The structural and functional configurations of glutamate, GABA, and catecholamine pre-synaptic terminals in the parvicellular neuroendocrine part of the paraventricular nucleus of the hypothalamus

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Content THE STRUCTURAL AND FUNCTIONAL CONFIGURATIONS OF
GLUTAMATE, GABA, AND CATECHOLAMINE PRE-SYNAPTIC
TERMINALS IN THE PARVICELLULAR NEUROENDOCRINE PART
OF THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS.
by
Caroline Johnson
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)
MAY 2017

Table of Contents
List of Figures and Tables ............................................................................................ iii
Abstract .......................................................................................................................... 1
Chapter 1: Introduction ................................................................................................ 4
Homeostasis and the Stress Response ........................................................................ 5
Hypothalamic-Pituitary-Adrenal Axis ............................................................................ 7
Pre-Motor Control Network ........................................................................................... 9
Glucocorticoids ........................................................................................................... 15
Research Design ........................................................................................................ 20
References ................................................................................................................. 22
Chapter 2: Neurotransmitter Diversity within Pre-Synaptic Terminals Located in
the Parvicellular Neuroendocrine Paraventricular Nucleus of the Rat and Mouse
Hypothalamus .............................................................................................................. 31
Introduction ................................................................................................................ 31
Materials & Methods .................................................................................................. 36
Results ....................................................................................................................... 45
Discussion .................................................................................................................. 56
References ................................................................................................................. 65
Chapter 3: Acute Glycemic Stressors Alter Terminal Activation Patterns in the
Paraventricular Nucleus of the Hypothalamus in a Stimulus-Intensity Dependent
Manner .......................................................................................................................... 74
Introduction ................................................................................................................ 74
Materials & Methods ................................................................................................... 77
Results ....................................................................................................................... 83
Discussion .................................................................................................................. 92
References ............................................................................................................... 108
Chapter 4: Corticosterone Maintains Excitatory Input to Corticotropin Releasing
Hormone Neurons in the Paraventricular Nucleus of the Hypothalamus ............. 116
Introduction ............................................................................................................... 116
Materials & Methods ................................................................................................. 122
Results ...................................................................................................................... 127
Discussion ................................................................................................................ 138
References ............................................................................................................... 154

i
Chapter 5: Summary and Conclusion ...................................................................... 163
Summary .................................................................................................................. 164
The Pre-Motor Control Network in the Absence of a Stressor ................................... 167
Excitatory/Inhibitory Balance in the PVH .................................................................. 172
In Response to Acute Stressors ............................................................................ 172
In Response to Altered Levels of Circulating Corticosterone ................................. 174
Adrenergic Response to Physiological Stressors ..................................................... 177
Conclusion ................................................................................................................ 180
References ............................................................................................................... 182
ii
List of Figures
Figure 1.1 Basic hypothalamic-pituitary-adrenal axis ...................................................... 7
Figure 1.2 The pre-motor control network ..................................................................... 10
Figure 1.3 Catecholamine biosynthesis .........................................................................13
Figure 1.4 Projections of the hindbrain catecholaminergic neurons in the control of the
HPA axis ........................................................................................................................ 14
Figure 1.5 HPA axis control with corticosterone negative feedback ............................... 19
Figure 2.1 Cytoarchitectural boundaries of the PVH in the rat and mouse ................... 41
Figure 2.2 Validation of analytical methods ................................................................... 45
Figure 2.3 VGluT2 and VGAT occur preferentially in pre-synaptic terminals ................. 48
Figure 2.4 Glutamatergic and GABAergic innervation patterns .................................... 49
Figure 2.5 Catecholaminergic innervation to the PVH ................................................... 50
Figure 2.6 VGluT2 and VGAT co-occur in a population of terminals in the PVHmpd
....................................................................................................................................... 51
Figure 2.7 Catecholamine terminals in the PVHmpd of the rat and mouse contain
VGluT2 .......................................................................................................................... 52
Figure 2.8 Co-occurrence of NPY with AgRP or catecholamines ................................. 54
Figure 2.9 Appositions to CRH neurons in the PVHmpd of the mouse .......................... 55
Figure 2.10 Pre-synaptic terminal diversity in the PVHmpd ........................................... 59
Figure 3.1 Insulin and 2DG elicit graded HPA axis responses ....................................... 84
Figure 3.2 Pre-synaptic terminal innervation is not altered by acute glycemic challenges
....................................................................................................................................... 85
Figure 3.3 Phosphorylation of synapsin I increases following an acute glycemic challenge
....................................................................................................................................... 86
Figure 3.4 Acute glycemic challenges alter detectable levels of VGluT2 and VGAT
...................................................................................................................................... 87
iii
Figure 3.5 2DG increases the co-occurrence of phospho-synapsin I and VGluT2 ........ 88
Figure 3.6 2DG reduces DBH-immunoreactivity in terminals in the PVHmpd ............... 90
Figure 3.7 Both 2DG and insulin increase detectable VGluT2 within adrenergic terminals
....................................................................................................................................... 91
Figure 3.8 Catecholaminergic inputs do not use phospho-synapsin I to relay stimulus
intensity ......................................................................................................................... 91
Figure 3.9 Acute glycemic challenges do not alter appositions to phospho-ERK1/2-labeled
soma ............................................................................................................................. 92
Figure 3.10 Stressors of different magnitudes differentially recruit afferent populations
...................................................................................................................................... 93
Figure 4.1
..................................................................................................................................... 127
Figure 4.2 The concentration of plasma corticosterone inversely affects thymus weight
..................................................................................................................................... 128
Figure 4.3 Circulating corticosterone alters CRH peptide detectability in the PVHmpd
..................................................................................................................................... 129
Figure 4.4 Corticosterone is necessary to maintain full pre-synaptic terminal innervation
to the PVHmpd ............................................................................................................. 130
Figure 4.5 Corticosterone maintains the excitatory/inhibitory balance within the PVHmpd
..................................................................................................................................... 131
Figure 4.6 Adrenalectomy reduces DBH-immunoreactivity in adrenergic structures ..........
..................................................................................................................................... 132
Figure 4.7 Figure 4.8 Corticosterone is required to maintain expression of VGluT2 within adrenergic
pre-synaptic terminals .................................................................................................. 134
Figure 4.9 Adrenalectomy decreases the co-occurrence of phospho-synapsin I and
VGluT2 in pre-synaptic terminals ................................................................................. 136
Figure 4.10 Corticosterone does not alter phospho-synapsin I detectability in
catecholaminergic pre-synaptic terminals .................................................................... 136
iv
Figure 4.11 Chronically high circulating corticosterone reduces excitatory innervation to
CRH soma ................................................................................................................... 137
Figure 5.1 Pre-motor control network .......................................................................... 171
List of Tables
Table 2.1 Primary antibodies used for immunohistochemistry ....................................... 38
Table 2.2 Fluorescently-conjugated secondary antibodies ............................................ 38
Table 3.1 Primary antibodies used for immunohistochemistry ....................................... 80
Table 3.2 Fluorescently-conjugated secondary antibodies ............................................ 80
Table 4.1 Primary antibodies used for immunohistochemistry ..................................... 124
Table 4.2 Fluorescently-conjugated secondary antibodies .......................................... 125
v
Abstract
Dysfunctional glucocorticoid secretion is a component of a wide range of clinical conditions.
Much of this dysfunction involves important but poorly understood neural control networks
in the hypothalamus. These networks integrate stress-related information to drive
ACTH secretion and peptide gene expression in a manner appropriate for the ongoing
as appropriate signaling cascades. In particular, glutamate, GABA, and catecholamine
(CA) inputs, which make up a “pre-motor control network,” are crucial for the regulation
of corticotropin releasing hormone (CRH) neuronal activity in response to a wide variety
of systemic stressors. Understanding how these inputs interact to relay the nature of
orchestrates the activity of the neuroendocrine CRH neurons, and allows for the dynamic
processing of different stressors.
In this dissertation we examine the structural and functional organization of this pre-motor
control network to determine and how its activity allows for an appropriate response from
afferent activity in the pre-motor control network affect how the CRH neuron “reads” these
inputs to result in an appropriate response to a stressor, in terms of both magnitude and
duration, and is tested using various in vivo procedures, as well as immunohistochemistry
mouse model.
1
In Chapter 2 organization of pre-synaptic terminals in the pre-motor control network in the absence of
a stressor, to establish a picture of this network in an unstimulated state. Using both a rat
(glutamate) and fast inhibitory (GABA) neurotransmitters. Furthermore, we show that at
least some of this excitatory innervation originates from the hindbrain CA neurons that
project to the PVHmpd. Within this CA population we found that the adrenergic subset is
the dominant input. Finally, we found that the vast majority of appositions to CRH neurons
occur at non-somal locations.
In Chapter 3 we investigate the effects of acute glycemic stressors of different intensities
on the functional organization of the pre-synaptic terminals examined in Chapter 2. We
show that stressors of different magnitudes differentially recruit afferent populations
in a stimulus intensity-dependent manner that favors excitation by altering detectable
vesicular glutamate transporter 2 (VGluT2) and vesicular GABA transporter (VGAT) within
pre-synaptic terminals. The increase in VGluT2 occurs within both CA and non-CA pre-
terminals.
In Chapter 4 we used bilateral adrenalectomy with either high corticosterone (CORT)
replacement or no CORT replacement to investigate the how CORT affects the organization
of pre-synaptic terminals in the absence of a stressor. We show that circulating CORT is
2
the adrenergic component that expresses VGluT2. Chronically high circulating CORT had
little effect on the overall structure of the pre-motor control network. This may be a way to
Overall, we show that the organization of pre-synaptic terminals of the pre-motor control
HPA axis response. In all cases the excitatory inputs were altered to a greater extent than
were the inhibitory inputs, underlining the importance of inhibitory control on the function
of the HPA axis. Finally, we show that adrenergic inputs appear to be the more salient CA
input in regards to physical challenges and changes in overall homeostasis.
3
Chapter 1: Introduction
Exposure to all stressors, real or perceived, evokes a fundamental and evolutionarily
conserved response from the hypothalamic-pituitary-adrenal (HPA) axis that helps
maintain long-term homeostasis. An acute HPA axis response to a stressor is adaptive,
but chronic activation of stress-responsive networks can be detrimental. Aberrant
secretion of adrenocorticotrophic hormone (ACTH) and/or glucocorticoids is a component
of many pathological conditions, including cardiovascular and metabolic diseases, as
well as anxiety, depression, and post-traumatic stress disorder (Ulrich-Lai and Herman,
2009). Much of this dysfunction originates from within important neural networks, though
how interactions between these networks and their components result in an appropriate
response from the HPA axis remains unclear.
Activity-dependent release of corticotropin-releasing hormone (CRH) neurons in the
medial dorsal parvicellular part (mpd) of the paraventricular nucleus of the hypothalamus
(PVH) ultimately underlies the secretion of glucocorticoids, and is a function of many
different integrative inputs. The actions of fast neurotransmitters glutamate and GABA,
and their interactions with catecholamine (CA) afferents to the PVHmpd, are well-studied
and important in the initiation of the HPA axis response (Daftary et al., 2000; Cole and
Sawchenko, 2002; Ritter et al., 2003).
Together these inputs constitute a control network that regulates the activity of
neuroendocrine CRH neurons. These particular CRH neurons directly control the activity
4
motor neurons (Watts, 2005). Therefore, this network can be described as a “pre-motor”
control network. The goal of this dissertation is to understand the structural and functional
network, and how their interactions allow for an appropriate response from CRH neurons
synaptic terminals in the pre-motor control network affect how the CRH neuron ‘reads’
these inputs to result in an appropriate response to a stressor, in terms of both magnitude
and duration.
Homeostasis and the Stress Response
Maintaining a relatively constant internal environment is crucial to the continued survival
was built upon the pioneering work of Claude Bernard in the 1800s (Bernard, 1858).
Homeostasis is now a central tenet in physiology. Crucially, Cannon also recognized that
separate and independent physiological mechanisms must be responsible for returning
a physiological variable into a homeostatic range depending on whether the variable
moved above or below this range (Cannon, 1932). This fundamental concept has since
variables.
5
endocrinologist Hans Selye (1936). The pattern of responses generated following
exposure to a wide variety of stimuli was termed “general adaptation syndrome,” and
later “stress” (Selye, 1956), a word which has since permeated the vocabulary of both
academic and colloquial language.
Exposure to a stressor, a real or perceived stimulus that jeopardizes homeostasis, rapidly
activates the sympathetic nervous system, resulting in the almost immediate release of
epinephrine (and some norepinephrine) from the adrenal medulla (Ulrich-Lai and Herman,
activation results in many changes throughout the body, including pupil dilation, increased
heart rate, and the inhibition of digestion, among other responses (Ulrich-Lai and Herman,
2009). However, many of these responses are short-lived.
To ensure a greater and more enduring response, the HPA axis is activated. This activity
is initiated by neuroendocrine CRH neurons in the PVHmpd, which release CRH (and
arginine vasopressin) into the hypophysial portal blood to reach the corticotropes of the
anterior pituitary (Dallman et al., 1987). CRH stimulates these cells to release of ACTH
into the general circulation, which reaches the adrenal cortex and stimulates the synthesis
and release of steroid hormones glucocorticoids: cortisol in humans or corticosterone
(CORT) in rodents (Melmed et al., 2015). Glucocorticoids target almost every organ/cell
6
in the body, where they play diverse roles in the maintenance of homeostasis. Notably,
glucocorticoids suppress the immune system, mobilize energy stores, and are a crucial
control element in negative feedback to the HPA axis (Keller-Wood and Dallman, 1984).
Hypothalamic-Pituitary-Adrenal Axis
CRH neurons are found in various nuclei throughout the brain (Swanson et al., 1983;
Dabrowska et al., 2013), but the highest concentration is found in the PVHmpd (Swanson
et al., 1983). CRH neurons sit atop the HPA axis, and their activity initiates its response
input from diverse regions throughout the brain. Different stressors activate distinct
neural networks as a way to ensure the appropriate response from CRH neurons, and
subsequently the HPA axis. Figure 1.1 presents a simple model of the HPA axis.
Figure 1.1 Hypothalamic-pituitary-adrenal axis
A simple model of the hypothalamic-pituitary-adrenal axis. In response to a stressor, corticotropin releasing
hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus release CRH into the median
eminence. CRH stimulates corticotropes in the anterior pituitary to release adrenocorticotropic hormone
(ACTH) into the general circulation. ACTH reaches the adrenal cortex where it stimulates the release of
glucocorticoids (GC) (cortisol in humans, corticosterone in rodents). These steroid hormones have myriad
effects on nearly every system of the body, as well as acting as a negative feedback signal to its own
release. (+) indicates stimulatory and (-) indicates inhibitory actions of corticosterone.
7
The HPA axis is activated in response to both real and perceived stressors. “Real” or
“systemic” stressors are generally considered to be those that present an immediate threat
to homeostasis and require a reaction for continued survival (Herman et al., 2003; Ulrich-
or infection, or marked changes in blood glucose concentration, among others. The HPA
axis can also be activated in the absence of a systemic stressor, in anticipation of a threat
to homeostasis (Herman et al., 2003). These anticipatory or “psychogenic” stressors can
either be learned (i.e., conditioned) or innate (instinctual, such as exposure to a predator),
and allow the animal to mount an HPA axis response in preparation to a possible threat
to homeostasis.
Respnoses to psychogenic stressors often require telencephalic processing and
activate many forebrain structures, including the medial pre-frontal cortex, hippocampus,
amygdala, and bed nuclei of the stria terminals (BST), among others (Ulrich-Lai and
Herman, 2009). Stress-induced activation of these regions is typically inhibitory to HPA
axis activity, though only some nuclei within the BST have substantial direct connections
with the PVH (Dong et al., 2001), and as such acts as an integrative hub for inputs from
these various forebrain structures (Ulrich-Lai and Herman, 2009).
Systemic stressors, including glycemic perturbations, activate the HPA axis by a more
direct route (Ulrich-Lai and Herman, 2009). Glucose is an obligate metabolic fuel in the
8
brain, though it cannot be synthesized by the brain (Cryer, 2001). Therefore, the
brain is entirely dependent on the glucose in circulation. Changes in glucose levels
outside of the physiological normal range (between 4 and 6 mM (Donovan and Watts,
2015)), and particularly those below the normal range, elicit swift counterregulatory
mechanisms, including activation of the sympatho-adrenal and HPA axis responses.
Systemic stressors activate both CA and non-CA neurons of the hindbrain, and the
adrenergic and noradrenergic neurons in the dorsomedial and ventrolateral medulla are
particularly important. These neurons directly innervate CRH neurons (Swanson et al.,
1981; Sawchenko and Swanson, 1982; Liposits et al., 1986a, 1986b; Cunningham and
Sawchenko, 1988; Cunningham et al., 1990), as well as other neural networks that affect
the HPA axis response, including other hindbrain nuclei, BST, amygdala, and the local
PVH glutamatergic and GABAergic networks.
Pre-Motor Control Network
CRH neurons directly control the activity of non-neuronal endocrine cells in the anterior
of CRH control would have each input received and summated by CRH neurons with little
to no integration, but evidence shows that this is unlikely. Rather, these networks appear
to be plastic and highly integrative (Herman et al., 2002; Watts, 2005; Wamsteeker and
Bains, 2010; Watts and Kahn, 2013). Studies have shown that interactions between CA,
CRH activity (Herman et al., 2004; Wamsteeker and Bains, 2010; Levy and Tasker, 2012).
9
Collectively these afferent components make up a “pre-motor control network” (Watts,
2005), a model of which is shown in Figure 1.2 (adapted from Watts and Khan, 2013).
Figure 1.2 The pre-motor control network
Figure 1.2 presents a simple model of the pre-motor control network. Hindbrain CA
neurons have been shown to express VGluT2 mRNA (Stornetta et al., 2002). These
neurons affect CRH neuronal activity directly, as well as through interactions with both
GABA and glutamate inputs. These inhibitory and excitatory inputs arise from several
regions within the brain, potentially including within the proximal hypothalamic nucleus
(GABA), and from within the PVH itself (glutamate).
10
A basic model of the pre-motor control network,
based on electrophysiological and tract tracing
studies, adapted from Watts & Khan (2013).
Projections from the hindbrain catecholamines
directly innervate CRH neurons via post-synaptic
alpha1 adrenergic receptors (gray triangle),
as well as interacting with glutamatergic and
GABAergic inputs via pre-synaptic alpha2
adrenergic receptors (gray triangle, black border).
Adrenergic inputs express both Neuropeptide Y
& VGluT2, conferring a glutamatergic phenotype.
Noradrenergic inputs also express VGluT2.
CRH neurons express NPY receptors (blue
triangle), and all glutamate ionotropic receptors,
and metabotropic receptors (green triangle).
GABAergic inputs from the periPVH region are
CRH neurons express GABA
A
receptors (red
triangle). Glutamatergic input from within the
PVH also controls CRH neuronal activity. Signals
from these inputs are integrated within the CRH
neuron to result in an appropriate response to a

GABA
Approximately half of the synapses in the PVH are GABAergic (Decavel and van den
Pol, 1990), and nearly 80% of the total GABAergic innervation to the PVH terminates
directly on CRH neurons (Miklós and Kovács, 2002). In the absence of stressors or other
perturbations, tonic engagement of GABAA receptors inhibits CRH neuronal activation
and the ensuing HPA axis activity (Cole and Sawchenko, 2002). Pharmacologically
axis (Bali and Kovács, 2003; Kovács et al., 2004), indicating that inhibition is a crucial
control element for CRH neuronal activity. This GABAergic innervation stems from distal
inputs such as the BST, but also apparently from the local peri-PVH region (Roland and
Sawchenko, 1993; Boudaba et al., 1996; Herman et al., 2004). While this peri-PVH region
houses many GABAergic neurons, and electrophysiological evidence suggests that this
(Bali and Kovács, 2003; Kovács et al., 2004), there is currently no anatomical evidence
to suggest that GABAergic neurons from the peri-PVH directly innervate the PVHmpd
(Risold et al., 1994; Vujovic et al., 2015).
Glutamate
Glutamate is the predominant fast excitatory neurotransmitter in the PVH (van den Pol et
al., 1990; Ziegler and Herman, 2000). CRH neurons express all of the various types of
ionotropic glutamate receptors, as well as metabotropic glutamate receptors (Herman et
11
effects on HPA axis activation (Makara and Stark, 1974; Darlington et al., 1989; Ziegler
and Herman, 2000; Cole and Sawchenko, 2002). However, blocking ionotropic glutamate
receptors abolishes the excitatory response evoked by norepinephrine (Daftary et al.,
neurons in the PVH, and that the drive provided by ascending catecholaminergic afferents
requires a glutamatergic component located either within or close to the PVH (Daftary et
al., 2000; Herman et al., 2002).
Catecholamines
Catecholaminergic neurons in the dorsomedial and ventrolateral medulla contain both
epinephrine and norepinephrine, and project to the PVH where they presumably release
these neuromodulators in response to systemic stressors (Pacak et al., 1995; Ritter et al.,
2011; Guyenet et al., 2013). These CA phenotypes can be differentiated by the biosynthetic
(PNMT) (Swanson et al., 1981; Siegel et al., 1999). Structures containing both enzymes
are adrenergic, while those containing DBH without PNMT are noradrenergic (Swanson
et al., 1981) (See Figure 1.3, adapted from Siegel et al.,1999).
Adrenergic neurons innervate the PVH from the C1, C2, and C3 nuclei (Cunningham et
al., 1990), while noradrenergic innervation originates almost exclusively from A1, A2, and
A6 (Sawchenko and Swanson, 1982) (Figure 1.4). CAs are both excitatory (by means of
12
within the parvicellular PVH (Plotsky et al., 1989; Daftary et al., 2000). Furthermore, these
CA neurons express vesicular glutamate transporter 2 mRNA (Stornetta et al., 2002), and
at least a subset of the adrenergic neurons use glutamate as a neurotransmitter (DePuy
et al., 2013). However, this has yet to be investigated for CA inputs to the PVH.
Figure 1.3 Catecholamine biosynthesis
While the role of norepinephrine in the brain has been extensively studied, the function
of epinephrine in response to physiological stressors remains unclear (Martin et al.,
2001; Ritter et al., 2011). Epinephrine constitutes only between 5-10% of the total CA
content of the brain (Martin et al., 2001). However, adrenergic inputs preferentially
innervate the parvicellular region of the PVH, particularly the PVHmpd (Swanson et al.,
1981; Cunningham et al., 1990), and are crucial in regulating a plethora of physiological
processes (Guyenet et al., 2013).
13
Catecholamine biosynthetic cascade. Dopamine is converted to
(DBH). In adrenergic neurons norepinephrine is then converted
to epinephrine via phenylethanolamine-N-methyltransferase
(PNMT). All catecholaminergic neurons express the enzyme
tyrosine hydroxylase (TH, not pictured). Cells expressing TH
and DBH are considered to be noradrenergic, while those
expressing all three enzymes are adrenergic.
Figure 1.4 Projections of the hindbrain catecholaminergic neurons in the control of the
HPA axis

