerebroventricular infusions of NPY produce more potent
hyperphagia compared to infusions of AgRP (Stanley & Leibowitz, 1984; Morley
et al, 1987), the hyperphagic actions of AgRP are longer lived compared to that of
NPY, lasting for several days as opposed to several hours (Hagan et al., 2000).
There is evidence suggesting that AgRP induces its hyperphagic effects
through melanocortin receptors 3 and 4 (MC3/MC4) of α-melanocyte stimulating
hormone (α -MSH), an anorexigenic compound. Alpha-MSH is thought to restrict
eating and constrain weight gain by acting as an agonist for MC3/MC4 receptors.
This implies an inhibitory role on eating by these melanocortin receptors. It is
demonstrated that AgRP acts as an antagonist for MC3/MC4 receptors (Ollmann et
al., 1997; Quillan, Sades, Wei, Jimenez, Ji et al., 1998) suggesting that the peptide
induces its effects on eating by inhibiting the satiogenic effects of melanocortin
receptors. Since the long-term actions of AgRP are still present following the
blockade of MC3 and MC4 receptors (Hagan et al., 2000), mechanisms other than
the α-MSH circuitry must aid in the orexigenic action of the peptide.
14
Galanin
In the ARC, NPY/AgRP and POMC/CART neurons (discussed in the
following section) have received considerable attention and have constituted the
focus of most research; however, galanin, another neuronal peptide that drives
feeding, also is localized in this hypothalamic structure (Bartfai, Fisone & Langel,
1992; Merchenthaler, Lopez & Negro-Vilar, 1993; Crawley, 1995). Three galanin
receptors have been identified and all three have been shown to densely populate
the hypothalamus (Bartfai et al., 1992; Smith, Walker, Artymyshyn, Bard,
Borowsky et al., 1998). Infusions of low amounts of the peptide into the lateral
(Schick, Samsami, Zimmermann, Eberl, Endres et al., 1993) and third (Dube,
Horvarth, Leranth, Karla, Karla, 1994) ventricles produce short-lived (~30 minutes)
increases in eating.
Although galanin also is produced in the periphery, in particular in the
pancreas, urogenital tract, and in ganglia innervating the heart and the gut
(Melander, Hokfelt, Rokaeus, Fahrenkrug, Tatemoto et al., 1985; McDonald,
Brooks, Rokaeus, Tinner & Staines, 1992), this effect of galanin on eating appears
solely to be mediated centrally because intraperitoneal injections of the agent have
no effect on consumption (de Pedro et al., 1995). As such, galanin is discussed in
this section as opposed to the section on peripheral signals that control eating. Site-
specific neural injections of galanin have shown that the peptide elicits its most
potent effects in the PVN (Kyrkouli, Stanley & Leibowitz, 1986; Corwin, Robinson
& Crawley, 1993) although the peptide also acts in the DMH (Kyrkouli et al.,
15
1986), VMH (Schick, Yaksh & Go, 1993), Amg (Kyrkouli et al., 1986), and the
AP/NST (Corwin et al., 1993). Although the peptide is not colocalized with other
neuropeptides in the ARC, it colocalizes with corticotrophin-releasing (CRH)
hormone and thyrotropin-releasing hormone (TRH) in the anterior PVN (reviewed
in Leibowitz, 1998), both of which produce reductions in eating (Arase, York,
Shimazu, Shargill & Bray, 1998 for CRH; Lin, Chu & Leu, 1983 for TRH). It may
be that galanin inhibits CRH and TRH neurons to preclude their anorexigenic
effects.
Galanin interacts with other chemicals in the CNS to exert its hyperphagic
effects. One of these agents is the neurotransmitter norepinephrine.
Administration of adrenergic receptor (α
2
) antagonist blocks the hyperphagic
effects of galanin (Kyrkouli, Stanley, Hutchinson, Seirafi & Leibowitz, 1990),
while leaving NPY-elicited feeding unaltered. Importantly, the authors showed
that infusions of the antagonists alone did not affect baseline measurements of
eating. Together, these suggest that galanin may produce its effects through the
release of norepinephrine. This finding is substantiated by an in vivo microdialysis
study, which showed that intra-paraventricular injections of galanin increased
hypothalamic norepinephrine release within the PVN (Kyrkouli, Stanley &
Leibowitz, 1992).
16
Catabolic Peptides within the Arcuate Nucleus
Proopiomelanocotin (POMC) and Cocaine-Amphetamine Related Transcript
(CART)
The second population of neurons in the ARC important in the regulation of
energy homeostasis involves the coexpressed anorexigenics POMC and CART
(Kristenson et al., 1998). It has been demonstrated that POMC also is produced in
the PVN and the DMH (Koylu, Couceyro, Lambert, Ling, DeSouza et al., 1997).
Proopiomelanocotin is the precursor for α-MSH and is classified as part of the
melancortin family (Cochet, Chang & Cohen, 1982). The inhibitory effect that
POMC exerts on eating appears to be mediated by α-MSH via MC4 receptors
(Poggioli, Vergoni & Bertolini, 1986; Fan, Boston, Kesterson, Hruby & Cone,
1997). Deletions targeted against POMC (Yaswen, Diehl, Brennan &
Hochgeschwender, 1999) and MC4 (Huszar, Lynch, Fairchild-Huntress, Dunmore
Fang et al., 1997) receptors both lead to increases in eating and obesity in mice,
suggesting the importance of this peptide in the inhibition of eating. Interestingly,
in humans, mutations in either the POMC (Krude, Biebermann, Luck, Horn,
Brabant et al., 1998) or the MC4 receptor gene (Vaisse, Clement, Durand,
Hercberg, Guy-Grand et al., 2000) are associated with obesity.
Most studies available on CART and eating suggest an anorexic role for the
peptide. Infusions of CART into the lateral ventricles consistently have been
shown to decrease food consumption in sated animals (Kristensen et al., 1998;
Edwards, Abbot, Sunter, Kim, Dakin et al., 2000; Abbott et al., 2001). On the
17
contrary, infusions of the peptide antiserum significantly increase eating in satiated
rats (Kristensen et al., 1998; Lambert, Couceyro, McGirr, Dall Vechia, Smith et al.,
1998). A comprehensive study by Abbott et al. (2001) has shown the more
dynamic effects of CART on eating behavior. The authors demonstrated that
intracerebroventricular infusions of the peptide produce reductions in eating in both
sated and fasted animals; however, the reductions were more dramatic in sated
animals. Furthermore, infusions of the peptide produced significant increases in
eating when injected into particular nuclei such as the LH, PVN, VMH, DMH, and
ARC. This increase was followed by a significant decrease in eating 4-24 hours
following the injection into the ARC and DMH. The significant decrease may be
either a compensatory response to the increase in eating or due to a delayed
anorectic effect of the peptide. This reduction of eating following an initial
increase in eating by CART is observed in both sated and fasted animals,
suggesting that the effect is not dependent on the energy state of the animal. Taken
together, these findings insinuate an anorectic and possibly a site-specific
orexigenic mechanism of CART.
Interaction between NPY/AgRP and POMC/CART Neurons
The two populations of neurons, NPY/AgRP and POMC/CART, form
connections with other hypothalamic nuclei. Within the ARC itself, it appears that
the two populations share reciprocal connections (Cowley, Smart, Rubinstein,
Cerdan, Diano et al., 2001), suggesting an interaction between these peptides.
Evidence for such an interaction has been demonstrated. For instance, it is reported
18
that one of the mechanisms by which NPY stimulates eating is by inhibiting
anorexigenic POMC/CART neurons (Roseberry, Liu, Jackson, Cai & Friedman,
2004). Additionally, both populations of neurons in the ARC send projections to
the PVN and to the LH (Bagnol, Lu, Kaelin, Day, Ollmann et al., 1999; Elias,
Aschkenasi, Lee, Kelly, Ahima et al., 1999). Specifically, they form synapses with
lateral hypothalamic neurons that contain the orexigenic agents melanin
concentrating hormone (MCH) and orexin or “hypocretin” (Elias et al., 1998b;
discussed in the section that follows). It could be purported then that NPY/AgRP
work in concert with these compounds to increase eating, while POMC/CART
inhibit the actions of these agents in an attempt to suppress eating. Indirect
evidence for this possibility is provided by the finding that CART is coexpressed
with MCH in the LH (Elmquist, Elias & Saper, 1999; Broberger, 1999; Yaswen et
al., 1999), suggesting a potential interaction by which both CART and MCH
influence energy intake.
1.3.1.2 Lateral Hypothalamus (LH)
The LH is a large nucleus comprised of several subnuclei, is highly
interconnected within the hypothalamus and with the rest of the brain, and is
traversed by the medial forebrain bundle (MFB) and the fornix (fx). The LH is
considered to play a major role in feeding behavior in part due to the orexigenic
neuropeptides that are synthesized within this particular nucleus. Both lesion
(Anand & Brobeck, 1951) and stimulation (Delgado & Anand, 1953) studies have
corroborated this hypothesis although the effects of these studies may have been
19
due to damage and/or stimulation of fibers of passage such as the MFB. Many
independently conducted studies have confirmed that the LH contains neurons that
express the orexigenic compounds melanin concentrating hormone (MCH;
Bittencourt & Elias, 1998) and orexin (Saper, 2000), otherwise known as
hypocretin (de Lecea, Kilduff, Peyron, Gao, Foye et al., 1998), which may better
define the role of the LH in eating behavior. Unlike NPY and AgRP in the ARC,
MCH and orexin are not coexpressed within the same neurons (Broberger et al.,
1998); however, the neurons synthesizing the two peptides are intermingled and
have similar wide-ranging projection sites (Bittencourt, Presse, Arias, Peto, Vaughn
et al., 1992; Peyron, Tighe, van de Poll, de Lecea, Hellar et al., 1998).
Additionally, unlike the ARC, the LH appears to contain only orexigenic agents
and no anorexic agents have been identified in the nucleus.
Anabolic Peptides within the Lateral Hypothalamus
Melanin Concentrating Hormone (MCH)
Studies spanning from intracerebroventricular infusions to transgenic MCH
null mice have repeatedly shown the stimulatory effect of MCH on eating.
Infusions of MCH into the ventricles increase food intake in both rats (Rossi et al.,
1997; Ludwig et al., 1998) and mice (Della-Zuana et al., 2002), which occur
throughout the light and dark phases of the day (Rossi & Bloom, 1997). Deletions
targeted against the MCH precursor protein prepro-MCH gene result in lean,
hypophagic animals (Shimada et al., 1998), while transgenic mice that overexpress
MCH are obese and hyperphagic (Ludwig et al., 2001). It must be taken into
20
account that because the prepro-MCH gene codes for other agents, this phenotype
cannot be attributed solely to the absence of MCH. Another piece of evidence
linking MCH with feeding behavior is the finding that both short-term (Fellmann et
al., 1993) and long-term (Presse et al., 1996; Herve & Fellmann, 1997) food
deprivation increase MCH immunoreactivity in the rat suggesting its role as an
anabolic signal to restore enery homeostasis.
Orexin
A second set of neuropeptides that are expressed in the LH and in the
adjacent PFA are orexins A and B (Sakurai et al., 1998), otherwise known as
hypocretins 1 and 2 (de Lecea, Kilduff, Peyron, Gao, Foye, et al., 1998),
respectively. Both orexin A and B appear to be involved in feeding behavior,
although the former has a more potent effect (Tritos, Vicent, Gillette, Ludwig, Flier
et al., 1998). It should be noted that genetically eliminated orexin leads to
hypophagia in addition to narcolepsy (Chemelli, Willie, Sinton, Elmquist,
Scammell, et al., 1999). This suggests a potential interaction between eating and
wakefulness. As such, it may be that orexin affects eating partly by affecting the
overall arousal state of the animals. Messenger RNA levels of the orexin precursor
protein, prepro-orexin, are upregulated during fasting states (Sakurai, Amemiya,
Ishii, Matsuzaki, Chemelli et al, 1998), a finding that is similar to the orexigenic
peptides discussed thus far. Injections of both orexin A and B into the lateral
ventricles produce a feeding response (Sakurai et al., 1998), although the
hyperphagic effect of orexin A outlasts that of orexin B. On the contrary, blocking
21
orexin A receptors decreases eating (Haynes, Jackson, Chapman, Tadayyon, Johns
et al, 2000). Evidence shows that although intracerebroventricular infusions of
orexin A produce increases in eating, they do not affect body weight (Yamanaka,
Sakurai, Katsumoto, Yanagisawa & Goto, 1999). This finding is not too surprising
given that orexin A has been shown also to increase energy expenditure (Wang,
Osaka & Inoue, 2001). In situ hybridization studies aiming to identify location of
the receptors for orexin report that orexin A receptor is most abundantly expressed
in the VMH, whereas orexin B receptor is predominantly found in the PVN
(Trivedi, Yu, MacNeil, van der Ploeg, Guan et al., 1998; Marcus, Aschkenasi, Lee,
Chemelli & Saper, 2001) although both types of receptors are localized outside of
the hypothalamus as well.
Although most of the aforementioned studies with orexin have been
performed in murine animals, the orexin circuitry within the hypothalamus is
involved in homeostatic mechanisms in non-human primates as well. Orexin-
synthesizing neurons are found exclusively in the LH-PFA area and in the DMH.
In fasted animals, there is increased c-fos activation in these hypothalamic orexin-
containing neurons (Diano, Horvarth, Urbanski Sotonyi & Horvarth, 1993). This
suggests that the orexin circuit in the hypothalamus of non-human primates also is
involved in signal transduction associated with metabolic alterations.
Orexin immunoreactive fibers from the LH and DMH form synapses on
ARC NPY/AgRP neurons (Horvath, Diano, Anthoney & van der Pol, 1999) and on
POMC cell bodies (Guan, Saotome, Wnag, Funahashi, Hori, et al., 2001),
22
suggesting a potential circuitry for orexin-induced feeding. Further, triple
immunofluorescence has revealed that NPY and POMC neurons contain orexin
receptors implying that these neurons are influenced by the peptide (Funahashi,
Yamada, Kageyama, Takenoya, Guan, et al., 2003). Lending support for an orexin-
NPY interaction is the finding that intracerebroventricular infusions of orexin
produce c-fos protein activation in ARC NPYergic neurons (Yamanaka, Kunii,
Nambu, Tsujino, Sakai, et al., 2000). In a review article, Funahashi, Takenoya,
Guan, Kageyama, Yada et al. (2003) report findings of unpublished physiological
studies that provide further evidence for the interaction of NPY and POMC with
orexin. They report that orexin increases [Ca]
2+
in isolated NPY neurons and
decreases [Ca]
2+
in POMC neurons in the ARC suggesting an excitatory effect of
orexin on NPY and an inhibitory effect on POMC.
The LH also has reciprocal connections with the NST where it receives
viscero-sensory information pertaining to food (Simpson & Raubenheimer, 2000).
In fact, the NST has been shown to receive dense orexin innervation from the LH
(Date, Ueta, Yamashita, Yamaguchi, Matsukura, et al., 1999; Peyron et al., 1998)
and orexin A depolarizes 90% of NST neurons tested (Yang & Ferguson, 2003).
Since the NST innervates the dorsal motor nucleus of the vagus, which in turn,
sends fibers that innervate the entire GI system (Grabauskas & Moises, 2003;
Hayakawa, Takanaga, Tanaka, Maeda & Seki, 2003; Wu, Gao, Yan, Owyang & Li,
2004), it is possible that the NST serves as the mediator between hypothalamic
orexin information and the periphery. Finally, in the rat LH, there are reciprocal
23
connections between the orexinergic and MCHergic neurons (Guan, Uehara, Lu,
Wang, Funahashi, et al., 2002) indicating that these two peptides interact with one
another in controlling food intake similar to the NPY/AgRP and POMC/CART
neuronal populations in the ARC.
1.3.1.3 Paraventricular Nucleus (PVN)
The role of the PVN in eating was discovered following the finding that
parasagittal knife cuts of what was thought to be at the level of the VMH produced
increases in eating and weight gain. It was discovered that the optimum site for
these knife cuts was at the PVN and not the VMH (Gold, Jones, Sawchenko &
Kapatos, 1977). Studies followed which showed that lesions of the PVN in the
absence of any damage to the VMH produced obese rats (Aravich & Scalfani,
1983; Leibowitz et al., 1981), a finding that was initially demonstrated in dogs
(Heinbecker, White & Rolf, 1944). This was the introduction of the PVN as a key
player in the eating system.
Supplementing the lesion studies are immunohistological staining
experiments that further implicate the PVN as an important structure in regulating
eating. Similar to the ARC and the LH, the role of the PVN in eating also is
characterized by the peptides that are synthesized within the hypothalamic nucleus.
Cocaine and amphetamine-regulated transcript (Koylu et al., 1997), POMC (Koylu
et al., 1997), corticotropin releasing hormone (CRH; Sawchenko, Swanson & Vale,
1984), thyrotropin-releasing hormone (TRH; Segersen, Kauer, Wolfe, Mobtaker,
Jackson & Lechan, 1987; Kow & Pffaf, 1991), and cholecystokinin (CCK; Mezey,
24
Reisine, Skirboll, Beinfeld & Kiss, 1986) are all synthesized in neurons residing in
the PVN and all produce reductions in eating (Arase, York, Shimazu, Shargill &
Bray, 1998 for CRH; Lin, Chu & Leu, 1983 for TRH). Taken together, these two
types of studies confer an inhibitory role of PVN in eating. On the other hand, the
ARC-PVN connection implicates the latter structure in the initiation of eating as
well. Arcuate orexigenic NPYergic neurons densely project to the PVN (Bagnol,
Lu, Kaelin, Day, Ollmann, Gantz, et al., 1999; Elias et al., 1998b), a connection
that has been implicated in the NPY correctional mechanism during states of
energy depletion (Sahu et al., 1988; Li et al., 1998; Smith, 1993; Lewis, Shellard,
Koeslag, Boer & McCarthy, et al., 1993). The stimulatory effect of noradrenaline
on eating (Leibowitz, 1978) and the inhibitory effect of serotonin (Smith, York &
Bray, 1999) when infused into the PVN further evinces the dual role this nucleus
plays in the eating system. The role of the PVN in serotonin hypophagia
(Leibowitz, 1990) is not surprising given that the structure is innervated by the
raphe nucleus (Larsen, Hay-Schmidt, Vrang & Mikkelsen, 1996), which is a rich
source of serotonin.
1.3.1.4 Dorsomedial Hypothalamus (DMH)
The DMH is another hypothalamic nucleus that is an important player in
food intake. Like most other neural structures discussed thus far, the dual role of
the DMH in eating has been confirmed through various methods. Although most
orexin/hypocretin cells are localized within the LH and in the adjacent PFA, some
also are localized in the DMH (de Lecea et al., 1998), suggesting the role of the
25
nucleus in feeding responses. The appetite suppressing neuropeptides
POMC/CART and the hypophagia-inducing neurotransmitter serotonin also is
produced in the DMH (Koylu et al., 1997 for POMC/CART; Frankfurt & Azmitia,
1983 for serotonin) demonstrating its role in satiation.
Ablation studies also implicate the DMH in energy regulation. Electrolytic
(Bellinger, Bernardis & Brooks, 1979; Bernardis, 1975), kainic acid (Bellinger &
Williams, 1983) or ibotenic acid (Belinger, 1987; Chou, Scammell, Gooley, Gaus,
Saper, et al., 2003) lesions of the DMH produce hypophagic animals that maintain
lower body weights. Dorsomedial hypothalamic lesioned (via ibotenic acid)
animals are able to respond to a 24-hour fasting period, which results in a further
decrease in body weight. They do so by increasing food intake until their post-
operative body weight is restored (Chou et al., 2003). This suggests that DMH
lesioned animals are still able to regulate intake and body weight, but just at a
reduced level.
Another interesting finding regarding the DMH and eating involves the role
of the nucleus in circadian rhythms. Chou et al. (2003) showed that ibotenic acid
lesions of the DMH disrupt the circadian rhythmicity of many behaviors including
eating. Animals in the experimental lesion group showed an overall decrease in
eating compared to controls; however, the pattern of eating throughout the
light/dark cycles also was disrupted. For instance, the animals exhibited increases
in eating during the light cycle and decreases during the dark phase compared to
controls. The same experiment showed that while lesions of the LH resulted in
26
overall reductions in eating, the ablations did not cause any circadian alterations.
That is, the animals continued to show the greatest eating during the dark phase.
The involvement of the DMH in circadian rhythms is significant considering that
the suprachiasmatic nucleus (SCN) of the hypothalamus is often thought to be the
sole regulator of circadian rhythmicity (Schwartz & Gainer, 1977; Silver, LeSauter,
Tresco & Lehman, 1996).
1.3.1.5 Ventromedial Hypothalamus (VMH)
Following the observations that lesions of the VMH lead to immediate,
voracious feeding, the VMH, at the time, was justifiably classified as the neural
satiety center. Since this area, once thought to be crucial for the induction of
satiety has been the focus of many debates, its history warrants some attention.
A group of researchers examining the effects of VMH lesions on eating
reported that the animals would initiate eating for several hours immediately
following surgery (Balagura & Devenport, 1970; Harrell & Remley, 1973; Becker
& Kissileff, 1974). One group reported that some of the lesioned rats started eating
prior to completely recovering from anesthesia and died as a result of choking
(Brooks, Lockwood & Wiggins, 1946). Soon after, evidence against this
hypothesis rapidly piled. Researchers claimed that VMH lesions lead to obesity,
not due to hyperphagia, but due to vagally mediated metabolic abnormalities such
as fat metabolism (Han, 1968; Cox & Powley, 1981), hyperinsulinemia (Hales &
Kennedy, 1964), and decreased gastric secretions (Ridley & Brooks, 1965;
Weingarten & Powley, 1980) that accompanied the lesions. This hypothesis gained
27
support from studies suggesting that the weight gain was not entirely due to
elevated levels of eating. In these studies, VMH-lesioned animals accumulated
more fat compared to pair-fed control animals (Han, 168; Cox & Powley, 1981).
The finding that restricted lesions of the PVN, which spare the entire VMH, result
in hyperphagia and obesity (Aravich & Scalfani, 1983; Leibowitz et al., 1981) put
an end to the ‘VMH as the satiety center’ claim. Additionally, Gold (1973)
illustrated that extremely circumscribed electrolytic lesions of the VMH did not
produce alterations in eating in male rats; however, when the lesions overflowed to
surrounding areas, the magnitude of the elevated eating and obesity was
proportional to the extent of overflow.
Despite the evidence against the VMH as a structure important in eating
behavior, the role of the nucleus in energy homeostasis has been resurrected. The
discovery of sexual dimorphism in VMH lesion weight gain provided explanation
for some of the disparate findings. It was shown that female rats and mice gained
substantially more weight following lesions compared to males (Cox, Kakolewski
& Valenstein, 1969; Kakolewski, Cox & Valenstein, 1968; King & Frohman, 1986;
Sanders et al., 1973), which explained why some research groups did not find
changes in eating following lesions since most animals used in lesion studies were
male. Although the role of the VMH as the satiety center of the brain is entirely
outdated, currently, the role of the VMH in eating should not be discarded.
28
1.3.1.6 Tuberomammillary Nucleus (TM)
This hypothalamic nucleus is seldom discussed as an important nucleus
involved in eating behavior; however, the TM is the major production site of the
neurotransmitter histamine (Garbarg, Barbin, Geger & Schwartz, 1974; Panula,
Yang & Costa, 1984; Steinbusch & Mulder, 1985; Wouterlood & Steinbusch,
1991), which has been shown to be a potent inhibitor of eating. Findings of
research manipulating both central and peripheral levels of histamine implicate this
neurotransmitter as a powerful appetite suppressor in cats, rats, goats, and chicks
(Machidori, Sakata, Yoshimatsu, Ookuma, Fujimoto, et al., 1992; Clineschmidt &
Lotti, 1973; Kawakami, Bungo, Ohgushi, Ando, Shimojo, et al, 2000). Ookuma
and colleagues (1993) have shown that infusions of -fluromethylhistamine
(FMH), a “suicide” inhibitor of histidine decarboxylase (HDC), into the third
cerebroventricle induces feeding behavior.
Studies investigating the significance of histamine in ingestive behaviors
were initiated following the observation that some antidepressant and antipsychotic
drugs stimulated appetite and led to weight gain in humans (Kalucy, 1980).
Examinations attempting to link these drugs inducing weight gain revealed that
they possess anti-histaminergic properties. In particular, binding assays have
shown them specifically to be potent histamine 1-receptor (H
1
R) blockers (Hill &
Young, 1978; Taylor & Richelson, 1980). The involvement of H
1
R in histamine’s
hypophagic abilities is substantiated by several studies. Intraperitoneal injections
of metorpine, which elevates brain histamine levels by inhibiting the conversion of
29
histamine to the inactive metabolite methylhistamine, suppresses food intake
(Leklin et al., 1994, Lelklin et al., 1995). Leklin and Tuomisto (1997)
demonstrated that this hypophagia is abolished with pretreatment of the H
1
R
antagonist mepyramine, suggesting that histamine exerts its depressive effects
through H
1
R. It is reported that H
1
R antagonists elicit feeding when they are
infused into the third cerebroventricle (Fukagawa et al., 1989), into the PVN
(Ookuma et al., 1989) or into the VMH (Sakata et al., 1988). Interestingly,
reduction of neuronal histamine appears to have a hyperphagic effect on eating.
1.3.2 Hindbrain Structures
1.3.2.1 Area Postrema (AP)
Neuroanatomical studies have established that the AP receives information
from the gut via the afferent vagus nerve (Leslie & Gwyn, 1984; Shapiro &
Miselis, 1985). This cranial nerve serves as a major afferent input to the brainstem
from the gastrointestinal system (Martin, Rogers, Novin & VanderWeele, 1977).
As such, it is an important structure in detecting gastric distention, malaise, gastric
emptying, among other gastrointestinal mediated responses that are pertinent for
eating behavior. The AP also receives input from hypothalamic structures such as
the PVN and the DMH (Shapiro & Miselis, 1985), which implies that this
circumventricular structure also receives information regarding eating from other
brain regions. In addition, it sends and receives information from the NST and the
PBN, predominantly the lateral subdivision, (van der Kooy & Koda, 1983; Shapiro
30
& Miselis, 1985), both structures that are integrally important in the energy
regulation (discussed below).
Direct evidence for the role of the AP in eating from lesion studies is
difficult to gather since ablations of the structure produce illness in animals.
Therefore, findings of such studies must be interpreted with caution. One study
showed that animals with lesions of the AP, which are allowed 6 weeks to recover,
displayed hypophagia and decreased weight (Lutz, Mollet, Rushing, Riediger &
Scharrer, 2001), which was interepreted as a role of the nucleus in feeding.
Unfortunately, since inactivation of this area potentially produced chronically sick
animals, this conclusion should be revisited. It may have been that the lesioned
animals were expressing illness-inudced food decrements and weight loss.
A second approach to determining the role of the AP in eating has been to
inspect the effects of anorexigenics in AP-lesioned animals. Studies have shown
that the appetite suppressors amylin (Lutz et al., 2001), calcitonin gene-related
peptide (Lutz, Senn, Althaus, del Prete, Ehrensperger, et al., 1998a), CCK
(Edwards, Ladenheim & Ritter, 1986), and estradiol (Bernstein, Courtney &
Braget, 1986) are rendered ineffective in animals with lesions of the AP and in
some of the cases the surrounding NST, implying that neurons in this area are
necessary for the expression of hypophagia produced by these substances. Once
again, although animals in the studies mentioned above were allowed a substantial
amount of time to recover following lesion surgeries, they all continued to express
hypophagia and lowered body weight, which taints the results of these studies. For
31
instance, since the animals were already at the lower end of their weight range, the
effects of the anorexigenics may have been masked by the decrease in eating and
body weight already produced by the lesions themselves (Roy & Wade, 1977).
One way to potentially circumvent this problem is through the use of
temporary AP lesions (approximately 1 hour inactivation), which may preclude the
illness of animals. One disadvantage to this approach is that the role of the AP in
short-term satiety agents can be best studied since the brief length of the
inactivation should overlap with the time the satiogenics produce their effects. A
possible approach for examining the role of the AP in long-term satiety agents
using temporary AP lesions exists. First, the length of time the agent produces its
reductions in eating needs to be determined. If the AP is going to be inactivated for
an hour and the agent produces reductions in eating up to 5 hours following its
administration, then the agent can be administered 4 hours before the inactivation
begins. This way, the final hour that the agent is active will overlap with the 1 hour
inactivation of the AP.
1.3.2.2 Nucleus of the Solitary Tract (NST)
The NST and the PBN (discussed in the section that follows) are major
relays for taste and malaise information, both of which play a critical role in eating
behavior. The taste and malaise pathways will be discussed in further detail in the
following chapter and therefore only a summary will be provided here. From the
cranial nerves, gustatory information is transmitted to the rostral portion of the NST
(Torvik, 1956). The information is then projected to the ipsilateral medial or
32
“gustatory” PBN (Norgren, 1978, 1984), which in turn sends this information to
forebrain structures through the thalamus. A second, less defined route by which
taste information reaches the forebrain involves mono-synaptic projections from
the medial PBN to the central nucleus of the Amg, the substantia innominata, the
LH, the BNST, and the insular cortex (Norgren, 1974, 1976; Saper & Loewy, 1980;
Shipley & Sanders, 1982; Fulwiler & Saper, 1984; Moga, Herbert, Hurley, Yasui,
Gray, et al., 1990; Bernard, 1993).
The pathway for malaise information follows very closely to the taste
pathway. This information is carried from the viscera by the vagus nerve to the AP
(Baldino & Wolfson, 1985), which transmits the information to the caudal NST
(Cechetto, 1987). The NST also receives direct connections from the vagus nerve
that carries gustatory (Hamilton & Norgren, 1984) and visceral information (Kalia
& Sullivan, 1982). Next, the caudal NST sends extensive projections to the
ipsilateral lateral PBN (Lowey & Burton, 1979; Saper & Lowey, 1980), which is
the relay station for ascending visceral information from the NST to the thalamus,
hypothalamus, and the Amg (Cechetto, 1987).
The caudal NST is thought to partially be involved in tumor anorexia. As
the name suggests, tumor anorexia is the subsequent decreases in eating and body
weight that are observed following the growth of tumors (Mordes & Rossini,
1981). A role for the peptide tumor necrosis factor (TNF) in tumor anorexia has
been proposed given that the agent causes reductions in eating and body weight
(Beutler & Cerami, 1987; Socher, Friedman & Martinez, 1988). Combined lesions
33
of the AP and the caudal NST are shown to attenuate anorexia produced both by
Leydig tumors, which cause elevations in estradiol, and by TNF (Bernstein, 1996);
however, since these two regions are sites of vagal afferent projections, any
attenuation could have been due to an interference with vagal function (Bernstein,
1996). The illness caused by lesions of the AP further complicates the
interpretation of these findings.
More compelling evidence for the role of the NST in eating is provided by
studies showing that the nucleus is a site of production for eating-related agents.
For instance, although the ARC is the main site for POMC synthesis, a small
population of neurons in the NST also manufactures the catabolic peptide
(Sawchenko, Swanson & Joseph, 1982). Similarly, the satiety-eliciting agents
glucagon-like peptides 1 and 2 (GLP-1/GLP-2) are produced in the NST (Jin, Han,
Simmons, Towle, Lauder, et al., 1988; Larsen, Tang-Christensen, Holst & Ørsky,
1997). As will be discussed shortly, the NST also is a key target for peripheral
signals that regulate meal initiation and termination and body weight, further
insinuating involvement of the nucleus in eating behavior.
1.3.2.3 Parabrachial Nucleus (PBN)
The Lateral Parabrachial Nucleus
The parabrachial nucleus, particularly the lateral subdivision, is implicated
in eating behaviors. However, the results of studies examining the effects of lateral
PBN lesions on eating and body weight have been contradictory. One of the
earliest studies on the lateral PBN and eating showed that electrolytic lesions of the
34
structure produced increases in eating and body weight in female Sprague-Dawley
rats 3 weeks following the surgery and obesity 12 weeks following surgery (Nagai,
Ino, Yamamoto, Nakagawa, Yamano, et al., 1987). This would suggest that the
lateral PBN plays a role in satiety; however, since the researchers used electrolytic
lesions, the possibility that the destruction of fibers of passage produced the
hyperphagia could not be discarded. More importantly however, histological
examination of the lesions revealed extensive damage which included the lateral
PBN, the superior cerebellar peduncle, the external and central nucleus of the
inferior colliculus, the sagulum nucleus, the dorsal nucleus of the lateral lemniscus,
and the ventral spino-cerebellar tract. In another study, neither lesions of the lateral
nor medial PBN affected eating during a 60-minute test following 24-hour food
deprivation. Surprisingly, in a companion paper, non-deprived medial PBN and
lateral PBN-lesioned rats showed reductions in baseline eating (Trifunovic &
Reilly, 2002), a finding that was inexplicable to the authors since in prior studies,
lesions had no effect on baseline levels of consumption. Finally, a study in which
lesions were restricted to the external portion of the lateral PBN found that
approximately a week following ibotenic acid lesions, animals did not exhibit an
increase in weight (Li, Spector & Rowland, 1994), but rather their intake and
weights were similar to those of the sham operated controls.
The role of the lateral PBN in eating behavior has been revealed by those
studies that have investigated the involvement of this structure in the effects of the
satiety agents CCK and serotonin. Excitotoxic lesions of the entire lateral PBN,
35
which do not affect baseline levels of consumption, abolish CCK-induced anorexia,
while destruction of the medial PBN leaves CCK hypophagia intact (Trifunovic &
Reilly, 2001). In addition, injections of CCK produce c-Fos-like immunoreactivity
(c-FLI) activation in the lateral PBN (Inagaki, Shiotani, Yamano, Shiosaka, Takagi,
et al., 1984) and lesions that include the lateral PBN abolish c-FLI normally
expressed in the VMH by an injection of the anorexigenic CCK (Nagai et al.,
1987). Taken together, these findings suggest that the decrease in consumption
produced by CCK involve the lateral PBN and possibly its projections to the VMH.
Studies implicate the lateral PBN in serotonin-induced hypophagia as well.
Unilateral infusions of the serotonergic agonist
D
-fenfluramine (serotonin releaser
and reuptake inhibitor) into the lateral PBN of male rats reduce food intake, while
infusions of the agent in surrounding brain regions have no effect (Simansky &
Nicklous, 2002). Administering the selective serotonin
1B
receptor agonist into the
lateral PBN also produces decrements in eating (Lee et al., 1998; Simansky &
Nicklous, 2002), which is blocked by a selective serotonin
1B
receptor antagonist
(Simansky & Nicklous, 2002), but not by serotonin
2C
antagonists (Kennett &
Curzon, 1988). These finding implicate the lateral PBN in serotonin-induced
anorexia. Unfortunately, the results of lesion studies have not been as unequivocal.
A study examining the effects of ibotenic acid lesions of the lateral PBN on
serotonin-induced anorexia found that the lesions blocked the hypophagia normally
observed following injection of a serotonin agonist dexfenfluramine (DFEN; Li et
al, 1994). This finding was contradicted in a study by Trifunovic and Reilly
36
(2001), which showed that excitotoxic lesions of the entire lateral PBN did not
affect DFEN hypophagia, while destruction of the medial PBN abolished the
serotonin agonist induced anorexia.
On the other hand, immunohistochemical studies specifically implicate the
external lateral PBN in DFEN-induced anorexia. The serotonin agonist normally
produces c-fos activation in the lateral PBN, central Amg, and the laterodorsal
BNST in intact animals, which is eliminated in lateral PBN-lesioned animals (Li et
al., 1994). This suggests that DFEN does not directly act on serotonin receptors in
the central Amg and the BNST and that its activation of these forebrain structures
is a secondary response to activation of neurons in the external lateral PBN.
Hence, the projections from the lateral PBN to the central Amg and the BNST may
be critical in serotonin-induced anorexia.
The lateral PBN also contains receptors shown to modulate feeding
responses. Stimulation of any of the opioid receptors (μ, κ, δ subtypes) produces
increases in eating in mammals, including rats (Polidori, de Caro & Massi, 2000;
Silva, Hadjimarkou, Rossi, Pasternak & Bodnar, 2001), mice (Asakawa Inui,
Momose, Ueno & Fujino, 1998), and rabbits (Gosnell & Lipton, 1986) and the
lateral PBN contains all three receptor subtypes (Mansour, 1995). A study by
Wilson, Nicklous, Aloyo & Simansky (2003) showed that infusions of a μ opioid
receptor agonist DAMGO into the lateral PBN increased food intake in male rats,
while infusions into the surrounding areas were ineffective. Additionally, the
authors showed that infusions of a non-selective opioid antagonist completely
37
blocked the hyperphagic effect of DAMGO without affecting baseline levels of
food consumption.
The Medial Parabrachial Nucleus
The medial PBN has been shown to be involved in the negative contrast
effect observed in consummatory behaviors. When animals are shifted from a low
to a high reward situation, they exhibit a suppressed level of the low reward
compared to animals that receive only the low reward (Flaherty & Checke, 1982;
Flaherty & Rowan, 1985; Capaldi & Sheffer, 1992). This negative contrast effect
is often discussed as an emotional response to shifted rewards. Similar results are
obtained when shifts are made from high concentration to low concentration of
sucrose (Becker & Flaherty, 1982; Becker et al., 1984). For instance, animals
given a 4% sucrose solution after being exposed to a 34% sucrose solution,
consume less of the 4% solution than control animals given 4% sucrose solution
alone. This negative contrast effect is eliminated in animals with ibotenic acid
lesions of the medial PBN (Grigson, Spector & Norgren, 1994) suggesting that an
intact medial PBN is essential for this consummatory effect and is involved in the
emotional responses to shifted rewards pertaining to food.
1.3.3 Forebrain Structures
1.3.3.1 Amygdala (Amg)
The role of the Amg in eating behavior is varied and complex. Electrolytic
lesions of various nuclei within the limbic structure have revealed disparate results.
Studies have shown that lesions of the basolateral Amg produce aphagia (Fonberg,
38
1971), while lesions of the basomedial nucleus result in hyperphagia (Fonberg,
1966). Predicated on these findings, Fonberg purported a dual control of eating by
the limbic structure (Fonberg, 1974). Since these studies used electrolytic lesioning
techniques, the possibility that the results are due to damage to fibers of passage
should be considered. Research using excitotoxic kainate acid lesions of the central
nucleus of the Amg have shown that destruction of this area results in decreases in
eating (Hajnal, Sandor, Jando, Vida, Czurko, et al., 1992). Some researchers
ascribe the effects on eating imposed by amygdaloid lesions to encroaching damage
to the striatum. To test this possibility, various groups of researchers have made
either discrete lesions of the central, lateral, and medial amygdala or combined
lesions of these nuclei with lesions of the striatum (Schoenfeld & Hamilton, 1981).
The results indicated that lesions restricted to the amygdaloid nuclei had no effect
on food intake or body weight, while lesions that encroached into the striatum did.
This finding is supported by a second study which illustrated that discrete damage
to the central nucleus did not affect eating or body weight, while damage to the
striatum decreased both (Dacey & Grossman, 1977). As such, these lesion studies
alone do not implicate the amygdala in eating behavior. Instead, other methods of
determining its role in eating are necessary.
Similar to the effects produced by medial PBN ablations, electrolytic
lesions of the medial, but not the lateral Amg, have been shown to disrupt the
negative contrast effect of consummatory behavior. Neurally intact animals
initially exposed to a 34% sucrose solution drink less of a 4% sucrose solution
39
compared to animals given only the 4% solution. This effect is abolished in
animals with medial amygdaloid lesions (Becker et al., 1984) suggesting a role for
the nucleus in food-related rewards.
Although the complications due to lesion studies cast some doubt on the
role of the Amg on eating, studies utilizing different techniques solidify the
structure’s role in eating. For instance, the central Amg has been shown to be
involved in the circuitry for serotonin-induced anorexia. Administration of the
serotonin agonist DFEN produces the induction of c-fos in the central Amg and the
laterodorsal BNST (Li et al., 1994). Further, activation in these areas is eliminated
following lesions of the external lateral PBN, which also eliminate the serotonin-
produced hypophagia. This finding shows that projections from the lateral PBN to
the central Amg and BNST are involved in the serotonin anorexia. Since lesion
studies have not confirmed the exact role of the Amg in eating, it is difficult to
decipher whether the activation of the central Amg following DFEN infusion in the
lateral PBN are indicative of activation or inhibition. If the central Amg is
involved in feeding behavior, it could be that the central Amg is inhibited following
lateral PBN administration of DFEN. If the central Amg is involved in satiation,
then the central Amg is probably being activated by the lateral PBN-infused DFEN.
Nevertheless, the loss of activation in the central Amg following lesions of the
lateral PBN and DFEN application implies that the nucleus is associated with the
decreases in eating produced by the serotonergic agonist.
40
There is evidence of an opioid signaling pathway between the NST and the
central Amg that solidifies the role of the central Amg in eating behavior. First,
bilateral infusions of an μ receptor agonist, DAMGO, in both the NST (Kotz,
Billington & Levine, 1997) and the central Amg (Gosnell, 1988) result in increases
in feeding behavior. Second, infusions of an opioid antagonist naloxone into the
central Amg blocks the feeding induced by DAMGO (Gosnell, 1988). Although,
this effect has not been demonstrated in the NST, it has been shown that naloxone
in the central Amg blocks NPY-induced feeding (Kotz, Billington & Levine, 1995).
Predicated on these findings, a study was conducted which showed that
hyperphagia induced by infusing DAMGO into the NST was inhibited by infusions
of an opioid antagonist injected into the central Amg (Giraudo, Kotz, Billington &
Levine, 1998). The reverse was true as well. These results suggest a potential
opioid-opioid signaling pathway between these two structures.
1.3.3.2 Bed Nucleus of the Stria Terminalis (BNST)
The involvement of the BNST in serotonin-induced anorexia has been
discussed in the previous section (Li, Spector & Rowland, 1994); however, this
structure has been shown to play an important role in the suppression of eating
produced by CRF as well (Ciccocioppo, Fedeli, Economidou, Policani, Weiss, et
al., 2003). Since the BNST contains receptors for both CRF, particularly the CRF
2
subtype (Chalmers, Lovenberg & de Souza, 1995) and receptors for the CRF
antagonist, N/OFQ, researchers purported the structure to be important in CRF
induced anorexia and for its reversal by N/OFQ (Ciccocioppo, et al., 2003). A
41
series of studies showed that microinjections of CRF into the BNST produced
marked hypophagia in food-deprived rats, while injections into the central Amg
had no effect. Further, injections of N/OFQ into the BNST blocked the anorexia
produced by infusions of CRF made into both the BNST and into the ventricles.
Injections of the antagonist into the central Amg, VMH or PVN had no effect on
CRF produced decrements in eating. Equally important is the finding that
injections of N/OFQ into the BNST did not affect basal levels of eating
(Ciccocioppo, et al., 2003; Ciccocioppo, Cippeitelli, Economidou, Fedeli & Massi,
2004).
The BNST also may be involved in reductions in eating produced by
estradiol. The hormone produced c-fos activation in the laterodorsal region of the
BNST 24 hours following its injection, which is precisely when reductions in
eating produced by estradiol are observed (Chambers, Hintiryan & So, unpublished
manuscript). The serotonin agonist DFEN also produces activation in this same
region, which is eliminated with lesions of the lateral PBN along with serotonin-
induced anorexia. This suggests that the conncections between the lateral PBN and
the BNST in serotonin anorexia. Whether lateral PBN lesions have the same effect
on estradiol-induced BNST activation and estradiol anorexia remains to be
determined.
Although not directly tested, evidence suggests a role for the BNST in the
orexigenic effects of both orexin and MCH. Immunohistochemical studies have
verified that orexin and MCH fibers originating from the PFA and LH innervate the
42
BNST (Peyron et al., 1998; Bittencourt et al., 1992). Given that this forebrain
structure receives projections from hypothalamic structures and neuropeptides that
are heavily involved in eating, a role for the BNST in eating behavior could be a
possibility.
1.4 Short-term Regulation of Eating
There are separate mechanisms that work in conjunction to control the size
of individual meals and to regulate body weight. Although the neural machinery is
highly conserved for these separate processes (control of meal size/frequency and
body weight), the molecular signals that initiate each process are disparate.
The phrase short-term control of eating refers to the initiation or termination
of an individual meal as opposed to long-term control, which refers to body weight
regulation over an extended period of time. There is a plethora of identified
peripheral satiety signals that are implicated in the cessation of a meal, while those
involved in the feeding response, termed here as peripheral feeding signals, are
more limited. Prior to discussing these signals individually, it should be clarified
that the term satiation refers to the physiological process that begins during a meal
and causes the termination of eating (Blundell, 1979; Smith, 1998). In the
literature, this is differentiated from postprandial satiety, which is the interval
between meals during which an animal does not engage in eating.
The central nervous system can gain access to information regarding the
initiation or termination of a meal carried by peripheral signals by at least two
mechanisms. The first involves the vagus nerve that terminates on the AP (Shapiro
43
& Miselis, 1985) and the NST (Cechetto, 1987) and the second involves the blood,
by diffusion through the blood-brain barrier or through areas where the blood-brain
barrier is poorly developed (i.e. the AP or ARC). From the NST, direct and
indirect projections through the PBN reach the PVN, DMH, ARC, LH, central
Amg and the BNST (Saper, 2000).
1.4.1 Glucose and the “Glucostat” Hypothesis
One of the first peripheral compounds speculated to be involved in the
regulation of eating was glucose. The popular “glucostat” hypothesis purported
that circulating levels of glucose serve as the negative feedback signal to the central
nervous system, such that decreases in the fuel initiate a meal, while the elevated
glucose levels following ingestion subsequently cause the termination of a meal
(Mayer, 1955; Anand, Chhina, Sharma, Dua & Singh, 1964). There is evidence to
support the involvement of glucose levels in the initiation and termination of a
meal. Infusions of 2-deoxyglucose (2DG), a competitor of glucose that causes
hypoglycemia, into the hepatic portal vein stimulate immediate eating (Novin,
VanderWeele & Zidek, 1973). Since the hepatic portal vein transports blood from
the small intestines into the liver, this finding suggests that the liver contains
receptors that are glucose sensitive, meaning they respond to decreases in the level
of glucose. Glucose responsive cells on the other hand are glucose receptors that
respond to increases in the level of glucose. The liver has been shown to contain
glucose responsive receptors as well since infusions of glucose into the hepatic
44
portal vein during a meal reduce the size and duration of the first meal of the dark
cycle (Langhans, Grossman & Geary, 2001).
Vagotomies have been shown to eliminate the feeding response elicited by
the infusion of 2DG into the hepatic portal vein (Novin et al., 1973), suggesting
that the information regarding initiation of a meal signaled by decreased glucose
levels is carried from the liver to the central nervous system via the Xth cranial
nerve, perhaps by the hepatic branch of nerve.
Since glucose is the sole source of fuel for the brain, it is intuitive then that
it too contains receptors that are directly sensitive to the availability of the
metabolic fuel. Glucose can pass through the blood-brain barrier (Nicholls &
Kuffler, 1964) thereby accessing the brain through the blood. Hindbrain areas such
as the AP (Bernstein et al., 1986) and the NST (Edwards et al., 1986) contain
glucose receptors (Bird, Cardone & Contreras, 1983 for the AP; Yettefti, Orsini &
Perrin, 1997 for the NST). As such, these areas can gain access to circulating
levels of glucose through their connection with the vagus nerve or directly via the
blood.
Originally, the VMH was hypothesized to be the locus of glucoreceptors or
the “glucostat” that initiated satiety. Immunohistochemical studies have revealed
that several other hypothalamic nuclei that are involved in energy homeostasis like
the LH, the ARC, the PVN, and the DMH all stain for glucose receptors (Dunn-
Meynell, Govek & Levine, 1997; Fukuda, Ono, Nishino & Sasaki, 1984). In vitro
electrophysiological studies have shown that about 40% of the neurons in the LH
45
are glucose sensitive, while glucose responsive neurons are sparse (Ashford, Boden
& Treherne, 1990). The situation is the opposite in the VMH, where most of the
neurons are glucose responsive, while glucose sensitive neurons are rare. These
findings would suggest that the glucose receptors in the LH are involved in
producing the feeding response, while those in the VMH are involved in the
inhibition process. In the section devoted exclusively to the role of the VMH in
eating behavior, it was mentioned that contradictory findings regarding the effects
of VMH lesions on eating had left researchers wondering if the structure even
played a role in the behavior. However, this study establishes the hypothalamic
structure as a glucose responsive site and provides evidence for its significance in
eating behavior.
Following the hypothesis of glucose regulation of eating, several other
peripheral compounds involved in eating were discovered. Among these, ghrelin is
involved in the initiating of eating, while cholecystokinin (CCK), peptide YY
(PPY), glucagon-like peptide-1 (GLP-1), and gastrin-releasing peptide (GRP) are
involved with the termination of a meal. Each of these agents, as will be discussed
in more detail shortly, is released from the gastrointestinal tract, but also is
produced in the brain. Thus, it is difficult to decipher whether the effects these
peptides exert on eating are primarily of a peripheral or central source.
46
1.4.2 Peripheral Feeding Signal
Ghrelin
Ghrelin, like the peptides discussed below, is a peripheral signal that
modulates food intake and recently has received considerable attention. Unlike
most of the gastrointestinal peptides that will be discussed in this section, ghrelin
drives the feeding response rather than suppressing it. Ghrelin is a growth hormone
releasing secretagogue (GHS) that is predominantly manufactured and released
from the stomach (Kojima, Hosoda, Date, Nakazato, Matsuo, et al., 1999; Date,
Kojima, Hosoda, Sawaguchi, Mondal, et al., 2000) and was initially discovered as a
ligand for the GHS receptor (Kojima et al., 1999). Lower amounts of the peptide
are produced by the kidneys (Mori et al., 2000), bowel (Date et al., 2000), and the
hypothalamus (Kagotani, Sakata, Yamazaki, Nakamura, Hayashi, et al., 2001),
specifically in the ARC (Cowley, Smith, Diano, Tschöpp, Pronchuck, et al., 2003).
The signals that stimulate ghrelin release are not known; however, since
ghrelin levels increase during fasting in rats (Kojima et al., 1999), peak pre-
prandially in humans (Cummings, Purnell, Frayo, Schmidova, Wisse, et al., 2001)
and decrease following a meal (Cox, 1998; Tschöpp, Smiley & Heiman, 2000), a
hunger-inducing role of the hormone is suggested. In humans, ghrelin levels rise in
response to weight loss due to dieting (Cummings, Weighle, Frayo, Breen, Ma, et
al., 2002) or due to pathological states such as cancer anorexia or during eating
disorders (Otto, Cuntz, Fruehauf, Wawarta, Folwaczny, et al., 2001). The finding
that provides direct evidence for its involvement in food intake is that
47
intracerebroventricular infusions of the peptide dose-dependently increase food
intake and subsequently lead to obesity in rats (Wren, Seal, Cohen, Brynes, Frost,
et al., 2001). More specifically, infusions of ghrelin into the PVN (Olszewski,
Grace, Billington & Levine, 2003) and into the ARC (Bagnasco et al, 2003),
produce increases in eating, thereby suggesting a role for the hormone in these
nuclei. The hormone also is effective in producing hyperphagia in humans (Wren
et al., 2001).
The endocrine hunger effect of ghrelin could be mediated via two
mechanisms. The first involves the vagus nerve since ghrelin receptors can be
found on vagal afferent neurons. Additionally, vagotomies abolish the initiation of
eating that is observed following intravenous injections of ghrelin (Date,
Murakami, Toshinai, Matsukura, Nijima, et al., 2002). The second route, which
has been overlooked in the literature, is via the blood stream and into the ARC.
This is a possibility considering the ARC is a site for ghrelin hyperphagia and has a
penetrable blood brain barrier (Broadwell & Brightman, 1978).
Studies attempting to elucidate the neural mechanisms underlying ghrelin-
mediated meal initiation have focused on the ARC and NPY for several reasons.
First, ghrelin may be produced in the ARC (Kojima et al., 1999). Second,
specifically NPYergic neurons in the ARC contain GHS receptors (Willensen,
Kristensen & Romer, 1999). Third, direct intra-arcuate infusions of ghrelin
produce increases in eating (Wren et al., 2001). Electron microscopy has revealed
that immunoreactive ghrelin dendrites receive afferent connections from unknown
48
terminals in the ARC (Lu, Guan, Wang, Uehara, Yamada, et al., 2002).
Intracerebroventricular administration of ghrelin increases NPY mRNA (Shintani
et al., 2001) and antagonism of NPY and AgRP abolish the feeding response
elicited by ghrelin (Nakazato, Murakami, Date, Kojima, Matsuo, et al., 2001).
Together, these findings suggest a possible relationship between ghrelin and NPY
and possibly AgRP.
Another hypothalamic pathway involved in ghrelin feeding involves the
orexin-synthesizing neurons of the LH. One study showed that ghrelin containing
axon terminals synapse on orexinergic neurons in the LH (Toshinai, Date,
Murakami, Shimada, Mondal, et al., 2003). Further, ghrelin is rendered ineffective
in the absence of orexin as illustrated by orexin knock-out mice (Toshinai, Date,
Murakami, Shimada, Mondal, et al., 2003). There is evidence of a second
orexigenic compound, namely NPY, which may be involved in ghrelin feeding.
For instance, blocking synthesis of orexin A and orexin B (via anti IgG) weakens
the effects of ghrelin on eating. Blocking NPY receptors in addition to blocking
orexin exaggerates this attenuation suggesting the involvement of NPY in the
ghrelin-orexin pathway. This combined attenuation is greater than the sum of the
independent reductions produced by each of the peptides alone, suggesting an
interaction between orexin and NPY rather than an additive effect. However,
ghrelin infused into the lateral ventricles produces activation in orexinergic
neurons, which is not reversed following administration of NPY anti IgG,
suggesting that NPY is not necessary for orexin-mediated ghrelin action on eating.
49
The role of MCH in ghrelin elicited feeding also has been examined. The
evidence to date suggests that ghrelin’s actions are independent of MCH. First,
ghrelin does not produce activation in MCH neurons like it does in orexin-
containing neurons (Toshinai et al., 2003). Second, pretreatment with anti-MCH
IgG does not affect ghrelin’s hyperphagic effects.
Although most studies examining the effects of ghrelin on eating have
focused on the ARC, hindbrain areas such as the AP and NST also may be
important in the hyperphagic effects of this peripheral peptide. Growth hormone
secretagogue receptors are also expressed in these structures (Guan, Yu, Palyha,
McKee, Geighner, et al., 1997) and infusions directly into the third and fourth
ventricles, which activate these hindbrain structures, produce increases in eating
comparable to those reported after infusions into the ARC (Faulconbridge,
Cummings, Kaplan & Grill, 2003).
1.4.3 Peripheral Satiety Signals
Cholecystokinin (CCK)
Cholecystokinin is a peptide that is released by the duodenum in response to
a meal (Moran & Schwartz, 1994) and serves as a negative feedback signal that
controls the size of a meal (Gibbs, Young & Smith, 1973). Cholecystokinin also is
produced neurally in the hypothalamus and in the neocortex (Rehfeld, 1978), in
quantities that exceed those produced in the small intestines. Specifically, CCK is
produced in PVN neurons that also express CRF (Mezey, Reisine, Skirboll,
Beinfeld & Kiss, 1986). The satiating effects of CCK have been thoroughly and
50
consistently demonstrated across various species and varying routes of
administration (Gibbs et al., 1973; Gibbs, Falasco & McHugh, 1976; Gibbs &
Smith, 1977). In the LH, CCK terminals release the peptide in response to both
vagal stimulation (Schick, Schusdziarra, Yaksh & Go, 1994) and after intragastric
meal load (Schick, Yaksh & Go, 1986).
Studies exploring the neural circuitry of CCK have led to some interesting
findings. For instance, when connections between the hindbrain and forebrain are
severed by supracollicular transections, peripherally administered CCK becomes
effective in decreasing intake of a sucrose solution in 24-hour fasted rats (Grill &
Smith, 1988). This result suggests that hindbrain structures are involved in the
satietogenic effects of the peptide. It also suggests that the termination of a meal
can occur in the absence of hypothalamic influences (Grill & Smith, 1988). This
hypothesis is corroborated by a separate study demonstrating that decerebrate
animals continue to decrease their intake of sucrose following intragastric infusions
of milk (Grill, 1980). Evidence to the contrary also exists. Bilateral PVN lesions
block the satiating effects of CCK (Crawley & Kiss, 1985). Additionally, central
infusions of CCK into discrete hypothalamic nuclei (i.e. DMH, PVN, VMH, and
LH) produce decreases in eating (Blevins et al., 2000) implicating a role for CCK
satiation in the diencephalic structure. Given the complexity of eating behavior, it
is not improbable that redundancy exists within the different levels of the eating
system. It may be that when the feedback signal CCK is released by the duodenum
due to food intake, the hindbrain structures are involved in terminating an
51
individual meal; however, hypothalamic structures come into play with the
peptide’s involvement in the circuitry of long-term satiety. For instance, leptin, a
compound involved in the regulation of body weight (discussed in later sections),
increases the satietogenic potency of CCK, which is accompanied by increased c-
fos activation in the AP and the NST (Emond, Schwartz, Ladenheim & Moran,
1999) than that produced by either CCK or leptin alone. Although c-fos expression
in the PVN does not differ from control levels in response to CCK administration,
the combination of leptin and CCK produces greater neuronal activation in the
PVN compared to that produced by leptin alone (Barrachina, Martinez, Wang, Wei
& Yvette, 1997; Wang et al., 1998; Emond et al., 1999). Together, these findings
suggest an interaction between the PVN and NST in the augmented satietogenic
response to CCK during weight regulation. This is not surprising considering that
reciprocal connections between the NST and PVN exist (ter Horst et al., 1989).
Evidence for the involvement of the NST in CCK is supplemented by lesion
studies. Inactivation of the commissural and medial NST, which is the target of
afferents from the gastric branch of the vagus nerve (Leslie, Gwyn & Hopkins,
1982; Shapiro & Miselis, 1985), eliminates the satietogenic effects of CCK
(Edwards et al., 1986). This peripheral information regarding energy state can
reach the hypothalamus via the NST, which has both direct and indirect
connections to the hypothalamus and other neural areas implicated in eating (Saper,
2002). For instance the NST sends projections directly or indirectly through the
52
PBN to the PVN, DMH, ARC and LH, the central amygdala, and bed nucleus of
the stria terminalis.
Peptide YY (PYY)
Peptide YY is a hormone released in the periphery that mediates food
intake. Peptide YY is synthesized in the endocrine L-cells of the ileum and is
released from the small intestines post-prandially in proportion to the caloric
content of a meal (Adrian, Savage, Sagor, Allen, Bacarese-Hamilton, et al., 1985;
Pedersen-Bjergaard, Host, Kelbaek, Schifter, Rehfeld, et al., 1996). One of the
major forms of PYY that is most commonly studied is PYY
3-36
. This form of PYY
is found in intestinal endocrine cells and in the circulation (Eberlein, Eysselein,
Schaeffer, Layer, Grandt, et al., 1989; Grandt, Schimiczek, Beglinger, Layer,
Goebell, et al., 1994). Exogenous administration of the hormone produces
hypophagia in both rats and humans (Batterham, Cowley, Small, Herzog, Cohen, et
al., 2002). Evidence thus far suggests that PYY produces hypophagia by acting on
NPY and POMC in the ARC (Batterham et al., 2002), which it can directly access
through the blood due to the poorly developed blood-brain barrier at the level of the
ARC (Broadwell & Brightman, 1978). Studies show that there is a significant
increase in c-fos expression in the lateral ARC and specifically in the POMC
neurons following a single injection of PYY
3-36
, which led researchers to further
investigate the role of the ARC in PYY
3-36
hypophagia. It was found that central
infusions of PYY
3-36
directly into the ARC produce reductions in eating (Batterham
et al., 2002; Batterham & Bloom, 2003) further corroborating this hypothesis.
53
Finally, Battterham et al. (2002) showed that infusions of PYY
3-36
depolarized 19
out of 22 ARC POMC neurons tested further solidifying the role of ARC POMC
neurons in the hormone’s satietogenic properties.
Peptide YY also has been shown to act on ARC NPYergic neurons. Studies
investigating the receptor subtype involved in PYY
3-36
satiation have speculated the
Y
2
receptor to be involved (Batterham et al., 2002). The Y
2
receptor is an
inhibitory receptor expressed on NPY neurons in the ARC (Broberger, Landry,
Wong, Walsh & Hökfelt, 1997). Additionally, PYY
3-36
is shown to be an agonist
for NPY Y
2
receptors (Grandt et al., 1994). Batterham et al. (2002) demonstrated
that infusions of aY
2
agonist into the ARC decreased eating, while infusions into
the PVN were ineffective, providing evidence for the possibility of ARC Y
2
receptor mediation in PYY
3-36
hypophagia. The finding that infusions of PYY
3-36
are ineffective in Y
2
knockout mice also substantiates this claim (Batterham et al.,
2002). However, although there is elevated neuronal activity in POMC neurons in
response to PYY
3-36
administration, the POMC neurons do not express Y
2
receptors
(Broberger et al., 1997). It could be that PYY initially inhibits NPY and this
inhibition in turn disinhibits POMC neurons. This NPY and POMC connection is a
strong possibility considering that these two populations of neurons send and
receive reciprocal collaterals (Cowley, Smart, Rubinstein, Cerdan, Diano, et al.,
2001).
The brainstem has been shown to express Y
2
receptors (Lynch, Walker &
Snyder, 1989; Dumont, Fournier, St-Pierre, Schwartz & Ouirion, 1990) raising the
54
possibility for PYY brainstem access in vivo. Patch-clamp studies have
demonstrated that the inhibitory effect of PYY in the brainstem is in part due to the
inhibition of glutamatergic transmission between the NST and DMH via the
activation of Y
2
receptors (Browning & Travagli, 1997).
Glucagon-Like Peptide-1 and 2 (GLP-1/GLP-2)
Like PYY, GLP-1 and GLP-2 are produced in the endocrine L-cells of the
ileum and are released post-prandially (Bell, Santerre & Mullenbach, 1983;
Drucker, 1998). This peptide has been shown to induce satiety in both rodents
(Turton, O’Shea, Gunn, Beak, Edwards, et al., 1996; Tang-Christensen, Larsen,
Goke, Fink-Jensen, Jessop, et al., 1996) and in humans (Flint, Raben, Astrup &
Holst, 1998; Näslund, Gutniak, Skogar, Rossner & Hellström, 1998), while
antagonism of the GLP-1 receptor has been shown to increase eating (Turton et al.,
1996).
Aside from being produced in the gastrointestinal system, GLP-1 also is
synthesized in the brain, specifically in the caudal NST and the medullary reticular
nucleus (Jin et al., 1988; Larsen et al., 1997), both of which receive vagal inputs
from the gastrointestinal system. It has been shown that the hypophagic actions of
GLP-1 are at least partially mediated via the vagus nerve (Balks, Holst, von zur
Mulen & Brabant, 1997). Glucagon-like peptide-1 immunoreactive terminals can
be found in the PVN, VMH, DMH, LH (Grossman, 1975; Jin et al., 1988), and in
the Amg (Jin et al., 1988). It has been demonstrated that all of these areas along
with structures in the brainstem (Shimizu, Hirota, Ohboshi & Shima, 1987) contain
55
GLP-1 receptors, suggesting a role for this hormone in each of these neural
structures. Intra-hypothalamic infusions of GLP-1 directly into discrete nuclei have
shown that the DMH, VMH, and the LH are all involved in the satietogenic effect
of GLP-1 (Schick, Zimmermann, vorm Walde & Schusdziarra, 2003). Infusions of
this hormone into the medial Amg however have no effect on eating (Schick et al.,
2003).
Interestingly, infusions of GLP-1 into the third ventricle produces c-fos
activation in the PVN, ARC, central Amg, AP, NST, and lateral PBN (van Dijk et
al., 1996). Since infusions of this peptide into the third ventricle could not
sufficiently diffuse to the brainstem, this finding suggests that the centrally infused
GLP-1 acts directly on one of the diencephalic nuclei and that the hindbrain
structures like the AP, NST, and the lateral PBN indirectly are involved in the
circuitry of the peptide.
It should be mentioned that the inhibitory effect of GLP-1 on eating may be
due to this hormone’s antagonistic effect on gastric emptying, which is the
movement of food from the stomach into the small intestines (Näslund et al., 1998;
Schirra, Leicht, Hildebrand, Beglinger, Arnold, et al., 1998). In humans,
administration of GLP-1 slows gastric emptying of both liquid (Wettergren,
Schjøldager, Mortensen, Myhre, Christiansen, et al., 1993) and solid (Näslund et
al., 1999) foods. Additionally, this peripheral peptide affects metabolism by
causing the release of insulin and inhibiting the breakdown of glucagon (Ørskov,
56
1992) each of which could produce a positive energy state and reduce the amount
of intake.
1.5 Long-term Regulation of Eating
The neural mechanisms that are involved in body weight regulation will be
discussed in this section. Not surprisingly, the neural structures involved in this
process are identical to those involved in short-term eating although the molecular
signals that activate the circuits of weight control are different.
Despite the fact that the amount and type of food ingested by individuals
varies from one meal to the next and fluctuates across days, most adults across a
wide range of mammalian species, including humans, have the ability to maintain
energy homeostasis (Edholm, 1977; Stallone & Stunkard, 1991). That is, adults are
capable of matching precisely their energy intake with their energy expenditure
over long intervals of time. Following a calorie-restricted diet, rats will show a
marked increase in eating when given ad libitum access to chow. This hyperphagia
ensues until body weight returns to baseline levels. Once baseline is achieved, the
rats revert to their normal levels of consumption (Harris, Kasser & Martin, 1986).
This maintenance of body weight set point has been demonstrated with force-fed
rats as well. When animals are force-fed to gain weight, they consequently reduce
their intake when allowed to consume at will until their body weights return to
baseline levels (Wilson, Meyer, Cleveland & Weigle, 1990). Additionally, when
animals are fed a diet that is either high or low in calorie, they will appropriately
adjust the amount of food that they eat in order to maintain their body weight
57
(Cabanac & Lafrance, 1991). Interestingly, peripheral satiety signals lose their
efficacy in calorically-restricted animals forced to lose weight (Cabanac &
Lafrance, 1991), indicating that signals regulating body weight work in conjunction
with gastric factors to exert their effects. Body weight regulation also is apparent
in humans. Evidence for this is provided by the Bioshpere 2 study. In this
experiment, eight volunteers (4 females and 4 males) maintained themselves within
a self-sustainable microcosm for 2 years. Due to the scarcity of harvested food, the
participants were subjected to severe caloric restriction for 2 years. The data
showed that within the first 6-9 months, all of the subjects lost weight, which
returned to baseline levels approximately 6 months following the end of the project
(Walford, Mock, MacCallum & Laseter, 1999).
1.5.1 The Set Point Theory of Body Weight
One largely debated theory about the control of body weight that is highly
influential is the set point theory. The set point theory of body weight purports that
each individual has a programmed weight that the body will battle to maintain in
the face of any changes. This theory asserts that body weight is controlled by an
“adipostat”, which receives a signal from adipose tissue, informing the central
nervous system about body mass. If this feedback signal from the body fat has
deviated from the programmed set point, regulatory mechanisms are activated in
order to correct for the error. Despite its vast influence, the validity of this set point
theory is controversial and is the subject of much debate (Berthoud, 2002).

