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Regulation of ethanol intake by purinergic P2X4 receptors
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Regulation of ethanol intake by purinergic P2X4 receptors
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Content
REGULATION OF ETHANOL INTAKE BY PURINERGIC P2X4 RECEPTORS
by
Letisha Renee Wyatt
__________________________________________________________________
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY & TOXICOLOGY)
May 2013
Copyright 2013 Letisha Renee Wyatt
ii
EPIGRAPH
“Nearly every man who develops an idea works it up to the point where it looks impossible
and then he gets discouraged.
That’s not the place to become discouraged.”
Thomas A. Edison
iii
DEDICATION
This is for
Mr. Hinds & Dr. Judith A. Hirsch
For always believing in me
iv
ACKNOWLEDGEMENTS
Special thanks to my mom and dad who have always emphasized the importance of hard work
and scholarly pursuits. I am also very grateful to my husband Jake Staniels, who without his
support, shoulder, love and laughs - I would not have survived. This work would not have been
possible without the guidance of my faculty advisors Dr. Ronald L. Alkana and Dr. Daryl L.
Davies. Their considerable enthusiasm and thoughtful input is very much appreciated. I would
also like to thank my dissertation committee members Dr. Ruth I. Wood and Dr. Marco
Bortolato who were always glad to lend me their expertise when questions would arise. I am
indebted to our collaborators Dr. Michael W. Jakowec, Dr. Roberta Diaz Brinton, Dr. Deborah
A. Finn, and Dr. Karen Szumlinski whom have been ever so helpful with technical guidance and
use of their equipment. I cannot forget family, friends, colleagues, or past and present lab
members as they have supplied countless hours of advice and encouragement. Lastly, I would
like to express my sincerest gratitude to Mr. Wade Harper-Thompson, Dr. Richard Andalon, and
the USC School of Pharmacy for significant academic and professional support throughout the
completion of this project.
Research Support
NIH National Institute on Alcohol Abuse and Alcoholism R01 AA013992 (D.L. Davies)
NIH National Institute on Alcohol Abuse and Alcoholism R01 AA03972 (R.L. Alkana)
NIH National Institute on Alcohol Abuse and Alcoholism F31 AA018926 (L.R. Wyatt)
The USC Graduate School & the USC School of Pharmacy
v
Statement of Contributions to works contained in this Dissertation
This dissertation is composed of the author’s original work and contains no material previously
published or written by any other individual except where due reference is made. All data
contained herein was collected and analyzed by L.R. Wyatt. The authorship on published
manuscripts is described in greater detail in the following page. Drs. Davies and Alkana provided
discussion and revisions to the manuscripts.
Authorships
Published and in press works by the Author incorporated into the Dissertation
Wyatt LR, Godar SC, Khoja S, Jakowec MW, Alkana RL, Bortolato M, Davies DL (in press)
Socio-communicative and sensorimotor impairments in male P2X4-deficient mice.
Neuropsychopharmacology. Incorporated as Chapter 2.
Wyatt LR, Yardley M, Asatryan L, Finn DA, Alkana RL, Davies DL (in revision). P2X4
receptors regulate ethanol intake in C57BL/6 mice. Incorporated as Chapter 3.
Ostrovskaya O, Asatryan L, Wyatt L, Popova M, Li K, Peoples RW, Alkana RL, Davies DL
(2011). Ethanol is a Fast Channel Inhibitor of P2X4 Receptors. J Pharmacol Exp Ther
337(1): 171-179. Incorporated as Chapter 4.
Additional works by the Author relevant to this Dissertation but not forming part of it
Yardley MM, Wyatt L, Khoja S, Asatryan L, Finn DA, Alkana RL, Louie SG, Bortolato M,
Davies DL (2012). Ivermectin reduces alcohol intake in mice. Neuropharmacology 63(2): 190
201.
Popova M, Asatryan L, Ostrovskaya O, Wyatt LR, Li K, Alkana RL, Davies DL (2010). A
point mutation in the Ectodomain-Transmembrane Domain Interfaces eliminates ethanol
response in P2X4 receptors. J Neurochem 112(1): 307-317, 2010.
vi
TABLE OF CONTENTS
Epigraph ii
Dedication iii
Acknowledgements iv
List of Figures viii
Abbreviations x
Abstract xii
Chapter 1: Introduction 1
Significance 1
Disorders Co-Occurring with Alcoholism 3
Alcohol Pharmacokinetics 5
Neurocircuitry of Abused Drugs 6
Ligand-Gated Ion Channels: Molecular Targets for Alcohol Action 9
Purinergic (P2X) Superfamily of LGICs 11
Ethanol Modulation of P2X4 Receptor Activity In Vitro 14
Indirect Evidence for a Role of P2X4Rs in Ethanol Behaviors 16
P2X4R Regulation of Ethanol Intake: Proof-of-Concept Studies with Ivermectin 17
Ethanol Studies with Mice – Utility and Limitations of Transgenic Knockouts 19
Conclusion: Gaps in Scientific Knowledge and Dissertation Hypotheses 21
Chapter 2: Socio-communicative and sensorimotor impairments
in male P2X4-deficient mice 24
Chapter 2 Abstract 24
Introduction 26
Results 27
Novel Open Field 27
Elevated Plus Maze 28
Light-Dark Box 28
Maternal Separation-Induced Ultrasonic Vocalizations 30
Sticky Tape Removal Test 30
Acoustic Startle Reflex and Prepulse Inhibition (PPI) of the Startle 30
Social interaction 31
Novel Object Interaction and Recognition 33
Olfactory Discrimination 34
Western Immunoblotting 35
vii
Discussion 37
Experimental Procedures 42
Chapter 3: P2X4 receptors regulate ethanol intake in C57BL/6 mice 50
Chapter 3 Abstract 50
Introduction 52
Results 55
Western Blot Protein Analysis of Brain Tissue 55
Experiment 1: 24-hr Access, Two-Bottle Choice 57
Experiment 2: Intermittent, Limited Access 59
Experiment 3: Behavioral Sensitivity to Ethanol 60
Discussion 61
Conclusion 66
Experimental Procedures 66
Chapter 4: Development and use of lentiviral vector technology to
investigate the role of accumbal P2X4 receptors in ethanol intake 73
Chapter 4 Abstract 73
Introduction 75
Results 78
Validation of Lentivirus-Based P2X4R Expression in
HEK293 Cells and Neurons 78
Confirmation of Functionality of Lentivirus-Delivered P2X4Rs 80
Validation of shRNA-based Lentiviral Knockdown of P2X4Rs in
Mouse Microglial (BV-2) Cells 82
Targeting and Lentivirus Expression in Brain Tissue 83
24-hr Access, Two-Bottle Choice
Ethanol and Tastant Drinking 84
Discussion 86
Conclusion 91
Experimental Procedures 92
Chapter 5: Overall discussion and conclusions 98
Summary of Overall Findings 98
Neurobiological Perspective 102
Limitations of the Study 103
Future Directions 104
Clinical Implications 107
Bibliography 110
viii
LIST OF FIGURES
Figure 1.1 Neurocircuitry of addiction in the mesolimbic pathway of the CNS 8
Figure 1.2 Proposed p2rx4 involvement in the GABAergic processes
within the VTA 13
Figure 1.3 Genetic associates of the p2rx4 gene 14
Figure 1.4 P2rx4 mRNA in the midbrain and striatum 17
Figure 2.1 Spontaneous locomotor activity in P2X4R HZ and KO mice 28
Figure 2.2 Anxiety-like behavior in P2X4R HZ and KO mice 29
Figure 2.3 Communication deficits and altered sensory function in
P2X4R KO mice 31
Figure 2.4 Reduction in social interaction by P2X4R KO mice 33
Figure 2.5 Normal cognitive function and olfactory discrimination in
P2X4R KO mice 34
Figure 2.6 P2X4R KO mice exhibit significant alterations in NMDA
and AMPA glutamate receptor subunit expression 36
Figure 2.7 Sample blots for significantly altered NMDA and AMPA
glutamate receptor subunits tested in P2X4R KO and WT mice 36
Figure 3.1 Western blot analysis of P2X4 protein expression in
P2X4R KO and WT mice 56
Figure 3.2 Western blot analysis of GABA
A
R α1 subunit expression in
P2X4R KO and WT mice 57
Figure 3.3 P2X4R genotype increases 24-hr, two-bottle choice ethanol intake
in male mice 58
Figure 3.4 P2X4R genotype does not alter 24-hr, two-bottle choice
saccharin intake and preference in male mice 59
Figure 3.5 P2X4R genotype increases intermittent, limited access ethanol intake 60
Figure 3.6 Effect of P2X4R KO genotype on the sensitivity to a hypnotic
Dose (3.6 g/kg) of ethanol 61
Figure 4.1 Lentiviral vectors efficiently transduce mammalian and
primary neuronal cultures 79
ix
Figure 4.2 Punctate fluorescence pattern of P2X4Rs in primary neurons
following lentiviral gene delivery 80
Figure 4.3 Lentivirus-mediated expression of functional P2X4Rs in neurons 81
Figure 4.4 shRNA-mediated reduction of P2X4Rs murine microglial cells
expressing endogenous P2X4RS 82
Figure 4.5 shRNA infection area and lentiviral expression in vivo 83
Figure 4.6 Ethanol intake is increased in C57BL/6 mice after
shRNA-mediated reduction of P2X4Rs in the nucleus accumbens 85
Figure 4.7 Saccharin but not quinine intake is increased in C57BL/6 mice
after shRNA-mediated reduction of P2X4Rs in the nucleus accumbens 86
x
ABBREVIATIONS
ADH, alcohol dehydrogenase
AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
ASD, autism spectrum disorder
ATP, adenosine-5'-triphosphate
AUD, alcohol use disorder
BAC, blood alcohol concentration
BBB, blood brain barrier
BDNF, brain-derived neurotrophic factor
BEC, blood ethanol concentration
BECRR, blood ethanol concentration at return of righting reflex
BZ, benzodiazepines
CNS, central nervous system
Cyp7b1, cytochrome P450 7B1
DA, dopamine
EtOH, ethanol
GABA, γ-amino-butyric acid
GFP, green fluorescent protein
HAP, high alcohol preferring
HEK, human embryonic kidney
HZ, heterozygous
iNP, inbred alcohol non-preferring
iP, inbred alcohol-preferring
IVM, ivermectin
xi
KO, knockout
LAP, low alcohol preferring
LGIC, ligand-gated ion channel
LORR, loss of righting reflex
LTM, long-term memory
MSN, medium spiny neuron
NAc, nucleus accumbens
NMDA, N-Methyl-D-aspartic acid
PCR, polymerase chain reaction
PFA, paraformaldehyde
PFC, prefrontal cortex
PK, pharmacokinetics
PPI, prepulse inhibition
PRCP, prolylcarboxypeptidase (angiotensinase C)
RNAi, interfering RNA
SEM, standard error of the mean
shRNA, short-hairpin RNA
siRNA, silencing RNA
SNP, single-nucleotide polymorphism
STM, short-term memory
TM, transmembrane
USV, ultrasonic vocalization
VTA, ventral tegmental area
WT, wildtype
xii
ABSTRACT
P2X receptors (P2XRs) are a family of cation-permeable, ligand-gated ion channels gated
by synaptically released extracellular adenosine-5'-triphosphate (ATP). Of the seven
P2XR subtypes (P2X1-P2X7), P2X4 is the most abundantly expressed subtype in the
central nervous system and to date is the most ethanol sensitive when measured in
recombinant expression systems. Previous work demonstrates that ethanol inhibits ATP-
activated currents in rat and mouse neurons, suggesting a role for P2X4Rs in ethanol-
related behaviors. A recent in vivo study identified p2rx4 as a candidate gene linked to
ethanol intake and/or preference in rodents. Despite these reports, the lack of specific
agonists and antagonists has hampered our ability to directly determine the role that
P2X4Rs play in regulating ethanol-induced behaviors. The availability of p2rx4 null
(i.e., knockout; KO) mice has partially overcome this limitation. However, transgenic
animal models do not allow for brain-regional analysis of P2X4R regulation on ethanol
drinking and the effect of constitutive p2rx4 gene deletion on general behavior has not
been assessed. This dissertation addresses these issues via a two-pronged approach
utilizing transgenic mice and lentiviral gene delivery techniques to investigate the
hypothesis that P2X4Rs play a role in regulating ethanol intake. Chapter 2 involves
characterization of inherent behaviors in P2X4R KO mice, specifically meant to capture
changes in general behavior and emotional reactivity. Chapter 3 examines differences
between wildtype C57Bl/6 and KO mice in ethanol intake and begins to elucidate the role
of P2X4Rs in the underlying mechanisms for differential drinking. Finally, Chapter 4
describes the development of lentiviral vectors for spatial and temporal control of P2X4R
expression and begins to investigate the regulation of ethanol intake by P2X4Rs in the
xiii
striatum (i.e., nucleus accumbens; NAc). Findings from each experimental chapter
support the notion that a deficiency in P2X4Rs 1) causes perceptual and socio-
communicative functions akin to autism spectrum disorder; 2) increases ethanol intake in
two drinking paradigms that model different degrees of alcohol consumption and 3)
suggests a specific role for accumbal P2X4Rs in regulating ethanol intake in mice.
Overall, these studies increase our knowledge regarding the broad spectrum of P2X4R
involvement in alcohol and non-alcohol behaviors and implicate P2X4Rs as a molecular
target for novel therapies to treat alcoholism and select endophenotypes related to
neurodevelopmental disorders.
1
CHAPTER 1:
INTRODUCTION
I.1 Significance
The consumption of alcohol (i.e., ethanol) has long since been a staple of history in many
cultures. Most individuals can enjoy a couple of drinks as a means for celebration,
socializing, or relaxation. But there also exists a substantial portion of individuals who
struggle with alcohol misuse. Resulting alcohol-related problems cost the U.S.
approximately 200 billion dollars annually due to medical costs related to the adverse
effects of alcohol (e.g. liver cirrhosis, heart disease, and fetal alcohol syndrome), vehicle
accidents, and violent crimes. Not only that, but alcohol drinking takes approximately
100,000 lives each year and is the leading cause of death for individuals between 15-24
years of age (Grant et al., 2004; Harwood, 2000).
Though alcoholics have common behavioral and drinking patterns, describing alcohol
use disorders (AUDs) can be challenging. In 1980, the American Psychiatric
Association's Diagnostic and Statistical Manual of Mental Disorders, 3
rd
Edition (DSM-
III) refined the definition of alcoholism by differentiating between alcohol abuse and
dependence. The current manual (DSM-IV-TR) outlines patterns of drinking behavior
that may be classified as risky, or resemble abuse or dependence. Risky drinking
behavior is defined for men as having >4 drinks and for women as having >3 drinks per
drinking occasion. Those considered alcohol abusers tend to drink despite negative
consequences including failure to fulfill major obligations, drinking when it’s physically
hazardous, and reoccurring drinking-related legal problems. The trademark symptoms of
2
dependent individuals are similar to those that abuse alcohol but also include tolerance,
withdrawal, failed attempts to control intake, and large amounts of time spent seeking or
recovering from drinking (Association, 2000).
In teens and young adults, binge drinking is becoming ever more prevalent and
gaining more attention. For example, the CDC reports that according to surveys in the
U.S. “90% of the alcohol consumed by individuals under 21 is in the form of binge
drinking” (Centers for Disease Control and Prevention, 2012). Binge drinking is defined
as having enough drinks to reach a blood ethanol concentration (BEC) of 80 mg% in a 2-
hour period (NIAAA, 2004). There are serious consequences to binge drinking including
intentional and unintentional injuries, alcohol poisoning, and neurological damage
(Centers for Disease Control and Prevention, 2012). Since young people are less
sensitive and tend to have a reduced response to alcohol (Schuckit et al., 2004), the risk
of overdrinking is increased. What is most troubling is that there is a strong correlation
between young individuals that binge drink and those diagnosed as suffering from AUDS
later in life (Schuckit et al., 2004).