Catecholaminergic projections from the hindbrain to the PVH originate predominately from noradrenergic
nuclei A1, A2, and A6 and adrenergic nuclei C1, C2, and C3. Shown here are the projections from these nuclei
to the hypothalamus, as well as to the amygdala and the bed nucleus of the stria terminals (BST), two regions
that receive input from the same catecholaminergic nuclei and are intricately linked to HPA axis function. These
catecholaminergic projections, and others, innervate many more regions throughout the brain, though these
regions have less direct control over HPA axis activity. Rostral is to the left of the image, caudal is to the right.
Noradrenergic inputs, on the other hand, innervate both the parvi- and magnocellular
divisions (Swanson et al., 1981; Cunningham and Sawchenko, 1988), and are involved
in the response to psychogenic (Rinaman, 2011). Furthermore, direct administration of
an equimolar dose of norepinephrine (Leibowitz et al., 1988). It is clear, though, that
the CA input to the parvicellular PVH is essential for a complete HPA axis response to
glycemic stressors. Chemical ablation of these afferents dramatically reduces the rise in
circulating CORT that occurs as a response to both insulin-induced hypoglycemia and
the administration of 2-deoxy-D-glucose (Ritter et al., 2003; Khan et al., 2011). The loss
of CA innervation does not alter the HPA axis response to psychogenic stressors, nor the
diurnal variations of CRH activity (Ritter et al., 2003).
14
Glucocorticoids
Glucocorticoids are secreted from the zona fasciculate of the adrenal cortex in a pulsatile
fashion throughout the day (Dallman et al., 1987; Watts et al., 2004). These steroid
hormones act on every cell and system in the body (Dallman et al., 1987). They bind
(Ruel and de Kloet, 1985; Reul et al., 2000). During the circadian peak, or following
II receptors, the glucocorticoid receptors (Ruel and De Kloet, 1985; Ruel et al., 1987).
While mineralocorticoid receptors exhibit limited patterns of expression, glucocorticoid
receptors are extensively expressed in the brain (Ruel and De Kloet, 1985).
Glucocorticoids signal through genomic and non-genomic mechanisms (Watts, 2005;
Stahn et al., 2007; Hill and Tasker, 2012). Classical genomic signaling occurs when
glucocorticoids bind to cytosolic receptors. The receptor-ligand complex translocates to
(GRE)) (Stahn et al., 2007). GREs can be either positive (inducing transcription) or
negative (suppressing transcription) (Stahn et al., 2007). Recently, non-genomic actions
of glucocorticoids have been recognized. As a fast feedback mechanism, glucocorticoids
apparently bind to membrane-bound glucocorticoid receptors, where they promote
endocannabinoid (eCB) synthesis and act as retrograde signals that, through eCB type 1
receptors, rapidly suppress excitation (Hill and Tasker, 2012).
15
Glucocorticoids affect nearly every system in the body, from metabolic and immune function
to cardiovascular responses and growth and development, as well as mood and cognitive
functioning (Melmed et al., 2015). In response to an acute stressor, glucocorticoids cause
the mobilization of stored energy, through an increase in gluconeogenesis and inhibition of
glucose uptake, as well as the stimulation of lipolysis in adipocytes (Melmed et al., 2015).
Furthermore, glucocorticoids suppress the immune system, by reducing lymphocytes,
synthesis. Glucocorticoids also increase blood pressure and intraocular pressure, along
with inhibiting linear growth and the uptake of Ca
2+
by osteoblasts, as well as inhibiting
reproductive functions (Melmed et al., 2015).
In response to an acute stressor these glucocorticoid-dependent changes are adaptive,
geared towards increasing the odds of survival. However, because of these wide-ranging
effects the overall impact of chronic glucocorticoid exposure is detrimental. To alleviate these
effects, glucocorticoids act as a powerful negative feedback signal to their own release by
inhibiting HPA axis activation. A major site of negative feedback is the corticotrope, where
glucocorticoids directly suppress ACTH synthesis and release (Dallman et al., 1987).
While glucocorticoids can have direct actions on CRH neurons (Kovács et al., 1986;
Sawchenko, 1987), their effect on HPA axis activity inhibition appears to occur primarily
at extra-PVH sites (Dallman et al., 1994), thereby emphasizing that glucocorticoids also
affect the components of the CRH pre-motor control network.
16
GABAergic innervation and release are modulated by glucocorticoids. The concentration
of circulating CORT directly affects the number of GABAergic synapses with CRH neurons,
2005). Chronic exposure to supraphysiological levels of circulating CORT decreases
mRNA levels of important GABA
A
receptor subunits in medial parvicellular PVH (Cullinan
and Wolfe, 2000), as well decreasing GABAergic tone over time (Verkuyl et al., 2004,
2005). Removing circulating CORT through adrenalectomy increases the frequency of
miniature inhibitory postsynaptic currents in the PVH (Verkuyl and Joëls, 2003), as well as
GABAergic suppression (Yang et al., 2007, 2008). Furthermore, the in vitro administration
(Verkuyl et al., 2005).
Glucocorticoids are also necessary for maintaining glutamatergic components in the brain,
and particularly the hippocampus (De Kloet et al., 1998; Joëls et al., 2001; Joëls, 2008),
although chronically high levels of the hormone cause degeneration of glutamatergic
hippocampal cells (Sapolsky et al., 1985). In the PVH, exposure to the chronic variable
stress paradigm increases the number of both glutamate and CA appositions to the CRH
neurons, suggesting an increase in excitatory signaling (Flak et al., 2009). This paradigm
also decreases mRNA expression of the NMDA receptor subunit NR2B (Ziegler et al.,
2005). The loss of this subunit increases Ca
2+
signaling, consistent with an increase in
excitatory signaling (Ziegler et al., 2005). Furthermore, adrenalectomy increases
17
noradrenaline-mediated excitation of PVH neurons (Yang et al., 2007), and given the
dependence of this noradrenergic input on functional ionotropic glutamate receptors
(Daftary et al., 2000), glutamate may be involved in this increase in excitation.
Glucocorticoids also directly affect the activity of hindbrain CA (Jhanwar-Uniyal and
Leibowitz, 1989), probably by way of glucocorticoid receptors in the CA neurons that
project to the PVH (Agnati et al., 1985; Fuxe et al., 1985; Härftstrand et al., 1986; Liposits
et al., 1987; Sawchenko, 1987; Sawchenko and Bohn, 1989). Adrenalectomy increases
the turnover of noradrenaline in the PVH (Pacak et al., 1995), which may be the result
of a glucocorticoid-induced increase in tyrosine hydroxylase, the rate limiting enzyme in
the CA biosynthetic cascade (Jhanwar-Uniyal et al., 1989). Consistent with an increase
in noradrenergic signaling (Pacak et al., 1995), the loss of circulating CORT increases
excitation in the PVH, through CA-mediated suppression of GABA inhibition (Yang et al.,
receptors on GABAergic pre-synaptic terminals (Feuvrier et al., 1999; Yang et al., 2007)
(Day et al., 1999), including CRH neurons (Cummings and Seybold, 1988). Furthermore,
glucocorticoids regulate both the development and maintenance of PNMT (Bohn, 1983;
Bohn et al., 1984; Jiang et al., 1989).
Catecholaminergic inputs are required for the negative feedback actions of glucocorticoids
on CRH mRNA levels when circulating CORT is high (Kaminski and Watts, 2012).
18
CORT also directly regulates CA signaling at CRH neurons by altering the number of
Feuvrier et al., 1998, 1999).
Figure 1.5 HPA axis control with corticosterone negative feedback
19
A more complex model of HPA axis control,
including those regions directly affected by
glucocorticoid negative feedback that further alters
CRH neuronal activity. CRH neurons receive direct
input from hindbrain catecholamines (blue), which
are affected by circulating glucocorticoids (GC,
including corticosterone (CORT)), as well as from
the bed nuclei of the stria terminalis (BST) (green).
The BST integrates input from the CORT-sensitive
hippocampus (purple) (and other regions, not
shown). CRH neurons of the PVH release CRH
(red), which result in the release of ACTH from
the anterior pituitary (orange), and ultimately the
release of glucocorticoids (GC/CORT, black). CORT
affects target organ systems, as well as ultimately
resulting in the inhibition of the HPA axis through
various neural targets and their down-stream
effects. Excitatory effects are represented by (+),
while inhibitory effects are represented by (-).
Catecholaminergic afferents, and especially the adrenergic subset, are important in the
transmission of information regarding various physiological stressors (Ritter et al., 2001,
2003; Li and Ritter, 2004; Khan et al., 2007; Guyenet et al., 2013; Lee et al., 2016).
Figure 1.5 presents a more complex model of the HPA axis, including the interactions
of corticosterone with various regions involved in HPA axis regulation. Given the wide-
ranging effects of glucocorticoids on different systems within the body, it is likely that
these changes also alter CA innervation and/or signaling to the PVH, thereby indirectly
altering HPA axis function (Watts, 2005).
Research Design
the pre-motor control network affect how the CRH neuron ‘reads’ these inputs to result in
an appropriate response to a stressor, in terms of both magnitude and duration. In Chapter
2 motor control network in the absence of a stressor, focusing on structural interactions
between glutamate, GABA, and CA, and their relationship to CRH neurons. This was
accomplished by applying immunohistochemistry with high resolution three-dimensional
image analysis to brain sections from both rats and the transgenic Crh-IRES-Cre;Ai14
Chapter 3
we used the same methods to asses how two different acute glycemic challenges alter
ability of this network to relay different stressor types and intensity. In Chapter 4 we
20
examined how changing the level of circulating CORT in the absence of a stressor altered
terminals in the pre-motor control network in the neuroendocrine PVHmpd, likely as a way
be uniquely involved in the relay of the stressors tested here. This dissertation will also
present a methodological model to investigate functional changes in network structure
applicable to many different regions within the brain.
21
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30
Chapter 2: Neurotransmitter Diversity Within Pre-Synaptic Terminals
Located in the Parvicellular Neuroendocrine Paraventricular Nucleus of the
Rat and Mouse Hypothalamus
Introduction
The dorsal medial parvicellular part (mpd) of the paraventricular nucleus of the
(Simmons and Swanson, 2009; Biag et al., 2012). These neurons send the majority of their
axons to the median eminence where their terminals release various neuropeptides into
the hypophysial portal vasculature to control hormone release from the anterior pituitary.
Of these neuropeptides, the neuroendocrine corticotropin-releasing hormone (CRH)
neurons are perhaps the most well-studied. These neurons sit atop the hypothalamic-
pituitary-adrenal (HPA) axis and control its response to all stressors, real or perceived.
CRH neuronal activity is controlled by a complex pre-motor network that relays information
from various parts of the brain to the PVHmpd (Swanson et al., 1981; Sawchenko and
Swanson, 1982, 1983; Swanson and Sawchenko, 1983; Liposits et al., 1986b; Watts,
2005; Watts and Khan, 2013). Each of these inputs use a myriad of transmitters and
peptidergic modulators (Sawchenko and Swanson, 1983; Swanson and Sawchenko,
1983). The hindbrain catecholamine (CA) neurons are among the most well-studied
inputs, and innervate all subdivisions of the PVH (Swanson et al., 1981; Sawchenko
and Swanson, 1982, 1983, Swanson and Sawchenko, 1983; Liposits et al., 1986b;
Cunningham and Sawchenko, 1988; Cunningham et al., 1990).
31
The CA phenotypes can be determined through the expression of the biosynthetic enzymes
N-methyltransferase (PNMT) (Swanson et al., 1981). Structures containing all three
enzymes are presumably adrenergic, while those containing DBH without PNMT are
noradrenergic (Swanson et al., 1981). Structures which contain only TH are ostensibly
parvocellular regions, with the greatest innervation to the PVHmpd (Swanson et al., 1981;
and magnocellular regions (Swanson et al., 1981; Sawchenko and Swanson, 1982).
The axon terminals of these catecholaminergic inputs make direct synaptic contact with
neurons in both the parvi- and magnocellular PVH (Liposits et al., 1986a), and direct
adrenergic synaptic contact to CRH neurons has been shown (Liposits et al., 1986b).
This suggests that adrenergic inputs exert direct control over the CRH neuronal activity,
and subsequently HPA axis activity.
These hindbrain-originating CA afferents are particularly important for relaying acute
physiological challenges to the PVHmpd (Ritter et al., 2001, 2011; Li and Ritter, 2004;
Guyenet et al., 2013), and are indispensible for the neuroendocrine response to insulin-
induced hypoglycemia, as well as the administration of 2-deoxy-D-glucose (2DG), as
well as GLP-1 agonists (Ritter et al., 2001, 2003; Khan et al., 2007; Lee et al., 2016).
They are also are required to maintain the sensitivity of CRH mRNA to elevated levels of
corticosterone (Kaminski and Watts, 2012). However, these inputs are expendable for the
32
daily rhythms of corticosterone secretion and for neuroendocrine responses to psychogenic
stressors (Ritter et al., 2003; Khan et al., 2011; Flak et al., 2014).
At least half of the pre-synaptic terminals in the PVH are GABAergic (Decavel & van den
Pol, 1992; Bali and Kovács, 2003), and CRH neurons receive direct GABAergic synaptic
innervation (Miklós and Kovács, 2002; Flak et al., 2009). Tonic activation of GABA
A

to activate the HPA axis (Cole and Sawchenko, 2002), indicating that GABA-mediated
inhibition is a crucial control element for CRH neurons. Numerous glutamate synapses
are also found within the PVH (Ziegler and Herman, 2000), and glutamatergic signaling is
involved in the activation the HPA axis (Ziegler and Herman, 2000). Glutamate terminals
appose CRH neurons (Wittmann et al., 2005), and the administration of norepinephrine
(NE) to the parvicellular PVH increases the frequency of excitatory post-synaptic potentials
in a subset of these cells (Daftary et al., 2000).
The simplest model for controlling CRH neuronal activity would be one wherein each input
is received and summated with little to no integration. However, this is unlikely. Rather,
it appears that these afferents are highly interactive (Herman et al., 2002, 2003; Watts,
2005; Yang et al., 2007, 2008; Wamsteeker and Bains, 2010; Watts and Khan, 2013).
Catecholamine-mediated excitation of neurons within the PVH requires a glutamatergic
component located in or around the PVH (Daftary et al., 2000; Herman et al., 2003). This
33
glutamate receptor antagonists (Daftary et al., 2000). Additionally, hindbrain CA neurons
contain vesicular glutamate transporter 2 (VGluT2) mRNA (Stornetta et al., 2002).
While a population of neurons may express protein and/or mRNA for a vesicular glutamate
transporter, this alone is not indicative of a glutamatergic phenotype (El Mestikawy et
al., 2011). In this study we directly investigated pre-synaptic terminals, marked with
synaptophysin, that express vesicular transporters in the PVHmpd. Activation of terminals
in this region has been shown to elicit glutamatergic excitatory post-synaptic potentials
(Daftary et al., 2000), therefore it is likely that that the VGluT2 expressed in the terminals
investigated in this study is related to pre-synaptic glutamate release, and therefore a
glutamatergic phenotype (Fremeau et al., 2001; Herzog et al., 2001).
The complete neuroanatomical framework of this pre-motor control network remains
elusive. Here we investigated the distribution and neurochemical phenotypes of pre-
synaptic terminals in the rat and mouse PVHmpd. We limited our analyses to markers of
the two main fast-acting neurotransmitters (glutamate and GABA), two catecholamines
(norepinephrine and epinephrine (E)), and two neuropeptides (neuropeptide Y (NPY) and
agouti-related peptide (AgRP)). Our goal was to elaborate on how these inputs contribute
to the anatomy of the pre-motor control network that controls neuroendocrine, and
particularly CRH, neuronal activity (Watts, 2005; Watts and Khan, 2013).
34
Although the functional components of the rat and mouse PVH are organized comparably
in terms of their relative locations, the distribution of the various peptidergic neurons
within these compartments is somewhat more homogenous in the rat as compared to the
mouse (Simmons and Swanson, 2009; Biag et al., 2012). In the rat most neuroendocrine
CRH neurons are clustered in the PVHmpd between levels 25 and 26 of the Swanson
Rat Brain Atlas (Swanson, 2004), where they are by far the most predominant peptide
phenotype (Simmons and Swanson, 2009; Watts and Khan, 2013). We therefore limited
our analyses to these two levels of the rat PVHmpd.
This organization is much less apparent in the mouse PVHmpd, where the different
chemical phenotypes are somewhat more heterogeneous (Biag et al., 2012). To analyze
this network in the mouse we used a transgenic line, Crh-IRES-Cre;Ai14, wherein all
an analysis of the pre-synaptic axon terminals that are immediately apposed to CRH
neurons.
We used immunohistochemistry (IHC) for markers of pre-synaptic terminals
(synaptophysin), adrenergic (PNMT and DBH) and noradrenergic (DBH only) processes,
vesicles associated with glutamate (VGluT2) or GABA (vesicular GABA transporter
(VGAT)), and neuropeptidergic (NPY and AgRP) processes. This was followed by high
resolution three dimensional (3D) image analyses to determine three parameters: the
relative abundance of different pre-synaptic terminal phenotypes within the PVHmpd;
35
appositions with CRH neurons.
Materials and Methods
Animals
Nine adult male Sprague Dawley rats (~300g) (Harlan) were housed two- or three-to-a-
cage in a climate-controlled room (20-22°C), on a 12h light/dark cycle (lights on 0600h)
with ad libitum access to food and water. Animals were acclimated and handled daily
adult male Crh-IRES-Cre;Ai14 mice were shipped from the University of Calgary and
immediately stored at -80°C until further processing. All procedures were approved by the
local Institutional Care and Use Committees.
Perfusion and Sectioning
Rats were transcardially perfused with 100mL of cold 0.09% saline, followed by 500mL of
cold 4% paraformaldehyde in a sodium borate solution (pH 9.5). Brains were removed and
sucrose. Brains were frozen in hexanes cooled by dry ice before being sectioned coronally
in a cryoprotectant solution (50% 0.05M sodium-phosphate buffer, 30% ethylene glycol,
20% glycerol) at -20°C until further processing. Seven series were reserved for IHC and
one series was reserved for thionin staining to verify cytoarchitecture. Mouse brains were
36
perfused and processed in the same manner (Wamsteeker et al., 2010).
Immunohistochemistry
each at room temperature (RT)), and allowed to incubate for two hours (RT) in a blocking
solution of TBS containing 2% (v/v) normal donkey serum (Chemicon, Temecula, CA,
S30-100ML) and 0.03% (v/v) Triton X-100 (Sigma Aldrich, 9002-93-1). Sections were
incubated in the same blocking solution containing various combinations of primary
antibodies (see Table 2.1) for 72 hours at 4°C. Sections were again washed in TBS and
incubated in a solution of appropriate and corresponding species-sourced secondary
before then mounted onto Superfrost slides and allowed to air dry in the dark at RT. Once
dry the slides were coverslipped with a solution of 50% TBS/50% glycerol, sealed with
clear nail polish, and stored in the dark at 4°C.
Immunohistochemistry Controls
A number of controls were run through the IHC procedure described above under the
following three conditions: incubation with neither primary nor secondary antibodies;
incubation with primary antibodies only; incubation with secondary antibodies only. There
data not shown), indicating that there was
37
Table 2.1 Primary antibodies used for immunohistochemistry
Primary Antibody Source, Species, & Clonality Titer
Anti-Agouti-related Peptide
Phoenix Pharmaceuticals Cat# H-003-53,
Rabbit Polyclonal
1: 5,000
Millipore, Cat# MAB308, Mouse Monoclonal 1: 20,000
ImmunoStar, Cat# 22806, Rabbit Polyclonal 1: 2,000
Anti-Neuropeptide Y Millipore, Cat# AB1583, Sheep Polyclonal 1: 5,000
Anti-Phenylethanolamine
N-Methyltransferase
M.C. Bohn, Northwestern University,
Rabbit
1: 10,000
Anti-Synaptophysin
R&D Systems, Cat# AF5555,
Goat Polyclonal
1: 5,000
Anti-Vesicular GABA Transporter
Synaptic Systems, Cat# 131 002,
Rabbit Polyclonal
1: 20,000
Anti-Vesicular Glutamate
Transporter 2
Millipore, Cat# AB2251,
Guinea Pig Polyclonal
1: 10,000
Anti-Vesicular Glutamate
Transporter 3
Millipore, Cat# AB5421,
Guinea Pig Polyclonal
1: 10,000

Table 2.2 Fluorescently-conjugated secondary antibodies
Secondary Antibody Source Titer
Alexa 488
Jackson ImmunoResearch
Cat# 715-545-151
1: 2,000
Alexa 488
Jackson ImmunoResearch
Cat# 711-545-152
1: 2,000