58
This ability of adult mammals to maintain neutral energy balance
compellingly indicates a robust biological mechanism involved in the regulation of
body weight set point. But how is the feeding system regulated and what cues are
involved in maintaining body weight over a long-term basis?
There are three main molecules that act as adiposity signals and are thought
to regulate body weight. It is agreed that in order for an agent to be considered an
adiposity signal, it must fulfill three requirements. It should (1) be secreted in
proportion to body fat mass, (2) affect both eating and body weight, and (3) act on
neural substrates implicated in eating (Schwartz & Seeley, 1997; Woods, Seeley,
Porte & Schwartz, 1998). Leptin, insulin, and amylin are three hormones that
meet these criteria and are considered to be the main signaling molecules regulating
body weight.
1.5.2 Peripheral Adiposity Signals
Leptin
Leptin is a hormone that is synthesized by adipocytes and is released into
the bloodstream (Zhang, Proenca, Maffei, Barone, Leopold, et. al, 1994). This
hormone becomes active after being cleaved of 21 amino acids, a process that
evidence suggests is activated by insulin secretion following increased glucose
levels after a meal (Zhang, Proenca, Maffei, Barone, Leopold, et al., 1994; Saladin,
de Vos, Guerre-Millo, Leturque, Girard, et al., 1995). Leptin is considered to be an
important adiposity signal that activates the central nervous system’s negative
feedback loop in the control of body weight. As an adiposity signal, leptin affects
59
both eating and body weight. Centrally administered leptin into the lateral
ventricles reduces food intake in both obese and lean mice (Campfield, Smith,
Guisez, Devos & Burn, 1995). Further, leptin administered Leptin deficient ob/ob
(Zhang et al., 1994), leptin resistant db/db (Campfield et al., 1995), and leptin
receptor deficient fa/fa Zucker (Chua, Chung, Wu-Peng, Zhang, Liu, et al., 1996)
mice exhibit profound hyperphagia and obesity, suggesting the hormone’s
involvement in body weight regulation.
In addition, levels of this hormone circulate in proportion to body fat
content in humans (Havel, Kasim-Karakas, Mueller, Johnson, Gingerich, et al.,
1996) and in animals (Ahren, Mansson, Gingerich & Havel, 1997; Landt,
Gingerich, Havel, Mueller, Schoner, et. al., 1998). For example, when body fat is
high, leptin levels are low, and eating is sustained at a lower level, while the
opposite is true when body fat is reduced. Although leptin levels generally
circulate in proportion to body fat, acute changes in the hormone level independent
of adiposity also occur. For instance, short-term fasting decreases leptin levels
(Weigle, Duell, Connor, Steiner, Soules, et al., 1997; Dubac, Phinney, Stern &
Havel, 1998), while re-feeding results in increases in the hormone (Weigle et al.,
1997). Although these short-term eating restrictions are not sufficient to affect
adiposity, they still produce reductions in leptin levels.
Since even very low doses of centrally administered leptin produce
reductions in eating and body weight (Campfield et al., 1995), it is speculated that
the brain is the main target for the actions of the hormone. This is supported by the
60
fact that leptin receptors have been localized in areas integrally involved in eating
such as on the NPY/AGRP neurons and POMC/CART neurons of the ARC
(Håkansson, Brown, Ghilardi, Skoda & Meister, 1998 and Shioda, Funahashi,
Nakjo, Yada, Maruta, et al., 1998 for NPY/ARGP; Joseph, Pilcher & Knigge, 1985;
Khachaturian, Lewis, Haber, Akil & Watson, 1984 for POMC; Broberger et al.,
1998; Kristensen et al., 1998; Vrang, Larsen, Clausen & Kristensen, 1999 for
CART). Leptin receptors also are localized on the orexin/hypocretin and MCH
neurons in the LH (Horvath et al., 1999 for orexin/hypocretin; Bittencourt et al.,
1992 for MCH), and on the CRH neurons in the PVN, VMH, DMH, AP, NST, and
PBN (Schwartz, Baskin, Bukowski, Kuijper, Foster et al.,1996 for PVN; Mercer,
Hoggard, Williams, Lawrence, Hannah, et al., 1996; Elmquist, Ahima, Elias, Flier
& Saper, 1998 for VMH and DMH; Elmqist, Ahima, Maratos-Flier, Flier & Saper,
1997 for AP; Mercer, Moar, Findlay, Hoggard & Adam, 1998 for the NST;Grill,
Schwartz, Kaplan, Foxhall, Breininger, et al., 2002 for PBN).
The mechanism by which leptin accesses the central nervous system
remains unclear. It is possible that the hormone gains access to the brain from
circumventricular organs lacking a blood-brain barrier such as the AP or median
eminence (Broadwell & Brightman, 1976). Alternatively, there may be a saturable
transport system for leptin through the blood-brain barrier (Banks, Kastin, Huang,
Jaspan & Maness, 1996) although potential neural access through the vagus also
should be considered.
61
Although all of the aforementioned brain areas contain leptin receptors, the
ARC is thought to be the central site for leptin action. Specifically, studies have
concentrated on leptin responsive NPY/AgRP and POMC/CART neurons in this
hypothalamic nucleus. Direct evidence for this hypothesis is provided by the
finding that infusions of leptin into the ARC produce marked hypophagia in rats
(Satoh, Ogawa, Katsuura, Hayase, Tsuji, et al., 1997) and that
intracerebroventricularly infused leptin is rendered ineffective by lesioning the
ARC (Tang-Christensen, Holst, Hartmann & Vrang, 1999; Dawson, Pelleymounter,
Millard, Liu & Eppler, 1997). Specifically, it is speculated that leptin induces
reductions in eating by inhibiting the NPY/AgRP neurons in the ARC. First, NPY
mRNA and leptin receptors are coexpressed within the ARC nucleus (Mercer et al.,
1996). Second, there is a decrease in NPY mRNA in the ARC following leptin
administration (Wang, Bing, Al-Barazanji, Mossakowaska, Wang, et al., 1997;
Stephens, Basinski, Bristow, Bue-Valleskey, Burgett, et al., 1995). On the
contrary, there is an increase in the expression of NPY mRNA when leptin levels
are low, for instance during fasting, or in ob/ob leptin deficient mice (Bray & York,
1979; Stanley et al., 1985; Chen, Charlat, Tartaglia, Woolf, Weng, et al., 1996;
Iida, Murakami, Ishida, Mizuno, Kuwajima, et al., 1996). Further, this ARC NPY
overexpression observed during leptin deficiency is eliminated upon leptin
administration (Ahima, Prabakaran, Mantzoros, Ou, Lowell, et al., 1996; Stephens
et al., 1995). Finally, electrophysiological studies have shown that leptin
hyperpolarizes and as such inhibits ARC NPY/AgRP neurons (Spanswick, Smith,
62
Groppi, Gogan & Ashford, 1997; Roseberry, Liu, Jackson, Cai & Friedman, 2004).
All of the evidence together implicates a role for NPY/AgRP in leptin regulation of
energy homeostasis.
The ARC POMC neurons are a second major target for leptin. Evidence for
this is provided by the finding that POMC neurons and leptin receptors are
coexpressed within the ARC (Cheung, Clifton, & Steiner, 1997). In leptin deficient
ob/ob mice, there is a decrease in POMC expression (Ahima et al., 1996) and
electrophysiological studies confirm that leptin depolarizes, and therefore activates,
POMC neurons in normal animals (Cowley et al., 2001). Further, deletions of
leptin receptors located only on POMC neurons in the ARC leads to animals that
are overweight (Balthasar, Coppari, McMinn, Lu, Lee, et al., 2004). However, the
extent of the weight gain in these mutated animals is less than the increase
observed in leptin resistant db/db mice, suggesting that leptin receptive POMC
neurons are not solely responsible for leptin mediated weight control.
A relationship between leptin and CART also has been identified.
Decreased levels of CART mRNA are found in situations of leptin deficiency such
as in the ob/ob mouse or during starvation (Kristensen et al., 1998). This reduction
in CART mRNA is reversed upon leptin administration indicating that the ARC
neuropeptide is regulated by the adiposity signal.
The DMH, VMH, and PVN also play an important role in the leptin
hypothalamic circuitry. Although intravenous administration of leptin produces c-
fos activation in the PVN, studies show that leptin receptors are sparse within this
63
hypothalamic nucleus, while they are most abundant in the ARC, VMH, and DMH
(Mercer et al., 1996; Fei, Okano, Li, Lee, Zhao, et al., 1997). It has been
hypothesized that injections of leptin activate the PVN through these other
hypothalamic leptin-responsive neurons. A double-labeling study has shown that
PVN afferents from leptin responsive neurons in the DMH and VMH activate the
PVN, which implicates a leptin circuit involving these three structures (Elmquist et
al., 1998b).
Given that brainstem areas such as the AP, NST, and the PBN contain leptin
receptors (Grill et al., 2002), these structures also are potential targets of leptin.
Intracerebroventricular infusions of leptin into the fourth ventricle, which probably
diffuse to the AP and NST, produce suppression of eating that is comparable to
those produced by infusions into the lateral ventricles, which diffuse to more
forebrain structures (Grill et al., 2002). In addition, a double labeling study has
shown that following the infusions of the retrograde tracer, cholera toxin-b, into the
PVN and intravenous infusions of leptin, double labeled cells are found in the
lateral PBN, while none are found in the NST (Elmquist et al., 1998a). This
suggests that direct or indirect activation of the PBN by leptin causes activation in
the PVN, indicating a potential PBN-PVN circuit in the central pathway of leptin.
It was mentioned in the beginning of this chapter that there are separate
neural mechanisms that work in conjunction to control the size of individual meals
and to regulate body weight. One example of this interaction involves leptin and
CCK. It has been shown that leptin increases the satietogenic potency of CCK,
64
which is accompanied by increased c-fos activation in the NST (Emond et al.,
1999). Although c-fos expression in the PVN does not differ from control levels in
response to CCK administration, the combination of leptin and CCK produces
greater neuronal activation in the PVN compared to that produced by leptin alone
(Barrachina, et al., 1997; Wang et al., 1998; Emond et al., 1999). Given that (1) the
NST contains both leptin receptors (Grill et al., 2002) and CCK synthesizing
neurons (Mantyh & Hunt, 1984; Herbert & Saper, 1990) and that (2) the NST
projects to the PVN (ter Horst et al., 1989), it is possible that leptin activation of
NST CCKergic neurons produces the augmented response, which in turn is
projected to the PVN. Together, these findings implicate a NST-PVN pathway in
the leptin-augmented CCK satiation.
Insulin
Although leptin is the most studied adiposity signal, insulin was the first
hormone discovered as a weight regulating compound. Insulin is a hormone
released primarily by the pancreas and much like leptin, circulates in proportion to
adiposity levels (Bagdade, Bierman & Porte, 1967). Insulin also is known to affect
eating and body weight and acts on the hypothalamus to do so. For instance,
infusions of the hormone into the ARC or into the third ventricle decrease food
intake (Woods, Lotter, McKay & Porte, 1979; McGowan, Andrews, Kelly &
Grossman, 1990), suggesting that insulin acts on the hypothalamus and also on
brainstem structures to produce its hypophagic response. There are dissimilarities
between leptin and insulin. Unlike leptin, insulin has a known transport system
65
into the brain (Schwartz, Sipols, Kahn, Lattermann, Taborsky, et al., 1990;
Schwartz, Bergman, Kahn, Taborsky, Fisher, et al., 1991). In addition, unlike
leptin, insulin deficiency does not lead to obesity. In both rats (Havel, Uriu-Hare,
Liu, Stanhope, Stern, et al., 1998) and humans (Hathout, Sharkey, Racine, Ahn,
Mace, et al., 1999), insulin deficient diabetes leads to hyperphagia (Leedom &
Meehan, 1989), but adiposity and circulating leptin levels remain low. This is
probably due to the fact that insulin is necessary for the lipid synthesis and storage
(Zhang et al., 1994; Saladin et al., 1995), which in turn produces leptin (Zhang et
al., 1994). Bringing leptin levels back to non-diabetic levels in this model
eliminates the hyperphagia, while infusions of insulin have no effect (Sindelar,
Havel, Seeley, Wilkinson, Woods, et al., 1999). This suggests that it is the
deficiency of leptin that results in hyperphagia and not deficiency in insulin in the
diabetes mellitus model. Evidence for the contrary also exists. One study showed
that insulin deficiency-induced hyperphagia was reversed upon central and
peripheral infusions of insulin (Sipols, Baskin & Schwartz). If this is true, then the
hyperphagia in a state of insulin deficiency could be a result of insulin deficit rather
than from a paucity of leptin.
Insulin, like leptin, potentially exerts its hypophagic effect through its
interactions with ARC NPYergic neurons. The ARC densely expresses insulin
receptors (Baskin, Wilcox, Figlewicz & Dorsa, 1988), suggesting a role for insulin
in the hypothalamic nucleus. Second, the hyperphagia resulting from insulin
deficient diabetes is accompanied by increases in NPY expression in the
66
hypothalamus (Williams, Grill, Lee, Cardosa, Okpere & Bloom 1989), which is
reversed upon both central and peripheral infusions of insulin (Sipols, Baskin &
Schwartz, 1995). This leads to the conclusion that insulin inhibits NPY neurons in
the ARC and thereby reduces eating. Arcuate AGRP also is a target for insulin.
Intracerebroventricular infusions of insulin eliminate ARC AGRP immunostaining
normally observed within the ARC in control animals (Dunbar, Lapanowski,
Barnes & Rafols, 2005) suggesting inhibition of this feeding-related neuropeptide
by insulin. Moreover, perifusion of hypothalamic tissue, including the anterior
hypothalamus and the PVN, with insulin blocks the release of AGRP (Breen,
Conwell & Wardlaw, 2005) further implicating an interaction between insulin and
AGRP.
There is evidence to suggest that insulin also exerts its effects on eating via
the short-term satiety agent CCK. Sub-threshold doses of intracerebroventricularly
administered insulin enhance the effectiveness of peripherally delivered sub-
threshold doses of CCK, such that there is a 50% reduction in intake (Figlewicz,
Stein, West, Porte & Woods, 1986). These findings suggest that insulin in the CNS
augments sensitivity to peripheral CCK. A follow up study has demonstrated that
centrally infused insulin also enhances sensitivity to central CCK (Figlewicz,
Sipols, Seeley, Chavez, Woods, et al., 1995), showing that the interaction between
these two agents occurs both centrally and perhaps peripherally. This provides
another example of how short-term satiety signals interact with adiposity signals to
regulate food intake and body weight.
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Amylin
Amylin is a hormone produced by the β cells of the pancreas (Westermark,
Wernstedt, Wilander & Sletten, 1986; Cooper, Willis, Clark, Turner, Sim, et al,
1987) in response to a meal and is the third adiposity signal discussed in this
chapter. It is released in proportion to body fat mass (Butler, Chou, Carter, Wang,
Bu, et al., 1990) and is transported through the blood-brain barrier (Banks, Kastin,
Maness, Huang & Jaspan, 1995). Denervation of the vagus (Lutz, del Prete &
Scharrer, 1994) or the splanchnic (Edwards, Power & Young, 1998) does not
disrupt amylin-induced anorexia. Injections of amylin reduce food intake by about
55% (Bhavsar, Watkins & Young, 1998), while blocking this hormone’s receptors
with an antagonist elevates eating (Rushing, Hagan, Seeley, Lutz, D’Alessio, et al.,
2001) and body fat mass (Rushing et al., 2001). Additionally, both male (Gebre-
Medhin, Mulder, Pekny, Zhang, Tornell, et al., 1997) and female (Devine &
Young, 1998) mice lacking the gene encoding for amylin become overweight,
suggesting an endogenous role of amylin in body weight regulation.
The effects of amylin appear to be predominantly centrally rather than
peripherally mediated. Intracerebroventricularly infused amylin more potently
produces reductions in eating compared to intraperitoneally administered amylin
(Bhavsar, Watkins & Young, 1997; Lutz, Rossi, Althaus, del Prete & Scharrer,
1998). Studies focused on localizing the central cite of amylin’s hypophagic effect
have reported that peripheral administration of amylin produces c-fos activation in
the AP, NST, PBN, the BNST, and central Amg (Riediger, Zuend, Becskei & Lutz,
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2004; Rowland, Crews & Gentry, 1997). Interestingly, c-fos immunoreactivity is
blocked in all of the aforementioned areas following AP lesions (Riediger et al.,
2004). This suggests that the circumventricular organ is the initial site of action for
the hormone and that the other brain regions are activated by amylin via their direct
or indirect connections with the AP. Further, inactivation of the AP blocks the
anorectic effect of peripherally delivered amylin (Edwards et al., 1998; Rowland &
Richmond, 1999), while dexfenfluramine anorexia remains unaltered. This is an
important finding because it suggests that the lesions of the AP do not destroy all
satietoogenic circuits. Further, the lesions of the AP do not affect amylin-produced
c-fos activation in rostral areas of the brain (Rowland & Richmond, 1999) and
lateral ventricle infusions of the hormone in these lesioned animals are effective in
producing marked reductions in eating (Lutz et al., 1998). These results suggest
that amylin also may directly act on hypothalamic sites independent of its effects
on the AP. In support of this hypothesis, direct intra-hypothalamic infusions of
amylin have been shown to reduce food intake (Chance, Balasubramaniam, Zhang,
Wimalawansa & Fischer, 1991), although the precise regions were not specified in
the report.
Investigations into the specific mechanisms underlying amylin anorexia
have found an interaction between the hormone and CCK once again suggesting
that these adiposity signals interact with meal-related signals to regulate meal size
and ultimately body weight. Doses of CCK and amylin that are individually
ineffective, result in very potent inhibition of intake when they are co-administered
69
(Bhavsar et al., 1998) and selectively blocking amylin receptors decreases the
satietogenic effectiveness of CCK (Lutz Tschudy, Rushing & Scharrer, 2000).
1.6 Conclusion
The biological basis of eating and body weight regulation is varied and
complex. Mechanisms that control short-term and long-term satiety work in
conjunction to maintain energy homeostasis. Research on eating behavior has
advanced tremendously from the time of the ‘dual center theory’ of ingestive
behavior, when the lateral hypothalamus was thought of as the feeding center,
while the ventromedial hypothalamus was considered the satiety center. Presently,
we know that the intricate connections between neural structures of the forebrain
telencephalon, forebrain diencephalon, and the hindbrain all work in concert to
control eating and to regulate body weight. Forebrain telencephalic structures
involved in eating include the amygdala and the bed nucleus of the stria terminalis.
Besides the lateral hypothalamus and the ventromedial hypothalamus, forebrain
diencephalic structures include other hypothalamic nuclei as well. These include
the arcuate, the paraventricular nucleus, the dorsomedial nucleus, and
tuberomammillary nucleus. Hindbrain structures like the area postrema, nucleus of
the solitary tract, and the parabrachial nucleus are all implicated in ingestive
behavior. These neural areas control eating behavior with the help of several
anabolic and catabolic peptides that are localized within the neural structures
themselves. The anabolic neural peptides that stimulate eating are plentiful and
include neuropeptide Y, agouti related protein, galanin, melanin-stimulating
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hormone, and orexin. Catabolic peptides that inhibit eating include
proopiomelanocortin, cocaine-amphetamine regulated transcript, corticotrophin
releasing hormone, and thyrotropin releasing hormone. These neural structures and
peptides are activated or inhibited via peripheral satiety and feeding signals in the
control of short-term satiety, which is satiety related to individual meals. These
satiety signals such as cholecystokin, peptide YY, and glucagon-like peptides 1 and
2 and feeding signals such as ghrelin are released mainly from the gastrointestinal
tract in response to the presence or the absence of a meal. In addition, there are
separate molecules that serve to control long-term satiety, which is the satiety that
regulates body weight. Although the neural machinery is highly conserved for
short-term and long-term satiety, the molecular signals that initiate each process are
disparate. The signals involved in long-term body weight regulation include the
adiposity signals leptin, insulin, and amylin, which act in conjunction with short-
term satiety signals to regulate body weight. Given that energy homeostasis is
essential for survival, the intricate and complex systems evolved to insure its
accurate functioning is expected.
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CHAPTER 2
Estradiol and Eating Behavior
Because the reduction in eating produced by estradiol is a subsidiary focus
of the experiments reported in this dissertation, a separate chapter is dedicated to
the discussion of the decreases in eating and body weight produced by this
hormone.
2.1 Estradiol: Overview
Estradiol is part of a group of steroid sex hormones called estrogens and is
present in both males and females. The primary source of estradiol in the
circulatory system is the granulosa cells in the ovaries of female rodents and the
aromatization of testosterone in the testes of male rodents (de Jong, Hey & Molen,
1973; Shoham & Schachter, 1996). As such, it is thought of primarily as a gonadal
hormone. However, evidence demonstrates that in humans and in rats estrogens
are synthesized outside of the gonads as well. For instance, in post-menopausal
women and in men, estradiol is produced as a product of androgen aromatization,
which occurs locally at various sites like the skin and adipose tissue (for review see
Nelson & Bulun, 2001; Grodin, Siiteri & MacDonald, 1973). In rats, the presence
of aromatase in bone suggests that estradiol is produced at this site as well (Eyre,
Bland, Bujalska, Sheppard, Stewart, et al., 1998). There also is evidence for the
neural synthesis of estradiol in rodents. There is a baseline concentration of
estradiol in the lateral parabrachial that is significantly increased by stimulation of
the cervical branch of the vagus nerve (Saleh, Saleh, Deacon & Chisholm, 2002).
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The vagal stimulation alone does not affect circulating levels of the hormone,
suggesting that the increase in estradiol observed in the lateral PBN is synthesized
neurally. In addition, aromatase activity is found in both the hypothalamic preoptic
area and in the amygdala (Balthazart, Foidart & Hendrick, 1990; Chambers,
Thornton & Roselli, 1991), suggesting that the hormone may be produced at these
sites. Recent research shows that estradiol may be produced in astrocytes in
response to brain injury (Peterson, Saldanha & Schlinger, 2001), suggesting that
estradiol may be synthesized generally throughout the brain and not simply in
discrete structures. Interestingly, aromatase activity also has been illustrated in
fetal and neonatal brains of vertebrates (MacLusky & Naftolin, 1981)
demonstrating the possible ubiquitous nature of estradiol.
In cycling female rats, physiological levels of estradiol vary across the
various phases of the reproductive cycle. Low physiological levels range from 6-
30 pg/ml and occur during the diestrous, metestrous, and the estrous phases, while
high levels range from 22-88 pg/ml during proestrous (Butcher, Collins & Fugo,
1974; Cecchini, Chattoraj, Fanous, Panda, Brennan, et al., 1983; Dupon & Kim,
1973; Hawkins, Freedman, Marshall & Killen, 1975; Henderson, Baker & Fink;
1977; Lu, LaPolt, Nass, Matt & Judd; 1985; for review, see Overpeck, Colson,
Hohmann, Applestine & Reilly, 1978). In adult male rats, physiological levels
range from 1.2 to 3.1 pg/ml (Hawkins et al., 1975; Södersten, DeJong, Vreeburg &
Baum, 1974). In humans, the range of circulating estradiol is between 31-164
pg/ml for females (Kim, Hosseinian & Dupon, 1974; Skinner, England, Cottrell &
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Selwood, 1974) and from 21-46 pg/ml for males (Angsusingha, Kenny, Nankin &
Taylor, 1974; Haug, Aakvaag, Sand & Torjesen, 1974).
2.2 Estradiol and Eating Behavior
There is a substantial amount of evidence illustrating the role of estradiol in
ingestive behaviors. Physiological levels of estradiol have been associated with
systemic variations in the amount of food intake across the reproductive cycle of
animals. Specifically, in rats (Wade, 1972), mice (Blaustein, Gentry, Roy & Wade,
1976), and hamsters (Morin & Fleming, 1978) decreases in consumption are
observed during proestrous, when estradiol levels are elevated. Cyclic changes in
food intake have been observed in female mammals as well where decreases in
consumption are observed during the follicular phase, while increases are
associated with the luteal phase in guinea pigs (Czaja & Goy, 1975), rhesus
monkeys (Czaja, 1975; Kemnitz, Eisele, Lindsay, Engle, Perelman, et al., 1984),
baboons (Gilbert & Gillman, 1956), and humans (Cohen, Sherwin & Fleming,
1987). These variations in eating observed across the reproductive cycles are
shown to be inversely associated with circulating levels of estradiol that accompany
the separate phases such that during the follicular phase, when endogenous
estradiol levels are highest, eating is at its lowest, while the opposite is true of the
luteal phase.
The role of estradiol as an anorectic hormone is further supported by studies
showing that ovariectomized rats demonstrate hyperphagia and increase in meal
size which are accompanied by increases in body weight (Blaustein & Wade, 1976;
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Asarian & Geary 2002). Similarly, ovariectomies in rhesus monkeys results in
increased eating which persists for 3 weeks and also results in weight gain
(Sullivan, Daniels & Cameron, 2003). Further, estradiol treatment, which mimics
physiological levels of the hormone, is sufficient to normalize both eating and body
weight in ovariectomized rats (Geary & Asarian, 1999b) and rhesus monkeys
(Czaja & Goy, 1975).
Administering estradiol exogenously in animals has validated that it is the
steroid hormone responsible for the decreases in eating. The ability of estradiol to
produce unconditioned decrements in food intake has been demonstrated across
different species, implementing various routes of administration, and different
forms of the estrogenic steroid (Wade, 1975; Bernstein, Courtney & Braget, 1986;
Sandberg, David & Stewart, 1982; Miceli & Fleming, 1983; Tritos, Segal-
Lieberman, Vezeridis & Maratos-Flier, 2004). The consistent results
unequivocally establish estradiol as a satiety-inducing agent. Further, in rats, the
decrease in eating produced by the hormone is expressed as a decrease in meal size,
which is absent from compensatory increases in meal frequency (Asarian & Geary,
2002), suggesting that the hormone regulates eating by advancing satiety.
Evidence strongly suggests that estradiol acts as a long-term satiety agent,
that is, it reduces eating as a means of regulating body weight (Tarttelin & Gorski,
1973; Wade 1972). After ovariectomy, there is a transient increase in food intake
and body weight; however, after the increase in eating subsides and returns to
control intact levels, body weight stabilizes and remains 20-25% higher than the
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weight of sham operated animals (Tarttelin & Gorski, 1973; Gentry & Wade,
1976). In addition, ovariectomies of rats and mice produce 10-30% increase in
body adiposity (Mystkowski & Schwartz, 2000; Wade & Gray, 1979). Finally,
estradiol fails to induce decreases in food intake in females that do not gain weight
after ovariectomy further suggesting that estradiol affects eating as a means of
regulating body weight (Wade, 1972). In addition, evidence shows that estradiol
affects body adiposity. Leshner and Collier (1973) showed that ovariectomy
double body fat content in female rats. Others have shown that treatments with
estradiol reverse the increase in adiposity produced by the ovariectomies (Roy &
Wade, 1977). Studies with aromatase knockout mice (ArKO), which cannot
synthesize endogenous estradiol, have shown that both male and female ArKO
mice progressively accumulate more adipose tissue compared to their wild type
littermates (Jones, Thorburn, Britt, Hewitt, Wreford, Proietto et al., 2000). This
finding corroborates the results of previous studies implicating estradiol in lipid
homeostasis.
Research conducted in mutant null mice implicates the importance of the α-
receptor subtype in estradiol-induced reductions in eating. While wild type (WT)
animals displayed hypophagia and reduced body weight following estradiol
injection, WT animals treated with oil and α-receptor knock out mice (αERKO) did
not show any alterations in their daily intake or in their body weight (Geary,
Asarian, Chan, Korach, Pfaff et al., 1999).
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Tonic and Phasic Effects of Estradiol
Based on the data discussed above, the inhibitory effect of estradiol on
eating and body weight can be divided into two categories: phasic and tonic
(Drewett, 1973, 1974). The phasic or “acute” effects refer to the ability of cyclic
estradiol to produce temporary decreases in eating, while the tonic or “chronic”
effects apply to the long-term suppression of body weight that is maintained by
estradiol and is eliminated upon ovariectomy. It is suggested that these two
separate effects operate via different physiological and neural mechanisms (Geary,
2001).
Estradiol and Anorexia Nervosa
According to the Diagnostic and Statistical Manual of Mental Disorders IV
(DSM IV; American Psychiatric Association, 1994), anorexia nervosa is
categorized as an Axis I disorder, which is thought to be a transient
psychopathological state that is superimposed on otherwise healthy individuals.
Anorexia nervosa is characterized by a failure to maintain 85% of ideal body
weight, an intense fear of gaining weight, unrealistic focus on weight, distortions of
body image, and physical manifestations that include, but are not limited to
amenorrhea. Eating disorders in general predominantly afflict females varying
from 12 to 22 years of age and anorexia nervosa is no exception. Not surprisingly
then adolescence has been identified as a major risk factor for the development of
this infirmity (Halmi, Casper, Eckert, Goldberg & Davis, 1979).
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Estradiol has now been implicated as a key player in anorexia nervosa
(Gustavson & Gustavson, Noller, Melton, O’Brien, et al, 1989; Young, 1991).
There are two facts that logically associate the hormone to the eating disorder.
First, the onset of anorexia nervosa parallels the drastic increase in estradiol that
occurs in females during the onset of puberty. At least 40% of identified cases of
anorexia occur in females 15-19 years of age according to the National Eating
Disorder Association. Second, the effects of this hormone mimic two of the
primary symptoms of the eating disorder; decreased eating and increased physical
activity. In addition to producing decreases in eating and body weight, injections
of the steroid cause increases in physical activity. Female rats given access to
running wheels generally run 10 km in 10 days (Ramsden, Berchtold, Kesslak,
Cotman & Pike, 2003). This level of activity varies across the estrus cycle, with
the highest levels of activity observed during proestrus when plasma estradiol is
elevated (Wollnik & Turek, 1988) despite the decreased caloric intake observed
during this phase. Further, ovariectomies decrease running wheel activity in female
rats and implantation of ovarian tissue returns activity levels to control levels or to
the levels of intact females (Slonaker, 1924). These findings indirectly implicate
estradiol in elevated activity levels. More directly, estradiol implants in
ovariectomized Swiss-Webster mice increase running-wheel activity (Garey,
Morgan, Frohlich, McEwen & Pfaff, 2001) solidifying the role of the hormone in
the regulation of locomotor activity.
78
Although scarce, evidence directly tying estradiol to anorexia nervosa also
exists. The first of this evidence came from studies conducted by Gustavson and
Gustavson, using conditioned taste aversion (CTA) as their paradigm and rats as
the animal model. Traditionally, a CTA is a form of associative learning where an
“aversion” is acquired toward a novel taste stimulus that has been paired with an
agent that produces aversive internal states such as nausea or gastrointestinal
malaise (Garcia, Kimmeldorf & Koelling, 1955; Barker, Domjan & Best, 1977).
Estradiol has been found to be capable of inducing CTAs in both female and male
rats. However, the dose required to induce CTA is greater for females than males.
Gustavson et al. (1989b) showed that subcutaneous injections of 0.4 mg/kg of
estradiol cypionate were sufficient to produce a CTA in males, but was ineffective
in females. This sexually dimorphic behavior involving estradiol and CTA led
these researchers to suggest estradiol as an originating cause of anorexia nervosa.
Since males appeared to be more sensitive to the hormone, they proposed that
during fetal and early development, the CNS of some females become partially
masculinized, which results in an increased sensitivity to the nauseating and thus
the conditioning effects of estradiol. A previous study by these investigators
provided evidence for this hypothesis, namely, that prenatal masculinization leads
to increased sensitivity to estradiol-induced food aversions. Female rats prenatally
masculinized due to exposure to testosterone showed a lower dose threshold for
developing estradiol-induced food aversions as adults (Gustavson, Gustavson,
Noller, Melton, O’Brien & Pumariega, 1989a), which led to persistent changes in
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food intake and body weight maintenance. They extended these results to humans
by suggesting that pollutants, maternal stress, or pharmacological drugs that elevate
androgen levels might cause fetal masculinization of female fetuses and
consequently increased sensitivity to the nauseating effects estradiol (Schou, 1968).
The researchers hypothesized that these masculinized females frequently get
nauseated when estradiol production rises during puberty and hence develop food
aversions, which limits the number of acceptable foods these girls consume.
Subsequently, this produces a general decrease in consumption and hence body
weight. Although this weight loss results in a reduction of estradiol production and
consequently an increase in ingestion, the resulting weight gain restores the
production of the hormone, which once again leads to decreases in eating. It was
speculated that this vicious cycle potentially manifests itself into a starvation
system, leading to the development of anorexia nervosa. Studies examining the
role of food aversions in various anorexia symptoms provide support for the food
aversion driven anorexia hypothesis. For example, it has been demonstrated that
continuous exposure to a target diet during a chronic illness state leads to a CTA to
that diet, which in turn leads to significant weight loss (reviewed in Bernstein &
Borson, 1986).
The hypothesis for the origin of anorexia proposed by Gustavson et al.
becomes clearer when the behavioral effects of hormones during fetal development
and during adulthood are considered. The presence or absence of prenatal
hormones dictates the neural development of fetuses. For instance, male fetuses
80
exposed to testosterone develop a “male” sexually dimorphic nucleus in the brain,
such that during adulthood, when these males are re-exposed to the hormone, the
hormones activate the “male” brain, which results in the exhibition of “male”
sexual behaviors. A disruption in the hormone balance during fetal development
disrupts behavior expression during adulthood. For instance, if male fetuses are not
exposed to testosterone during fetal development, they develop more of a “female”
brain or a smaller sexually dimorphic nucleus. When these males are exposed to
estrogen and progesterone during puberty or adulthood, the “female” brain is
activated and they display “female” sexual behaviors and prefer male partners.
Similar disruptions can occur in female fetuses. In utero estrogenic exposure has
major organizational effects on the brain development of female fetuses such that
when these females are re-exposed to the hormones in adulthood, their behavior is
affected. Exposure to estrogens in utero leads to masculinization of the female
brain or a larger sexually dimorphic nucleus. As such, in adulthood, when these
females are exposed to estrogens and progesterone, they exhibit male sexual
behaviors such as mounting (masculinization) and decreased lordosis and impaired
ovarian function (defeminization: Hines, Alsum, Roy, Gorski & Goy, 1987; Hines
& Goy, 1985). Gustavsons’ hypothesis of anorexia is clearer once it is
superimposed on this hormonal model of development and adulthood exposure.
They propose that prenatal exposure to hormones that masculinize the female brain
during the organizational period leads to masculinized behavior in adulthood when
these females are exposed to estrogen during puberty. In their hypothesis, this
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masculinized behavior is a sensitized reaction estrogen, which serves as a potent
unconditioned stimulus for the acquisition of food aversions and hence reduced
food intake. Although the examples used to demonstrate the effects of prenatal
hormones and adulthood behavior have focused on sexual behavior, one study has
showed the effects prenatal hormonal alteration in food preference. For instance,
female fetuses exposed to TCDD, a dioxin that produces hormonal changes,
showed reduced preference for saccharin as adults (Amin, Moore, Peterson &
Schantz, 2000). Their preference for saccharin was reduced to levels of saccharin
preference exhibited by males. This study shows that alterations in prenatal
hormones affect eating behaviors in addition to reproductive behaviors and supplies
credence to Gustavsons’ hypothesis of the origins of anorexia.
Synthetic estrogens also are shown to produce masculinization effects
during fetal development. For instance, diethylstilbestrol (DES), a synthetic non-
steroidal estradiol, is known to produce masculinization syndrome in female rats.
One study demonstrated that the sexually dimorphic nucleus in the preoptic area of
female rats was masculinized by treatment with either testosterone or DES. The
size of this nucleus in the females was similar to size of the sexually dimorphic
nucleus of normal male rats (Dohler, Coquelin, Davis, Hines, Shryne & Gorski,
1984). Although the researchers did not test for sexual behaviors expressed as
adults, these females probably would display male sexual behaviors when exposed
to estrogen and progesterone in adulthood. Female guinea pigs exposed to
synthetic estrogens in utero display masculine typical sexual behaviors as adults,
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instead of female sexual behaviors, when they are exposed to estradiol and
progesterone (Hines, Alsum, Roy, Gorski & Goy, 1987; Hines & Goy, 1985). The
Gustavson et al. study (1989) discussed above, which produced masculinized
brains in female rats by prenatally treating them with testosterone also had
produced masculine females by prenatally exposing them to DES. These female
rats exhibited behavior identical to those prenatally treated with testosterone;
namely, they developed estradiol-induced food aversions at lower doses compared
to non-treated controls. It should be mentioned that these findings raise the
possibility that the aromatization of testosterone to estradiol may be producing the
“masculinization” effects since no differences are detected between the brains of
female rats treated prenatally with testosterone or DES. Evidence for this
hypothesis exists. One study showed that female guinea pig fetuses exposed to
testosterone in utero displayed male sexual behaviors in adulthood. However,
exposing these females to both testosterone and ATD, an aromatase inhibitor,
prevented the display of masculine sexual behaviors by the female guinea pigs
(Roy, 1992; Choate & Resko, 1994). This suggests that the aromatization of
testosterone into estradiol is primarily responsible for the masculine behaviors
displayed by these females. This possibility is further bolstered by the fact that
prenatal aromatase activity in vertebrates has been demonstrated (MacLusky &
Naftolin, 1981). Along these same lines, it would be interesting to see whether in
utero exposure to dihydrotestosterone (DHT), which does not aromatize into
estradiol, would preclude females from displaying masculine behaviors in
83
adulthood. If masculine behaviors are not displayed in these females, this too
would suggest that prenatal exposure to estradiol is responsible for masculinized
behaviors exhibited during adulthood.
Evidence from human research supports the hypothesis regarding the
origins of anorexia laid out by Gustavson et al. (1991). This evidence can be pulled
from the daughters of women who were treated with DES during pregnancy. If
exposure to DES masculinizes the brains of female fetuses such that during
adulthood, these women display an increased sensitivity to the aversive affects of
estrogen in adulthood, then the daughters of women exposed to DES during
pregnancy should be at an increased risk for developing anorexia. Physicians
began to prescribe DES to pregnant women from about 1947-1971 in hopes of
avoiding miscarriages and circumventing other complications associated with
pregnancy (Hammes & Laitman, 2003). The FDA encouraged the discontinuation
of its use in 1971 when the synthetic estrogen became associated with vaginal and
cervical cancer. Children of mothers who were administered DES also were
affected, suffering from reproductive tract abnormalities, increased infertility, and
eating disorders (Bibbo, Al-Naqeeb, Baccarini, Gill, Newton, et al., 1975;
Gustavson et al., 1991). Interestingly, as Chambers and Bernstein (2003, 2005)
point out, the time of DES usage corresponds to the increase in the reported
incidence of anorexia nervosa in 14-20 year old females in the United States
beginning in 1965 and continuing through 1976 (Jones, Fox, Babigian & Hutton,
1980). More direct evidence for the role of DES in eating disorders is provided by
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the findings that women prenatally exposed to DES were 5 times more likely to
show inexplicable weight loss and to be diagnosed with an eating disorder such as
anorexia nervosa or bulimia nervosa, compared to women who are not exposed to
the drug (Gustavson, Gustavson, Noller, O’Brien, Melton et al., 1991).
Evidence that anorexics show an exaggerated anorexic effect of estradiol
provides strong support for the role of this hormone in the eating disorder. It has
been reported that in anorexic patients, estrogen therapy increases the vomiting
related to estrogenic vaginal smears (Moulton, 1942). How this increased
sensitivity to estradiol is produced is still in question. As stated above, Gustavson
et al. (1989a,b) proposed in utero “masculinization” produced by various agents
like testosterone and DES as potential culprits. However, there is evidence that
prenatal exposure to testosterone may serve a protective role. For instance, young
women with twin brothers have more healthy eating behavior than women with
twin sisters (Culbert, Breedlove, Burt & Klump, 2008). Moreover, disordered
eating is lower in both females and males with a male twin, suggesting that
exposure to embryonic testosterone may play a preventative role. These findings
also suggest that prenatal exposure to estradiol may provoke disordered eating since
both males and females with a female twin exhibited disordered eating. However,
these findings seem contradictory since evidence of prenatal aromatase activity
exists (MacLusky & Naftolin, 1981). If in utero testosterone is capable of
aromatizing into estradiol and estradiol is playing a facilitatory role, then how
would in utero testosterone be serving a protective role against the eating disorder?
85
One possibility for the contradictory results may lie in the prenatal aromatizability
of testosterone. The MacLusky and Naftolin (1981) study showed aromatase
activity in the brains of fetuses and neonates. Although testosterone may aromatize
within the fetus, it may not be able to convert to estradiol inside of the embryonic
fluid, if the embryonic fluid is the only way twin fetuses are exposed to each
other’s hormones. As such, female twins exposed to testosterone due to their male
siblings may be protected. Even still, the logic of this hypothesis that testosterone
protects females by making them more masculine contradicts the hypothesis set
forth by Gustavson et al. (1989a,b), who claim that making the females more
masculine is precisely the culprit in predisposing them to disordered eating.
Taken together, all of these data demonstrate a role for estradiol in anorexia.
It is clear that females with anorexia do exhibit increased sensitivity to the effects
of estradiol; however, the originating cause of this sensitization to estradiol remains
in question although prenatal exposure to estradiol continues to be a strong
possibility. Studies showing the masculinization of female brains treated prenatally
with testosterone or DES provide evidence for the masculinization hypothesis
although further investigation is warranted. The effects of prenatal exposure to
pollutants and maternal stress remain unexplored. Progress towards testing this
hypothesis demands identification of neural areas that mediate the anorectic effects
of estradiol. In the next section, existing research that has investigated possible
neural sites for this effect of estradiol will be discussed.
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2.3 Neural Substrates of Estradiol Hypophagia
To date, the neural site(s) of estradiol action regarding its ability to inhibit
feeding have not been equivocally identified; however, there have been many
studies implicating several brain regions as potential candidates. These structures
include the area postrema (AP), the nucleus of the solitary tract (NST), the
parabrachial nucleus (PBN), the paraventricular nucleus of the hypothalamus
(PVN), and the amygdala (Amg).
C-fos Like Immunoreactivity (c-FLI) Studies
Immunohistological studies conducted in our lab suggest a role for the AP,
lateral PBN and the PVN in estradiol-elicited reductions in consumption.
Ovariectomized female rats were injected with sesame oil or 50μg/kg dose of
estradiol benzoate at the end of the light phase of the light/dark cycle and their
brain were processed for c-FLI 24 hours later. Estradiol activated c-FLI expression
in the AP, the lateral PBN, the PVN, and in the amygdala (central, lateral,
basolateral, and medial subnuclei) 24 hours following its administration, which is
precisely the time when reductions in eating produced by exogenous administration
of this hormone are initially expressed (Chambers, Hintiryan & So, unpublished
manuscript; Tartellin & Gorski, 1973). Decreases in eating also are observed 24
hours following the increase in plasma estradiol levels in cycling rats (Asarian &
Geary, 2002; Griffin & Ojeda, 1996). Although c-fos activation provides only an
indirect association between estradiol and these neural structures, it provides a
good starting point in identifying neural substrates of estradiol hypophagia.
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Immunohistological studies conducted in other labs have shown that in
food-deprived ovariectomized rats, eating increases the expression of c-FLI in the
NST, PVN, and the central nucleus of the Amg (ceAmg), all areas that are involved
in consumption (discussed in Chapter 1, “Neural Regulation of Eating”).
Activation of c-FLI is found within these same neural areas when ovariectomized
females are pretreated with a dose of estradiol that produces blood levels of
estradiol observed during proestrous (Eckel & Geary, 2001). Since c-FLI
activation may be indicative of activation or inhibition, estradiol may be inhibiting
feeding-induced neuronal activation within these brain regions. Unfortunately,
immunohistochemical staining of the lateral PBN was excluded from this study.
Central Implant Studies
Initial studies delving into the neural areas involved in estradiol hypophagia
included the medial Amg. Bilateral implants of estradiol benzoate into the medial
Amg produced decreases in food intake and body weight over a period of 72 hours
(Donohoe & Stevens, 1981) implicating this limbic structure in estradiol