Despite what we know about typical behaviors related to alcohol misuse, alcoholism is
a complex disease that is undeniably difficult to study due to 1) genetic and
environmental differences that influence individual drinking patterns, 2) the range of
behaviors associated with acute and chronic alcohol exposure, some of which are
reversible and some of which are not, and 3) differential effects of exposure to alcohol at
various ages in life (Tabakoff and Hoffman, 2000).
3
I.2 Disorders Co-Occurring with Alcoholism
The consequences of alcohol abuse and dependence are burdensome alone but much
worse for those with dual diagnoses of AUD and mood, anxiety, or psychiatric disorders.
In the general population co-morbidity of mental disorders and substance use disorders
occur at higher than chance levels (Kessler et al., 2005). The general thought is that there
may be overlap in genes or neural pathways that mediate some addictive patterns and
psychiatric disorders. Not surprisingly, the co-morbidity of general anxiety disorder,
aggression, depression, and bipolar disorder is high in alcoholics. Two major studies
looking at the rates of psychiatric disorders in those with AUDs reported that of those
classified as alcohol abusers or alcohol dependent, approximately 12-30% also had mood
disorder and 29-37% also suffer from anxiety (Petrakis et al., 2002). The level of alcohol
misuse had a greater effect on the lifetime rate of schizophrenia and those that were
identified as alcohol dependent were 3.8 times more likely to have schizophrenia than
someone who is not alcohol dependent (Petrakis et al., 2002).
Tracking the age of onset for mood and psychiatric disorders and the occurrence of
alcohol misuse is difficult. The aforementioned study found that the median age for the
onset of the co-morbid disorder was ten years earlier than the median age of onset for the
alcohol use disorder. Though, 72% of the males classified as alcohol-abusers reported
their alcohol misuse to occur prior to the onset of a first mood disorder (Petrakis et al.,
2002). Knowing for certain is challenging since the symptoms for the disorders often
overlap and since they typically begin early in life, adults rely on memory to
retrospectively determine the onset of each disorder (Bukstein et al., 1989). As discussed
above, alcohol use and psychiatric disorders tend to occur in one’s early years. Autism-
4
spectrum disorder (ASD) is a neurodevelopmental condition that shares some biological
and behavioral similarities to schizophrenia, largely related to social function (Gilman et
al., 2012; Sasson et al., 2011). Individuals with ASD display characteristic behaviors,
often-which are used for diagnosis, including difficulties with social interaction and
communication. In addition, those with ASD suffer from uncontrolled repetitive
behavior (i.e., perseveration) and inflexibility during day-to-day routines (Association,
2000).
Recent evidence suggests a link between ASD and alcoholism (Miles et al., 2003;
Schumann et al., 2011). The AUTS2 gene has been identified as a potential candidate for
autism but is also implicated as a regulator of ethanol consumption (Schumann et al.,
2011). The meta-analysis study provides evidence for the role of AUTS2 in mediating
ethanol preference and sensitivity in both mice and drosophila. The authors argue that
gene expression in the prefrontal cortex of human post-mortem tissue as well as the
quantitative trait locus of this gene being mapped to chromosome 5 in high alcohol
preferring (HAP) mice, provides additional support for a role in the reinforcing effects of
alcohol (i.e., preference and impulsivity). Another study investigated the prevalence of
AUDs in families of autistic individuals and concluded that there is a significant
association between alcoholism and autism that appeared to be genetic (Miles et al.,
2003). Though the molecular mechanisms for ASD have not been completely elucidated,
one idea is that neurodevelopmental deficits are the result of alterations in the
excitatory/inhibitory balance in the brain (Gogolla et al., 2009). Matrisciano et al. (2012)
show that the neurodevelopmental deficits occurring prior to the onset of schizophrenia
5
involve enzymatic hypermethylation of genes for maturation and function of GABAergic
and glutamatergic neurons.
I.3 Alcohol Pharmacokinetics
Ethanol is a small, polar molecule. As such, it has the capability to passively diffuse
across biological membranes, including the vascular endothelium (Kalant, 1996;
Ramchandani et al., 2001). These properties allow ethanol to distribute in organs and
tissues based on water content (Ramchandani et al., 2001) and studies have indicated a
high correlation between ethanol volume of distribution and total body water (Norberg et
al., 2003). Circulating plasma ethanol rapidly reaches equilibrium in tissues with high
blood flow rates such as the brain, kidney, and lung. Since ethanol bioavailability
directly affects circulating blood alcohol concentrations (BACs) (Kalant, 1996), the
influence of alcohol on brain function is largely dependent on alcohol pharmacokinetic
(PK) factors.
Alcohol PK begins with and is largely determined by absorption. Several factors
influence the rate of absorption across the stomach and small intestine after oral
administration and many of these can be attributed to intra- and inter-individual
variability (Norberg et al., 2003). Following absorption, ethanol undergoes first pass
metabolism in the liver allowing for initial removal of some of the drug before entry into
the systemic circulation, and is then distributed throughout the body and eventually
eliminated by processes of metabolism or excretion (Norberg et al., 2003). Intra-
individual differences such as sex/gender and allelic differences in metabolic enzymes
(i.e., alcohol dehydrogenase; ADH) and other associated proteins also play a significant
6
role in alcohol PK. Studies show that women typically have a faster rate of alcohol
absorption and subsequent higher peak BAC when compared to males having a drink of
equivalent concentration (Li et al., 2000). This effect is thought to be attributed to
women having a lower proportion of total body water and decreased ADH activity during
first pass metabolism (Ramchandani et al., 2001). Mounting data indicates that women
have higher rates of alcohol clearing possibly partially influenced by sex hormones that
may modulate ADH activity or total body water (Norberg et al., 2003). Following a
human study examining liver mass per kilogram body weight in males and females of
differing ethnicities, Li and colleagues (Li et al., 2000) concluded that gender differences
in lean body mass influences the perceived higher clearance rates in women. These
studies provide evidence supporting the capability for females to exhibit higher alcohol
disappearance rates (g/L/hr) compared to men (Mumenthaler et al., 1999). Alcohol
research utilizing animal models must keep such factors in mind as they determine the
concentration of ethanol circulating in the blood and how this may influence initial and
subsequent behavioral responses.
I.4 Neurocircuitry of Abused Drugs
There are five classes of abused drugs: opiates, psychostimulants, nicotine, cannabinoids,
and ethanol (Pierce and Kumaresan, 2006). Several anatomical structures have been
identified as neural substrates of drugs of abuse, including the ventral tegmental area
(VTA), prefrontal cortex (PFC), nucleus accumbens (NAc), amygdala, and hippocampus.
These areas of the brain have distinct features and interconnected nuclei where cellular
7
modulation by drugs may elicit obvious or subtle changes in behavior. Studies in this
area have shown that the VTA and NAc are likely mediators of addictive behaviors.
The VTA, a midbrain structure distinct from the substantia nigra, is arguably one of
the most studied brain areas with respect to drug reward (Fig. 1.1, Top). The VTA is
largely comprised of dopamine (DA)-releasing cells. As theorized, the normal
physiological function of these dopaminergic neurons involves 1) the release of DA as a
function of pleasure or “liking”, 2) a mechanism to aid in associative learning by
encoding predictions for future rewards that are necessary for survival through firing
rates and the release of DA, and 3) incentive salience or “wanting” (Ikemoto, 2007).
Drugs of abuse are capable of corrupting the system through stimulating the release or
slowing down the re-uptake of DA in the VTA, thereby eliciting a high or extreme
pleasure response that becomes reinforcing for drug use (Ikemoto, 2007).
The NAc is designated as having a specific role in directly mediating behavioral
output by controlling information flow to the thalamus, striatum, and PFC via the ventral
pallidum (Fig. 1.1, Bottom) (Pierce et al., 2006). Some investigations differentiate the
role of the accumbens shell versus core for the rewarding effect of drugs, with the shell
being the most involved in limbic processes and the core in motor functions (Ikemoto,
2007; Pierce et al., 2006).
8
FIGURE 1.1
Figure 1.1: Neurocircuitry of addiction in the mesolimbic pathway of the CNS. (Top) Mesolimbic
pathways in the brain. Image credit: Wikipedia Commons. (Bottom) Neurocircuitry of addiction (Pierce et
al., 2006).
The mesolimbic pathway specifically describes afferent connections between the VTA
and regions like the NAc, amygdala, hippocampus, and medial PFC (Pierce et al., 2006).
This pathway is involved with the emotional, motivational, and contextual influence on
behaviors (Pierce et al., 2006). Drugs of abuse, including ethanol, can affect both
9
pathways thereby altering behavior and creating a vicious cycle that results in addiction.
Mounting evidence illustrates the capability of ethanol to alter dopamine
neurotransmission in the mesolimbic system by elevating DA levels in the NAc.
Bustamante and colleagues (2008) demonstrated this through investigations comparing
alcohol-preferring versus alcohol-avoiding rats that self-administered ethanol to the NAc
via a microdialysis technique. Some studies argue that local administration into the NAc
doesn’t increase DA but rather may enhance the upstream firing rate of dopaminergic
neurons in the VTA (Pierce et al., 2006). There also exists other reports supporting the
notion that ethanol can modulate neural activity within other areas of the mesolimbic
system like the VTA and the hippocampus (McCool, 2011).
I.5 Ligand-Gated Ion Channels: Molecular Targets for Alcohol Action
On a molecular level, less is understood regarding the initial sites and mechanisms of
ethanol action. Originally, the method of action for general anesthetics, including
ethanol, was theorized to involve disruption of the fluidity of lipids within the cell
membrane (Kar, 2005). The lipid theory is largely based on observations by H.H. Meyer
and C.E. Overton, who independently identified a direct relationship between the
partition coefficient and potency of alcohols and anesthetics (Meyer, 1901; Overton,
1901). More recently, this mechanism of action has been challenged by evidence
supporting an interaction of anesthetics with the function of particular cell membrane-
bound proteins (Kar, 2005).
It is now well established that ligand-gated ion channels (LGICs) are key mediators of
the behavioral effects of alcohol (Grant, 1994). The three major classes of LGICs include
10
the cys-loop, glutamate, and purinergic superfamilies. With regard to alcohol
pharmacology, the cys-loop: γ-amino-butyric acid (GABA), glycine, and nicotinic
acetylcholine receptor-containing superfamily and the glutamate superfamily consisting
of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-Methyl-D-
aspartic acid (NMDA), and kinate receptors, have been studied for some time. On the
other hand, the purinergic (P2X) superfamily is a relatively new family of LGICs. Given
that GABA and glycine neurotransmitters evoke inhibition of cellular processes within
the central nervous system (CNS), and ethanol has been shown to positively potentiate
this inhibitory action, these receptors have been extensively studied with regard to the
behavioral effects of alcohol. GABA receptors have been implicated as mediators of a
variety of ethanol-induced behaviors including, sedation, anxiety-reduction, and motor
impairments (Kumar et al., 2009). Furthermore, reducing GABA receptor subunits in the
NAc of male rats causes a decrease in ethanol consumption and preference (Rewal et al.,
2009). Similar behavioral effects of ethanol by interaction with glycine receptors have
also been documented (for a review, see (Perkins et al., 2010). Glutamate and associated
receptors are regulators of excitatory neurotransmission, which are also considered
mediators of alcohol behaviors due to ethanol inhibition of neuroexcitation,
reinforcement, and addiction; particularly due to glutamatergic involvement in synaptic
plasticity and learning and memory (Gass and Olive, 2012).
Current pharmacological therapies for the treatment of AUDs exploit some of the
receptor systems described above. The three most commonly prescribed treatments are
Disulfiram (Antabuse), Naltrexone, and Acamprosate (Campral). Antabuse employs a
metabolic strategy for reducing drinking, namely by blocking the breakdown of
11
acetaldehyde. Build-up of this toxic byproduct results in nausea and vomiting. The
mechanism for Naltrexone and Campral involve antagonism of opioid and NMDA
receptors, respectively (Petrakis et al., 2002). Both therapies are intended to reduce
cravings and withdrawal symptoms and eliminate drinking in abstinent individuals.
However, because the molecular mechanisms of ethanol are poorly understood and
patient compliance is low, these drugs are largely ineffective (Petrakis et al., 2002).
Furthermore, direct modulation of receptors that are historically linked to alcohol’s
behavioral effects (e.g., GABA, glycine, and glutamate), present a significant challenge
as such compounds like benzodiazepines (BZ) have behavioral consequences such as
psychomotor retardation, changes in appetite, and disinhibition for those with borderline
mental disorders. In addition, compounds like BZs have significant addictive liability
thus reducing the utility of GABA and glutamate effectors for treatment of AUDs (Longo
and Johnson, 2000).
I.6 Purinergic (P2X) Superfamily of LGICs
Of late, ATP has been recognized as a fast-acting neurotransmitter and modulator of
other neurotransmitters in certain cell types (Choi et al., 2009). P2X receptors are a fairly
unexplored superfamily of cation-permeable LGICs (Davies et al., 2005). Seven P2X
receptor subunits, named P2X1-P2X7, have been identified to date (Burnstock, 2004).
All subtypes except P2X6 are able to form homotrimeric receptors while six
heterotrimeric receptors have been documented: P2X1/2, P2X1/4, P2X1/5, P2X2/3,
P2X2/6 and P2X4/6 (Davies et al., 2005) (Khakh, 2001). The recently published crystal
12
structure for the zebrafish P2X4R has provided atomic resolution confirmation of the
proposed physiology of the receptor.
P2XR function is mediated by the opening of the channel through the binding of
extracellular ATP. This action allows a non-selective influx of cations. P2XRs located
presynaptically have a direct role in the release of neurotransmitters including GABA and
glutamate and ATP is often co-released with these neurotransmitter vesicles (Burnstock,
2004; Khakh and North, 2006). Since P2XRs facilitate a high cellular entry of calcium,
postsynaptic receptors are thought to have a modulatory role in synaptic transmission
(Khakh et al., 2006) (Jarvis and Khakh, 2009).
P2X receptor subtypes have been localized to different areas in the body and have
been linked to various physiological processes. P2X1 and P2X3 receptors are fast
desensitizing channels that are predominately expressed in smooth muscle and pain-
sensing neurons, respectively (Burnstock, 2004). The P2X2 subtype is highly expressed
in autonomic and sensory systems and is often post-synaptically localized (Xin et al.,
2011). P2X5 and P2X7 have been found to have highest expression in neurons and
microglia of the CNS, particularly in the medulla, cerebellum, and pons (Lein and al.,
2007).
The P2X4 subtype is also highly expressed pre- and post-synaptically in most neurons
and glia of the central and peripheral nervous systems (Fig. 1.2) (Choi et al., 2009).
13
FIGURE 1.2
Figure 1.2: Proposed p2rx4 involvement in the GABAergic processes within the VTA. P2rx4 as a
candidate gene to modulate GABA release in VTA dopaminergic neurons from HXB/BXH recombinant
inbred rat strain microarray analysis. Modified from (Tabakoff et al., 2009).
Currently, the importance or physiological roles of P2X4Rs are beginning to be defined.
For example, using p2rx4 null mice, recent investigations suggest that P2X4Rs may play
a role in neuroinflammation, neuroendocrine function, and learning and memory (Sim et
al., 2006; Tsuda et al., 2003).
Using the string database (stringdb.org), genetic associations between the p2rx4 gene
and other molecules can be made via “textmining”. This resource revealed links with
genes that have regulatory roles in glycogen storage, body weight regulation, and
neurosteroid metabolism, including cytochrome P450 7B1 (cyp7b1) and
prolylcarboxypeptidase (angiotensinase C) (PRCP) (Fig. 1.3). The cyp7b1 gene produces
monooxygenases that catalyze many reactions related to drug metabolism and the
biochemical synthesis of steroids and lipids (Tang et al., 2006), thus this gene is involved
VTA
14
in neurosteroid metabolism and sex hormone synthesis. The PRCP gene encodes the
enzyme, lysosomal prolylcarboxypeptidase, responsible for cleaving the C-terminus
amino acids linked to proline (e.g., angiotensin II). This enzyme is important for
regulation of blood pressure. Interestingly, current studies are investigating a role for
mercury in autism pathology. An analysis of urinary porphoryins to measure Hg body-
burden identified elevated PRCP levels in autistic children (Kern et al., 2011). However,
to date, a complete understanding of the role of P2X4Rs in behavioral organization is
unclear.