Cy3
Jackson ImmunoResearch
Cat# 705-165-147
1: 2,000

Cy3
Jackson ImmunoResearch
Cat# 711-165-152
1: 2,000
Alexa 647
Jackson ImmunoResearch
Cat# 706-605-148
1: 2,000
Alexa 647
Jackson ImmunoResearch
Cat# 713-605-003
1: 2,000
38
Confocal Microscopy
Images were captured using a Zeiss 700 Laser Scanning Confocal Microscope equipped
with a Zeiss AxioImager Z1 camera (Carl Zeiss MicroImaging, Inc., Thorwood, NY), using
a 40x (numerical aperture 1.3) oil-corrected objective. An optical zoom of 1.6 was applied
Images were taken using a unidirectional line scan at 1024 x 1024 pixels captured on an
8 bit gray scale. Each pixel was scanned twice and the result was averaged to reduce
optical noise.
On this scale the intensity of a pixel is given a value ranging from “0” (pure black, no
(FocalCheck Fluorescence Microscope Test Slide #1, Invitrogen, F36909) expressing
by blocking the light path completely and verifying that all pixels registered a value of 0.
This was adjusted using the “Offset” function in the Zeiss Zen software, and was done
the Digital Gain function to ensure that all labeled pixels registered a value of 255, as the
physiological tissue. By ensuring that the camera is capable of capturing every possible
39
intensity.
The airy unit was set to ~1.0 for all laser channels, allowing for an optimal signal to noise
applied to prevent bleed-through between optical channels. 3D images were constructed
throughout the entire depth of the tissue section. Images were analyzed using Volocity
software (PerkinElmer, version 6.3, Waltham, MA), operating on a MacBook Pro (Apple,
Inc.).
The images presented of the rat PVHmpd correspond to Level 26 of the Rat Brain Atlas
(Swanson, 2004), and images of the mouse PVHmpd correspond to Level 60 of the Allen
Reference Atlas (Dong, 2008; Biag et al., 2012). Anatomical regions of interest were
Image Adjustments
beads were analyzed to ensure that all channels were properly aligned. At the scale of
pre-synaptic terminals, any misalignment of images caused by light properties, external
vibrations, or other sources of interference has serious consequences on accurate
40
alignment was corrected using the Registration Correction feature in Volocity. Subsequent
experimental images were corrected using the same settings. To reduce optical aberrations
caused by the diffraction of light, particularly along the Z axis, image deconvolution was
achieved through the Iterative Restoration feature using the Measured Point Spread
Function in Volocity.
Figure 2.1 Cytoarchitectural boundaries of the PVH in the rat and mouse
Sections containing the PVH of the rat (a) and mouse (c) were stained with thionin to establish
cellular architecture. Borders were adapted from the Swanson Rat Brain Atlas, Level 26 (a) and the
Allen Brain Institute Online Coronal Reference Atlas, Image 61 (c). DBH (b) and CRH (d) labeling
shown for reference. White boxes indicate the sampling region for all images. Scale bars indicate
dp, dorsal parvicellular part; mm, medial magnocellular part; mpd, medial parvicellular part,
dorsal zone; mpv, medial parvicellular part, ventral zone; pml, posterior magnocellular part, lateral
zone; pv, periventricular part.
Image Analysis
of colocalization. Dunn et al. (2011) have noted that there are two components when
occurrence, where two or more markers label the same physical structure, and correlation,
correlated manner.
41
those structures that were double- and/or triple-labeled, using the Volocity software
expression. Thresholds were set to reduce aberrant signaling in both the volume and
3
3
for pre-synaptic
terminals. Thresholds for volume were determined through comparisons with previously
published methods (Bouyer and Simerly, 2013). Furthermore, a minimum threshold of two
3
3

3
throughout the depth of the tissue.
The Intersect function in Volocity was applied to determine the co-occurrence of
containing DBH that did not also contain PNMT) the Intersect function was applied
labeled structures that also contained PNMT, leaving only single-labeled structures in
the analysis. To determine appositions to neurons the subtractive methods developed by
Bouyer and Simerly (2013) were followed and adjusted to measure the appositions per
42
unit of surface area, rather than per unit of volume. To determine appositions to the soma,
3
3
was
set as the upper volume limit.
3
, using the total measured
2
of measured neuronal
surface area. To determine the percent of co-occurrence of a single population with
another population the total number of double-labeled structures was divided by the total
number of single-labeled structures measured from each of the populations.
Excel Analysis
Measurements from Volocity were transferred to Microsoft Excel for further analysis. The
3
, using the total measured volume
of the tissue. To determine the percent of co-occurrence of each population with another
population the number of double-labeled structures was divided by the total number of
structures measured from each of the two populations. Appositions were normalized to
2
of CRH neuronal surface area.
43
Validation Controls for the Methods of Assessing Co-Occurrence
We performed two sets of controls on rat brain sections to validate the methods developed
for assessing co-occurrence: theoretical, where the co-occurrence of two different
antibodies against DBH in labeled structures was analyzed; and technical, to verify that
we could differentiate between structures containing both DBH and PNMT and those
containing DBH only.
Theoretical. Sections containing the PVHmpd were labeled using two different antibodies
against DBH (mouse anti-DBH and rabbit anti-DBH) using the methods described
above. Because these antibodies should identify the same structures there should be
no discernable difference between the percent of mouse anti-DBH-labeled structures
that express rabbit anti-DBH and the percent of rabbit anti-DBH-labeled structures that
express mouse DBH. Analysis indicated that there was no difference (Figure 2.2).
Statistics
hoc test, as appropriate (JMP Pro, version 12.1, SAS Institute Inc., Cary, NC). All data are
expressed as the mean ± the standard error of the mean (SEM). A p value of 0.05 or less
44
Figure 2.2 Validation of analytical methods
Images show an orthogonal view of a section of the rat PVHmpd double-labeled with two different
antibodies against DBH (b-e) or DBH and PNMT (g-j).There is no difference in the co-occurrence of
DBH antibodies (a). All PNMT occurs with DBH, but not all DBH occurs with PNMT (f). White lines
indicate a representative double-labeled structure in the X,Y, and Z dimensions. Scale bar represents
p<0.005. Error bars indicate SEM.
Results
Establishing the Validity of the Immunohistochemical and Quantitative Analyses
Theoretical Validity. Figure 2.2 shows structures in the PVHmpd of the labeled by the
mouse- or rabbit-generated antibody against DBH. We found that 86.0 ± 3.7% of the
structures labeled by the mouse anti-DBH antibody were also labeled by the rabbit anti-
DBH antibody, and that 81.0 ± 5.1% of the structures labeled by the rabbit anti-DBH
antibody were also labeled by the mouse anti-DBH antibody. There was no statistical
difference in the percent of co-occurrence between the two measurements (p<0.4),
indicating that the methods of calculating percent co-occurrence are theoretically valid.
45
Theoretical Validity. Adrenergic structures are characterized by the presence of the
enzymes DBH and PNMT, while noradrenergic structures contain only DBH, without
PNMT (Goldstein et al., 1972; Swanson et al., 1981). Therefore, all PNMT should co-
occur with DBH, but not all DBH will co-occur with PNMT. We double-labeled sections
containing the PVHmpd with DBH and PNMT (Figure 2.2) and calculated the percent
co-occurrence of each antibody with the other. Virtually all PNMT co-occurs with DBH
(97. 0± 3.2%) while only 70.0 ± 6.5% of DBH-labeled structures also occur with PNMT. This
VGluT2 and VGAT Occur in Pre-Synaptic Terminals in the PVHmpd
To determine if structures labeled by antibodies against the vesicular transporters were
pre-synaptic terminals, we assessed the percent co-occurrence of VGluT2 and VGAT
labeling with labeling for synaptophysin in the rat and the mouse (Figure 2.3, images for
VGAT not shown). Synaptophysin is a ubiquitous, vesicular membrane-bound protein
(Wiedenmann et al., 1985), and its presence indicates pre-synaptic terminals (Masliah et
al., 1990; Thiel, 1993; Flak et al., 2009; Kwon et al., 2011; Chavan et al., 2015).
We found that 97.0 ± 2.9% of the VGluT2-labeled structures in the PVHmpd also were
labeled for synaptophysin (Figure 2.3), indicating that VGluT2 in the rat is found almost
may account for the approximately 3% of VGluT2 structures unrecognized as co-occurring
with synaptophysin. 47.0 ± 10.2% of the terminals labeled by synaptophysin also
46
contained VGluT2, indicating that nearly half of the terminals innervating the PVHmpd
contained glutamate. As in the rat, virtually all of the VGluT2 in the PVHmpd of the mouse
(Figure 2.4) also contained synaptophysin (94.0 ± 1.3%). Similarly, 46.9 ± 6.8% of the
synaptophysin co-occurred with VGluT2, indicating that nearly half of the terminals
innervating the mouse PVHmpd are glutamatergic as well.
We analyzed the distribution of VGluT3 in the rat PVHmpd (Figure 2.3) and found that it
co-occurred with synaptophysin far less frequently than did VGluT2. Previous work has
shown that not all VGluT3 is exclusive to presynaptic terminals (Seal and Edwards, 2006;
Santos, 2009). Accordingly, we found that 34.2 ± 9.7% of the VGluT3 in the rat PVHmpd
co-occurred with synaptophysin and that only 9.7 ± 2.4% of the synaptophysin was also
labeled for VGluT3.
95.0 ± 5.1% of the measured VGAT also occurred with synaptophysin in the PVHmpd of the
rat (Figure 2.3), indicating that the majority of VGAT-containing structures are pre-synaptic
terminals. Furthermore, 53.9 ± 10.9% of the synaptophysin-labeled structures were also
labeled for VGAT, indicating substantial GABAergic innervation. Again similar to the rat,
95.0 ± 2.3% of VGAT-labeled structures in the PVHmpd of the mouse also occurred with
synaptophysin, indicating the preferential expression of VGAT in pre-synaptic terminals.
Finally, 67.0 ± 8.7% of all synaptophysin-labeled terminals also contained VGAT, indicating
47
Figure 2.3 VGluT2 and VGAT occur preferentially in pre-synaptic terminals
Synaptophysin (green) and VGluT2 (blue) in the PVHmpd of the rat (a-d) and mouse (e-h), and terminals
containing both transporters (c, g). Images d and h highlight co-occurrence in white. CRH neurons are
expressed in red. All VGluT2 (i) and VGAT (k) is found within pre-synaptic terminals, though VGluT3
is not preferential to terminals (j). White lines indicate a representative terminal in the X, Y, and Z
48
Glutamatergic and GABAergic Innervation Patterns in the PVH of the Rat and Mouse
Sections from the rat brain were labeled with antibodies against either VGluT2, VGluT3, or
3
of tissue in the
3
3
,
p 3
) than either VGluT2- (p<0.05) or VGluT3-labeled (p<0.0001) terminals.
These results indicate that the majority of the terminals in the PVHmpd contain VGAT
(53%) and are presumably inhibitory. As there is little to no VGluT1 in the hypothalamus
(Fremeau et al., 2004), our results indicate that VGluT2 is the dominant type of glutamate
transporter in afferents to the PVHmpd.
Figure 2.4 Glutamatergic and GABAergic innervation patterns
Photomicrographs of VGluT2 (a), VGluT3 (b), and VGAT (c) innervation to the PVH and surrounding
glutamate transporter (e p p<0.0001. Error bars represent SEM.
49
Catecholaminergic Innervation Patterns in the PVH of the Rat and Mouse
regions of the rat. We analyzed the number of terminals containing synaptophysin and
either DBH-only or PNMT in the PVHmpd and found four times more adrenergic (44.0 ± 5.0
3
3
, p<0.0001) terminals.
This indicates that the majority of the catecholaminergic input to the neuroendocrine
region of the PVH is adrenergic.
Figure 2.5 Catecholaminergic innervation to the PVH
DBH (a) and PNMT (b) innervation to the PVH and surrounding regions in the rat. Within
c).
p<0.0001. Error bars indicate SEM.
VGluT2 and VGAT Co-Occur in a Population of Terminals in the PVHmpd
Because VGluTs and VGAT have been found in the same terminals (Ottem et al., 2004;
Zander et al., 2010) we analyzed the co-occurrence of these transporters in the rat (Figure
2.6). We found a small population of terminals that contained both vesicular transporters
3
), corresponding to approximately 8% of the total measured
single-labeled labeled for either VGluT2 (p<0.0001) or VGAT (p<0.0001).
50
Figure 2.6 VGluT2 and VGAT co-occur in a population of terminals in the PVHmpd
VGluT2 (a) and VGAT (b) co-occur in the same pre-synaptic terminals (c,d) in the PVHmpd of the rat. There
only one transporter (e). White lines indicate a representative pre-synaptic terminal in the X, Y, and Z
p p<0.005. Error bars represent SEM.
Catecholaminergic Terminals in the PVHmpd of the Rat and the Mouse Contain VGluT2
Sections containing the PVHmpd from the rat were triple-labeled using antibodies against
DBH, PNMT, and VGluT2 (Figure 2.7). We found that VGluT2 is found in CA terminals in
3
3
, p<0.0001).
Approximately 25% of the total VGluT2-containing terminals also occurred with CA
markers. Of this 25%, approximately 4% contained only DBH, indicating a noradrenergic
phenotype, while the remaining 21% contained PNMT, indicating an adrenergic phenotype.
We also analyzed the co-occurrence of VGluT2 and DBH in the mouse (Figure 2.7).
3
3
, p<0.005).
51
3
) than either VGluT2-only (p<0.0001) or
CA-only (p and CA-only than double-labeled terminals (Fig. 2.7, open circles). The PNMT antibody
was not viable in the mouse, thus we could not analyze adrenergic inputs.
Figure 2.7 Catecholamine terminals in the PVHmpd of the rat and mouse contain VGluT2
Sections containing the PVHmpd of the rat (a-h) and mouse (j-m) were labeled with antibodies
against CA markers DBH (a,j) and PNMT (e) and VGluT2 (b,f,k). The mean number of double-labeled
terminals (d,g,l) was analyzed. Images d,h, and m highlight co-occurrence in white. In the rat there are
i). White lines indicate
a representative terminal in the X, Y, and Z dimensions. CRH soma are presented in red in images
j-m p p<0.0001.
whether non-VGluT2-labeled DBH structures were pre-synaptic terminals.
52
Catecholamines and NPY Co-Occur in Terminals in the PVHmpd of the Rat
Approximately two-thirds of the NPY innervation to neuroendocrine CRH neurons
originates from the catecholaminergic cell groups of the hindbrain (Bai et al., 1985;
Sawchenko et al., 1985; Füzesi et al., 2007). The remainder of the NPY innervation comes
from the arcuate and dorsomedial nuclei of the hypothalamus (Bai et al., 1985; Füzesi et
al., 2007). The NPY innervation originating from the arcuate nucleus also contains AgRP
(Hahn et al., 1998) and is GABAergic (Tong et al., 2008).
To determine if our methods produced consistent results with previously reported
innervation patterns we measured the percent of NPY objects co-occurring with AgRP,
DBH, and PNMT (Figure 2.8). We found that 62.0 ± 5.2% of the NPY co-occurs with
one or both of the CA markers, while the remaining 38.1 ± 1.1% co-occurs with AgRP.
Furthermore, 97.0 ± 2.0% of the structures labeled with AgRP also contained NPY.
Appositions to the Soma of CRH Neurons in the Mouse PVHmpd
3
) than
3
, p<0.005) or DBH appositions (5.1 ± 9.4
3
, p<0.005) (Figure 2.9). Because we only had the brains of two male
mice we could not perform statistical analyses. However, the data obtained from these
animals are presented alongside that of the female data (Fig. 2.9, open circles). Similar to
the female mice there appear to be more VGAT appositions than either VGluT2 or DBH
appositions in the male.
53
Figure 2.8 Co-occurrence of NPY with AgRP or catecholamines
Sections were labeled with antibodies against NPY (a,e,i) and either AgRP (b) or DBH (f) and PNMT (j). The
number of double-labeled objects (c,g,k) were analyzed (m), as was the percent co-occurrence (n). Images
d,h, and l highlight the co-occurrence in white. White lines indicate representative terminals in the X, Y, and
p p p p<0.0001.
54
Figure 2.9 Appositions to CRH neurons in the PVHmpd of the mouse
Sections were labeled with antibodies against synaptophysin (a-c), DBH (d-f), VGluT2 (g-i), and
VGAT (j-l) and analyzed for appositions of each type against the soma of the CRH neuron (red).
White lines indicate representative appositions in the X, Y, and Z dimensions. Images c,f,i, and
l terminals than DBH or VGluT2 (m as well (n (o p p<0.01,
p p<0.001.
55
We next calculated the number of appositions made to either the somal or non-somal
regions of the CRH neuron (Figure 2.9, o). Because tdTomato labels the entire CRH
neuron, all apposition measurements made when the entirety of the image was selected
indicate appositions without specifying the physical location on the CRH neurons. We
therefore calculated the percent of appositions to either the somal or non-somal regions
by dividing the number of somal appositions by the total number of measured appositions
for each terminal marker, and then dividing the number of non-somal appositions by the
as well as VGluT2 and VGAT appositions, to non-somal parts of the CRH neuron (Figure
2.9). However, we were not able to analyze DBH appositions to non-somal regions
because we did not run the antibodies against DBH and synaptophysin in the same tissue
sections.
Discussion
catecholamine pre-synaptic terminals within the pre-motor control network that regulates
neuroendocrine CRH activity. To do this we utilized immunohistochemistry followed by
dependent network that is necessary to relay acute physiological stressors to the PVHmpd
(Ritter et al., 2001, 2003, 2011; Khan and Watts, 2004; Li and Ritter, 2004; Khan et al.,
2007, 2011; Kaminski and Watts, 2012; Guyenet et al., 2013; Lee et al., 2016).
56
It is now understood that neurons containing more than one type of neurotransmitter is the
rule rather than the exception (El Mestikawy et al., 2011), and this applies to the afferents
innervating the neuroendocrine PVH as well. Here we analyzed the chemical phenotypes
of pre-synaptic terminals and found that the inputs to the PVHmpd can be divided almost
evenly between glutamatergic (47%) and GABAergic (53%), and that there is greater
adrenergic than noradrenergic innervation. Furthermore, we found substantial physical
relationships between these, and other, afferent populations. Notably, approximately 25%
of the total glutamatergic input to the PVHmpd is found within catecholaminergic terminals,
and that VGluT2 is more prevalent in the adrenergic population. We also found an overlap
in VGluT2 and VGAT expression in a small population of pre-synaptic terminals. While
these populations have been considered mutually exclusive it is becoming increasingly
clear that this is not always the case (Boudaba et al., 1996; Ottem et al., 2004; Zander et
al., 2010). This suggests that the role of vesicular transporters is multifaceted.
While it is clear that these vesicular transporters package fast neurotransmitters into
synaptic vesicles (McIntire et al., 1997; Chaudhry et al., 1998; Fremeau et al., 2001/2?;
Herzog et al., 2001), their expression does not always directly correlate with the ability
for vesicular release (El Mestikawy et al., 2011). Along with packaging glutamate into the
vesicle, VGluTs can augment the packaging ability of other transporters (monoaminergic,
cholinergic, etc.) as well in a process known as vesicular synergy (El Mestikawy et al.,
2011). VGluT expression is also developmentally regulated (Bérubé-Carrière et al., 2009).
57
Additionally, VGluTs may be involved in the transport of inorganic phosphate into the
vesicle, independent of glutamate transport (Ni et al., 1994; Aihara et al., 2000; Juge et
al., 2006). VGluT1 and -2 mRNA have been found in cholinergic motor neurons in the rat
spinal cord, but the corresponding protein is not present at all motor endplates (Herzog
et al., 2004a; Kraus et al., 2004). Additionally, VGluT3 protein occurs in populations not
traditionally considered glutamatergic (Fremeau et al., 2002; Gras et al., 2002; Herzog et
al., 2004b; Somogyi et al., 2004), and not only in pre-synaptic axon terminals, but in cell
bodies and dendrites as well (Gras et al., 2002; Commons, 2009).
Therefore, while a neuronal population may contain VGluT protein and/or mRNA, this alone
is not indicative of glutamate release at the terminal. However, we directly investigated
pre-synaptic terminals, marked with synaptophysin, that contain vesicular transporters in
the PVHmpd. Activation of terminals in this region has been shown to elicit glutamatergic
excitatory post-synaptic potentials (Daftary et al., 2000), and therefore it is likely that that
the VGluT2 expressed in the terminals investigated in this study is related to pre-synaptic
glutamate release.
Finally, while there are overwhelmingly more appositions to the non-somal regions of the
or catecholaminergic appositions to the soma (Figure 2.9).
58
Figure 2.10 presents a summary of the glutamate, GABA, and CA (and other) components
number of pre-synaptic terminals in a selected region of the PVHmpd, represented by
the black box in column one. From this number we determined the proportion of other
labeled terminals within this population. The height of each box represents the proportion
of the labeled population out of the total synaptophysin-labeled terminals. The second
third column are the populations found to co-occur with the populations in column two.
with each population, but were not investigated in this study.
Figure 2.10 Pre-synaptic terminal diversity in the PVHmpd
59
The left portion indicates the measured
transmitters (white boxes) as a portion of
(black box). Within each major population
the type and proportion of other co-
occurring transmitters is indicated by the
overlapping gray boxes. The portion on the
right shows other neuroactive agents that
may occur in each population, but were
not investigated in the current experiment.
See text for details.
of terminals contained both VGluT2 and VGAT. Because we were unable to determine if
this population is predominately glutamatergic or GABAergic, or some other biochemical
phenotype, this population of pre-synaptic terminals is represented by the (??) between
While we did not investigate serotonergic inputs directly, evidence from the literature
indicates that this population from the hindbrain raphe nuclei contains VGluT3, and is
separate from glutamatergic and GABAergic populations innervating the PVH (Gras et
al., 2002; Herzog et al., 2004b).
A large portion of the total pre-synaptic terminals contained VGluT2. Within this population
we found a small number of terminals also containing DBH without PNMT, indicating a
noradrenergic phenotype, as well a population containing PNMT, indicating adrenergic
innervation. Within this adrenergic population, NPY was present in nearly all of the input.
Terminals containing VGAT made up another large portion of the pre-synaptic terminal
innervation. Within this population we found a number of terminals that also contained
DBH. As the antibodies against VGAT and PNMT were both from the rabbit, we could not
determine if this population was adrenergic or noradrenergic. While we did not directly
investigate the co-occurrence of VGAT with NPY, the majority of NPY innervation comes
from the hindbrain CAs and the remainder from within the hypothalamus (Bai et al., 1985;
Sawchenko et al., 1985; Füzesi et al., 2007). The population of NPY originating from the
60
arcuate nucleus co-occurs with AgRP, and has been determined to be GABAergic (Tong
et al., 2008).
VGluT3, which occurs in many populations not traditionally considered glutamatergic
(Fremeau et al., 2002; Gras et al., 2002; Herzog et al., 2004b; Somogyi et al., 2004) has
also been shown to occur in the predominately GABAergic BST (Dong et al., 2001; Herzog
et al., 2004b), which projects to the PVHmpd (Dong et al., 2001), and VGluT3 has been
shown to co-occur in terminals containing VGAT (Hioki et al., 2004; Stensrud et al., 2013).
Therefore, while we did not directly analyze the co-occurrence of VGAT and VGluT3,
it is probable that at least a small population of inputs to the PVHmpd contains both
transporters. Finally, the last column indicates other neuroactive agents as determined
through the literature that most likely affect the output of the CRH neuron as well, but
were not analyzed in this study.
GABAergic Afferents are the Most Prevalent Input to the PVHmpd
VGAT-immunoreactive terminals are the most numerous inputs in the PVHmpd, and this
of the presynaptic terminals in the PVHmpd contained VGAT, mirroring previous reports
indicating that approximately half of the synapses in the PVH are GABAergic (Decavel
and van den Pol, 1990). GABAergic terminals make direct contact with CRH neurons
(Bali and Kovács, 2003) and exert control over the CRH neuron and subsequently the
HPA axis (Cole and Sawchenko, 2002; Miklós and Kovács, 2002; Bali and Kovács, 2003).
61
GABA
A
receptors are found in CRH neurons (Cullinan, 2000; Cullinan et al., 2008), and
administration of a GABA
A
receptor antagonist to the PVH results in HPA axis activity,
including the up-regulation of CRH gene expression and an increase in the concentration
of circulating plasma adrenocorticotropic hormone (ACTH) (Bali and Kovács, 2003) and
corticosterone (Cole and Sawchenko, 2002). On the other hand, administration of GABA
into the third ventricle decreases ACTH secretion into the general circulation (Makara
and Stark, 1974) and CRH secretion into the hypophysial portal vein (Plotsky et al., 1987;
Hillhouse et al., 1989). These data indicate that CRH neuronal activity is under tonic
GABAergic inhibition, and that the removal of this inhibition is crucial for HPA axis activity.
Our results corroborate these reports indicating that GABAergic afferents are a crucial
component in controlling the activity of the CRH neuron.
Catecholaminargic Innervation of the PVHmpd is Predominantly Adrenergic
While E constitutes approximately 5-10% of the total CA content of the brain (Martin et al.,
2001), the role of E as opposed to NE in response to physiological stressors is unclear
(Martin et al., 2001; Ritter et al., 2011). Adrenergic inputs preferentially innervate the
parvicellular region of the PVH, including the PVHmpd (Swanson et al., 1981; Cunningham
et al., 1990), and are crucial in regulating a plethora of physiological processes (Guyenet
et al., 2013), whereas noradrenergic inputs innervate both the parvi- and magnocellular
divisions (Swanson et al., 1981; Cunningham and Sawchenko, 1988), and are also
involved in the response to psychogenic stressors (Rinaman, 2011).
62
We found that a greater number of catecholaminergic terminals in the PVHmpd contain
PNMT than DBH only, which is consistent with a greater innervation of adrenergic
greater release of corticosterone than the molar equivalent of NE (Leibowitz et al.,
1988). Catecholaminergic inputs are necessary for the HPA axis response to glycemic
challenges (Ritter et al., 2001, 2003; Khan et al., 2007, 2011), and adrenergic neurons
are preferentially recruited in response to 2DG-induced glucoprivation (Ritter et al., 1998).
Furthermore, hindbrain adrenergic neurons are heavily involved in regulating vasomotor
that adrenergic inputs to the neuroendocrine PVH are more salient than noradrenergic
inputs for relaying purely physiological stressors. It should be noted, however, that it has
(Guyenet et al., 2013).
While NE is certainly involved in activating the HPA axis following physiological stressors,
it is also involved the emotional and cognitive aspects of the stress response (Rinaman,
2011). Hindbrain NE cell groups not only project to the PVH, but also to the amygdala
(Petrov et al., 1993; Rinaman, 2011) and the BST (Banihashemi and Rinaman, 2006;
Rinaman, 2011) as well, both of which are involved in the processing of psychogenic
stressors. Thus, while both E and NE contribute to CRH neuronal activity, E may be more
salient in relaying physiological stressors.
63
Catecholaminergic Pre-Synaptic Terminals in the PVHmpd Contain VGluT2
VGluT2 mRNA is found in most of the adrenergic cell groups, as well as in A2 neurons,
though its expression appears preferential to C1 neurons (Stornetta et al., 2002).
Neurons from C1 project heavily to the PVHmpd (Swanson et al., 1981; Cunningham et
al., 1990) and make asymmetric synapses with their targets, which are structurally similar
to glutamatergic synapses (Sheng and Kim, 2011; DePuy et al., 2013).
We found VGluT2 protein in CA terminals in the PVHmpd, indicating a glutamatergic
phenotype (Stornetta et al., 2002; Guyenet et al., 2013). Of the total VGluT2 population,
approximately 25% also contained PNMT and/or DBH. Roughly 4% of the total VGluT2
population contained DBH only (indicating noradrenergic terminals) while the remaining
21% occurred in adrenergic PNMT-containing terminals. Previous reports suggest that
catecholaminergic neurons engage glutamatergic interneurons (Herman et al., 2003), but
our results indicate that at least a subset of the CA inputs contain glutamate and directly
innervates the PVHmpd.
Finally, the populations analyzed in this study contain a number of other neurochemicals
(Sawchenko et al., 1985; Ceccatelli et al., 1989; Fekete et al., 2004; Das et al., 2007;
Parker et al., 2013) that may be recruited differentially in response to various stressors
(Figure 2.10). Thus, it is likely that particular combinations of activated inputs act to convey
the appropriate magnitude.
64
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73
Chapter 3: Acute Glycemic Stressors Alter Terminal Activation Patterns in
the Paraventricular Nucleus of the Hypothalamus in a Stimulus-Intensity
Dependent Manner
Introduction
Exposure to a stressor, real or perceived, initiates the hypothalamic-pituitary-adrenal (HPA)
axis through the activation of corticotropin-releasing-hormone (CRH) neurons in the medial
dorsal parvocellular part (mpd) of the paraventricular nucleus of the hypothalamus (PVH),
ultimately resulting in the release of glucocorticoids from the adrenal cortex (Ulrich-Lai and
Herman, 2009; Aguilera and Liu, 2012; Bains et al., 2015). This response is fundamental
for maintaining homeostasis. Studies from our lab and others have established the critical
role that hindbrain catecholaminergic neurons play in HPA responses to acute glycemic
challenges (Ritter et al., 2001, 2003; Khan et al., 2011). Ablation of these inputs abolishes
both the HPA axis response and the phosphorylation of extracellularly-regulated kinases
1&2 (pERK1/2) in CRH neurons that follows exposure to a acute glycemic perturbations
(Khan et al., 2011).
In the CRH neuron ERK1/2 are phosphorylated in response to a number of stimuli (Khan et
al., 2011) and in turn phosphorylate other protein kinases, as well as synapsin I (Jovanovic
et al., 1996, 2000; Cesca et al., 2010; Campos et al., 2013). Synapsins are neuronal
phosphoproteins involved in the modulation of synapse function and neuronal plasticity
with immunohistochemistry has been used to determine pre-synaptic terminal activation
(Campos et al., 2013). Upon axon terminal depolarization, synapsin I is phosphorylated
74
by various protein kinases at a number of different phosphorylation sites (Cesca et al.,
2010; Giachello et al., 2010; Campos et al., 2013). Phosphorylation by pERK1/2 at
serines 62 and 67 alters the interaction of synapsin I with both G-actin and synaptic
vesicles (Jovanovic et al., 1996, Cesca et al., 2010), releasing the vesicles from the actin
cytoskeleton and increasing the number of vesicles available for release (Chi et al., 2003;
Cesca et al., 2010; Kushner et al., 2005; Valente et al., 2012).
Both glutamate (Makara and Stark, 1974; Darlington et al., 1989; Ziegler and Herman,
2000; Herman et al., 2002; Ziegler et al., 2005) and GABA (Boudaba et al., 1996; Cole and
Sawchenko, 2002; Herman et al., 2002, 2004; Cullinan et al., 2008) play important roles
in the initiation of the HPA axis in response to a stressor. Glutamate terminals appose the
CRH neuron (Wittmann et al., 2005), and glutamate signaling is important for the initiation
of HPA axis activity (Daftary et al., 2000; Ziegler and Herman, 2000). Tonic activation of
GABA
A
receptors during exposure to a stressor inhibits CRH activity in the PVH (Kovács
(Cole and Sawchenko, 2002).
Catecholamine (CA) afferents from the hindbrain are crucial for transmitting information
about acute physiological stressors to the PVHmpd (Ritter et al., 2001, 2003, 2011; Li and
Ritter, 2004; Khan et al., 2007; Guyenet, et al., 2013; Lee et al., 2016). Both adrenergic
and noradrenergic afferents innervate the PVHmpd (Swanson et al., 1981; Sawchenko
and Swanson, 1982, 1983; Swanson and Sawchenko, 1983; Liposits et al., 1986b,
75
Cunningham and Sawchenko, 1988; Cunningham et al., 1990). The CA phenotypes
can be differentiated through the presence of certain biosynthetic enzymes; structures
containing both PNMT and DBH are likely adrenergic, while those containing DBH without
PNMT are noradrenergic (Swanson et al., 1981). Furthermore, the administration of
norepinephrine (NE) to the parvicellular PVH increases the frequency of excitatory post-
ionotropic glutamate receptor antagonists (Daftary et al., 2000).
Exposure to a stressor is communicated at the synapse through the release of different
combinations of chemical transmitters, which act on both pre- and post-synaptic receptors
(Wamsteeker and Bains, 2010). These release patterns depend on the population(s) of
afferents stimulated, past exposure to stressors, and the present and antecedent levels of
circulating glucocorticoids (Watts, 2005; Kuzmiski et al., 2010; Wamsteeker et al., 2010;
Inoue et al., 2013; Bains et al., 2015).
While much is known about the physiological effects of the stress response on the body
(Melmed et al., 2015), the mechanisms involved in the initiation of the HPA axis at the level
of the neuroendocrine CRH neuron are less clear (Watts, 2005). The simplest models of
CRH control would suggest that each input is received and summated independently
of other inputs, though it is evident that these afferents are highly interactive (Watts,
2005; Wamsteeker and Bains, 2010; Watts and Khan, 2013). How activity in pre-synaptic
terminals, and the interactions between these terminals, conveys information about the
76
response of the appropriate magnitude and duration remains unclear.
We used immunohistochemistry (IHC) combined with high-resolution image analysis
synaptic terminals in the PVHmpd in response to acute glycemic challenges. We show
that stressors of different magnitudes differentially recruit afferent populations in a stimulus
pre-synaptic terminals.
Materials and Methods
Animals
Twelve adult male Sprague-Dawley rats (~300g at time of surgery) (Harlan) were housed
two-to-a-cage in a climate controlled room (20-22°C) on a 12h light/dark cycle (lights on
at 0600h) with ad libitum access to food and water. Animals were allowed to acclimate
and were handled daily for at least ten days before any procedures began. All procedures
were approved by the local Institutional Care and Use Committee.
In Vivo Procedures
Surgery
Under anesthesia (mixture of 50% Ketamine, 25% Xylazine, 10% Acepromazine (KXA)),
dissolved in 0.9% sterile saline (v/v), delivered intramuscularly at 1 mL/kg body weight,
77
No. 508-001) terminating near the atrium, as previously described (Khan and Watts, 2004;
Khan et al., 2007, 2011). Animals were allowed to recover to pre-surgical body weight,
Induction of Glycemic Challenge and Blood Collection
Rats were administered an intravenous bolus of either 0.9% saline (1 mL/kg BW; n=4),
insulin (Regular Iletin II, 100 U/mL stock solution, 2 U/kg/mL, Eli Lilly; n=4), or 2DG
(250 mg/kg in 0.9% saline, Sigma-Aldrich; n=4). Food was removed at least 2 hours
before administration of any solution, and all procedures occurred between 1000h and
1400h. Blood samples for corticosterone (CORT) were collected into EDTA-coated tubes
immediately before (T0) and 30 minutes after (T30) administration of the experimental
solution. Immediately following the T30 blood collection animals received a bolus of
anesthesia (KXA, 1 mL/kg BW) directly down the catheter and were perfused. Plasma
was separated by centrifugation at 4°C and stored at -20°C until use.
Tissue Perfusion and Sectioning
Rats were transcardially perfused with 0.09% saline followed by 4% paraformaldehyde
in a sodium borate solution (pH 9.5), as described in Chapter 2. Brains were removed
sucrose (w/v). Brains were frozen with hexanes on dry ice before being cut coronally at
78
stored at -20°C in a cryoprotectant solution as described in Chapter 2 until further
processing. Seven series were used for IHC and one series was reserved to verify
cytoarchitectonics.
Assays
Blood glucose concentrations were determined using a commercially available blood
glucose monitor (AlphaTrak, Zoetis, Florham Park, NJ) according to the manufacturer’s
recommendations. A commercially available ELISA kit was used according to the
manufacturer’s protocols to determine circulating CORT (Abcam, Cambridge, MA, USA,
ab108821). The lower sensitivity limit each CORT ELISA was 0.3ng/mL, with an intra-
assay variability of 5%. All samples were measured in a single assay.
Immunohistochemistry
Sections were washed free of cryoprotectant with Tris-buffered saline (TBS, pH 7.4) (six
containing 2% normal donkey serum (Chemicon, Temecula, CA) and 0.3% (v/v) Triton
X-100 (Sigma Aldrich, 9002-93-1), and then incubated in the same blocking solution
containing combinations of primary antibodies (Table 3.1) for 72 hours at 4°C. Sections
were again washed in TBS and incubated overnight at 4°C in a solution of appropriate and
corresponding species-sourced secondary antibodies (Table 3.2). Sections were washed
79