hypophagia. Seminal studies on the neural localization of estradiol hypophagia
also focused on the ventromedial nucleus of the hypothalamus (VMH) since at the
time this area was branded the ‘satiety center’ (Stellar, 1954) of the brain.
Although there is evidence contrary to the now archaic notions of ‘satiety’ and
‘hunger’ centers, nevertheless, the VMH has been shown to be involved in satiety
processes (Cox, Kakolewski & Valenstein, 1969; King & Frohman, 1986). These
studies showed that microinjections of pure estradiol into the VMH (Palmer &
88
Gray, 1986) and the VMH/arcuate (ARC; Butera & Czaja, 1984) decreased eating
in ovariectomized rats. Based on these findings, it was concluded that the VMH
was a neural area involved in mediating decreases in consumption produced by
estradiol. Further, it was demonstrated that amygdaloid implants continue to cause
suppression in eating and body weight in ovariectomized rats sustaining lesions of
the VMH/ARC region (Donohoe & Stevens, 1981). Together, these results imply
that the steroid may act independently on both structures to produce its hypophagic
effects.
Subsequent studies however, showed that infusing a more diluted form of
estradiol (3:1 or 10:1 mixture of estradiol to cholesterol), which restricts steroid
diffusion within the brain, into the VMH had no effect on eating and body weight.
On the other hand, implants of this diluted estradiol into the PVN decreased both
intake and body weight (Butera & Beikirch, 1989). Consistent with this failure to
find an involvement of the VMH, are the results of studies showing that neither
VMH electrolytic lesions in female rats (Kemnitz et al., 1977) nor gold thioglucose
lesions of the VMH in mice (Blaustein, Gentry, Roy & Wade, 1976) block
estradiol-induced reductions in food intake. Retroactively, it was speculated that
the neural administration of pure estradiol produced a vast number of changes that
could have independently affected eating. This may have also been true for the
medial Amg implant studies since the researchers used the undiluted form of the
steroid.
89
Lesion Studies
Lesion studies also implicate the AP in hypophagia that is induced by
estradiol. Male rats were given a target diet the night before they were placed under
chronic vehicle treatment or estradiol treatment that produced supraphysiological
blood levels of estradiol (116.2 or 169.4 pg/ml). The estradiol-treated males
showed a decrease in consumption of their target diet across 5 days while the
control animals did not. A subsequent study showed that these estradiol-treated
males did not express hypophagia to the target diet following thermal lesions of the
AP (Bernstein et al., 1986).
The results of inactivation studies examining the role of the PVN in
estradiol-induced hypophagia have been mixed. Some evidence has been provided
by the finding that PVN lesions render estradiol ineffective in producing food
decrements (Butera, Willard & Raymond, 1992). On the other hand, these findings
have been inconsistent with the findings of Dagnault and Richard (1994) and
Hrupka and Geary (unpublished data as cited in Geary, 2000), which show that
lesions of this hypothalamic nucleus do not block the effect of the steroidal
hormone on eating. The reason for these inconsistencies remains unexplored;
however, despite these incongruous findings, the central implant studies discussed
above and the studies examining the neuropeptides involved in estradiol
hypophagia discussed below, strongly suggest a role for the PVN in estradiol
hypophagia.
90
Because the primary focus of this dissertation is the role of the PBN in
estradiol hypophagia, a separate section is dedicated to the interaction of estradiol
and this neural structure.
2.4 Estradiol and the Lateral Parabrachial Nucleus (PBN)
As discussed in chapter 1, “Neural Regulation of Eating”, the PBN has been
implicated in eating. In particular, evidence suggests that the lateral PBN is
involved in satiation (Nagai et al., 1987). Although there is no direct evidence that
the lateral PBN plays a role in the hypophagic effects of estradiol, several pieces of
evidence are suggestive. First, both α and β estrogen receptors have been identified
in the lateral PBN (Shughrue, Lane & Merchenthaler, 1997), suggesting a role for
the hormone in this neural area. Second, although research on the neural
production of estradiol is scarce, one study suggests that this hormone either is
produced in the lateral PBN or that it accumulates in this structure following vagal
stimulation (Saleh, Saleh, Deacon & Chisholm, 2002). The authors showed that
there is a baseline concentration of estradiol in the lateral PBN that can be changed
by stimulation of the cervical branch of the vagus nerve. Estrogen significantly
increases in response to vagal stimulation in ovariectomized female and intact male
rats, reaching a maximum 90 minutes after stimulation is initiated, while it remains
constant in non-stimulated controls. The authors also showed that vagal
stimulation alone does not affect circulating levels of the hormone, suggesting that
the increase in estradiol observed in the lateral PBN is not due to peripheral
increases in the hormone. The third piece of indirect evidence links the PBN
91
specifically to estradiol hypophagia. This evidence involves the neural connectivity
of the structure. The PVN, which remains the most likely candidate for
involvement in the decrements in eating produced by estradiol
,
is predominantly
innervated by the lateral PBN (Krukoff, Harris & Jhamandas, 1993), suggesting a
potential interaction between these two areas in mediating estradiol produced
changes in eating. In fact, this PVN-lateral PBN connection has been shown to be
involved in serotonin-induced anorexia (Li, Spector & Rowland, 1994). Fourth, it is
demonstrated that c-FLI is activated in the central, crescent, and external lateral
subnuclei of the PBN 24 hours after injection of 50μg/kg of estradiol benzoate,
which is precisely the time when reductions in eating produced by endogenous and
exogenous estradiol are expressed (Chambers, Hintiryan & So, unpublished
manuscript; Griffin & Ojeda, 1996). Although the lateral PBN appears to be a good
candidate for the site of estradiol hypophagia, no studies examining this possibility
have been conducted.
2.5 Estradiol, Neuropeptides and Eating
Although most research involved in determining the neural regulation of
estradiol hypophagia has focused on establishing the site of the hormone’s action,
some attention has been centered on the neurochemical mediation of estradiol-
induced reductions in eating. This research, which also gives insight into the neural
structures important for the hormone’s actions, shows that there is an interaction
between estradiol and CCK, NPY, and MCH.
92
Cholecystokinin (CCK) and Estradiol
A vast amount of research has established CCK as a satiety-inducing agent
(Moran & Schwartz, 1994). Further, it is suggested estradiol produces its
hypophagic effects by increasing the potency of CCK (for review, see Geary,
2001). For instance, chronic estradiol injections augment the satiating potency of
CCK in ovariectomized animals (Lindén, Uvnäs-Moberg, Forsberg, Bednar &
Södersten, 1990; Butera, Campbell & Cataldo, 1993; Geary, Trace, McEwen &
Smith, 1994). This augmented decrease in eating produced by CCK in estradiol
treated animals could be the additive hypophagic effects of the two anorectic
compounds; however, the decrease in eating produced by the CCK and estradiol
combination was greater than the decrease produced by adding the reduction in
eating produced by each of these agents alone. Therefore, the augmented decrease
was most likely not due to the additive satiogenic effects of CCK and estradiol.
Studies using estradiol treatment that mimics the reproductive cycles of rats have
revealed corroborating evidence for the augmented decrease in eating when CCK is
added to the estradiol treatment regimen (Asarian & Geary, 1999a). Further support
for an estradiol-CCK interaction is provided by the results of three additional
studies. Estradiol treatment increases the satiating effects of intralipid, an agent
whose satiating action is mediated partly by CCK (Yox, Brenner & Ritter, 1992).
On the other hand, estradiol treatment is ineffective in altering the satiating effect of
l-phenylalanine, an agent whose effects on eating are independent of CCK action
(Greenberg, 1998). These results suggest that estradiol potentiates the satiating
93
properties of agents that release CCK in female rats. Another study which
confirms the interaction of estradiol

and CCK in satiety showed that the
administration of a CCK
A
antagonist during a day that mimics estrus estradiol
levels, prevented the expected decrease in the size of meals, while administration of
the intestinal peptide on a day that mimics diestrus estradiol levels, had no effect
(Asarian & Geary, 1999b). These data indicate that the satiogenic CCK signaling
is increased during a period when estradiol levels are elevated. Studies
investigating c-FLI protein activation also substantiate the interaction between
estradiol and CCK and suggest potential neural substrates for the site for this action.
Eckel, Houpt and Geary (1998) showed that injections of CCK octapeptide induced
c-FLI activation in the NST, PVN and in the central Amg, which also were
increased in animals treated with estradiol.
Research conducted in mutant null mice has revealed the nature of the
interaction between CCK and estradiol. Estradiol or oil treatments were given to
either wild type (WT) or α-estradiol receptor knock out (αERKO) mice. WT
animals treated with estradiol displayed hypophagia and reduced body weight,
while eating remained unaltered in WT animals treated with oil and in αERKO
mice (Geary, Asarian, Chan, Korach, Pfaff et al., 1999). In addition, a CCK
A
antagonist prevented the decrease in food intake in the WT mice injected with
estradiol, but it did not have an effect in oil treated control animals. The CCK
A
antagonist also did not affect eating in αERKO mice treated with estradiol, which is
what one would expect since estradiol did not affect eating in these animals. These
94
data suggest a role for CCK in estradiol hypophagia. Moreover, the caudal NST is
implicated as one of the sites of this estradiol-CCK interaction. Central implants of
estradiol benzoate above the caudal NST decreased eating and increased CCK-
induced c-FLI in the estradiol alpha receptors of the caudal NST (Thammacharoen,
Lutz, Geary & Asarian, 2008).
Neuropeptide Y (NPY) and Estradiol
It has been consistently and thoroughly demonstrated that NPY is a potent
appetite stimulator (Clark, Karla, Crowley & Karla, 1984; Morley, Hernandez &
Flood, 1987; Pau, Pau & Spies, 1985). Hypothalamic sites such as the ARC, PVN
and VMH that contain NPY synthesizing neurons have been shown to concentrate
estradiol following administration of the radioactively labeled hormone (Sar &
Stumpf, 1981; Morell, Krieger & Pfaff, 1986; Sar, Sahu, Crowley & Karla, 1990).
This morphological inter-relationship suggests a potential link between the two
peptides. A direct examination of this possibility was conducted by Bonavera,
Dube, Karla and Karla (1994) in a two-part experiment. The first study
demonstrated a decrease in the expression of NPY in the PVN and in the
surrounding PFA in ovariectomized female rats treated with estradiol for 18 days
via a Silastic capsule and behaviorally showing decreases in eating and in body
weight compared to the control oil animals. Hypothalamic levels of β-endorphin,
another orexigenic agent (Morley, Levine, Yim & Lowy, 1983), were unaffected by
estradiol treatment, although these animals also showed reductions in eating and
body weight. These results suggest that (1) the effects of estradiol were specific to
95
NPY and (2) the reductions in the neuronal peptide were not a result of a negative
energy state produced by the estradiol-produced decreases in eating. If the changes
in NPY expression were in response to general decreases in eating in body weight,
one would expect that there would be an increase in this orexigenic peptide since
NPY levels increase in response to energy depletion (Sahu, Sninsky, Phelps, Dube,
Karla, et al., 1992).
The second study in the two-part experiment examined the effects of
estradiol treatment on the release of NPY from the PVN and VMH. Increases in
eating observed during the onset of the dark phase are accompanied by increases in
NPY release in the PVN in the rat (Karla, Dube, Sahu, Phelps & Karla, 1991; Sahu
& Karla, 1993). As such, the animals treated with estradiol for 18 days via a
Silastic capsule were sacrificed at the onset of the dark phase. Results showed that
both basal and KCl-induced release of NPY in the PVN was significantly decreased
in rats treated with estradiol, while levels of the neuropeptide release in the VMH
remained unchanged. Together, the findings of this two-part experiment suggest
that estradiol produces its hypophagic effects by decreasing the release of NPY in
the PVN. Whether estradiol acts directly on the PVN to reduce NPY release or
whether the hormone acts indirectly through another neural structure such as the
ARC is not known and warrants further investigation.
Melanin-Concentrating Hormone (MCH) and Estradiol
Research also suggests an interaction between estradiol and the orexigenic
peptide MCH (Rossi, Choi, O’Shea, Miyoshi, Ghatel et al., 1997; Ludwig, Tritos,
96
Mastaitis, Kulkami, Kokkotou et al., 2001). Murray, Baker, Levy and Wilson
(2000) showed that subcutaneous administration of estradiol benzoate significantly
decreased the expression of MCH precursor protein in the zona incerta and in the
LH 24 and 48 hours following the injection, which is the time when decreases in
eating are elicited by estradiol following its administration (Chambers, Hintiryan &
So, unpublished manuscript). This finding was corroborated by a separate study
which investigated the effects of chronic estradiol treatment on the expression of
MCH (Mystkowski, Seeley, Hahn, Baskin, Havel et al., 2000). The results showed
that estradiol treatment for 18 days resulted in decreased eating and body weight,
which were accompanied by decreases in hypothalamic MCH expression. This
decreased hypothalamic MCH expression was not observed in pair-fed controls,
which were given the same decreased amount of food as the estradiol group, but
did not receive estradiol benzoate. In fact, there was a significant increase in MCH
expression in pair-fed animals. This is in line with results showing an increase in
MCH in response to energy depletion (Fellmann, Risold, Bahjaoui, Compagnone,
Bresson, et al., 1993; Herve & Fellmann, 1997).
97
CHAPTER 3
Conditioned Taste Aversion Learning
3.1 Conditioned Taste Aversion (CTA): Overview
Attributes of Conditioned Taste Aversion Learning
When consumption of a novel tasting substance is followed by administration
of a chemical agent that produces physiological changes indicative of malaise, animals
will reduce their consumption of the substance during subsequent encounters. This
learned response is traditionally referred to as a conditioned taste aversion (CTA). A
CTA is acquired through a learning process that is similar to the traditional classical
conditioning paradigm. For instance, pairing a distinctively flavored taste stimulus (the
conditioned stimulus or CS) with an agent that normally elicits nauseating effects (the
unconditioned stimulus or US), will lead to the subsequent avoidance of the taste (the
conditioned response or CR). Although CTAs follow the general scheme of classical
conditioning, there are two important differences that distinguish them from most other
types of the paradigmatic learning and render them a very powerful form of learning.
First, aversions to novel taste stimuli can be learned easily in a single trial,
while most classically conditioned responses develop following several to numerous
pairings of the CS with the US (Mackintosh, 1974). In the classic illustration of
Pavlov’s experiment, a CR of salivation was elicited by the CS tone only after several
pairings of the meat powder (US) and the tone (Pavlov, 1927). Early studies of eye-
blink conditioning where the CS tone is paired with a US air puff to the eye show that
learning in rabbits occurs after approximately 140 trials (Thompson & Krupa, 1994)
98
and after 60 pairings in humans (Reynolds, 1945). The one-trial attribute of CTAs has
been demonstrated extensively in studies using lithium chloride (LiCl). As a powerful
poison, LiCl is considered to be the putative illness-inducing agent and is the most
commonly used US in studies investigating the underlying mechanisms of CTA
learning. Lithium chloride is a salt of lithium and has properties similar to sodium,
although the precise mechanism by which this agent acts is not quite understood.
The second characteristic of CTAs that separates them from other forms of
Pavlovian conditioning involves the temporal contiguity between the CS and US
presentations. In CTAs, associations between the CS tastants and US toxins still occur
if the interval between the CS and US is as long as 4-12 hours, while in traditional
classical conditioning, delays of over a few minutes or even seconds can greatly disrupt
learning. The earliest studies exploring this phenomenon showed that an apomorphine
CTA towards a saccharin solution could be formed with an inter-stimulus interval as
long as 75 minutes (Garcia, Ervin & Koelling, 1966). Laboratories have reported
significant aversions to a 4% sucrose solution when exposure to x-radiation was
administered 6 hours following the CS presentation and gustatory aversions were
readily established toward a 0.1% saccharin solution when the CS and the x-radiation
US were separated by as much as 12 hours (Smith & Roll, 1967). The mechanisms
underlying such a lengthy interval remain undetermined; however, studies show that it
is neither due to regurgitation nor due to lingering aftertaste. The regurgitation
hypothesis cannot be upheld in the case of rapidly metabolized substances such as
sucrose solutions, which are no longer present in the system throughout the 1-12 hour
99
delay (Revusky, 1968). Additionally, this hypothesis cannot be applied to rats since
the emetic response is blocked by the cardiac sphincter in this species. The aftertaste
hypothesis has not received support either, since CTA to a novel solution is not
disrupted when either the animal’s pallet is rinsed or when other more familiar flavors
are ingested during the CS-US interval (Kalat & Rozin, 1971). Such a lengthy CS-US
interval is extraordinary considering that traditional conditioning experiments deal in
seconds and fractions thereof. For instance, in a study that paired the sound of a buzzer
to an electric shock, researchers found that the most efficient conditioning occurred
when the inter-stimulus interval was 667 milliseconds (Kappauf & Schlosberg, 1937).
Optimal CS-US interval delay in eye-blink conditioning is approximately 400-450
milliseconds in humans (Kimble, 1947; Reynolds, 1945) and 250 milliseconds in
rabbits (Coleman & Gormezano, 1971).
Evolutionary Significance of Conditioned Taste Aversion
The aforementioned qualities of CTAs designate them as a potent form of
learning, which is common to most animals including humans (Bernstein, 1978, 1985;
Gustavson, Gustavson, Young, Pumariega & Nicolaus, 1989). Evolutionarily, this type
of learning is imperative for the survival of a species. Omnivorous animals that
survive after ingestion of a novel toxic substance and learn quickly and unequivocally
to avoid that distinctive taste will increase their chances of survival. Possessing the
capability to bridge very long CS-US intervals and to disregard interpolated familiar
CSs as culprits for the illness further confers selective advantage.
100
The classic study by Garcia and Keolling (1966) illuminates the biological
predisposition of animals to associate particular tastes with illness. In this study, male
rats were presented with both audiovisual and gustatory cues. The audiovisual cues
consisted of “bright-noisy” water, which was provided by connecting an incandescent
lamp (5 watt) and a clicking relay into the drinkometer circuit such that each time the
animal licked, a bright flash was accompanied by the emission of a noise. The
gustatory stimulus was “tasty” water, which was water mixed with flavors. Both
audiovisual and gustatory stimuli were paired with radiation. Aversions developed
only to the gustatory cues (tasty water) and not to the audiovisual stimulus (bright-
noisy water). On the other hand, when both cues were paired with a foot shock,
aversions were formed only to the audiovisual stimuli. These findings suggest that
biologically, animals are predisposed to associate taste with internal malaise. Auditory
and visual cues, on the other hand, are not so readily associated with gastrointestinal
discomfort, but are readily associated with peripheral pain.
3.2 Gustatory (CS) and Malaise (US) Information
Since the formation of a CTA requires the association between taste (CS) and
malaise (US) information, the neural areas involved in individually processing both of
these stimuli become potential candidates as mediators of the learned behavior.
Neural Gustatory Pathway (CS Pathway)
From taste receptors on the tongue, gustatory information is transmitted to the
trigeminal (V), to the lingual branch of the glossopharyngeal (IXth) and to the chorda
tympani and superficial petrosal branches of the facial (VIIth) cranial nerves (Torvik,
101
1956; Hamilton & Norgren, 1984). From these nerves, gustatory information is
transmitted to the rostral portion of the nucleus of the solitary tract (NST; Torvik,
1956) located in the brainstem. The information is then projected to the ipsilateral
medial or “gustatory” parabrachial nucleus (PBN; Norgren, 1978, 1984), which
transmits the information to the contralateral ventroposterior medial thalamus (VPMpc;
Saper & Loewy, 1980). The VPMpc is the final relay of taste information before its
final destination in the gustatory cortex of the dysgranular region of the insular cortex
(IC; Cechetto & Saper, 1987).
A second, yet less defined, route by which taste information reaches the
forebrain involves mono-synaptic projections from the medial PBN to the central
nucleus of the amygdala (Amg), the substantia innominata, the lateral hypothalamus
(LH), the bed nucleus of stria terminalis (BNST), and the IC (Norgren, 1974, 1976;
Saper & Loewy, 1980; Shipley & Sanders, 1982; Fulwiler & Saper, 1984; Moga,
Herbert, Hurley, Yasui & Gray, 1990; Bernard, 1993).
Neural Visceral Pathway (US Pathway)
There are three routes by which malaise information produced specifically by
LiCl reaches the central nervous system (CNS). First, malaise information is
transmitted via the vagus nerve (parasympathetic) second, through the splanchnic nerve
(sympathetic) and third, through a humoral route via the area postrema (AP).
Visceral information from the gut, liver and cardiorespiratory system is carried
by the vagus nerve (Baldino & Wolfson, 1985) to the caudal NST (Cechetto, 1987).
Next, the caudal NST sends extensive projections to the ipsilateral lateral PBN (Lowey
102
& Burton, 1979; Saper & Lowey, 1980), which is the relay station for ascending
visceral information from the NST to the thalamus, hypothalamus, and Amg (Cechetto,
1987). Specifically, the lateral PBN has a contralateral projection to the thalamus, in
particular, to the ventroposterior lateral parvocellular nucleus of the thalamus (VPL
pc
;
Cechetto & Saber, 1987). The VPL
pc
is the final relay station of visceral information
being carried to the granular IC (Cechetto & Saper, 1987).
The brain-gut axis involving the vagus and the AP also has been established as
a potential route through which LiCl-induced malaise information is carried into the
CNS. Neuroanatomical studies have well established that the AP receives visceral
input via the afferent vagus nerve (Leslie & Gwyn, 1984; Shapiro & Miselis, 1985), a
major afferent input to the brainstem from the gastrointestinal system. In addition, it
sends and receives information from the NST and the PBN, predominantly the lateral
subdivision, (van der Kooy & Koda, 1983; Shapiro & Miselis, 1985), thereby
transmitting information regarding malaise.
A second route through which malaise information is carried to the brain is via
the splanchnic nerves. Application of LiCl into the intestines or the duodenum elicits
discharges in the afferent splanchnic nerve (Niijima & Yamamoto, 1994).
Additionally, the magnitude of the LiCl-elicited neural discharges is greater for the
splanchnic than for the vagus nerve. The authors ascribed this effect to a more
important role of the splanchnic compared to the vagus in carrying information
regarding LiCl produced malaise information. The greater activation produced by LiCl
in the splanchnic however could also be due to the transmission of pain information
103
produced by the injection and not necessarily due to the transmission of malaise
information. One study examining the effects of sympathectomy on emetic responses
showed that bilateral sympathectomy alone had no effect on copper sulfate-induced
emesis (Wang & Borison, 1951); however, the combined vagotomy and
sympathecotomy increased the threshold for the drug produced emesis above that
observed by vagotomies alone. This suggests that communication between the vagus
and splanchnic or the concomitant transmission of information of the two play a critical
role in emetic responses. To our knowledge, there is no study showing the effect of
splanchnic denervation specifically on the acquisition of a CTA.
Although the projections of the splanchnic still need to be elucidated, research
examining various behaviors has shown a potential connection between the splanchnic
and the NST. Microinfusions of GABA into the commissural NST markedly reduce
sympathetic splanchnic nerve activity (Sato, Colombari & Morrison, 2001). Studies
examining the connection between peripheral osmoreceptors and the CNS have also
focused on the targets of the afferent splanchnic nerve. The researchers showed that
gastric infusions of hypertonic saline produce c-fos activation in the AP and the NST,
which was attenuated following bilateral splanchnic denervation (Carlson & Osborn,
1998), suggesting possible direct or indirect connections between the nerve and the
brainstem areas.
Finally, information regarding the US can reach the CNS through the blood.
The blood-brain-barrier is poorly developed at the level of circumventricular organs
such as the AP (Lang & Marvig, 1989) and arcuate nucleus of the hypothalamus
104
(Broadwell & Brightman, 1978). As such, the structure serves as a potential passage
for circulating toxins from the blood into the CNS (Grant & Borison, 1988; Lang &
Marvig, 1989). Evidence for the involvement of this structure in CTAs is provided in
the following section.
3.3 Neural Substrates of Conditioned Taste Aversion Learning
Lesioning techniques provide valuable information regarding the role of a
particular neural structure in CTAs; however, prior to delving into evidence that have
utilized lesioning techniques, it is important to mention a few important general
limitations of the procedure. When lesioning a brain area results in the subsequent
abolition of a behavior, this may mean one of several things. First, the lesion may have
caused a deficit in motivation such that the animal no longer cares to learn the task.
Second, there could be an attention deficit so that the subject is no longer able to focus
on the stimuli. These two possibilities could be eliminated if the lesioned subjects are
able to show a different type of learning suggesting that the animals are motivated to
learn and are capable of attending to stimuli. Finally, it may be that there is an initial
loss of function, but with time, the behavior may return due to subsiding stress or
swelling caused by the mechanics of the lesion. To avoid this, subjects must be given
sufficient time to recover following lesions prior to proceeding with behavior testing.
Behavior testing also could be repeated several months following the lesioning to
ensure that the result of the ablation is a permanent chronic effect, rather than a
temporary acute one.
105
When interpreting the results of lesion studies involving CTAs, there are further
considerations that need to be addressed, some of which differ from the
aforementioned explanations. The abolition of a CTA following a lesion may mean
one of several things. All of these possibilities must be given serious consideration
when interpreting the results of lesion effects on the acquisition of CTAs.
(1) The ability to perceive the CS tastant is destroyed.
(2) The ability to process the gustatory stimulus is destroyed. Reilly, Grigson and
Norgren (1993a) explain this as the ability to use the CS to predict important events in
the internal or external environment. For instance, the animals should be able to
perceive a taste as novel or familiar in order to predict the future consequences of
ingesting that particular food.
(3) The ability to perceive the US may be damaged.
(4) The ability to process the US stimulus is destroyed. This refers to the ability of
the US to predict important events in the internal and external environment. For
example, the ability to perceive the specific negative sensory properties of a malaise-
inducing agent as familiar allows the animal to predict that a novel taste consumed
after experiencing those negative sensations is unlikely to be the cause of them even
though they return after consuming the novel taste.
(5) The ability to associate the CS with the US is eliminated.
(6) The ability to bridge the temporal interval between the offset of the CS and the
onset of the US may be affected.
(7) The ability of the animal to express the learned behavior is destroyed.
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Two brain access routes have been identified as important for conveying US
information during the acquisition of a CTA, the AP and the vagus. Although the
splanchnic is also a possible route, as indicated above, information regarding its role in
CTAs is non-existent. Three neural areas have been implicated in transmitting both CS
and US information. These areas are (1) the parabrachial nucleus (PBN), (2) the
amygdala (Amg), and (3) the insular cortex (IC). Since the focus of this proposal is the
PBN, more attention will be given to this nucleus compared to any of the others.
3.3.1 The Vagus Nerve and the Nucleus of the Solitary Tract (NST)
As mentioned previously, the vagus nerve carries information regarding LiCl
from the gastrointestinal system into the CNS. Intraperitoneally and intraduodenally
administered LiCl causes gradual discharges in the afferent vagus nerve (Nijima &
Yamamoto, 1994). This information regarding malaise is then carried from the vagus
to the NST. Evidence corroborating this comes from a study which showed that the
expression of the protein c-fos found in the NST after LiCl administration is eliminated
following bilateral vagotomies (Yamamoto, Shimura, Sako, Azuma & Bai, 1992).
Additionally, aside from LiCl, various other agents used as USs in CTAs, such as
hypertonic saline and copper sulfate, also produce c-fos in the caudal NST (Sakai &
Yamamoto, 1997). Moreover, the expression of the protein is correlated with the
strength of the CTA produced following the administration of these agents. That is,
stronger CTAs elicited by higher doses of LiCl result in higher expression of the
protein in the caudal NST. Given that this region is essential for respiration, lesions of
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the caudal NST would result in death. As such, ablation studies examining the role of
the caudal NST in CTAs have not been conducted.
Both intraoral and intragastric infusions of sucrose produce activation of c-fos
in the rostral NST (Yamamoto & Sawa, 2000). This is not unexpected considering that
the rostral region of the NST is important for processing gustatory information;
however, this region does not appear to be involved in the acquisition of CTAs. Rats
with electrolytic lesions of the rostral NST express impaired gustatory processing, but
they are still capable of developing a CTA (Shimura, Grigson & Norgren, 1997;
Grigson, Shimura & Norgren, 1997a,b). As such, it remains unclear how gustatory
information important for CTAs gets from the taste receptors to the PBN.
3.3.2 The Area Postrema (AP)
The AP is located in the caudal region of the brainstem, more specifically at the
ventral midline of the IV ventricle near the obex. This area has many features that
would implicate its involvement in CTAs. First, the blood-brain barrier is poorly
developed at the level of the AP. As such, the structure serves as a passage for
circulating toxins from the blood into the CNS (Grant & Borison, 1988; Lang &
Marvig, 1989). Second, the neural connections of this circumventricular structure also
suggest a function of this structure in CTAs. As indicated above, neuroanatomical
studies have well established that the AP receives visceral input via the afferent vagus
nerve (Leslie & Gwyn, 1984; Shapiro & Miselis, 1985), which is a major afferent input
to the brainstem from the gastrointestinal system. The AP also sends and receives
information from the NST and the lateral PBN (van der Kooy & Koda, 1983; Shapiro
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& Miselis, 1985), two structures that are important in the formation of a CTA (Chang
& Scott, 1984; Swank & Bernstein, 1994; Grigson, Shimura & Norgren, 1997a,b for
the NST; DiLorenzo, 1988; Ivanova & Bures, 1990b; Flynn, Grill, Schulkin &
Norgren, 1991 for the PBN). Additionally, the AP receives input from structures
integrally involved in eating such as the paraventricular nucleus (PVN) and the
dorsomedial (DMH) hypothalamus (Shapiro & Miselis, 1985) suggesting that the
structure may receive information regarding ingested foods. Third, in humans, the AP
is regarded as a chemoreceptor trigger zone for emetic responses (Borison & Wang,
1953). This is supported by findings showing that thermal lesions of the AP in humans
relieve intractable vomiting (Lindstrom & Brizzee, 1962). Finally, electrical
stimulation of the AP alone can serve as the US in a CTA paradigm (Gallo, Arnedo,
Agüero & Puerto, 1988), thus implicating the circumventricular structure in
conditioned gustatory aversions.
Given these characteristics, the AP is speculated to be a US agent detector in a
CTA model. C-fos immunoreactivity is evident in the AP following the intraperitoneal
administration of LiCl (Yamamoto et al., 1992; Swank, Schafe & Bernstein 1995;
Thiele, Roitman & Bernstein 1996; Sakai & Yamamoto, 1997; Yamamoto & Sawa,
2000) indicating that the chemical produces some increased activity in this region of
the brain. As mentioned earlier, the AP could potentially detect US agents either
through the blood (Coil, Rogers, Garcia & Novin 1978; Grant & Borison, 1988; Lang
& Marvig, 1989) or from the vagus (Coil et al., 1978). Examination of this possibility
through electrophysiological studies shows that the AP contains neurons that respond
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to LiCl that is administered directly into the blood (Adachi, Kobashi, Miyoshi &
Tsukamoto, 1991) or into the IV ventricle (Adachi et al., 1991) suggesting a more
humoral route for the illness-inducing agent. Additionally, vagotomies do not disrupt
acquisition of LiCl CTAs and based on this result it has been suggested that for this
learned behavior, LiCl acts directly on the AP, via the bloodstream, rather than
indirectly through the vagus nerve (Martin, Cheng & Novin 1978); however, one
cannot eliminate the possibility that the AP mediates LiCl CTAs when activated by a
vagal as well as a humoral stimulus. If LiCl CTAs can be induced via two pathways,
vagus to AP and humoral to AP, then blocking the AP would eliminate the ability to
acquire CTA but blocking the vagus would not because the humoral to AP pathway
would still be intact. Since the humoral pathway was not blocked in the study
mentioned above, all one can say is that CTAs are not supported by vagal input alone.
Lesion studies substantiate the importance of the AP in the acquisition of CTAs
induced by various agents. Permanent lesions of the AP eliminate CTAs caused by
LiCl (Ossenkopp, 1983; Rabin, Hunt & Lee, 1983a; Wang, Lavond & Chambers,
1997a), gamma radiation (Ossenkopp, 1983; Rabin et al., 1983a), histamine (Rabin et
al., 1983a), and estradiol (Bernstein, Courtney & Braget, 1986). Temporary cooling
lesions of this structure also block LiCl CTAs (Wang et al., 1997a). The AP however,
does not affect ethanol- (Rabin et al., 1983a), amphetamine- (Berger, Wise & Stein,
1973; Rabin et al., 1983a), nicotine- (Ossenkopp & Giugno, 1990) or apomorphine
(Wang, Lavond & Chambers, 1997b) CTAs. These latter findings are important, since
they suggest that the inability of an animal to develop a CTA when LiCl, radiation,
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histamine and estradiol are used as USs is not due to a deficit in motivation or attention
since lesioned animals are able to learn CTA when certain USs are used.
Several of the aforementioned possible explanations for the disrupted CTA
following the lesions of the AP can be eliminated due to some important studies. It
has been proposed that the abolition of a CTA following AP lesions may be due to the
inability of the rats to detect the CS or to recognize it as novel, thereby blunting the
acquisition of the learned response (Kosten & Contreras, 1989). A study using the
reversible cooling lesion technique has shown evidence against both of these
hypotheses by inactivating the AP only during the duration of US (Wang et al.,
1997a,b). Since the AP is functional when the animals first encounter the novel CS
before acquisition and after acquisition on extinction day 1, blocking of a LiCl CTA
cannot be due to the inability of an animal to detect the CS nor to an inability to
process it as novel. The lesion of the AP also is probably not due to the ability of an
animal to express the learned behavior since the structure is intact during extinction test
1. Additionally, permanent lesions of the AP made following acquisition of a LiCl or
radiation CTA, but before extinction day 1, have no effect (Rabin et al, 1983b), on the
acquisition of a CTA, implying that the animals are still able to express the learned
behavior with a dysfunctional AP.
Since the AP is an important structure implicated in human emetic responses,
destruction of the AP probably interferes with the detection or the processing of illness
responses. The temporary lesion of the AP made exclusively throughout the duration
of the US (Wang & Chambers, 1997) suggests that the disruption of the LiCl CTA is
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due to either an interference with malaise information processing or even the
associative processing, since the formation of a LiCl CTA is thought to be quite rapid
(within 15 minutes; Houpt & Berlin, 1999). Evidence supports the former hypothesis.
Lesions of the AP not only block LiCl CTAs, but they also eliminate some behavioral
and physiological effects normally produced by the agent. For instance, following the
administration of a high dose of LiCl, AP-lesioned animals do not display ‘lying on the
belly’ or delayed gastric emptying compared to sham operated controls (Bernstein,
Chavez, Allen & Taylor, 1992). Since emesis in rats is blocked by the cardiac
sphincter, behavioral responses like ‘lying on the belly’ and physiological responses
such as delayed stomach emptying, are commonly used as indices of malaise.
Although this finding suggests that the lesions disrupt the ability of animals to detect or
process malaise information rather than interfering with the association process, it is
conceivable that the lesions of the AP could have disrupted both. Emphasis has been
placed on the former hypothesis, namely that lesions of the AP disrupt malaise
information processing, but it is worth noting that the AP also receives some gustatory
information via the lingual-tonsilar branch of the IXth cranial nerve (Hamilton &
Norgren, 1984) implicating it as a possible site of association. In addition, lesions of
the AP do not affect the development of a CTA when the CS (saccharin) and the US
(hypertonic NaCl) are administered concurrently (Arnedo, Gallo, Agüero & Puerto,
1990), which could mean that the AP is necessary for bridging the time interval
between the CS and US when the CS and US are administered sequentially.
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3.3.3 The Parabrachial Nucleus (PBN)
A second neural structure important for CTA acquisition is the PBN, which is
also located in the brainstem. This pontine nucleus is separated into two main
subdivisions by the superior cerebellar peduncle or brachium conjuctivum: the medial
“gustatory” PBN, which receives taste information (Norgren & Leonard, 1971, 1973;
Perrotto & Scott, 1976; Cechetto & Saper, 1987) and the lateral “visceral” PBN, which
receives malaise information in addition to information regarding cardiovascular,
respiratory, and nociceptive processes (Chamberlin & Saper, 1992; Bernard, Huang &
Besson, 1994; Bester, Menendez, Besson & Bernard, 1995).
It is primarily the lateral subdivision of the PBN that sends information to and
receives inputs from the AP (van der Kooy & Koda, 1983; Shapiro & Miselis, 1985),
making it the second neural structure potentially involved in US detection in the CTA
circuit. Administration of LiCl is known to evoke c-fos immunoreactivity in the
central, crescent dorsal, and external lateral subnuclei of the PBN and the external
medial PBN (Yamamoto et al., 1992; Chambers & Wang, 2004). The external lateral
nucleus is thought to be the putative lateral PBN target zone for ascending LiCl-
induced viscerosensory information (Sakai & Yamamoto, 1997; Yamamoto et al.,
1992). More importantly, there is strong correlation between the strength of a CTA
developed to a sweet solution and c-fos activation in the central, crescent, and external
lateral PBN and the external medial PBN following radiation, rotation, and LiCl (Sakai
& Yamamoto, 1997; Chambers & Wang, 2004). On the other hand, substances that do
not induce a CTA such as electric foot shock, strychnine, and physiological saline,
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elicit only very modest induction of the protein in the PBN (Sakai & Yamamoto, 1997)
suggesting activation in the region is indicative of US processing.
Initial studies investigating the role of the PBN in CTAs performed lesions of
the entire nucleus and examined the effects of the ablations on the learned behavior. It
has been found that bilateral temporary tetrodotoxin (TTX) lesions (Ivanova & Bures,
1990b) and permanent electrolytic lesions (Hill & Almli, 1983; Di Lorenzo, 1988;
Yamamoto & Fujimoto, 1991; Yamamoto, Shimura, Sako, Yasoshima & Sakai, 1994)
of the entire PBN consistently abolish the formation of a LiCl CTA, which implicates
involvement of the whole neural structure in the acquisition process. This finding
sparked interest in the role of the individual subdivisions of the pontine nucleus in the
acquisition of CTAs.
The Lateral Parabrachial Nucleus
Inactivation of the lateral subdivision of the PBN using various lesioning
techniques has yielded congruous results. Confined electrolytic (Sakai &Yamamoto,
1998) or ibotenic acid (Reilly & Trifunovic, 2000a,b, 2001; Trifunovic & Reilly, 2002)
lesions of the nucleus block the acquisition of a LiCl CTA. Reversible TTX lesions
(Ivanova & Bures, 1990a,b; Bielavska & Bures, 1994) or cooling lesions (Wang &
Chambers, 2002) made after the CS such that it overlaps with the US duration also
disrupt the associative learning. The lateral PBN is also involved in CTAs induced by
different USs other than LiCl. Cytotoxic lesions of the pontine nucleus block the
acquisition of a morphine-induced CTA (Nader, Bechara & van der Kooy, 1996), while
cooling lesions block apomorphine CTAs (Chambers & Wang, 2004).
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The lesions of this subnucleus have been shown to either attenuate (Reilly &
Trifunovic, 2001) or have no effect (Sakai, Tanimizu, Sako, Shimura & Yamamoto,
1994) on neophobic responses to the novel CS. In both cases however, the lateral PBN
lesion still blocks the LiCl CTA. In the first study, the authors claimed that the
disruption in CTA was not due to the effect of the lesion on neophobia. They argued
that lesions of the lateral PBN do not affect neophobic responses to a capsaicin
solution, (Reilly & Trifunovic, 2001) yet animals with these lesions are unable to
develop a LiCl CTA to this solution (Reilly & Trifunovic, 2001). As such, the authors
claimed that the acquisition of a CTA is not predicated on neophobic responses to the
taste, rather, it is independent of these responses because the lesions disrupt the
learning despite the effects on neophobia.
A direct argument against the neophobia hypothesis is provided by reversible
lesions of the lateral PBN (Wang & Chambers, 2002). Cooling lesions made between
the CS and the US such that the lesion overlaps only with the US duration, also disrupt
the formation of a CTA. If the disruption in CTAs with lateral PBN lesions was due to
abolished neophobia, then inactivation of the nucleus made only during the US should
not disrupt the learning. Additional support for this hypothesis comes from a study
finding that animals with lesions of the lateral PBN continue to develop strong
aversions when intragastric hypertonic sodium chloride serves as the US (Cubero,
Lopez, Navarro & Puerto, 2001). This finding is important not only because it
substantiates that lateral PBN lesions leave gustatory capabilities intact but also
because it demonstrates that these lesions leave the attentional state and motivational
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state of the animal undisturbed. The temporary cooling lesion study also provides
evidence against the hypothesis that the lesions of this subdivision may be due to
disruptions in the ability of the animal to express the learned behavior since the nucleus
is functional during the first extinction trial. Permanent lesions of this nucleus made
after the CS-US pairing do not affect the expression of the learned behavior (Sakai &
Yamamoto, 1998) further negating this hypothesis.
Although the evidence above allows for the dismissal of most alternative
explanations, it leaves open the possibility that the blockade of the CTA is either due to
a disruption in the transmission of malaise information or a disruption in associative
processes. The evidence available thus far suggests that lesions of the lateral PBN
abolish CTAs by causing a disruption in ascending malaise information rather than
affecting the associative process. First, the lateral PBN (especially the external
subnucleus) receives the greatest part of the afferents projecting from the AP (Lanca &
van der Kooy, 1985; Shapiro & Miselis, 1985; Milner, Joh & Pickel, 1986; Miceli, Post
& van der Kooy, 1987). In neurally intact animals, electrical stimulation of the AP can
serve as a US in a CTA paradigm; however, this is disrupted in lateral PBN lesioned
animals (Agüero, Arnedo, Gallo & Puerto, 1993). Given that the AP is critical for
malaise information and emetic responses (Borison & Brizzee, 1951), this finding
would suggest that the lateral PBN is important in processing the malaise information it
receives from the AP. Second, animals with lesions of the lateral PBN are also unable
to learn other behaviors that involve LiCl-toxicosis. For instance, animals with lateral
PBN lesions are also unable to develop a CTA towards a mild capsaicin solution
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(Reilly & Trifunovic, 2000a), which the authors claim is non-gustatory trigeminal
stimulus (Silver & Finger, 1991). As such, since the lateral PBN does not receive
information regarding the trigeminal stimulus, disruption of the CTA cannot be due to
a disruption in association. It should be noted that although capsaicin is considered by
researchers to be solely a trigeminal stimulant (Reilly & Trifunovic, 2000a; Shimura et
al., 1997) there is some evidence that the agent may also activate taste receptors (Silver
& Finger, 1991; Travers, Urbanek & Grill, 1999). To our knowledge, no studies
investigating the effect of capsaicin on medial or lateral PBN activation have been
conducted. Therefore, the possibility that some neurons in the lateral PBN mediate
both trigeminal and US information cannot be completely dismissed. A third piece of
evidence that suggests the abolition of CTAs following lateral PBN lesions are due to
disruption in the transmission of malaise information is provided by a study that used
morphine as a US. Ibotenic acid lesions of the lateral PBN precluded the formation of
a CTA (Nader et al., 1996). These same animals also were not able to develop a
morphine-elicited conditioned place aversion, and on the basis of these results it was
suggested that the lesions had interrupted the transmission of malaise information
rather than disabling the associative process. This conclusion assumes that the lateral
PBN does not process information regarding place. If the lateral PBN does process
place information, then the inability of lesioned animals to acquire a conditioned place
aversion may be due to the inability of the animals to process information regarding
place and not the US properties of morphine. This hypothesis however has not been
investigated.
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Fourth, lesions of the lateral PBN impair CTAs produced by intraperitoneal
injections of an AP-dependent muscarinic receptor antagonist methyl-scopolamine
(Gallo, Arnedo, Agüero & Puerto, 1990; Berger, Wise & Stein, 1993), which crosses
the blood-brain barrier; however, the lesions are ineffective against CTAs produced by
intraventricularly infused methyl-scopolamine (Cubero & Puerto, 2000). If the lateral
PBN was involved in association, then the lesions should disrupt CTAs produced by
the US delivered by both routes of administration. On the contrary, it only disrupts the
CTA produced by the route that involves the malaise afferent pathway and not the
intracerebroventricular one that bypasses it. Once again, based on one study, one
cannot completely discard the association possibility. It could be that the lateral PBN,
in addition to other areas like the amygdala and the insular cortex, serves as a place of
association. Therefore, in the absence of the PBN, these forebrain structures could
substitute as the association areas. As a result, a CTA could still be formed when a US
is infused intraventricularly despite a dysfunctional PBN.
The Medial Parabrachial Nucleus
Lesions of the medial subdivisions of the PBN also disrupt CTAs. Both
electrolytic (DiLorenzo, 1988; Flynn et al., 1991; Reilly et al, 1993a) and excitotoxic
(Spector, Norgren & Grill, 1992; Sakai et al., 1994; Sakai & Yamamoto, 1998;
Trifunovic & Reilly, 2002) lesions of the medial PBN block CTAs even when very
high doses of LiCl are used (DiLorenzo, 1988; Grigson, Reilly, Shimura & Norgren,
1998; Flynn et al., 1991; Spector et al., 1992; Reilly et al, 1993a; Sakai et al., 1994;
Sakai & Yamamoto, 1998; Trifunovic & Reilly, 2002). Additionally, medial PBN
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lesioned animals fail to learn a second CTA 8 months following the first, indicating the
absence of recovery following permanent lesions of this area (Scalera, Spector &
Norgren, 1995). Lesions of the medial PBN are not involved in morphine-induced
CTAs (Bechara, Martin, Pridgar & van der Kooy 1993). This suggests that lesions of
the area cannot be due to the absence of attentional capacities or motivational factors of
the subjects.
It has been demonstrated that the blockade of CTA following medial PBN
lesions is not due to the inability of animals to bridge the temporal interval between the
offset of the CS and the onset of the US. This possibility was directly tested and
rejected by using a methodology that minimizes the demand on memory.
Neurologically intact animals injected with LiCl immediately before intraoral infusions
of a CS that is delivered every 5 minutes over 30 minutes shows a reversal of taste
reactivity from ingestive to aversive. On the contrary, medial PBN lesioned animals do
not show these changes in taste reactivity responses (Spector et al., 1992).
Additionally, in a concurrent CTA design, i.e. when a LiCl solution is used both as the
CS and US thereby minimizing the inter-stimulus interval, animals with medial PBN
lesions still do not acquire a CTA (DiLorenzo, 1988).
The abolition of CTAs following medial PBN lesions also is not due to a
disruption in motivation nor in the ability of animals to express the learned behavior.
Permanently lesioning this area after the acquisition of a CTA does not affect the
learned behavior (Grigson, Shimura & Norgren, 1997b). This finding also argues
against the hypothesis the medial PBN lesions disrupt the ability of the animals to
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detect the CS since post-acquisition lesioned animals continue to show the gustatory
aversion learning.
Another possible explanation for the blockade of CTAs in medial PBN lesioned
animals is that the ablations interfere with gustatory processing. Taste reactivity
testing of orofacial responses in non-deprived animals with medial PBN lesions shows
that these animals display blunted responses to alanine, a sweet tasting amino acid
(Flynn et al., 1991). These animals also display increased intake of highly
concentrated sucrose solution and quinine, which neurally intact animals normally
avoid (Flynn et al., 1991). Sakai and Yamamoto (1998) demonstrate that animals with
medial PBN lesions also show equal preferences for quinine, HCl, and sucrose
solutions, while animals with lateral PBN lesions do not exhibit this impaired
sensibility for sapid stimuli. Animals with medial PBN lesions also show attenuation of
neophobia to sucrose, which could potentially explain the abolished CTA following its
inactivation (Scalera, Grigson, Shimura, Reilly & Norgren, 1992; Reilly et al., 1993a;
Sakai et al., 1994). These findings are not surprising given that the medial PBN is a
major relay for ascending taste information.
Cleverly designed experiments suggest that the blockade of a CTA by lesions
of the medial PBN is not due to disruption in either gustatory or visceral information
processing, but due to a selective disruption in the associative mechanism responsible
for taste aversion learning. In a series of experiments, Grigson et al. (1998) show that
rats with electrophysiologically guided ibotenic acid lesions of the medial PBN fail to
acquire a CTA to an alanine solution that is paired with LiCl. These same animals
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however are able to develop a conditioned taste preference for a flavor that is enhanced
with a non-caloric sweetner. A conditioned flavor preference (CFP) procedure pairs
two flavors: one distinctive flavor, the CS+, which is mixed with a nutrient, and a
second distinctive flavor, the CS-, which is not mixed with a nutrient. Following
several acquisition trials during which animals get single-bottle presentations of either
the CS+ or CS-, the animals are given a two-bottle test of preference for the two flavors
but the nutrient is excluded from the CS+. The animals choose the flavor that is paired
with the nutrient (CS+). The ability of lesioned animals to develop a CFP indicates
that the animals are able to detect and process gustatory information. Further, these
same animals are able to learn an aversion to a dilute (0.01mM) capsaicin solution
paired with LiCl, indicating that these animals also are able to process malaise
information. These findings are consistent with other experiments. Animals with
excitotoxic lesions of the medial PBN, which fail to develop a LiCl CTA, are able to
develop a conditioned place aversion with the identical US (type and dose), which is
ineffective in producing a CTA (Reilly et al., 1993a). These findings provide strong
evidence that medial PBN lesions preclude the integration of the CS with US stimulus.
Supporting evidence for this hypothesis can be found in studies that do not
involve lesions. Electrophysiological studies show that there are neurons in the medial
PBN that respond to both gustatory and visceral information. Hermann and Rogers
(1985) implicate the interstitial zone of neurons in the caudal medial PBN and the
subadjacent parvocellular reticular formation in containing such neurons. In a separate
study, they also show that these two specific areas receive projections from both the
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gustatory and visceral NST further validating the existence of these co-responsive
neural cells (Hermann, Kohlermann & Rogers, 1983). It is mainly for this reason that
in the realm of CTA learning, the PBN is thought to be the first area where integration
of CS and US information.
The evidence presented thus far suggests that the PBN is critically involved in
the acquisition process of a CTA. Interestingly, although an intact PBN is necessary, it
is not sufficient to produce a CTA. Decerebrated rats with an intact PBN do not
acquire CTAs (Grill & Norgren, 1978). Additionally, when the bilateral decerebration
is made following the CS presentation either 15 or 5 minutes before the US
presentation, animals remain capable of learning the behavior (Buresova & Bures,
1973). This suggests that CS gustatory processing at the level of the brain stem (i.e. in
the NST and the medial PBN) is insufficient for CTA and that gustatory processing in
the forebrain is essential for the development of a gustatory aversion.
3.3.4 The Amygdala (Amg)
A third structure important for CTA acquisition is the amygdala (Amg).
Studies investigating the involvement of the Amg in CTAs have mainly focused on two
amygdalar nuclei: the central and basolateral. Intraperitoneal injection of LiCl induces
c-fos activation in the central (Yamamoto et al., 1992; Gu, Gonzalez, Chen & Deutsch,
1993; Lamprecht & Dudai, 1995; Yamamoto, Sako, Sakai & Iwafune, 1997; Spencer
& Houpt, 2001) and basolateral (Koh & Bernstein, 2005) nuclei although the
expression is higher in the former. C-fos induction is also present in the central Amg
following sucrose consumption, while the expression of the proto-oncogene is
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relatively low in the basolateral nucleus relative to the central nucleus (Yamamoto et
al., 1997). Additionally, expression of c-fos is also found in both of these amygdaloid
nuclei following the pairing of a novel CS and US (Koh & Bernstein, 2005) further
suggesting their potential role in CTAs.
Despite the initial contradictory findings across studies due to differences in
lesioning techniques and behavioral testing, some conclusions have been drawn
regarding the roles of the central and basolateral nuclei in CTAs. Bilateral ibotenic
acid lesions of the central Amg do not affect the acquisition of a CTA (Yamamoto &
Fujimoto, 1991; Sakai & Yamamoto, 1999; Morris, Frey, Kasambira & Petrides,
1999), while lesions of the basolateral Amg, which spare the bi-directional PBN-
insular fibers that pass through the central nucleus (Frey, Morris & Petrides, 1997),
abolish CTAs (Yamamoto, Fujimoto, Shimura & Sakai, 1995; Morris et al., 1999;
Sakai & Yamamoto, 1999).
A study looking at the role of the Amg in different stages of CTAs shows that
bilateral temporary lesions of the Amg made by TTX infusions before the CS so that
the inactivation overlaps only during the CS duration do not disrupt the development of
a CTA, while temporary lesions of the area made after the CS presentation, during the
US activation, block the CTA (Gallo, Roldan & Bures, 1992; Roldan & Bures, 1994).
The findings of this experiment suggest several things. It means that the blockade of
CTA following amygdalar lesions is not due to the ability of animals to (1) detect or
process gustatory information, (2) express the learned behavior, (3) pay attention to CS
or US stimuli, (4) be motivated to perform the learned behavior, or (5) bridge the
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temporal gap between the CS and the US presentations. In this fashion, temporary
lesions provide extremely valuable information that permanent lesions are unable to
provide. On the other hand, the findings do suggest that the Amg is involved in either
the processing of malaise information or in the CS-US association. Investigations that
disassociate the latter two possibilities unfortunately have not been conducted.
3.3.5 The Insular Cortex (IC)
A fourth structure implicated in CTA acquisition is the IC. The IC is generally
divided into three layers that are hierarchically organized. The granular is the most
dorsal, the dysgranular is the middle layer, and the agranular, most ventral. Moreover,
the three layers of the IC are stratified into rostral and caudal areas (for review, see
Sewards, 2004). Studies (Hanamori, Kunitake, Kato & Kannan, 1998a,b) show that
the rostral area is exclusively gustatory, while the caudal portion is predominantly
visceral. In addition, in between these two regions, there is an area that receives
convergent gustatory, chemoreceptive, baroreceptive, and nociceptive information.
Given the complexity of the IC organization, interpreting IC lesion studies has been
difficult when the precise location of the lesions has not been specified.
There has been one study that attempted to show the differential effect of the
rostral, central, and caudal IC lesions on CTA learning. The results showed that
ibotenic acid lesions of the central IC, but not the rostral or caudal, disrupted formation
of the CTA (Nerad, Ramirez-Amaya, Ormsby & Bermudez-Rattoni, 1996). For the
remainder of the IC lesion studies discussed in this section, the area that is lesioned
will be considered rostral, central or caudal depending on the stereotaxic coordinates
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set forth for each of these areas by Nerad et al. (1996). Some researchers report that
bilateral lesions of the central IC made prior to the CS presentation impair, but do not
abolish, the acquisition of a LiCl CTA (Braun, Lasitar & Kiefer, 1982; Dunn & Everitt,
1988; Bermudez-Rattoni & McGaugh, 1991; Sakai & Yamamoto, 1999). This means
that the lesions accelerated the extinction rate of the CTA, but did not completely block
the learning. On the other hand, different laboratories show that permanent (Braun,
Slick & Lorden, 1972) or temporary TTX (Gallo et al., 1992) lesions of the central IC
made before the CS, but not after (Gallo et al., 1992; Roldan & Bures, 1994),
completely abolish the CTA. The temporary lesion study provides valuable
information regarding the role of the IC lesions in gustatory aversions. For instance, it
shows that the blockade of a CTA following the IC lesions is not due to the animal’s
inability to (1) detect or process visceral information, (2) express the learned behavior,
(3) pay attention to the CS or US stimuli, (4) be motivated to perform the learned
behavior, or (5) bridge the temporal gap between the CS and the US presentations. On
the other hand, the findings do suggest that the IC is involved in either the detection or
processing of gustatory information or in the association process. Follow up studies
show that animals with permanent IC ablations are able to detect the saccharin solution
since they showed a preference for the CS solution over water, similar to the neurally
intact control animals (Braun et al., 1972; Dunn & Everitt, 1988). Taken together,
these studies suggest that the lesions of the IC do not interfere with the detection of the
CS, but rather with the processing of the gustatory information, i.e. the lesions destroy
the ability to process the CS as novel. In fact, there is some evidence for this
125
attenuated neophobia hypothesis. The study by Dunn & Everitt (1988) shows that both
electrolytic and ibotenic acid lesions of the IC reduce the neophobic response of
animals to the CS. It should be mentioned that since the authors used a different rat
atlas than Nerad et al. (1996), it is difficult to compare the area they lesioned to those
mentioned above; however, given the description of the histology, it is likely that the
caudal, central, and rostral areas of the IC as described by Nerad et al (1996) sustained
damage.
In summary, the IC appears to be involved in the acquisition of CTAs, most
probably by affecting the processing of gustatory information. This idea is further
supported by the fact that bilateral cortical spreading depression or anesthesia induced
before the CS presentation abolishes a CTA, without interfering with the ability of
animals to show unconditioned signs of illness (Buresova & Bures, 1973; Bures &
Buresova, 1989b). In contrast, bilateral cortical spreading depression or anesthesia
applied after the CS, such that the depression overlaps with the US, does not affect the
associative learning process (Buresova & Bures, 1973; Bures & Buresova, 1989).
These results suggest that the acquisition of a CTA requires cortical involvement for
the processing of gustatory information, while subcortical areas are sufficient for
illness processing.
3.3.6 Configuring the Conditioned Taste Aversion Neural Pathway
Although the individual aforementioned neural areas play an important role in
CTAs, acquisition of this associative learning involves the interaction of all of these
brain structures. Lesioning of individual neural structures is very informative in terms
126
of determining whether or not the structure alone is involved; however, individually,
they do not give a complete picture of the CTA neural circuitry. Thus far, it is known
that the AP, PBN, Amg, and the IC are all involved in the acquisition process of LiCl
CTAs, but how are these areas interconnected? Figure 2 depicts the possible neural
circuitry that underlies conditioned taste aversion learning when LiCl is used as the
US.
It is well established that the AP and the PBN, primarily the lateral subdivision,
have reciprocal connections (van der Kooy & Koda, 1983; Shapiro & Miselis, 1985;
Miceli et al., 1987), establishing the initial circuitry of CTA learning. It should be
mentioned that lesions of the lateral PBN disrupt CTAs produced by AP-dependent
(i.e. methyl-scopolamine) and AP-independent (i.e. ethanol) USs (Cubero et al., 2001)
suggesting that CTA-producing agents may act directly on the lateral PBN or indirectly
via a different structure. Studies have demonstrated the importance of the connections
between the PBN, IC, and the Amg in CTAs by performing combined lesions of these
areas. It appears to be the case that the intact ipsilateral connections between the PBN
and the IC, between the PBN and Amg, and between the Amg and the IC are important
in the acquisition of CTAs.
The first study in a series conducted by Gallo and Bures (1991) shows that
neither unilateral de-cortication of the IC made before the CS presentation nor
unilateral TTX lesions of the entire PBN made during the inter-stimulus interval
overlapping the US disrupt a CTA, an effect that has been substantiated by other
studies (Buresova & Bures, 1973). More interestingly, combined ipsilateral lesions of
127
the PBN and the IC do not block the formation of a CTA, while contralateral lesions of
these structures inhibit the learning. Together, these findings suggest that ipsilateral
connections between the IC and the PBN are critical in the acquisition of a CTA.
A series of studies conducted by Bielavska and Roldan (1996) shows the
significance of the connections between the PBN and the Amg. The initial part of the
study demonstrates that separate unilateral TTX lesions of the individual structures do
not affect CTA acquisition. The following study demonstrates that ipsilateral
concomitant destruction of both structures during the CS-US interval do not impair
CTA development, while contralateral lesions of the two structures abolish CTAs.
This implicates the importance of ipsilateral connections between the PBN and Amg in
CTA acquisition. If contralateral connections between the PBN and Amg were critical,
the animals with lesions of the PBN and the Amg on the same side of the brain should
not have acquired a CTA since this pattern of lesion would have effectively disrupted
the contralateral communication between the two structures.
Identical results are obtained when the connections between the Amg and IC
are examined. Namely, the results show that neither separate unilateral TTX lesions of
the IC prior to CS presentation nor unilateral TTX lesions of the Amg made between
the CS-US interval have an effect on the acquisition of a LiCl CTA. Similarly,
unilateral lesions of the Amg and the IC made on the same side of the brain do not
disrupt a CTA; however, when the lesions of each of the structures are made on
opposite hemispheres, CTA formation is precluded, once again suggesting that
128
ipsilateral connections between the Amg and the IC are necessary for the formation of
the gustatory aversion.
One more piece of evidence that stresses the importance of the connections
between the basolateral Amg and the IC in CTA acquisition is provided by a study that
involves long-term potentiation (LTP). Long-term potentiation is an increase in the
excitability of a neuron to a certain input due to repeated exposure to that particular
input (Bliss & Lømo, 1973). Long-term potentiation has been proposed as a model for
synaptic plasticity underlying learning and memory (Bliss & Collingridge, 1993;
Escobar & Bermudez-Rattoni, 2000). Tetanic stimulation of the basolateral Amg
induces LTP in the IC of rats in vivo (Escobar, Chao & Bermudez-Rattoni, 1998).
More interestingly, inducing LTP in the IC in this manner prior to CTA training
enhances retention of the learned task (Escobar & Bermudez-Rattoni, 2000).
Studies investigating the connections between the lateral PBN and the
basolateral Amg have assessed their importance in CTA acquisition. Tracing studies
have established at least 4 pathways through which the lateral PBN projects to the
Amg. The first is a direct connection to the central Amg (Norgren, 1984; Sakai &
Yamamoto, 1999). The second involves a cortical route via the IC (Saper & Loewy,
1980; Sakai & Yamamoto, 1999) to the basolateral Amg (Saper & Loewy, 1980;
Saper, 1982; Yamamoto, Azuma & Kawamura, 1984; Turner & Herkenham, 1991).
The third route involves projections from the lateral PBN to the zona incerta (ZI;
Krukoff et al., 1993; Bianchi, Corsetti, rodella, Tredici & Gioia, 1998; Sakai &
Yamamoto), which projects to the midline and intralaminar thalamic nuclei (MITC;
129
Sakai & Yamamoto, 1999), which in turn projects to the basolateral Amg
(Groenewegen & Berendse, 1994). Although not replicated in the research of
Yamamoto, one study showed that the dorsal PBN cells accumulate Evans Blue
following the infusion of the retrogradely transported fluorescent label into the
basolateral Amg (Woolf & Butcher, 1982). This suggests that there potentially is a
direct connection from the lateral PBN to the basolateral Amg.
In order to assess the importance of these connections from the lateral PBN to
the Amg in CTA acquisition, single and combined lesions of these areas have been
conducted. Sakai &Yamamoto (1999) showed that bilateral ibotenic acid lesions of the
ZI, IC, central Amg, and MITC alone did not abolish LiCl CTAs, while individual
lesions of the lateral PBN and the basolateral Amg completely blocked the learning.
Additionally, combined lesions of the ZI with either the central Amg or the IC blocked
the CTA, while lesions of both the IC and central Amg did not. On the basis of these
results, it was concluded that the route from the lateral PBN through the ZI and MITC
to the basolateral Amg is of significant importance for CTA acquisition followed by
the cortical route. Figure 2 depicts the possible neural circuitry that underlies
conditioned taste aversion learning when LiCl is used as the US.
130
Figure 2. Neural Circuitry of Conditioned Aversions.
Insular Cortex