FIGURE 1.3
Figure 1.3: Genetic associates of the p2rx4 gene. Image credit: stringdb.org.
I.7 Ethanol Modulation of P2X4 Receptor Activity In Vitro
In vitro studies demonstrate P2X receptor sensitivity to ethanol with some subtypes
showing potentiation of ATP-gated currents following ethanol application and some
showing inhibition of ATP-gated currents (Davies et al., 2006). Mounting evidence
15
suggests some involvement of P2X receptors in the observed behavioral effects of
alcohol. Xiao et al. (2008) was able to show that P2XRs located at GABAergic synapses
of the VTA are targets for ethanol action.
The P2X4R subtype is challenging to study due to the lack of subtype-specific
pharmacological agonists and antagonists in the purinergic superfamily. Studies of
recombinant homomeric P2X2 and P2X4 receptors in a Xenopus oocyte expression
system show that ATP-gated currents of P2X4Rs are inhibited by ethanol in a reversible
and concentration-dependent manner (Davies et al., 2005; Davies et al., 2002b). In vitro
investigations also show that P2X4Rs are sensitive to ivermectin (IVM) (Jelinkova et al.,
2006). IVM is a commonly used anti-parasitic that is a semisynthetic derivative of
naturally occurring avermectins (Spinosa et al., 2002). The insecticidal action of IVM is
attributed to increases in glutamate-gated chloride conductance by enhancing receptor
binding. In mammals, IVM stimulates GABA release from nerve terminals (Khakh et
al., 1999b). Khakh et al. (1999b) reports IVM to be an allosteric modulator of P2X4Rs,
capable of significantly potentiating ATP-gated currents.
Recent studies from our lab and others suggest that IVM interacts with P2X4Rs at the
transmembrane interface (Asatryan et al., 2010; Silberberg et al., 2007). Mutagenic
studies by Asatrayan et al. (2010) allowed for modeling of potential interactions between
IVM and ethanol based on the zebrafish P2X4R crystal structure. This work revealed
overlapping sites of action between ethanol and IVM in residues within the ectodomain-
TM interface and TM-2 domain. In further support of this idea, our lab has identified
IVM as a subtype-specific antagonist to ethanol inhibition in P2X4R ATP-gated currents
in Xenopus oocytes and primary cultures of hippocampal neurons (Asatryan et al., 2010)
16
(Ostrovskaya et al., 2011; Popova et al., 2010). IVM represents a useful P2X4R-specific
pharmacological tool to study how changes to P2X4R function regulate ethanol behaviors
in vivo.
I.8 Indirect Evidence for a Role of P2X4Rs in Ethanol Behaviors
Localized brain stimulation studies support a role for the mesolimbic dopaminergic
system in positive reinforcement and motivation with specific emphasis on DA release as
a learning mediator (Alcaro et al., 2007; Cador et al., 1991; Ikemoto and Panksepp, 1999;
Koob, 1992; Weiss et al., 1993). Interestingly, intracranial self-administration studies
have shown that rodents will self-administer ethanol to the posterior VTA and that the
reinforcing effect can be measured by maintenance and discrimination of an “active”
(ethanol-producing) lever press in operant paradigms (Li et al., 2001). In the NAc,
ethanol has been shown to increase dopamine levels (Doyon et al., 2003), while
modulation of circulating DA dopamine levels can alter operant ethanol intake – an effect
that is considered a mediator of reward in rodents (Price and Middaugh, 2004).
Furthermore, the negative effect of ethanol on the hippocampus has been demonstrated in
vivo and in vitro and is attributed to suppression of pyramidal cell activity, the output cell
of the hippocampus (White et al., 2000).
P2X4Rs have been identified in mesolimbic brain regions, including the VTA, NAc,
and hippocampus (Fig. 4) (Pierce et al., 2006; Rubio and Soto, 2001) and so it is probable
that expression of P2X4Rs in these brain regions may regulate some aspects of the
behavior effects of ethanol mediated by these different brain regions.
17
FIGURE 1.4
Figure 1.4: P2rx4 mRNA in the midbrain and striatum. Left red circles denote the VTA and on the right
red circles denote the NAc. Image credit: Allen Institute for Brain Science.
P2X4Rs have been indirectly implicated as playing a role in alcohol consumption or
alcohol induced behaviors through genetic analyses in rodents. Studies by Kimpel et al.
(2007) and Tabakoff et al. (Tabakoff et al., 2009) found an inverse relationship between
p2rx4 expression and alcohol drinking phenotypes, reporting that rats consuming high
amounts of alcohol had a reduction in the p2rx4 gene. Based on support from the
literature, Tabakoff and colleagues hypothesized that P2X4Rs localized on GABAergic
interneurons within the VTA may be involved with regulation of alcohol intake (Fig.
1.2). The 2007 study was able to localize differential gene expression in brain regions
that are often studied for their role in mediating the rewarding effects of abused drugs.
I.9 P2X4R Regulation of Ethanol Intake: Proof-of-Concept Studies with Ivermectin
Few studies have investigated the effect of IVM administration on rodent behavior.
Moreover, of the existing studies the results are conflicting. Davis et al. (1999) found
strain-dependent increases in locomotor activity, exploratory tendencies, and acoustic
Midbrain
18
startle responses following IVM administration when mice were tested in open field and
acoustic startle assays. For these studies the mice orally self-administered IVM by free
choice access to an 8 oz. water bottle containing 0.008 mg/ml IVM or a bottle containing
tap water. In a later study, testing the behavioral effects of IVM on rats, a 1.0 mg/kg i.p.
dose of IVM significantly reduced locomotor activity and the duration of immobility in
open field tests while 0.5 mg/kg IVM did not, indicating a dose-dependent sedative effect
of IVM. The investigation also revealed that administration of IVM reversed picrotoxin-
induced anxiety suggesting a GABAergic mechanism of IVM action (Spinosa et al.,
2002).
Early reports suggested that IVM did not cross the blood brain barrier (BBB) in
mammals suggesting that it may indirectly influence behavior through sensory relay to
the brain after modulating receptors found in the peripheral nervous system (Davis et al.,
1999). On the other hand, in vivo IVM studies from our laboratory demonstrated reduced
ethanol consumption in mice treated with IVM compared to saline injected mice (Yardley
et al., 2012). This finding suggested that IVM could cross the BBB. In support of this
latter hypothesis, follow-on PK studies by Yardley and colleagues (2012) found a
correlation between the level of IVM in the brain and the degree of reduction of ethanol
intake. Despite these advances in understanding how ethanol relates to P2X4R function
in vitro and expression in vivo, there was no direct evidence linking P2X4Rs to the
behavioral effects of ethanol.
19
I.10 Ethanol Studies with Mice – Utility and Limitations of Transgenic Knockouts
To date there are several animal models to study various aspects of AUDs and
alcoholism, though no “all encompassing” model exist. Rodents do not readily consume
large amounts of ethanol when other fluids are available and once pharmacologically
relevant levels are reached, some behavioral responses are difficult to measure
(Kliethermes, 2005). Ethanol-induced phenotypes include motor and coordination
deficits (ataxia), impaired cognitive function, heightened preference and motivation for
ethanol, and changes in emotionality - all of which may be tested by a variety of
behavioral assays evaluating an animal’s response to ethanol (Kliethermes, 2005).
Alcohol researchers typically employ a variety of assays to study various degrees of
alcohol consumption in rodents (NIAAA, 2012). Home cage voluntary drinking is a
popular choice to assess intake and with two-bottle procedures, preference for alcohol
over water can be easily measured. Using these methods mice do not consume large
volumes of ethanol and do not become intoxicated, so two-bottle choice paradigms
measure of a level of ethanol intake akin to social drinking (Blednov et al., 2010).
Protocols have evolved to induce higher levels of drinking in rodents. These methods
typically involve intermittent ethanol access periods, alcohol presentation during the
circadian dark and the availability of only ethanol during testing. All of these factors
cause measurable intoxication in mice and elevate BECs (Rhodes et al., 2005), thus
allowing for modeling of excessive ethanol intake or binge-like drinking with high face
validity. Home cage drinking does not allow for the analysis of motivation for ethanol
but this can be achieved with operant chambers where rodent responses illuminate the
rewarding effects of the drug.
20
The use of a gene knockout approach to directly elucidate genetic determinants of
behavior has been demonstrated to be very useful. The targeted null mutation provides
specificity that sometimes cannot be achieved pharmacologically, since test compounds
are likely to have off-target effects. Knockouts (KO) are also beneficial in that they can
be global or designed to be cell-type specific (Stephens et al., 2002). Regardless, this
strategy is not without its drawbacks and difficulties in interpretation of results. Issues
that may arise with KO mice are 1) lethality or poor breeding, 2) subunit
substitution/compensation that may arise during early development to offset the effects of
gene deletion in the KO animals, 3) lack of brain region or anatomical specificity, and 4)
genetic background interference with mutant/desired measured behavior.
Alternative strategies to help overcome complications associated with gene knockout
technology include the use of genetic knock-in, point mutations, gene silencing, inducible
transgenes, and gene rescue. An approach that has become increasingly popular in
genetic engineering is the use of interfering RNA (RNAi), including silencing (siRNA)
and short-hairpin (shRNA) methods. These types of RNAi are handy due to their ability
to eliminate genes with temporal and anatomical specificity. Functioning under the idea
that these RNA sequences act as magnets for endogenous mRNA for the protein of
interest, binding to such mRNA prevents the translation of the target protein. shRNA and
siRNA sequences can be delivered to the cell in a variety of ways with the most popular
being viral vectors, peptide, or nanoparticulate delivery (Cazzin and Ring, 2010).
Direct injection of viral vectors stays mostly local to the site of delivery and requires a
relatively short time course for activation. Crittenden et al. (2007) reported that lentivirus
diffusion is minimal due to vector size, which is unable to traverse major structural
21
delineations. A study by Hommel et al. (2003) showed an anterior to posterior diffusion
of about 800 μm with an AAV vector, which are generally smaller than lentiviral vectors.
Nonetheless, compensation is still a potential limitation to this type of technology.
Currently, Cre/loxP and tetracycline on/off gene inducible systems have been
popularized in order to provide greater temporal and regional control of gene expression.
Target DNA is flanked with loxP sequences, which is excised by the enzyme cre
recombinase allowing the utility of Cre/loxP recombinase for gene deletion or insertion.
This technology is attractive because it allows for tissue and cell-specific mutation and is
also reversible for gene rescue experiments (Stephens et al., 2002). Though quite
valuable, these systems are more costly due to the need to produce two transgenic strains
of mice, one each expressing the Cre and lox sites.
I.11 Conclusion: Gaps in Scientific Knowledge and Dissertation Hypotheses
Building evidence supports a role for P2X4Rs in the action of alcohol, but direct
evidence is lacking. Furthermore, how modulation of P2X4R activity may relate to
behavioral changes related to alcohol is also unknown. The experiments contained
within the chapters of my dissertation begin to fill this gap by testing the hypothesis that
P2X4Rs play an important role in modulation of ethanol consumption. This will be
accomplished by employing several inter-related strategies. First, using transgenic mice,
in Chapter 2 will begin to characterize the general behavior and emotional reactivity of
P2X4R heterozygous (HZ) and KO mice. Our laboratory has an established P2X4R KO
colony at USC. Experimentation in this chapter will be to test the sub-hypothesis that
22
deletion of the p2rx4 gene will result in behavioral perturbations which are associated
with disorders co-occurring with (AUDs) (as outlined in section 1.2).
Chapter 3 experiments directly test the hypothesis that P2X4Rs play a role in
regulation of ethanol intake by investigating the effects of p2rx4 gene deletion (i.e.,
P2X4R KO) on ethanol intake. As individuals with AUDs or alcoholism display varying
degrees and patterns of alcohol intake, I tested P2X4R KO mice versus WT controls
using two paradigms of ethanol intake that modeled different degrees of alcohol drinking
(i.e., social and binge-like drinking). In order to begin to assess the potential mechanisms
for changes in drinking comparing P2X4R KO versus WT controls, I also utilized a loss
of righting reflex (LORR) test and a measure of BECs at the return of righting reflex
(BECRR). These investigations provide a measure of behavioral sensitivity to ethanol.
Chapter 4 tested the prediction that P2X4R knockdown in the NAc would lead to
changes in ethanol intake. This was accomplished using site-specific lentiviral gene
delivery for P2X4R knockdown in an effort to circumvent some of the challenges
associated with the gene knockout strategy (see section 1.9). To this end, shRNA-based
lentiviral vectors were employed. The lentiviral vectors were constructed and
subsequently extensively tested in validation studies for efficacy in expression and
knockdown capabilities in both cell culture and CNS tissue. Once the vectors were
developed, male C57Bl/6 mice received microinjections targeting the NAc and then
tested for ethanol intake in a 24-hr, two-bottle choice paradigm. This study focused on
the NAc due to relatively high expression of P2X4R mRNA (Fig. 1.4) and also because it
is the output center for behaviors in the neural circuitry of addiction (Fig. 1.1, Bottom). I
predicted that a study of ethanol intake in mice receiving shRNA-based lentiviral vectors
23
should provide new information not only regarding the role of accumbal P2X4Rs in the
regulation of ethanol intake but also the effect of compensation, if any, in the KOs.
Chapter 5 summarizes the conclusions from my dissertation work. These include a
discussion of the multi-pronged approach utilized in my dissertation and how this work
added key insights into the role of P2X4Rs in general behavior and ethanol intake
phenotypes. In addition, I discuss mechanisms that support the behavioral relevance of
changes in P2X4R function. I also discuss future studies that may shed new insights for
additional understanding the role of P2X4Rs in the regulation of ethanol intake.
24
CHAPTER 2:
SOCIO-COMMUNICATIVE AND SENSORIMOTOR IMPAIRMENTS
IN MALE P2X4-DEFICIENT MICE
CHAPTER 2 ABSTRACT
Purinergic P2X receptors are a family of ligand-gated ion channels gated by extracellular
adenosine 5’-triphosphate (ATP). Of the seven P2X subtypes, P2X4 receptors (P2X4Rs)
are richly expressed in the brain, yet their role in behavioral organization remains poorly
understood. In the present study, we examined the behavioral responses of P2X4R
heterozygous (HZ), and knockout (KO) mice in a variety of testing paradigms designed
to assess complementary aspects of sensory functions, emotional reactivity and cognitive
organization. P2X4R deficiency did not induce significant alterations of locomotor
activity and anxiety-related indices in the novel open field and elevated plus-maze tests.
Conversely, P2X4R KO mice displayed marked deficits in acoustic startle reflex
amplitude, as well as significant sensorimotor gating impairments, as assessed by the
prepulse inhibition of the startle. In addition, P2X4R KO mice displayed enhanced
tactile sensitivity, as signified by a lower latency in the sticky-tape removal test.
Moreover, both P2X4R HZ and KO mice showed significant reductions in social
interaction and maternal separation-induced ultrasonic vocalizations in pups. Notably,
brain regions of P2X4R KO mice exhibited significant brain-regional alterations in the
subunit composition of glutamate ionotropic receptors. These results collectively
document that P2X4-deficient mice exhibit a spectrum of phenotypic abnormalities
partially akin to those observed in other murine models of autism-spectrum disorder. In
conclusion, our findings highlight a putative role of P2X4Rs in the regulation of
25
perceptual and socio-communicative functions and point to these receptors as putative
targets for disturbances associated with neurodevelopmental disorders.