Table 3.1 Primary antibodies used for immunohistochemistry
Primary Antibody Source, Species, & Clonality Titer
Millipore, Cat# MAB308
Mouse Monoclonal
1: 20,000
Anti-Neuropeptide Y
Millipore, Cat# AB1583
Sheep Polyclonal
1: 5,000
Anti-Phenylethanolamine
N-Methyltransferase
M.C. Bohn, Northwestern University
Rabbit
1: 10,000
Anti-phospho-p44/42 MAPK
(ERK1/2) (Thr202/Tyr204)
Cell Signaling Technology, Cat# 9106
Mouse Monoclonal
1 : 1,000
Anti-phospho-p44/42 MAPK
(ERK1/2) (Thr202/Tyr204)
Cell Signaling Technology, Cat# 9101
Rabbit Polyclonal
1: 1,000
Anti-phospho-Synapsin I
(pSer62/67)
Acris Antibodies San Diego,
Cat# Ap08742PU-N,
Rabbit Polyclonal
1 : 10,000
Anti-Synaptophysin
R&D Systems, Cat# AF5555,
Goat Polyclonal
1 : 1,000
Anti-Vesicular GABA
Transporter
Synaptic Systems, Cat# 131 002,
Rabbit Polyclonal
1 : 20,000
Anti-Vesicular Glutamate
Transporter 2
Millipore, Cat# AB2251,
Guinea Pig Polyclonal
1: 10,000
Table 3.2 Secondary Antibody Source Titer
(H+L) DyLight 405
Jackson ImmunoResearch
Cat# 705-475-147
1: 2,000
(H+L) Alexa 488
Jackson ImmunoResearch
Cat# 715-545-151
1: 2,000
(H+L) Alexa 488
Jackson ImmunoResearch
Cat# 711-545-152
1: 2,000

(H+L) Cy3
Jackson ImmunoResearch
Cat# 705-165-147
1: 2,000
(H+L) Cy3
Jackson ImmunoResearch
Cat# 711-165-152
1: 2,000
(H+L) Alexa 647
Jackson ImmunoResearch
Cat# 706-605-148
1: 2,000
(H+L) Alexa 647
Jackson ImmunoResearch
Cat# 713-605-003
1: 2,000
80
a solution of 50% TBS/50% glycerol and sealed with clear nail polish. Methodological
controls were run as in Chapter 2.
Confocal Microscopy
Laser Scanning Confocal Microscope, equipped with Zeiss AxioImager Z1 (Carl Zeiss
MicroImaging, Inc., Thorwood, NY). Three dimensional (3D) images were constructed
beads (FocalCheck Fluorescence Microscope Test Slides, Invitrogen, F36909) expressing
Images of the PVHmpd correspond to Level 26 of the Swanson Rat Brain Atlas (2004).
Image Analysis
Analysis of images was performed using the Volocity software Analysis Suite module
(version 6.3, PerkinElmer, Waltham, MA), as described in Chapter 2. The total number of
Nissl-stained sections.
81
3
pSynI, vesicular glutamate transporter 2 (VGluT2), and/or vesicular GABA transporter
3
3
3
, and were counted both through the
Volocity software and by hand through the extent of the Z stack. Only those structures at
We analyzed the number of co-occurring labels (Dunn et al., 2011) by identifying double-,
triple-, and quadruple-labeled structures and terminals using the methods described in
Chapter 2. The total number of structures (single-, double-, triple-, and quadruple-labeled),
3
.
Administration of saline did not elicit pERK1/2 activity, thus appositions could not be
voxel, with no unlabeled space between) contact with a labeled soma, and were counted
2