C

Central Amg Basolateral
Amg
Zona Incerta Midline & Intralaminar
Thalamic Nuclei

Medial PBN Lateral PBN

Area Postrema
Hindbrain Midbrain Forebrain
Figure 2. Depicts the possible neural circuitry that underlies conditioned taste aversion learning
when LiCl is used as the US. The figure illustrates ipsilateral connections among the structures,
which are thought to be necessary in the learned gustatory aversion. Not all connections are
depicted. Double arrows indicate reciprocal connectivity ( ).
3.4 Neurotransmitters and Conditioned Taste Aversion Learning
Methodological Issues
Methodologically, studying the neurochemical basis of CTAs is extremely
difficult. Two issues have hampered progress in this area of study and must be taken
into account when designing CTA neurochemical studies: (1) US pre-exposure effect
and (2) state-dependent learning.
131
Generally, the way in which the role of a neurochemical has been examined in
CTAs has involved blocking the receptors of interest prior to the CS-US pairing by
peripherally administering the appropriate antagonist. For instance, it has been
hypothesized that histamine mediates estradiol-induced CTAs via its H
1
receptors
(Rice, Lopez & Garcia, 1987; Rice, 1989; Hintiryan et al., 2005). As such, an H
1
antagonist would be peripherally administered prior to the CS-estradiol pairing to
determine whether blocking the histamine receptors affect the subsequent formation of
a CTA; however, pre-exposure effects may confound the interpretation of these results.
In CTA experimental designs, exposing animals to an agent (exposure agent) capable
of producing a CTA before either the same (intra) or a different (inter) agent
(conditioning agent) is paired with a taste solution on acquisition day attenuates or
blocks the subsequent formation of the CTA. The experience of pretreatment is
referred to as the US pre-exposure effect. This effect can be found when pretreatment
is given less than 24 hours before conditioning (proximal) or greater than 24 hours
before (distal). It is generally believed that only agents that produce CTAs themselves
can serve as pre-exposing agents. Preliminary studies suggest that for agents capable
of inducing CTAs, doses that are too low to promote acquisition can still produce a
pre-exposure effect (Merwin & Doty, 1994). Pre-exposure effects have been illustrated
with a wide range of agents. Proximal pre-exposure to LiCl 30 minutes before the
pairing of saccharin with a second injection of LiCl significantly disrupts the formation
of a LiCl CTA (Domjan & Best, 1977). Distally pre-exposing animals to LiCl blocks a
LiCl CTA when identical doses of LiCl are used (Domjan & Best, 1977).
132
Correspondingly, distal pre-exposure to radiation blocks a radiation-induced CTA
(Rabin, Hunt & Lee 1989), distal pre-exposure to ethanol blocks ethanol-induced CTA
(Berman & Cannon, 1974), and distal pre-exposure to estradiol blocks CTAs produced
by estradiol (Merwin & Doty, 1994). Inter-agent pre-exposure studies have shown that
previous experience with LiCl is also effective in disrupting CTAs produced by
radiation (Rabin et al., 1989), ethanol (Cannon, Baker & Berman, 1977; Rabin et al.,
1989), and estradiol (De Beun, Peeters & Broekkamp, 1993), and that pre-exposure to
estradiol accelerates the extinction of LiCl CTA (Chambers & Hayes, 2002; Yuan &
Chambers, 1999).
One way to show whether an antagonist is acting as a pre-exposing agent or an
antagonist is to have two groups that receive the antagonist prior to the CS-US pairing:
one group is given the CS-US pairing while the antagonist is actively blocking the
receptors and a second group receives the pairing while the antagonist is no longer
active. If the CTA is blocked in both cases, then it would suggest that the antagonist is
acting as a pre-exposure agent rather than a true antagonist. A second way to possibly
avoid pre-exposure effects is to administer the antagonists centrally rather than
peripherally; however, studies on pre-exposure and state-dependent effects (discussed
in the following paragraph) of neurally infused drugs are non-existent. As such, pre-
exposure effects should be kept in mind when examining the effects of centrally
infused drugs and CTAs.
The histamine and estradiol study mentioned above is complicated by one other
factor: state-dependent learning. The state dependent learning hypothesis states that
133
the failure to provide identical states during the training and testing days may interfere
with the retrieval of a previously learned avoidance response on conditioning day
(Overton, 1974). For instance, in the study where an H
1
antagonist is peripherally
administered prior to the CS, the animals are conditioned to the novel tastants in a drug
state (the H
1
antagonist), but are tested for saccharin preference in a non-drug state.
Therefore, the failure to acquire a CTA may be due to a state-dependent learning
process. One way to avoid state-dependent learning is to administer the antagonist
AFTER the CS presentation. This way, the animals will be trained and tested under
identical, non-drug states. Although this may eliminate state-dependency and pre-
exposure effects, it does not eliminate possible conditioning effects if the antagonist
has conditioning abilities. When two separate 1.5meq/kg doses of LiCl are given after
the CS, separated in time by 90-120 min, the subsequent CTA is stronger than when
only one 3.0meq/kg dose injection of LiCl is given. Likewise, it has been shown that
administering an H
1
antagonist after the CS, but before the US estradiol augments the
CTA (Hintiryan et al., 2005). As such, in an attempt to circumvent state-dependency
and pre-exposure effects the neurotransmitter antagonist may be delivered after the CS;
however, if the agent has conditioning capabilities, then the data will be confounded
with an augmented CTA.
Due to such methodological issues, studies investigating the involvement of
neurotransmitters in CTAs have been sparse. Of those studies performed, most of the
attention has been focused on the muscarinic cholinergic receptors, while histamine has
been the focus of fewer studies.
134
3.4.1 Acetylcholine (ACh)
Synthesis of Acetylcholine
Acetylcholine is synthesized by the combination of choline and acetate, an ion
which is provided by the cofactor acetyl-CoA with the aid of the enzyme choline
acetyltransferase (ChAT). Post-synaptic potentials induced by ACh are short-lived and
are enzymatically deactivated by acetylcholinesterase (AChE). Research examining
the effects of ACh on various behaviors has used these enzymes to manipulate levels of
the neurotransmitter. Acetylcholine is synthesized in many regions of the brain
including those involved in CTA learning such as the AP (Tago et al., 1989), PBN
(Tago et al., 1989; Tafti et al., 1997), Amg (Ichikawa et al., 1997) and possibly the IC
(Lopez-Garcia, Bermudez-Rattoni & Tapia, 1990; Berman, Hazvi, Neduva & Dudai,
2000).
Cholinergic Receptors and Receptor Sites
Cholinergic neurons project extensively throughout the brain. Due to the
limited scope of this paper, the discussion regarding these cholinergic projections will
be restricted to the previously mentioned brain areas that are involved in CTA learning,
namely the AP, PBN, Amg, and IC.
Two types of ACh receptors exist: nicotinic receptors, which are ionotropic in
nature and muscarinic, which are metabotropic. Most research on ACh and CTAs has
focused on the role of muscarinic receptors. There is scarce information regarding the
role of nicotinic receptors in memory formation as it relates to CTAs. As such, the
discussion in this section will focus only on the metabotropic receptors. The
135
muscarinic ACh receptors are classified as M1-M5 according to their biochemical
properties (Bonner, Buckley, Young & Brann, 1987; Bonner, Young, Brann &
Buckley, 1988; Buckley, Bonner & Brann, 1988; Buckley, Bonner, Buckley & Brann,
1989). Scopolamine, a commonly used antagonist in CTAs, which also will be used in
the experiments proposed in this proposal, is a non-selective muscarinic receptor
antagonist that blocks all subtypes.
A high density of muscarinic receptors, identified by radiolabeled
3
H-QNB
(quinuclidinyl benzilate) binding, can be found in the AP (Pedigo & Brizzee, 1985) and
the dorsal nucleus of the lateral PBN (Wamsley, Lewis, Young & Kuhar, 1981). It is
not surprising to find these receptors in the AP given that it is considered to be a trigger
zone for emetic responses (Borison & Brizzee, 1951) and that the muscarinic receptor
antagonist scopolamine is agreed to be the most effective treatment against motion
sickness-induced emesis (Brand & Perry, 1966; Wood, 1979; Waldrop, 1982). With
respect to the PBN, the M1, M2, and M3 receptors have been found in both the medial
(Mallios, Lydic & Baghdoyan, 1995) and the entire lateral PBN (Christie & North,
1988; Mallios et al., 1995). A high density of M1 receptors can be found in the
basolateral Amg and the IC (Spencer, Horvarth & Traver, 1986; Wall, Yasuda, Hory,
Flagg, Martin et al., 1991; Flynn & Mash, 1993) as well. The IC also contains a large
number of M2 receptors (Spencer et al., 1986; Wall et al., 1991; Flynn & Mash, 1993).
Given that the M3 receptor distribution frequently coincides with that of M1 sites, this
subtype also is probably found in the basolateral Amg and the IC (Flynn & Mash,
1993).
136
Acetylcholine and Learning
In general, cholinergic mechanisms are implicated in learning, memory, and
attention (Decker & McGaugh, 1991; Fibiger, 1991; Hasselmo & Bower, 1993; Muir,
Page, Sirinathsinghji, Robbins & Everitt, 1993). Specifically, there is evidence to
suggest the involvement of ACh in habituation (Pearson, 1973), and acquisition of
classical eye-blink conditioning (Downs, Cardozo, Schneiderman, Yehle, Vandercar et
al., 1972), passive avoidance (Buresova, Bures, Bohdanecky & Weiss, 1964), active
avoidance (Meyers, Roberts, Riciputi & Domino, 1964), and maze learning
(Whitehouse, 1967). It is not surprising then that cholinergic mechanisms are also
reported to be involved in CTAs (Deutch, 1978; Bermudez-Rattoni, Coburn,
Fernandez, Chavez & Garcia, 1987; Lopez-Garcia et al., 1990).
Acetylcholine and Conditioned Taste Aversions
One piece of evidence that would suggest that ACh is a mediator of CTAs, is if
the neurotransmitter administered alone produces a CTA. Acetylcholine has been
shown to be capable of producing the learned gustatory response. Intraperitoneal
injections of cholinergic agonists that cross the blood-brain barrier are capable of
producing CTAs (Preston & Schuster, 1981; Romano & King, 1987). Intracerebral
infusion of these agonists into the medial septum and lateral ventricles also result in
CTAs, whereas infusions into the ventral hippocampus do not (Myers & de Castro,
1977). This site-specific effect suggests that ACh is not producing some general effect
that is acting as the US.
137
Several studies using ACh receptor antagonists claim to provide support for the
involvement of cholinergic mechanisms in CTAs. The muscarinic receptor antagonist
atropine delivered peripherally before the intraoral infusions of saccharin solution,
blocks the CTA normally produced by a high dose of LiCl (Deutsch, 1978). The CTA
however, is not blocked when atropine is given after the CS and during exposure to
LiCl. Although anti-cholinergic activity appears to interfere with fear (Plotnik,
Molenauer & Snyder, 1974) and nociception (Pert, 1975), this blocking effect of
atropine is not due to an antidote effect. That is, atropine sulfate does not attenuate the
effects of LiCl since atropine sulfate co-administered with LiCl produces an aversion
that is equal in strength to a CTA produced by LiCl alone (Deutch, 1978). Although
the authors claim that these studies show the involvement of ACh in LiCl CTAs, pre-
exposure and state-dependency may be confounding the findings. In fact, atropine
sulfate administered peripherally has been shown to produce a CTA (Preston &
Schuster, 1981; Romano & King, 1987), which suggests that the agent may serve as a
pre-exposure agent.
Interestingly, ACh antagonists also interfere with radiation CTAs when
backwards conditioning is employed, that is, when the US is administered prior to the
CS. Atropine sulfate, but not atropine methylnitrate, given subcutaneously 30 minutes
prior to radiation, blocks the formation of a radiation CTA (Gould & Yatvin, 1972;
Gould & Yatvin, 1973). Since atropine sulfate crosses the blood-brain barrier, while
atropine methylnitrite does not, this suggests central rather than peripheral effects of
the antagonist. A different study has shown that atropine administered after the
138
radiation also blocks the CTA (Gould & Yatvin, 1973). This suggests that the
abolition of the CTA is not due to pre-exposure effects. Equally important, the
researchers showed that the half-life of peripherally injected atropine is 3 days. This
would mean that the animals were both trained and tested for a CTA under the
influence of the drug, thus also eliminating the state-dependency hypothesis as a
possible explanation for the ability of atropine to block CTA.
Central effects of atropine on CTAs have also been investigated. Atropine
sulfate delivered into the lateral ventricles prior to the CS presentation blocks a LiCl
CTA (Deutch, 1978), an effect that seems to be centrally, rather than peripherally
mediated, since intraperitoneal injections of atropine methylnitrite does not interfere
with the LiCl CTA (Deutch, 1978). The same study showed that the intraperitoneal
injection of a LiCl and atropine sulfate mixture produced a CTA comparable in
strength to LiCl injection alone (Deutch, 1978). This implies that in this study,
atropine did not produce a CTA and thus the effects may not be due to pre-exposure
effects; however a direct test of this possibility was not conducted using a method
similar to distal pre-exposure. If distal pre-exposure to atropine blocked a LiCl CTA,
then this would suggest that pre-exposure effects may have played a role since the
blockade of the CTA would have occurred even after the ACh receptors were no longer
actively blocked. Table 1 summarizes the effects of peripherally and centrally
administered atropine on CTAs.
139
Table 1. Effects of Peripherally and Centrally Administered Cholinergic
Antagonists on Conditioned Taste Aversions.
ACh Antagonist Infusion Site Outcome
Atropine Peripheral CS ---> ACh antagonist ---> LiCl CTA Deutsch, 1978
Peripheral ACh antangonist ---> CS--> LiCl No CTA Deutsch, 1978
Peripheral ACh antagonist ---> Radiation ---> CS No CTA Gould & Yatvin, 1973
Peripheral Radiation ---> ACh antagonist ---> CS No CTA Gould & Yatvin, 1973
Lateral Ventricles No CTA Deutsch, 1978
Scopolamine Peripheral ACh antagonist ---> CS ---> LiCl No CTA Evenden et al., 1992
Peripheral CS --> ACh antagonist ---> LiCl CTA
Amygdala ACh antagonist ---> CS ---> LiCl No CTA Castellanos et al., 2000
Amygdala CS ---> ACh antagonist ---> LiCl CTA Castellanos et al., 2000
Insular Cortex ACh antagonist ---> CS ---> LiCl No CTA Ferreiro et al., 2002
Insular Cortex CS ---> ACh antagonist ---> LiCl CTA Ferreiro et al., 2002
ACh antagonist ---> CS ---> LiCl
Methodology Reference
Kral, 1971
Similar results have been obtained using the muscarinic receptor antagonist
scopolamine. Intraperitoneal injection of scopolamine (0.1 and 0.3mg/kg) given 30
minutes prior to (Evenden, Lavis & Iversen, 1992), but not after (Kral, 1971) exposure
to sucrose solution, blocks a LiCl CTA. Microinjection of the muscarinic ACh
antagonist scopolamine into the gustatory IC before, but not during LiCl exposure
impairs the CTA (Castellanos, Salas, Gonzalez, Roldan & Garcia, 2000; Ferreira,
Gutierrez, de la Cruz & Bermudez-Rattoni, 2002) suggesting the importance of ACh in
the acquisition of taste memory and further implicating the role of cortical ACh in
gustatory information processing. Similarly, scopolamine administered into the Amg
10 minutes before the CS presentation also blocks a LiCl CTA indicating the role of
the cholinergic system in the limbic structure as well (Castellanos et al., 2000). On the
contrary, infusing the antagonist during the inter-stimulus interval so that it is present
during LiCl exposure does not affect the learned behavior. Microinjection of
scopolamine into the IC 20 minutes prior to CS presentation on extinction day 1 only,
140
does not affect the CTA, suggesting that the muscarinic antagonist does not affect the
sensation of taste, the hedonic valence of the taste or other faculties required to perform
the task (Naor and Dudai, 1996). As discussed previously, this blockade potentially
could be due to either state dependency or due to drug pre-exposure effects. Table 1
summarizes the effects of peripherally and centrally administered scopolamine on
CTAs.
Despite possible pre-exposure effects and state-dependent learning that may
account for the findings in the previous studies, there is sufficient evidence to suggest
the involvement of cholinergic mechanisms in the processing of gustatory stimuli.
Microdialysis studies show that there is a release of ACh in the IC in response to a
novel saccharin or quinine taste stimulus in comparison to rats that drink familiar water
(Shimura, Zuzuki & Yamamoto, 1995; Miranda, Ramirez-Lugo & Bermudez-Rattoni,
2000). Further, this increment is attenuated as the novel stimuli become familiar
(Miranda et al., 2000) indicating an inverse relationship between the levels of ACh and
the familiarity of taste. This suggests that ACh in the IC is involved in assessing the
novelty of a particular taste. Corroboration for this hypothesis is provided by a study
that shows the involvement of ACh in neophobic responses. Blocking muscarinic ACh
receptors with scopolamine in the IC before the CS presentation prevents a novel
solution from becoming familiar (Gutierrez, Tellez & Bermudez-Rattoni, 2003). That
is, the animals continue to show neophobia toward the sweet solution when cholinergic
receptors are blocked. This effect is not due to state-dependency since scopolamine
administered both before training and test days revealed the same finding.
141
Additionally, scopolamine administered after the CS presentation also prevents the
solution from becoming familiar. Finally, this effect also is not due to the aversive
qualities of scopolamine, since infusions of the cholinergic antagonist into the IC do
not produce a CTA.
Interestingly, ACh may modulate the induction of LTP that is observed in the
IC following the tetanic stimulation of the basolateral Amg (Escobar et al., 1998).
Microinfusions of atropine (Jones, French, Bliss & Rosenblum, 1999) or scopolamine
(Ramirez-Lugo, Miranda, Escobar, Espinosa & Bermudez-Rattoni, 2003) into the IC
reduces the induction of LTP in the basolateral-IC projection and simultaneously
blocks the increased retention of LiCl CTA that is normally observed with the
induction of LTP.
Although studies have shown that infusions of muscarinic antagonists into the
IC and Amg following the CS do not affect a LiCl CTA, no studies have examined the
effect of these antagonists in the PBN. The evidence presented thus far implicates
cholinergic mechanisms in gustatory information processing; however, the
neurotransmitter may be involved in the processing of malaise information as well.
Stimulation of the vagus nerve normally produces excitatory potentials in the
“visceral” region of the IC (Barnabi & Cechetto, 2001). Administration of atropine, a
muscarinic receptor antagonist, attenuates these vagally evoked cortical responses,
suggesting a cholinergic mechanism in the transmission of visceral information. On
the other hand, GABA
A
and α-adrenergic receptor antagonists have no effect on
cortical excitatory outputs induced by vagal stimulations.
142
Further, ACh has been implicated in emetic responses. The muscarinic
antagonist scopolamine is one of the most effective treatments for motion or space
sickness (Wood, 1979; Waldrop, 1982). Ex juvantibus reasoning suggests the
involvement of ACh in human emetic responses. As mentioned previously, one of the
brainstem areas that mediates emetic responses is the AP (Borison & Brizzee, 1951),
which has connections with both the medial and lateral PBN (Leslie & Gwyn, 1984;
Shapiro & Miselis, 1985). It would not be surprising then to find that cholinergic
mechanisms are involved in the processing of US stimuli in the brainstem, particularly
in the PBN.
3.4.2 Histamine (HA)
Synthesis of Histamine
Histamine is synthesized by the combination of the amino acid histidine
through pyridoxal phosphate and histidine decarboxylase. It is broken down into its
major inactive catabolite 3-(tele)-methylhistamine through the combined actions of
hitamine-N-methyltransferase, aldehyde dehydrogenase, and S-adenosyl-methionine
(SAM). Histamine may also be catabolized into imidazole acetic acid by diamine
oxidase. The 3-(tele)-methylhisamine can be further metabolized to N-methyl-
imidazoleacetic acid by monoamine oxidase as well.
Several investigators have implemented immunohistological methods in
attempts to localize and map the distribution of HA in the CNS using neural tissue of
rats and guinea pigs (Garbarg, Barbin, Feger & Schwartz, 1974; Steinbusch & Mulder,
1985; Wouterlood & Steinbusch, 1991). The data are summarized only briefly here.
143
Histamine cell bodies are generally confined to a region in the hypothalamus,
specifically in the tuberomammillary nucleus. Additionally, scattered HA cell bodies
are found throughout the posterior hypothalamus that extend to the middle portion of
the third ventricle. Some histaminergic cell bodies also are localized in the medial
forebrain bundle. A similar organization is described in humans, with the exception
that the neurons are greater in number (Airaksinen, Paetau, Paliarui, Reinikainen,
Riekkinen, et al., 1991).
Histaminergic Receptors and Receptor Sites
Four types of HA receptors have been identified up to date: H
1-4
, the H
4
being
the most recent. All four are G-protein coupled receptor subtypes (Gantz, Munzert,
Tashiro, Schäffer, Wang, et al., 1991; Lovenberg, Rowland, Wilson, Jiang, Pyati, et al.,
1999; Hough, 2001). Localization of the H
4
receptor in the central nervous system
remains to be established; however, studies show that both the medial and lateral PBN
contain H
1
(Palacios et al., 1981; Lintunen, Sallmen, Karlstedt, Fukui, Eriksson, et al.,
1998 et al., 1998) and H
2
(Ruat, Traiffort, Buthenet, Schwartz, Hirschfeld, et al., 1990)
receptor subtypes. The H
3
receptor is an autoreceptor that inhibits the release of HA
(Arrang, Garbarg & Schwartz, 1983, 1987). H
1
(Palacios et al., 1981; Lintunen et al.,
1998; Lozeva, Tuomisto, Sola, Plumed, Hippeläinen, et al., 2001), H
2
(Martinez-Mir,
Pollard, Moreau, Traiffort, Ruat, et al., 1993) and H
3
(Pillot, Heron, Cochois, Tardivel-
Lacombe, Ligneau, et al., 2002) are also found in the AP, Amg, and the IC.
144
Histamine and Learning
The role of HA in learning has been contradictory. Initial studies have shown
that depletion of neuronal HA retards the learning in an active avoidance response
(Kamei, Okumura & Tasaka, 1993), and this deficit is reversed upon infusions of HA
into the lateral ventricles. Histamine 1 receptor blockers have been shown to cause
decrements in the same learning paradigm, suggesting that HA facilitates learning via
H
1
receptors (Kamei & Tasaka, 1991). Similar facilatory effects are observed with H
3
antagonists, which increase the production of neuronal HA. This elevated performance
is observed with different learning paradigms including passive avoidance (Meguro,
Yanai, Sakai, Sakurai, Maeyama, et al., 1995), object recognition (Giovannini,
Bartolini, Bacciottini, Greco & Blandina, 1999), elevated plus maze (Onodera et al.,
1998), and object recognition (Giovannini et al., 1999). In contrast, other researchers
have shown that intraperitoneal injections of the HA precursor (
L
-Histidine) impairs
acquisition in the active avoidance paradigm, while an inhibitor of HA synthesis has
the opposite effect (Rubio, Begega, Santin, Miranda, & Arias, 2001). Similar results
have been obtained when the neurotransmitter is infused into the basolateral Amg and
ventral hippocampus (Alvarez & Ruarte, 2002). A study using the elevated plus-maze
illustrates that an infusion of HA into the ventral hippocampus inhibits learning, which
is blocked by an H
1
antagonist (Alvarez, Ruarte & Banzan, 2001).
Interestingly, it is speculated that HA may facilitate learning through its
interactions with acetylcholine. Histamine 3 agonists, which decrease neuronal
histamine cause the inhibition of cortical ACh release (Clapham & Kilpatrick 1992;
145
Arrang, Drutel & Schwartz, 1995), implicating a relationship between the two agents.
This hypothesis is supported by the finding that administration of HA antagonizes the
learning deficits caused by the ACh antagonist scopolamine (Miyazaki, Imaizumi &
Onodera, 1995).
Histamine and Conditioned Taste Aversions
Much less attention has been focused on the involvement of the
neurotransmitter HA in CTAs. Studies investigating the cellular basis of CTAs have
targeted peritoneal mast cell degranulation as a potential mediator of the gustatory
learning (Persinger & Fiss, 1978). Mast cells, which contain biological agents such as
serotonin and dopamine, also are known to be large depositories for HA (Persinger,
1977). Evidence supporting the mast cell hypothesis is provided by three different
findings. First, ionizing radiation, which has been used in many CTA studies as an
unconditioned stimulus (Garcia et al., 1955; Garcia, Ervin & Koelling, 1967; Levy,
Carrol, Smith & Hofer, 1974; Rabin, Hunt & Lee, 1982) activates degranulation of
peritoneal mast cells (Seyle, 1965). Second, this radiation-induced degranualtion is
abolished by the intraperitoneal administration of pyrilamine, an H
1
antagonist (Smith,
1958). Third, administration of another H
1
antagonist, chlorpheniramine maleate,
before pairing a saccharin solution with gamma radiation, prevents the formation of a
CTA normally produced by that dose of radiation (Levy, Carrol, Smith & Hofer, 1974).
Additional evidence for the involvement of HA in mediating CTAs comes from
the fact that the neurotransmitter itself is capable of inducing the learned response.
Both intraperitoneal injections (Rabin et al., 1983a) and intraventricular infusions into
146
the IV ventricle (Rabin et al., 1982) of HA produce CTAs. This is not surprising
considering that like ACh, HA, specifically through its H
1
receptor subtype, is also
implicated in emetic responses. Histamine 1 receptor antagonists that cross the blood-
brain-barrier are effective in preventing motion sickness in humans (Brand & Perry,
1966; Wood & Graybiel, 1970; Graybiel, Wood, Knepton, Hoche & Perkins, 1975),
while those that do not, remain ineffective (Kohl, Homick, Cintron & Calkins, 1987),
suggesting a central role of the histaminergic system in malaise.
Administration of chlorpheniramine maleate (CM), before pairing a saccharin
solution with gamma radiation, prevents the formation of a CTA normally produced by
that dose of radiation (Levy et al., 1974); however, this effect is probably due to state-
dependent learning since the CTA is no longer abolished when the anti-HA is given
after the sucrose presentation on acquisition day (Levy, Carrol & Smith, 1975). The
same result is found in a separate study that also used radiation as the US.
Administration of CM before the CS on acquisition day completely blocked the
radiation CTA; however, when CM was given before the CS on both acquisition and
extinction day 1, the CTA was no longer abolished (Rabin et al., 1982).
3.5 Estradiol and Conditioned Taste Aversion Learning
There are a considerable number of chemical agents that are capable of
inducing CTAs. One agent that has been studied extensively as an unconditioned
stimulus in CTA experiments is the hormone estradiol. The ability of estradiol to
produce a conditioned decrement in food intake has been demonstrated across different
species, routes of administration, and conjugated forms for both males and females
147
(Mordes, Longscope, Flatt, MacLean & Rossini, 1984; Bernstein et al., 1986; Rice et
al., 1987; Miele, Rosellini & Svare, 1988; Ganesan & Simpkins, 1990; Ganesan &
Simpkins, 1991; De Beun, Jansen, Smeets, Niesing, Slangen et al., 1991; Peeters et al.,
1992; De Beun et al., 1993; Merwin & Doty, 1994; Ossenkopp, Rabi & Eckel, 1996;
Yuan & Chambers, 1999). The successful treatments have included subcutaneous
injection of estradiol, estradiol benzoate, and estradiol cypionate, subcutaneous
implantation of a melted estradiol pellet, an estradiol-filled Silastic capsule, and an
osmotic mini-pump filled with estradiol benzoate, oral administration of 17α-ethinyl
estradiol, and intravascular administration of a brain-enhanced estradiol chemical
delivery system. The species have included Sprague-Dawley, Wistar, and Long-Evans
rats, Crl: CD-1(ICR)BR and Rockland-Swiss mice, and humans. Although it
unequivocally has been found that high supraphysiological doses of estradiol produce
strong CTAs, low physiological doses appear to be ineffective in both females and
males. In one study, a supraphysiological dose of 17β-estradiol produced strong CTAs
to a saccharin solution, while physiological doses, such as 0.4 and 2μg/kg, did not (De
Beun et al., 1991).
Conditioned Taste Aversions Revisited
There is an assumption that those agents capable of inducing CTAs produce
some type of illness and thus all reductions in consumption that occur after pairing an
agent with a novel taste are based on aversive conditioning; however, this assumption
has been challenged. It has been suggested that conditioned reductions in intake can
result from states that differ from illness (Booth, 1977). For instance, pairing a food
148
with a satiety-producing agent will elicit a learned reduction in the consumption of that
particular food (Booth, 1985). More recently, it has been suggested that CTAs induced
by some agents are qualitatively different than those produced by the classic illness
agent LiCl (Hunt & Amit, 1987; Parker, 1988; Zalaquett & Parker, 1989). For
instance, animals conditioned with LiCl avoid sucrose on subsequent exposures. These
animals also increase their aversive orofacial responses to the CS when the solution is
forcibly infused into their oral cavities via a cheek fistula. On the other hand, although
morphine (Parker, 1988) and amphetamine (Zalaquett & Parker, 1989) produce
reductions in the consumption of the taste CS, the animals do not show an increase in
aversive orofacial responses when exposed to the CS. Because of these concerns and
the fact that estradiol is a long-term satiety hormone, our lab has decided to use the
terminology conditioned taste avoidance for estradiol CTAs since it is behaviorally
descriptive and is not laden with inferences as to its illness producing properties. The
learned gustatory aversions produced by LiCl will be referred to as aversions.
Neural Substrates of Estradiol Conditioned Taste Avoidance
Neural mechanisms of CTAs have primarily focused on CTAs produced by
LiCl. Limited attention has been paid to CTAs produced by estradiol. One study
demonstrated that estradiol-induced CTAs may be mediated by the AP. Male rats
placed under chronic estradiol treatment decrease their food consumption.
Additionally, when offered a novel diet while under estradiol treatment, animals show
a preference for the novel diet, while the control animals prefer their familiar diet,
suggesting that the estradiol treated animals develop a CTA to the diet they were
149
exposed to under estradiol treatment. Following thermal lesions of the AP, these
animals no longer express estradiol-induced hypophagia or CTA (Bernstein et al.,
1986).
A preliminary study in our lab shows that injections of 10- and 50μg of
estradiol induces the expression of c-FLI in the crescent, external, and dorsal nuclei of
the lateral PBN, and the external medial PBN 2- and 24 hours following injection
(Chambers, unpublished data). Expression is found in the central lateral PBN for
50μg/kg when measured 2 and 24 hours after injection, but for 10μg/kg it is found only
when measured 2 hours following injection. We speculate that the 2-hour
measurement is associated with acquisition of a CTA, while the 24-hour measurement
is probably associated with the unconditioned effects of estradiol since decrements in
eating are observed 24 hours following subcutaneous injection.
Neurochemical Mediation of Estradiol Conditioned Taste Avoidance
Studies examining the neurochemical basis of estradiol CTAs are extremely
limited. A preliminary study conducted in our laboratory shows that pre-treating
animals with peripherally administered scopolamine methyl nitrate, which does not
cross the blood-brain barrier, 20 minutes prior to implantation of a 30mm estradiol
capsule, attenuates a CTA normally produced by estradiol (Ikramullah & Chambers,
unpublished data). This attenuation could in fact be due to the blockade of muscarinic
ACh receptors and not due to pre-exposure effects or state-dependent learning since the
antagonist was administered after the CS. Further, the study was not contaminated by
conditioning effects of the antagonist. If scopolamine methyl nitrate had conditioning
150
properties, the CTA would have been augmented, but instead, it was attenuated.
Together, these suggest that ACh may be mediating the conditioning effects of
estradiol.
An association between estradiol and HA also has been demonstrated.
Application of 17-β estradiol increases the secretion of HA that has been triggered by
compound 48/80, a mast cell degranulator (Vliagoftis, Dimitriadou, Boucher,
Rozniecki, Correia et al., 1992). This elevated release of HA is blocked by the
application of the estrogen receptor antagonist, tamoxifen (Vliagoftis et al., 1992).
Predicated on these findings, it is purported that CTAs produced by estradiol are
mediated by HA. An experiment was conducted in our lab to directly test this
hypothesis. Peripheral administration of the H
1
receptor antagonist chlorpheniramine
maleate (CM) 24 hours or 20 minutes before pairing sucrose with estradiol blocked the
acquisition of a subsequent CTA (Hintiryan et al., 2005). Three explanations were
suggested to account for this result: (1) the anti-histaminergic properties of CM
antagonize the action of HA, which mediates CTAs elicited by estradiol (the
histaminergic hypothesis), (2) CM given before acquisition but not before each
extinction test creates a state-dependency that precludes retrieval of the CTA during
post-acquisition testing if CM is not present (the state-dependency hypothesis), or (3)
CM given before acquisition acts as a pre-exposure agent and consequently blocks the
CTA (Hintiryan et al., 2005).
Two additional studies were conducted in order to clarify which of these
hypotheses best explained the findings. To examine the possibility of state-dependent
151
learning, CM was administered before the CS on both acquisition and extinction days.
The estradiol CTA was blocked, providing evidence against the state-dependent
hypothesis. Since CM has a depressive effect on drinking, its administration prior to
the CS in each of these experiments resulted in decreased consumption of the CS on
acquisition day. In order to circumvent this problem and to provide additional
evidence against the state-dependent hypothesis, CM was injected after sucrose
consumption on acquisition day. As such, the animals were not under a drug state
during their exposure to sucrose and the drug did not compromise their drinking.
Further, if HA mediated estradiol CTAs, CM should block the CTA whether it was
given before or after the CS presentation on acquisition day. Administering CM after
sucrose consumption actually augmented the CTA rather than blocking or attenuating
it. This provided evidence against the histaminergic hypothesis as well as the state-
dependent hypothesis. Taken together, the results suggest that some other explanation
than a histaminergic or state-dependency hypothesis must account for the blocking of
the estradiol CTA observed when CM is administered before sucrose consumption
during acquisition. A summary of the HA and estradiol studies is provided in Table 2.
Table 2. Histamine and Estradiol Conditioned Taste Avoidance.
Exp erim e nt D ista l Pre-exp osure Proxim al Pre-e xpo su re Pre -expo sure O utcom e
1 N o C T A
C M N o C T A
2 C M N o C T A
3 Stro ng er C T A
Su crose
Su crose
Su cro se Estra diol Su crose
C M
C M
Su cro se
Su cro se
Su cro se
Estra diol
Estra diol
C M ---> Estrad io l
Su crose
A c quis ition E xtinctio n
T a ste Stim ulu s Post-exposure T aste Stim u lus
152
Closer examination of the literature suggests that the effects of CM follow a
pattern that supports its role as a pre-exposure and conditioning agent. The pattern of
results for the first two experiments is similar to the pattern found with proximal pre-
exposure to LiCl in CTAs induced by LiCl (Domjan & Best, 1977) and in CTAs
induced by estradiol (De Beun et al., 1993; Chambers & Hintiryan, unpublished data).
When CM was given 20 minutes before sucrose consumption on acquisition day, it
blocked the estradiol-induced CTA. In the third experiment, CM was injected
immediately after sucrose consumption and estradiol was implanted 20 minutes later.
This post-exposure to CM augmented the estradiol-induced CTA. This finding is
similar to what has been observed with LiCl. A stronger CTA is produced when two
separate doses of 1.5meq/kg LiCl are injected immediately and 35 or 70 minutes after
sucrose consumption than when a single dose of 3.0meq/kg LiCl is injected
immediately after consumption (Domjan, Foster & Gillan, 1979).
All of these results taken collectively suggest that peripheral administration of
CM has aversive properties and that its effect on estradiol CTAs is due to these
properties. It serves as a pre-exposure agent to the conditioning agent estradiol, thus
blocking the subsequent formation of the CTA, and it does not act as an anti-
histaminergic agent. Recent evidence in our lab showing that CM can induce a CTA is
consistent with this hypothesis. As such, it is not possible to examine the
neurochemical basis of estradiol CTAs using peripheral administration of antagonists.
On the other hand, there are agents that selectively induce CTA when infused centrally,
that is, they induce CTA when infused into some neural sites but not others. This
153
suggests that central infusions of neurotransmitter antagonists may be a potential
solution to the problem of studying the chemical mediation of conditioned gustatory
aversions.
154
CHAPTER 4
Conducted Experiments
4.1 Outline of Experiments
The experiments conducted and discussed in the present chapter addressed the
following two Primary Aims:
(I) to determine the ability of estradiol to produce a conditioned taste
avoidance
(CTA) by testing
- the unconditioned hypothesis
- the negative contrast hypothesis
- the aversive hypothesis
(II) to conduct preliminary experiments that would allow the examination of
the neurochemical basis of estradiol elicited CTA.
A total of five experiments, comprising of nine studies, were conducted in
order to address each of these aims. In addition to the two Primary Aims, the
experiments addressed the following two Secondary Aims:
(i) to compare CTAs produced by lithium chloride and estradiol and
(ii) to investigate the neural basis of estradiol anorexia
4.2 Primary Aims
4.2.1 Primary Aim 1
Primary Aim 1 is to test the capability of estradiol to induce a conditioned
taste avoidance (CTA).
155
In this dissertation, the ability of estradiol to produce a CTA is challenged.
We introduce two alternative hypotheses that could account for reductions in sucrose
consumption observed after pairing it with estradiol. The first is termed the
unconditioned hypothesis and it purports that reductions in CS consumption are a
result of the unconditioned effects of estradiol on eating. The second is the negative
contrast hypothesis, which proposes estradiol conditioning based on a relative
preference model rather than an avoidance model. Under Primary Aim 1,
experiments were designed to determine whether either of these hypotheses could
account for the reduction in CS consumption after the CS has been paired with
estradiol.
Specific Aim 1.1: To test the unconditioned hypothesis by determining whether the
unconditioned effects of estradiol on food consumption contribute to the apparent
reduction in CS consumption observed after pairing the hormone with the novel
tastant.
When consumption of a novel tasting substance is followed by administration
of a chemical agent that produces physiological changes indicative of malaise,
animals will reduce their consumption of the substance during subsequent
encounters. This learned response is traditionally referred to as a conditioned taste
aversion (CTA). The ability of estradiol to produce a CTA when administered
exogenously has been demonstrated across different species, routes of administration,
and forms of the hormone in both males and females (Mordes, Longscope, Flatt,
MacLean & Rossini, 1984; Bernstein et al., 1986; Rice et al., 1987; Miele, Rosellini
156
& Svare, 1988; Ganesan & Simpkins, 1990; Ganesan & Simpkins, 1991; De Beun,
Jansen, Smeets, Niesing, Slangen et al., 1991; Peeters, Smets & Broekkamp, 1992;
De Beun et al., 1993; Merwin & Doty, 1994; Ossenkopp, Rabi & Eckel, 1996; Yuan
& Chambers, 1999).
There also is substantial evidence illustrating the role of estradiol in the
unconditioned regulation of ingestive behaviors. Physiological levels of estradiol
have been associated with systemic variations in the amount of food intake across the
reproductive cycle of certain mammals (Wade, 1972; Blaustein, Gentry, Roy &
Wade, 1976; Morin & Fleming, 1978; Czaja & Goy, 1975; Czaja, 1975; Kemnitz,
Eisele, Lindsay, Engle, Perelman, et al., 1984; Gilbert & Gillman, 1956; Cohen,
Sherwin & Fleming, 1987). These variations in eating are shown to be inversely
associated with circulating levels of estradiol that accompany the separate phases.
During the follicular phase, when endogenous estradiol levels are highest, eating is at
its lowest, while the opposite is true of the luteal phase. Administering estradiol
exogenously in ovariectomized female rats has validated that it is estradiol that is
responsible for the decreases in eating, a finding likewise demonstrated across
various species, methods of administration, and forms of the estrogenic steroid
(Wade, 1975; Bernstein, Braget & Courtney, 1986; Sandberg, David & Stewart,
1982; Miceli & Fleming, 1983; Tritos, Segal-Lieberman, Vezeridis & Maratos-Flier,
2004). The consistent results unequivocally establish estradiol as an anorexigenic
agent.
157
Due to the timing of the unconditioned effects exerted on eating behavior by
the steroid, the role of estradiol as a true unconditioned stimulus in a CTA paradigm
is called into question. The traditional CTA procedure consists of an acquisition
(training) phase during which animals consume a novel substance such as a sweet
solution (CS), before exposure to the unconditioned stimulus (US) and a post-
acquisition (testing) phase during which the animals are again exposed to the CS but
not the US. The formation of a CTA is assessed by comparing the amount of sweet
solution drunk during the acquisition trial and the first post-acquisition test. A
significant reduction in intake across tests indicates that conditioning has occurred.
In studies using estradiol as the US, reductions in consumption of the CS have been
observed when the first post-acquisition test is given 24 (Miele, Rosellini & Svare,
1988; Merwin & Doty, 1994), 48 (Hintiryan, Hayes & Chambers, 2005; Rice et al.,
1987) or 72 (DeBeun et al., 1991; 1993; Ossenkopp, Rabi & Eckel, 1996) hours
following the CS-US pairing. However, the unconditioned decreases in food
consumption produced by estradiol also have been shown to occur 24-72 hours
following the administration of the hormone. For instance, decreases in eating have
been observed 24 hours following the increase in plasma estradiol levels in cycling
rats (Asarian & Geary, 2002; Griffin & Ojeda, 1996) and 20 (Riviera & Eckel, 2005),
24 (Tartellin & Gorski, 1973), and 48 hours (Asarian & Geary, 2002; Santollo, Wiley
& Eckel, 2007) following the exogenous administration of estradiol in
ovariectomized rats. In addition, a study conducted in our laboratory showed that a
50μg/kg dose of estradiol benzoate produced food decrements 24 hours following its
158
subcutaneous administration, which was sustained at least 72 hours following the
injection (Chambers, Hintiryan & So, unpublished manuscript). Consequently, for
estradiol-induced CTAs any decrease observed in CS consumption during the post-
acquisition test potentially may be due to the unconditioned reduction in eating
produced by estradiol and not due to the US stimulus properties of the hormone.
Two approaches were used to determine whether the expression of CTA after
pairing estradiol with a novel taste solution is due to the unconditioned reduction in
eating induced by this hormone. In the first approach, experiments were designed to
test the hypothesis that there is a correlation between expression of estradiol
hypophagia and estradiol CTA such that CTA is not expressed unless hypophagia is
activated. Specifically, if the unconditioned hypothesis is correct, then one would
expect each of the following to be evident:
(1) For a dose of estradiol that is capable of inducing CTA, the expression of the
CTA should be present only during the time of unconditioned suppression of
food intake (Experiment 1).
(2) A dose of estradiol that does not induce CTA also should not trigger
unconditioned suppression of food intake at the time of the post-acquisition
test, while a dose of estradiol that does induce CTA should also trigger
unconditioned suppression (Experiment 2).
(3) Contingent pairing of the taste solution with estradiol should not be a
requirement for expression of the apparent CTA (Experiment 3).
159
In the second approach, experiments were designed to test the hypothesis that
the same neural areas mediate estradiol hypophagia and estradiol CTA. Specifically,
if the unconditioned hypothesis is correct, then one would expect both the
unconditioned and conditioned reductions in food consumption to be mediated by the
same neural areas. Experiments 5a and 5b were designed to begin testing this
hypothesis by determining whether lesions of the lateral parabrachial nucleus block
expression of both estradiol CTA and hypophagia.
Specific Aim 1.2: To test the negative contrast hypothesis by determining whether an
estradiol CTA could be formed if an inter-stimulus interval greater than 30 minutes
is employed.
The ability of estradiol to produce a CTA has been challenged by a second
hypothesis. Recently, it has been suggested that the CTAs produced by reinforcing
agents are qualitatively different than those induced by the illness agent LiCl (Hunt
& Amit, 1987; Parker, 1988, 1995). Reinforcing agents are a class of agents that are
effective positive reinforcers in a drug self-administration paradigm and that produce
conditioned place preferences (Reicher & Holman, 1977; Spyraki, Fibiger & Phillips,
1982; van der Kooy, Swerdlow & Koob, 1983; Wise, Yokel & DeWitt, 1976). These
agents can induce CTAs at the same doses that are rewarding (van der Kooy et al.,
1983; Wise et al., 1976), but they do not produce conditioned aversive taste reactions
(Parker, 1995; Parker & Brosseau, 1990). One hypothesis that has been suggested to
account for this apparent contradiction is based on the results of reward-comparison
studies, which have revealed an anticipatory negative contrast effect (Grigson, 1997).
160
In these studies, animals are given access to a less preferred solution (e.g., 15%
sucrose) followed by access to a more preferred solution (e.g., 32% sucrose; Capaldi
& Sheffer, 1992; Flaherty & Checke, 1982; Flaherty & Grigson, 1988; Lucas,
Timberlake, Gawley & Drew, 1990). Following several pairings, they learn that the
presence of the less preferred solution predicts the future availability of the more
preferred solution, and consequently reduce their consumption of the less preferred
solution. Accordingly, it has been suggested that when consumption of the CS is
followed by an injection of a reinforcing agent, the CS becomes devalued so that on
subsequent exposures, animals reduce their consumption in anticipation of receiving
the more rewarding stimulus, the reinforcing drug (Grigson, 1997). Given the recent
finding that intermediate doses of intracranially administered estradiol might be
reinforcing in male Syrian hamsters (DiMeo & Wood, 2006), one might argue that
reductions in the CS following injections of estradiol are due to negative contrast
effects (Grigson, 1997). Although it is difficult to make comparisons between doses
administered intracranially and those administered peripherally in CTA studies, the
possibility that the doses of estradiol used to induce CTA potentially may be
reinforcing precludes the abandonment of the negative contrast hypothesis.
As discussed in an article by Reilly, Bornovalova, and Trifunovic (2004), in
order for the anticipatory negative contrast effect to be expressed, the time interval
between the presentation of the less preferred solution and the more preferred
solution should be of reasonable length. This would allow the animals to effectively
make comparisons between the solutions. This is supported by the findings that
161
anticipatory contrast effects are most evident when the solutions are presented
sequentially with no delay, while the effect is eliminated when the interval between
the two presentations approaches 30 minutes (Flaherty & Checke, 1982; Flaherty,
Grigson, Checke & Hnat, 1991).
If the CTAs formed by estradiol are due to negative contrast effects, then CS-
US intervals greater than 30 minutes should not result in the gustatory avoidance.
Therefore, one of the purposes behind Experiment 4 was to test the effect of CS-US
intervals that were longer than 30 minutes on the formation of an estradiol CTA.
This experiment also was designed to determine the longest CS-US interval that
would support an estradiol CTA (see Primary Aim 2). As such, several CS-US
intervals were tested in six separate studies, all of which included intervals much
longer than 30 minutes.
Specific Aim 1.3: To test the aversive hypothesis by determining whether lesions of
the lateral parabrachial nucleus block estradiol CTA.
If estradiol truly conditions (Specific Aim 1.1) and it is not do so based on its
reinforcing properties (Specific Aim 1.2), then we will test to see if the hormone
produces a CTA based on its aversion producing properties (Specific Aim 1.3). This
hypothesis may be examined by determining the role of the lateral PBN in estradiol
CTA. The lateral PBN is a relay station for visceral information that is carried from
the vagus to the area postrema. The area postrema is a neural substrate that processes
information regarding malaise and emesis (reference) and it sends strong projections
to the lateral PBN (van der Kooy & Koda, 1983; Shapiro & Miselis, 1985). This
162
suggests that information regarding aversive states is projected and processed in the
lateral PBN. In addition, lesions of the lateral PBN block CTAs produced by the
putative illness inducing agent LiCl (Sakai &Yamamoto, 1998; Reilly & Trifunovic,
2000a,b). More importantly, it is determined that lesions of the pontine nucleus
block the CTA learning not because of a disruption in the associative process, but
because of a disruption in the US processing (reference). Taken together, if lesions
of the lateral PBN block estradiol CTA, then this would suggest that estradiol too
conditions avoidance to a sucrose solution based on its aversion-producing
properties. This possibility is tested in Experiment 5.
4.2.2 Primary Aim 2
Primary aim 2 is to conduct preliminary experiments that would allow for the
examination of the neurochemical basis of estradiol CTA.
In order to study the neurochemical basis of estradiol CTAs, it is critical to
determine the length of time the neurotransmitter receptors of interest would have to
remain inactivated following the estradiol injection. With LiCl CTAs, the receptors
are kept inactive for 2 hours, which is the estimated time that LiCl remains in the
system before it is cleared below functional levels. However, a 50μg/kg injection of
estradiol benzoate produces estradiol levels that remain in circulation for an extended
period of time, thus requiring neurotransmitter receptors to be blocked for greater
lengths of time (Wooley & McEwen, 1993). In addition, the relationship between
plasma levels of estradiol and the time a CTA is developed has not been established,
thereby making it difficult to determine the amount of time necessary to keep
163
neurotransmitter receptors inactivated. These same issues have precluded the use of
temporary lesions in the investigation of the neural circuitry of estradiol CTAs. A
possible solution to this problem may be to implement chronic versus acute
inactivation of the receptors to ensure the antagonism of the receptors throughout the
duration of estradiol activation. However, this chronic blockage of receptors could
directly or indirectly affect the behaviors of interest and thus confound the data.
Such chronic inactivation could also result in aversive effects in the animals. As
such, this option was considered, but discarded.
The deterioration of the CS trace potentially could be used to circumvent this
problem. In male rats, CTA learning can support up to a 12-hour delay between the
taste and toxin when radiation is used as the US (Garcia et al., 1966; Smith & Roll,
1967). Presumably, a CTA cannot be formed with intervals greater than 12 hours
because the CS trace deteriorates and consequently cannot be associated with the US.
The longest inter-stimulus interval allowed for the formation of an estradiol CTA has
not been established. Once this information is obtained, then it could be used to
determine when the neurotransmitter antagonists need to be infused in order to assess
the agents’ influence on estradiol CTAs. For instance, suppose we wanted to
examine the effect of the muscarinic receptor antagonist scopolamine on estradiol
CTAs and we know that scopolamine infused into the brain inactivates receptors for
at least 1 hour. If the maximum time interval allowed for an estradiol CTA is 6
hours, then scopolamine and estradiol would be administered 6 hours following the
CS presentation (see Figure 3). In this fashion, the cholinergic receptors would be
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blocked during the time that is critical for the formation of the estradiol CTA. The
high blood levels of estradiol would not matter since a CTA cannot be formed due to
the extended interval between the CS and the US. This same methodology also could
be employed to study the effects of temporary lesions of the lateral PBN on estradiol
CTA. In the above example, the injection of estradiol would be made 5 hours after
the CS exposure period and the lateral PBN would be blocked for 1 hour.
Figure 3. Methodology for Determining Chemical Mediation of Estradiol
Conditioned Taste Avoidance.