26
INTRODUCTION
Purinergic ionotropic P2X receptors are hetero- and homotrimeric cation-permeable
channels activated by extracellular adenosine 5’-triphosphate (ATP) (Khakh et al., 2006;
North, 2002). Of the seven P2X subunits characterized to date (named P2X1 through
P2X7), P2X4 receptors (P2X4Rs) are the most abundant in the central nervous system
(Buell et al., 1996a; Soto et al., 1996b), and are expressed in neurons across multiple
regions of the brain and spinal cord, as well as in microglia (Burnstock and Knight, 2004;
Ulmann et al., 2008). Recent studies have pointed to the implication of P2X4 in the
regulation of multiple nervous functions, including neuropathic pain (Tsuda et al., 2003;
Ulmann et al., 2008), neuroendocrine functions (Zemkova et al., 2010) and hippocampal
plasticity (Baxter et al., 2011; Lorca et al., 2011; Sim et al., 2006). In addition, P2X4
receptors have been recently shown to modulate the function of other major ionotropic
targets, such as N-methyl-D-aspartate (NMDA) glutamate receptors (Baxter et al., 2011).
These ion channels have been implicated in the organization of emotional and cognitive
responses (Newcomer and Krystal, 2001), as well as in the pathophysiology of
neurodevelopmental conditions, such as autism-spectrum disorder (ASD) (Carlson,
2012). Thus, it is likely that P2X4 receptors (P2X4Rs) may be implicated in the
modulation of behavioral responses and in neurodevelopmental processes.
To begin to test this possibility, our group recently investigated the behavioral effects
of P2X4R activation in mice. In particular, we found that ivermectin, a potent positive
allosteric modulator of P2X4Rs, induced anxiolytic-like effects, reduced sensorimotor
gating and reduced alcohol intake in mice (Bortolato et al., 2012b). Nevertheless, the
lack of P2X4R selectivity of this drug and the lack of potent P2X4R antagonists in vivo
27
limit greatly our knowledge on the behavioral functions of these targets. Whereas the
role of P2X4Rs in the regulation of nociception and inflammation has been examined in
prior studies, the involvement of these targets in information processing, emotional
reactivity and cognitive organization remains elusive.
Here we show, for the first time, that P2X4R KO mice exhibit a number of phenotypic
alterations highly reminiscent of neurodevelopmental problems, including socio-
communicative deficits and impairments in acoustic startle response.
RESULTS
Novel Open Field
In the open field assay, we found no significant differences in total distance (Fig. 2.1a)
[F(2, 24) = 1.68; NS] or percent locomotor activity in the center (Fig. 2.1b) [F(2, 24) =
1.41; NS] between the groups. Conversely, a significant difference in time spent in the
center (Fig. 2.1c) [F(2, 24) = 3.99; P<0.05] was detected. Neuman-Keuls post-hoc
comparison revealed a pronounced reduction in center duration between HZ mice
(P<0.05), but not KO mice, compared to WT counterparts.
28
FIGURE 2.1
Figure 2.1 No differences between WT and P2X4R KO mice in spontaneous locomotor activity
measured in the open field arena. (a) Total distance and (b) % locomotor activity in the center was
similar between WT, HZ, and P2X4R KO mice. (c) Time in center was significantly reduced in HZ mice.
Data are shown as mean ± SEM. *P<0.05 compared to WT mice.
Elevated Plus Maze
We ascertained whether P2X4R mutant mice exhibit anxiety-like responses in the
elevated plus maze. No significant differences were detected between genotypes in the
total entries (Fig. 2.2g) [H(2, 29) = 5.13; NS], open arm entries (Fig. 2.2a) [F(2, 26) =
0.12; NS), closed arm entries (Fig. 2.2b) [F(2, 26) = 0.12; NS], open arm duration (Fig.
2.2d) [F(2, 26) = 0.54; NS] and closed arm duration (Fig. 2.2e) [F(2, 26) = 0.09; NS].
Conversely, a significant trend was found in the time spent in the center (Fig. 2.2f) [F(2,
26) = 3.23; P<0.06].
Light-Dark Box
The absence of anxiety-related behaviors was also tested using a light-dark box
paradigm. In contrast to WT and P2X4R KO mice, HZ mice appeared to show some
changes in the latency to enter the light compartment (Fig. 2.2j) [H(2, 27) = 9.69;
P<0.01]. The differences were not significant as determined using a post-hoc analysis.
29
For HZ mice, but not KO, we also observed a significant increase in the number of light
compartment entries (Fig. 2.2h) [F(2, 24) = 4.29; P<0.05]. Finally, there was no
significant difference between WT, HZ or P2X4R KO mice in the light compartment
duration (Fig. 2.2i) [F(2, 24) = 3.08; NS].
FIGURE 2.2
Figure 2.2 The effect of genotype on anxiety-like behavior measured in the elevated plus maze and
light-dark box. No differences between WT, HZ, and P2X4R KO mice in (a, d) open arm entries and
duration; (b, e) closed arm entries and duration; (c, f) center platform entries and duration; and (g) total
entries. (h) Light box entries was significantly greater in HZ mice while there was no differences between
the groups in (i) time spent in the light box and (j) latency to exit the dark box. Data are shown as mean ±
SEM. *P<0.05 compared to WT mice. #P<0.05 compared to HZ mice.
30
Maternal Separation-Induced Ultrasonic Vocalizations
We investigated the frequency of maternal separation-induced vocalizations in pups to
determine whether the P2X4 receptor was involved in mediating communicative
behaviors. We found a significant reduction in vocalization frequency (Fig. 2.3a) [H(2,
26) = 8.69; P<0.05]. Specifically, P2X4 HZ and KO lines exhibited approximately 30%
and 90% reduction in overall vocalizations compared to their WT counterparts.
Sticky Tape Removal Test
To examine sensorimotor integration, we tested the behavioral responses of P2X4R
mutants in the sticky tape task. We found that P2X4R KO mice displayed a significant
reduction in the latency to remove the tape (Fig. 2.3b) [F(2, 21) = 7.37; P<0.01]. Post-
hoc analysis revealed a significant difference in latency for sticky tape removal between
HZ (P<0.01) and KO mice (P<0.01) compared to WT animals.
Acoustic Startle Reflex and Prepulse Inhibition (PPI) of the Startle
We examined acoustic sensitivity and informational processing in P2X4R mutants using
the prepulse inhibition of the acoustic startle paradigm. P2X4R KO animals displayed a
significant reduction in startle amplitude (Fig. 2.3c) [F(2, 24) = 11.24; P<0.001]. Post-
hoc comparisons showed that P2X4R KO mice exhibited a significant reduction in the
startle reflect compared to both WT (P<0.001) and HZ (P<0.01) littermates. We found a
significant effect of genotype on percent PPI (Fig. 2.3d) [F(2, 24) = 5.76; P<0.01]. A
post-hoc analyses revealed significant alterations in KO mice compared to WT (P<0.05)
and HZ (P<0.01) littermates. In order to account for the baseline changes in acoustic
31
sensitivity, we measured the delta prepulse inhibition (Fig. 2.3e) [F(2, 24) = 4.74;
P<0.05]. Post-hoc tests analyses revealed that P2X4 KO (P<0.05) and HZ (P<0.05) lines
displayed a significant decrease in the delta prepulse inhibition parameter compared to
WT mice.
FIGURE 2.3
Figure 2.3 P2X4R KO mice exhibit communication and sensory deficits. (a) Maternal separation-
induced ultrasonic vocalizations are reduced in P2X4R KO mice compared to their WT counterparts. (b)
P2X4R HZ and KO mice exhibit significant reductions in latency to remove sticky tape compared to WT
controls. (c) Acoustic startle amplitude was significantly lower in P2X4R KO mice compared to HZ and
WT mice. P2X4R KO mice exhibit altered (d) % PPI and (e) delta PPI compared to WT and HZ mice. Data
are shown as mean ± SEM. ***P<0.001, **P<0.01, and *P<0.05 compared to WT mice. ##P<0.01 and
#P<0.05 compared to HZ mice.
Social interaction
We assessed social responses in P2X4 mutants using the social interaction paradigm. We
found a significant reduction in social exploration in both frequency (Fig. 2.4a) [F(2, 23)
32
= 9.13; P<0.01] and duration (Fig. 2.4e) [F(2, 23) = 8.32; P<0.01]. In particular, the
number of social approaches was significantly reduced in both P2X4 lines (P<0.01).
Similarly, the overall duration of social exploration was markedly decreased in HZ
(P<0.01) and KO (P<0.05) mice compared to their WT counterparts.
To provide a more detailed analysis of the different types of social exploratory
patterns, we defined and measured investigative behaviors targeting the frontal,
abdominal, and anogenital regions. Oftentimes frontal and anogenital bouts seemed to
occur much more passively in the KOs and in fact a strong trend toward significance in
reduced frontal sniffing bouts [F(2, 23) = 3.15; P=0.062] and duration [F(2, 23) = 2.84;
P<0.08] was observed in KO mice. Taking a closer look at this, we found a significant
effect of genotype on total number of abdominal sniffing bouts [F(2, 23) = 6.65; P<0.01]
and duration [F(2, 23) = 12.96; P<0.001]. Post-hoc analysis revealed that compared to
WT mice, P2X4R HZ and KO mice had significantly fewer sniffing bouts in the
abdominal area (P<0.01 and P<0.05, respectively) and reduced abdominal sniffing
duration compared to controls (P<0.01 and P<0.001, respectively). No significant
differences were observed for anogenital bouts though we did observe a trend toward
significance for the effect of genotype on anogenital sniffing duration between the three
groups [F(2, 23) = 3.14; P=0.063].
33
FIGURE 2.4
Figure 2.4 P2X4R KO mice display reductions in social interaction. (a, e) Total sniffing bouts and
duration and (c, g) abdominal sniffing bouts and duration were significantly lower in P2X4R KO mice
compared to their HZ and WT counterparts while (b, f) frontal sniffing bouts and duration and (d, h)
anogenital sniffing bouts and duration were unchanged. Data are shown as mean ± SEM. ***P<0.001,
**P<0.01, and *P<0.05 compared to WT mice.
Novel Object Interaction and Recognition
We examined novel object interaction as well as short-term and long-term recognition in
order to asses working memory. The number of novel object exploratory approaches was
similar between WT, HZ, and P2X4R KO mice (Fig. 2.5a) [F(2, 24) = 1.60; NS]. There
were also no significant differences between the genotypes for the number of contacts
with the novel object measured at 1.5 hours (STM) [H(2, 21) = 1.47; NS] and 24 hours
(LTM) [F(2, 24) = 0.70; NS] after the initial object interaction test (Fig. 2.5b-c).
34
Olfactory Discrimination
We assessed changes in olfactory discrimination in order to investigate olfactory deficits
that may affect social behavior or impair pups from being able to perceive the proximity
of the dam during maternal separation experiment. We found no differences between
WT, HZ, and KO mice in the amount of time spent sniffing the novel scent measured by
novelty scent index for frequency [F(2, 26) = 1.61; P = NS] and duration [F(2, 26) = 1.11;
P = NS] (Fig. 2.5d-e).
FIGURE 2.5
Figure 2.5 No differences between WT, HZ, and P2X4R KO mice in cognitive function and olfactory
discrimination. (a) Novel object approach number, (b) novel object recognition index for STM and (c)
novel object recognition index for LTM were similar between the two groups. Odor discrimination
measured as the index of frequency (d) and duration (e) for sniffing the novel scent did also not differ
between WT, HZ, and P2X4R KO mice. Data are shown as mean ± SEM.
35
Western Immunoblotting
Because findings from the current study mirror some of the impairments commonly seen
in mouse models of ASD, we next assessed the expression of NMDA and AMPA
glutamate receptor subunits in brain regions isolated from WT and P2X4R KO mice. As
shown in Figure 6, we identified significant alterations between WT and KO mice in
glutamate receptor expression in the prefrontal cortex and hippocampus. Specifically,
there was a significant decrease in expression of both the GluN2A subunit (46.77%;
P<0.05) and GluN2B subunit (38.35%; P<0.05), while GluN1 subunit expression did not
differ in the prefrontal cortex of P2X4R KO compared to WT mice (p>0.05; NS). In
contrast to the prefrontal cortex, GluN1 subunit expression was significantly reduced in
the hippocampus by 65.65% (P<0.01) and in the cerebellum by 64.90% (P<0.05) of KO
mice. Hippocampal GluN2A and GluN2B subunit expression was similar in WT and KO
mice. We also found that there was no significant difference between WT and KO mice
in GluN2B expression in the cerebellum (P>0.05; NS).
Conversely, we found that GluA1 subunit expression was significantly increased in
the hippocampus (66.51%; P<0.05) of P2X4R KO mice compared to WT controls.
However, we did not observe any significant difference in expression of GluA1 in the
prefrontal cortex of P2X4R KO mice compared to WT (P>0.05). KO mice also had
significantly increased GluA2 subunit expression in the hippocampus (31.34%; P<0.01)
and in the cerebellum (88.29%; P<0.05) compared to their WT counterparts. Similar to
GluA1, we did not detect any differences between WT and P2X4R KO mice in GluA2
subunit expression in the prefrontal cortex (P>0.05; NS).
36
FIGURE 2.6
Figure 2.6 P2X4R KO mice exhibit significant alterations in NMDA and AMPA glutamate receptor
subunit expression. NMDA receptor subunit expression was significantly reduced while AMPA subunit
expression was significantly increased in P2X4R KO mice compared to WT controls in the (a-e) prefrontal
cortex and (f-j) hippocampus. Data are shown as mean ± SEM for 3-8 mice/genotype. *P<0.05 and
**P<0.01
FIGURE 2.7
Figure 2.7 Sample blots for significantly altered NMDA and AMPA glutamate receptor subunits
tested in WT and P2X4R KO mice. (a-b) GluN2A and GluN2B in the prefrontal cortex and (c-e) GluN1,
GluA1, and GluA2 in the hippocampus. Data are shown as mean ± SEM for 3-8 mice/genotype. WT and
KO samples were run on the same blot for each region.
37
DISCUSSION
The findings of the present study showed that genetic ablation of P2X4Rs results in a
spectrum of abnormal behavioral phenotypes, including deficits in social interaction and
maternal separation-induced ultrasonic vocalizations. Furthermore, P2X4R KO mice
displayed significant alterations in startle reactivity and sensorimotor gating, as indicated
by the PPI impairments. These results were accompanied (and may have been partially
contributed) by auditory and tactile, but not by olfactory impairments. Notably these
changes were not paralleled by overt alterations in locomotor activity and anxiety-like
responses across several testing paradigms based on approach-avoidance conflicts, such
as the novel open field, elevated plus-maze, light-dark box and novel object exploration.
Finally, P2X4R deficiency did not appear to disrupt cognitive function based on the
object-recognition memory test.
These findings collectively point to the implication of P2X4 receptors in the
modulation of socio-communicative and sensorimotor functions, and extend previous
evidence on the phenotypes of P2X4R KO mice (Sim et al., 2006; Tsuda et al., 2003;
Ulmann et al., 2008). In the absence of overt alterations in anxiety-related responses and
motoric abnormalities, the reduced startle amplitude and increased tape-removal latency
in P2X4R KO mice may suggest that the deficiency of these receptors may lead to
auditory deficits and tactile enhancement. It is worth noting that our observation of
enhanced tactile sensitivity by P2X4R HZ and KO mice is in line with reports of
increases (and not only deficits) in tactile sensitivity within the autism spectrum (Kern et
al., 2006).
38
The possibility of acoustic impairments in P2X4R KO mice is in agreement with
recent data indicating the relevance of this receptor in the modulation of the blood flow
of spiral ligament capillaries in the cochlear lateral wall (Wu et al., 2011). In view of the
importance of ATP-mediated currents to attune the endocochlear potential, it is likely that
the deficiency of P2X4Rs may result in marked auditory impairments. Alternatively,
tactile and acoustic alterations in P2X4R KO mice may reflect a broad role of these
receptors in sensory transmission. P2X4R KO mice display reduced pain sensitivity
(Tsuda et al., 2003). Furthermore, P2X4Rs are abundant in key regions for the regulation
of sensory functions, such as the spinal cord, as well as the perikarya and dendrites of
thalamic and cortical neurons (Le et al., 1998).