as electron microscopy would need to be employed. However, the use of IHC to evaluate
putative contacts with a labeled neuron has been established in the literature (Wittmann
82
et al., 2005; Flak et al., 2009; Bouyer and Simerly, 2013).
Statistics
followed by Tukey’s multiple comparison post hoc test (JMP Software, SAS Institute, Inc.,
Cary, NC). A p Results
Insulin and 2DG Elicit Graded HPA Axis Responses
In accordance with the known physiological outcomes of these two challenges (Cryer,
1997; Ritter et al., 2001), as well as previous results from our lab (Khan et al., 2011), we
p p<0.0001) blood
p<0.005)
p<0.05). While the
plasma corticosterone concentration was elevated in response to insulin, there was no
statistical difference between saline and insulin (Figure 3.1). This likely is due to the
variance of data points in the insulin condition.
83
3
, p 3
, p greater following 2DG as compared to insulin (p<0.0001) (Figure 3.1), consistent with
previous results (Khan et al., 2011).
Figure 3.1 Insulin and 2DG elicit graded HPA axis responses
Insulin and 2DG elicited graded glycemic responses in terms of both blood glucose (a) and the
level of plasma CORT (b). Insulin resulted in a decrease in blood glucose, while 2DG resulted in a
dramatic increase in blood glucose (a). Insulin and 2DG increased the level of plasma CORT in a
graded fashion (b). The number of pERK1/2-labeled soma mirrored the graded increase in plasma
CORT (c,a). Photomicrographs show pERK1/2-labeled soma in the PVH (c,b-d). Scale bar represents
p p<0.005, ++p<0.0005, +++p<0.0001, ns Acute Glycemic Challenges Do Not Alter Do Not Alter the Number of Terminals
Containing Synaptophysin in the PVHmpd
Synaptophysin is a glycoprotein found in the membrane of synaptic vesicles (Wiedenmann
84
and Franke, 1985), and is widely used as a marker of pre-synaptic terminals (Masliah et
al., 1990; Thiel, 1993; Flak et al., 2009; Kwon and Chapman, 2011; Chavan et al., 2015).
3
3
3
) altered the number
of pre-synaptic terminals labeled for synaptophysin (Figure 3.2).
Figure 3.2 Pre-synaptic terminal innervation is not altered by acute glycemic challenges
There was no difference in the number of pre-synaptic terminals in the PVHmpd between
conditions (a). Photomicrographs illustrate an orthogonal view of synaptophysin expression in
a selected region of interest in the PVHmpd in the X, Y, and Z dimensions following treatment
(b-d 2
ns Phosphorylation of Synapsin I by phospho-ERK1/2 in the PVHmpd Increases Following
Acute Glycemic Challenge
Detecting phosphorylated synapsin I (pSynI) with IHC has been used to indicate activity in pre-
synaptic terminals (Chi et al., 2003; Cesca et al., 2010; Campos et al., 2013). Compared to control
3
) we found that the number of pre-synaptic terminals
3
, p 3
, p<0.005).
85
There was no difference in the number of terminals containing pSynI between insulin and 2DG
(Figure 3.3).
Figure 3.3 Phosphorylation of synapsin I increases following an acute glycemic challenge
a). Images illustrate an orthogonal view of pSynI expression
in the PVHmpd in the X, Y, and Z dimensions following treatment (b-d p<0.005.
Acute Glycemic Challenges Alter Detectable Levels of VGluT2 and VGAT
and VGAT terminals in the PVHmpd (Figure 3.4). The total number of detectable VGluT2
3
, p<0.0001)
3
, p<0.005) compared to saline-treated animals
3
). There was no difference in detectable VGluT2 between
3
) compared to both saline
3
, p 3
,
p<0.05). There was no difference in detectable VGAT between saline and insulin.
86
2DG, but not Insulin, Increases the Co-Occurrence of pSynI and VGluT2 in the PVHmpd
and the total number of terminals containing VGluT2 following the administration of either
insulin or 2DG, only administration of 2DG increased the number of terminals in which
3
), compared to saline
3
, p 3
,
p<0.01) (Figure 3.5). There was no difference in the number of double-labeled terminals
between saline and insulin.
Figure 3.4 Acute glycemic challenges alter detectable levels of VGluT2 and VGAT
Acute glycemic challenges differentially altered the number of detectable vesicular transporters (a,e).
Insulin and 2DG equally increased VGluT2 expression as compared to saline (a), while only 2DG
e). Images illustrate
an orthogonal view of VGluT2 (b-d) and VGAT (f-h) expression in the PVHmpd following treatment.
p p p<0.005, +++p<0.0001.
87
Figure 3.5 2DG increases the co-occurrence of phospho-synapsin I and VGluT2
88
2DG increased the co-occurrence of pSynI and VGluT2 in terminals in
the PVHmpd (a). Images show an orthogonal view of pSynI (b,f,j) and
VGluT2 (c,g,k) expression the PVHmpd in the X, Y, and Z dimensions
following treatment. Images d,h, and l show both pSynI and VGluT2
and Images e,i, and m illustrate only those terminals that express
p<0.01
The Number of Pre-Synaptic Terminals Containing DBH is Reduced Following 2DG
3
3
, p 3
, p<0.05) (Figure
3.6). There was no difference in DBH-immunoreactive terminals between saline and
3
3
, p<0.05)
3
, p<0.05). Again, there was no difference
between insulin and saline. There was no change in PNMT labeling in terminals between
any condition.
Acute Glycemic Challenges Increase the Co-Occurrence of Catecholamines with VGluT2,
but not with pSynI
number of terminals that contained both DBH and VGluT2 following the administration
3
, p 3
, p 3
) (Figure
3.7). There was no difference in the total number of terminals in which both DBH and
VGluT2 occurred between insulin and 2DG, nor was there any difference in the number
of noradrenergic terminals that co-occurred with VGluT2 between any condition. There
89
3
3
3
).
Figure 3.6 2DG reduces DBH-immunoreactivity in terminals in the PVHmpd
(a,b). This was apparent in both adrenergic (a) and noradrenergic (b) pre-synaptic terminals.
There was no change in PNMT in pre-synaptic terminals (c p<0.05, ns 3
) the number of
3
, p<0.05). There was no statistical difference in the number
90
3
).
Finally, there was no difference in the number terminals that contained both DBH and
pSynI between any condition (Figure 3.8).
Figure 3.7 Both 2DG and insulin increase detectable VGluT2 within adrenergic terminals
The co-occurrence of VGluT2 with total DBH increased following either insulin or 2DG (a), but only
b). Neither challenge altered the
number of detectable VGluT2 in noradrenergic pre-synaptic terminals (c p<0.05, ns Figure 3.8 Catecholaminergic inputs do not use phospho-synapsin I to relay stimulus
intensity
The Number of Appositions to pERK1/2-Expressing Soma in the PVHmpd is not Altered
by Acute Glycemic Challenges
As administering saline did not elicit phosphorylation of ERK1/2, we could only evaluate
appositions following insulin or 2DG to pERK1/2-labeled soma. The number of pSynI
91
The number of pre-synaptic terminals labeled for both DBH
and pSynI was not altered in response to either insulin or
2DG, indicating that the pERK1/2-mediated phosphorylation
of synapsin I is not a mechanism used by the hindbrain
catecholamines to relay stressor intensity to the PVHmpd.
ns
2
)
2
). Furthermore, there was no difference in
2
)
2
), nor was there a difference in VGluT2
2
appositions/100um
2
) (Figure 3.9).
Figure 3.9 Acute glycemic challenges do not alter appositions to phospho-ERK1/2-
labeled soma
VGAT appositions were most prevalent to soma. There was no statistical difference in the number of
pSynI (a), VGluT2 (b), or VGAT (c) appositions with pERK1/2-labeled soma in the PVHmpd following
either insulin or 2DG. Appositions to soma in the saline condition could not be analyzed, as there was
no pERK1/2 labeling. ns Discussion
We used IHC and high-resolution image analysis techniques to analyze changes in the
response to stimuli of different magnitudes to determine if the afferent terminals of the
different stimuli to the neuroendocrine PVH.
92
We found that acute glycemic stressors differentially alter inputs to the PVHmpd in a
stimulus-intensity dependent manner (Figure 3.10). Notably, the number of detectable
VGluT2 and VGAT in pre-synaptic terminals is altered to favor excitation, without altering
the total number of pre-synaptic terminals. We also found that acute glycemic challenges
increase pERK1/2-induced phosphorylation of synapsin I (Cesca et al., 2010; Campos
et al., 2013). This increase occurs at least in partly in glutamatergic terminals following
2DG, but not insulin, suggesting that 2DG may recruit additional pathways apart from the
well-documented CA inputs (Ritter et al., 1998). The increase in pSynI occurs in a non-CA
population in both cases. While insulin and 2DG utilize the hindbrain CA afferents (Ritter
et al., 1998, 2001, 2003; Khan et al., 2007, 2011), 2DG appears to result in a greater
response from these neurons (Figure 3.6). We also found an increase in the number of
both catecholaminergic and non-catecholaminergic terminals containing VGluT2.
Overall, our results indicate that the components of the pre-motor control network are
recruited differentially and dynamically as a function of the intensity of the stimulus to
result in an post-synaptic output of the appropriate magnitude (Figure 3.10).
Figure 3.10 Stressors of different magnitudes differentially recruit afferent populations
93
Different afferent populations are
recruited in a stimulus intensity-
dependent manner. Both insulin and
2DG increase the separate VGluT2-
(green triangle) and pSynI-containing
(orange triangle) populations. 2DG
further increases a population of
terminals containing both VGluT2 and
pSynI (yellow triangle), along with
resulting in a large response from CA
terminals (blue triangle). 2DG, but
not insulin, decreases the number of
detectable VGAT (red triangle).
Insulin and 2DG Elicit Graded HPA Axis Responses
Both insulin and 2DG activate the HPA axis through initiation of CRH activity in the
PVHmpd (Brown, 1962; Ritter et al., 1998; Donovan and Watts, 2014). Administration of
indicating the initiation of the counterregulatory HPA axis response. We found a dramatic
increase in blood glucose following intravenous administration of 2DG, and both insulin
and 2DG increase the number of soma containing pERK1/2 in a graded manner in the
PVHmpd (Figure 3.1). Our lab has shown that phosphorylation of these kinases is crucial
for CRH peptide release and synthesis following a glycemic challenge (Khan et al., 2007),
and accurately tracks the amount of circulating adrenocorticotropic hormone (ACTH)
(Khan et al., 2011). Because ACTH triggers the release of corticosterone from the adrenal
received 2DG as compared to both saline and insulin, though there was no statistical
difference in corticosterone concentration between saline and insulin (Figure 3.1). The
small sample size (n=4/group) and high SEM in the insulin-treated group may contribute
to the unexpected lack of statistical difference between the saline and insulin conditions.
Additionally, it is worth noting that unlike ACTH, plasma corticosterone is not a direct
measure of HPA axis activity. The concentrations of the two in the blood are not invariably
linked, as the release of corticosterone relies on both ACTH-dependent and –independent
94
mechanisms (Bornstein and Chrousos, 1999, Bornstein et al., 2008). As such, a
dissociation between the levels of ACTH and corticosterone can occur due to a number
of different factors, including the metabolism of circulating ACTH and the amount of
corticosterone bound to its binding protein, transcortin (Bornstein et al., 2008). When
bound, circulating corticosterone is rendered physiologically inactive (Lippman and
Thompson, 1974). Therefore, while the lack of statistical difference plasma corticosterone
concentration between the saline and insulin conditions was unexpected, given that the
average corticosterone concentration was around 60 ng/mL in response to saline and 300
ng/mL in response to insulin, the most likely reason for the lack of difference is the small
sample size.
The Total Number of Pre-Synaptic Terminals in the PVHmpd is not Altered in Response
to Acute Glycemic Challenges
There was no change in the number of terminals labeled for synaptophysin following
administration of either insulin or 2DG (Figure 3.2), indicating that the total number of pre-
synaptic terminals was not altered. Both acute and chronic exposure to a stressor can
alter the number of total pre-synaptic inputs to the neuroendocrine PVH (Nikonenko et al.,
2003; Verkuyl et al., 2004, 2005; Flak et al., 2009; Levy and Tasker, 2012). In organotypic
hippocampal slice cultures, exposure to a combination of anoxia and hypoglycemia
followed by electrical stimulation has been show to result in axonal outgrowths which
contact post-synaptic densities in as little as 30 minutes (Nikonenko et al., 2003). Exposure
to the chronic variable stress paradigm results in an increases in total synaptophysin
95
expression in the PVH, interpreted as an increase in total presynaptic terminal innervation
(Flak et al., 2009).
Acute Glycemic Challenges Increase pERK1/2-Mediated Phosphorylation of Synapsin I
In response to either insulin or 2DG,the number of terminals containing pSynI increased
the number of pSynI-labeled terminals between the two challenges (Figure 3.3). While
pERK1/2 phosphorylates SynI (Jovanovic et al., 1996, 2000), SynI is phosphorylated by
many other kinases as well (Cesca et al., 2010). The pSynI antibody used in this study only
labels SynI that has been phosphorylated at serines 62 and 67 by pERK1/2. This indicates
that while the phosphorylation of synapsin I by pERK1/2 may be a mechanism involved in
the response to a glycemic challenge at the level of the PVHmpd, this phosphorylation is
not part of the mechanism by which differences in stimulus intensity are conveyed.
Acute Glycemic Challenges Alter Detectable Vesicular Transporters to Favor Excitation
Glutamate and GABA are integral components in the initiation of the stress response
and synaptic plasticity (Boudaba et al., 1996; Ziegler and Herman, 2000; Cole and
Sawchenko, 2002; Herman et al., 2002, 2004; Ziegler et al., 2005; Cullinan et al., 2008).
While synaptic plasticity is most often studied in the hippocampus, neurons of the PVH
exhibit substantial plasticity following chronic stress (Herman et al., 2008; Flak et al.,
2009; Wamsteeker and Bains, 2010). Less is known about fast homeostatic plasticity in
response to an acute stressor (Liu, 2003; Hartman et al., 2006). In this study we found
96
that acute glycemic challenges increased the number of detectable VGluT2 and decreased
the number of detectable VGAT terminals, as compared to controls. These changes
appear to be modulated by the strength of the stimulus.
The quantity of neurotransmitter released by both excitatory (Liu et al., 1999; Liu, 2003;
al., 2006; Hartmann et al., 2008) pre-synaptic terminals varies in response to changes
in network activity. One mechanism that appears to be involved in the quantity of
neurotransmitter released at both glutamatergic and GABAergic synapses is the number
Wilson et al., 2005).
Increasing the number of VGluT1 on the synaptic vesicles increases the quantity of
glutamate within the vesicle, and subsequently the quantity released upon terminal
activation (Wilson et al., 2005), as measured by an increase in the amplitude of the
al., 2005; Erickson et al., 2006). Reducing the number of VGluT1s on vesicles has the
opposite effect (Wilson et al., 2005; Moechars et al., 2006), and in the total absence of
Hyper-excitation in differentiated cultures leads to an increase in both VGluT2 mRNA and
protein expression, while decreasing the level of excitation reduces the levels of both
97
(De Gois et al., 2005). This indicates that VGluTs are crucial components of glutamatergic
responses, and that VGluT2 in particular dynamically responds to the strength of the
stimulus, potentially as a way to result in a response of the proper magnitude from the
post-synaptic cell.
While the aforementioned studies examined changes in VGluTs ranging from 3-11 days
in vitro, we found that the number of detectable VGluT2s in the PVHmpd increased in
vivo within 30 minutes of administering either insulin or 2DG. There was no difference in
detectability between the two conditions (Figure 3.4). Therefore, while VGluT2 is involved
in the response to stressors, the lack of difference in detectability suggests that this
work together with other mechanisms.
GABA-mediated synaptic transmission (Hartmann et al., 2008) and the number of VGAT
(Kang et al., 2003) dynamically change as a function of local synaptic activity in the
hippocampus. Reducing the number of VGATs drastically reduces neurotransmitter
release in GABAergic neurons, by lowering the rate of spontaneous release frequency
quantity of transmitter released. We found a substantial decrease in the number of
detectable VGATs in the PVHmpd following 2DG, but not insulin (Figure 3.4). This would
then suggest that similar to VGluT2, VGAT expression is altered in response to a stressor,
98
and that the two vesicular transporters are altered dynamically to relay the strength of the
stimulus.
The mechanisms involved in mediating changes in vesicular transporters during this
time frame remain unclear. Changes in vivo could be the result of an alteration in the
number of synaptic vesicles within the terminal (through vesicular exo- and endocytosis),
or the incorporation of additional individual transporters into existing synaptic vesicle
membranes (Santos et al., 2009). Synaptic vesicle membrane proteins are transported
from the trans-Golgi network to the plasma membrane in either constitutive or regulated
secretory vesicles, providing the opportunity for the incorporation of individual vesicular
transport proteins to new or existing synaptic vesicles (Santos et al., 2009). However, the
mechanisms involved in incorporating individual proteins into synaptic vesicles remain
unclear (Santos et al., 2009).
Another potential method for altering the expression of vesicular transporters may be
through activity-dependent bulk endocytosis (Cousin, 2009). During periods of high activity
large areas of the plasma membrane are retrieved to form endosomes, as opposed to
the retrieval of single vesicles (Cousin, 2009). Recycling vesicles through an endosome
may allow synaptic proteins and/or cargo to be redistributed as necessary (Santos et
al., 2009). However, elucidating the mechanisms behind short-term changes in vesicular
transporter expression will require further studies.
99
receptors for glutamate (Liu et al., 1999; McAllister and Stevens, 2000; Liu, 2003; Wilson
et al., 2005) and GABA (Engel et al., 2001; Liu, 2003). When post-synaptic receptors
are not saturated by ligand binding, changes in the amount of neurotransmitter released
could be a mechanism to alter the post-synaptic response (Liu et al., 1999; Liu, 2003;
De Gois et al., 2005; Wilson et al., 2005; Hartman et al., 2006; Hartmann et al., 2008).
Altering the number of glutamate and GABA vesicular transporters on the synaptic vesicle
may be one way to dynamically alter the response of the post-synaptic target to stimuli of
varying intensities.
Overall, our present results, taken with the literature, suggests that VGluT2 and VGAT are
magnitude from the post-synaptic target. The overall degree of excitation may be a
function of both increased glutamate and decreased GABA activity (Figure 3.10).
Although the number of terminals labeled for both pSynI and VGluT2 were increased
following insulin or 2DG administration, the co-occurrence of the two increased only in
response to 2DG. While ablation of the hindbrain CAs results in the total loss of pERK1/2
of pERK1/2 in the PVH following 2DG (Khan et al., 2011). Phosphorylation of ERK1/2 is
not exclusive to CA activity, and occurs in response to a wide variety of stimuli (Khan
100
et al., 2011). 2DG is an antimetabolite that impairs glucose uptake by the cell through
competitive inhibition of a glycolytic enzyme (Brown, 1962; Ritter and Dinh, 1994), and
induces Fos expression in many different brain regions outside of the hindbrain CA nuclei
(Ritter and Dinh, 1994), as well as many non-catecholaminergic neurons in the A2 and
A7 nuclei (Ritter et al., 1994).
Our results indicate that insulin increases the pERK1/2-mediated phosphorylation
of synapsin I in a non-glutamatergic population, while 2DG increases pSynI in both
glutamatergic and non-glutamatergic populations, indicating that the afferents recruited
by the two stressors are not identical, supporting a model in which 2DG may recruit
additional pathways alongside the hindbrain CAs (Ritter and Dinh, 194; Ritter et al., 1994).
2DG Elicits a Greater Response from Catecholaminergic Neurons than does Insulin-
Induced Hypoglycemia
Hindbrain CAs are necessary for a full neuroendocrine response to glycemic challenges
(Ritter et al., 2001, 2003; Khan et al., 2011), but are not required for the response to
psychogenic stressors, such as forced swim (Ritter et al., 2003) or the chronic variable
stress paradigm (Flak et al., 2009), nor are they necessary for circadian rhythms of HPA
axis activity (Ritter et al., 2003). We analyzed the CA biosynthetic enzymes DBH and
PNMT to examine stimulus-induced alterations in the CA response to glycemic challenges
saline and insulin. There was no change in PNMT content in terminals following any
101
treatment (Figure 3.6).
DBH is found within the synaptic vesicle in two forms, both soluble and bound to the
membrane (Kobayashi et al., 1994). Along with the release of neurotransmitters, some
of the soluble DBH is released into the synaptic cleft during synaptic vesicle exocytosis
and as such a decrease in detectable DBH expression has been interpreted to indicate
an increase in CA exocytosis (Sorimachi and Yoshida, 1979; Li and Ritter, 2004). Given
that we were able to label less DBH in CA terminals following 2DG but not insulin, this
suggests that these two glycemic stressors may differentially recruit CAs, either in the
timing or magnitude (or both) of the CA response. However, by evaluating only DBH we
afferents.
To address this we evaluated changes in CA terminals that both contained and lacked
insulin, drastically reduced DBH content in noradrenergic terminals, indicating a role for
NE, and potentially E. In response to an acute glycemic challenge, we saw no change in
PNMT (Figure 3.6). Unlike DBH, PNMT is located only in the cytoplasm and is therefore not
a lack of change was not unexpected, as 30 minutes is not long enough to translate and
transcribe new PNMT in the CA soma and transport it approximately 13
102
millimeters to axon terminals in the PVHmpd (Swanson, 2004), given that PNMT is
transported along the axon at the rate of 2 mm/day (Abrahamsson, 1979).
We were unable to determine if insulin and 2DG differentially recruit NE or E with this
preparation. However, it is clear that adrenergic neurons are crucial for physiological
responses to stimuli that threaten systemic homeostasis (Guyenet et al., 2013), and that
1998).
Detectable VGluT2 Increases in both Adrenergic and Non-Catecholaminergic Populations
VGluT2 in response to both insulin and 2DG (Figure 3.4) that appears to occur both within
in catecholaminergic and non-catecholaminergic populations. Despite the decrease in the
increase in the number of terminals that contained both DBH and VGluT2 in response
inputs (Figure 3.7). Adrenergic neurons of the hindbrain express VGluT2 mRNA in the
soma (Stornetta et al., 2002) and VGluT2 protein is present in pre-synaptic terminals in
the PVH (Wittmann et al., 2005; Chapter 2). These neurons make asymmetric synapses,
suggestive of conventional glutamate synapses (Milner et al., 1988), and at least a subset
of C1 neurons use glutamate as a transmitter in the dorsal motor nucleus of the vagus
(DePuy et al., 2013). Whether this is the case in the PVH remains unclear.
103
The number of VGluT2-positive CA terminals did not account for all of the VGluT2-
positive terminals within the PVHmpd, however, indicating that VGluT2 increases in non-
catecholaminergic populations as well. VGluT2 is ubiquitous throughout the diencephalon
post-synaptic potential frequency, suggesting that CAs increase excitatory drive (Daftary
receptor antagonists or ionotropic glutamate receptor antagonists (Daftary et al., 2000).
Both of these receptros are present within the CRH neuron (Cummings and Seybold, 1988;
al., 2002) and functional (Daftary et al., 2000) relationships between glutamate and CAs.
Overall, an increase in the number of detectable VGluT2 in both catecholaminergic and
non-catecholaminergic terminals suggests an overall increase in excitatory tone in the
PVHmpd.
pERK1/2-Mediated Phosphorylation of Synapsin I is Not Used by Catecholamines to
Convey Stimulus Strength
We evaluated the co-occurrence of DBH with pSynI to determine if the CA afferents utilized
pERK1/2 phosphorylation of SynI. There was no change in co-occurrence following either
insulin or 2DG as compared to the saline condition, indicating that the pERK1/2-
104
-mediated increase in pSynI (Figure 3.3) did not occur in a catecholaminergic population
following either challenge. As stated above, synapsin I is phosphorylated by many other
kinases as well (Cesca et al., 2010). Therefore, while these results indicate that synapsin
I is not phosphorylated by pERK1/2 in the CA neurons, it is still possible that synapsin
I is phosphorylated by other phospho-kinases as part of the signaling cascade within
catecholaminergic terminals.
The overall analysis of afferent terminals indicates that the network controlling the
neuroendocrine neurons within the PVHmpd is multifaceted and complex. While we
stressor to the PVH.
Appositions to Soma in the PVHmpd are Not Altered Following Acute Glycemic Challenges
The chronic variable stress paradigm increases the number of both glutamatergic and
catecholaminergic appositions with the soma of CRH neurons in the PVH, interpreted
to mean an increase in excitatory innervation through an increase in synaptic contact to
pSynI appositions to pERK1/2-labeled soma in the PVHmpd following either insulin or
2DG administration but found no statistical differences between the conditions (Figure
3.8). We could not evaluate appositions following saline, as there was no pERK1/2
105
The most likely explanation for these results is that there was no change in the number
of synaptic contacts. We found no change in the total number of terminals innervating
the PVHmpd (Figure 3.2), therefore we would not expect to see a change in the number
of appositions. This suggests that any changes in excitatory or inhibitory signaling at the
synapse are occurring through alterations within the existing pre-synaptic terminals, such
as through VGluT2 and VGAT (Figure 3.4) and other signaling properties, and not with
overall synaptic contact.
Conclusion
In summary, this study aimed to address how pre-synaptic terminals within the pre-motor
control network relay stressors of different intensities to the neuroendocrine PVH to result
in an HPA axis response of the appropriate magnitude. Acute glycemic challenges do
not alter the total number of pre-synaptic terminals in the PVHmpd or the number of
appositions to the soma of neurons within the PVHmpd. These stressors do, however,
alter the detectable numbers of both VGluT2 and VGAT molecules within pre-synaptic
terminals. Presumably this leads to changes in glutamatergic and GABAergic activity in a
way that favors excitation and increases the dynamic range of glutamatergic activity, in both
catecholaminergic (predominantly adrenergic) and non-catecholaminergic populations.
We also show that both insulin and 2DG increase MAPK-induced phosphorylation of
synapsin I, though this appears to occur in two different populations, neither of which are
106
neurons (Ritter et al., 1998), indicating that this stressor activates afferents other than the
crucial hindbrain CAs (Ritter et al., 1998, 2001, 2003; Khan et al., 2007, 2011). Additionally,
2DG appears to result in a greater response from the hindbrain CA inputs (Figure 3.6).
In conclusion, it appears as though components of the pre-motor control network are
recruited differentially and dynamically as a function of the intensity of the stimulus to
result in an post-synaptic output of the appropriate magnitude. Future studies will need to
be employed to clarify the mechanisms underlying these changes.
107
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115
Chapter 4: Corticosterone Maintains Excitatory Input to Corticotropin-
Releasing Hormone Neurons in the Paraventricular Nucleus of the
Hypothalamus
Introduction
Glucocorticoid release is the end result of hypothalamic-pituitary-adrenal (HPA) axis
activation. Activity of the HPA axis is initiated by neuroendocrine corticotrophin-releasing-
hormone (CRH) neurons in the medial dorsal parvocellular part (mpd) of the paraventricular
nucleus of the hypothalamus (PVH) (Rivier and Vale, 1983; Aguilera and Liu, 2012).
Activity-induced release of CRH at the median eminence stimulates corticotropes in the
anterior pituitary to release adrenocorticotropic hormone (ACTH), which travels through
the bloodstream to reach the adrenal cortex, resulting in the release of glucocorticoids
(Keller-Wood and Dallman, 1984). The adrenal cortex releases corticosterone (CORT) in
response to stimulation (Dallman et al., 1987). This steroid hormone has diverse actions
within the body, including mobilizing energy stores, suppressing the immune system, and
acting as a negative feedback signal to both the CRH neurons and the anterior pituitary
(Keller-Wood and Dallman, 1984; Dallman et al., 1987; Charmandari et al., 2005). In
response to acute stressors this response is fundamental, but the effects of chronic
exposure to high levels glucocorticoids are deleterious to the long-term maintenance of
homeostasis within the body.
Appropriate levels of circulating CORT are important for maintaining the integrity of stress-
response mechanisms within in the PVH (Watts and Sanchez-Watts, 1995; Tanimura and
116
the circadian cycle (Dallman et al., 1987; Watts et al., 2004) they operate within a
narrow physiological range (Akana et al., 1985). Alterations outside of this range have
consequences on both stress-response mechanisms and overall homeostasis (Keller-
Wood and Dallman, 1984; Akana et al., 1985; Tanimura and Watts, 1998; Tanimura et al.,
1998; Charmandari et al., 2005).
CORT acts as a negative feedback signal to its own release, and this feedback varies
as a function of time (Keller-Wood and Dallman, 1984). Fast feedback occurs within
seconds to minutes and inhibits the release, but not the synthesis, of CRH (Keller-Wood
and Dallman, 1984). This likely occurs through non-genomic actions, which implies the
existence of different receptor mechanisms than the traditional glucocorticoid receptors
(Keller-Wood and Dallman, 1984). Fast negative feedback actions may occur through
glucocorticoid-induced activation of the endocannabinoid (eCB) system (Hill et al., 2010;
Tasker and Herman, 2011; Hill et al., 2012). CORT also acts in an intermediate time
domain of between 2-10 hours (Keller-Wood and Dallman, 1984). During this time frame
both the release and synthesis of CRH are inhibited (Keller-Wood and Dallman, 1984).
Both fast and intermediate feedback are likely to occur under physiological conditions,
but slow CORT feedback is more likely under pathological or pharmacological conditions.
Slow CORT feedback occurs when CORT concentrations are elevated over a number of
days, and appears to inhibit not only CRH, but the release and synthesis of ACTH as well
(Keller-Wood and Dallman, 1984; Akana et al., 1985; Swanson and Simmons, 1989;
117
Watts and Sanchez-Watts, 1995; Dallman et al., 2004; Watts, 2005). On the other hand,
the loss of circulating CORT through adrenalectomy (ADX) increases CRH mRNA and
peptide in the PVH, as well as increasing ACTH synthesis and release in the pituitary
(Sawchenko et al., 1984; Akana et al., 1985; Sawchenko, 1987a; Swanson and Simmons,
1989 Watts and Sanchez-Watts, 1995; Brown and Sawchenko, 1997).
Glucocorticoids bind to both mineralocorticoid (type I) and glucocorticoid (type II)
receptors, approximately 10x greater than to glucocorticoid receptors (Reul et al., 2000).
Approximately 80% of the mineralocorticoid receptors are occupied at the circadian
nadir (Ruel and de Kloet, 1985; Reul et al., 2000). At higher levels of circulating CORT,
such as those seen during the circadian peak or in response to a stressor, glucocorticoid
receptors become occupied (Ruel and De Kloet, 1985; Ruel et al., 1987). Glucocorticoid
receptors are ubiquitous throughout the brain (Fuxe et al., 1985; Ruel and De Kloet,
1985, 1986; Chao et al., 1989), including in CRH neurons (Agnati et al., 1985; Fuxe et
al., 1985; Liposits et al., 1987; Sawchenko, 1987b; Sawchenko and Bohn, 1989) and
hindbrain catecholamine (CA) neurons (Agnati et al., 1985; Fuxe et al., 1985; Härftstrand
et al., 1986; Liposits et al., 1987; Sawchenko, 1987b; Sawchenko and Bohn, 1989).
While glucocorticoids are capable of directly affecting CRH activity (Kovács et al., 1986;
Sawchenko, 1987b), their actions on the inputs to the PVHmpd appear to be more likely
to alter CRH activity (Dallman et al., 1994; Popoli et al., 2011; Myers et al., 2014).
118
Both the current and antecedent CORT environments affect CRH neuronal responses to
stressors (Watts, 2005) by altering the glutamate, GABA, and CA components of the pre-
motor control network within the PVHmpd (Cullinan and Wolfe, 2000; Miklós and Kovács,
2002; Verkuyl and Joëls, 2003; Verkuyl et al., 2004, 2005; Herman et al., 2005; Ziegler
et al., 2005; Yang et al., 2007, 2008). Glucocorticoid-mediated changes in the activity of
these afferents subsequently alter how the CRH neuron “reads” the inputs to produce a
response (Tanimura and Watts, 1998; Tanimura et al., 1998; Watts, 2005).
Physiological levels of CORT appear to be crucial for the maintenance of glutamatergic
components in the hippocampus (De Kloet et al., 1998; Joëls et al., 2001; Joëls, 2008),
though chronic exposure to supraphysiological levels of glucocorticoids also results in
hippocampal cell degeneration (Sapolsky et al., 1985). However, CORT-mediated effects
on neuronal activity is not the same across various nuclei within the brain (Swanson and
Simmons, 1989; Watts and Sanchez-Watts, 1995), and as such CORT-mediated changes
in hippocampal structure and/or function may not be applicable to the PVH.
Exposure to the chronic variable stress paradigm alters the glutamatergic component of
the pre-motor control network. In this model, animals are exposed to unpredictable bouts
of various psychogenic stressors for several days. Exposure to this paradigm increases
VGluT2-labeled appositions to CRH neurons, consistent with an increase in excitatory
signaling (Flak et al., 2009). This paradigm also decreases mRNA expression of the
NMDA receptor subunit NR2B, which increases Ca
2+
signaling and is also consistent with
119
an increase in excitatory signaling (Ziegler et al., 2005). However, this model utilizes
physiological stressors (Herman et al., 2003; Ulrich-Lai and Herman, 2009). ADX without
CORT replacement does not change the mRNA expression of any NMDA receptor
subunit in the PVHmpd (Ziegler et al., 2005). Little is known about pre-synaptic changes
in glutamate function as a result of CORT.
The effects of CORT on GABAergic mechanisms in the PVH are more clear. ADX
enhances GABAergic tone in the PVHmpd and increases the number of GABAergic
synapses with the CRH neuron (Majewska et al., 1985; Miklós and Kovács, 2002; Verkuyl
and Joëls, 2003). In the absence of a stressor, the loss of circulating CORT increases
GABAergic suppression (Yang et al., 2007, 2008). Chronic stress, on the other hand,
decreases inhibitory tone within the PVHmpd (Cullinan and Wolfe, 2000; Verkuyl et al.,
2004, 2005), consistent with an increase in excitatory tone (Flak et al., 2009). However,
the effects of CORT on pre-synaptic GABAergic input in the absence of a stressors are
less clear.
Catecholaminergic neuronal activity is also affected by the concentration of circulating
CORT (Jhanwar-Uniyal and Leibowitz, 1989; Kaminski and Watts, 2012). These neurons
contain glucocorticoid receptors at the soma (Agnati et al., 1985; Fuxe et al., 1985;
Härftstrand et al., 1986; Liposits et al., 1987; Sawchenko, 1987b; Sawchenko and Bohn,
120
1989), but are also affected by indirect glucocorticoid-mediated changes. Hindbrain CA
neurons are important for the transmission of systemic stressors to the PVHmpd (Ritter
et al., 2001, 2003, 2011; Li and Ritter, 2004; Khan et al., 2007; Guyenet et al., 2013;
Lee et al., 2016). Given the important role CORT plays in overall metabolic functioning
(Dallman et al., 1993), it is possible that changes in peripheral energy metabolism alter CA
innervation of the PVH as well (Watts, 2005). CORT also regulates interactions between
the CRH neuron (Jhanwar-Uniyal and Leibowitz, 1986; Cummings and Seybold, 1988;
Feuvrier et al., 1998, 1999).
While many studies have investigated the role of CORT in the regulation of the HPA axis in
terminals in the PVHmpd in the absence of a stressor. To address this, we performed
bilateral ADX with either no CORT or high CORT replacement. We found that CORT is
necessary to maintain excitatory innervation to the PVHmpd as a whole, and particularly
that from adrenergic inputs. Chronically high circulating CORT, however, decreases the
number of VGluT2-labeled appositions to CRH soma without altering total input to the
PVHmpd.
121
Materials and Methods
Animals
Twenty-four adult male Sprague-Dawley rats (~300g at time of surgery) were housed two-
to-a-cage in a climate-controlled room (20-22°C), on a 12h light/dark cycle (lights on at
6:00am) with ad libitum access to food and water. Animals were acclimated and handled
daily for at least ten days before any procedures. All procedures were approved by the
local Institutional Care and Use Committee.
In Vivo Procedures
Surgery
Animals were randomly assigned to one of three groups: ADX without CORT replacement,
oxygen anesthesia (2-3%) we performed bilateral ADX and immediately implanted
animals with either 0mg (ADX+0) or 100mg (ADX+100) slow-release (21 days) CORT
pellets (Innovative Research of America, Sarasota, FL, USA). The third group received a
sham operation in which the adrenal glands remained intact (sham ADX). Animals healed
individually overnight before returning to two-to-a-cage housing for the duration of the
experiment. Animals were weighed daily and allowed unrestricted access to food and
water, and a 0.9% saline solution.
122
Tissue perfusion, blood collection, and tissue sectioning
Rats were transcardially perfused seven days after the surgery with 100mL of cold 0.09%
saline, followed by 500mL of cold 4% paraformaldehyde in a sodium borate solution
(pH 9.5). Blood samples were collected into EDTA-coated tubes directly from the aorta
immediately before exsanguination with saline. Plasma was separated by centrifugation
at 4°C and stored at -20°C until use. The thymus gland was removed following perfusion
and placed in 0.9% saline, then blotted dry and weighed. The weight of the thymus gland
was normalized to mg/100g body weight.
the addition of 12% sucrose (w/v). Brains were frozen and cut with a sliding microtome
extent of the PVH and stored in a cryoprotectant solution (50% 0.05 M sodium-phosphate
buffer, 30% ethylene glycol, 20% glycerol, v/v) at -20°C until further processing. Seven
series were reserved for immunohistochemistry (IHC) and one series was reserved for
thionin staining, to verify cytoarchitectonics.
Corticosterone Assay
The concentration of circulating CORT was determined using a commercially available
ELISA kit (Abcam, Cambridge, MA, USA, ab108821) following the manufacturer’s
protocol. The lower sensitivity limit for the CORT ELISA was 0.3ng/mL, with an intra-
assay variability of 5%. All samples were run in a single assay.
123
Immunohistochemistry
2 hours at RT in a blocking solution of TBS plus 2% normal donkey serum (v/v) (Chemicon,
Temecula, CA) and 0.3% Triton X-100 (v/v) (Sigma Aldrich, 9002-93-1). Sections were
incubated for 72 hours at 4°C in the same blocking solution containing combinations of
primary antibodies (Table 4.1), and then incubated in a solution of corresponding species-
sourced secondary antibodies (Table 4.2) overnight at 4°C. Sections were washed in TBS
were coverslipped with a solution of 50% TBS/50% glycerol and sealed with clear nail
polish. Methodological controls were run as in Chapters 2 and 3.
Table 4.1 Primary antibodies used for immunohistochemistry
Primary Antibody Source, Species, & Clonality Titer
Millipore, Cat# MAB308,
Mouse Monoclonal
1 : 20,000
Anti-Phenylethanolamine
N-Methyltransferase
M.C. Bohn, Northwestern University,
Rabbit
1 : 10,000
Anti-phospho-p44/42 MAPK
(ERK1/2) (Thr202/Tyr204)
Cell Signaling Technology,
Cat# 9106, Mouse Monoclonal
1: 1,000
Anti-phospho-Synapsin 1
(pSer62/67)
Acris Antibodies San Diego,
Cat# Ap08742PU-N, Rabbit Polyclonal
1: 10,000
Anti-Synaptophysin R&D Systems, Cat# AF5555,
Goat Polyclonal
1: 5,000
Anti-Vesicular GABA
Transporter
Synaptic Systems, Cat# 131 002,
Rabbit Polyclonal
1: 20,000
Anti-Vesicular Glutamate
Transporter 2
Millipore, Cat# AB2251,
Guinea Pig Polyclonal
1: 10,000
124
Table 4.2 Fluorescently-conjugated secondary antibodies
Secondary Antibody Source Titer