CTA No CTA
Time receptors will be
inactivated

CS trace
2 3 4 5 6 7
Estradiol
Time between CS-US interval (hrs)
Figure 3. Depicts the methodology that will be used to test the role of acetylcholine and histamine
in estradiol induced CTAs. If, hypothetically, 6 hours is the longest CS-US interval that supports an
estradiol CTA, then acetylcholine and histamine antagonists that are active for at least 1 hour, will
be infused 6 hours following the CS. The estradiol injections will be made immediately following
the infusions of the antagonists.
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Specific Aim 2.1: To determine the longest CS-US interval that supports an estradiol
CTA.
No studies to date have been conducted on the effects of CS-US length on
estradiol CTA. As such, several interval lengths were tested to hone in on our target
inter-stimulus interval. In Experiment 4, four studies with six different interval
lengths and one replication study were conducted in order to find an interval that
produced a strong estradiol CTA. It is important that the interval chosen produces a
CTA in the majority of the animals tested. If we block an estradiol CTA with the
antagonists, we want to be certain that it was because the infusions blocked the
conditioning and not because the interval did not produce a CTA in some of the
control animals. In order to find a site of infusion for the antagonists, Experiments
5a and 5b were conducted in order to see if lesions of the lateral PBN affected
estradiol CTA with the CS-US interval determined in Experiment 4. For this lesion
study, it also is important that the majority of the control animals acquire a CTA with
the predetermined inter-stimulus interval. If lesions abolish the estradiol CTA, we
want to be able to say with certainty that it was due to the lesion and not due to the
possibility that those animals did not acquire a CTA in the first place due to the
lengthy CS-US interval.
Specific Aim 2.2: Do lesions of the lateral parabrachial nucleus block estradiol CTA
when an extended inter-stimulus interval is used?
As discussed earlier, one of the purposes behind Experiment 4 was to
determine the maximum CS-US time interval that supported an estradiol CTA. The
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next step after determining this critical interval was to find the neural site of infusion.
In other words, we needed to determine a neural site that is involved in estradiol CTA
where the neurotransmitter antagonists could be infused. Since the lateral PBN
extensively has been shown to be involved in the acquisition of LiCl CTA, it is
hypothesized that lesions of the lateral PBN would also prevent acquisition of
estradiol CTA.
4.3 Secondary Aims
Examination of the role of the lateral PBN in estradiol CTA and anorexia
warrants investigation for two additional reasons. These constitute the two
secondary aims below.
4.3.1 Secondary Aim 1
Secondary aim 1 is to determine whether estradiol CTAs are similar to
gustatory aversions produced by the putative illness inducing agent lithium chloride.
Lesioning the lateral PBN will show whether the neural circuitry of CTAs
produced by estradiol and LiCl are similar. By doing so, we may be able to shed
light into the underlying mechanism of estradiol CTAs. For instance, it would be
valuable to know if estradiol, like LiCl, is able to condition an avoidance to sucrose
based on its ability to produce an aversion. Lithium chloride (LiCl) is considered the
putative illness inducing agent and all other USs used in CTA studies are compared
to this chemical agent. As an illness producing agent, conditioning to the CS when
LiCl is used is thought to be due to an aversive state. Estradiol, like LiCl, has been
shown to be aversive. For instance, it has been demonstrated that at high doses the
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hormone produces aversive orofacial responses in male rats (Ossenkopp et al., 1996)
and it produces nausea in humans (Gustavson et al., 1989). However, CTAs formed
when the hormone is used as a US may be due to states other than illness, such as
satiety (Booth, 1977), and therefore different from LiCl. Investigating the
similarities and differences in the neural substrates between the two agents will shed
some light on the mechanism by which estradiol produces CTA. Specifically,
comparing the neural substrates involved in estradiol and LiCl CTAs would test the
aversive hypothesis of estradiol CTA.
Conditioned taste avoidance induced by estradiol shares some similarities
with those produced by LiCl. First, the area postrema (AP) is necessary for CTAs
produced by both the steroid hormone (Bernstein et al., 1995) and by LiCl
(Ossenkopp, 1983; Rabin et al., 1983; Wang et al., 1997a,b). Second, both produce
c-fos activation in similar brain regions i.e. the AP, the NST, crescent lateral PBN,
and medial PBN (Yamamoto et al., 1992; Chambers & Wang, 2004). Third, they
each can serve as pre-exposure inter-agents for one another. That is, distal and
proximal pre-treatment with LiCl attenuates an estradiol CTA (Hintiryan &
Chambers, 2003), while pre-exposure to estradiol attenuates a LiCl CTA (Chambers
& Hayes, 2002). Researchers use this cross-familiarization of drugs to suggest
common underlying neural mechanisms for the chemical agents. Fourth, the dose-
response curve for LiCl is linear and there is some evidence that this is true for
estradiol as well. After pairing different doses of estradiol with a saccharin solution,
consumption is reduced in fluid deprived rats given high doses of estradiol (50- and
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250μg/kg). Consumption is not reduced in those given a low dose of 0.4μg/kg and
for animals given intermediate doses of 2- and 10μg/kg, the consumption amount
falls in between the high and low doses, although the consumption amounts for these
doses were not statistically different than either the high or the low dose consumption
levels (De Beun et al., 1991). Fifth, a parametric study conducted in our laboratory
has revealed that the strength of the CTA produced by LiCl and estradiol varies as a
function of CS-US interval length such that the shorter CS-US intervals produce
stronger CTAs compared to the longer CS-US intervals (Chambers, Hintiryan &
Foster, 2006 unpublished data).
On the other hand, the two agents produce CTAs that appear to be
qualitatively different. Only high supraphysiological doses of estradiol (100μg/kg)
have been shown to produce aversive orofacial responses to saccharin following
conditioning (Ossenkopp et al., 1996), while even very low doses of LiCl (2 ml/kg)
have been shown to produce aversive orofacial responses (Parker, 1995) during taste
reactivity testing. This is despite the fact that a 100μg/kg dose of estradiol probably
conditions more strongly than a 2ml/kg dose of LiCl. Unpublished studies in our
laboratory indicate that LiCl doses between 2- and 4ml/kg produce CTAs similar in
strength to a 50μg/kg dose of estradiol. Although low supraphysiological doses like
those comparable to 50μg/kg dose, produce CTAs, they do not produce negative
orofacial responses in female rats (Flanagan-Cato, Grigson & King, 2001).
It has been demonstrated consistently that both excitotoxic (Reilly &
Trifunovic, 2000a,b, 2001; Trifunovic & Reilly, 2002) and electrolytic (Sakai
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&Yamamoto, 1998) lesions of the lateral PBN eliminate CTAs produced by LiCl.
Reversible TTX lesions (Ivanova & Bures, 1990a,b; Bielavska & Bures, 1994) or
cooling lesions (Wang & Chambers, 2002) made after the CS such that it overlaps
with LiCl duration also disrupt the associative learning. The lateral PBN also is
involved in CTAs induced by different USs other than LiCl. Cytotoxic lesions of the
pontine nucleus block the acquisition of morphine-induced CTA (Nader, Bechara &
van der Kooy, 1996), while cooling lesions block apomorphine CTAs (Chambers &
Wang, 2004). Given that the lateral PBN is involved in CTAs induced by LiCl (as
well as other various US agents), determining whether this area is involved in
estradiol CTAs will provide evidence for or against the hypothesis that estradiol
CTAs are similar to those produced by LiCl.
4.3.2 Secondary Aim 2
Secondary Aim 2 is to determine whether the lateral parabrachial nucleus is
critical for estradiol elicited anorexia.
Since the role of estradiol in anorexia nervosa is reported in detail in the
“Estradiol and Anorexia Nervosa” section of Chapter 2, only a summary of the data
will be presented here. Physiological levels of estradiol produce both marked
reductions in food consumption (Geary & Asarian, 1999) and increases in physical
activity (Wollnik & Turek, 1988). Because the symptoms of the eating disorder
anorexia nervosa can be mimicked by high doses of estradiol, this hormone has been
implicated in the eating disorder. Briefly, it has been suggested that environmental
estrogen-like agents contribute to the onset of anorexia nervosa in pubertal girls by
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sensitizing areas of the brain that control estradiol-regulated eating responses
(Gustavson et al., 1991). Associations have been found between exposure to such
agents and an increased incidence of the eating disorder (Bibbo, Al-Naqeeb,
Baccarini, Gill, Newton, et al., 1975; Gustavson et al., 1991). However, progress
towards testing this hypothesis demands identification of neural areas that mediate
the anorectic effects of estradiol. Attempts have been made to identify these neural
areas in the forebrain, but its localization remains unestablished. It is well
documented that the lateral PBN of the brainstem contains estrogen receptors
(Shughrue, Lane & Merchenthaler, 1997) and is involved in ingestive behaviors
(Nagai, Ino, Yamamoto, Nakagawa, Yamano, et al., 1987) thereby making the lateral
PBN a potential mediator of estradiol induced anorexia. Preliminary studies in our
laboratory show that estradiol produces protein activation in the lateral PBN 24 and
48 hours following its administration, which is precisely the time when decreases in
food intake are observed after injections of estradiol. Conducting lesions of the
lateral PBN and testing for estradiol produced suppression of eating possibly will
help identify a neural area for estradiol anorexia.
4.4 General Methodology
4.4.1 Subjects and Husbandry
Female Sprague-Dawley rats obtained from Harlan Laboratories (San Diego,
CA) were used for each of the experiments. The subjects were 60 days old and
weighed approximately 190 grams at the start of each experiment. They were housed
individually in a room that was temperature (21-22
o
C), humidity (51%), and light
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controlled (12 hr light:12 hr dark cycle). The animals in Experiment 4 series were
housed in pairs and a divider was used to separate the animals during behavior
testing. Each cage (North Kent Plastics) measured 58 x 38 cm and had a solid
bottom that was covered with wood chips. The rats were allowed at least 1 week to
adapt to their living conditions before surgery. Rats had ad libitum access to rat
chow (Rodent Blox; protein 24%, fat 4%, fiber 4.5%; Harlan Teklad 8604) and tap
water throughout the duration of the experiments. The experiments were conducted
according to the standards set by the National Institutes of Health Guide for the Care
and Use of Laboratory Animals (DHEW Publication 80-23, Revised 1985, Office of
Science and Health Reports, DRR/NIH, Bethesda, MD) and the institutional
guidelines of the University of Southern California.
4.4.2 Chemical Agents
Conditioning Agents
Crystalline estradiol and estradiol benzoate were purchased from Steraloids
(Wilton, NH). Each dose of estradiol was dissolved in 0.25ml of sesame oil and was
injected subcutaneously in the amounts of 1- (Experiment 2), 10- (Experiment 1), and
50µg/kg of body weight (Experiments 1-5). Sesame oil in the same amount was used
as vehicle. In Experiment 4a, each of the animals received an intraperitoneal
injection of 0.9% NaCl in addition to their assigned drug. The reason for this is
discussed in the methods section for Experiment 4a. Lithium chloride dissolved in
0.9% NaCl was administered in the dose of 10ml/kg as a conditioning agent in
Experiment 5.
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Lesioning Agent
Ibotenic acid (Ascent Scientific, Princeton, New Jersey) was dissolved in PBS
to achieve a concentration of 20μg/μl. Due to the low solubility of ibotenic acid in
PBS, the ibotenic acid was titrated with 1M NaOH until the solution was completely
dissolved. Approximately 5μl of NaOH was needed to thoroughly dissolve a 250μl
solution of ibotenic acid. The final pH of the solution was approximately 7.5 and the
bottle was kept refrigerated throughout the experiment. The same bottle of ibotenic
acid was utilized across 10 days of surgery since the solution is stable for two weeks
under proper conditions of storage (Filer, Lacy & Peng, 2005; Nielson Schousboe,
Hansen, Krogsgaard-Larsen, 1985) and does not get decarboxylated to muscimol.
Pilot studies in our laboratory have shown that a bottle of ibotenic acid is effective 11
days after it is dissolved. Surgeries for Experiment 5a were conducted over a 10 day
period, suggesting that the batch of dissolved ibotenic acid used throughout the
surgeries was still effective.
4.4.3 Surgical Procedures
Anesthetics and Surgical Techniques
(1) Ovariectomies
(a) Anesthesia. Animals in each of the experiments were ovariectomized.
Animals in Experiments 1-4, and 5b were ovariectomized using intraperitoneal
injections of a ketamine-xylazine solution, which was a mixture of 5 parts of Ketaject
(equivalent to 100mg of ketamine: Phoenix Pharmaceutical, Inc) to 1 part xylazine
(Lloyd Laboratories) in the dose of 0.12ml/100g of body weight. For Experiment 5a,
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the ovariectomies were performed under halothane anesthesia (Halocarbon
Laboratories, River Edge, NJ). The halothane was vaporized (Fluotec 3) and mixed
with oxygen by a Fraser Harlake VMC small animal inhalation anesthesia machine
(Datex-Ohmeda, Madison, WI). This gas mixture was delivered to each rat through a
nose cone. The percent of halothane in the gas mixture was maintained between 1.5
and 2.0 and the oxygen flow rate was maintained between 3.5 and 4.0 L/min, while
the animals were ovariectomized.
(b) Surgical Technique. Ovariectomies included bilateral incision of the skin and
muscles (1-2 cm from the midline and anterior to the hip), removal of the ovaries,
and tying of the fallopian tubes. For Experiment 5a, the ovariectomies were
performed the same day as the excitotoxic lesions. For Experiment 5b, the
ovariectomies were performed several weeks after the electrolytic lesions. Since
recovery from electrolytic lesions is more difficult and longer in length than
excitotoxic lesions, we decided not to overwhelm the animals with an additional
surgery. As such, the ovariectomies were performed after the animals had fully
recovered from their electrolytic lesions.
(2) Neural Lesions
(a) Anesthesia. Excitotoxic (Experiment 5a) and electrolytic (Experiment 5b)
lesions were performed under ketamine-xylazine anesthesia. In Experiment 5a, once
the ovariectomy was completed, the animals were removed from the halothane.
Once the animals started to recover from the halothane, they were injected
intraperitoneally with 5 to 1 ketamine-xylazine mixture in the dose of 0.06ml/100g of
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body weight to prepare for neural surgeries. In order to minimize possibility of
overdose, the injection anesthesia was administered after the animals started to
recover from the halothane and in half of the dose normally required to anesthetize an
animal. The animals were given supplemental injections of ketamine-xylazine in
amounts ranging from 0.02-0.04ml to maintain deep anesthetic state throughout the
remainder of the neural surgeries in both experiments.
(b) Surgical Technique
(i) Coordinates. For Experiment 4, the lateral parabrachial (PBN) nucleus
was approached at a 30° angle in an attempt to avoid the transverse sinus. The
coordinates according to Paxinos and Watson (1998) 12.84 (AP), 2.0 (ML), and
7.275 (DV) were used for the lateral PBN. For Experiment 5b, these same
coordinates were used in 3 of the animals and 13.12 (AP), 2.0 (ML), and 7.85 (DV)
were used in 3 other animals. To increase the accuracy of placement, the ML for
bregma was measured by calculating the midpoint of the coronal suture. This was
accomplished by placing the micropipette (Experiment 5a) or electrode (Experiment
5b) in the groove on left side of skull above the end of the coronal suture and
recording the coordinate. The pipette or electrode was then moved into the groove of
the right side above the coronal suture and the coordinate recorded. The two
coordinates were subtracted. The midpoint was shifted 0.125ml to the right. The
length of the skull from groove to groove calculated from each arm never differed
more than 60µm. In addition, for Experiment 5a, the surgery was performed using a
microscope to increase accuracy of the placement.
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(ii) Excitotoxic Lesions (Experiment 5a). Excitotoxic lesions of the entire
lateral PBN were made. The excitotoxic lesioning technique with ibotenic acid was
chosen in order to avoid damaging fibers of passage such as the superior cerebellar
peduncle and the ventral spinocerebellar tract or any other fibers passing through the
pontine nucleus. When infused into the brain, ibotenic acid causes selective
degeneration of cell bodies while sparing not only fibers of passage, but also nerve
terminals from extrinsic origin and glial elements (Schwarcz, Kohler, Fuxe, Hokfelt
& Goldstein, 1979; Markowaska, Bakke, Walther & Ursin, 1985; Edwards &
Johnson, 1991).
Ibotenic acid was delivered via non-filamented micropipettes (outside tip
diameters 40-60μm) in the amount of 0.2μl/10minutes (Reilly & Trifunovic, 2000a,b;
2001). To minimize the spread of the neurotoxin along the track, the micropipette
remained in situ for an additional 10 minutes following the 10-minute infusion.
Control sham lesioned animals were treated the same way as lesioned animals except
infusions of phosphate buffered saline (PBS) in the place of ibotenic acid were made.
Two new micropipettes were used for each rat. The micropipettes were
attached and glued to PE100 tubing, which was attached and glued to PE20, which
then was attached to a 10μl Hamilton syringe situated in a micro-injector pump
(CMA/100, Carnegie Medicin, Stockholm, Sweden). The PE tubes, micropipettes,
and Hamilton syringe were filled with mineral oil to ensure the creation of an airtight
seal. The tips of the pipettes were painted with red nail polish for better visibility and
thus improved accuracy during surgery. This procedure was tested in pilot animals
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and did not have any effect neurally or behaviorally. A microscope was used during
the surgery to increase visibility of the micropipette tips. The ibotenic acid and PBS
were backfilled manually into the pipettes via a micro-injector.
(iii) Electrolytic Lesions (Experiment 5b). An insect pin coated with
epoxylite was used as the electrode for the electrolytic lesions. A current of
0.1mAmps was delivered for 10 seconds by a lesion maker (Grass Instruments, West
Warwick, RI). The tip of the electrode was exposed approximately 0.3mm. Control
sham lesioned animals were treated the same way as lesioned animals except no
electric current was passed through the electrode.
Analgesics, Antibiotics, and Post-Operative Care
For Experiment 5, the analgesic buprenex (buprenorphine hydrochloride:
Reckitt & Colman Products: Hull, England) and the antibiotic dualcillin (penicillin
benzathine: Western Medical, Arcadia, CA) were administered subcutaneously in the
amounts of 0.02ml (0.017mg/kg) and 0.05ml (43,000 units/kg), respectively, to each
rat the day of surgery. The same dose of buprenex also was given the day following
surgery in order to alleviate any persisting discomfort. No buprenex was
administered in Experiments 1-4 after ovariectomies. Generally, it has been our
observation that animals remove their stitches after ovariectomies if their pain is
alleviated with analgesics. Consequently, when only ovariectomies are performed,
buprenex is not administered to the animals; however, it was our concern that the
double surgeries (the neural lesion and ovariectomy) would cause too much pain and
discomfort to the animals not to administer analgesics. As such, each animal in
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Experiment 5 received an injection of buprenex. Additional post-operative care for
Experiment 5 consisted of weighing and general inspection of the animals. Each
animal also was given Saltine crackers following surgery. Since the rats find the
novel crackers palatable, they begin eating shortly after recovering from the
anesthesia. This is not the case with their Rodent Blox. We also have observed that
the Saltine crackers increase the animals’ water intake immediately after surgery.
Since eating and drinking are signs of recovery, the crackers were given after surgery
to aid in the animals’ return to physiological baseline.
For Experiment 5b, whenever necessary, intraperitoneal injections of Ringer
solution, which contained calcium, potassium, and magnesium, were made to assist
the recovery process. Those animals that were otherwise immobile and lethargic,
started to display increased activity following the injections of the Ringer solution.
4.4.4 Behavior Testing
Behavioral testing commenced following a 1-week recovery period after
ovariectomies for Experiments 1-4, while a 2-3 week recovery period was allowed
for Experiments 5a and 5b.
Conditioned Taste Avoidance/Aversion (CTA) Procedure
For Experiments 1-4, animals were subjected to one CTA procedure, which
was divided into the following four periods: (1) preconditioning, (2) acquisition, (3)
post-acquisition recovery, and (4) post-acquisition test. For Experiment 5, two of the
groups in study a and all of the animals in study b were subjected to a second CTA
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procedure. Each of the CTA procedures consisted of (1) preconditioning, (2)
acquisition, (3) post-acquisition recovery, and (4) extinction.
All drinking solutions used during each period were stored under refrigeration
and were given to the rats at the beginning of the dark portion of the light/dark cycle.
During the preconditioning phase, a 100ml cylinder containing refrigerated tap water
was added to the cages of the animals for 1 hour. It has been our observation that
preconditioning rats with cold water immediately after the lights switch off increases
the likelihood that non-deprived rats will drink the novel sucrose solution presented
during acquisition. On acquisition day, a 100ml cylinder containing a novel sucrose
solution (10% w/v) or chocolate milk (Experiment 5) was added to the cages. One
hour later, the amount of fluid consumed was recorded, the cylinder was removed,
and each rat was administered its assigned conditioning agent. The amount of time
between the exposure to the CS and the US injection was 0 minutes in Experiments
1, 2, and 5 while it ranged from 3-12 hours in Experiment 4 and was 24 hours in
Experiment 3. Depending on the experiment, the post-acquisition test was conducted
35 to 215 hours after acquisition. Animals were left undisturbed during the interval
between acquisition and post-acquisition testing or extinction. For Experiment 5,
daily extinction tests were initiated two days after acquisition. The extinction tests
were conducted in the same manner as the acquisition tests except no conditioning
agent was administered. Extinction tests were given daily until group differences, if
present, were no longer statistically significant.
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Measurements of Fluid Intake
For Experiment 5a, water consumption was measured at the same time as
sucrose intake for the first CTA (CTA1, described in methods section for Experiment
5a). If any significant changes in sucrose consumption were detected across the
groups, the water intake data would help determine whether the changes were due to
factors related to the CTA, changes in neophobic responses (Reilly & Trifunovic,
2001), or due to alterations in general drinking behavior (i.e. adipsia or polydipsia;
Edwards & Johnson, 1991).
Measurements of Food Intake
In addition to CTA testing, food intake measurements were conducted in the
same animals for Experiments 1, 2, and 5. In Experiments 1and 2 food intake
measurements were made every 24 hours just prior to each period of the CTA
procedure while in Experiment 5, baseline measurements of food intake were
initiated at least two weeks following the final estradiol injection of the CTA
procedure. For Experiment 5a, food measurements were made every 12 hours, once
immediately before the lights turned on and once immediately before the lights
turned off. For Experiment 5b, food measurements were made every 24 hours,
immediately before the lights turned off. For both Experiments 5a and 5b, two
injections of estradiol were administered 24 hours apart. Each injection was made
immediately before the lights turned off. Given the natural variability in eating data
and the fact that we occasionally have observed decreases in eating in control animals
following oil injections, we adopted this two injection methodology to increase the
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reliability of obtaining differences between the oil and estrogen groups following
injections. This two injection method was not used in Experiments 1 and 2 since
CTA and eating were tested at the same time; only a single estradiol injection is
required for CTA testing and two injections would interfere with the CTA procedure.
4.4.5 Histological Staining
Cresyl violet staining is typically used to assess the accuracy and extent of
excitotoxic lesions. However, due to difficulties we encountered during the infusion
of ibotenic acid (discussed in Experiment 5a section), the lesions were going to be
extremely difficult to read with cresyl violet. Further, gliosis from damaged cells
typically serves as the marker for reading lesions with cresyl violet staining and
gliosis fades over the course of time. One study showed that gliosis had faded
dramatically 7 weeks following excitotoxic lesions of the lateral PBN (Calingasan &
Ritter, 1993). Since the behavior testing for Experiment 5a lasted up to 5 months
following the lesioning, cresyl violet staining was not a viable option, assuming that
gliosis would not be present at the conclusion of the experiment. As such, we
decided to stain for neuron-specific nuclear protein (NeuN), a protein found
exclusively in neurons. The absence of neurons due to the lesion would be indicative
of a lesion, thus simplifying localization and extent of the lesion.
Neuron Specific Nuclear Protein (NeuN) Staining
At the conclusion of the behavior testing of Experiment 5, each rat was
anesthetized with an overdose injection of ketamine:xylazine (5:1) and transcardially
perfused with 300ml of room temperature 0.9% NaCl followed by 300ml of a
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refrigerated 4% paraformaldehyde solution. The brains were extracted and post-fixed
in 4% paraformaldehyde overnight at 4°C. For cryoprotection, they were transferred
to a jar filled with a solution of 20% sucrose PBS for 24-48 hours. Coronal brain
sections corresponding to the lateral PBN (according to the coordinates of Paxinos
and Watson, 1998) were sliced at 36-40µm thickness with a microtome (American
Optical Company, Buffalo, New York).
The NeuN staining protocol adopted by our laboratory was a modified
version of a protocol provided by Millipore (Temecula, CA). The free-floating
method was used and each step was performed on a rotator. The sections were
transferred to a 3% hydroxide absolute methanol for 20 minutes to inactivate the
endogenous peroxidases. Following a no soak wash, they were rinsed in PBS 3 times
(2 minutes each) and treated with 3% horse serum (Vector Laboratories, Inc.
Burlingame, CA) and 0.2% Triton (Sigma, St Louis, MO) PBS for 30 minutes.
Without rinsing, the sections were transferred to a 1% horse serum and 0.4% Triton
PBS with 1:1000 primary anti-NeuN antibody (biotinylated, Mouse IgG, Millipore,
Temecula, CA) for 48-72 hours at 4 C. After 48-72 hours of incubation and a no
soak wash, the slices were rinsed 3 times with PBS (2 minutes each) and were
transferred to an avidin-biotin complex reagent solution (ABC Standard kits, Vector
Laboratories, Inc. Burlingame, CA) for 1 hour at room temperature. Next, the
sections were soaked and rinsed 3 times with PBS (2 minutes each). A peroxidase
substrate kit was used to visualize the antigen-antibody reaction sites in the brain.
The sections were placed in a solution made by 3,3'-diaminobenidine and nickel
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enhancer (DAB kit; Vector Laboratories, Burlingame, CA) for 5-7 minutes
depending upon the color of the sections and were rinsed 10 times with PBS (2
minutes each) to stop the reaction. The sections were individually mounted onto
gelatin-coated glass slides coated, air- dried, and cover slipped with Cytoseal
(Stephens Scientific, Kalamazoo, MI).
4.4.6 Statistical Analyses
Description of Analyses
Our data were analyzed by taking advantage of modern statistical procedures
that avoid the problems of traditional statistics. Conventional statistical procedures
such as the ANOVA are fraught with deleterious problems when assumptions of
normality and equal variances are violated. Violations of these assumptions lead to
poor probability coverage, low power, and inability to control for Type I errors and
bias, which means that the probability of rejecting can drop as the population means
become more unequal (Wilcox, 2001, 2003). Traditional approaches to achieve
normality and equality of variances (i.e. transformations) also have been shown to be
inadequate (Wilcox & Keselman, 2003). Reliable modern statistical techniques have
been developed to replace such conventional methods of analyses. Due to the non-
normality and heteroscedasticity of our data, more modern robust statistical
methodologies were implemented in order to attain reliable results. A percentile
bootstrap with 20% trimmed means, which is not predicated on the assumptions of
normality or equal variances, was performed to analyze these data. In general, this
method randomly samples with replacement from the actual data to produce a data
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set called a bootstrap sample. Next, the trimmed mean is computed. In the two-
sample case, the p-value is the probability that a bootstrap trimmed mean from the
first group is less than the bootstrap trimmed mean of the second. The value of p is
computed by repeatedly generating bootstrap samples. This procedure can be
generalized to a repeated measures design (Wilcox, 2003b). Additionally, this
method controls the family- wise error rate using adjusted p-values. It has been
found to perform well among a range of methods when working with real data,
including when working with small sample sizes (Wilcox, 2003b; Wu, 2002).
For each comparison, a critical significance level is reported along with a
significance level. A statistically significant result is obtained when the significance
level is lower than the critical significance value. The p-value was set at 0.05 for all
comparisons. The percentile bootstrap equivalent of dependent-t and ANOVA with
repeated measures was performed on the data. This type of robust analysis
previously has been used to analyze CTA data (Hintiryan, Hayes & Chambers, 2005,
2006).
Assessment of CTA Formation
For Experiments 1-4, within-group analyses of sucrose consumption across
acquisition and the post-acquisition test was used to determine whether a CTA had
been acquired. For Experiment 5, within-group analyses of sucrose consumption
across acquisition and the first extinction test was used to assess acquisition. The
post-acquisition test in Experiments 1-4 is equivalent to the extinction test 1 for
Experiment 5. We have used different designations to prevent possible confusion
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since, as indicated above, extinction of the CTA was assessed in Experiment 5 but
not in the other experiments.
Between-group comparisons of sucrose consumption were made for both the
acquisition trial and the post-acquisition test (Experiments 1-4) or extinction test 1
(Experiment 5) to assess differences in acquisition strength.
Assessment of Extinction
Only between-group analyses were performed on the extinction data of
Experiment 5. Due to the nature of the bootstrap analyses, the extinction trials for
Experiment 5a were divided into phases. The estradiol CTA (discussed in
Experiment 5a methods) consisted of 4 phases and each of the phases included 5
extinction days. The sum of the extinction trials in each phase was calculated and
group-wise comparisons were performed across all of the phases.
For Experiment 5a, the extinction data for the second CTA where LiCl was
used as the US was not divided into phases. Analyses were performed on individual
days since the animals extinguished from the CTA in 4 days.
Assessment of Fluid Intake
For Experiment 5a, water intake was analyzed in the same manner as the
sucrose data for CTA1. Water intake was divided into 4 phases and each phase
consisted of 5 days. These 5 days for each phase were the same for the sucrose data
from CTA1. The sum of the water intake in each phase was calculated and group-
wise comparisons were performed across all of the phases.
185
Assessment of Food Decrement
Experiments 1 and 2
During acquisition and post-acquisition testing, the animals had access to
both food and a sucrose solution. As such, a pattern of food intake developed such
that following the injection, there was a decrease in food consumption in each of the
groups, including the oil groups. Investigation into this pattern revealed that the
control oil animals were voluntarily decreasing their food intake in order to
compensate for the caloric intake of the sucrose solution. Therefore, the food and
sucrose intake data was converted into calories and the total calorie intake of the
individual days was used in the statistical analysis. Sucrose has 3.75 Cal/g, which
translates to 0.375 Cal/ml and the chow pellets have 3.3 Cal/g. Baseline day for each
of these experiments was the average of the caloric intake 24 and 48 hour periods
prior to the estradiol injection.
Within- and between-group analyses were performed on the total calories
consumed during baseline days compared to the total calories consumed in the 24
hour period starting on the post-acquisition trial to assess any changes in caloric
intake within and between groups.
Experiment 5
Predicated on our experience in Experiments 1 and 2, food intake
measurements were made independently of the CTA behavior testing. Therefore,
food intake was not converted into calories and analyses were performed on raw data
measurements (grams). Baseline day for Experiments 5a and 5b was the average of
186
the 4-day intake prior to the first estradiol injection. Within-group analyses were
performed to assess differences between baseline and post injection days. Between-
group comparisons were performed to detect differences in the amount of food
consumed on each of the days.
Additional Analyses
Two additional types of analyses were performed that were specific to
Experiments 4 and 5. They are introduced here, but are discussed in further detail in
the methods sections for the respective experiments.
Comparison of Number of Animals that Acquired CTA across Groups
In Experiment 4 series, it was necessary to statistically compare the number
of animals that acquired a CTA across the different groups. A percentile bootstrap
equivalent to binomial analysis was performed and Rom’s method was used to
control the family-wise error rate. Details regarding the criteria for CTA
quantification are provided in the methods section for Experiment 4.
Comparisons of Lesion Size between Lesioned Groups
In Experiment 5a, comparisons of lesion size were made between the lesioned
groups that received oil and estradiol. The lesion size of each animal was determined
by the number of cells each animal had below the lower limit set for its
corresponding AP level (discussed in detail in the methods for Experiment 5a).
Comparisons between the two groups were made for the right and left sides.
Between-group analyses were performed to assess differences in lesion size between
the two groups.
187
4.5 Presentation of Experiments
4.5.1 Experiment 1
PRIMARY AIM 1- SPECIFIC AIM 1.1: Testing the Unconditioned Hypothesis -
Correlation between expression of hypophagic and CTA based on time of testing
(Strategy 1).
In Experiment 1, we employed the first of the three strategies outlined in the
introduction to test the hypothesis that the unconditioned effects of estradiol on food
consumption contribute to the apparent CTA. This strategy involved correlating the
period of time during which a particular dose of estradiol triggers unconditioned
reductions in consumption with its ability to induce CTA. To do this, we used two
different time intervals between acquisition and post-acquisition testing, a 2-day
interval, when unconditioned reductions in consumption produced by estradiol
presumably are evident and a 9-day interval, when these reductions in intake
presumably are no longer evident. If the unconditioned effects of estradiol are the
reason for CTA expression, than both CTA and unconditioned reduction of food
consumption should be expressed at the 2-day time period but neither should be
expressed at the 9-day time period.
Methods
Sixty-five animals were randomly divided into one of the following six
groups: (1) Oil-D2, (2) EB10-D2, (3) EB50-D2, (4) Oil-D9, (5) EB10-D9, and (6)
EB50-D9, with n=11 for all groups except the Oil-D9 group which had n=10. Both
of the Oil, EB-10, and EB-50 groups received an injection of sesame oil, 10μg/kg of
188
estradiol benzoate (EB), or 50μg/kg of EB, respectively, immediately following the 1
hour drinking of sucrose on acquisition day. The post-acquisition test was held 2
days (47 hours) after acquisition for the three D2 groups and 9 days (215 hours)
following acquisition for the three D9 groups. Body weight and food measurements
were made throughout the duration of the experiment.
Results
Assessment of CTA Formation
Groups Oil-D2, EB10-D2, and EB50-D2. Both of the 2-day estradiol groups
developed a CTA, while the oil group did not (see Figure 4). Within-group analysis
showed that groups EB10-D2 and EB50-D2 drank significantly less sucrose during
the post-acquisition test than the acquisition trial (critical significance=0.05,
significance=0.00 in each case).
Between-group comparisons revealed that the estradiol groups did not differ
from the oil group in the amount of sucrose consumed during the acquisition trial, but
during the post-acquisition test, the EB10-D2 and EB50-D2 groups drank
significantly less sucrose than the Oil-D2 group (critical significance=0.05,
significance=0.00 in each case). In addition, comparisons between each of the
estradiol groups and the oil group revealed significant interaction effects across
acquisition and the post-acquisition test (critical significance=0.05 and
significance=0.00 in each case).
Moreover, the EB50-D2 group formed a stronger CTA than the EB10-D2
group. Although these two groups consumed similar amounts of sucrose during
189
acquisition, the EB50-D2 group consumed less sucrose during the post-acquisition
test than the EB10-D2 group (critical significance=0.05, significance=0.013). There
also was a significant interaction effect across acquisition and the post-acquisition
test in sucrose consumption when the EB10-D2 and EB50-D2 groups were compared
(critical significance=0.05, significance=0.014).
Groups Oil-D9, EB10-D9, and EB50-D9. As was true for the 2-day groups, both of
the 9-day estradiol groups developed a CTA, while the oil group did not. Within-
group analyses showed that the EB10-D9 and EB50-D9 groups drank significantly
less sucrose during the post-acquisition trial than the acquisition trial (critical
significance=0.05, significance=0.00; see Figure 4).
Although both the EB10-D9 group and the EB50-D9 group did not differ
statistically from the Oil-D9 group in the amount of sucrose they drank during the
acquisition trial they drank significantly less sucrose than the control group during
the post-acquisition trial (critical significance=0.05, significance=0.00 in each case).
Comparisons between each of the estradiol groups and the control oil group also
showed a significant interaction effect across acquisition and the post-acquisition test
(critical significance=0.05, significance=0.00 in each case).
Similar to the 2-day estradiol groups, the EB50-D9 group acquired a stronger
CTA than the EB10-D9 group. Although the EB50-D9 group consumed significantly
more sucrose during the acquisition trial than the EB10-D9 group, this group also
showed a greater reduction in sucrose consumption during the post-acquisition trial
than the EB10-D9 group (critical significance=0.05, significance=0.002).
190
Furthermore, there was a significant interaction effect across acquisition and post-
acquisition test sucrose consumption between these two estradiol groups (critical
significance=0.00, significance=0.05). Therefore, the difference found between the
two estradiol groups in consumption during the acquisition trial does not warrant
consideration.
D2 Groups versus D9 Groups. Comparisons between the Oil-D2 and Oil-D9 groups
revealed that these two oil groups did not differ in the amount of sucrose consumed
during the acquisition or post-acquisition trials and they showed no interaction effect
across these two tests. Similarly, comparisons made between the two EB10 groups
(EB10-D2 versus EB10-D9) and the two EB50 groups (EB50-D2 versus EB50-D9),
revealed no differences in sucrose consumption during or across both of these trials.
As such, whether the post-acquisition test was held 2 days or 9 days following
acquisition and whether a 10- or 50μg/kg dose was used, CTAs of comparable
strength (relative to dose of estradiol) were acquired.
Assessment of Caloric Decrement
Groups Oil-D2, EB10-D2, and EB50-D2. Both doses of estradiol produced
decreases in caloric intake across baseline and the post-acquisition test day while oil
did not (see Figure 4). Within-group analyses showed that both the EB10-D2 and
EB50-D2 groups consumed fewer calories on the day of the post-acquisition test
compared to the baseline time period (critical significance=0.254-0.038,
significance=0.00 in each case; see Figure 4). On the other hand, the Oil-D2 group
did not reduce its caloric intake from the baseline time to the post-acquisition day.
191
In addition, between-group analyses revealed that both the EB10-D2 and
EB50-D2 groups ingested significantly fewer calories during the post-acquisition test
day than the Oil-D2 group (critical significance=0.0254, significance=0.00 in each
case). Furthermore, there was a significant interaction effect across baseline and
post-acquisition levels of consumption between the Oil-D2 and EB10-D2 groups and
the Oil-D2 and EB50-D2 groups (critical significance=0.017, significance=0.00 in
each case).
The evidence for differences between the two estradiol groups in the extent of
the decrease across baseline and the post-acquisition test day was not clearly
discernable. These two groups did not differ in baseline levels of consumption, but
showed differences in consumption during the post-acquisition test period (critical
significance=0.025, significance=0.02). However, there was no significant
interaction effect detected across the two time periods between the two estradiol
groups.
Groups Oil-D9, EB10-D9, and EB50-D9. Unlike the 2-day groups, neither dose of
estradiol produced decreases in calorie intake. Within-group analyses showed that
caloric intake was not reduced during the post-acquisition test day compared to the
baseline time period for any of the 9-day groups. The three groups also did not differ
in the amount of calories ingested during the baseline time period or during the post-
acquisition test day and there were no significant interactions across these two time
periods.
192
D2 Groups versus D9 Groups. Comparisons made between the two oil groups (Oil-
D2 and Oil-D9) showed no differences in calorie intake during either the baseline
time period or during the post-acquisition trial day. Although the two EB10 groups
consumed comparable calories during the baseline time period, the EB10-D2 group
ingested significantly fewer calories during the post-acquisition test day than the
EB10-D9 group (critical significance=0.05, significance=0.00). There also was a
significant interaction effect across the two time periods for these two EB10 groups
(critical significance=0.05, significance=0.01). Similarly, groups the two EB50
groups did not differ in calorie intake during the baseline time period, but group
EB50-D2 ingested significantly fewer calories during the post-acquisition test day
than group EB50-D9 (critical significance=0.05, significance=0.00). Finally,
comparisons of intake across the baseline and post-acquisition time periods between
these two EB50 groups revealed a significant interaction effect (critical