We also documented that P2X4R KO mice exhibit a significant reduction in PPI, a
highly reliable operational index of sensorimotor gating. PPI measures the reduction of
the startle reflex that occurs when the eliciting acoustic burst is immediately preceded by
a weak, non-startling prestimulus (Ison and Hoffman, 1983). Because the computation of
this parameter is inherently dependent on the amplitude of the startle reflex, the
interpretation of variations in %PPI in P2X4R KO mice is complicated by potential
“floor effects” consequent to the decrement in their baseline startle amplitude (Swerdlow
and Geyer, 1993). However, the possibility that the %PPI changes may be artifactual
was challenged by our finding of significant reductions in ΔPPI (Bortolato et al., 2004;
Devoto et al., 2011). Thus, the reduction in PPI is likely to signify a deficit in the ability
of P2X4R-deficient animals to extract and filter relevant information from the external
milieu, possibly pointing to a role for this receptor in the regulation of information
processing. In keeping with this concept, it is worth noting that P2X4Rs have been
39
recently shown to modulate synaptic strength in hippocampal neurons (Baxter et al.,
2011; Sim et al., 2006), which play a central role in preserving the integrity of PPI and
sensorimotor gating (Swerdlow et al., 2004; Zhang et al., 2006). Furthermore, the
significant enhancement in tactile sensitivity in the sticky tape removal test may suggest a
poor ability of P2X4R KO mice to filter out sensory stimuli.
We recently showed that the anthelminthic IVM induced a dose-dependent PPI deficit
in C57BL/6 mice through activation of P2X4 receptors (Bortolato et al., 2012b). In
addition, IVM reduced anxiety-like behavior and had no affect on tactile sensitivity,
findings that were not reproduced in our studies with P2X4R KO mice. These
differences may represent IVM-mediated behavior effects that are the result of interaction
of IVM with other receptor systems and future studies should fully investigate the
influence of IVM on affective behavior. Since this drug acts as a positive allosteric
modulator of P2X4Rs, the PPI reduction observed in P2X4R KO mice may result from
neurodevelopmental changes. This possibility is supported by several observations: first,
we found that acute injection with IVM did not affect PPI in P2X4R KO mice (Bortolato
et al., 2012b); second, P2X4Rs have been shown to be expressed from postnatal day 1
onward in rodents (Cheung et al., 2007); third, some of the other behavioral alterations of
P2X4R KO mice documented in the present study, including early communication
impairments and social deficits, are also often associated with neurodevelopmental
disorders such as autism-spectrum disorder. Accordingly, it is worth noting that PPI
deficits are common, albeit not pathognomonic, features of ASD (Perry et al., 2007).
Although P2X4R KO mice failed to exhibit significant behavioral alterations across
most anxiety-related paradigms, we cannot rule out that the observed deficits in social
40
interaction and maternal separation-induced ultrasonic vocalizations may reflect aspects
of social anxiety. However, while a reduced interaction with conspecifics is normally
interpreted as an index of greater social anxiety (Bailey and Crawley, 2009), decreased
levels of ultrasonic emissions in pups are typically reflective of lower separation anxiety.
The complexity of this scenario warrants future studies to parse the potential contribution
of P2X4Rs to different facets of sociability.
Several limitations of the present study should be acknowledged, including the lack of
behavioral analyses on P2X4R KO females, as well as pharmacological characterization
of the neurochemical bases of the observed behavioral abnormalities. Several
neurochemical mechanisms may account for the function of P2X4Rs in brain and
behavioral functions, as well as its potential implication in neurodevelopmental and
autism-related alterations. P2X4Rs have substantial calcium permeability (Egan and
Khakh, 2004; North, 2002; Soto et al., 1996b); thus, the lack of P2X4Rs is likely to result
to alterations of calcium homeostasis, which may in turn lead to alterations in synaptic
transmission and higher vulnerability to abnormalities of neural plasticity (Kostyuk,
2007). P2X4Rs have been shown to exert complex modulatory effects on the function
and/or composition of other receptors, including NMDA (Baxter et al., 2011) and AMPA
(Andries et al., 2007) receptors. In addition, activation of P2X4Rs stimulates the release
of brain-derived neurotrophic factor (BDNF) (Trang et al., 2009), and dysregulation of
this neurotrophin have been widely implicated in ASD (Hashimoto et al., 2006; Miyazaki
et al., 2004). Interestingly, in some measures we observed a biphasic affect of hetero-
and homozygosity on behavior which may reflect the modulatory role of P2X4Rs on
biologically antagonistic neurotransmitter systems like glutamate and GABA.
41
In support of this notion, we observed significant alterations in the expression of
subunits that comprise NMDA and AMPA glutamate receptors in P2X4R KO mice.
Reductions of GluN1 subunits in the hippocampus suggest an overall reduction of
NMDA receptor expression in these mice that is not uniform across brain regions. In line
with reports published using mouse models of ASD with altered NMDA receptor
function, P2X4R KOs show deficits in social behavior, communication, and PPI (Duncan
et al., 2004; Gandal et al., 2012; Won et al., 2012). Although reports of the involvement
of AMPA receptors in ASD are less understood, changes in AMPA receptor subunit
expression have also been linked to neurodevelopmental deficits. Similar to our findings
with P2X4R KO mice, increased AMPA subunit expression has been documented in a
recent study of post-mortem analysis of brain tissue from autistic individuals (Purcell et
al., 2001). The molecular findings, coupled with behavioral endophenotypes of ASD in
P2X4R-deficient mice, implicate a role for P2X4Rs in neurodevelopment.
The translational validity of the present results remains unknown. To the best of our
knowledge, the only currently available evidence on P2X4 deficiency in humans comes
from studies on a single-nucleotide polymorphic (SNP) variant of the p2rx4 gene
(Tyr315>Cys mutation; rs 28360472), which has been found to lead to the disruption of
ATP binding to the receptor, and ultimately to loss of function of P2X4Rs (Stokes et al.,
2011). The only phenotypic alteration that was assessed in carriers of this variant was an
increase in pulse pressure (Stokes et al., 2011). However, that study did not include any
psychological/psychiatric assessment and was mainly limited to heterozygous carriers of
the variant itself (Stokes et al., 2011). Thus, further research will be necessary to verify
42
the behavioral implications of partial and/or total congenital deficiency of P2X4 in
humans and the existence of convergent cross-species endophenotypes.
Irrespective of these limitations, the present set of results underscore the implication
of P2X4Rs in a broad array of behavioral functions, and highlight these ionotropic
channels as putative therapeutic target for perceptual and socio-communicative
disturbances.
EXPERIMENTAL PROCEDURES
Animals
We used 3-5 month old experimentally naïve male P2X4R KO, heterozygous (HZ) and
wildtype (WT) mice from breeding colonies at the University of Southern California
(USC) in all behavioral studies. Generation of this line was described elsewhere (Sim et
al., 2006). In brief, breeding colonies were produced by re-derivation of frozen HZ
embryos obtained from an original, previously established, P2X4R KO mouse colony
designed on a C57BL/6 background (Sim et al., 2006). This procedure was performed by
the USC Transgenic Core and resulted in 7 HZ mice, which were backcrossed to
C57BL/6J mice to produce the first generation of offspring at USC. HZ offspring are
backcrossed every three generations to WT C57BL/6J mice (Jackson Laboratory; Bar
Harbor, ME) and maintained on a C57BL/6 background. Mice from our 5
th
generation
were used for this study. WT mice were generated from HZ x HZ crosses. Prior to
initiation of experiments, mice were housed in groups of 4-5 per cage in a facility
maintained at 22ºC with a 12 h-12 h light/dark cycle (lights on at 06:00 hours) and ad
43
libitum access to food and water. The order of animals in each test was counter-balanced
throughout the study for mice exposed to more than one testing paradigm. In these cases,
behavioral analyses were always performed from least to most stressful (e.g., open field
to social interaction) with a minimum interval of one week between subsequent
paradigms to minimize carry-over stress and potential confounds of repeated testing. All
handling and experimental procedures were performed in compliance with the National
Institute of Health guidelines and approved by the Institutional Animal Care and Use
Committee of USC.
Genotyping
Animal genotyping was performed by PCR. Samples of genomic DNA were extracted
from tail biopsies acquired from mice at weaning (postnatal day 21). Primers were used
to identify LacZ (5’GCGAACGCGAATGGTGCAGC 3’) and P2X4R
(5’TCGCTCTCTGGGTCTGGGGC 3’). Reaction conditions were 5 min at 95°C
followed by 32 cycles of 15 sec at 95°C, 15 sec at 60°C, 15 sec at 72°C 15 sec.
Novel Open Field
Locomotor behaviors in a novel open field were tested using a modified version of the
protocol adopted in (Bortolato et al., 2011). The apparatus consisted of a square, grey
arena (40 x 40 cm) surrounded by four 40 cm high black Plexiglas walls. The floor was
divided into two zones of equivalent areas: a central square compartment and a
concentric peripheral frame. Mice (7-11/genotype) were placed in the center and their
behavior was monitored for 10 min. The light level was maintained at 12 lux in the
44
center of the open field arena. Spontaneous locomotor activity was assessed with
Ethovision software (Noldus Instruments, Wageningen, The Netherlands). Behavioral
measures included: total distance traveled, time spent in the center, and percent
locomotor activity in the center (calculated as the distance traveled in the center divided
by the total distance traveled).
Elevated Plus Maze
Elevated plus-maze behavior was tested as described elsewhere (Bortolato et al., 2009b).
The apparatus consisted of two open (25 × 5 cm) and two closed arms (25 × 5 × 5 cm)
extending from a central platform (5 × 5 cm). The maze was constructed from black
Plexiglas with a light grey floor. Mice (9-11/genotype) were placed on the central
platform facing an open arm and allowed to freely explore the apparatus for 5 min. Light
and sound were maintained at 20 lux and 70 dB respectively. Behaviors were video-
recorded and subsequently scored. Behavioral measures included the number of entries
and time spent in the open and closed arms, as well as central platform. An arm entry
was defined as all four paws in the section.
Light-Dark Box
Testing was performed as described previously (Bourin and Hascoet, 2003). The
apparatus consisted of a Plexiglas cage (20 x 30 x 20 cm) comprising of a dark area (20 x
10 x 20 cm) and an adjacent brightly lit compartment (20 x 20, x 20 cm; illumination:
100 lux). The two compartments were separated by a Plexiglas divider, providing a 7 x 4
cm opening. Briefly, mice (8-10/genotype) were individually placed in the corner of the
45
dark area, and allowed to freely explore either compartment for 10 minutes. Behavior
was video-recorded, and the latency to exit the dark compartment as well as the number
and total duration of light compartment-entries were scored.
Maternal Separation-Induced Ultrasonic Vocalization
Vocalizations were recorded as previously described (Bortolato et al., 2012a). Because
of the stressful nature of the test, measurements were conducted in a separate cohort of
animals that did not undergo additional behavioral testing. Assessment was conducted on
pups (8-10/genotype) at age P6-P7. This age was selected based on previous studies
conducted in our laboratories, which highlighted that this developmental stage is optimal
to capture differences in maternal separation-induced ultrasonic vocalizations. Pups were
individually placed on the test platform in a sound-proof cabinet and the total number of
vocalizations was recorded for 5 min. The test platform was placed on top of a heating
pad to maintain a consistent temperature of 35°C and eliminate any potentially aversive
thermal effects.
Sticky Tape Removal Test
Sensorimotor integration was tested using the sticky tape test similar to as described
elsewhere (Bortolato et al., 2012b; Bouet et al., 2009). Each mouse (7-10/genotype) was
briefly restrained and a circular piece of tape was placed on each forepaw. The latency to
remove the second piece of tape was recorded.
46
Acoustic Startle Reflex and Prepulse Inhibition (PPI) of the Startle
Acoustic startle reflex and PPI were tested as previously described (Kerstetter et al.,
2012). We used startle reflex detection (San Diego Instruments, San Diego, CA)
consisting of one standard cage placed in sound-attenuated chambers with fan ventilation.
Each cage consisted of a Plexiglas cylinder of 3 cm diameter, mounted on a piezoelectric
accelerometric platform connected to an analog-digital converter. Background noise and
acoustic bursts were conveyed by two separate speakers, each one properly placed so as
to produce a variation of sound within 1 dB across the startle cage. Both speakers and
startle cages were connected to a main PC, which detected and analyzed all chamber
variables with specific software. Before each testing session, acoustic stimuli were
calibrated via specific devices (San Diego Instrument). Mice (8-10/genotype) were
placed in a cage for a 5-min acclimatization period with a 70 dB white noise background,
which continued for the remainder of the session. Each session consisted of three
consecutive sequences of trials (periods). Unlike the first and the third period - during
which mice were presented with only five pulse-alone trials of 115 dB - the second
period consisted of a pseudorandom sequence of 40 trials, including 12 pulse-alone trials
and 30 trials of pulse preceded by 73, 76 and 82 dB pre-pulses (respectively defined as
PP3, PP6 and PP12; 10 for each level of pre-pulse loudness). Percent PPI was calculated
as 100 - (mean startle amplitude for pre-pulse trials/mean startle amplitude for pulse-
alone trials) x 100. Delta PPI was calculated as mean startle amplitude for pulse-alone
trials - [mean startle amplitude for prepulse trials].
47
Social Interaction
Testing was performed with the protocol employed by (Bortolato et al., 2011). Test mice
(8-10/genotype) were paired with stranger age- and weight-matched male WT
conspecifics. The test animal and novel conspecific were placed in a new cage for 10
minutes. The frequency and duration of social exploration towards the frontal, abdominal
and anogenital regions were measured.
Novel Object Interaction and Recognition
Similar to prior studies (Bortolato et al., 2009a), mice (8/genotype) were individually
exposed to two identical novel objects, affixed to the floor and symmetrically placed 6
cm from the two nearest walls of a cubed Plexiglas box (20 x 20 x 20) lit to 120 lux.
Mice were placed in a corner facing the center, equidistant from each object, with the
starting position rotated and counter-balanced for genotype. The behavior of each mouse
was recorded for 15 minutes in order to attenuate any potential confounds related to
neophobia. Sniffing behavior was scored as number of approaches and duration of
exploration with the novel object. 1.5 hours and 24 hours later, mice were placed in the
same cage for an additional 15 minutes to assess short- and long-term memory (STM and
LTM, respectively). The time intervals for STM and LTM testing were selected based on
previous experiments in our laboratory, which highlighted them as optimal to capture
potential mnemonic deficits, in relation to a 15 minute exposure.
At this time, one of the objects was replaced by a novel object, different in color, size
and texture from the novel object in the previous session. For both STM and LTM, novel
object recognition index was calculated as the number of novel object contacts/ (number
48
of novel object contacts + number of familiar object contacts). These test sessions were
also video-recorded and the behavior scored as the novel object frequency and duration.
Olfactory Discrimination
Mice (8-10/genotype) underwent five training trials of 5-minute exposure to two identical
objects of the same scent. The objects were cylinders wrapped in tape and evenly scented
with diluted almond or lemon oil. On the subsequent (sixth) test trial, one of the
cylinders was replaced with another sprayed with a novel scent (i.e., almond during
training with lemon as the novel scent). The test scent was counter-balanced between the
groups. The test was performed in dim light (50 lux) and the behavior was video-
recorded and olfactory discrimination was measured as the novel scent index for
frequency (sniffing frequency novel scent/sniffing frequency for old scent + novel scent)
and the novel scent index for duration (sniffing duration novel scent/sniffing duration for
old scent + novel scent).