IgG (H+L) DyLight 405
Jackson ImmunoResearch,
Cat# 705-475-147
1 : 2,000
IgG (H+L) Alexa 488
Jackson ImmunoResearch
Cat# 715-545-151
1 : 2,000
IgG (H+L) Cy3
Jackson ImmunoResearch
Cat# 711-165-152
1 : 2,000
Pig IgG (H+L) Alexa 647
Jackson ImmunoResearch,
Cat# 706-605-148
1 : 2,000
Confocal Microscopy
Images were captured with a 40x (numerical aperture 1.3) oil-corrected objective, as
previously described (Chapters 2 and 3), using a Zeiss 700 Laser Scanning Confocal
Microscope, equipped with Zeiss AxioImager Z1 (Carl Zeiss MicroImaging, Inc., Thorwood,
absence of a light source (pixel value of 0 on an 8 bit scale) as well as at a level of light
that fully saturated the pixels (pixel value of 255 on an 8 bit scale) to allow us to capture
remained the same for the duration of the imaging session to be able to analyze different
the entire depth of the tissue section. Images of the PVHmpd correspond to Level 26 of
the Swanson Rat Brain Atlas (2004).
125
Image Analysis
Images were analyzed and adjusted in the same manner as Chapters 2 and 3 using
the Volocity software Analysis Suite module (version 6.3, PerkinElmer, Waltham, MA).
well as those structures that were double- and triple-labeled, and the co-occurrence of
3
as structures labeled for synaptophysin, pSynI, VGluT2, and/or VGAT, with a maximum
3
3
3
, and were counted both through the software and by hand
through the extent of the Z axis. Only those structures two standard deviations or above
The number of structures, pre-synaptic terminals, and soma was counted and normalized
3
. Appositions to labeled CRH soma were counted using the
methods of Bouyer and Simerly (2013), and normalized to the number of appositions per
2
neuronal input, making direct (voxel-to-voxel, with no unlabeled space between) contact
structural analysis such as electron microscopy would need to be employed. However,
the use of IHC to evaluate putative contacts with a labeled neuron has been established
126
in the literature (Wittmann et al., 2005; Flak et al., 2009; Bouyer and Simerly, 2013).
Statistics
followed by Tukey’s multiple comparison post hoc test (JMP Software, SAS Institute, Inc.,
Cary, NC). A p Results
The Concentration of Circulating CORT Affects Body and Thymus Weight
compared to both the sham ADX and ADX without CORT replacement (ADX+0) animals.
There was no difference in body weight between the sham ADX and ADX+0 animals
(Figure 4.1).
Figure 4.1 127
By day 3, chronically high levels
of circulating CORT (black circles)
compared to controls only (gray circles).
total body weight as compared to both
control and ADX conditions (open circles),
despite ad libitum access to food and
water, and 0.09% saline, for the duration
of the experiment. All data presented as
mean ± SEM. +p<0.005, +++p<0.0001
Clamping circulating CORT either above or below the physiological range inversely
altered the weight of the thymus gland. Compared to the sham ADX animals (160.41 ±
gland (197.71 ± 7.65 mg/100mg body weight, p thymus weight (43.03 ± 5.13 mg/100mg body weight) compared to both sham ADX
(p<0.0001) and ADX+0 (p<0.0001) animals (Figure 4.2).
Figure 4.2 The concentration of plasma corticosterone inversely affects thymus weight
Levels of Circulating CORT Alter the Detectability of CRH Peptide in the PVHmpd
3
3
, p<0.01).
However, there was no difference in the number of detectable CRH soma between sham
128
The concentration of plasma CORT (a) inversely
gland (b). ADX without CORT replacement
circulating CORT, but did not abolish it completely.
*p<0.05, ***p<0.01, +p<0.005, +++p<0.0001
3
) and ADX+0, nor between sham ADX and ADX+100.
(147.62 ± 8.16 mean gray units) compared to both sham ADX (72.46 ± 3.22 mean gray
units, p<0.005) and ADX+100 (59.56 ± 4.44 mean gray units, p<0.005). There was no
Figure 4.3 Circulating corticosterone alters CRH peptide detectability in the PVHmpd
Circulating CORT Increases Synaptophysin Labeling in the PVHmpd
3
) compared to sham ADX animals
3
, p<0.005). There was no difference in the number of
synaptophysin-labeled terminals between the sham ADX and ADX+100 animals (87.11 ±
3
). There was no difference in the number of pre-synaptic terminals
129
ADX increased the detectable
number of soma expressing
CRH peptide (a), as well as the
(b). There was no difference in
either measurement between
the sham and high CORT
conditions. Images (c-e) show
the population of CRH neurons
in the PVH, by condition. Images
were taken using a 20x objective,
Medial is to the right. ***p<0.01,
+++p<0.0001.
between the ADX+0 and ADX+100 animals (Figure 4.4).
Figure 4.4 Corticosterone is necessary to maintain full pre-synaptic terminal innervation
to the PVHmpd
ADX reduced synaptophysin expression in the PVHmpd compared to the sham condition (a).Increasing the
amount of circulating CORT had no effect. Images (b-d) show an orthogonal view of synaptophysin-labeled
terminals in a selected region of interest in the PVHmpd in the X, Y, and Z dimensions. All images are
p<0.005.
Circulating CORT Alters VGluT2 and VGAT Detectability in the PVHmpd
3
3
,
p 3
, p=0.001). There was
no difference in the number of detectable VGluT2 between sham ADX and ADX+100
(Figure 4.5).
ADX+0 reduced the number of detectable VGAT in the PVHmpd (126.73 ± 6.30
3
3
, p<0.05).
There was no statistical difference in the number of detectable VGAT between ADX+0
130
ADX+0 Sham
ADX
ADX+100
0
20
40
60
80
100
Terminals/100 m
3
+
ADX+0 Sham ADX ADX+100
Z
Z
Y
X
a)
b
c d
3
), nor between the sham ADX and
ADX+100 animals (Figure 4.5).
Figure 4.5 Corticosterone maintains the excitatory/inhibitory balance within the PVHmpd
and high CORT conditions (a following ADX as compared to the high CORT condition (b). Images c-e show an orthogonal view of
VGluT2-labeled terminals and Images f-h of VGAT-labeled terminals in the PVHmpd in the X, Y, and
p<0.05, **p=0.01, +++p<0.0001.
Loss of Circulating CORT Alters the Catecholaminergic Structures in the PVHmpd
3
3
,
p 3
, p<0.01). There was no
difference in the number of total DBH-labeled structures between sham ADX and ADX+100
(Figure 4.6).
131
Figure 4.6 Adrenalectomy reduces DBH-immunoreactivity in adrenergic structures
both the sham ADX and high CORT conditions (a). However, there was no change in DBH-labeling
in noradrenergic terminals (b), indicating that the change occurs only in adrenergic structures. There
was no change in the total number of structures that contained PNMT (c). ***p<0.01, +++, p<0.0001.
Because DBH is found in both noradrenergic and adrenergic neurons, but PNMT is found
only in adrenergic neurons (Swanson et al., 1981), we analyzed DBH-labeled structures
3
),
there was no change in DBH in noradrenergic structures (containing DBH but not PNMT)
3
) or ADX+100 animals (37.43 ±
3
). Furthermore, there was no change in PNMT in adrenergic
3
) or ADX+100 (53.05 ±
3
) conditions compared to the sham ADX animals (53.46 ± 1.59
3
) (Figure 4.6).
PNMT-labeled structures in ADX+0 animals (65.26 ± 9.17 mean gray units) compared
to ADX+100 (101.59 ± 7.30 mean gray units, p<0.05), but not to the sham ADX animals
132
PNMT label between sham ADX and ADX+100, nor was there any difference in the
Figure 4.7 Circulating CORT Increases the Co-Occurrence VGluT2 in Catecholamine Pre-Synaptic
Terminals
ADX+0 decreased the co-occurrence of total DBH and VGluT2 in pre-synaptic terminals
3
3
,
p 3
). There was no difference in
double-labeled pre-synaptic terminals between sham ADX and ADX+100. There was no
change in the number of noradrenergic pre-synaptic terminals (labeled with DBH but not
3
)
3
) compared to sham ADX (27.48 ± 1.64
3
) (Figure 4.8).
ADX+0 decreased the number of adrenergic pre-synaptic terminals containing VGluT2
3
) as compared to both sham ADX (54.39 ± 2.32
3
, p 3
, p<0.05).
133
label as compared to the high CORT
condition (a). Presumably this indicates
a decrease in total PNMT content.
intensity of the DBH labeling (b).
*p<0.05
There was no difference in the number of adrenergic pre-synaptic terminals containing
VGluT2 between the sham ADX and ADX+100 (Figure 4.8).
Figure 4.8 Corticosterone is required to maintain expression of VGluT2 within adrenergic
pre-synaptic terminals
Circulating CORT Alters the Presence of phospho-Synapsin I in Glutamatergic, but not in
Catecholaminergic, Pre-Synaptic Terminals
While there was a trend towards a decrease in the total number of pre-synaptic terminals
3
) compared to
3
) and ADX+100 (139.65 ± 12.89
3
134
contained VGluT2 (a e). There was no decrease
in VGluT2 labeling in noradrenergic pre-synaptic terminals (not shown). Images b-d show
an orthogonal view of the co-occurrence of DBH and VGluT2, and images f-h show co-
p<0.05, ***p<0.01.
However, the effect of CORT on pSynI-containing pre-synaptic terminals became
total number of pre-synaptic terminals labeled for both pSynI and VGluT2 (86.08 ± 3.11
3
3
,
p 3
).
There was no difference in the number of double-labeled pre-synaptic terminals between
ADX+100 and ADX+0 animals (Figure 4.9).
3
) there was no
difference in the number of terminals abeled for both pSynI and DBH in either the ADX+0
3
).
Furthermore, there was no difference in the number of terminals that contained both
3
), ADX+0 (38.75 ± 3.28
3
3
) animals. There was no
change in the number of terminals containing pSynI, DBH, and VGluT2 between any
condition (Figure 4.10).
Elevating CORT Above the Physiological Range Reduces the Number of Both
Synaptophysin and VGluT2 Appositions with the Soma of CRH Neurons
There was no statistical difference in the number of synaptophysin appositions with the
2
) and either sham
2
2
).
135
Figure 4.9 Adrenalectomy decreases the co-occurrence of pSynI and VGluT2 in
pre-synaptic terminals
Figure 4.10 Corticosterone does not alter phospho-synapsin I detectability in
catecholaminergic pre-synaptic terminals
136
Removing circulating CORT decreased
the number of pre-synaptic terminals
containing pSynI in the PVHmpd, but it
a). As compared to the
reduced the co-occurrence of pSynI and
VGluT2 in pre-synaptic terminals (b).
Images show an orthogonal view of pSynI
(c,g,k) and VGluT2 (d,h,l) by condition, as
well as the co-occurrence of the two labels
(e,i,m). Images f,j,n highlight only the co-
occurrence in white. Scale bars represents
p<0.05.
Altering the level of circulating
CORT had no effect on the co-
occurrence of pSynI with DBH
pre-synaptic terminals (a), or
with DBH pre-synaptic terminals
also containing VGluT2 (b). ns,