significance=0.05, significance=0.00).
193
Figure 4. Effect of Two Different Time Intervals between Acquisition and Post-
acquisition Testing on Estradiol Conditioned Taste Avoidance and Calorie
Intake.
Acq Post-Acq
0
4
8
12
16
20
Oil-D2 EB10-D2 EB50-D2
ab
abc
Trimmed Mean Sucrose
Intake (ml)
Acq Post-Acq
Oil-D9 EB10-D9 EB50-D9
d
ab
abc
Base Post-Acq
50
60
70
80
90
Oil-D2 EB10-D2 EB50-D2
ab
abc
Trimmed Mean Total
Calorie Intake
Base Post-Acq
Oil-D9 EB10-D9 EB50-D9
Acquisition Trial & Post-Acquisition Test
Baseline & Post-Acquisition Test
Figure 4 Caption.
Upper Graphs. Trimmed mean sucrose consumption of ovariectomized female rats during the
acquisition (Acq) trial and the post-acquisition (Post-Acq) test. The acquisition trial consisted of
pairing a 10% sucrose solution with a subcutaneous injection of either sesame oil vehicle (Oil) or a 10-
or 50μg/kg dose of estradiol benzoate (EB10 or EB50, respectively). The post-acquisition test was
given either 2 days (D2) or 9 days (D9) following acquisition for each of the three groups.
a
Significant
reduction in sucrose consumption across Acq and Post-Acq.
b
Significantly lower than the Oil group
during Post-Acq and significant interaction across Acq and Post-Acq when compared to the Oil group.
c
Significantly lower than the EB10 group during Post-Acq and significant interaction across Acq and
Post-Acq when compared to the EB10 group.
d
Significantly lower than the EB50 group during Acq.
Lower Graphs. Trimmed mean total calorie intake of ovariectomized female rats during the baseline
time period (Base) and the post-acquisition (Post-Acq) test day. For the Base time period, caloric
intake was measured during the 24 and 48 hour periods prior to the injection of either sesame oil (Oil-
D2) or estradiol benzoate in the doses of 10- (EB10-D2) or 50μg/kg (EB50-D2) and a mean for these
two days was computed. For the Post-Acq test day, caloric intake was measured during the 24 hour
period that occurred 2 days following injection and that started at the onset of the post-acquisition
trial. Caloric intake during the Post-Acq test day included consumption of lab chow and sucrose while
caloric intake during the Base time period included only lab chow.
a
Significant reduction in total
calorie intake across Base and Post-Acq days.
b
Significantly lower than Oil-D2 group during the Post-
Acq day and significant interaction across Base and Post-Acq days when compared to the Oil group.
c
Significantly lower than EB10-D2 group during the Post-Acq day.
194
Discussion
The results of Experiment 1 revealed that estradiol benzoate, at both 10- and
50µg doses, is able to condition an avoidance of sucrose whether the unconditioned
effects of the hormone on eating are present or absent. Groups tested for CTA 2 days
after receiving estradiol benzoate exhibited both CTA to sucrose and a general
diminution of caloric intake. Although groups assessed for CTA 9 days after
estradiol benzoate injection also displayed CTAs, they no longer displayed reduced
caloric intake. Unlike the 2-day groups, the 9-day groups had returned to their
baseline level of caloric consumption during their post-acquisition test day. These
results are inconsistent with the hypothesis that the hypophagic effects of estradiol
contribute to the expressed CTA and they are strongly supportive of the notion that
estradiol is a true conditioning agent. Furthermore, the fact that the CTAs developed
by the 2-day and 9-day groups were of comparable strengths (within each dose)
suggests that the conditioned and unconditioned effects of estradiol are independent,
because if they were not, one would expect the unconditioned effects to contribute to
the CTA strength in the 2-day groups.
Examination of differences between the two estradiol doses provides
additional support for dissociation between the CTA observed after pairing this
hormone with a sucrose solution and the unconditioned effects of estradiol on calorie
intake. The high dose of estradiol clearly induced a stronger CTA than the low dose
in the 2-day groups, as evidenced by the higher dose group showing a greater extent
of decrease in sucrose consumption across acquisition and the post-acquisition test.
195
However, there was no difference between the two estradiol groups in the extent of
decrease in calorie intake across acquisition and post-acquisition days.
Despite the strength of these results, there is one other possibility that should
be addressed. One cannot discount the slim possibility that the unconditioned effect
of estradiol specifically inhibit sucrose consumption for a much longer period of time
than it affects lab chow intake. However, given the high palatability valence of
sucrose and the fact that rats will forgo lab chow for sucrose (observed in the Oil
groups), we consider this possibility to be unlikely. In addition, the results of
Experiment 3 increase the unlikelihood of this possibility (see the discussion of that
experiment).
Before delving into the details of the next experiment, it is worth noting that
there are inconsistencies in the ability of a 10µg/kg dose of estradiol to induce a
CTA. DeBeun et al. (1991) showed in a previous study that this dose of estradiol
was ineffective in producing a CTA when the post-acquisition test was given 3 days
after acquisition. We failed to replicate this in Experiment 1, demonstrating that the
10µg/kg dose produced a CTA whether the test was held 2 days or 9 days following
acquisition. There are three major dissimilarities in the methodologies between the
two studies. DeBeun et al. used fluid deprived Wistar rats and estradiol while the
present study used Sprague-Dawley rats that had ad libitum access to water
throughout the duration of the experiment and were injected with estradiol benzoate.
Because fluid deprivation reduces sensitivity to the US, this factor could account for
why the 10µg/kg dose did not induce CTA in fluid deprived rats but produced CTA
196
in nondeprived rats (Chambers, Sengstake, Yoder & Thornton, 1981; Grote and
Brown, 1973; Hamdani & White, 2007; Sengstake, Chambers & Thrower, 1978).
Moreover, Sprague-Dawley rats have been found to be less sensitive to LiCl than
Fischer-344, as evidenced by the finding that Sprague-Dawley rats require a
substantially higher dose of LiCl to achieve the same slow rate of extinction as
Fischer-344 (Brownson, Sengstake & Chambers, 1994), which raises the possibility
that a similar difference in sensitivity exists between Sprague-Dawley and Wistar rats
when estradiol is used as the US. Finally, the conjugated form of estradiol, estradiol
benzoate, has been shown to have varying kinetic and dynamic properties compared
to estradiol (McEwen, 1981), which could alter the effects the hormone has on
behavior. However, additional evidence does not support any of these differences as
a reason for the disparate findings regarding CTAs and a 10µg/kg dose of estradiol.
In another study conducted in our lab, we failed to induce CTA in nondeprived
Sprague-Dawley female rats with a 10µg/kg dose of estradiol benzoate even after 7
sucrose-estradiol pairings (Chambers, Hintiryan & So, unpublished manuscript). At
the present time, analysis of all of the evidence suggests that the best explanation for
the differences in the ability to acquire a CTA with a 10µg/kg dose of estradiol is that
this dose may be a threshold dose for inducing CTA.
4.5.2 Experiment 2
PRIMARY AIM 1- SPECIFIC AIM 1.1: Testing the Unconditioned Hypothesis -
Correlation between expression of hypophagic and CTA based on varying doses of
estradiol (Strategy 2).
197
Although the results of Experiment 1 make a strong argument against the
hypothesis that the unconditioned effects of estradiol on food consumption contribute
to the apparent CTA, reproducing these findings using a different methodology
would further solidify our finding. This leads us to the second strategy, which
involved correlating the ability of doses of estradiol to induce CTA with their ability
to trigger unconditioned reductions in consumption at the time of CTA expression.
To do this, we chose to try a 1µg/kg dose, basing our decision on previous findings
that doses as low as 0.5µg/kg reduce food intake (Zucker, 111969), but a 2µg/kg does
not induce CTA (DeBeun et al, 1991). Additionally, we chose to use unconjugated
estradiol instead of estradiol benzoate to verify that despite the form of estradiol used,
the conditioned and unconditioned effects of the hormone are dissociable.
Methods
Thirty animals were randomly assigned to one of the three following groups
(n=10 per group): Group (1) Oil (sesame oil), (2) E
2
1 (1μg/kg dose of estradiol), and
(3) E
2
50, (50μg/kg dose of estradiol). Oil and estradiol injections were given
immediately following the 1 hour sucrose exposure on acquisition day. The post-
acquisition trial was given 2 days (47 hours) following acquisition.
Results
Assessment of CTA Formation
The high dose of estradiol induced a CTA while the low dose of estradiol and
oil vehicle did not. Within-group comparisons showed that the E
2
50 group drank
significantly less sucrose during the post-acquisition test compared to the acquisition
198
trial (critical significance=0.05, significance=0.00; see Figure 5), while groups Oil
and E
2
1 drank comparable amounts across both trials. Between-group analysis
revealed that while the E
2
50 animals did not differ from the Oil and E
2
1 animals in
the amount of sucrose they consumed during the acquisition trial, they did drink
significantly less sucrose than the Oil and E
2
1 animals during the post-acquisition
trial (critical significance=0.05 in each case, significance=0.00-0.02). In addition,
there was significant interaction effect across acquisition and post-acquisition trials
between the E
2
50 group and both the Oil and E
2
1 groups (critical significance=0.05
in each case, significance=0.00-0.019). The Oil and E
2
1 groups did not differ in the
amount sucrose consumed during either the acquisition trial or the post-acquisition
test and there was no significant interaction effect across acquisition and the post-
acquisition test for these groups.
Assessment of Caloric Decrement
Both estradiol groups showed decreases in caloric intake while the Oil group
did not. Within-group analysis revealed that compared to baseline, group Oil did not
show reductions in calorie on the post-acquisition day, while groups E
2
1 and E
2
50
ingested significantly fewer calories (critical significance=0.05, significance=0.00 in
each case; see Figure 5). Between-group analysis revealed that the caloric intake of
the Oil group during the baseline time period was comparable to that of both the E
2
1
and E
2
50 groups. However, both of the estradiol groups differed significantly from
the Oil group in the amount of calorie intake on the post-acquisition test day (critical
significance=0.033-0.05, significance=0.004-0.018). In addition, there was a
199
significant interaction effect across the baseline and post-acquisition time periods
between groups Oil and E
2
1 (critical significance=0.02, significance=0.04) and Oil
and E
2
50 groups (critical significance=0.02, significance=0.012). The caloric intake
of the two estradiol groups did not differ during the baseline or the post-acquisition
time periods. In addition, no significant interaction was detected between the two
groups across the two days.
Figure 5. Effect of Two Different Doses of Estradiol on Conditioned Taste
Avoidance and Calorie Intake.
Acq Post-Acq
0
4
8
12
16
20
Oil E
2
1 E
2
50
ab
Acquisition Trial & Post-Acquisition
Test
Trimmed Mean Sucrose
Intake (ml)
Base Post-Acq
60
70
80
90
Oil E
2
1 E
2
50
ab
ab
Baseline & Post-Acquisition Test
Trimmed Mean Total
Calorie Intake
Figure 5 Caption.
Left Graph. Trimmed mean sucrose consumption during the acquisition (Acq) trial and the post-
acquisition (Post-Acq) test for animals that received either sesame oil (Oil) or estradiol in the dose of
either 1- (E
2
1) or 50μg/kg (E
2
50) on Acq day.
a
Significant reduction in sucrose consumption across
Acq and Post-Acq.
b
Significantly different sucrose consumption than Oil and E
2
1 groups during Post-
Acq and significant interaction across Acq and Post-Acq when compared to the Oil and E
2
1 groups.
Right Graph. Trimmed mean total calorie intake of ovariectomized female rats during the baseline
time period (Base) and the post-acquisition (Post-Acq) test day. For the Base time period, caloric
intake was measured during the 24 and 48 hour periods prior to the injection of either sesame oil (Oil)
or estradiol in the doses of 1-(E
2
1) or 50μg/kg (E
2
50) and a mean for these two days was computed.
For the Post-Acq test day, caloric intake was measured during the 24 hour period that occurred 2 days
following injection and that started at the onset of the post-acquisition test. Caloric intake during the
Post-Acq test day included consumption of lab chow and sucrose while caloric intake during the Base
time period included only lab chow.
a
Significant reduction in total calorie intake across Base and
Post-Acq .
b
Significantly lower than Oil group during the Post-Acq trial day and significant
interaction across Acq and Post-Acq when compared to the Oil group.
200
Discussion
The results of Experiment 2 provide additional evidence against our
hypothesis that the unconditioned effects of estradiol contribute to the conditioning
effects of the hormone. Specifically, the findings indicate that although a low dose of
estradiol produces significant reductions in eating, it does not produce a CTA. It is
the case that the 1µg group did show a small, albeit non-significant, drop in sucrose
consumption. However, all of the statistical outcomes strongly indicate that this slight
drop is unlikely to be meaningful. First, the amount of sucrose consumed by the Oil
and E
2
1 groups during the acquisition and post-acquisition trials did not differ,
neither the Oil nor the E
2
1 group decreased consumption across acquisition and post-
acquisition testing, and there was no significant interaction across these two trials
between these two groups. Second, the sucrose consumption of both of the Oil and
E
2
1 groups differed from the E
2
50 group during the post-acquisition test but not the
acquisition trial and there was a significant interaction across these two trials between
the E
2
50 group and both the Oil group and the E
2
1 group. Taken together, these
results clearly indicate that the E
2
1 group did not develop a CTA.
In contrast, the 1µg dose of estradiol produced significant reductions in
calorie intake across the baseline and post-acquisition time periods. Furthermore, in
contrast to the CTA findings, the caloric intake of both the E
2
1 and E
2
50 groups
differed from the Oil group during the post-acquisition time period but not the
baseline time period, both the E
2
1and E
2
50 group decreased intake across the two
time periods but the Oil group did not, and there was a significant interaction across
201
these two time periods between each of the estradiol groups and the Oil group.
Therefore, the results of Experiment 2 show that a 1μg dose of estradiol produces
significant reductions in eating without producing a CTA, while a 50µg/kg dose of
estradiol produces significant decreases in caloric intake and produces a CTA.
Similar to the findings in Experiment 1, these data suggest that the unconditioned
effects of estradiol do not account for the conditioning effects of the hormone.
As a final note, utilization of both estradiol and estradiol benzoate revealed
that they produced similar effects. At 50µg/kg, both types of estradiol were shown to
diminish calorie intake and to condition an avoidance of sucrose. This consistency
across hormone variants increases confidence in the veracity of the effects we have
demonstrated.
4.5.3 Experiment 3
PRIMARY AIM 1- SPECIFIC AIM 1.1: Testing the Unconditioned Hypothesis -
Correlation between expression of hypophagic and CTA based on non-contingent
pairing of the CS and US (Strategy 3).
As a final means of determining whether the unconditioned effects of
estradiol on eating contribute to the CTA observed after pairing this hormone with a
novel sucrose solution, we employed the third strategy outlined in the introduction.
This strategy involved manipulating the length of the interval between consumption
of sucrose and administration of estradiol. Contingent pairing of the taste solution
with estradiol should not be a requirement for expression of the apparent CTA if
unconditioned effects of estradiol play a role in this expression. In CTAs,
202
associations between the CS tastants and US toxins still occur if the interval between
the CS and US is as long as 4-12 hours. The earliest studies exploring this
phenomenon showed that an apomorphine CTA towards a saccharin solution could
be formed with an inter-stimulus interval of 1.4 hours (Garcia, Ervin & Koelling,
1966). Other laboratories reported significant aversion to a 4% sucrose solution
when exposure to x-radiation was administered 6 hours following the CS presentation
and gustatory avoidance was readily established toward a 0.1% saccharin solution
when the CS and the x-radiation US were separated by as much as 12 hours (Smith &
Roll, 1967). However, administration of a US after a certain interval fails to result in
CTA. For instance, CS-US intervals greater than 12 hours do not support radiation
CTAs (Smith & Roll, 1967) and a 24 hour inter-stimulus interval prevents acquisition
of a lithium chloride CTA (Houpt & Berlin, 1999).
Predicated on this feature of CTAs, we proposed to find an interval that does
not support an estradiol CTA. A 50μg/kg dose of estradiol was used as the US since
this dose was demonstrated to produce strong unconditioned effects. Based upon
pilot data, the CS-US interval 24 hours was chosen and the post-acquisition test was
held approximately 2 days following the US administration. If the strength of the
unconditioned effect is the only factor determining whether a CTA is detected 47
hours later, then regardless of the CS-US interval, an injection of a 50μg/kg dose of
estradiol should produce significant reductions in sucrose consumption. On the other
hand, if this interval fails to result in an estradiol CTA, just as they do with radiation
and LiCl CTAs, then this would support a conditioning hypothesis instead.
203
Methods
Twenty two animals were randomly assigned to one of the following two
groups (n=11 per group): (1) E
2
50-24, which received an injection of estradiol 24
hours following the 1 hour exposure to the CS on acquisition day or (2) Oil-24,
which received an injection of sesame oil 24 hours following the CS on acquisition
day. The post-acquisition test for this experiment was held 47 hours following the
injections administered on acquisition day.
Results
A 24 hour CS-US interval did not support an estradiol CTA (see Figure 6).
Analysis of the acquisition and post-acquisition test data revealed that neither the Oil-
24 nor the E
2
50-24 showed a difference in the amount of sucrose drunk across the
two days. In addition, the E
2
50-24 group did not differ statistically from the Oil-24
group in the sucrose consumption levels during the acquisition or post-acquisition
tests and no interaction effects were detected between the two groups across the two
days.
204
Figure 6. Effect of a 24 hour CS-US Interval on Estradiol Conditioned Taste
Avoidance.
0 Acq Post-Acq
0
4
8
12
16
20
Oil-24 E
2
50-24
Acquisition Trial & Post-Acquisition
Test
Trimmed Mean Sucrose
Intake (ml)
Figure 6 Caption. Trimmed mean sucrose consumption on acquisition (Acq) and post-acquisition
(Post-Acq) days for animals that received either a 50μg/kg dose of estradiol (E
2
50-24) or sesame oil
(Oil-24) 24 hours following the 1 hour exposure to sucrose on Acq day. No significant differences
were found.
Discussion
Consistent with Experiments 1 and 2, the results of Experiment 3 also failed
to support the hypothesis that the hypophagic effects of estradiol on eating contribute
to CTAs induced by this hormone. Instead, they strongly suggest that CTA
expression solely is due to the conditioning capability of estradiol. If reductions in
sucrose consumption observed during post-acquisition testing were due entirely to
the unconditioned effects of the hormone, then uncoupling sucrose and estradiol
should still result in reductions of sucrose consumption when tested during the time
that estradiol produces its unconditioned hypophagic effects, especially when a
potent 50µg/kg dose of estradiol is used. The results of Experiment 3 demonstrate
that this is not the case. A 24 hour CS-US interval prevented the expression of a
CTA. The post-acquisition test for this interval was held 47 hours following the
205
injection of estradiol, when the unconditioned effects of estradiol are still evident.
The results of Experiments 1 and 2 demonstrated that a 50μg/kg dose of estradiol
produces significant reductions in caloric intake 47 hours after injection and other
labs have shown hypophagia 24 to 72 hours following its administration (Tartellin &
Gorski, 1973; Rivera & Eckel, 2005; Asarian & Geary, 2002; Santollo, Wiley &
Eckel, 2007), suggesting that the unconditioned effects of the hormone were present
at the time of post-acquisition testing. The lack of a CTA during a time when
unconditioned effects of estradiol were present strongly suggests a dissociable
mechanism for estradiol conditioning and estradiol hypophagia.
The results of Experiment 1 showed that if the post-acquisition test is
conducted at a time when the unconditioned effects of estradiol on calorie intake are
no longer expressed (9 days after estradiol injection) animals show significant
reductions in sucrose consumption, which indicates the acquisition of a CTA. As
mentioned in the discussion of Experiment 1, these results could alternatively be
accounted for by a slower decay in hypophagia expressed specifically towards
sucrose than the hypophagia expressed towards lab chow. This hypothesis is refuted
by the findings of Experiment 3. The animals with a 24 hour CS-US interval did not
show a reduction in sucrose consumption during their post-acquisition test, which
was given at a time when estradiol hypophagia is expressed. With estradiol not
having an effect upon sucrose consumption in the near-term when the unconditioned
effects are probably evident (47 hours after estradiol injection), it is unlikely that it
has an effect in the long-term either (9 days after estradiol injection).
206
The results of Experiment 3 also address Secondary Aim 1 of this
dissertation, which is to compare CTAs produced by estradiol to those produced by
the putative illness inducing agent LiCl. It has been demonstrated that a 24 hour
inter-stimulus interval precludes the acquisition of a LiCl (Houpt & Berlin, 1999). In
Experiment 3, we demonstrated that this interval also was ineffective in producing a
CTA, providing an additional piece of evidence as to how the CTAs produced by the
two USs are similar.
4.5.4 Experiment 4
PRIMARY AIM 1 – SPECIFIC AIM 1.2: Testing the Negative Contrast Hypothesis
and
PRIMARY AIM 2 – SPECIFIC AIM 2.1: Determining the Longest CS-US Interval
Supporting Estradiol CTA.
Experiment 4 was conducted for two reasons. The first reason was to test the
negative contrast hypothesis of estradiol conditioning (PRIMARY AIM 1-SPECIFIC
AIM 1.2). Anticipatory negative contrast effects are not established when the
interval between the presentation of the two stimuli exceed 30 minutes (Flaherty &
Checke, 1982; Flaherty, Grigson, Checke & Hnat, 1991). If the reductions in sucrose
observed after pairing it with estradiol are due to negative contrast, then CS-US
intervals greater than 30 minutes should not result in the gustatory avoidance. In this
experiment, intervals much greater than 30 minutes were used.
The second rationale behind Experiment 4 was to determine the longest CS-
US interval that supports an estradiol CTA (PRIMARY AIM 2-SPECIFIC AIM 2.1),
207
which would allow for the examination of the neurochemical basis of estradiol CTA.
Since no data to date exists on estradiol CTA and extended CS-US intervals, several
interval lengths needed to be tested to hone in on the interval of interest. As such,
inter-stimulus lengths of 3, 6, 7, 8, 9, and 12 hours were tested in Experiment 4.
Methods
Groups
One hundred seventy two animals were used in 4 separate studies to assess a
3, 6, 7, 8, 9, and 12, CS-US interval on the formation of an estradiol CTA. For each
interval, two groups of animals were tested; one group was injected with sesame oil
and another group was injected with 50μg/kg of estradiol after 1 hour access to a
sucrose solution on acquisition day.
Procedures
Normally in experiments with no CS-US interval, the CS is given for the first
hour of the dark cycle and immediately following this, each animal is injected with
the US. The post-acquisition test is held 47 hours after the pairing. However, in
order to keep the conditions during acquisition and post-acquisition trials as similar
as possible for these studies with varying inter-stimulus intervals, the time between
the US injection and post-acquisition testing had to be shortened for each group in
proportion to the length of its CS-US interval.
Experiment 4a. 3 and 6 hours
Forty eight animals were randomly assigned to one of the following four
groups (n=12 per group): (1) E
2
50-3 or (2) E
2
50-6, which received estradiol 3 or 6
208
hours, respectively, following CS exposure or (3) Oil-3 or (4) Oil-6, which received
oil 3 or 6 hours, respectively, following CS exposure. The post-acquisition test for
the 3-hour and 6-hour CS-US interval was held 44 and 41 hours, respectively,
following the injection administered on acquisition day as opposed to our standard 47
hours.
The four groups in this study were a part of a larger study that included
animals that received injections of LiCl. In order to minimize the number of animals
used in the study, one control group was used for both the estradiol and LiCl
experimental groups. As such, the Oil-3 and Oil-6 control groups received injections
of both sesame oil and saline. In order to equate the control and experimental
groups, the E
2
50-3 and E
2
50-6 groups also were given an additional injection of
saline. The additional injection of saline did not affect the sucrose consumption of
the Oil-3 or Oil-6 since these groups behaved similar to the other control oil groups.
Sucrose consumption across acquisition and post-acquisition trials either stayed the
same or increased for all of the Oil groups. For instance, the Oil-3, Oil-6, Oil-7, Oil-
8, and Oil-12 groups of Experiment 4 showed no changes in their sucrose
consumption across the 2 days, while the Oil-9 group showed a significant increase
in sucrose consumption across the 2 days (see Figure 7).
Experiment 4b. 7 and 8 hours
Fifty two animals were randomly assigned to one of the following four groups
(n=13 each): (1) E
2
50-7 or (2) E
2
50-8, which received estradiol 7 or 8 hours,
respectively, following CS exposure or (3) Oil-7 or (4) Oil-8, which received oil 7 or
209
8 hours, respectively, following CS exposure. Post-acquisition testing was held 40
and 39 hours, respectively, following the injection administered on acquisition day.
Experiment 4c. 9 and 12 hours
Forty nine animals were randomly assigned to one of the following four
groups: (1) E
2
50-9 (n=13) or (2) E
2
50-12 (n=12), which received estradiol 9 or 12,
respectively, following CS exposure or (3) Oil-9 (n=12) or (4) Oil-12 (n=12), which
received sesame oil 9 or 12 hours, respectively, following CS exposure. Post-
acquisition testing was held 38 and 35 hours following the injections administered on
acquisition day.
Experiment 4d. 6 hours (Replication)
Statistical analyses (discussed below) revealed that the 6 hour interval was the
longest interval that supported a strong estradiol CTA in the majority of the animals
tested. Since all of our neurochemical studies were predicated on this interval, it was
imperative to the reliability of this finding. Therefore, another study was conducted
to test the reproducibility of the results. Twenty three animals were randomly
assigned to one of the following two groups: (1) E
2
50-6R (n=12), which received
estradiol 6 hours following CS exposure or (2) Oil-6R (n=11), which received oil 6
hours after CS exposure. The post-acquisition test was held 41 hours following the
injections administered on acquisition day.
Additional Statistical Analyses
As discussed in the beginning of the chapter, the longest CS-US interval was
going to be used to ascertain whether neurotransmitter antagonists would block an
210
estradiol CTA. If we block an estradiol CTA with the antagonists, we want to be
certain that it was because the infusions blocked the conditioning and not because the
interval did not produce a CTA in some of the animals. Also, in order to find a site
of infusion for the antagonists, we conducted Experiment 5, where we tested to see if
lesions of the lateral PBN affected estradiol CTA with the CS-US interval determined
in Experiment 4. At this point, it too is important that the majority of the animals
acquire a CTA with the predetermined inter-stimulus interval. If lesioned animals do
not express an estradiol CTA, we want to say with certainty that it was due to the
lesion and not due to the possibility that those animals had not acquired a CTA. For
instance, in the lateral PBN lesion study, we could have a situation in the intact
estradiol control group where only 6 out of 12 animals develop a strong estradiol
CTA, which would lead to a significant group decrease across acquisition and post-
acquisition day. This would indicate that the estradiol control group developed a
CTA; however, this situation could lead to complications in the interpretation of our
results. If we showed that the lateral PBN lesioned experimental group did not
develop an estradiol CTA, we would not be able to conclusively state that the lesions
obliterated the CTA since it is possible that some of those animals would not have
developed the CTA to begin with due to the interval. In order to circumvent such a
situation, we needed to choose an interval that would allow the majority, if not all, of
the animals in the control estradiol CTA group to develop a CTA. As such, solely
examining group effects in this experiment did not suffice and the number of animals
that developed a CTA within each group needed to be considered.
211
Thus, the following secondary analyses were performed on the data. A strict
set of criteria, outlined below, were set forth for the qualification of a CTA. A
percentile bootstrap equivalent to a binomial analysis was used to compare the
number of animals that developed a CTA in each experimental group to both its
respective oil group and to other experimental groups. It should be mentioned that
using this analysis has an important limitation that needs to be addressed. It allows
only two groups to be compared at a time and does not control for family-wise error
rate when multiple comparisons are made. This limitation is not catastrophic since
methods, such as Rom’s method, have been developed for adjusting p-values in such
situations (Wilcox, 2003). Since multiple comparisons were made, Rom’s method
was used to adjust p-values for this part of the analysis. Thus, for this analysis, a
significance level and a critical significance level based on Rom’s method is
reported. A significant result is obtained when the significance level is lower than
the critical significance level.
Criteria for CTA Qualification
The amount of sucrose drunk from acquisition to post-acquisition test and, if
necessary, extinction test 2 was used to determine whether or not an animal acquired
a CTA. These criteria were based mainly on data collected on estradiol CTAs and
from control oil or saline groups in both the present studies and past studies
conducted in our laboratory.
CTA. A CTA was acquired if the animal displayed a decrease greater than 2ml
across acquisition and post-acquisition test 1. If the animal had decreased by 2ml or
212
less on post-acquisition test 1, then on post-acquisition test 2, the animal had to
display a decrease greater than or equal to 1ml.
No CTA. An animal did not acquire a CTA if it displayed an increase, no
change, or a decrease by 1ml in sucrose consumption across acquisition and the post-
acquisition test 1. There was also no CTA if an animal displayed a decrease by 2ml
across acquisition and post-acquisition test and displayed no change or an increase on
post-acquisition test 2.
These specifications are summarized in Table 3 and work well given the
present data. As will be discussed shortly, based on these criteria, the majority of the
animals in the control groups did not develop a CTA. This does not appear to be due
to the fact that the criteria for CTA were too stringent since for the shortest intervals,
most of the experimental animals acquired a CTA. Also, it would be expected that as
the CS-US interval increases, the strength of the CTA decreases since after a certain
interval length, a CTA is no longer acquired (i.e. 12 hours for radiation (Smith &
Roll, 1967). Based on our criteria, the data showed that as the inter-stimulus interval
increased, the number o f animals acquiring a CTA decreased (for summary see
Table 4).
213
Table 3. Criteria for Acquisition of a CTA: Changes in Sucrose Consumption
Across Acquisition & Either Post-Acquisition Test 1 or Post-Acquisition Test 2.
Results
Assessment of CTA Formation Experiments 4a-4d
Analysis of the acquisition and post-acquisition test 1 data for each group
revealed that none of the control groups developed a CTA (see Figure 7). The Oil-3,
Oil-6, Oil-7, Oil-8, and Oil-12 groups showed no difference in the amount of sucrose
drunk across the two days. The Oil-9 group showed an increase in sucrose
consumption across acquisition and post-acquisition test 1 (critical significance=0.05,
significance=0.018).
Analysis of the experimental groups revealed that groups E
2
50-3, E
2
50-6, and
E
2
50-7 developed a CTA (critical significance=0.05, significance=0.00 for E
2
50-3
and E
2
50-6; critical significance=0.05, significance=0.04, for E
2
50-7), while groups
E
2
50-8, E
2
50-9, and E
2
50-12 did not (see Figure 7).
Comparisons of the experimental groups with their respective oil groups
across acquisition and post-acquisition test 1 revealed that although the E
2
50-3 and
E
2
50-6 groups did not differ from their respective control groups (Oil-3 and Oil-6,
Acquisition to
Post-Acquisition Test 1
Acquisition to
Post-Acquisition Test 2
Acquired a CTA Decrease > 2 ml ------------------------------
Decrease = 2ml Decrease > 1
Did Not Acquire a CTA No change ------------------------------
Any increase ------------------------------
Decrease = 1 ml ------------------------------
Decrease = 2 ml No change or increase
214
respectively) in their acquisition trial sucrose consumption, they significantly differed
in their levels of consumption during post-acquisition test 1 (critical
significance=0.05, significance=0.00 in each case). Although according to between-
group analysis the E
2
50-7 group acquired a CTA, it did not significantly differ from
its respective Oil group (Oil-7) during acquisition or post-acquisition test 1
consumption. Similarly, the E
2
50-8, E
2
50-9, and E
2
50-12 groups did not differ
statistically from their respective control groups in their sucrose consumption levels
during acquisition or post-acquisition test 1.
Since within-group analysis revealed that a 7 hour inter-stimulus interval
produced a CTA, but between-group analyses showed that this group did not differ
from its control group in sucrose consumption, it was questionable whether this
interval should be used as the longest interval to produce a CTA. As mentioned in
the methods section to this experiment series, it was imperative that the majority of
the animals in the estradiol control groups of future lesion and neurochemical studies
acquire a CTA. Therefore, a secondary set of analyses was warranted.
215
Figure 7. Effect of Various CS-US Intervals on Estradiol Conditioned Taste
Avoidance.
0 Acq Post-Acq
0
4
8
12
16
20
Oil-3 E
2
50-3
ab
Trimmed Mean Sucrose
Intake (ml)
0 Acq Post-Acq
E
2
50-6 Oil-6
ab
Acquisition Trial & Post-Acquisition
Test
0 Acq Post-Acq
E
2
50-7 Oil-7
a
0 Acq Post-Acq
0
4
8
12
16
20
E
2
50-8 Oil-8
Trimmed Mean Sucrose
Intake (ml)
0 Acq Post-Acq
E
2
50-9 Oil-9
Acquisition Trial & Post-Acquisition
Test
0 Acq Post-Acq
Oil-12 E
2
50-12
Figure 7 Caption. Trimmed mean sucrose consumption during acquisition (Acq) and post-acquisition
(Post-Acq) test 1 for animals that received a 50μg/kg dose of estradiol either 3 (E
2
50-3), 6 (E
2
50-6), 7
(E
2
50-7), 8 (E
2
50-8), 9 (E
2
50-9), or 12 (E
2
50-12) hours following the 1 hour exposure to sucrose on
Acq day with their respective control oil groups.
a
Significant reduction in sucrose consumption across
Acq and Post-Acq tests for E
2
50-3, E
2
50-6, and E
2
50-7 groups.
b
Significant difference in sucrose
consumption during Post-Acq test from Oil group for E
2
50-3 and E
2
50-6.
Results of Binomial Analyses
Binomial analysis of the data revealed that the number of animals acquiring a
CTA in the E
2
50-3 and E
2
50-6 groups significantly differed from the number of
animals that developed a CTA in the Oil-3 and Oil-6 groups, (critical
significance=0.0051, significance=0.003 and 0.0002, respectively). There were no
significant differences between the number of animals that developed a CTA in the 7,
8, 9, and 12 hour interval groups and their respective controls.

216
Additional analyses with the 6 hour interval group revealed that the number
of animals that developed a CTA in this group significantly differed from the number
of animals that developed a CTA in the E
2
50-8, E
2
50-9, and E
2
50-12 groups, each of
which did not show a CTA using within-group analysis (critical significance=0.0051
for each, significance=0.00009-0.00132; see Table 4 and Figure 9). The number of
animals that acquired a CTA in the E
2
50-6 did not differ from the number of animals
that developed a CTA in the E
2
50-3 group, which did acquire a CTA according to
within-group analysis.
Comparison of the E
2
50-7 group with each of the other experimental groups
revealed that the number of animals that developed a CTA in this group did not differ
from the number of animals that developed a CTA in groups E
2
50-8, E
2
50-9, and
E
2
50-12 each of which did not acquire a CTA according to within-group analyses
(see Table 4 and Figure 9).
Finally, there was a significant difference in the number of animals that
acquired a CTA in the E
2
50-6 and E
2
50-7 groups (critical significance=0.05,
significance=0.03). More animals acquired a CTA with the 6 hour interval than the 7
hour interval (see Table 4 and Figure 9).
6 hour CS-US Replication Study
The findings for the E
2
50-6 group were replicated in the E
2
50-6R group. The
E
2
50-6R group showed a significant decrease in sucrose consumption across
acquisition and post-acquisition test 1 (critical significance=0.05, significance=0.00),
while Oil-6R did not (critical significance=0.05, significance= 0.432; see Figure 8).
217
Between-group analysis revealed that although the groups did not differ in the
amount of sucrose they drank during the acquisition test, the differed significantly on
the amount they consumed during post-acquisition test 1 (critical significance=0.05,
significance=0.024). In addition, there was a significant interaction effect between
the Oil-6R and E
2
50-6R groups across these two tests (critical significance=0.05,
significance=0.038). Binomial analyses showed that the number of animals that
developed a CTA in the E
2
50-6R group significantly differed from the number of
animals that developed a CTA in the Oil-6R group (critical significance=0.05,
significance=0.00, see Table 4 and Figure 9). Finally, for both the E
2
50-6 and E
2
50-
6R groups, 11/12 animals acquired a CTA.
Figure 8. Replication of the Effect of a 6 hour CS-US Interval on an Estradiol
Conditioned Taste Avoidance.
0 Acq Post-Acq
0
4
8
12
16
20
Oil-6R E
2
50-6R
ab
Acquisition Trial & Post-acquisition
Test
Trimmed Mean Sucrose
Intake (ml)
Figure 8 Caption. Trimmed mean sucrose consumption during acquisition (Acq) and post-acquisition
(Post-Acq) test 1 for animals that received a 50μg/kg dose of estradiol 6 (E
2
50-6R) hours following
the 1 hour exposure to sucrose on Acq day with its respective control oil group (Oil-6R).
a
Significant
reduction in sucrose consumption across Acq and Post-Acq tests.
b
Significant difference in sucrose
consumption during Post-Acq test from Oil group.
218
Table 4. Number of Animals that Acquired or Did Not Acquire an Estradiol
Conditioned Taste Avoidance Based on Criteria for Conditioned Taste
Avoidance Qualification.
Group CTA No CTA
Oil-3 2 10
E
2
50-3
10 2
Oil-6 2 10
E
2
50-6
11 1
Oil-7 2 11
E
2
50-7
7 6
Oil-8 3 10
E
2
50-8
3 10
Oil-9 0 12
E
2
50-9
2 11
Oil-12 0 12
E
2
50-12
3 9
Replication Study
Oil-6R 3 8
E
2
50-6R
11 1
Figure 9. Number of Animals in each Estradiol and Oil Group that Acquired or
Did Not Acquire Estradiol Conditioned Taste Avoidance for Each CS-US
Interval.
E -3 E -6 E -6R E -7 E -8 E -9 E -12
0
5
10
15
CTA No CTA
2 2 2 2 2 2 2
Number of Animals
O-3 O-6 O-6R O-7 O-8 O-9 O-12
0
5
10
15
CTA No CTA
Groups
Figure 9 Caption.
Left Graph. Number of animals in each of the estradiol groups in Experiments 4a-d that acquired or
did not acquire a CTA when the CS-US interval was 3 (E
2
-3), 6 (E
2
-6, E
2
-6R), 7 (E
2
-7), 8 (E
2
-8), 9
(E
2
-9) or 12 (E
2
-12) hours. All animals received the same dose of estradiol (50µg/kg). As the interval
increases, the number of animals that acquired a CTA decreased, while the number of animals that did
not acquire a CTA increased.
219
Right Graph. The number of animals in each of the oil groups in Experiments 4a-d that acquired or
did not acquire a CTA when the CS-US interval was 3 (O-3), 6 (O-6, O-6R), 7 (O-7), 8 (O-8), 9 (O-9)
or 12 (O-12) hours. Regardless of interval length, the number of animals that acquired or did not
acquire a CTA stayed relatively the same.
Discussion
One of the goals of Experiment 4 was to test whether intervals greater than 30
minutes would still result in an estradiol CTA. It was determined that even an inter-
stimulus interval of 3 and 6 hours resulted in a strong CTA. This finding refutes the
hypothesis that reductions in sucrose consumption following its pairing with a CS are
due to negative contrast effects. This finding further solidifies estradiol as an
effective US in a CTA learning paradigm.
The second goal of the present experiment was to determine the longest CS-
US interval that would support an estradiol CTA. Although the E
2
50-7 group
showed significant reductions in sucrose consumption across acquisition and post-
acquisition test 1, it was decided that 6 hours is the longest CS-US interval to support
an estradiol CTA given that
(a) between-group analyses did not find a difference in sucrose consumption
between E
2
50-7 and Oil-7 on acquisition and post-acquisition test days,
(b) a binomial analysis revealed that the number of animals that acquired a
CTA in the E
2
50-7 group did not differ from the number of animals that
acquired a CTA in the Oil-7 group, and
(c) the number of animals that acquired a CTA in E
2
50-7 did not differ from
the number of animals that acquired a CTA in E
2
50-8, E
2
50-9, and E
2
50-12,
220
all of the groups that did not acquire a CTA according to within-group
analyses conducted on the data.
Therefore, the decision was made to use the 6 hour interval as the longest
interval that supported a strong estradiol CTA. This interval was tested in two
different batches of animals and in both experiments this interval produced a strong
CTA. In fact for both studies, 11 out of 12 animals acquired a CTA.
In conducting the study to ascertain the effects of antagonists on the estradiol
CTA, the receptors of interest probably should be administered 5.75 hours after the
CS exposure (see Figure 10). The antagonists will be delivered 5.75 hours after the
CS presentation instead of 6 hours since ideally one should allow some time for the
antagonist to take effect in case the effect is not immediate. This inactivation should
last for 2 hours or 7.5 hours after CS exposure. Since there is some evidence of CTA
with a 7 hour CS-US interval, extending the receptor blockage time to cover the 7
hour time period would be wise. The estradiol injection should be made 6 hours
following the CS and 15 minutes following infusion of the antagonist. As such, we
can be certain that at any time when estradiol is capable of producing a CTA, the
receptors of interest will be inactivated.
Although not a specific goal of the experiment, the results of Experiment 4
also failed to support the hypothesis that the hypophagic effects of estradiol on eating
contribute to CTAs induced by this hormone. Like Experiment 3, they strongly
suggest that CTA expression solely is due to the conditioning capability of estradiol.
The results of the present study demonstrated that an 8, 9, and 12 hour CS-US
221
interval prevented the expression of a CTA. The post-acquisition tests for these
intervals were held 35-39 hours following the injection of estradiol, which is during
the time when the unconditioned effects of the hormone still are evident (results of
Experiment 1, Tartellin & Gorski, 1973; Rivera & Eckel, 2005; Asarian & Geary,
2002; Santollo, Wiley & Eckel, 2007). This suggests that a CTA was not acquired
despite the unconditioned effects of the hormone providing evidence for the
dissociation between estradiol conditioning and estradiol hypophagia.
Figure 10. Methodology for Determining Chemical Mediation of Estradiol
Conditioned Taste Avoidance.