Western Immunoblotting
Mice were euthanized by CO
2
asphyxiation, followed by cervical dislocation. The
prefrontal cortex, hippocampus and cerebellum were dissected within 2 minutes of
euthanasia and frozen on dry ice. The tissues were stored in -80
o
C until use. Brain tissue
was homogenized using 200 µl of a homogenization buffer (50mM Tris-HCl pH 8.0, 150
mM NaCl, 1mM EDTA, 0.1% SDS, 1/100 dilution protein inhibitor cocktail) and
sonicated. Protein concentrations were determined by Pierce BCA protein assay kit
(Thermo Scientific, Rockford, IL). 50µg/lane of protein samples were made to run on
49
12% tris-glycine gels (Lonza, Rockland, ME) for 1 hour at room temperature. The
samples (3-8 mice/genotype) were then transferred to nitrocellulose membrane for 2
hours at 4
o
C (Licor Biosciences, Lincoln, NE). Membranes were incubated with
Odyssey blocking buffer for 1 hour at 4
o
C (Licor Biosciences, Lincoln, NE) to prevent
non-specific antibody binding, followed by incubation with mouse monoclonal
antibodies. The following antibodies were used: for NMDA receptor subunits, anti-
GluN1, GluN2A and GluN2B (1:1000; NIH Neuromab Facility, UC Davis, CA), for α-
amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, anti-GluA1 and
GluA2 (1:1000; NIH Neuromab Facility, UC Davis, CA), and α-tubulin (1:10,000;
Millipore, Temecula, CA) primary antibodies for 2 hours at room temperature. The
membranes were then incubated with secondary antibodies conjugated with IR dye
800CW for 1 hour at room temperature. Bands were visualized using the Odyssey
Infrared Imaging system (Licor Biosciences, Lincoln, NE). Blots were imaged using
700nm and 800nm channels in a single scan at a resolution of 169µm and quantified by
software provided with imaging system (Licor Biosciences, Lincoln, NE).
Statistical Analyses
Parametric and non-parametric statistical analyses on behavioral parameters were
performed by a one-way ANOVA or Kruskall-Wallis test, followed by Neuman-Keuls or
Nemenyi’s test for post-hoc comparisons, respectively. Expression levels of the NMDA
and AMPA receptor subunits were analyzed by unpaired, two-tailed student’s t-test.
Normality and homoscedasticity of data were verified by the Kolmogorov-Smirnov and
Bartlett’s test. Significance was set at P = 0.05.
50
CHAPTER 3:
P2X4 RECEPTORS REGULATE ETHANOL INTAKE IN C57BL/6 MICE
CHAPTER 3 ABSTRACT
P2X receptors (P2XRs) are a family of cation-permeable ligand-gated ion channels
activated by synaptically released extracellular ATP. The P2X4 subtype is abundantly
expressed in the CNS and is sensitive to low intoxicating ethanol concentrations. Genetic
meta-analyses identified the p2rx4 gene as a candidate gene for innate alcohol intake
and/or preference. Based on these findings, we hypothesized that P2X4Rs play an
important role in regulating alcohol intake. Adult male mice lacking the p2rx4 gene
(knockout; KO) and wildtype (WT) C57BL/6 controls were used. Ethanol intake was
measured using 24-hr access, two-bottle choice and intermittent, limited access
procedures. As an extension of the 24-hr study, saccharin was used to assess the
selectivity of KO modulation on intake. Loss of righting reflex (LORR) was used to
measure behavioral sensitivity to ethanol. Blood ethanol concentrations were measured
at the return of righting reflex to investigate potential mechanism(s) for observed changes
in LORR duration. P2X4R KO mice drank significantly more ethanol than controls in
the 24-hr, two-bottle choice paradigm. Furthermore, saccharin intake and preference did
not significantly differ. Using an intermittent, limited access procedure, we found that
ethanol intake was greater in P2X4R KO mice compared to WTs on the first drinking
session. Finally, P2X4-deficient mice also had altered expression of GABA
A
α 1 subunit
expression in brain regions associated with regulation of ethanol behaviors. These
findings provide the first direct evidence that P2X4Rs can regulate ethanol intake and
51
indicate that there is a complex interaction between P2X4Rs, ethanol, and other
neurotransmitter receptor systems.
52
INTRODUCTION
Alcohol (i.e., ethanol) is the number one abused drug in the United States. In the U.S.
alone, the misuse of alcohol affects approximately 18 million people, causes 100,000
deaths, and results in an annual loss of greater than 200 billion dollars (Grant et al., 2004;
Harwood, 2000). Current pharmacological and psychological strategies to treat alcohol
use disorders (AUDs) have provided limited success, due in part to the numerous
neurochemical systems affected by alcohol and a limited understanding of its initial
molecular site(s) of action in the brain.
With regard to the behavioral effects of ethanol, the cys-loop: γ-amino-butyric acid
(GABA) and the glutamate superfamilies have been studied for some time. Given that
GABA neurotransmission evokes inhibition of cellular processes within the CNS.
Notably, ethanol has been shown to positively potentiate the inhibitory action of GABA
A
receptors (GABA
A
Rs) have been extensively studied with regard to the behavioral effects
of alcohol. GABA
A
Rs have been implicated as mediators of a variety of ethanol-induced
behaviors including, sedation, reducing anxiety, and motor impairments (Kumar et al.,
2009). Furthermore, reduced GABA
A
R subunit expression in the NAc of male rats has
been associated with decreased ethanol consumption and preference (Rewal et al., 2009).
Excitatory neurotransmission by glutamate and associated receptors are also considered
mediators of alcohol behaviors due to ethanol inhibition of neuroexcitation,
reinforcement, and addiction, in particular, glutamatergic involvement in synaptic
plasticity and learning and memory (Gass et al., 2012). Moreover, a reduction in
excitatory control of GABA inhibition facilitated by an interaction between ethanol and
53
N-methyl-D-aspartate (NMDA) receptors, have been linked to the sedative/depressive
effects of alcohol intoxication (for a review see (Tsai and Coyle, 1998)). On the other
hand, the purinergic (P2X) superfamily is a relatively new family of LGICs.
Purinergic P2X receptors (P2XRs) have been identified as potential targets for ethanol
action (Davies et al., 2002a; Davies et al., 2005; Weight et al., 1999). P2XRs are fast-
acting, cation-permeable ion channels that are gated by synaptically released extracellular
adenosine 5’-triphosphate (ATP) (Burnstock, 2004). In the central nervous system
(CNS), ATP can affect the function of other neurotransmitters (e.g. GABA, glycine and
glutamate) known to play important roles in alcohol drinking and related behaviors by
acting on P2XRs located on pre- and postsynaptic membranes (Chizh and Illes, 2001;
Deuchars et al., 2002; Hugel and Schlichter, 2002; Jo and Schlichter, 1999; Khakh, 2001;
Tabakoff et al., 2009; Xiao et al., 2008).
Current knowledge regarding possible roles of P2XRs in physiology and behavior
comes primarily from work using dissociated neuronal cultures and slice preparations
(Coddou et al., 2011; Surprenant and North, 2009). In addition, several P2XR knockout
(KO) mouse models have been used to support and extend in vitro predictions of P2XR
function. This work indicates that learning and memory (Labrousse et al., 2009; Sim et
al., 2006; Wang et al., 2004), depression, and anxiety (Basso et al., 2009) are modulated
by P2XRs. P2XRs have also been linked to hormonal control of temperature regulation,
food and water intake (Stojilkovic, 2009; Ulmann et al., 2008), pain perception (Honore
et al., 2006; Jarvis et al., 2002; Tsuda et al., 2000; Tsuda et al., 2009; Ulmann et al.,
2008; Ulmann et al., 2010), and vascular tone (Yamamoto et al., 2006). Many of these
54
physiological and behavioral functions linked to P2XRs are also known to be affected by
ethanol.
Of the seven P2XR subtypes, P2X4 is the most abundantly expressed in the CNS
(Buell et al., 1996b; Soto et al., 1996a). Several lines of evidence suggest that P2X4Rs
can modulate a spectrum of the effects of ethanol. In vitro studies report that ethanol
concentrations starting at approximately 5 mM modulate ATP-activated currents in
neurons (Li et al., 1993, 1994, 1998; Weight et al., 1999; Xiao et al., 2008) and
recombinant models (Asatryan et al., 2010; Asatryan et al., 2008; Davies et al., 2005;
Davies et al., 2002b; Xiong et al., 2000; Xiong et al., 2001). This concentration of
ethanol is well below the 17 mM (i.e., 0.08%) blood ethanol concentration (BEC) that is
considered “under the influence” in the U.S. In addition, P2X4Rs are located in brain
regions that have been identified as neural substrates of alcohol (e.g., hippocampus,
cerebellum, ventral tegmental area, and nucleus accumbens) (Gonzales et al., 2004;
McCool, 2011; Pankratov et al., 2009; Sim et al., 2006).
Converging evidence suggests a possible role for P2X4Rs in modulating ethanol
intake. These investigations, using microarray techniques, found an inverse relationship
between p2rx4 gene expression and innate rodent consumption and preference for
ethanol. Kimpel et al. (2007) examined gene expression in brain areas associated with
reward in inbred alcohol-preferring (iP) and non-preferring (iNP) rat lines and found that
functional p2rx4 expression was significantly reduced in iP rats. Along similar lines,
Tabakoff and colleagues (2009) found lower levels of whole brain expression of p2rx4
mRNA in inbred rats that display a high ethanol-drinking phenotype compared to those
with a lower ethanol-drinking phenotype. Furthermore, pre-treatment with ivermectin
55
(IVM), a drug that antagonizes ethanol-mediated inhibition of recombinant P2X4Rs in
vitro (Asatryan et al., 2010), significantly reduced two-bottle choice and operant ethanol
self-administration in mice (Yardley et al., 2012).
Collectively, the findings outlined above suggest that P2X4Rs can regulate ethanol
intake and that there is an inverse relationship between P2X4R activity and ethanol
intake. Yet, direct evidence is lacking. The present study tests these hypotheses using a
gene knockout strategy in combination with measures of ethanol intake and sensitivity.
In support of these hypotheses, we found that male mice lacking the p2rx4 gene drank
significantly more ethanol and altered ethanol-induced loss of righting reflex (LORR)
duration compared WT controls. These findings have implications for future therapeutic
development targeting P2X4Rs for the treatment of AUDs.
RESULTS
Western Blot Protein Analysis of Brain Tissue
Prior to using mice in these studies, initial immunoblotting in a group of mice assessed
the levels of P2X4 protein in the brain and liver of P2X4R KO and WT littermates. As
shown in Figure 3.1a, we detected P2X4 protein at the expected molecular weight (60
kDa) in the brain of WT mice which was absent in the KOs. Similarly, P2X4 protein was
determined to be in the liver (Fig. 3.1a) of WT mice and not in the KOs. These results
support the lack of P2X4R protein in KO mice. Subsequently, we used PCR to confirm
the P2X4R genotype.
56
FIGURE 3.1
Figure 3.1 Western blot analysis of P2X4 protein expression in P2X4R KO and WT mice.
Confirmation of the presence of P2X4R protein in WT mice and absence in P2X4R KO mice in the (a)
brain and (b) liver. The lanes represent normalized protein samples (50 μg/lane).
Since inhibitory neurotransmission has been historically linked to ethanol behaviors,
we next assessed the expression of GABA
A
R α1 subunits in brain regions isolated from
WT and P2X4R KO mice. As shown in Figure 3.2, we identified significant brain-
regional alterations between WT and KO mice in GABA
A
R expression. Specifically,
there was a significant increase in α1 subunit expression in the cerebellum
(81.67%;P<0.05) of P2X4R KO compared to WT mice. In contrast to these regions,
GABA
A
R
α1 subunit expression in the prefrontal cortex and striatum (dorsal and ventral)
was similar in WT and KO mice.
57
FIGURE 3.2
Figure 3.2 Western blot analysis of GABA
A
R α1 subunit expression in P2X4R KO and WT mice.
Increased expression of GABA
A
R α1 in the (a) cerebellum of P2X4R KO mice. No differences between
WT and KO mice in the (b) prefrontal cortex, (c) dorsal striatum, and (d) ventral striatum. The lanes
represent normalized protein samples (50 μg/lane). *p<0.05
Experiment 1: 24-hr Access, Two-Bottle Choice
P2X4R KO and WT mice were provided 24-hour access to 10E and water as described in
the methods. As shown in Fig. 3.3a, knocking out the p2rx4 gene significantly increased
ethanol intake compared to WT controls. Ethanol intake (averaged across the 4
consecutive days) in KO mice was increased 22% versus WTs. Repeated measures, two-
way ANOVA for daily consumption revealed a significant main effect of genotype on
ethanol intake [F(1,60) = 5.42, p<0.05] with no effect of day or significant genotype x
day interaction. The 10E preference did not differ significantly between P2X4R KO and
WT controls and was approximately 82.1 + 3.8% and 82.1 + 2.0%, respectively (Fig
3.3b). Water intake was significantly affected by day [F(3,60) = 12.50, p<0.001], but not
genotype (Fig. 3.3c). Conversely, there was a strong non-significant trend [F(1,60) =
4.13, p=0.056] for the effect of genotype on total fluid intake and a main effect of day
[F(3,60) = 7.42, p<0.001] (Fig. 3.3d). There was no genotype x day interaction on any of
these measures.
58
FIGURE 3.3
Figure 3.3 P2X4R genotype increases 24-hr, two-bottle choice ethanol intake in male mice. (a) P2X4R
KO increases 10% ethanol (10E) intake compared to WT controls. (b) There was no significant effect of
genotype on preference and (c) water intake with (d) a significant trend for the effect of genotype on total
fluid intake in male mice. The insets represent the cumulative over the four consecutive days. Values
represent mean + SEM for 12 WT and 10 KO mice. *p<0.05 compared to WT.
Saccharin intake and preference for a 0.033% and 0.066% solution did not
significantly differ between controls and P2X4R KO mice (Figure 3.4a-b). Repeated
measures, two-way ANOVA revealed a significant effect of saccharin concentration on
intake [F(1,16) = 337.10, p<0.001] and preference [F(1,16) = 26.19, p<0.001] at both
concentrations, with no effect of genotype and no concentration x genotype interaction.
59
FIGURE 3.4
Figure 3.4 P2X4R genotype does not alter 24-hr, two-bottle choice saccharin intake and preference in
male mice. WT and P2X4R KO mice did not differ in their (a) intake and (b) preference for a 0.033% and
0.066% saccharin solution. Values represent mean + SEM averages across 4 consecutive days of intake at
each concentration for 6 WT and 12 KO mice.
Experiment 2: Intermittent, Limited Access
Male P2X4R KO and WT mice were exposed to an intermittent, limited (4-hr) access
paradigm to determine if removal of P2X4Rs altered ethanol consumption in a model of
high alcohol intake. There was no significant difference in baseline total water intake
between P2X4R KO versus WT mice measured three drinking sessions prior to initiating
the experiment (Fig. 3.5a). Ethanol intake for 10E and 20E were analyzed separately.
Repeated measures two-way ANOVA of 10% ethanol (g/kg) consumed revealed a
significant interaction between genotype and drinking session [F(5,100) = 4.98, p<0.001],
a significant effect of drinking session [F(5,100) = 4.96, p<0.001], with no main effect of
genotype (Fig. 3.5b). Bonferroni post-hoc analysis identified a significant increase in
10E intake by KO mice on the first drinking session (p<0.01; Fig. 3.5b). There was no
60
significant effect of genotype, drinking session, and no genotype x drinking session
interaction on 20E intake.
FIGURE 3.5
Figure 3.5 P2X4R genotype increases intermittent, limited access ethanol intake. (a) Water intake did
not differ between P2X4R KO and WT mice measured for 3 drinking sessions prior to exposure to ethanol.
(b) Ethanol intake was measured for a 6 drinking sessions at each concentration (10% and 20%). P2X4R
KO mice drank more 10% ethanol on their first exposure (drinking session 1) compared to WT controls.
Values represent mean + SEM for 12 WT and 10 KO mice. *p<0.05 compared to WT.
Experiment 3: Behavioral Sensitivity to Ethanol
We used LORR duration in combination with blood ethanol concentration at return of
righting reflex (BECRR), measured in a subset of mice tested for LORR duration, to
begin assessing whether a null mutation of P2X4Rs affected brain sensitivity to ethanol.