of synaptophysin appositions to CRH soma by approximately 20% compared to the sham
ADX condition (p<0.05) (Figure 4.11).
ADX+0 did not alter the number of VGluT2 appositions with the soma of CRH neurons
compared to the sham ADX animals, though supraphysiological CORT reduced the
2
) compared to both
2
, p<0.05) and ADX+0 (17.44 ± 1.03
2
, p<0.05) (Figure 4.11). We were not able to analyze DBH appositions
pSynI, or PNMT appositions.
Figure 4.11 Chronically high circulating corticosterone reduces excitatory innervation to
CRH soma
Chronically high levels of circulating CORT reduced the number of synaptophysin appositions to CRH
soma as compared to the sham ADX condition (a). The number of VGluT2 appositions was also
reduced (b), indicating that chronically high CORT reduces excitatory input at the soma. Adrenalectomy
did not alter the number of either type of apposition to the soma, therefore any changes in innervation
may occur at non-somal locations. We could not evaluate catecholaminergic appositions. *p<0.05.
137
Discussion
of glutamate, GABA, and CA pre-synaptic terminals in the pre-motor control network
synaptophysin and found that circulating CORT is necessary for the maintenance of pre-
synaptic terminal innervation to the PVHmpd, but has no effect on pre-synaptic terminal
innervation when chronically high. We also found that CORT is required to maintain the
excitatory/inhibitory balance within the PVHmpd, in both CA and non-CA populations,
but has less of an effect on this balance at chronically high concentrations. Pre-synaptic
glutamate components appear to be more sensitive to change in the CORT environment
than do pre-synaptic GABA components. While the loss of CORT did not alter the number
response to high CORT.
These data corroborate other reports indicating that CORT affects a plethora of inputs
to CRH neuron (Mulders et al., 1997; Miklós and Kovács, 2002; Verkuyl et al., 2004,
2005; Yang et al., 2007, 2008; Flak et al., 2009; Kaminski and Watts, 2012). Furthermore,
circulating concentrations (Kaminski and Watts, 2012).
138
Levels of Circulating CORT Have Diverse Effects on the Maintenance of Homeostasis
The effects of CORT on the immune system, including the thymus gland, are well-
documented (Dougherty, 1952; Akana et al., 1985; Watts et al., 1995). The concentration
of circulating CORT is inversely related to thymus gland weight (Akana et al., 1985;
Dallman et al., 1987; Watts et al., 1995, 2002). The thymus gland expresses only very low
levels of mineralocorticoid receptors, thus the glucocorticoid receptor is the dominant type
of corticosteroid receptor (Dhabhar et al., 1993; Dallman et al., 1994). Glucocorticoids
are crucial for regulated apoptosis in the thymus gland, and chronically high levels of
these hormones result in increased apoptosis and the subsequent involution of the gland
(Talaber et al., 2015). Loss of circulating glucocorticoids reduces apoptosis and leads
to hypertrophy of the thymus gland (Talaber et al., 2015). As would be expected, we
CORT plays an important role in metabolic regulation (Dallman et al., 1993), and altering
levels of circulating CORT outside of the physiological range affects body weight (Akana
et al., 1985; Kaminski and Watts, 2012). At high doses CORT suppresses feeding, as
well as mobilizing energy stores from the liver, adipose tissue, and muscle (Dallman et
al., 1993). Conversely, CORT stimulates feeding at low doses (Dallman et al., 1993).
body weight compared to both the sham ADX and ADX+0 animals, but that there was no
difference in body weight between the sham ADX and ADX+0 animals (Figure 4.1).
139
25 ng/mL of CORT was still detectable (Figure 4.1). While this is below the normal
physiological range (Akana et al., 1985), this may be the reason there was no difference
in body weight between the sham and ADX+0 animals.
of the labeling (Figure 4.3). Fluorescent intensity has been used as an indicator of the
quantity of labeled antigen present in a structure (Bohn et al., 1984; Khan et al., 2011),
an increase in peptide accumulation. This is consistent with previous reports showing
enhanced immunostaining and an increase in the number of neurons in the PVHmpd
that contain the CRH peptide (Sawchenko, 1987a), as well as an increase in CRH mRNA
(Young et al., 1986; Swanson and Simmons, 1989; Watts et al., 1995), following ADX.
High levels of circulating CORT reduced the number of soma in which CRH was detected
Chronically high levels of circulating CORT reduces CRH mRNA in the PVH (Swanson
and Simmons, 1989). Dexamethasone, a synthetic CORT analog, reduces the ADX-
induced increase in CRH peptide in the PVHmpd (Sawchenko, 1987b). The PVH is most
sensitive to CORT concentrations less than 120ng/mL (Watts and Sanchez-Watts, 1995),
and CORT-mediated effects begin to plateau around 130ng/mL (Swanson and Simmons,
1989).
140
CORT is Required to Maintain Total Pre-Synaptic Terminal Innervation to the PVHmpd
in the PVHmpd (Figure 4.4), but did not change in the total number of synaptophysin-
labeled appositions to CRH soma (Figure 4.11). This suggests that the loss of total pre-
synaptic innervation affects both CRH and non-CRH neurons in the PVHmpd, and that
changes in innervation likely occur at non-somal CRH regions, which receive the majority
appositions (Chapter 2).
Chronic exposure to supraphysiological levels of CORT had no effect on total pre-synaptic
terminal innervation, but did reduce total synaptophysin appositions. Since there was no
substantial loss in the number of pre-synaptic terminals within the PVHmpd as a whole this
may suggest that 1) that chronically high levels of circulating CORT only affect innervation
to CRH neurons, rather than other populations within the PVHmpd, 2) that while there is
a loss of pre-synaptic terminal innervation to the soma of CRH neurons, there may be
a compensatory increase in innervation to the non-somal regions and/or other neuronal
populations. Further studies will need to be employed to clarify which, if either, of these
possibilities is correct.
Physiological levels of CORT are necessary to maintain innervation patterns to many
regions involved in HPA axis regulation, including the hippocampus and the bed nuclei
of the stria terminals (BST) (Sapolsky et al., 1991; Mulders et al., 1997). The loss of
circulating CORT results in apoptotic-like degeneration of both cell bodies and dendrites
141
in hippocampal regions (Sapolsky et al., 1991; de Kloet et al., 1998; Wossink et al., 2001;
Joëls, 2008) and decreases the density of BST-originating afferents to the whole PVH,
with the greatest loss being to the PVHmpd (Mulders et al., 1997). Our data suggest
that CORT is required to maintain full pre-synaptic terminal innervation patterns to the
PVHmpd as well, and supports data indicating that low CORT operates using different
mechanisms than both normal and high CORT conditions (Swanson and Simmons, 1989;
Watts, 2005; Kaminski and Watts, 2012).
One caveat to note is that this particular lot of the antibody against synaptophysin appears
to have had reduced sensitivity, as the number of synaptophysin-labeled pre-synaptic
the control conditions of the previous two chapters. However, this lot was the only one
used within this particular experiment, therefore the results are internally consistent.
Furthermore, there appeared to be no aberrant labeling of non-pre-synaptic terminal
structures.
Circulating CORT Alters the Excitatory/Inhibitory Balance within the PVHmpd
The concentration of circulating CORT has an effect on the balance of excitatory and
Miklós and Kovács, 2002; Verkuyl and Joëls, 2003; Verkuyl et al., 2004, 2005; Herman et
al., 2005; Ziegler et al., 2005; Yang et al., 2007, 2008). Both glutamatergic and GABAergic
mechanisms are important in the regulation of the HPA axis (Daftary et al., 2000;
142
Cole and Sawchenko, 2002; Herman et al., 2004), and are highly plastic in response to
stressors (Flak et al., 2009; Kuzmiski et al., 2010; Bains et al., 2015; Chapter 3).
PVHmpd compared to both the sham and high CORT animals (Figure 4.5). This loss may
be consistent with the loss of total pre-synaptic terminal innervation. However, there was
no loss in the number of VGluT2 appositions to the soma of CRH neurons following ADX,
suggesting that any down-regulation of VGluT2-expressing afferents to the CRH neuron
occurs at non-somal sites. ADX does not alter NMDA receptor subunit mRNA expression
in the PVH, nor is it expected to alter the NMDA receptor itself either in number or ability
(Ziegler et al., 2005), which may indicate that pre-synaptic glutamate function is more
amenable to change than are post-synaptic mechanisms.
Chronically high levels of circulating CORT had no effect on the total number of detectable
with CRH soma (Figure 4.5). While exposure to chronic stress may increase excitatory
tone through an increase in VGluT2 appositions (Flak et al., 2009), the present study
aimed to examine changes in the absence of a stressor. A reduction in VGluT2 appositions
likely indicates either changes in vesicular transporter number or a loss of pre-synaptic
terminals, and may be an adaptive mechanism to decrease excitatory tone, but to allow
for an increase in excitatory tone if faced with a subsequent stressor. There are presently
no studies that have examined the effects of ADX with or without CORT replacement on
143
glutamate input to CRH neurons in the PVHmpd. Therefore, the relationship between pre-
synaptic VGluT2 expression and post-synaptic glutamate receptors (Ziegler et al., 2005)
GABAergic tone in the PVHmpd by increasing the frequency of GABAergic signaling
(Majewska et al., 1985; Miklós and Kovács, 2002; Verkuyl and Joëls, 2003). ADX also
increases the number of GABAergic synaptic contacts with the CRH neuron, as well
as the area of those pre-synaptic terminal boutons (Miklós and Kovács, 2002). Due to
soma.
to the PVHmpd (Mulders et al., 1997), consistent with an increase in CRH activity
(Sawchenko, 1987a) and potentially a loss of VGAT expression, as seen here. Given
that we saw no statistical change in the number of pre-synaptic terminals containing
VGAT, despite a reported loss of innervation (Mulders et al., 1997), this may indicate
that changes in innervation from the BST may be compensated for by other mechanisms
and/or that overall changes in GABAergic signaling may be mediated primarily by post-
synaptic rather than pre-synaptic mechanisms.
144
Supraphysiological levels of CORT did not alter VGAT as compared to the sham condition,
but did increase the number of detectable VGAT as compared to the ADX condition (Figure
4.5). Chronic stress reduces the frequency of miniature inhibitory post-synaptic currents
in the parvocellular PVH (Verkuyl et al., 2004), most likely through a decrease in release
probability (Verkuyl et al., 2005). Furthermore, chronic stress down-regulates GABA
A
and Wolfe, 2000). However, these results were reported in response to chronic stress,
rather than in the absence of a stressor. Whether the increase in VGAT reported here is
due to an increase in the number of vesicular transporters or an increase in GABAergic
Taken together, these data indicate that glutamatergic mechanisms in pre-synaptic
structures may be more prone to change in response to the CORT environment than are
pre-synaptic GABAergic mechanisms. While GABA mechanisms are altered following
both ADX and chronic stress (Majewska et al., 1985; Cullinan and Wolfe, 2000; Miklós
and Kovács, 2002; Verkuyl and Joëls, 2003; Verkuyl et al., 2004; Verkuyl et al., 2005), we
report here that VGAT remains fairly stable. This may be a function of the important role
GABA plays in the overall maintenance of HPA axis activity (Cole and Sawchenko, 2002;
Kovács et al., 2004).
145
The Adrenergic Input to the PVHmpd Requires Circulating CORT
It is well-understood that glucocorticoids interact both directly and indirectly with hindbrain
CA neurons to affect both CA and CRH neuronal activity (Fuxe et al., 1985; Härftstrand
et al., 1986; Jhanwar-Uniyal and Leibowitz, 1989; Plotsky et al., 1989; Liposits et al.,
1987; Sawchenko, 1987; Sawchenko and Bohn, 1989; Yang et al., 2007, 2008). We
investigated the effects of CORT on catecholaminergic pre-synaptic terminals to the
immunoreactivity, but did not alter its detectability in structures that did not also contain
PNMT (noradrenergic structures) (Figure 4.6).
CORT directly affects hindbrain CA neurons by activating glucocorticoid receptors in
the soma (Fuxe et al., 1985; Härftstrand et al., 1986; Liposits et al., 1987; Sawchenko,
1987; Sawchenko and Bohn, 1989). Additionally, CORT affects overall metabolic function
(Dallman et al., 1993; Charmandari et al., 2005), and these hindbrain CA neurons are
crucial for relaying systemic (including metabolic) stressors to the PVH (Ritter et al., 2001,
2003, 2012; Li and Ritter, 2004; Khan and Watts, 2007; Guyenet et al., 2013; Lee et
al., 2016). It therefore seems likely that CORT-induced modulation of peripheral energy
metabolism peripheral modulation induced by CORT affects the signals sent to CRH
neurons (Watts, 2005).
CORT also affects the interaction between CA and CRH neurons through glucocorticoid
146
are the dominant subtype of adrenergic receptor in the PVH and are located both pre- and
post-synaptically, and appear to be most sensitive to the CORT environment (Jhanwar-
1986; Cummings and Seybold, 1988; Plotsky et al., 1989). This may be consistent with an
increase in CRH activity following ADX (Sawchenko, 1987a), and indicates that altering
adrenergic receptor number and function is another mechanism involved in producing an
appropriate CRH neuronal response to circulating CORT.
ADX increases the turnover of norepinephrine in the PVH through a glucocorticoid-
dependent mechanism (Rastogi et al., 1978; Pacak et al., 1995). We found a decrease
in the total detectable DBH content in adrenergic neurons, which is consistent with an
increase in CA exocytosis (Sorimachi and Yoshida, 1979; Li and Ritter, 2004). Epinephrine
plays an important role in relaying physiological stressors (Guyenet et al., 2013), and both
the development and maintenance of PNMT mRNA in the adrenal medulla and superior
cervical ganglia is dependent on circulating CORT (Bohn, 1983; Bohn et al., 1984; Jiang
et al., 1989). Furthermore, there is no immunochemical difference between the PNMT in
the adrenal medulla and that in the brain (Park et al., 1986), indicating that the antigenic
properties of the enzyme do not differ with location.
147
structures in any condition (Figure 4.7). Fluorescent intensity has been linked to the
amount of labeled antigen present in a labeled structure (Bohn et al., 1984; Khan et al.,
label in culture, and increases total PNMT in a glucocorticoid-dependent manner (Bohn
et al., 1984).
Removing circulating CORT also alters CA interactions with components of the pre-
motor control network, including GABA inputs (Yang et al., 2007, 2008). ADX increases
mediated excitation and a norepinephrine-mediated suppression of GABAergic inhibition
release (Yang et al., 2007), consistent with increased CRH activity (Sawchenko, 1987a).
VGluT2 following ADX as compared to the sham condition, particularly within the
adrenergic subset (Figure 4.8), highlighting the relationship between CA and glutamate
excitatory signaling in the PVH (Daftary et al., 2000).
148
At this time we are unable to say if the loss of VGluT2 is due to a decrease in vesicular
transporters or a loss of adrenergic pre-synaptic terminals. However, given the dependence
of PNMT development and maintenance in the adrenal medulla (Bohn, 1983; Bohn et
al., 1984; Jiang et al., 1989), and the lack of antigenic difference between PNMT in the
adrenal medulla and the brain (Park et al., 1986), it is possible that ADX may result in
the loss of adrenergic pre-synaptic terminals. However, further studies will need to be
employed.
Though chronically high levels of circulating CORT reduce CRH mRNA in the PVH, the
HPA axis response to a subsequent stressor is not diminished (Watts and Sanchez-
Watts, 2002). Exposure to the chronic variable stress paradigm increases both glutamate
and CA innervation to CRH neurons, consistent with an enhanced HPA axis response to
subsequent stressors (Flak et al., 2009; Zhang et al., 2010). This enhanced response
appears to be facilitated by the CA input (Zhang et al., 2010). While complex psychogenic
stressors such as the chronic variable stress paradigm appear to utilize the CA inputs
(Flak et al., 2009), these afferents are dispensable for many other psychogenic stressors
(Ritter et al., 2003), but are necessary for the HPA axis response to systemic stimuli (Ritter
et al., 2003; Li and Ritter, 2004; Khan and Watts, 2007; Guyenet, 2013; Lee et al., 2016).
Our lab has shown that these catecholaminergic inputs are crucial for the CORT-mediated
suppression of CRH activity when circulating levels of CORT are high (Kaminski and
Watts, 2012). However, in the absence of a subsequent stressor, chronically high levels
149
an increase in the PNMT enzyme, and subsequently epinephrine biosynthesis.
CORT is Required for Phosphorylation of Synapsin I in Glutamate Pre-Synaptic Terminals
We examined phosphorylated synapsin I (pSynI) in the PVHmpd. Synapsins are neuronal
phosphoproteins that modulate synapse function and plasticity (Cesca et al., 2010).
Quantifying pSynI-labeled pre-synaptic terminals with IHC has been used to determine the
activation state of those pre-synaptic terminals (Campos et al., 2013). The pSynI antibody
used here only labels synapsin I that has been phosphorylated by the phosphorylated
forms of extracellularly-regulated kinases 1&2 (pERK1/2) (Jovanovic et al., 1996, 2000;
Cesca et al., 2010), which are important protein kinases in cell signaling in the PVH in
response to glycemic stressors (Khan et al., 2011).
While ADX appears to have decreased the detectable amount of pSynI in pre-synaptic
terminals compared to both the sham and ADX+100 animals, this reduction was not
magnocellular PVH, but not in the parvicellular region (Nomura et al., 2000), while chronic
stress increases unphosphorylated synapsin I protein and mRNA (McEwen et al., 1987;
Wu et al., 2007) in the hippocampus. While these data are not from the parvicellular PVH,
they indicate CORT can alter the phosphorylation state of synapsins. However, we found
150
no difference in the number of pSynI-labeled pre-synaptic terminals between sham
ADX and the high CORT conditions in the PVHmpd. This indicates that information
about chronically high levels of circulating CORT are not conveyed to the PVHmpd in
pre-synaptic terminals that show changes in the phosphorylation state of synapsin I.
Furthermore, changes in pERK1/2-dependent synapsin I phosphorylation may not be
part of the mechanism that conveys information about metabolic changes that result from
chronically high concentrations of CORT.
There was a decrease in the total number of pre-synaptic terminals labeled for both pSynI
and VGluT2 following ADX as compared to the high CORT condition, but neither the ADX
nor high CORT conditions were statistically different than the sham ADX condition (Figure
4.9). A loss of pre-synaptic terminals double-labeled for pSynI and VGluT2 suggests that
glutamatergic pre-synaptic mechanisms may be particularly sensitive to CORT.
There was no change in the number of pre-synaptic terminals in which both pSynI and DBH
occurred, or in the number or pre-synaptic terminals labeled for pSynI, DBH, and VGluT2
between any condition (Figure 4.10). In Chapter 3 we show that pSynI labeling occurs
in both CA and non-CA populations, but that acute stressors affect pERK1/2-mediated
phosphorylation of synapsin I only in the non-CA population. The present results suggest
that pERK1/2-mediated phosphorylation of synapsin I in CA afferents is also unaffected
by circulating CORT, and may not be a mechanism involved in relaying stimuli from the
hindbrain CA neurons to the PVHmpd.
151
Conclusion
terminals in the PVHmpd in the absence of a stressor. CORT is required to maintain
full excitatory innervation to the PVHmpd, particularly that coming from the adrenergic
afferents. However, the loss of CORT does not appear to alter direct excitatory innervation
to CRH soma. It has been documented that ADX increases norepinephrine turnover in the
PVH through a glucocorticoid-dependent mechanism (Rastogi et al., 1978; Pacak et al.,
1995). However, the effects of glucocorticoids on epinephrine in the brain remain unclear.
Our data suggest that the VGluT2-containing adrenergic inputs (Stornetta et al., 2002)
require CORT for full innervation to the PVHmpd, indicating a relationship between CORT
and the excitatory components of the pre-motor control network.
Chronically high levels of circulating CORT, on the other hand, decrease VGluT2
innervation to CRH soma without altering total pre-synaptic input to the PVHmpd. Though
chronically high CORT reduces CRH mRNA in the PVH, the HPA axis response to a
subsequent stressor is not diminished (Watts and Sanchez-Watts, 2002). While we found
PNMT enzyme.
Overall, our data suggest that excitatory inputs to the PVHmpd are altered in response to
changing CORT environments to a greater degree than are inhibitory inputs. Furthermore,
152
while CA mechanisms are directly affected by CORT, the adrenergic subset appears to be
more sensitive to changes in the CORT environment than does the noradrenergic subset.
Given that ADX+100 did not alter the structure of pre-motor control network in a manner
opposite of that caused by ADX, it is likely that CORT utilizes different mechanisms at
low and high circulating concentrations to alter CRH neuronal activity (Swanson and
Simmons, 1989).
153
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Chapter 5: Summary and Conclusion
Dysfunctional adrenocorticotropic hormone (ACTH) and/or glucocorticoid secretion is
a feature of many clinical conditions. Much of this dysfunction involves important but
poorly understood neural control networks in the hypothalamus that integrate stress-
related information to drive ACTH secretion and GC output in a manner appropriate for
the stimulus, in terms of both the magnitude and duration of the response. The release of
glucocorticoids is ultimately initiated by the activity of the neuroendocrine corticotropin-
releasing hormone (CRH) neurons in the medial dorsal parvocellular part (mpd) of the
paraventricular nucleus of the hypothalamus (PVH), and dysfunctions in CRH activity can
lead to the abnormal glucocorticoid responses seen in a wide variety of diseases.
CRH neuronal activity is initiated through the input of various afferent sets, including
interactions between glutamate, GABA, and the hindbrain catecholamine (CA) afferents.
These particular inputs are important components of the CRH “pre-motor” control network,
and are involved in the response to many stressors. These inputs are critical in relaying
information about systemic stressors such as cardiovascular, immune, and metabolic
afferents, and the integration of these inputs by the pre-motor control network allows for
terminals within the pre-motor control network, any interactions between the components,
understand how the CRH neuron functions in both health and disease.
163
Summary
The work presented in this dissertation relied upon three experimental paradigms, a
transgenic animal model, the intravenous administration of insulin or 2DG to induce
acute glycemic stress, and long-term adrenalectomy with either no or high corticosterone
(CORT) replacement, coupled with high resolution three dimensional (3D) image analysis
GABA, and CA pre-synaptic terminals in the pre-motor control network, as well as to
various stimuli. To elucidate both the structural and functional interactions between these
synaptic terminals in the absence of a stressor. We used high resolution 3D image analysis
Crh-IRES-Cre;Ai14 mouse, wherein all CRH neurons
glutamate, GABA, and CA within the brain have been examined using electrophysiology
remains unclear.
We found that pre-synaptic terminals in the PVHmpd are divided almost evenly between
a glutamatergic and GABAergic phenotype, though GABAergic innervation is moderately
more prevalent. This is compatible with previous studies emphasizing the importance of
GABAergic inhibition in hypothalamic-pituitary-adrenal (HPA) axis regulation (Cole and
164
afferents, which appear to be the dominant CA input to the PVHmpd. These data are
consistent with reports showing the preferential innervation of adrenergic inputs to
we also show that that vast majority of appositions to CRH neurons occur at non-somal
locations.
After analyzing the structural relationships between the pre-synaptic terminal components
of the pre-motor control network in the absence of a stressor, we investigated functional
systemic stressors. We show that stressors of different magnitudes differentially recruit
afferent populations in a stimulus intensity-dependent manner that favors excitation by
altering detectable VGluT2 and VGAT within pre-synaptic terminals. Changes in the
number of vesicular transporters may correspond to changes in neurotransmitter release
As such, the mechanisms involved in this shortened time frame remain unclear. The
increase in VGluT2 occurs within both CA and non-CA pre-synaptic terminals. Within
underlining the importance of the relationship between glutamate and CA in excitatory