CTA Weak CTA  No CTA
Time receptors will be
kept inactivated

CS trace
2 3 4 5 6 7 8
Estradiol
Time between CS-US interval (hrs)
Figure 10. Depicts the methodology that will be used to test the role of acetylcholine and histamine
in estradiol induced CTAs. Six hours is the longest CS-US interval that supports an estradiol CTA.
As such, acetylcholine and histamine antagonists that are active for at least 2 hours, will be infused
5.75 hours following the CS. The estradiol injections will be made approximately 15 minutes
following the infusions of the antagonists.
222
4.5.5 Experiment 5A
PRIMARY AIM 1- SPECIFIC AIM 1.1: Testing the Unconditioned Hypothesis -
Determining Whether Lateral Parabrachial Lesions Block Estradiol CTAs and
Hypophagia
and
PRIMARY AIM 1: Testing the Aversive Hypothesis of Estradiol CTA
and
PRIMARY AIM 2 – SPECIFIC AIM 2.2: Determining the Ability of Lateral
Parabrachial Lesions to Block Estradiol CTAs with Extended CS-US Intervals.
In Experiment 5, animals with excitotoxic (Experiment 5a) and electrolytic
(Experiment 5b) lesions of the lateral PBN were tested for estradiol CTA, LiCl CTA,
and estradiol anorexia. Conducting this study was important for 4 reasons. First, it
would determine whether lesions of the lateral PBN could eliminate estradiol CTA
and/or estradiol anorexia thereby lending further support to the conclusion that the
conditioned and unconditioned effects of the hormone are dissociated (PRIMARY
AIM 1-SPECIFIC AIM 1.1). Second, it would help ascertain whether lesions of the
lateral PBN would block an estradiol CTA when a 6 hour CS-US interval was used
(PRIMARY AIM 2-SPECIFIC AIM 2.2). If so, then this would provide an infusion
site for the neurotransmitter antagonists in the experiment examining the chemical
mediation of estradiol CTA. In addition, if the lateral PBN is involved in estradiol
CTA, then this provides one more piece of evidence that likens estradiol CTA to LiCl
CTA (SECONDARY AIM 1) and will give insight into the underlying mechanism of
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estradiol CTA (i.e. if estradiol conditions on the basis of its aversion-inducing
properties; PRIMARY AIM 1-SPECIFIC AIM 1.3). Finally, Experiment 5 could
potentially reveal a neural substrate for estradiol anorexia (SECONDARY AIM 2).
Methods
Groups
Prior to histology, sixty-five rats were randomly assigned to one of the
following four groups:
(1) Intact Oil (n=12), which were neurally intact animals that received oil
(2) Intact E (n=13), which were neurally intact animals that received estradiol
(3) Lesion Oil (n=20), which were lesioned animals that received oil
(4) Lesion E (n=20), which were lesioned animals that received estradiol
Following histology, fifty-three rats were revealed to be in one of the
following six groups:
(1) Intact Oil (n=11), which were neurally intact and received oil
(2) Intact E (n=13), which were neurally intact and received estradiol
(3) Unilateral Oil (n=7), which were unilaterally lesioned and received oil
(4) Unilateral E (n=6), which were unilaterally lesioned and received estradiol
(5) Bilateral Oil (n=7), which were bilaterally lesioned and received oil
(6) Bilateral E (n=9), which were bilaterally lesioned and received estradiol
One Intact Oil, 6 Lesion Oil and 5 Lesion E animals were excluded from the
statistical analyses due to either misplaced lesions, suboptimal lateral PBN
sectioning, or poor staining.
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Technical Difficulties with the Lesioning Technique
Prior to discussing the method used to assess lesion accuracy, the technical
difficulties encountered during the lesioning process will be addressed. In addition,
we faced several other technical challenges that also are addressed in this section.
1. The Infusion Process
1a. The Problem. Laboratories that have performed ibotenic acid lesions of the
lateral PBN have used micropipettes with a 40-60µm tip and a 10 minute infusion
rate. However, for our experiment, blood dried at the tip of our micropipettes, thus
clogging it. The clogging was facilitated by the slow infusion rate. As a
consequence, insufficient amounts of ibotenic acid were infused into the lateral PBN.
The Solution. In subsequent excitotoxic pilot studies, our solution to this
problem was to abandon the micropipettes for 30G cannulae with beveled tips. The
wider tip allowed more ibotenic acid to be infused, while the beveled tips
circumvented the problem of clogging the tip with blood or brain tissue. In addition,
a faster infusion rate (i.e. 0.2µl/1min) was used which averted the problem of
clogging due to dried blood without producing physical lesions at the infusion site.
The faster infusion rate did not allow sufficient time for blood to clog at the tip and if
it did, the faster rate probably helped to unclog the debris.
1b. The Problem. A second reason for our incomplete infusions was that our pump
was not exerting enough pressure to push out the ibotenic acid from the clogged
pipettes. This probably was because we had smaller PE tubing attached to the
Hamilton syringe in the pump and that was attached to larger PE tubing attached to
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the micropipette. Going from the smaller PE to larger PE was potentially drowning
the pressure that was being applied into the micropipette.
The Solution. We attached the Hamilton syringe in the pump to the cannula via a
single PE size tubing. As such, the pressure was not diluted.
2. Cresyl Violet Staining and Patchy NeuN Staining
2a. The Problem. Cresyl violet staining is typically used to assess the accuracy and
extent of excitotoxic lesions. However, lesions are often difficult to read using this
type of staining. In addition, due the complications faced during the infusion
process, we anticipated small lesions, which would make the reading of the lesions
even more difficult with cresyl violet. Finally, due to our lengthy behavior testing,
gliosis, which is used as a marker for lesions with cresyl violet staining, would have
faded and exacerbated the problem.
The Solution. We needed to adopt a new staining method and needed to perfect
the technique for optimal staining. We decided to stain for a compound that is found
exclusively in neurons. As such, we chose to stain for neuron-specific nuclear
protein (NeuN).
2b. The Problem. The lateral PBN is naturally heavily myelinated, which makes
even NeuN staining of the pontine nucleus difficult. Reading lateral amygdala
lesions with NeuN staining works flawlessly (see Figure 11). However, this was not
the case with the lateral PBN.
The Solution. Our protocol for NeuN staining had to be tailored specifically for
the lateral PBN. The sections were sliced at 36-40μm instead of the usual 50μm
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thickness. The incubation period for the primary antibody was extended (from 1
hour to 48-72 hours), the concentration was lowered (from 1:600 to 1:1000), and the
temperature at the time of incubation was decreased (from room temperature to 4 C).
Figure 11. Neuron-Specific Nuclear Protein (NeuN) Stain of an Ibotenic Acid
Lesion of Lateral Amygdala.
Figure 11 Caption. Magnified (4x) photomicrograph of a NeuN stained lateral amygdala from a rat
with a unilateral ibotenic acid lesion. The extent of the lesion is outlined with dashed lines.
3. Insufficient Neuronal Death
3a. The Problem. In follow up pilot studies in which 30G cannulae and a 1 minute
infusion rate were used to infuse ibotenic acid, the concentration of ibotenic acid was
insufficient to kill all of the neurons in the lateral PBN. NeuN staining revealed
surviving cells in the region, which made it difficult to read lesions.
The Solution. We tested 3 potential solutions to this problem: (a) we increased
the concentration of the ibotenic acid. Increasing the concentration helped kill more
cells although there still was not a complete annihilation of the neurons at the
infusion site, (b) we dissolved the ibotenic acid in a ringer solution that contained
calcium. We hoped to facilitate neuronal death by increasing the influx of calcium
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into the neuron. This did not alter the number of cells affected by the ibotenic acid,
(c) we changed the neurotoxin from ibotenic acid to NMDA (0.02µl, 10µg/µl;
dissolved in PBS). Unlike the ibotenic acid, the NMDA dissolved rapidly in PBS.
No NaOH was necessary. The animals rapidly recovered from the anesthesia, i.e. 20
minutes following the surgery compared to the 2 hour recovery when ibotenic acid
was used, and the NMDA killed all of the neurons at its point of infusion.
Predicated on these pilot studies, for our next lateral PBN excitotoxic lesion
study we will use 0.2µl/1minute NMDA (10µg/µl; solvent PBS) infused via 30G
cannulae attached to a Hamilton syringe via PE1 tubing (Scientific Commodities Inc.,
Lake Havasu, AZ).
Assessing Lesion Accuracy
Due to the malfunction of the micropipettes, clear lesions of the lateral PBN
were not obtained. Excitotoxic lesions should be easy to read with NeuN staining
and the area intended for a lesion should not contain any neurons (see Figure 11).
Although a paucity of cells could be seen in our lateral PBN sections, complete
annihilation of the neural area was not obtained (see Figure 12). As a result, cell
counts were performed for the lateral PBN to determine whether an animal was
considered lesioned.
Following NeuN staining, the lateral PBN sections of each animal were
examined under a light microscope. For each animal that received infusions of
ibotenic acid, the section that contained the maximum reduction in neurons in the
lateral PBN was chosen for analysis. The option of analyzing more sections to assess
228
the extent of the lesions was considered, but eventually discarded since the lesions
were not sufficiently extensive in the majority of the ibotenic acid animals. In 3
randomly selected ibotenic acid animals, a reduction in the number of cells
(compared to a control) was observed only on the section that contained the
maximum lesion. Differences in the number of neurons were not obtained in the
sections anterior or posterior to the section with the maximum lesion. As such, only
the section with the maximum reduction of neurons was analyzed.
After choosing the section with maximum lesion for both the left and right
sides, the AP level of each section was recorded (AP range -9.18 to -9.80) according
to the Paxinos and Watson (1998) rat atlas. Next, sections corresponding to the same
AP levels (AP range -9.18 to -9.8) were chosen from the PBS-infused animals. A
minimum of ten sections from the control animals for each of the AP levels for each
side was chosen. For instance, for animals that had their maximum lesion at AP -9.8
on the left side, 10 sections corresponding to AP level -9.8 on the left side of PBS-
infused animals were chosen for cell count analysis. The same was done for the right
side.
Once the appropriate sections were chosen, magnified (10x)
photomicrographs were taken and printed. The lateral PBN of each animal was
divided into 5 subnuclei: dorsal, ventral, central, external, and crescent. Because the
intermediate nucleus was difficult to identify in most sections, it was included in the
central subsection of the lateral PBN. The neurons in each of the subnuclei were
manually counted although only the total number of cells in the lateral PBN was used
229
for the analysis. During the cell counts, the experimenter was blind to the condition
of the animal. Once the cells were counted, the photomicrographs were identified as
either the ibotenic acid or PBS control and the data was recorded.
Given the complications faced during the infusion process and given the
subjective nature of performing cell counts, we decided to choose a conservative
method of identifying lesions. For each AP level (both left and right sides) of the
PBS-infused animals, the range, mean, 20% trimmed mean, median, and the 25%
inter-quartile range was computed in order to assess the most conservative method of
deciding whether an animal was considered lesioned. Based on these calculations,
the lower end of the computed range was the most conservative number. Hence, an
ibotenic acid animal was considered bilaterally lesioned if the cell count of that
animal was lower than the lower end of the range of the PBS animals at that AP level
for both the left and right sides.
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Figure 12. Neuron-Specific Nuclear Protein (NeuN) Stain of Ibotenic Acid
Lesions of Lateral Parabrachial Nucleus.
Figure 12 Caption. Magnified (4x) photomicrograph of a NeuN stained parabrachial nucleus of a (A)
sham lesioned PBS-infused animal , (B) unilaterally lateral PBN lesioned animal, and (C) bilaterally
lateral PBN lesioned animal.
B
C
A
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Procedures Followed During CTA Testing
Two separate CTA procedures were conducted in these animals (CTA1 and
CTA2).
CTA1: Estradiol
CTA1 consisted of 7 acquisition trials: 4 trials with a CS-US interval of 6
hours and 3 trials with 0 minute CS-US interval.
6 hour CS-US interval. 4 acquisition trials
Initially, this study was set up to assess the involvement of the lateral PBN in
estradiol CTA where the inter-stimulus interval was 6 hours. This interval was
chosen based on Experiment 4 where it was discovered that 6 hours was the
maximum CS-US interval that supported a strong estradiol CTA. Although in our
previous experiment 6 hours was sufficient to produce a CTA and this was replicated
in a second independent study, the Intact E control animals, which received an
injection of estradiol

6 hours following the 1 hour access to sucrose, did not develop
a CTA in the present experiment. Repeated acquisition trials with the 6 hour CS-US
interval were given since it is demonstrated that multiple acquisition trials strengthen
CTA (Garcia, Kimeldorf & Hunt, 1956). On each acquisition day, animals were
given access to a 10% sucrose solution and 6 hours later were injected with either
sesame oil or estradiol depending on their group assignment. Acquisition trials were
held every 48 hours.
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0 hour CS-US interval. 3 acquisition trials
Because the Intact E animals did not acquire a CTA after 4 acquisition trials
with the 6 hour inter-stimulus interval, it was decided that a 0 minute CS-US interval
would be employed. Three additional acquisition trials were given where each
animal received a 10% sucrose solution for 1 hour. Following the 1 hour CS
exposure, each animal was immediately injected with either sesame oil or estradiol.
Two days after the final acquisition trial, animals were given one extinction trial each
day for 21 days.
Measurements of Fluid Intake
Throughout the CTA1 procedure, measurements of water intake also were
recorded. Water intake from a 100ml cylinder bottle was measured every 24 hours
immediately before the lights turned off. Some researchers have shown that lesions
of the lateral PBN produce increases in general fluid intake (Edwards & Johnson,
1991), while others have reported reductions in neophobic responses to novel
solutions like sucrose (Reilly & Trifunovic, 2001). As such, if lesioned animals
drank increased amounts of sucrose on acquisition day compared to non-lesioned
animals, the fluid intake data would help to decipher between these two possibilities.
Additionally, if the lesioned animals drank more sucrose across extinction tests, the
water intake data would help to determine whether this increased drinking was due to
a disruption in general drinking behavior (i.e. polydipsia) or whether it was due to an
attenuated CTA.
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CTA2: Lithium Chloride. 1 acquisition trial
After animals were extinguished from their first CTA, the eating portion of
the experiment was conducted (discussed below). Following food consumption
testing, each animal was given a second CTA. Two studies on second CTAs
conducted in our laboratory have revealed that when animals extinguish from their
first CTA, they can acquire a second CTA of equal strength (Ycaza, Lavond, and
Chambers, unpublished data).
All of the animals in the Lesion E group acquired an estradiol CTA although
they drank significantly more sucrose than the Intact E group during extinction, a
finding that was apparent before histology was conducted. At the time, this lack of
acquisition effect was considered to be due to two possibilities: (a) our lesions were
incomplete since we discovered many problems with our glass pipettes after the
surgeries were conducted or (b) we made sufficient lesions, but unlike LiCl, an
estradiol CTA does not work through the lateral PBN, which was part of our rationale
for conducting the study. Since it consistently has been demonstrated that LiCl
CTAs are eliminated following lesions of the lateral PBN, we decided to inject the
Lesion E group with LiCl to see whether or not they could acquire a LiCl CTA. The
following two groups were used: (a) Intact animals that had received estradiol during
CTA1 and were given LiCl during CTA2 (Intact E-Li) and (b) Lesioned animals that
had received estradiol during CTA1 and were given LiCl during CTA2 (Lesion E-
Li).
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The second CTA with LiCl was conducted in the same manner as estradiol
CTA1 with a few differences. First, only one acquisition trial was given because all
of the animals acquired a CTA. Second, during acquisition, the animals were given
access to NesQuik Reduced Fat Chocolate milk for 1 hour. Pilot data from our
laboratory has shown that despite previously having developed a CTA to sucrose,
animals will readily drink the chocolate milk. Additionally, we have shown that it
can be used as an effective CS in a CTA paradigm when LiCl or estradiol is used as
the US. Third, immediately following access to the CS, each animal received a
10ml/kg injection of 0.15M LiCl. Daily extinction tests were initiated 47 hours later
and commenced until the groups had extinguished.
Measurements of Food Intake
After completion of CTA testing, the animals had to be moved to a holding
vivarium and then to a new permanent vivarium in a new building. Therefore, in
order to allow sufficient time for them to adapt to their new environment, food intake
measurements were not initiated until approximately one month after the last
extinction test for CTA1. The measurements were made every 12 hours, once
immediately before the lights turned off and once immediately before the lights
turned on, for 5 consecutive days.
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Results
CTA1: Estradiol: Assessment of CTA Formation
6 hour CS-US interval. 4 acquisition trials
None of the estradiol groups acquired a CTA. However, while none of the
estradiol groups exhibited a change in sucrose consumption across the first 4
acquisition trials, all of the oil groups showed an increase in consumption (see Figure
13). Within-group analyses revealed an increase in sucrose consumption from
acquisition 1 (A1) to post-acquisition test 4 for each of the oil groups (PA4; critical
significance=0.005 in each case, significance=0.00). For all of the estradiol groups,
sucrose consumption during A1 was not significantly different from their
consumption during PA4.
Between-groups analyses of consumption during PA4 confirmed the
difference in sucrose consumption between the estradiol and oil groups. For all but
one comparison, sucrose consumption was higher in the oil groups than the estradiol
groups during PA4 (critical significance=0.05, significance=0.28 for Bilateral E vs.
Intact Oil; critical significance=0.02-0.033, significance=0.00-0.032 for remaining 8
estradiol and oil comparisons).
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Figure 13. Sucrose and Water Intake for Animals with Excitotoxic Lesions of
the Lateral Parabrachial Nucleus Across 7 Acquisition Trials of an Estradiol
Conditioned Taste Avoidance.
0 A1 PA1/A2 PA2/A3 PA3/A4 PA4/A5 PA5/A6 PA6/A7 PA7
0
2
4
6
8
10
12
14
16
18
20
22
24
Intact Oil
Intact E
Uni Oil
Uni E
Bi Oil
Bi E
a
a
b
Acquisition & Post-Acquisition Tests
Trimmed Mean Sucrose
Intake (ml)
0 A1 PA1/A2 PA2/A3 PA3/A4 PA4/A5 PA5/A6 PA6/A7 PA7
0
10
20
30
40
50
Intact Oil
Intact E
Uni Oil
Uni E
Bi Oil
Bi E
c
Acquisition & Post-Acquisition Tests
Trimmed Mean Water
Intake (ml)
Figure 13 Caption. The amount of sucrose (upper graph) or water (lower graph) drunk by animals
with no (Intact), unilateral (Uni), or bilateral (Bi) lesions of the lateral parabrachial nucleus across the
7 acquisition trials of CTA1 that received either estradiol (E) or sesame oil (Oil). The first 4
acquisition trials (A1 to A4) consisted of a 6 hour CS-US interval, while the final 3 acquisition trials
(A5 to A7) had a 0 hour CS-US interval. The post-acquisition trials for each acquisition training day
(PA1 to PA7) were held 48 hours following the acquisition trial.
a
Signficant decrease in sucrose
following the 0 hour pairing (PA4/A5-PA5/A6) for Intact E and Uni E group and across PA5/A6-
PA6/A7 for the Bi E group.
b
Signficantly less water than each of the Oil groups on PA7.
0 Hour CS-US interval. 3 acquisition trials
None of the Oil groups showed a decrease in sucrose consumption across the
final 3 acquisition trials (see Figure 13). According to within-group analyses, no
237
statistically significant changes in sucrose consumption were detected across the 3
acquisition trials for any of the Oil groups. Between-group analyses revealed that the
three Oil groups did not differ from one another in the amount of sucrose consumed
during any of the acquisition trials.
Each of the estradiol groups developed a CTA but they differed in the number
of trials required to do so. The Intact E and Unilateral E groups immediately
developed a CTA following the first acquisition trial. Both groups showed decreases
in sucrose consumption across acquisition 5 (PA4/A5) and post-acquisition test 5
(PA5/A6; critical significance=0.017, significance=0.001 for Intact E; critical
significance=0.014, significance=0.00 for Unilateral E). On the contrary, the
Bilateral E group required two acquisition trials before developing a CTA. This
group did not show changes in sucrose consumption across A5 and PA5/A6 but
showed a significant decrease in sucrose consumption across acquisition 6 (A6) and
extinction 6 (PA6/A7; critical significance=0.014, significance=0.00) and across
acquisition 7 (A7) and post-acquisition test 7 (PA7; critical significance=0.017,
significance=0.015).
Between-group analyses corroborated within-group analysis, namely that
unlike the Intact E and Unilateral E groups, the Bilateral E group did not acquire a
CTA after the first paired acquisition trial, but did develop a CTA following the
second sucrose-estradiol pairing. None of the estradiol groups differed in the amount
of sucrose they drank during acquisition of the first immediate pairing (PA4/A5).
However, the Bilateral E group drank significantly more sucrose during post-
238
acquisition test 5 (PA5/A6) compared to the Intact E and Unilateral E groups (critical
significance=0.01 in each case, significance=0.01-0.011). The estradiol groups
however did not differ in their sucrose consumption levels during tests PA6/A7 or
PA7. No significant interaction effects were detected between the estradiol groups
across PA4/A5 through PA7. In addition, the Bilateral Oil group did not differ from
the Intact Oil group in the amount of sucrose consumed across any of the acquisition
trials. This suggests that the greater sucrose consumption of the Bilateral E group
was not due to the effects of the lesions on sucrose consumption per se.
Although the Bilateral E group did not acquire a CTA on the first 0 hour CS-
US interval acquisition trial, similar to the Intact E and Unilateral E groups, it did
significantly differ from each of the oil groups in the amount of sucrose it drank on
days PA4/A5 and PA5/A6. The Intact E, Unilateral E, and Bilateral E groups drank
significantly less sucrose on these days compared to the each of the Oil groups
(critical significance=0.02-0.025, significance= 0.00-0.012 for PA4/A5; critical
significance= 0.01-0.038, significance=0.00-0.021 for PA5/A6).
Relationship between Sucrose and Water Intake
For most comparisons, changes in sucrose consumption were not
accompanied by concomitant changes in water intake. However, whenever a
relationship between sucrose and water consumption was found, it was an inverse
relationship.
239
6 hour CS-US interval
None of the significant changes in sucrose consumption observed in the oil
groups across A1 and PA4/A5 were accompanied by changes in water intake (see
Figure 13). Similarly, on PA4 when each of the oil groups drank more sucrose than
each of the estradiol groups (except for the Bilateral E vs. Intact Oil comparison),
they each showed comparable amounts of water intake compared to the estradiol
groups.
0 Hour CS-US interval
The significant decreases in sucrose consumption across the first paired
acquisition trial (PA4/A5 and PA5/A6) for the Intact E and Unilateral E groups were
not accompanied by significant changes in water intake. The Bilateral E group did
not show changes in sucrose consumption across PA4/A5 and PA5/A6, but did show
significant reductions in sucrose across PA5/A6 and PA6/A7 and PA6/A7 and PA7.
Water intake for this group did not differ across PA4/A5 and PA5/A6 and PA5/A6
and PA6/A7, but the Bilateral E group showed significant increase in water intake
only across PA6/A7 and PA7 (critical significance=0.014, significance=0.00).
In addition to not acquiring a CTA following the first 0 hour CS-US interval
acquisition trial, the Bilateral E group drank more sucrose than the Intact E and
Unilateral E groups during PA5/A6. However, the Bilateral E group drank similar
amounts of water as the two other estradiol groups on PA5/A6. Similarly, no
relationship between sucrose and water intake were found when the Bilateral E group
was compared to each of the oil groups. The Bilateral E group drank less sucrose
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than each of the oil groups on PA4/A5 and PA5/A6, but showed no difference in
water intake compared to the control groups.
During the test that followed the final acquisition trial (PA7), an inverse
relationship was found in the amount of sucrose and water consumed by the estradiol
and oil groups. The estradiol groups drank less sucrose but more water than the oil
groups (see Figure 13). Between-group analyses showed that on PA7, each of the
estradiol groups drank significantly less sucrose than each of the Oil groups (critical
significance=0.012, significance=0.00 in each case), but drank significantly more
water than the Oil groups (critical significance=0.012 in each case,
significance=0.00-0.01). No statistical differences in sucrose or water consumption
were detected among the Oil groups or among any of the estradiol groups on PA7.
CTA1: Assessment of CTA Extinction
Lesions of the lateral PBN alone had no effect on sucrose consumption.
Between-group analyses revealed that the Bilateral Oil group did not differ from the
Intact Oil in the amount of sucrose consumed during each of the 4 extinction phases
(see Figure 14). In addition, no difference between these two groups was detected in
the amount of sucrose consumed across the 4 extinction phases, that is, there was no
significant interaction.
Each of the oil groups drank significantly more sucrose than each of the
estradiol groups during each of the 4 extinction phases (critical significance=0.025-
0.05 for Phases 1-4, significance=0.0 for Phase 1; significance=0.00-0.004 for
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Phase 2; significance=0.0-0.008 for Phase 3; significance=0.0-0.03 for Phase 4, see
Figure 14).
Although the estradiol groups did not differ from each other in the amount of
sucrose they drank during Phases 1 and 2, the Bilateral E group drank significantly
more sucrose than the Intact E and Unilateral E groups during extinction Phases 3
and 4, (critical significance=0.025-0.05, significance=0.0-0.02). However, no
significant differences in extent of the increase across phases 3 and 4 were detected
among the estradiol groups. In addition, no significant interactions were detected
across extinction phases 1 and 2 either for any of the estradiol groups.
The higher levels of sucrose consumption displayed by the Bilateral E group
during Phases 3 and 4 were not due to a general increase in fluid consumption. No
statistical differences were detected in the amount of water consumed between the
Bilateral E group and the Intact E or Unilateral E groups during Phases 3 and 4 (see
Figure 14).
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Figure 14. Sucrose and Water Intake for Animals with Excitotoxic Lesions of
the Lateral Parabrachial Nucleus Across Extinction Phases of an Estradiol
Conditioned Taste Avoidance.
0 Phase1 Phase2 Phase3 Phase4
0
5
10
15
20
25
30
35
40
Intact Oil
Intact E
Uni Oil
Uni E
Bi Oil
Bi E
a
a
Extinction Phases
Average Trimmed Mean
Sucrose Intake (ml)
0 Phase1 Phase2 Phase3 Phase4
0
5
10
15
20
25
30
35
40
45
Intact Oil
Intact E
Uni Oil
Uni E
Bi Oil
Bi E
Extinction Phases
Average Trimmed Mean
Water Intake (ml)
Figure 14 Caption. Amount of sucrose (upper graph) or water (lower graph) drunk by animals with no
(Intact), unilateral (Uni), or bilateral (Bi) lesions of the lateral parabrachial nucleus across extinction
phases of CTA1 that received either estradiol (E) or sesame oil (Oil). Each extinction phase consisted
of 5 extinction days. Data on graph represents the trimmed mean of the sum for each phase.
a
Significantly more sucrose consumed compared to the two other estradiol groups on extinction phases
3 and 4.
Relationship between Sucrose and Water Intake
As was true for the acquisition data, there was an inverse relationship between
the amount of water and sucrose consumed by the oil and estradiol groups during
243
extinction. During Phases 1 and 2, when each of the Oil groups drank significantly
more sucrose than each of the estradiol groups, they also drank significantly less
water than each of the estradiol groups (critical significance=0.025-0.05 for Phases 1
and 2, significance=0.00-0.16 for Phase 1, significance=0.0-0.026 for Phase 2; see
Figure 14).
Similarly, although the Bilateral E group showed increased sucrose
consumption during Phases 3 and 4 compared to the Intact E and Unilateral E groups,
no statistical differences were detected in the amount of water consumed between
these groups during these phases of extinction.
CTA2: Lithium Chloride (LiCl): Assessment of CTA Formation
Each of the groups developed a CTA following a single pairing of the CS
with LiCl (see Figure 15). Within-group analyses showed that the Intact E-Li,
Unilateral E-Li, and the Bilateral E-Li groups each showed a significant drop in
chocolate milk consumption across acquisition and extinction day 1 (E1;
significance=0.0, critical significance=0.05 in each case).
Each of the groups reduced their drinking of the CS to the same level on E1
(see Figure 15). Between-group analyses showed that the Unilateral E-Li and
Bilateral E-Li groups drank significantly more chocolate milk on acquisition day
compared to the Intact E-Li group (critical significance=0.05 in each case,
significance=0.26-0.049). However, despite this difference in acquisition day
consumption, the three groups did not differ in the amount of CS consumed on E1.
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In addition, no significant interaction was detected among the groups across
acquisition day and E1.
CTA2: Lithium Chloride: Assessment of Extinction
The Unilateral E-Li and Bilateral E-Li groups drank significantly more
chocolate milk than the Intact E-Li group on extinction days 2-4 (E2-E4; critical
significance=0.033-0.05 for E2, significance=0.03-0.49; critical significance=0.033-
0.05 for E3, significance=0.03-0.04; critical significance=0.025 in each case for E4,
significance=0.02-0.025; see Figure 15). No significant differences in the rate of
increase in consumption were detected.
Figure 15. Chocolate Milk Intake in Animals with Excitotoxic Lesions of the
Lateral Parabrachial Nucleus During Acquisition and Extinction of a Lithium
Chloride Conditioned Taste Aversion.
0 Acq E1 E2 E3 E4
0
2
4
6
8
10
12
14
16
18
20
Intact E-Li Uni E-Li Bi E-Li
a
a
a
Acquisition Trial & Extinction Tests
Trimmed Mean
Chocolate Milk Intake
(ml)
b
b
b
b
b
b
Figure 15 Caption. Amount of chocolate milk consumed by animals with no (Intact), unilateral (Uni),
or bilateral (Bi) lesions of the lateral parabrachial nucleus across acquisition (Acq) and each extinction
test (E1-E4) of CTA2 that received lithium chloride (LiCl).
a
Significantly less sucrose from Acq.
b
Significantly more sucrose than the Intact E-Li group.
245
Comparison of Lesion Size between Lesioned Groups (Bilateral E versus Bilateral
Oil)
Comparisons of lesion size were made between the Bilateral E and Bilateral
Oil-E groups. The lesion size of each animal was determined by the number of cells
each animal had below the lower limit set for its corresponding AP level.
Comparisons between the two groups were made for the right and left sides.
Analysis revealed that the two groups did not differ in amount of damage the animals
sustained in the left or side of the brain (see Figure 16).
Figure 16. Size of Lateral Parabrachial Nucleus Lesion in Bilateral E and
Bilateral Oil Groups on Right and Left Sides of the Brain.
-100 -80 -60 -40 -20 0
Left
Right
Number of Cells Below Lower Range
Bilateral E
Bilateral Oil-E
Figure 16 Caption. Represents the average number of cells below the lower limit set. No significant
differences were detected in lesion size between the two groups.
Assessment of Food Decrement: 24 hour
Within-group analysis of the 24 hour food measurement revealed that
compared to baseline (Base) each of the groups, including the Oil groups, decreased
their food consumption 24 (24E
2
1) and 48 (48E
2
1) hours following the first injection
246
(48E
2
1 was also 24 hours after the second injection, 24E
2
2; critical
significance=0.017-0.02 for 24 hours, significance=0-0.004; critical
significance=0.014-0.02 for 48 hours, significance=0.00 in each case; see Figure 17).
However, 72 hours following the first estradiol injection (72E
2
1/48E
2
2), the Oil
groups returned to baseline levels of eating, while food consumption for each of the
estradiol groups remained significantly lower than baseline (critical
significance=0.012-0.017 for 72 hours, significance=0.00 in each case).
Between-group analysis showed that none of the groups differed from one
another in the amount of food consumed during baseline. However, although 48
hours following the first estradiol injection (48E
2
1/24E
2
1), each of the oil groups
showed reductions in eating compared to baseline, they still consumed more food
compared to each of the estradiol groups during that time period (critical
significance=0.02-0.025; significance=0.00-0.01). That is, the estradiol groups
showed greater reductions in eating at this time period compared to each of the Oil
groups. None of the Oil groups differed from one another in their food consumption
levels during this time period. Similarly, the estradiol groups did not differ in their
food consumption level during this 48E
2
1/24E
2
1 time period.
Additional between-group analysis showed that each of the Oil groups
consumed more food than each of the estradiol groups 72 hours following the first
estradiol injection (72E
2
1/48E
2
2; critical significance=0.017-0.02, significance=0.0
in each case). Each of the estradiol groups sustained their reduced consumption until
247
96 hours after the first injection (96E
2
1/72E
2
2; critical significance=0.011-0.03,
significance=0.00-0.013).
Figure 17. Amount of Food Consumed During Twenty Four Hour Periods that
Followed Estradiol Injections in Animals with Excitotoxic Lesions of the Lateral
Parabrachial Nucleus.