P2X4R KO mice were tested for LORR following an acute i.p. injection of 3.6 g/kg
ethanol. There was no significant difference between P2X4R KOs and WT controls in
the latency to LORR (Fig. 6A). In contrast, LORR duration was 27% longer in P2X4R
KO versus WT mice (p<0.05; Fig. 6B). BECRR was determined to be 2.83 + 0.16 in WT
controls compared to 3.12 + 0.077 in the KOs and did not reach statistical significance.
61
FIGURE 3.6
Figure 3.6 Effect of P2X4R KO genotype on the sensitivity to a hypnotic dose (3.6 g/kg) of ethanol. (a)
There was no difference in latency to LORR in KO compared to WT controls. (b) LORR duration was
increased in KO compared with respective WT mice. (c) BECRR was increased in KO when compared
with respective WT mice. Values represent mean + SEM for 24 WT and 25 KO mice (LORR) and 11 WT
and 12 KO mice (BECRR). *p<0.05 compared to WT.
DISCUSSION
The current study tested the hypotheses that P2X4Rs play an important role in the
regulation of ethanol intake and that there is an inverse relationship between P2X4R
activity and ethanol consumption. In support of these hypotheses, we found that male
P2X4R KO mice drank significantly more ethanol in the 24-hr ethanol preference
drinking paradigm than did WT controls. This effect appeared to be specific for ethanol
intake since KO and WT mice did not differ in saccharin intake or preference. In
addition, P2X4R KO mice consumed more ethanol on the first exposure (drinking session
1) during the intermittent, limited access procedure modeling high alcohol intake. KO
mice also differed significantly in their behavioral response to a hypnotic dose of ethanol.
Collectively, these findings provide the first direct evidence of a role for P2X4Rs in
regulating ethanol intake.
62
A large body of in vitro evidence demonstrates that P2X4Rs are inhibited by ethanol
(Davies et al., 2005; Ostrovskaya et al., 2011; Popova et al., 2010; Xiong et al., 2005).
Moreover, these studies identified amino acid residues that play an important role in the
action of ethanol (Asatryan et al., 2010; Yi et al., 2009). While these investigations
begin to shed light on the mechanism of ethanol action on P2X4Rs, the findings are
derived from isolated cellular preparations and thus do not provide insight into how
ethanol affects P2X4R function in vivo or how these effects translate to behavioral
change.
Typically, the link between specific receptor actions of a drug and the downstream
behavioral cascade is investigated using selective pharmacological agonists and
antagonists. At present, such tools are not available for P2X4Rs. IVM has been used to
help fill this gap. IVM is an allosteric modulator of several ligand-gated ion channels
(LGICs) including GABA
A
, glycine, and nicotinic acetylcholine receptors (Khakh et al.,
1999a; Toulme et al., 2006). Within the P2X superfamily, IVM is selective for P2X4Rs
and has been used to link these receptors to specific behavioral responses (Bortolato et
al., 2012b; Sim et al., 2006). Asatryan et al. (2010) found that IVM could antagonize the
inhibitory effects of ethanol on recombinant P2X4Rs. Their findings suggested that the
antagonism reflected interaction of IVM at a site of ethanol action in the TM1-TM2
region of the receptor. Yardley and colleagues (2012) extended this work to mice. They
found that IVM significantly reduced ethanol intake in both male and female mice.
Although receptors other than P2X4RS might be involved in IVM-mediated reduction of
ethanol intake, their findings provide evidence suggesting that positive modulation of
P2X4R function can alter reduce ethanol intake.
63
Two genetic investigations provide another line of evidence that P2X4Rs play a role
in the regulation of ethanol intake. The first used a microarray technique to compare
gene expression across brain regions associated with reward in alcohol-preferring versus
non-preferring selectively bred rats (Kimpel et al., 2007). The findings revealed that
p2rx4 gene expression was markedly lower in the alcohol-preferring versus non-
preferring rat lines, particularly in the amygdala and caudate-putamen. Similarly,
Tabakoff et al. (2009) used microarray analysis to identify gene expression differences
among 26 recombinant inbred rat strains that differed in 24-hr, two-bottle choice ethanol
intake. Interestingly and in agreement with Kimpel et al. (2007), there was a strong
negative correlation between p2rx4 gene expression and the high alcohol-drinking
phenotype of rats. In other words, high 24-hr ethanol preference drinking was associated
with low p2rx4 expression.
The present findings provide two lines of direct evidence that P2X4Rs play a role in
regulating ethanol intake. P2X4R KO mice drank 22% more 10% ethanol than WT mice
over 4 days using the 24-hr, two-bottle choice paradigm. Interestingly, although high
variability may have obscured a significant effect of drinking day on the repeated
measures ANOVA, the data presented in Fig. 3.3a suggests that the strongest effect of
P2X4R KO on intake was during the first two days of exposure. The findings using the
intermittent ethanol exposure paradigm provide similar results. Knocking out the
P2X4Rs significantly increased ethanol intake on their first exposure (drinking session 1),
but did not have a significant effect on subsequent drinking sessions. Hence, the impact
of knocking out P2X4Rs on ethanol intake appears to primarily affect the early drinking
experience. Collectively, these findings demonstrate that eliminating P2X4Rs
64
significantly increases ethanol intake across two drinking paradigms designed to model
low levels of ethanol intake similar to those achieved with social drinking and high levels
of intake that are associated with binge-like drinking.
The current studies provide some insight into the mechanistic complexity by which a
reduction of P2X4R function might regulate ethanol intake. LORR duration accompanied
by the BECRR measurement after a hypnotic dose of ethanol is a way by which to assess
brain sensitivity, acute tolerance, or ethanol clearance (Radcliffe et al., 2005). Our data
suggest that eliminating P2X4Rs alters sensitivity to a hypnotic dose of ethanol and that
this effect may be due in part to changes in ethanol pharmacokinetics in the KO mice.
This is evidenced by 1) the modest, albeit significant, increase in LORR duration in the
KO mice and 2) that the KO mice regained their righting reflexes at similar BECs than
the WT controls. If the rate of metabolism and other PK factors were the same in KOs
and controls, one would expect the KOs to regain function with lower BECs. The most
likely explanation is that ethanol clearance, or metabolism, was somewhat slower in the
KO mice than in controls. Reduced metabolic function in P2X4R KO mice may
contribute to the transient effect of increased ethanol consumption on day 1 in the
intermittent limited access paradigm, representing an aversive effect after the initial
experience. However, it is unlikely that the potential change in metabolism observed in
the LORR test, would affect the 24-hr, two-bottle choice intake as mice do not become
intoxicated with this drinking procedure; thus, allowing for KO mice to be able to sustain
their increased ethanol intake over several days.
It should be mentioned that the interpretation of LORR duration and BECRR becomes
difficult when subtle genotype-based differences are observed. We found the LORR
65
duration to be significantly longer in P2X4R KO mice but differed by only 14 minutes.
In this small window of time, it is unlikely that significant differences in BECRR would
be captured. These results coupled with greater ethanol intake by KO mice that was
consistent across two drinking paradigms, supports the notion of an overall reduction in
brain sensitivity to ethanol in P2X4R KO. We cannot rule out the influence of a potential
modest reduction in the rate of ethanol metabolism on intake, which may explain why
ethanol intake appeared to normalize between the two groups in both the two-bottle
choice and intermittent, limited access procedures. It is clear from this study that there is
a complex interaction between the effects of the p2rx4 null mutation on ethanol
sensitivity, metabolism, and ethanol drinking which warrant further investigation.
Finally, P2XRs localized on microglia and interneurons in the ventral tegmental area
(VTA) and other areas in the mesolimbic system have been suggested to play a role in the
modulation of GABA and glutamate neurotransmitter release (Baxter et al., 2011, Xiao et
al., 2008). As such, P2X4R modulation of these receptor systems could regulate ethanol
intake by altering VTA neurotransmission and downstream dopamine release in the
nucleus accumbens (NAc) (Tabakoff et al., 2009; Xiao et al., 2008). This is supported by
the high number of P2X4Rs in medium spiny neurons and interneurons of the striatum
(Amadio et al., 2007) and the proposed role that P2X4Rs play in synaptic plasticity
(Pankratov et al., 2009).
Consistent with this notion, we found increased GABA
A
R α1 subunit expression
in the cerebellum of P2X4R KO mice. These data may provide insight into the
mechanism of P2X4R regulation of ethanol intake via changes in GABAergic activity in
a brain-region specific manner. That is, the change in expression of α1 GABA
A
R
66
subunits may lead to a dysregulation of inhibitory pathways due to a loss of regulatory
control via P2X4Rs. This change could then lead to downstream effects on ethanol
drinking phenotype. Preliminary results from our lab suggest that shRNA-based
lentiviral knockdown of P2X4Rs in the NAc increases ethanol intake in male C57BL/6
mice, further implicating a specific role for P2X4Rs in the regulation of the consumption
of ethanol.
CONCLUSION
The present findings in P2X4R KO mice provide the first direct evidence that P2X4Rs
play a role in the regulation of alcohol intake and that this effect may be mediated by a
reduction in behavioral sensitivity to ethanol. Thus, understanding the basic underlying
neurobiological processes for alcohol intake and behaviors in relation to P2X4R
expression could provide key information regarding the development of new therapeutics
for AUDs targeting this relatively new and unexplored family of LGICs.
EXPERIMENTAL PROCEDURES
Animals
The present study utilized male homozygous p2rx4 null (i.e., P2X4R KO) mice from a
breeding colony that we have established at University of Southern California (USC; Los
Angeles, CA). Re-derivation of P2X4R heterozygous (HZ) mice was performed by the
USC transgenic core using frozen HZ embryos obtained from a previously established
P2X4R KO colony (Sim et al., 2006). This effort resulted in 7 HZ mice, which were
67
backcrossed with C57BL/6J mice to produce the 1
st
generation of offspring. HZ
offspring are backcrossed every three generations with wildtype (WT) C57BL/6J mice
purchased from Jackson Laboratory (Bar Harbor, ME). We are currently in our 5
th
generation of mice. Quantitative real time polymerase chain reaction was used to
genotype DNA extracted from tail biopsies probing with primers specific for LacZ
(5’GCGAACGCGAATGGTGCAGC 3’) and P2X4 (5’TCGCTCTCTGGGTCTGGGGC
3’).
All studies were conducted with male mice that were at least 2 months and no more
than 6 months. Animals of similar age were used within each test. We used C57BL/6
mice from our colony or from Jackson Laboratory as WT controls. Pilot studies indicated
that C57BL/6 mice from these different sources did not differ significantly in ethanol or
total fluid intake. Upon weaning, all animals were separated by sex and group housed at
4-5 per polycarbonate cage until testing. Prior to the start of experiments, mice were
acclimated to individual housing and a reverse light-dark cycle (12/12 h; lights off at
1200 h) for one week. Mice received ad libitum access to food and water bottles fitted
with ball-bearing sippers. All handling and experimental procedures were performed in
accordance to protocols approved by USC’s Institutional Animal Care and Use
Committee.
Drugs
Ethanol solutions were prepared from USP grade ethyl alcohol (Gold Shield Chemical
Company, Hayward, CA) (200 proof) diluted (v/v) in tap water for drinking or 0.9%
sodium chloride for systemic intraperitoneal (i.p.) injection. Saccharin (Sigma-Aldrich,
68
St. Louis, MO) was prepared as a 0.033% and 0.066% w/v solution in tap water.
Drinking solutions were prepared and refreshed once per week, while solutions for
systemic administration were prepared fresh on the day of the experiment.
Western Blot Protein Analysis of Brain Tissue
Mice were euthanized by CO
2
inhalation. Whole brain and liver tissue were excised from
mice for quantification of P2X4R subunit expression. Similar to methods described by
Sim and colleagues (2006), tissues were homogenized in ice-cold hypotonic
homogenization buffer (10 mM Tris-HCl, pH 7.4, 1 mM MgCl
2
and protease inhibitor
cocktail). Sodium chloride was added to the final concentration of 155 mM and the
homogenates cleared by centrifugation. Membranes were recovered from the supernatant
by ultracentrifugation (45 minutes at 100,000 g, 4°C). Pelleted membranes were
solubilized by incubation in PBS containing 1% Triton X100. Solubilized protein was
cleared by centrifugation at 13,000 rpm at 4°C for 10 minutes and the protein
concentration determined using a protein assay kit (BCA, Pierce Biotechnology,
Rockford, IL). Samples (50 μg/lane) were run on 10% SDS-PAGE and transferred to
polyvinylidene difluoride PVDF membranes. The cerebellum, prefrontal cortex, and
striatum (dorsal and ventral) were dissected for quantification of GABA
A
R α1 subunit
expression. The membranes were blocked in 5% dry milk and incubated overnight at 4°C
with a rabbit anti-P2X4 primary antibody (1:2000 dilution, Alomone Labs, Israel) and
rabbit anti-GABA
A
α 1 antibody (1:1000 dilution, Phosphosolutions, Aurora, CO).
Protein bands were visualized using enhanced chemiluminescence (Pierce
69
Biotechnology) after incubation with the secondary anti-rabbit and anti-mouse antibody
(1:10000 dilution).
24-hr Access, Two-Bottle Choice
The 24-hr access, two-bottle choice (i.e., preference) ethanol drinking model (Belknap et
al., 1993; McClearn, 1959; Middaugh et al., 1999; Rodgers, 1966; Yoneyama et al.,
2008) is widely used to assess changes in drinking and preference and is considered a
model of “social drinking” behavior in rodents since sustained elevated ethanol intake,
BECs, and intoxication are not achieved (Blednov et al., 2010). We utilized a
modification of the procedure described previously by Yoneyama et al. (2008) and
(Yardley et al., 2012). In this experiment, we used assessed ethanol intake in male
P2X4R KO mice, using C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) as the
WT controls. Briefly, the alcohol naïve mice had 24-hr access to a 25 mL bottle
containing a solution of ethanol and another containing tap water. The ethanol bottle
contained a 10% (10E) solution and intake was measured for a period of 4 consecutive
days. Bottle volumes were measured in the morning on each day with positions switched
every other day to avoid side preferences. Body weights were measured for each mouse
to calculate the g/kg/24-hr intake of ethanol. The percent preference for ethanol was
calculated as the volume of the ethanol solution consumed divided by the total fluid
volume consumed (i.e., ethanol + water) x 100.
A separate group of male C57Bl/6 and P2X4R KO mice were tested for saccharin
intake using the same two-bottle choice paradigm used in the ethanol study. These
animals had a choice between a 25 mL bottle containing 0.033% saccharin or tap water
70
for a period of four consecutive days. This was followed by a choice between 0.066%
saccharin solution and tap water for four consecutive days.
Intermittent, Limited Access
The intermittent, limited access procedure was used to model high ethanol drinking, as
described previously by our lab and others (Neasta et al., 2010; Yardley et al., 2012).
Briefly, each alcohol naïve WT and KO mouse was tested with two concentrations of
ethanol (10% then 20%, 10E and 20E respectively) according to the following
procedures. Mice were given access to one bottle containing an ethanol solution for six
drinking sessions, followed by a one week wash out period between the two ethanol
concentrations in which only tap water was available. Ethanol access periods were every
other day for 4-hrs beginning three hours into the circadian dark. Drinking session days
were Monday, Wednesday, and Friday. Water was available continuously between
ethanol sessions. Ethanol intake was determined by measuring bottle volumes
immediately prior to and after the 4-hr drinking period. Body weights were also
measured prior to each drinking session. Water intake over four hours was measured
during the time frame of the ethanol drinking period for three drinking sessions prior to
initiating the experiment.
Loss of Righting Reflex (LORR) and Blood Ethanol Concentration at Return of
Righting Reflex (BECRR)
Ethanol sensitivity in WT and KO mice was assessed by measuring the duration of
LORR and then determining the blood ethanol concentration at return of righting reflex
71
(BECRR) via a retro-orbital blood sample (Alkana et al., 1988). Mice were injected (i.p.)
with 3.6 g/kg of ethanol and returned to the cage until they appeared ataxic. Each mouse
was placed on its back in a V-shaped trough and the LORR latency and duration was
measured. The time from injection to LORR and the time from LORR to return of
righting reflex were recorded. Return of righting reflex was defined as the animal’s
ability to right itself on all 4 paws, three times in 60 seconds.