D-glucose (2DG) apparently also recruits non-CA neurons in the hindbrain (Ritter et al.,
1998), allowing for at least attenuated activation in the PVH following the removal of
are recruited in response to 2DG that are not recruited by insulin. Together these data
indicate that the components of the pre-motor control network are recruited differentially
and dynamically as a function of the type and intensity of the stimulus to result in an post-
synaptic output of the appropriate magnitude.
control network changes as a function of the CORT environment over a number of days.
PVHmpd, and particularly the adrenergic component that contains VGluT2. Chronically
high circulating CORT had little effect on the overall structure of the pre-motor control
glucocorticoids for both the development and maintenance of phenylethanolamine-N-
methyltransferase (PNMT), though this has only been investigated in the adrenal medulla
166
The loss of circulating CORT did not alter glutamatergic appositions to CRH soma. We
were unable to investigate appositions to non-somal locations in this study, and therefore
cannot say if changes in glutamatergic innervation are occurring away from the soma.
innervation to CRH soma without altering innervation patterns to the PVHmpd as a whole.
Again, without being able to investigate non-somal appositions we cannot say if the
reduction in glutamatergic innervation was exclusive to somal regions. Furthermore, we
only investigated CRH neurons, though it is probable that changes in innervation occur at
other types of neurons within the PVHmpd.
not noradrenergic pre-synaptic terminals, which may correspond to an increase in CA
absence of a stressor.
The Pre-Motor Control Network in the Absence of a Stressor
We began by evaluating the physical relationships between three major components of

the pre-motor control network in the absence of a stressor. We found that pre-synaptic
terminals were divided almost evenly between those that contain VGluT2 and those
that contain VGAT, indicating major innervation by glutamatergic and GABAergic inputs,
respectively. This is consistent with previously reported patterns of innervation (Decavel
excitatory/inhibitory balance controlling CRH neuronal activity.
We found that just over half of the pre-synaptic terminals innervating the PVHmpd
contained VGAT, indicating a GABAergic phenotype (Chaudhry et al., 1998). GABA is the
primary inhibitory neurotransmitter in the brain and we, like others (Decavel and van den
These GABAergic inputs originate both from both distal and local sources (Roland and
plays an important role in regulating the activity of CRH neurons, and the majority of
somal and non-somal locations. While appositions were overall more numerous away
from the soma, at the soma VGAT accounted for the majority of appositions.
Glutamate is the major excitatory neurotransmitter in the hypothalamus (van den Pol,
168
pre-synaptic terminals within the PVHmpd, consistent with previous reports (van den
Glutamatergic axon terminals make synaptic contact with both dendrites and cell bodies in
to the soma.
While VGluTs 1 and 2 are found exclusively in pre-synaptic terminals, VGluT3 has
been found in many neurons not traditionally assumed to be glutamatergic (Fremeau
GABAergic neurons of the bed nucleus of the stria terminals (BST), as well as GABAergic
forebrain structures, while VGluT2 is the dominant vesicular glutamate transporter in the
PVH, we found that VGluT2 is the dominant glutamate transporter in these pre-synaptic
terminals, indicating that inputs to this region originate predominantly from diencephalic
and hindbrain regions.
co-occurred with other measured markers less often. Of the total VGluT2 pre-synaptic
169
epinephrine to the PVH results in a greater CORT response than does administration of an
may be more salient in producing an HPA axis response. However, very few studies
address differences between norepinephrine and epinephrine in the initiation of the HPA
subset of adrenergic neurons use glutamate as a signaling mechanism (DePuy et al.,
the PVHmpd. We also show that the co-occurrence of VGluT2 within CA pre-synaptic
terminals is preferential to the adrenergic input. However, it is worth pointing out that it
is not clear if adrenergic neurons release epinephrine from pre-synaptic terminals in the
in Chapter 1 (Figure 1.2). Systemic stressors activate both CA and non-CA neurons in the

hindbrain. Both of these populations contain VGluT2, and are presumably excitatory. The
CA population, which contains NPY, projects directly to CRH neurons, as well as to many
other nuclei that further affect CRH activity. The hindbrain CA neurons project to regions
proximal to CRH neurons, including within the hypothalamus and within the PVH itself,
as well as more distal regions, including the primarily GABAergic BST. This population
we found in the PVH.
Figure 5.1 Pre-motor control network
An updated model of the pre-motor control
network of that presented in Figure 1.2.
Distal inputs arise from the dorsomedial and
ventrolateral hindbrain, as well as the bed
nucleus of the stria terminalis. These inputs
affect signaling at downstream locations
located both within the hypothalamus
(Proximal) and the PVH itself (PVH).
These inputs are highly interactive. VGAT
and VGluT2 by green. VGluT3 is presented
in light blue. Catecholaminergic input
is expressed by black (norepinephrine)
or gray (epinephrine). These neurons
also contain NPY (blue), as does an
AgRP (orange) population in the arcuate
nucleus of the hypothalamus. Receptors
are expressed by appropriately-colored
triangles, key presented below.
CRH neurons may also receive GABAergic input from the periPVH region, although there
appears to be present in a subset of GABAergic terminals. Further GABAergic innervation
arises from NPY/AgRP neurons of the arcuate nucleus, which are innervated by hindbrain
innervation arises from both distal (hindbrain) and proximal (PVH) sites (Herman et al.,
There are certainly many other neuroactive substances that affect the activity of the HPA
axis in response to both systemic and psychogenic stressors. These are likely recruited
terminals in the pre-motor control network in the absence of a stressor provides a model
as well as the ability to apply the underlying methods to evaluate other neural networks.
In Response to Acute Stressors
An appropriate balance between excitatory and inhibitory signaling is crucial for the
overall function of the central nervous system, and dynamic responses of glutamatergic
and GABAergic neurons are crucial for the appropriate response to various stressors.

What is clear, though, is that glutamate is excitatory to neuroendocrine neurons in the
component is absolutely critical.
We found that acute glycemic challenges altered the number of detectable vesicular
transporters in a way that appears to be modulated by the strength of the stimulus. While
not traditionally used as a measure of activity-dependent changes, changes in the number
in the number of detectable VGluT2, but there was only a decrease in the number of
detectable VGAT in response to 2DG. This is consistent with the idea that stressors of
increasing magnitude differentially recruit components of the pre-motor control network

may then decrease to allow for greater excitation at the post-synaptic neuron (Wojcik et
neurons in response to various stressors.
remain unclear, as to date there have been no studies investigating in vivo changes
in vesicular transporter expression during this period. Thirty minutes may not be long
enough to translate, transcribe, and transport new protein to the pre-synaptic terminal.
Changes in the number of vesicular transporter in vivo could be the result of changes in
the number of synaptic vesicles within the pre-synaptic terminal through vesicular exo-
and endocytosis, or the incorporation of additional individual transporters into existing
in incorporating individual proteins into synaptic vesicles remain unclear.
In Response to Altered Levels of Circulating Corticosterone
Chronic exposure to high levels of circulating CORT affects the balance of excitation and
inhibition within the PVHmpd as a whole, as well as in CRH neuronal activity (Cullinan
provides evidence that circulating CORT affects glutamatergic and GABAergic

pre-synaptic mechanisms in the PVH in the absence of a stressor. We show that circulating
glutamatergic innervation to CRH neurons is likely occurring at non-somal locations, given
that we found no decrease in VGluT2 appositions at the soma. Given that NMDA receptor
Dallman et al., 1986).
The effects of reduced CORT on GABA components within the PVH are more clear. While
adrenalectomy (ADX) enhances GABAergic tone within the PVHmpd (Majewska et al.,
in VGAT-immunoreactivity in pre-synaptic terminals in the PVHmpd, as compared to the
ADX + high CORT replacement condition. This is consistent with reports indicating that
condition. The loss of circulating CORT increases the number of GABAergic synaptic
potential loss of GABA input is compensated for by an increase in innervation from other
GABA appositions to CRH neurons, but previous reports have indicated an increase in

alternatively may allow for an increase in the total number of detectable VGAT molecules.
While many studies examine the effects of the loss of glucocorticoids on both the PVH
effects of chronically high circulating CORT in the absence of an external stressor are less
understood. Many studies investigate the effects of chronic psychogenic stressors, which
utilize telencephalic processing, whereas systemic stressors generally do not (Herman
considerably more physiologically relevant than chronically high levels of circulating
CORT in the absence of a stressor, a condition that generally occurs only pathologically
or pharmacologically.
in high CORT environments, is vitally important to survival. This is evident by the fact that
content within the PVHmpd, but did reduce the number of VGluT2 appositions to CRH
for an overall change in innervation. Furthermore, no net change in glutamate innercation
may be a way to retain the excitatory/inhibitory balance, thereby allowing a response to

the glutamate response is not enhanced purely in response to chronically high circulating
Chronically supraphysiological levels of CORT did not alter VGAT content compared
to the sham condition, though there was a small increase compared to the ADX
condition. This suggests that chronically high CORT alone has little effect on GABAergic
mechanisms in the PVHmpd. Similar to glutamatergic mechanisms, this may be way
change in glutamatergic and GABAergic pre-synaptic functioning purely in response to
high circulating CORT may allow for an HPA axis response even when circulating CORT
is already high (i.e., during exposure to a chronic stressor).
Adrenergic Response to Physiological Stressors
The effects of both acute and chronic stress on hindbrain CA neurons are well-
understood. Hindbrain CA projections are necessary for the neuroendocrine response
components, and particularly the adrenergic subset, of the pre-motor control network are
preferentially affected by both acute systemic stressors and the level of circulating CORT.

Both insulin and 2DG activate neurons in the A1/C1 region, while 2DG further activates
immunoreactivity in both adrenergic and noradrenergic pre-synaptic terminals, which
presumably corresponds to an increase in CA exocytosis in both populations (Sorimachi
increased in both CA and non-CA populations. Within CA pre-synaptic terminals the
increase in VGluT2 was preferential to the adrenergic input. Given that glutamate signaling
is necessary for adrenergic activation of neurons in the parvicellular PVH (Daftary et al.,
PVHmpd.
but did not alter DBH-immunoreactivity in noradrenergic structures, indicating that the
change in DBH content occurred preferentially in the adrenergic population. This may
mean that a decrease in detectable DBH in the adrenergic subset indicates an increase
in adrenergic signaling, which is consistent with reports that indicate that the removal of

that there have been no studies investigating whether epinephrine is in fact released from
linked to the PNMT antibody following ADX as compared to the high CORT condition.
PNMT is found only in the cytoplasm, and unlike DBH, is not released during exocytosis.
as would occur in response to a loss of PNMT, has been shown to enhance CRH-
immunoreactivity in neurons (Mezey et al., 1984), which is consistent with the increase
increasing total PNMT in a glucocorticoid-dependent manner (Bohn et al., 1984). These
results, taken with the work of this dissertation, may suggest that glucocorticoids are
important for the maintenance of the CA, and particularly the adrenergic, component of
the pre-motor control network.

VGluT2 within the adrenergic input appears to be altered by the loss the circulating CORT
loss of VGluT2 appeared to correlate with a loss in overall pre-synaptic terminals, we
were unable determine whether adrenergic pre-synaptic terminals were among those that
were lost due to the decreased sensitivity of the synaptophysin antibody. Therefore, we
cannot be sure that there was a decrease only in VGluT2 content within the adrenergic
pre-synaptic terminals, or if there was an overall loss of adrenergic pre-synaptic terminals.
adrenal and brain PNMT (Park et al., 1986), it is possible that a loss of circulating CORT
structures within the PVH. This is likely a mechanism to avoid saturation of receptors and
Conclusion

that the excitatory input to the PVH was altered to a greater degree than was the inhibitory
input in response to physiological challenges, highlighting the importance of the stability
of inhibitory input in the control of the HPA axis. Finally, we show that adrenergic inputs,
important in the regulation of physiological processes and the transmission of information
regarding systemic stressors, appear more sensitive to challenges that jeopardize
homeostasis than do the noradrenergic inputs.
181
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Asset Metadata

Creator Johnson, Caroline Sinclair (author)

Core Title The structural and functional configurations of glutamate, GABA, and catecholamine pre-synaptic terminals in the parvicellular neuroendocrine part of the paraventricular nucleus of the hypothalamus

Contributor Electronically uploaded by the author (provenance)

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 04/18/2017

Defense Date 01/30/2017

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

Tag catecholamine,corticotropin-releasing hormone,GABA,glutamate,Hypothalamus,oai:digitallibrary.usc.edu:usctheses,OAI-PMH Harvest

Language English

Advisor Swanson, Larry (committee chair), Donovan, Casey (committee member), Simerly, Richard (committee member), Watts, Alan (committee member)

Creator Email carolinj@usc.edu,somethingcaroline@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-357419

Unique identifier UC11255949

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Legacy Identifier etd-JohnsonCar-5204.pdf

Dmrecord 357419

Document Type Dissertation

Rights Johnson, Caroline Sinclair

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Abstract (if available)

Abstract Dysfunctional glucocorticoid secretion is a component of a wide range of clinical conditions. Much of this dysfunction involves important but poorly understood neural control networks in the hypothalamus. These networks integrate stress-related information to drive ACTH secretion and peptide gene expression in a manner appropriate for the ongoing situation. Specific stimuli are relayed by unique combinations of active afferents as well as appropriate signaling cascades. In particular, glutamate, GABA, and catecholamine (CA) inputs, which make up a “pre-motor control network,” are crucial for the regulation of corticotropin releasing hormone (CRH) neuronal activity in response to a wide variety of systemic stressors. Understanding how these inputs interact to relay the nature of a specific stressor is important in understanding how this pre-motor control network orchestrates the activity of the neuroendocrine CRH neurons, and allows for the dynamic processing of different stressors. ❧ In this dissertation we examine the structural and functional organization of this pre-motor control network to determine and how its activity allows for an appropriate response from CRH neurons. The underlying hypothesis of this dissertation is that specific patterns of afferent activity in the pre-motor control network affect how the CRH neuron “reads” these inputs to result in an appropriate response to a stressor, in terms of both magnitude and duration, and is tested using various in vivo procedures, as well as immunohistochemistry and high resolution 3D image analysis techniques in both a rat model and a transgenic mouse model. ❧ In Chapter 2 we use high resolution image analysis techniques to examine the organization of pre-synaptic terminals in the pre-motor control network in the absence of a stressor, to establish a picture of this network in an unstimulated state. Using both a rat and a transgenic mouse model we show almost equal innervation of the fast excitatory (glutamate) and fast inhibitory (GABA) neurotransmitters. Furthermore, we show that at least some of this excitatory innervation originates from the hindbrain CA neurons that project to the PVHmpd. Within this CA population we found that the adrenergic subset is the dominant input. Finally, we found that the vast majority of appositions to CRH neurons occur at non-somal locations. ❧ In Chapter 3 we investigate the effects of acute glycemic stressors of different intensities on the functional organization of the pre-synaptic terminals examined in Chapter 2. We show that stressors of different magnitudes differentially recruit afferent populations in a stimulus intensity-dependent manner that favors excitation by altering detectable vesicular glutamate transporter 2 (VGluT2) and vesicular GABA transporter (VGAT) within pre-synaptic terminals. The increase in VGluT2 occurs within both CA and non-CA pre-synaptic terminals. Within CA terminals the increase in VGluT2 is specific to adrenergic terminals. ❧ In Chapter 4 we used bilateral adrenalectomy with either high corticosterone (CORT) replacement or no CORT replacement to investigate the how CORT affects the organization of pre-synaptic terminals in the absence of a stressor. We show that circulating CORT is required to maintain full excitatory innervation to the PVHmpd. This is particularly true of the adrenergic component that expresses VGluT2. Chronically high circulating CORT had little effect on the overall structure of the pre-motor control network. This may be a way to ensure that exposure to a subsequent stressor elicits a response. ❧ Overall, we show that the organization of pre-synaptic terminals of the pre-motor control network are highly interactive and plastic. Furthermore, the configuration of these components varies uniquely as a function of the specific stimulus to allow for an appropriate HPA axis response. In all cases the excitatory inputs were altered to a greater extent than were the inhibitory inputs, underlining the importance of inhibitory control on the function of the HPA axis. Finally, we show that adrenergic inputs appear to be the more salient CA input in regards to physical challenges and changes in overall homeostasis.