Base/ 24 48 72 96 120
10
15
20
25
Intact Oil
Intact E
Uni Oil
Uni E
Bi Oil
Bi E
DAYS
E
2
1 E
2
1 E
2
1 E
2
1 E
2
1 E
2
1
24E
2
2 48E
2
2 72E
2
2 96E
2
2
E
2
2
a
a
a
ab
ab
ab
a
a
a
a
a
a
Trimmed Mean 24 hr
Food Consumption (g)
Figure 17 Caption. Twenty-four hour eating data for animals with no (Intact), unilateral (Uni), or
bilateral (Bi) lesions of the lateral parabrachial nucleus that received either a 50µg/kg dose of estradiol
(E
2
) or sesame oil (Oil). Baseline (Base) consisted of the average of the two 24 hour periods before
the first estradiol injection (E
2
1). A second estradiol injection (E
2
2) in the same dose was
administered 24 hours after E
2
1. Comparisons in 24 hour food consumption were made between Base
and 24 (24 E
2
1), 48 (48 E
2
1/24 E
2
2), 72 (72 E
2
1/48 E
2
2), and 96 (96 E
2
1/72 E
2
2) hour post E
2
1.
a
Signficantly different compared to baseline.
b
Significantly different from control Oil groups.
Assessment of Food Decrement: 12 hour Dark Phase
Within-group analysis of the 12 hour eating period for the dark phase revealed
that when compared to baseline, each of the groups, including the Oil groups,
decreased their food intake during the dark phase of the 48E
2
1/24E
2
2 hour period
(critical significance=0.011-0.014, significance=0.00 in each case; see Figure 18).
However, during the dark phase of the 72E
2
1/48E
2
2 hour period, the Oil groups
either returned to baseline or surpassed their baseline levels of consumption, while
each of the estradiol groups maintained a reduced consumption level. Statistical
248
analyses revealed that the Unilateral-Oil group had returned to baseline, while the
Intact Oil and Bilateral Oil groups showed increased consumption levels (critical
significance=0.013-0.017, significance=0.00-0.012) while each of the estradiol
groups sustained their suppressed consumption (critical significance=0.011-0.14,
significance=0.00-0.004).
Between-group analysis showed that none of the groups differed in the
amount of food they consumed during the baseline period. At the 48E
2
1/24E
2
1 time
period, only the Intact E and Unilateral E groups showed lower food intake than at
least one of the Oil groups (significant difference between Intact E and Intact Oil,
critical significance=0.02, significance=0.00 and significant difference between
Unilateral Oil and each of the Oil groups, critical significance=0.017-0.02,
significance=0.00-0.016).
Additional between-group analysis showed that each of the Oil groups
consumed more food than each of the estradiol groups 72E
2
1/48E
2
2 (critical
significance=0.017-0.02, significance=0.0-0.014). The estradiol groups maintained
the reduced intake until 96E
2
1/72E
2
2 (critical significance=0.011-0.014,
significance=0.00-0.004).
249
Figure 18. Amount of Food Consumed During Twelve Hour Dark Phase
Periods that Followed Estradiol Injections in Animals with Excitotoxic Lesions
of the Lateral Parabrachial Nucleus.
0 Base/ 24 48 72 96 120
10
15
20
Intact Oil
Intact E
Uni Oil
Uni E
Bi Oil
Bi E
E
2
1 E
2
1 E
2
1 E
2
1 E
2
1 E
2
1
24E
2
2 48E
2
2 72E
2
2 96E
2
2
Days
E
2
2
a
a
a
a
ab
ab
ab
ab
ab
Trimmed Mean 12hr
Food Consumption (g)
Dark Phase
Figure 18 Caption. Twelve hour dark phase eating data for animals with no (Intact), unilateral (Uni),
or bilateral (Bi) lesions of the lateral parabrachial nucleus that received either a 50µg/kg dose of
estradiol (E
2
) or sesame oil (Oil). Baseline (Base) consisted of the average of the two 24 hour periods
before the first estradiol injection (E
2
1). A second estradiol injection (E
2
2) in the same dose was
administered 24 hours after E
2
1. Comparisons in 12 hour food consumption during the dark phase
were made between Base and 24 (24 E
2
1/ E
2
2), 48 (48 E
2
1/24 E
2
2), 72 (72 E
2
1/48 E
2
2), and 96 (96
E
2
1/72 E
2
2) hour post E
2
1.
a
Signficantly different compared to baseline.
b
Significantly different from
control Oil groups.
Assessment of Food Decrement: 12 hour Light Phase
Analysis of the 12 hour eating period for the light phase revealed that
compared to baseline each of the groups, including the Oil groups, decreased their
food intake during the light phase of the 24 hour period following the first injection
(critical significance=0.014-0.03, significance=0.00-0.012; see Figure 19). For the
Bilateral Oil group, this decrease lasted until the 48E
2
1/24E
2
2 hour period (critical
significance=0.02, significance=0.00). However, the Intact Oil and Unilateral Oil
groups had returned to their baseline levels of consumption by the 48E
2
1/24E
2
2 hour
period, while the Bilateral Oil group returned to baseline by the 72E
2
1/48E
2
2 hour
250
period. On the other hand, the Intact E group maintained a reduced consumption level
until the 72E
2
1/48E
2
2 hour period while the Unilateral E and Bilateral E groups
showed reduced consumption until the 96E
2
1/72E
2
2 hour period (critical
significance=0.012-0.017, significance=0.00 in each case).
Between-group analysis showed that none of the groups differed in the
amount of food they consumed during the baseline period. During the 48E
2
1/24E
2
1
period, each of the estradiol groups consumed less food than the Intact Oil and the
Unilateral Oil groups (critical significance=0.02-0.025, significance=0.00-0.008). No
significant differences were detected between the estradiol groups and the Bilateral
Oil group during the 48E
2
1/24E
2
1 period.
Additional between-group analysis showed that each of the Oil groups
consumed more food than each of the estradiol groups in the light phase of the 72
hour period following the first injection (critical significance=0.017-0.025,
significance=0.00-0.02).
251
Figure 19. Amount of Food Consumed During Twelve Hour Light Phase
Periods that Followed Estradiol Injections in Animals with Excitotoxic Lesions
of the Lateral Parabrachial Nucleus.
0 Base/ 24 48 72 96 120
0
2
4
Intact Oil
Intact E
Uni Oil
Uni E
Bil Oil
Bi E
E
2
1 E
2
1 E
2
1 E
2
1 E
2
1 E
2
1
24E
2
2 48E
2
2 72E
2
2 96E
2
2 E
2
2
ab
ab
ab
ab
ab
ab
ab
Days
Trimmed Mean 12hr
Food Consumption (g)
Light Phase
Figure 19 Caption. Twelve hour light phase eating data for animals with no (Intact), unilateral (Uni),
or bilateral (Bi) lesions of the lateral parabrachial nucleus that received either 50µg/kg dose of
estradiol (E
2
) or sesame oil (Oil). Baseline (Base) consisted of the average of the two 24 hour periods
before the first estradiol injection (E
2
1). A second estradiol injection (E
2
2) in the same dose was
administered 24 hours after E
2
1. Comparisons in 12 hour food consumption during the light phase
were made between Base and 24 (24 E
2
1/ E
2
2), 48 (48 E
2
1/24 E
2
2), 72 (72 E
2
1/48 E
2
2), and 96 (96
E
2
1/72 E
2
2) hour post E
2
1.
a
Signficantly different compared to baseline.
b
Significantly different from
control Oil groups.
Discussion
Due to the complications encountered during the experimentation process for
Experiment 5a, solid conclusions could not be made based solely on the findings of
this study. A more complete and clear picture develops when the results of
Experiment 5a are combined with the results of Experiment 5b. As such, the findings
of these two experiments are discussed in combination following the results of
Experiment 5b.
252
4.5.6 Experiment 5B
Since the lesioning technique in Experiment 5a was fraught with problems,
subsequent pilot studies were conducted in order to correct for some of the
complications. One of these pilot studies was conducted in order to determine
whether electrolytic lesions would affect LiCl CTA, estradiol CTA, and estradiol
anorexia. With the results of Experiment 5a, it was difficult to conclusively state
whether the very confined bilateral lesions of the lateral PBN affected acquisition of
the CTAs although they did accelerate extinction of both an estradiol and LiCl CTAs.
Therefore, one of the purposes behind Experiment 5b was to determine whether more
extensive lesions of the lateral PBN would preclude CTA acquisition when estradiol
and LiCl were used as the USs. Further, in Experiment 5a, weak lesions did not
appear to affect estradiol anorexia. As such, the secondary purpose of Experiment 5b
was to determine whether the inability of lesions to affect estradiol hypophagia was
due to insufficient lesions. If so, then the more extensive lesions should inhibit
estradiol induced decrements in eating.
Methods
Groups
Eight ovariectomized rats were assigned to one of the two following groups:
(1) Bilateral E (n=6), which received bilateral electrolytic lesions or
(2) Intact E (n=2), which received sham lesions
253
Following histology, the animals were divided into three of the following groups:
(1) Bilateral E (n=3), which had accurate, extensive bilateral lesions of the lateral
PBN
(2) Unilateral E (n=3), which had accurate and extensive unilateral lesions of the
lateral PBN
(3) Intact E (n=2), which received sham lesions
Assessing Lesion Accuracy
Following NeuN staining, each of the sections was mounted on gelatin coated
slides and cover slipped. Each section was then examined under a light microscope
for physical damage in the lateral PBN (see Figure 20). Sections represented contain
the maximum damage that was observed on any of the sections.
Conditioned Taste Aversive Procedure
Two separate CTA behavior tests were conducted in these animals. The
second CTA was conducted after each animal had extinguished from the first CTA (if
acquired).
CTA1: Lithium Chloride. 1 acquisition trial
Since it consistently has been demonstrated that lesions of the lateral PBN
block LiCl CTA and since one of the purposes behind conducting Experiment 5a was
to insure accuracy of placement, the decision was made to administer LiCl first. The
first CTA was a LiCl CTA that consisted of a single acquisition trial. On acquisition
day, each of the animals was given 1 hour access to a 10% sucrose solution.
254
Immediately following the CS exposure, each animal was administered an
intraperitoneal injection of LiCl (10ml/kg of body weight).
CTA2: Estradiol. 3 acquisition trials
The second CTA was an estradiol CTA and consisted of three acquisition
trials in an effort to keep things as consistent as possible between Experiment 5a and
5b. After animals were extinguished from their first CTA, preconditioning for the
second CTA began. One month separated the final extinction trial of CTA1 and the
first acquisition trial of CTA2.
Conditioned taste avoidance 2 was conducted in the same manner as CTA1
with some minor differences: (a) three acquisition trials were given as opposed to one
and (b) during acquisition the animals were given access to NesQuik Reduced Fat
Chocolate milk in place of 10% sucrose for 1 hour. Immediately following access to
the CS, each animal received a 50µg/kg injection of estradiol. Acquisition trials were
held every 48 hours. Extinction tests were initiated 48 hours following the final
acquisition trial and continued until the groups had extinguished, if a CTA was
acquired.
Measurements of Food Intake
Two weeks following the final extinction test of CTA2, baseline
measurements of food intake were initiated. Measurements were made every 24
hours immediately before the lights turned off. Five days of baseline intake were
taken before the first estradiol injection. Two estradiol injections separated by 24
hours were made. Measurements were taken until animals reached baseline levels of
255
intake. The intake of the 5 baseline days was averaged and used as the baseline level
of food consumption.
Statistical Analyses
Due to the low number of animals in each group (n=3 for bilaterally and
unilaterally lesioned and n=2 for sham lesioned), formal statistical analyses could not
be performed on the data. Despite the lack of analyses, the graphs and the
percentages of the animals in each group that acquired a CTA reveal the clear deficits
produced by electrolytic lesions. In addition, the criteria set for CTA in Experiment 3
were used to assess whether the animals acquired a CTA.
Explanation of Procedures
Prior to discussing the results, there is an important issue that should be
addressed regarding Experiment 5b. First, we tested an estradiol and LiCl CTA with
a 0 hour CS-US interval. We made the decision not test the effect of a 6 hour CS-US
interval on estradiol CTA in these animals. Experiment 5b was conducted as a pilot
study to insure that making more extensive lesions of the lateral PBN with the same
coordinates as those used in Experiment 5a would block LiCl CTA. This would
show that the coordinates we used in Experiment 5a were accurate. When the LiCl
CTA was blocked, the decision was made to test for an estradiol CTA. Since the 6
hour CS-US interval did not produce a CTA in Experiment 5a, the decision was made
to test for an estradiol CTA with no interval. If we had used the 6 hour CS-US
interval and the animals did not acquire a CTA, it would have been unclear whether it
was due to the lesions or whether that particular batch of animals was not going to
256
acquire a CTA with this longer CS-US interval. The reason for this is because the
control group only included 2 animals, thus making it difficult to decipher between
these two possibilities. Although we could have used a 6 hour CS-US interval in a
third CTA, we have no information on the ability of animals to acquire a third CTA,
so interpretation of the results would have been problematic.
Results
About the Electrolytic Lesions
In addition to sustaining complete bilateral lesions of the lateral PBN, each of
the lesioned animals sustained some collateral damage. These included unilateral
destruction of the superior cerebellar peduncle (see Figure 20, C, D, & E), unilateral
and potentially bilateral damage to the ventral portion of the medial PBN (see Figure
20, E), and potentially unilateral damage of the medial external PBN (see Figure 20,
D). The lesions did not extend anteriorily past the lateral PBN.
257
Figure 20. Neuron Specific Nuclear Protein (NeuN) Stain of Electrolytic Lesions
of the Lateral Parabrachial Nucleus.
A.
B.
C.
D.
Lateral
PBN
Superior Cerebellar
Peduncle
Medial Ext
PBN
Medial
PBN
258
Figure 20 Continued. Neuron Specific Nuclear Protein (NeuN) Stain of
Electrolytic Lesions of the Lateral Parabrachial Nucleus.
Figure 20 Caption. Magnified (4x) photomicrographs of NeuN stained brains of animals with a sham
lesion (A), an electrolytic unilateral lesion (B), or electrolytic bilateral lesions (C, D, & E) of the
lateral parabrachial nucleus. Dashed lines were used to outline the lesioned structure and solid lines
were used to outline the intact structure. Bilaterally lesioned animals also sustained damage to the
superior cerebellar peduncle on one (C & D) or both (E) sides, potential unilateral lesion to the medial
external PBN (D), and damage to the ventral portion of the medial PBN (E). Sections represented
contain the maximum lesion that was observed on any of the sections.
CTA1: Lithium Chloride
Assessment of CTA Formation
As depicted in the graph, animals that sustained extensive electrolytic lesions
of the lateral PBN did not show reductions in sucrose consumption across acquisition
(Acq) and post-acquisition test (PA) and did not acquire a CTA (see Figure 21). On
the contrary, the Intact Li and Unilateral Li groups both show reductions in sucrose
consumption across the two days.
According to our criteria set for CTA in Experiment 3, none (0/3) of the
animals in the Bilateral Li group acquired a CTA. On the other hand, 100% of the
animals in the Unilateral Li group (3/3) and the Intact Li group (2/2) developed a
CTA. It should be mentioned that there was overlap between the Bilateral Li group
E.
259
and the other two groups in the amount of sucrose consumed during the acquisition
trial but no overlap during the post-acquisition test.
Figure 21. Sucrose Intake in Animals with Electrolytic Lesions of the Lateral
Parabrachial Nucleus During Acquisition and the First Post-Acquisition Test of
a of a Lithium Chloride Conditioned Taste Aversion.
0 Acq Post-Acq
0
5
10
15
20
25
30
Intact Li Uni Li Bi Li
Acquisition Trial & Post-Acquisition Test
Mean Sucrose Intake
(ml)
Figure 21 Caption. Mean sucrose consumption across acquisition (Acq) and post-acquisition (Post-
Acq) for animals with either a sham (Intact) lesion or a unilateral (Uni) or bilateral (Bi) electrolytic
lesion of the lateral parabrachial nucleus. These animals received a 10ml/kg dose of lithium chloride
(Li) as the US.
CTA1: Lithium Chloride
Assessment of Extinction
All of the Bilateral Li animals continued to drink more sucrose than the Intact
Li and Unilateral Li animals during each of the extinction trials (see Figure 22). In
addition, the data suggest that the Unilateral Li group drank more sucrose in the later
days of extinction than the Intact Li group. In the Unilateral Li group, all (3/3) of the
animals had reached acquisition level consumption by extinction day 6 (E6). On the
contrary, neither (0%) of the Intact Li animals had reached acquisition consumption
260
levels by E6. One of the 2 in this group extinguished on extinction test 9 (E9), while
the other continued to show decreased sucrose consumption on E9.
Figure 22. Sucrose Intake in Animals with Electrolytic Lesions of the Lateral
Parabrachial Nucleus During Extinction of a Lithium Chloride Conditioned
Taste Aversion.
0 E2 E3 E4 E5 E6 E7 E8 E9
0
5
10
15
20
25
30
35
40
Intact Li Uni Li Bi Li
Extinction Tests
Mean Sucrose Intake
(ml)
Figure 22 Caption. Mean sucrose consumption across extinction tests for animals with either a sham
(Intact) lesion or a unilateral (Uni) or bilateral (Bi) electrolytic lesion of the lateral parabrachial
nucleus. These animals received 10ml/kg dose of lithium chloride (Li) as the US.
CTA2: Estradiol
Assessment of CTA Formation
Animals that did not acquire a LiCl CTA in CTA1 also did not develop an
estradiol CTA even after 3 pairings (see Figure 23). In the Bilateral E group, none
(0/3) of the animals developed a CTA after the 3 pairings. Interestingly, 66% (2/3) of
the animals in the Unilateral E group acquired a CTA after the first pairing, and
100% (3/3) of the animals acquired a CTA after the second pairing (similar to the
261
findings of the Bilateral E group of Experiment 5a). In the Intact E group, all (2/2) of
the animals acquired a CTA after first pairing.
Unlike the LiCl CTA1, all of the animals in the Bilateral E group drank more
sucrose during both the acquisition trial and the post-acquisition test than all of the
animals in the Intact E and Unilateral E groups.
Figure 23. Chocolate Milk Intake in Animals with Electrolytic Lesions of the
Lateral Parabrachial Nucleus During Acquisition and Post-Acquisition Tests of
an Estradiol Conditioned Taste Avoidance.
0 A1 PA1/A2 PA2/A3 PA3
0
5
10
15
20
25
30
35
40
45
Intact E Uni E BiE
Acquisition & Post-Acquisition Tests
Mean Chocolate Milk
Intake (ml)
Figure 23 Caption. Mean chocolate milk consumption across acquisition (Acq) and post-acquisition
(Post-Acq) tests for animals with either a sham (Intact) lesion or a unilateral (Uni) or bilateral (Bi)
electrolytic lesion of the lateral parabrachial nucleus. These animals received 50µg/kg dose of
estradiol (E) as the US.
CTA2: Estradiol
Assessment of Extinction
All of the animals in the Bilateral E group continued to drink more sucrose
than all of the animals in the Intact E and Unilateral E groups throughout extinction
testing (see Figure 24). Similar to the LiCl CTA extinction, the Unilateral E group
drank more sucrose in the later stages of extinction compared to the Intact E group.
262
Each of the animals in the Unilateral E group drank more sucrose on extinction days
11 and 12 compared to each of the Intact E animals.
Figure 24. Chocolate Milk Intake in Animals with Electrolytic Lesions of the
Lateral Parabrachial Nucleus During Extinction of an Estradiol Conditioned
Taste Avoidance.
Figure 24 Caption. Mean chocolate milk consumption across extinction days (E2-E12) for animals
with either a sham (Intact) lesion or a unilateral (Uni) or bilateral (Bi) electrolytic lesion of the lateral
parabrachial nucleus. These animals received 50µg/kg dose of estradiol (E) as the US.
Assessment of Estradiol Hypophagia
All of the groups reduced their food consumption after estradiol injection (see
Figure 25). For the Intact E, Unilateral E, and Bilateral E groups, 100% of the
animals had reduced their food intake 48 (48E1/24E1) and 72 (72E1/48E2) hours
following the first estradiol injection.
0 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12
0
5
10
15
20
25
30
35
40
45
Intact E Uni E Bi E
Extinction Tests
Mean Chocolate Milk
Intake (ml)
263
Figure 25. Amount of Food Consumed During Twenty-Four Hour Periods that
Followed Estradiol Injections in Animals with Electrolytic Lesions of the Lateral
Parabrachial Nucleus.
0 Base/ 24 48 72 96 120
12.5
15.0
17.5
20.0
22.5
25.0
Intact E
Uni E Bi E
E
2
1 E
2
1
E
2
2
E
2
1 E
2
1 E
2
1 E
2
1
24E
2
2 48E
2
2 72E
2
2 96E
2
2
Days
Mean Food
Consumption (g)
Figure 25 Caption. Twenty four hour eating data for animals with no (Intact), unilateral (Uni), or
bilateral (Bi) lesions of the lateral parabrachial nucleus that received a 50µg/kg dose of estradiol (E
2
).
Baseline (Base) consisted of the average of the five 24 hour periods before the first estradiol injection
(E
2
1). A second estradiol injection (E
2
2) in the same dose was administered 24 hours after E
2
1.
Discussion
The results of the behavioral tests for Experiment 5b are discussed in
conjunction with those of Experiment 5a in the following section. However, the
electrolytic lesions themselves need to be addressed prior to discussing the
behavioral results. As aforementioned, in addition to sustaining complete bilateral
lesions of the lateral PBN, each of the lesioned animals sustained some collateral
damage. These included unilateral destruction of the superior cerebellar peduncle,
unilateral damage to the ventral portion of the medial PBN, and potentially unilateral
damage of the medial external PBN. The effect of unilateral lesions of structures on
our results is not problematic. A previous study has demonstrated that unilateral
lesions of even the entire PBN (the lateral and medial subdivisions combined, along
264
with damage to the superior cerebellar peduncle) did not affect acquisition of CTA
(Gallo & Bures, 1991). As such, these extraneous unilateral damages probably were
not responsible for the blocked acquisition of CTA (discussed below in results
section) we obtained.
The collateral damage that may be more worrisome is the potential bilateral
damage caused to the superior cerebellar peduncle. It is difficult to speculate on the
effect of such lesions on CTA acquisition. However, all 3 bilaterally lesioned
animals behaved identically. That is, none of them acquired a CTA after a single
pairing of LiCl with sucrose or after three pairings of estradiol with chocolate milk.
If the bilateral lesions of the superior cerebellar peduncle were responsible for the
abolition of CTA in one of the animals, then the two other animals that sustained
only unilateral damage to this structure should have exhibited CTA. Therefore, the
potential bilateral lesion of this structure does not change the conclusions of the
study, namely that complete bilateral lesions of the lateral PBN block CTA whether
LiCl or estradiol is used as the US.
4.5.7 Experiment 5 Discussion
CTA1: Estradiol: 6 Hour CS-US Interval
One of the reasons for conducting Experiment 5a was to determine whether
inactivation of the lateral PBN would block an estradiol CTA if an extended CS-US
interval was utilized. In the series of studies conducted in Experiment 4, it was
determined that 6 hours was the longest inter-stimulus interval that supported a
strong and reliable estradiol CTA. As such, Experiment 5a was conducted with the
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intention of determining whether the lateral PBN was critical for an estradiol CTA
when a CS-US interval of 6 hours was used. However, in Experiment 5a the first
injection of estradiol did not produce a CTA in the Intact E control animals when it
was administered 6 hours after the 1 hour exposure to sucrose. Given that multiple
injections increase the strength of CTA (Garcia, Kimeldorf & Hunt, 1956), 3
additional injections were given in hopes of acquiring a CTA. Analysis of the data
revealed that even after 4 injections, none of the estradiol groups developed a
gustatory avoidance toward the CS.
Although none of the estradiol groups developed a CTA, they also did not
exhibit a change in sucrose consumption across the first 4 acquisition trials. On the
contrary, each of the oil groups showed an increase in consumption across this same
time period, a behavior that is often observed in control animals most likely due to
attenuated neophobia, the palatability of the sucrose, and learned preference due to
calories provided by the CS. As such, although the estradiol group did not show
reductions in CS consumption, they also failed to show increases in sucrose
consumption like the oil groups. This finding is indicative of a negative effect of the
hormone injections that was not experienced by the control animals. This negative
impact was sufficient to prevent increases, but insufficient in producing decreases.
We can only provide speculations as to why the Intact E animals did not
acquire a CTA with a 6 hour inter-stimulus interval. This interval was tested in two
separate studies in Experiment 4 and in both instances produced a CTA in 11 out of
12 animals. One possible explanation is batch effects. Perhaps the batch of animals
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used for Experiment 5a was less sensitive to estradiol and as a consequence, the
inter-stimulus interval was too long to sustain a CTA. As mentioned above, the
estradiol appeared to have some negative effect although it was not sufficient to
produce decreases in sucrose. This result is consistent with the suggestion that the
animals may have been less sensitive to the US injections. A second possibility is
that the PBS infused into the lateral PBN produced some damage or alteration in the
functioning ability of the neurons such that the CTA with the 6 hour CS-US interval
was obliterated. It has been the observation of some researchers that
iontophoretically infused PBS produces damage to neurons. A control group that
received no infusion into the brain would have answered this question; however, no
such group was included. An alternative way of conducting this study is discussed in
the Future Directions section below and will include this control group.
CTA1: Estradiol: 0 Hour CS-US Interval
Given that none of the groups developed a CTA after 4 acquisition trials with
a 6 hour CS-US interval, the decision was made to administer estradiol immediately
following exposure to sucrose. Three such paired acquisition trials were given. The
analysis revealed that unlike the Intact E group, the Bilateral E group did not develop
a CTA after the first pairing. In fact, the sucrose consumption level of the Bilateral E
group following the first paired acquisition trial lay between the Intact E and Intact
Oil control groups. So even though the Bilateral E group differed from the Intact E
group, it also did not behave like the Intact Oil control group. This may have been
due to the fact that each of the oil animals had increased their consumption across the
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first 4 acquisition trials, while the estradiol injections had precluded such alterations
across the same time period. Nevertheless, animals with bilateral lesions did not
acquire a CTA after one pairing. The second pairing was sufficient to produce a
CTA in the Bilateral E group and the amount of sucrose the Bilateral E and Intact E
groups drank on the second pairing did not differ from one another.
Prior to discussing the potential reasons for the inability of the Bilateral E
group to acquire a CTA following the first pairing, it is important to point out three
factors that do not explain this finding. First, it is highly unlikely that this finding
was due to the effects of lesions per se on sucrose consumption. The Bilateral Oil
and Intact Oil groups did not differ in the amount of sucrose they drank across the
acquisition trials. This means that the lesions alone did not increase sucrose
consumption such that the Bilateral E group drank more sucrose in general and as a
consequence did not express CTA. One also cannot argue that the Bilateral E group
sustained more neural damage than the Bilateral Oil and as a consequence the lesions
may have affected drinking since analyses performed on lesion size between these
two groups revealed no significant difference. Finally, the lack of CTA in the
Bilateral E group was not due to a general effect in drinking produced by the lesions.
The animals in the Bilateral E group did not show changes in the amount of water
they drank across the same time period.
There are four possible hypotheses that could explain the inability of the
Bilateral E group to acquire a CTA after the first pairing. The first two hypotheses
involve the role of the lateral PBN in pre-exposure effects. First, this finding simply
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may be due to either CS and/or US pre-exposure effects. Because the 6 hour CS-US
interval was too long to support acquisition of a CTA in this batch of animals, the
Bilateral E group, in essence, had received 4 non-contingent exposures to sucrose and
4 non-contingent injections of estradiol. Both types of pre-exposure regimens (CS
and US) have been shown to attenuate CTAs. For example, non-contingent pre-
exposure to the CS, attenuates subsequent formation of CTA in neurally intact
animals (Benedict & Ayres, 1971; Rescorla, 2000). Similarly, distal pre-exposure
(greater than 24 hours) to estradiol blocks a CTA produced by estradiol in neurally
intact animals (Merwin & Doty, 1994). However, the data do not support this
hypothesis. If pre-exposure effects precluded the Bilateral E group from acquiring a
CTA after the first pairing, then this should have been the case for the Intact E group
as well. The Intact E group also received 4 non-contingent CS and US exposures,
but this group immediately acquired a CTA, suggesting that pre-exposure effects
could not explain the inability of the Bilateral E group to develop a CTA after the
first pairing.
The second hypothesis also involves pre-exposure effects. This hypothesis
purports that bilateral lesions of the lateral PBN either allow for the expression of
pre-exposure effects or they enhance pre-exposure effects. As such, the Bilateral E
group did not acquire a CTA following the first pairing while the Intact E group did.
This hypothesis assumes that the lateral PBN exerts some inhibitory effect on pre-
exposure effects such that when it is intact and activated, pre-exposure effects are not
expressed and a normal CTA is acquired. On the other hand, its inactivation removes
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the inhibition and allows the expression of pre-treatment effects. This assumption
faces some opposition from the fact that neurally intact animals pre-exposed to
estradiol show attenuated estradiol CTA (Merwin & Doty, 1994). If an intact lateral
PBN prevented the expression of pre-exposure effects, then these neurally intact
animals should not exhibit attenuated CTA when pre-treated with estradiol.
A third possible explanation for the inability of the Bilateral E group to
acquire a CTA after the first pairing is that this finding was a chance occurrence and
that the finding is not reproducible. This is presented as a possibility due to the
acquisition findings of LiCl CTA2, which are discussed below.
The fourth hypothesis to account for the inability of the Bilateral E group to
acquire a CTA after the first pairing is that the lateral PBN is involved in estradiol
CTA. It is easier to discuss this possibility when the results of Experiment 5b also
are taken into consideration. In Experiment 5b, complete electrolytic lesions of the
lateral PBN were achieved and the extensive lesions prevented the acquisition of an
estradiol CTA even after 3 acquisition trials were administered. Taken together, the
results of Experiment 5a and 5b suggest that incomplete ablations of the lateral PBN
weaken estradiol CTA such that more pairings are required to acquire the learned
avoidance, while complete inactivation of the nucleus entirely obliterates the CTA.
Although determining the particular role the pontine nucleus plays in estradiol CTA
was outside the scope of Experiment 5, conjectures could be made based on
extensive research that has been conducted on the role of the lateral PBN in LiCl
CTA. These are discussed in the General Discussion section.
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A solid conclusion as to which of these last two hypotheses best explains the
acquisition finding should not be drawn in the absence of the extinction data.
Examination of the extinction data revealed that the Bilateral E group drank
significantly more sucrose than the Intact E group during the final 2 phases of
extinction. As was true for the acquisition data, the increased consumption in
sucrose was not an effect of the lesions per se since there were no differences
detected in CS consumption between the Intact Oil and Bilateral Oil groups during
these phases of extinction. Once again, the argument could be made that the Bilateral
E group may have sustained more neural damage than the Bilateral Oil and as a
consequence the lesions may have affected drinking; however, the two groups did not
differ in lesion size. Furthermore, this increase in sucrose consumption during
extinction was not due to a disruption in the general drinking mechanism. The
Bilateral E and the Intact E groups did not differ in the amount of water consumption
during these final 2 phases of extinction. Given that we have eliminated these
possibilities as potential causes for increases in CS consumption during extinction
and given that the Bilateral E group required more acquisition trials to develop a
CTA, the conclusion that increases in sucrose during extinction are a reflection of an
attenuated learned gustatory avoidance is viable. Taking the acquisition and
extinction findings in combination, that more pairings are required to acquire the
learned avoidance and that animals show increased CS consumption during
extinction, the data lend support to the hypothesis that partial lesions of the lateral
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PBN weaken estradiol CTA. This hypothesis is further supported by the finding that
complete lesions inhibit CTA learning all together.
CTA2: Lithium Chloride: 0 Hour CS-US Interval
The intact and bilaterally lesioned animals that had received an estradiol CTA
(Intact E and Bilateral E) also received a LiCl CTA as their second gustatory
aversion (Intact E-Li and Bilateral E-Li). Analysis revealed that both of the groups
acquired a LiCl CTA after one pairing of the chocolate milk with LiCl. Furthermore
on acquisition day, the Bilateral E-Li groups drank more chocolate milk than the
Intact E-Li but consumed similar amounts during the first post-acquisition test. In
addition, there was no difference in the extent of decrease following the pairing,
which suggests that the strength of acquisition for both groups was similar. The
results of a previous study support this conclusion (Chambers and Wang, 2004). In
that study, CTAs were induced with three different doses of LiCl (2, 10, and 20
ml/kg). The animals showed dose-dependent differences in the rate of increase in
sucrose consumption during extinction such that the rate increased significantly with
each decrease in dose. Comparisons between the two lowest doses revealed that
there were no differences in the amount of sucrose consumption during the first four
extinction tests but those animals given the lowest dose consumed more sucrose
during the later stages of extinction. Thus, increased consumption of the CS during
the later phases of extinction can reflect a weaker avoidance.
Although water intake was not measured during the CTA2 procedure, the
LiCl CTA2 animals already had been tested for changes in their drinking patterns
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during estradiol CTA1. The data showed that partial bilateral lesions did not lead to
increases in water intake. As such, the increases in CS consumption observed across
the 4 extinction days in these animals cannot be due to alterations in the general
drinking system.
Once again, conclusions as to what these data mean are more easily drawn
taking the results of Experiment 5b into consideration. Experiment 5b showed that
complete electrolytic lesions of the lateral PBN eliminated LiCl CTAs. Taken
together, the data suggest that partial lesions of the lateral PBN attenuate LiCl CTAs,
which was expressed as increased CS consumption during extinction, while complete
lesions eliminate the aversion produced by the putative illness-inducing agent all
together.
Estradiol (CTA1) and Lithium Chloride (CTA2)
Direct statistical comparisons between the estradiol CTA1 and the LiCl CTA2
were not made even though they were tested in the same animals. There were three
reasons for this decision. First, it was not known whether comparable doses of
estradiol and LiCl were used. That is, we did not know whether a 50µg/kg dose of
estradiol and a 10ml/kg dose of LiCl produced CTAs that were comparable in
strength. The 10ml/kg dose of LiCl was chosen not on the basis of a comparison
with the estradiol dose used, but on the basis of previous studies which have shown
that lesions of the lateral PBN block LiCl CTAs when this dose is used. Second,
three pairings of the CS with estradiol were made, while only a single pairing was
made between the CS and LiCl. Third, the animals had received 4 non-contingent
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sucrose and estradiol exposures immediately prior to receiving contingent pairings of
sucrose and estradiol. Regardless of the lack of direct statistical comparisons, two
differences between the Bilateral E and the Bilateral E-Li groups were clear.
The first of these differences was that animals with identical neural lesions
showed different behaviors for estradiol and LiCl CTAs. Previous research indicates
that the doses of estradiol and LiCl used in Experiment 5 readily induce CTA after
only one pairing. However, for the estradiol CTA in Experiment 5a, lesioned
animals did not acquire a CTA after the first pairing, while one pairing was sufficient
to acquire a CTA for LiCl. This suggests that the LiCl CTA was more easily
acquired than the estradiol CTA. There are 4 possible explanations for this finding.
One potential explanation for this was discussed in “CTA1: 0 Hour CS-US
Interval” section above, namely that the finding that the Bilateral E group did not
acquire a CTA after the first pairing was by chance and irreproducible. However, as
discussed above, given all of the data, this possibility is not very likely. A second
reason is that the area that was lesioned is involved in different aspects of estradiol
and LiCl CTA. For instance, the area lesioned affects acquisition for estradiol CTA,
but does not for LiCl CTA. This would suggest that the subnuclei of the lateral PBN
that control estradiol and LiCl CTA acquisition differ. A third reason for the
difference between the estradiol and LiCl acquisition behavior could be accounted for
by the fact that the estradiol CTA was tested first, followed by the LiCl CTA. That
is, the fact that the LiCl CTA was a second CTA might have affected the behavior of
the animals. Research in our lab indicates that when the time interval between two
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CTAs is short (1 week), the second CTA is weaker than the first CTA but when the
time interval is long (9 weeks), the second CTA is stronger than the first CTA
(Ycaza, Lavond, and Chambers, unpublished manuscript). The CS and US were the
same for both CTAs and for both intervals. Given that the time interval between the
two CTAs in Experiment 5a was 8.5 weeks, these second CTA data suggest that the
greater facility with which the LiCl CTA was acquired was a function of the long
interval. The fourth and most likely explanation for the difference in the number of
acquisition trials required for estradiol and LiCl CTAs is dose effects. Studies in our
lab have shown that a 50µg/kg dose of estradiol produces a CTA that is comparable
in strength to a CTA produced by a 2-4ml/kg dose of LiCl. The dose used for LiCl
CTA2 was 10ml/kg, which should produce a stronger CTA compared to the 50µg/kg
dose of estradiol. Given that lesions of the lateral PBN probably attenuated the
CTAs by reducing the effectiveness of both USs, the effectiveness of the 50µg/kg of
estradiol potentially was reduced such that it necessitated a second acquisition, while
the CTA produced by LiCl was strong enough and not sufficiently reduced to affect
acquisition. Given this rationale, if a lower dose of LiCl had been used, we would
have increased our likelihood of blocking the acquisition in CTA2.
A second clear difference between the Bilateral E and Bilateral E-Li animals
was that the animals recovered from the LiCl CTA much sooner than from the
estradiol CTA. On average, the animals recovered from the LiCl CTA in four days,
while it took about 15 days for them to recover from the estradiol CTA. This delayed
extinction in the estradiol animals probably could be explained by the multiple
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pairings the Bilateral E group received versus the single pairing of the Bilateral E-Li,
although differences in dose strength could have played a role as well.
Unilateral Lesions and CTAs
In Experiment 5a, animals with unilateral lesions did not acquire an estradiol
CTA after four 6 hour CS-US interval acquisition trials. However, this group
acquired a CTA immediately following the first 0 hour CS-US acquisition. This
finding is not surprising given that research on unilateral lesions of the PBN has
shown that these lesions alone are ineffective in abolishing acquisition of CTA
(Bielavska & Roldan, 1996). These unilateral lesions also did not affect extinction of
the estradiol CTA.
Not surprisingly, unilateral lesions did not affect acquisition of LiCl CTA
either. Both the Unilateral E-Li and Intact E-Li groups acquired a CTA after a single
pairing of the chocolate milk with LiCl and they did not differ in the extent to which
they each decreased their consumption from acquisition to post-acquisition day.
However, animals with these lesions showed increases in CS consumption during
extinction when LiCl was used as the US. The Unilateral E-Li animals, similar to the
Bilateral E-Li group, drank significantly more chocolate milk compared to the Intact
E-Li group on each of the 4 extinction days. To our knowledge, the effect of
unilateral lesions on LiCl has not been studied in previous investigations. This
finding suggests that partial unilateral lesions attenuate LiCl CTA. The results of
Experiment 5b corroborate this conclusion. The results showed that each of the
animals in the Unilateral Li group had reached acquisition consumption levels by
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extinction day 6, while each of the animals in the Intact Li group continued to show
reductions in sucrose consumption up until at least extinction day 9. Although a
strong possibility, the effects of unilateral lesions on LiCl CTA should be tested in a
more extensive study which includes a larger number of animals (discussed in the
Future Directions section below).
Complete unilateral lesions of the lateral PBN also may attenuate estradiol
CTA. This could be observed by the separation in the extinction data between the
Intact E and Unilateral E groups in Experiment 5b, where the unilaterally lesioned
group showed increased CS consumption in the later days of extinction. Each of the
animals in the Unilateral E group drank more chocolate milk on extinction days 11
and 12 compared to each of the Intact E animals. This was observed despite the fact
that the animals had received 3 pairings of estradiol with the CS. In addition, the
effect of the unilateral lesion on LiCl CTA probably was more evident due to the fact
that the animals had received only a single pairing. However, once again, the effect
of unilateral lesions on estradiol CTAs should be tested in a more complete study
with more animals.
It is curious that in Experiment 5a animals with identical unilateral lesions of
the lateral PBN showed different behaviors for estradiol and LiCl CTAs. For
instance, partial unilateral lesions of the lateral PBN accelerated extinction of LiCl
CTA but not estradiol CTA. There are two possible explanations for this. First, the
Unilateral E animals received 3 acquisition trials compared to the one pairing the
Unilateral E-Li animals received. The multiple acquisitions may have produced a
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stronger CTA such that it would take a longer time to extinguish. Perhaps we would
have observed an attenuated estradiol CTA in the Unilateral E group if we had
extended extinction testing. The second explanation for this difference in the
estradiol and LiCl extinction in unilaterally lesioned animals is that the area lesioned
in the Unilateral E group affects extinction of LiCl CTAs, while it has no effect on
estradiol CTA extinction. Given the findings of Experiment 5b, namely that
complete lesions of the lateral PBN possibly attenuate both LiCl and estradiol CTA,
this would suggest that the parabrachial subnuclei involved in the extinction of CTA
are not different for the two USs.
One finding from Experiment 5a that is difficult to explain is the fact that
both partial bilateral and unilateral lesions of the lateral PBN affected LiCl CTA
similarly. Both the Unilateral E-Li and Bilateral E-Li groups showed elevated CS
consumption on acquisition day and increased sucrose consumption across the 4
extinction days compared to the Intact E-Li groups. However, this was not the case
with the Unilateral Li and the Bilateral Li groups from Experiment 5b. The LiCl
CTA was completely blocked in bilaterally lesioned animals. On the other hand, the
unilaterally lesioned animals acquired the LiCl CTA, although it appeared to be
weaker than the CTA exhibited by the intact controls as evidenced by higher levels of
sucrose consumption in the later days of extinction. As such, one can argue that the
reason the bilateral and unilateral lesions of Experiment 5a yielded similar results
was due to some aspect of the lesions. It could be that the unilateral lesions were
extensive enough on one side to produce attenuated CTAs comparable to those
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produced by the less extensive bilateral lesions. However, examination of the extent
of the lesions showed that the unilateral lesions were not more extensive than the
lesions on either the left or the right side of the bilateral lesions. Another possibility
involves our conservative method of determining lesions. It is possible that critical
cells were lesioned on the “non-lesioned” side of the unilateral animals and as such
the bilateral and unilateral lesions showed the same effect on LiCl CTA. Once again
the specific location of the lesions in unilaterally lesioned animals may have played a
role. Finally, the extinction rate for the LiCl was generally fast. The animals
extinguished from the CTA in 4 days. As such, it may be that the extinction rate was
too fast to detect a just-noticeable difference.
Experiment 5b and CTAs: Some Important Issues
One issue with Experiment 5b that should be addressed is that only a single
pairing was made for the LiCl CTA. It has been argued that if lesions of certain
structures block CTA learning when only one pairing is given, it is possible that the
lesions only reduced the sensitivity of the animals to the US. One way researchers
have circumvented this problem is to perform multiple acquisition trials. However,
although these animals received one LiCl pairing, they did receive 3 estradiol
acquisition trials. Despite the multiple pairings, the animals did not acquire an
estradiol CTA even, suggesting that the lateral PBN lesions did not simply reduce
sensitivity to the US. Nevertheless, replication of the study should include multiple
pairings of the CS with LiCl (discussed below).
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A second issue with Experiment 5b is that the bilaterally lesioned group drank
more sucrose (CTA1) and chocolate milk (CTA2) during their first exposure to these
CSs on acquisition day compared to the intact and unilaterally lesioned animals for
both the LiCl and estradiol CTAs, respectively. Since water intake was not measured
in these animals, it is difficult to tell whether these bilaterally lesioned animals drank
more during the acquisition test due to attenuated neophobia or due to an alteration in
their general drinking mechanism. Similarly, our study did not include a lesioned
group that received an injection of oil or saline. As such, it is difficult to say whether
the lesions per se affected sucrose consumption such that these animals did not
express CTA. Based on the available data, it is not possible to ascertain why the
bilaterally lesioned animals drank more sucrose on acquisition day. There is
contradictory evidence in the literature regarding lesions of the lateral PBN and
neophobic responses to novel CSs. Some studies have shown that lesions eliminate
neophobic responses (Reilly & Trifunovic, 2001), while others have shown no effect
(Sakai, Tanimizu, Sako, Shimura & Yamamoto, 1994). However, even if the
bilateral lesions blocked neophobic responses to the CS, evidence in the literature
suggests that the absence of a CTA in lateral PBN lesioned rats is not predicated
upon the absence of a neophobic response (Reilly & Trifunovic, 2001). One study
showed that lesions of the lateral PBN eliminated neophobia to alanine, but had no
effect on neophobic reactions to capsaicin. However, the lesions blocked the CTA to
both capsaicin and alanine. This finding suggests that the neophobia deficit in
lesioned animals is independent of, rather than responsible for, the absence of
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conditioned aversions in lateral PBN lesioned rats. If the absence of neophobia was
responsible for the absence of CTA acquisition, then only the animals in which the
neophobic response was eliminated (the alanine) should have shown CTA deficits.
Therefore, in Experiment 5b, even if the increased CS consumption during the first
acquisition test was due to elimination of the neophobic response, the bilaterally
lesioned animals still should have been able to express a CTA. The fact that they did
not express a CTA is consistent with the hypothesis that they were unable to acquire
a CTA.
Although it is difficult to ascertain whether complete lesions of the lateral
PBN produced increases in sucrose consumption, evidence suggests that this too
should not preclude the acquisition of a CTA. In general, there is no relationship
between the amount of sucrose consumed during the first acquisition test and the
ability to acquire a CTA. For instance, the Bilateral E-Li and Unilateral E-Li groups
in CTA2 of Experiment 5a drank significantly more sucrose than the Intact E-Li
group during the first acquisition test. However, the three groups consumed
comparable amounts during extinction test 1 and no significant interaction was
detected among the groups across acquisition and extinction test 1. Similarly, in
Experiment 1, the EB50-D9 group drank more sucrose on acquisition day compared
to the two other groups; however, this group still acquired a strong CTA. This
suggests that elevated acquisition test consumption levels do not preclude the
acquisition of a CTA if the learning has taken place nor do they predict the strength
of a CTA that is acquired. Therefore, even if the increased amount of sucrose and
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chocolate milk that were consumed by the bilaterally lesioned rats during the
acquisition test of Experiment 5b were the result of increases in general levels of CS
drinking, this should not have prevented them from acquiring the CTA.
Summary of CTA Findings
There are a few conclusions that can be drawn based on the combined CTA
data of Experiments 5a and 5b. First, the data suggests that partial bilateral lesions of
the lateral PBN attenuate both estradiol and LiCl CTA, while extensive lesions of the
nucleus block acquisition of a CTA induced by both agents. Second, our data
suggests that unilateral lesions of the lateral PBN also attenuate LiCl and possibly
estradiol CTAs, although this claim warrants further investigation. Our data on CTA
and the lateral PBN thus far replicate, but also extend on previous research. Research
consistently has shown that both excitotoxic (Trifunovic & Reilly, 2002) and
electrolytic (Sakai &Yamamoto, 1998) lesions block acquisition of a LiCl CTA.
However, no study in the literature has examined the effect of lateral PBN lesions on
estradiol CTA. To our knowledge, no study to this point has examined the effects of
unilateral lesions of the lateral PBN on extinction of CTAs produced by either agent.
Estradiol Hypophagia
Relationship between CTA and Hypophagia. Each animal from Experiment 5a also
was tested for the hypophagic effects of estradiol. The data showed that the lesions
of the lateral PBN alone did not affect baseline levels of consumption since none of
the animals differed in the amount of food they consumed during baseline. This
probably was not due to the fact that the lesions in Experiment 5a were incomplete
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since complete lesions of the nucleus did not affect baseline consumption levels
either. Furthermore, the data revealed that despite exhibiting an attenuated estradiol
CTA, the animals that sustained partial bilateral ibotenic lesions did not express
attenuated hypophagic effects following the hormone injections. The Intact E,
Unilateral E, and Bilateral E groups each significantly reduced their food
consumption following estradiol injections and each recovered simultaneously from
the suppressed levels of eating. This decrease in consumption was observed in the 12
hour dark, 12 hour light, and in the 24 hour food intake. This provides some
evidence for the dissociative nature of the conditioned and unconditioned effects of
estradiol. The lack of lesion effect on estradiol hypophagia may be due to two
factors. First, once again it could have been due to the fact that our excitotoxic
lesions of the pontine nucleus were not extensive enough to affect eating.
Nevertheless, the limited area of the lateral PBN that was lesioned was sufficient to
attenuate the estradiol CTA, but not the estradiol hypophagia, suggesting separate
neural substrates for the two behaviors. The results of Experiment 5b provide
evidence for this possibility. Even complete lesions of the lateral PBN left estradiol
hypophagia undisturbed suggesting that the partial lesions of Experiment 5a could
not account for why those animals continued to exhibit estradiol hypophagia. To our
knowledge, no other study has examined the effects of the lateral PBN in estradiol
anorexia. Second, food intake measurements for Experiment 5a were made one
month following the end of the CTA1 procedure. This means that measurements of
food intake were made approximately 73 days after the lesions were produced.
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Given this lengthy period time between the lesions and the testing for food
consumption, the possibility of recovery of function cannot be dismissed. The results
of Experiment 5b do not support this possibility. Food intake measurements for
Experiment 5b were initiated approximately 37 days following the electrolytic
lesions. Although the interval between the lesions and the time of testing was much
shorted in Experiment 5b compared to Experiment 5a, the effects on estradiol
hypophagia were the same, namely that lateral PBN lesions left estradiol hypophagia
undisturbed. As such, finding an effect of lateral PBN lesions on CTA but not
hypophagia substantiates the conclusions of Experiments 1-3 that the conditioned and
unconditioned effects of estradiol are dissociable. If the unconditioned effects of
estradiol were responsible for the hormone’s ability to condition, then lesions of the
lateral PBN should have eliminated both CTA and hypophagia induced by estradiol.
However, the lesions eliminated CTA without affecting hypophagia.
Oil Groups and Reductions in Food Intake. As reported in the results section of
Experiment 5a, for the 24-hour eating measurement, each of the estradiol and oil
groups showed significant reductions in food intake in the 24 and 48 hour periods
following the first estradiol injection compared to baseline. However, in the 48 hour
period following the first injection, each of the estradiol groups showed significantly
greater reduction in eating compared to each of the oil groups. In addition, each of
the oil groups recovered by the 72 hour time period following the first injection,
while the estradiol groups continued to show reductions in eating during this time
period. So, clearly there was a difference between the oil and estradiol groups. In
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pilot studies, we have observed fluctuations in day-to-day consumption of
ovariectomized female rats and statistical analysis of these increases and decreases
sometimes yielded significance. It is likely that the reduction in consumption
exhibited by the oil animals was simply part of the typical pattern of fluctuations.
Experiment 5b and Estradiol Hypophagia: 24 hour Measurements. An important
issue regarding Experiment 5b and estradiol hypophagia that should be addressed is
that only 24 hour food measurements were made in place of 12 hour measurements
as was done in Experiment 5a. The results of Experiment 5a indicated that the
separate 12 hour dark and 12 hour light measurements did not contribute any
noteworthy information that was not revealed in the 24 hour measurements. The
lateral PBN lesioned animals in Experiment 5b showed significant reductions in food
intake following estradiol injections and as such the lack of having dark and light
phase eating data was not critical.
Summary of Experiment 5 Findings
In summary, Experiment 5 answered three out of the four questions it set out
to answer. First, the findings determined that complete lesions of the lateral PBN
block an estradiol CTA without affecting estradiol anorexia and hence the two
behaviors are dissociable (PRIMARY AIM 1-SPECIFIC AIM 1.1). Second,
experiment 5 provides one more piece of evidence that likens estradiol CTA to LiCl
CTA (SECONDARY AIM 1) namely that partial lesions of the lateral PBN
attenuate, while complete lesions eliminate CTAs. Third, it provided information
regarding the neural substrates of estradiol anorexia (SECONDARY AIM 2), namely
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that the lateral PBN is either not involved or it is one of several areas that can
mediate estradiol anorexia and as such is not essential. Unfortunately, with
Experiment 5, we were unable to ascertain whether lateral PBN lesions affect
estradiol CTA when a 6 hour CS-US interval is used (PRIMARY AIM 2-SPECIFIC
AIM 2.2). As such, we have not determined an infusion site for the neurotransmitter
antagonists in the experiment in which we will examine the chemical mediation of
estradiol CTA.
4.6 General Discussion
Dissociating Estradiol CTA from Estradiol Hypophagia
The results of all five experiments support the conclusion that the effects of
estradiol on conditioning are separable from the effects of estradiol on food
consumption. Experiment 1 showed that an estradiol CTA is formed at a time when
unconditioned effects of the hormone have subsided. Experiment 2 demonstrated
that even though a low dose of estradiol produced unconditioned reductions in eating,
it did not produce a CTA. Experiments 3 and 4 showed that non-contingent pairing
of sucrose with estradiol did not result in a CTA even though the post-acquisition test
was held at a time when unconditioned effects of the hormone are still evident.
Finally, Experiment 5 showed that lesions of the lateral PBN attenuate (Experiment
5a) or block (Experiment 5b) estradiol CTA without affecting estradiol hypophagia.
Each of these findings provides evidence against the hypothesis that the
unconditioned effects of estradiol contribute to the conditioning capability of
estradiol.
286
It should be mentioned that research conducted with different species of
animals also has attempted to demonstrate that estradiol’s hypophagic effect is
separate from its conditioning abilities. Predicated on the finding that progesterone
attenuates the unconditioned hypophagic effects of estradiol (Wade, 1975), Ganesan
(1994) tested to see whether progesterone antagonized the conditioning ability of
estradiol benzoate in Rockland-Swiss albino mice. If the unconditioned and
conditioned effects of the hormone were not dissociable, then progesterone should
antagonize a CTA produced by estradiol benzoate. The results of the study showed
that progesterone did not alter an estradiol CTA, suggesting independent underlying
mechanisms for the unconditioned and conditioned effects of estrogen. Although a
clever approach, this study suffered from one major flaw. The study demonstrating
the antagonistic effects of progesterone on estradiol hypophagia used female rats as
subjects, while Ganesan used female mice. It could be that progesterone does not
have the same attenuating effects on estradiol hypophagia in mice as it does in rats.
However, this possibility was not tested in control animals in the Ganesan study.
Ganesan (1994) also used another approach to establish the true conditioning
ability of estradiol. As aforementioned, estradiol has been shown to produce
reductions in eating across many species of animals Wade, 1972; Blaustein, Gentry,
Roy & Wade, 1976; Morin & Fleming, 1978; Czaja & Goy, 1975;Czaja, 1975;
Kemnitz, Eisele, Lindsay, Engle, Perelman, et al., 1984; Gilbert & Gillman, 1956;
Cohen, Sherwin & Fleming, 1987). Interestingly however, this hormone has the
opposite effect on eating in the Mongolian gerbil (Ganesan, 1994; Maass & Wade,
287
1977; Raible & Gorzalka, 1985). In a three part study, Ganesan (1994) showed that
despite the hyperphagic effect of estradiol benzoate in these gerbils, the hormone
produces a CTA in this species indicating that reductions in sucrose consumption
cannot be due the unconditioned effects. However, one problem with this conclusion
is that even if these results do represent a conditioning effect of estradiol, one is left
wondering whether gerbils are an exception in showing dissociation of the
unconditioned and conditioned effects of estradiol on eating as they are in exhibiting
hyperphagia as the unconditioned response to estradiol. Another problem is that the
dose of estradiol benzoate used to induce a CTA in one of the three experiments was
an extremely high dose (50mg), while the doses used to test for the hormone’s effects
on eating in a separate experiment were much lower (10mg). In the study examining
the conditioning abilities of estradiol, the group used to control for unconditioned
effects of the hormone (water paired with estradiol) drank significantly less sucrose
compared to the control oil group (sucrose paired with oil) on test day, suggesting
that the high dose of estradiol benzoate may have suppressed the consumption of
sucrose. It should be mentioned that the lower sucrose consumption of the water-
estradiol group on test day compared to the sucrose-oil group also could be a mere
reflection of a neophobic response. Test day constituted the second sucrose exposure
for the sucrose-oil group whereas it was the first exposure for the water-estradiol
group. However, comparing sucrose consumption on first exposure for both of these
groups suggests that the water-estradiol group showed a greater neophobic response
to the CS than the sucrose-oil group, indicating a possible contribution of estradiol to
288
the reduced consumption. In any case, it is difficult to draw unambiguous
conclusions from this study. Our research extends these previous conclusions by
clearly demonstrating the conditioning ability of estradiol using doses that were
shown to produce both conditioned and unconditioned effects in rats.
What is Estradiol CTA?
In this dissertation, we have demonstrated that the unconditioned effects of
estradiol on eating do not account for the ability of the hormone to produce a
gustatory avoidance. We also have discussed the unlikelihood that estradiol produces
an avoidance to sucrose based on its reinforcing properties. So what property of
estradiol allows it to induce avoidance? There are two alternative hypotheses that
could account for the conditioning ability of estradiol. One is based on satiety while
the other is based on the ability of estradiol, like LiCl, to produce an aversion.
The first hypothesis of estradiol CTA is one based on conditioned satiety. It
has been suggested that conditioned reductions in intake can result from states that
differ from illness (Booth, 1977). For instance, pairing a food with a satiety-
producing agent will elicit a learned reduction in the consumption of that particular
food (Booth, 1985). Some of these agents include cholecystokinin, cocaine-
amphetamine regulated transcript, and glucagon-like peptide-1 (Deutsch & Hardy,
1977; Aja, Robinson, Mills, Ladenheim & Moran, 2002; Thiele et al., 1997). Given
that estradiol is considered a long-term satiety agent, it is possible that it conditions
on the basis of its satiety-inducing properties. That is, following its administration, it
produces feelings of satiety rather than illness and as such produces conditioned
289
reductions in sucrose consumption. It has been demonstrated that CTAs are formed
rapidly following the pairing of the CS with the US (Houpt & Berlin, 1999). A CTA
is expressed 15 minutes following the pairing of saccharin with lithium chloride. If
this holds true for estradiol as well, then in order for estradiol to condition on the
basis of satiety, the animals must be in a state of satiation quickly following estradiol
injection. However, a study conducted in our laboratory has shown that a 250µg/kg
dose of estradiol benzoate does not produce reductions in eating during the interval
beginning immediately following its administration until 1.5 hours or 11.5 hours after
it was administered (Chambers and Hintiryan, 2009). Because females show
hypophagia when measurements are made 24 hours after estradiol administration,
satiety must be triggered sometime between 11.5 hours and 24 hours after estradiol
administration. This would suggest that animals are not in a state of satiation
immediately following the injection of estradiol. Based on the information at hand,
one would assume that CTAs acquired following the pairing of a CS with estradiol
probably are not due to conditioned satiety although further experiments are required
to make that determination. For instance, the speed at which an estradiol CTA is
formed would need to be determined. It may be that unlike LiCl, an estradiol CTA
takes a much longer time to be established, which could bring the conditioned satiety
hypothesis back into contention. However, the results of Experiment 5 provide
evidence against the conditioned satiety hypothesis. We showed that despite
estradiol anorexia expression, the hormone was ineffective in conditioning an
avoidance to sucrose. If estradiol CTA was conditioned satiety, then an estradiol
290
CTA should have been expressed in lateral PBN lesioned animals since satiety by
estradiol was still expressed in these animals.
The second more probable hypothesis is that estradiol conditions on the basis
of its aversion-producing properties. This aversive hypothesis purports that estradiol
produces aversive “internal” feelings like nausea and malaise such that when a CS is
paired with the hormone, the animals feel ill. As a consequence, the animals will
acquire a conditioned taste aversion. In male rats, high doses of estradiol (100µg/kg;
Ossenkopp, Rabi & Eckel, 1996) have been shown to produce aversive orofacial
reactions such as gaping, a gag-like response which evidence suggests is a marker for
internal aversive states (Parker, 1995). Therefore, the aversive hypothesis remains a
possibility when high doses of estradiol are used as a US in a CTA paradigm. In
addition, estradiol has been shown to produce nausea and vomiting in humans
(Schou, 1968), which corroborates the aversive hypothesis. It is not known whether
lower doses of estradiol (i.e. 50µg/kg) produce aversive orofacial responses. In
addition, the taste reactivity study conducted with the high dose of estradiol used
male rats, which have been shown to be more sensitive to estradiol compared to
females (Gustavson, Gustavson, Young, Pumariega & Nicolaus, 1989). Therefore,
this dose may not produce the same negative orofacial responses in females as it did
in males.
The results of Experiment 5 provide evidence for the conditioned aversive
hypothesis for estradiol CTA. First, we showed that the lateral PBN was necessary
for expression of estradiol CTA. The lateral PBN receives information regarding the
291
viscera from the area postrema, which processes information regarding nausea and
malaise. As such, the lateral PBN is one of the relay stations in the ascending
visceral pathway. Second, the lateral PBN also is essential for the expression of CTA
produced by the putative illness inducing agent LiCl. This blockade of LiCl CTA
following lateral PBN lesions is not due to a disruption in the associative process, but
due to a disruption in the US information processing (see discussion below). Taken
together, the data suggest that CTAs produced by estradiol are based on the
hormone’s aversion producing properties. This is not surprising considering that the
hormone produced nausea and emesis in humans (Schou, 1968).
How is the Lateral PBN Involved in Estradiol CTA?
The results of Experiment 5 demonstrate that an intact lateral PBN is essential
for the expression of an estradiol CTA. Although the role this pontine nucleus plays
in estradiol CTA was beyond the scope of the experiment, comprehensive research
conducted on the lateral PBN and different agents suggest a disruption in US
processing rather than an associative role. The most obvious of the evidence is the
fact that the pontine nucleus is the second relay station for afferent visceral
information (Lowey & Burton, 1979; Saper & Lowey, 1980). The lateral PBN
(especially the external subnucleus) receives the greatest part of the afferents
projecting from the AP (Lanca & van der Kooy, 1985; Shapiro & Miselis, 1985;
Milner, Joh & Pickel, 1986; Miceli, Post & van der Kooy, 1987), which is critical for
malaise information and emetic responses (Borison & Brizzee, 1951). Based on this
292
information, one could make a strong argument that the lateral PBN plays an
essential role in US information processing for estradiol CTA as well.
A second piece of evidence for this hypothesis is provided by research that
has investigated the role of the nucleus in LiCl CTA. In addition to not acquiring a
CTA, animals with lesions of the lateral PBN also are unable to learn other behaviors
that involve LiCl-toxicosis. For instance, they are unable to develop a CTA towards
a mild capsaicin solution (Reilly & Trifunovic, 2000), which the authors of the study
claim is a non-gustatory trigeminal stimulus (Silver & Finger, 1991). As such, since
the lateral PBN does not receive information regarding the trigeminal stimulus,
disruption of the CTA cannot be due to a disruption in association. It should be
noted that although capsaicin is considered by researchers to be solely a trigeminal
stimulant (Reilly & Trifunovic, 2000; Shimura et al., 1997) there is some evidence
that the agent may also activate taste receptors (Silver & Finger, 1991; Travers,
Urbanek & Grill, 1999). To our knowledge, no studies investigating the effect of
capsaicin on medial or lateral PBN activation have been conducted. Therefore, the
possibility that some neurons in the lateral PBN mediate both trigeminal and US
information cannot be dismissed.
Another piece of evidence that suggests the abolition of CTAs following
lateral PBN lesions is due to disruption in the transmission of malaise information is
provided by a study that used morphine as a US. Ibotenic acid lesions of the lateral
PBN precluded the formation of a CTA (Nader et al., 1996). These same animals
also were unable to develop a morphine-elicited conditioned place aversion, and on
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the basis of these results it was suggested that the lesions had interrupted the
transmission of malaise information rather than disabling the associative process.
This conclusion assumes that the lateral PBN does not process information regarding
place, a possibility that has not been investigated. If the lateral PBN does process
place information, then the inability of lesioned animals to acquire a conditioned
place aversion may be due to the inability of the animals to process information
regarding place and not the US properties of morphine.
A final piece of evidence is that lesions of the lateral PBN impair CTAs
produced by intraperitoneal injections of an AP-dependent muscarinic receptor
antagonist methyl-scopolamine (Gallo, Arnedo, Agüero & Puerto, 1990; Berger,
Wise & Stein, 1993), which crosses the blood-brain barrier; however, the lesions are
ineffective against CTAs produced by intraventricularly (lateral ventricles) infused
methyl-scopolamine (Cubero & Puerto, 2000). If the lateral PBN was involved in
association, then the lesions should disrupt CTAs produced by the US delivered by
both routes of administration. On the contrary, it only disrupted the CTA produced
by the route that involves the malaise afferent pathway and not the
intracerebroventricular route that bypasses it. Once again, based on one study, one
cannot completely discard the association possibility. It could be that the lateral
PBN, in addition to other areas like the amygdala and the insular cortex, serves as a
site of association. If this is the case, then in the absence of the lateral PBN, these
forebrain structures could substitute as the association areas. As a result, a gustatory
294
aversion could still be formed when a US is infused intraventricularly despite a
dysfunctional PBN.
What is Meant by “Attenuated CTA”?
In Experiment 5a, it was reported that animals with weak bilateral lesions of
the lateral PBN exhibited an attenuated estradiol CTA because they showed increased
sucrose consumption across the final two phases of extinction. But what exactly is
meant by the phrase “attenuated CTA” when there is increased consumption during
extinction? There was a time when extinction was viewed as a function of the
acquisition process. That is, the strength of acquisition was reflected in the
extinction process such that stronger CTAs took longer to extinguish compared to
weaker CTAs. Taking this view, it could be argued that the Bilateral E group from
estradiol CTA1 exhibited increased sucrose consumption during the final 2 phases of
extinction because the animals showed weakened acquisition, which also was
evidenced by the fact that it took 2 pairings for this group to acquire an estradiol
CTA. As indicated above, dose response studies with LiCl have shown that for some
dose comparisons, the only difference detected is in the later phases of extinction and
this most certainly can be interpreted as a difference in strength of acquisition
(Chambers and Wang, 2004). Currently, however, a more complex view of
extinction has been adopted, which must be taken into consideration. Extinction is
no longer viewed as a function of just acquisition, but is a reflection of (1) the taste-
malaise association, (2) the ability to retain the association in memory, (3) the facility
with which the memory of the association is retrieved, and (4) the ability to relearn
295
that consumption of the taste substance is followed by positive consequences. This
final point about extinction representing the ability of an animal to relearn replaces
the older view that extinction is simply forgetting or unlearning the previous
association. Evidence for this assertion comes from the observation that rate of re-
acquisition is faster after extinction, spontaneous recovery of the conditioned
behavior after extinction training has been completed, and reinstatement of the
extinguished behavior with unpaired presentation of the CS after the US is given
alone (Bouton, 1993; Delamater, 1996; Rescorla, 2001). Taking all of this into
consideration then, the increased CS consumption during phases 3 and 4 of extinction
in the Bilateral E group might be a reflection of the strength of the avoidance
acquired, but the possibility of it being a reflection of the extinction process alone
should not be dismissed. Although animals in LiCl CTA2 (Bilateral E-Li) acquired
CTAs comparable in strength to the control animals, they also showed increased CS
consumption across their 4 days of extinction. This finding could mean that
extinction was independent of acquisition strength since despite similar acquisitions,
the animals showed different extinction behavior. On the other hand, it is the case
that even though statistical analyses did not reveal significant differences between the
bilaterally lesioned and intact animals in the strength of CTA acquisition, floor
effects could have obscured any possible differences that otherwise would have been
observed.
296
4.7 Future Direction
Due to the complications we experienced in Experiment 5a and the low
numbers of animals in Experiment 5b, a follow up experiment (Experiment 6), which
will comprise of two studies, will be conducted. There are two reasons for
completing such a study. First, it is important to verify our conclusion that the lateral
PBN is essential for acquisition of estradiol CTAs when the CS-US interval is 0
hours but it is not essential for estradiol anorexia (Experiment 6a). Second, given
that lesions of this structure abolish estradiol CTAs, we also need to determine
whether this structure mediates estradiol CTAs when the CS-US interval is longer
than 0 hours (Experiment 6b). If it does, this will give us a neural site for infusion of
antagonists and will allow us to proceed with determining the chemical mediation of
estradiol CTA. As such, the 6 hour CS-US interval, along with two other intervals,
will be tested.
Excitotoxic Lesions
Excitotoxic lesions of the lateral PBN will be performed. However, in this
experiment, NMDA (10µg/µl; solvent PBS) infusions in the amount and rate of
0.2µl/1minute will be made via a 30G cannulae. The cannulae will be attached to a
Hamilton syringe via PE1 tubing (Scientific Commodities Inc., Lake Havasu, AZ),
which will be situated in a micro-pump.
Ibotenic acid will be replaced with NMDA since this chemical has been less
problematic for us in the lab. For instance, it easily dissolves in PBS, it is stable and
does not breakdown into a different compound like ibotenic acid, and NMDA
297
produces complete annihilations of the neurons at the infusion site in the lateral PBN.
We recognize that other studies that have examined lesions of the lateral PBN on
CTA have used ibotenic acid as the neurotoxin. However, if complete lesions of the
lateral PBN are made (as indicated with NeuN staining) then replacing the toxin
should not make a difference as long as there are no surviving neurons.
Prior to conducting this major study, a pilot study will be conducted to test all
parameters and coordinates for the lateral PBN and to ensure that the volume of
NMDA is sufficient to lesion the entire lateral PBN and that NMDA is destroying all
of the neurons at the site of infusion.
Groups for Experiment 6a: 0 Hour CS-US Interval
Sixty-four ovariectomized rats will randomly be assigned to one of the
following 3 groups:
(1) Intact Oil (n=12), which will be PBS infused animals that receive oil
(2) Intact E (n=12), which will be PBS infused animals that receive estradiol
(3) Lesion Oil (n=20), which will be NMDA lesioned animals that receive oil
(4) Lesion E (n=20), which will be lesioned animals that receive estradiol
Groups for Experiment 6b: 6 Hour CS-US Interval
Seventy-four ovariectomized rats will randomly be assigned to one of the
following 5 groups:
(1) Intact Oil (n=12), which will be PBS infused animals that will receive oil
(2) Intact E (n=12), which will be PBS infused animals that receive estradiol
(3) Lesion Oil (n=20), which will be NMDA lesioned animals that receive oil
298
(4) Lesion E (n=20), which will be lesioned animals that receive estradiol
(5) Intact Interval Test Group1 (n=10), which will be randomly selected from
the larger batch.
(6) Intact Interval Test Group 2 (n=10), which will be randomly selected from
the larger batch.
Before neural surgery is performed, we will test the 6 hour CS-US interval in
these animals first to determine whether the interval works in that particular batch of
animals. If this interval fails to produce an estradiol CTA, a 5 hour CS-US interval
will be tested. If this fails, a 4 hour interval will be tested in the Intact Interval Test
Group 2.
Also, there was concern that PBS infusions may be producing some
alterations in the brain that affect CTAs when an extended CS-US interval is
employed. The Intact Interval Test Group also would control for this possibility.
The animals will not receive neural infusions of any sort. The behavior of this group
could then be compared to the behavior of the Intact E group, which will receive
infusions of PBS into the lateral PBN, to determine whether the PBS infusions affect
behavior.
CTA Behavioral Procedure
Experiment 6a: 0 Hour CS-US Interval
Each animal will be given 2 CTAs. An estradiol CTA with a 0 hour CS-US
interval will be given as CTA1. The second CTA will be a LiCl CTA to enable
299
comparisons between estradiol and LiCl CTAs. Three acquisition trials will be given
for both estradiol and LiCl.
Experiment 6b: 6 Hour CS-US Interval
The interval that produces a CTA in the Intact Interval Test Groups will be
tested in the lesioned and sham-lesioned animals. Each of these animals will be
given 2 CTAs, an estradiol CTA first and be a LiCl CTA second. Three acquisition
trials will be given for both estradiol and LiCl.
Measurements of Food and Fluid Intake
Measurements of water intake will be made throughout the CTA testing.
After the completion of CTA testing, food intake measurements will be made in the
same animals.
Additional Considerations
It should be mentioned that potentially we could have a situation where the
longest CS-US interval for the animals in Experiment 6b is 6 hours and that lesions
of the lateral PBN block the estradiol CTA with this interval. However, due to batch
effects, in the infusion study, it may be that 5 hours is the longest CS-US interval
supporting an estradiol CTA, an interval that has not been tested lateral PBN lesioned
animals. Although this type of situation may present itself, it is not necessarily a
problem since it is the longest interval producing a CTA that is critical and not the
specific amount of time.

300
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Abstract (if available)

Abstract When consumption of a novel tasting substance is followed by administration of a chemical agent that produces physiological changes indicative of malaise, animals will reduce their consumption of the substance during subsequent encounters. This learned response is traditionally referred to as a conditioned taste aversion (CTA). Studies have shown that the hormone estradiol is capable of producing this learned gustatory aversion. In addition, estradiol produces reductions in food intake and body weight, a phenomenon that is referred to as its anorectic effects. As a consequence of this anorectic effect, we question whether estradiol truly can induce CTA learning. Therefore, one of the purposes of the experiments presented in this dissertation was to test the dissociability of estradiol CTA and estradiol anorexia. The second purpose of this thesis was to examine the neural basis of estradiol CTA and estradiol anorexia. Four approaches were adopted to test the ability of estradiol to condition independent of its ability to produce reductions in eating. First, we show that estradiol can produce a CTA in the absence of its anorectic effects. Second, we demonstrate that a low dose of estradiol that produces reductions in eating does not produce CTA. Next, we show that contingent pairing is necessary for a CTA since non-contingent pairing does not result in the gustatory aversion. Finally, we dissociate the conditioning effect of estradiol from its anorectic effect by showing that both excitotoxic and electrolytic lesions of the lateral parabrachial nucleus (PBN) either attenuated or blocked an estradiol CTA, while leaving estradiol anorexia unaffected. Together, all of the data suggest that estradiol can condition a gustatory aversion. The data also suggest that estradiol elicits a CTA based on its aversion inducing properties since lesions of the lateral PBN, an area that processes visceral information, blocked the CTA.

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Creator Hintiryan, Houri (author)

Core Title The neural basis of estradiol conditioned taste aversion learning and estradiol anorexia

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Psychology

Publication Date 05/04/2009

Defense Date 03/12/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag anorexia,associative learning,conditioned taste aversion learning,estradiol,female rats,lateral parabrachial nucleus,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Chambers, Kathleen C. (committee chair), Greene, Ernest (committee member), Lavond, David G. (committee member), Watts, Alan G. (committee member), Wilcox, Rand R. (committee member)

Creator Email hintirya@gmail.com,hintirya@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2130

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