Blood samples of 20 μL were obtained from a subset of ethanol-injected mice via
retro-orbital sinus immediately upon return of righting reflex. Blood obtained was
processed by dilution in storage vials containing a matrix (500 µl) comprised of n-
propanol (Sigma-Aldrich, St. Louis, MO) and deionized water. BECRRs were analyzed
by head-space gas chromatography at Oregon Health & Science University, using routine
procedures (Finn et al., 2007). Mice that did not lose their righting reflex in less than five
minutes post-ethanol administration or those that had an LORR duration or BECRR
greater than two standard deviations from the group mean were excluded from the
analysis (Parker et al., 2008; Radcliffe et al., 2005).
Statistical Analysis
Experiments were conducted using a between-subjects design. All data were analyzed
with GraphPad Prism software (San Diego, CA) and are presented as mean + SEM for
each experiment. Expression levels of GABA
A
α1 receptor subunits were analyzed by
unpaired, two-tailed student’s t-test. Repeated-measures, two-way ANOVA were used to
analyze the effect of genotype and day/drinking session on two-bottle choice and
intermittent, limited access ethanol intake and genotype and concentration on two-bottle
72
choice saccharin intake. LORR and BECRR data were analyzed by unpaired, two-tailed
student’s t-test. Statistical significance was set at p<0.05.
73
CHAPTER 4:
DEVELOPMENT AND USE OF LENTIVIRAL VECTOR TECHNOLOGY
TO INVESTIGATE THE ROLE OF ACCUMBAL P2X4 RECEPTORS IN ETHANOL INTAKE
CHAPTER 4 ABSTRACT
Recent work indicates that P2X4 receptors (P2X4Rs), a member of the P2X superfamily
of ligand-gated ion channels, play an important regulatory role in alcohol intake and
behavior. In particular, studies using microarray techniques that focused on alcohol (i.e.,
ethanol) intake in high and low consuming male rats found an inverse relationship
between ethanol intake and expression of the p2rx4 gene. Likewise, we recently found
that male P2X4R knockout (KO) mice drink significantly greater amounts of ethanol
compared to wildtype (WT) C57BL/6 controls. Conversely, saccharin intake in male
P2X4R KO mice did not significantly differ compared to controls, suggesting that the
changes in ethanol intake did not generalize to other tastants. While KO mouse models
are an invaluable tool for understanding genetic contributions to behavior in vivo, results
from KO studies may be complicated by compensatory mechanisms arising during
development and/or non-specificity of the gene deletion. The present study used a
lentiviral gene delivery strategy to begin investigating the effect of brain-region specific
modification of P2X4R expression on ethanol intake. Based on our data from P2X4R
KO mice, we predicted that p2rx4 suppression in the nucleus accumbens (NAc) would
lead to an increase in ethanol consumption. To this end, we constructed, optimized, and
validated several lentiviral vectors in murine and mammalian cell lines and primary
neuronal cultures for targeted p2rx4 expression and suppression. Lentiviral particles for
P2X4R knockdown were generated from a shRNA vector and microinjected into the NAc
74
of male C57Bl/6 mice. In agreement with our results from KO mice, we found that
experimentally reduced expression of P2X4Rs in the NAc increased ethanol intake
compared to WT mice measured over three weeks using a 24-hr, two-bottle choice
paradigm. The shRNA-injected mice also consumed significantly greater levels of
saccharin compared to controls. Overall, the findings agree with evidence from the
P2X4R KO mouse studies and add new support for the hypothesis that P2X4Rs play a
role in regulating ethanol intake. Continued investigation with brain-regional knockdown
of P2X4Rs will provide insight into the role of these receptors in other areas of the brain.
Such studies may aid the development of novel therapeutics for the treatment of alcohol
use disorders.
75
INTRODUCTION
Building evidence supports a role for P2X receptors (P2XRs) in modulating the cellular
and behavioral effects of alcohol (i.e., ethanol). ATP-activated P2XRs are ion channels
that allow the influx of sodium and potassium, and are highly permeable to calcium. In
addition, P2XRs are sensitive to ethanol at intoxicating concentrations and are located in
brain regions reported to be neural substrates of alcohol. Thus ethanol may cause some
of its behavioral effects by directly modulating P2XR function. Recent studies have
shown that ATP activation of presynaptic P2XRs can alter GABA and glutamate
neurotransmitter release, which are widely believed to play key roles in ethanol-induced
behaviors (Chizh et al., 2001; Deuchars et al., 2002; Hugel et al., 2002; Jo et al., 1999;
Khakh, 2001; Tabakoff et al., 2009; Xiao et al., 2008). Taken together, these reports
suggest that P2XRs may directly or indirectly alter the cellular and behavioral effects of
ethanol via modulation of these other ethanol sensitive systems.
Of the seven P2XR subtypes (i.e., P2X1-P2X7), P2X4Rs are the most abundantly
expressed in the central nervous system (CNS) and are found in both neurons and
microglia. P2X4Rs are also the most ethanol sensitive subtype to be identified (Buell et
al., 1996b; Soto et al., 1996a). For example, physiological concentrations of ethanol
(e.g., 5 mM) reduces P2X4R function when expressed in Xenopus oocytes and tested
using two-electrode voltage clamp (Davies et al., 2005; Davies et al., 2002b). Moreover,
support for the importance of P2X4R in vivo can be taken from recent genomic studies in
rats, reporting that the p2rx4 gene may be linked to alcohol intake and/or preference
(Kimpel et al., 2007; Tabakoff et al., 2009).
76
Work from our laboratory with ivermectin (IVM) provides another line of evidence
supporting a role for P2X4Rs in regulating alcohol intake (Yardley et al., 2012). IVM is
an FDA-approved antiparasitic which, when administered to mice, significantly reduces
ethanol consumption. This effect is believed to be linked, in part, to the capacity of IVM
to block ethanol-induced inhibition of P2X4R function. More recently our laboratory
also has shown that male P2X4R knockout (KO) mice drink more ethanol compared to
wildtype (WT) controls (Wyatt et al., in revision). Collectively, these studies provide
support for the hypothesis that P2X4Rs play a role in regulating ethanol intake and
related behaviors.
While KO mouse models provide information about genetic contributions to behavior
in vivo, results from KO studies may be complicated by compensatory mechanisms
arising during development and/or non-specificity of the gene deletion. Lentiviral vector
gene delivery technology is an alternative strategy beneficial for efficient, stable, and
long-lasting delivery of transgenes and interfering RNA (RNAi) in vitro and in vivo
(Lasek and Azouaou, 2010). Additionally, RNAi technologies are applicable during
adult stages ameliorating issues associated with developmental differences in gene
expression or compensatory mechanisms (Lasek et al., 2010).
Delivery of RNAi (e.g., short hairpin RNA; shRNA) has been demonstrated to be a
useful tool for targeted protein knockdown in brain-regions implicated in behaviors
associated with addiction (Lasek et al., 2010) and is also more cost- and time-efficient
than other strategies when targeted expression is required (i.e., Cre/loxP). The targeted
down regulation of P2X4Rs, in a brain-region specific manner, allows for testing the role
of P2X4Rs in ethanol behaviors primarily through actions in the targeted region. Others
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have established the utility of viral-mediated methods to demonstrate the importance of
various proteins in ethanol-induced behaviors. Recent examples include investigating the
effect of: 1) Down regulation of the mu opioid receptor by RNAi in the VTA (Lasek et
al., 2007); 2) Knockdown of the 4 subunit in accumbal GABA
A
Rs (Rewal et al., 2009)
and 3) Knockdown or over-expression of NAc Homer2b levels (Cozzoli et al., 2012;
Cozzoli et al., 2009; Goulding et al., 2011; Lominac et al., 2005; Szumlinski et al., 2008)
on ethanol intake in mice.
Likewise, it is probable that P2X4Rs in different regions of the brain regulate ethanol
intake and related behaviors. Traditionally, studies centered on drug-induced changes in
behavior focus on the mesolimbic dopamine (DA) reward system, including the ventral
tegmental area (VTA) and the nucleus accumbens (NAc) (Foster et al., 2004; Gonzales et
al., 2004; McCool, 2011). There are a high number of P2X4Rs in medium spiny
neurons, interneurons, and microglia of the striatum (Amadio et al., 2007; Lein et al.,
2007). As well, Pankratov et al. (2009) has previously proposed a role for P2X4Rs in
synaptic plasticity. Furthermore, P2X4Rs localized on microglia and interneurons in the
VTA and other areas in the mesolimbic system are reportedly involved in the modulation
of GABA and glutamate neurotransmitter release (Baxter et al., 2011; Xiao et al., 2008).
As such, P2X4R modulation of these receptor systems could regulate ethanol intake by
altering VTA neurotransmission and downstream DA release in the NAc (Tabakoff et al.,
2009; Xiao et al., 2008). Collectively, this information supports a role for brain-region
specific contribution of P2X4Rs in ethanol-induced behaviors. However, such studies
are impeded by the lack of potent subtype specific P2X4R agonists and antagonists
necessary for defining the role of these receptors in ethanol responses.
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The present study develops and utilizes a lentiviral gene delivery as an alternate
strategy to investigate the role of P2X4Rs expressed in the NAc in regulating ethanol
intake. Based on previous addiction studies utilizing lentiviral vector strategies and of
investigations of P2X4R KO mice, we hypothesized that lentiviral vectors would be
effective at reducing P2X4R expression in brain tissue and that knocking down P2X4Rs
in the NAc would reduce ethanol intake. A 24-hr, two-bottle choice procedure was used
to test the role of accumbal P2X4Rs on intake of ethanol and non-ethanol tastants.
RESULTS
Validation of Lentivirus-Based P2X4R Expression in HEK293 Cells and Neurons
HEK293 cells and isolated hippocampal neurons were used to validate the construction
and applicability of the lentiviral vectors expressing GFP and P2X4R-GFP in mammalian
cell culture and primary neurons. The lentiviral vector successfully infected mammalian
cultures (i.e., HEK293 cells) as indicated by GFP fluorescence. Lentiviral transduction
did not cause any overt signs of cytotoxicity (Fig. 4.1a, 4.1e). Primary neuronal cultures
are difficult to transfect. Therefore, we next tested the effectiveness of lentiviral gene
delivery in hippocampal neurons isolated from embryonic (day 18) rat pups. As
illustrated in figures 4.1b and 4.1f, hippocampal neurons also showed a high transduction
efficiency using the GFP lentiviral vector. Next, we tested the lentiviral vector
containing p2rx4 as our gene of interest targeted for P2X4R expression. HEK293 cells
tolerated incubation with P2X4-expressing lentiviral particles, exhibiting less cell death
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than did primary cultures (Fig. 4.1g-h). However, both cultures showed acceptable levels
of GFP fluorescence (Fig. 4.1c-d).
FIGURE 4.1
Figure 4.1 Lentiviral vectors efficiently transduce mammalian and primary neuronal cultures.
Lentiviral vector expressing GFP and P2X4 in HEK293 cells (a and c, respectively) and primary cultures of
embryonic E18 rat hippocampal neurons (b and d, respectively). Corresponding phase contrast images
below.
Additionally, we observed a punctate GFP fluorescence pattern in hippocampal
neurons infected with P2X4-expressing lentivirus, representing P2X4R expression at the
cell membrane, which is also visible in the axonal projections at a higher magnification
(Fig. 4.2a-b).
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FIGURE 4.2
Figure 4.2 Punctate fluorescence pattern of P2X4Rs in primary neurons following lentiviral gene
delivery. (a) 10X magnification and (b) 20X magnification illustrate a fluorescence pattern typical for a
membrane-localized ion channel in lentivirus transduced primary hippocampal neurons.
Confirmation of Functionality of Lentivirus-Delivered P2X4Rs
We sought to test the functionality of P2X4Rs expressed by the lentiviral vectors. This
was achieved by first comparing the level of P2X4 protein in primary neuronal cultures
before and after lentiviral infection and second by assessing ATP-gated currents in
lentivirus-delivered P2X4Rs. Western immunoblotting revealed that hippocampal
neurons from embryonic day 18 rat pups that were infected with GFP and P2X4
expressing lentivirus, have protein bands at the expected molecular weights when probed
with an anti-GFP primary antibody (Fig. 4.3a). When measuring the level of endogenous
P2X4 protein in primary hippocampal cultures, we found that these neurons do not
express a detectable amount of P2X4 protein (Fig. 4.3a). As shown, probing
hippocampal neurons with anti-P2X4 primary antibody resulted in bands at 90kDa, the
expected molecular weight for cells infected with P2X4-expressing lentiviral vectors
(Fig. 4.3a).
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FIGURE 4.3
Figure 4.3 Lentivirus-mediated expression of functional P2X4Rs in neurons. (a) Detection of GFP at
30 kDa and P2X4-GFP fusion protein at 90 kDa in lentivirus-infected neurons. (b) 50 mM and (c) 100 mM
reduced ATP-induced currents in E18 rat hippocampal neurons transduced with lentivirus to express
P2X4RS. (d) Dose-dependent ethanol inhibition of P2X4Rs expressed by lentiviral vectors. Figure
modified from (Ostrovskaya et al., 2011).
In addition to demonstrating lentivirus-mediated delivery of P2X4Rs, we also
evaluated the presence of functional receptors. ATP-induced currents and ethanol
modulation of receptor activity was measured by whole-cell patch-clamp
electrophysiology (Ostrovskaya et al., 2011). In accordance with the lack of detectable
P2X4 protein in hippocampal neurons from embryonic day 18 rat pups, ATP did not
elicit currents in non-transduced neurons (data not shown). However, tracings obtained
from lentivirus-infected neurons were typical of P2X4Rs (Fig. 4.3b-c) and showed
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ethanol inhibition of ATP-induced currents in a reversible and concentration-dependent
manner (Fig. 4.3d).
Validation of shRNA-based Lentiviral Knockdown of P2X4Rs in Mouse Microglial
(BV-2) Cells
The capability of shRNA-based lentivirus to knockdown P2X4Rs was tested in the BV-2
cell line. BV-2 cells have high levels of endogenous P2X4 protein (Fig. 4.4a-b).
Comparatively, control virus caused a minimal reduction in P2X4 expression.
Densitometry quantification of P2X4 protein levels normalized to β-actin revealed that S-
1 and S-2, which targeted different regions of P2X4 mRNA, caused a marked reduction
in P2X4 protein of approximately 30-40%. S-1 and S-2 mixed in equal parts (S-1+2) and
used to transduce BV-2 cells significantly reduced P2X4 protein to 90% of non-
transfected cells (p=0.018) (Fig. 4.4a-b).
FIGURE 4.4
Figure 4.4 shRNA-mediated reduction of P2X4Rs murine microglial cells expressing endogenous
P2X4RS. (a) shRNA lentiviral vector targeting P2X4 mRNA is capable of significantly reducing P2X4
protein expression in BV-2 cells. (b) Protein knockdown analyzed by densitometry with bands normalized
to β-actin. Values represent mean + SEM compared to NT BV2. *p<0.05.
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Targeting and Lentivirus Expression in Brain Tissue
Injections of methylene blue dye in a subset of mice illustrate that the stereotaxic
coordinates used in these experiments target the NAc (Fig. 4.5, Left). At the conclusion
of this experiment, histological assessment of GFP fluorescence in mice that underwent
two-bottle choice ethanol drinking revealed infection of NAc tissue. GFP-positive tissue
was unilateral in the shRNA-injected mice (Fig. 4.5, Center). Presumably due to a
technical issue, fluorescence was not detected for one animal in the shRNA treatment
group and mice that received Scr virus. Sections from virus-injected mice demonstrate
that bolus lentiviral injections are capable of cellular transduction in brain tissue (Fig. 4.5,
Right).
FIGURE 4.5
Figure 4.5 shRNA infection area and lentiviral expression in vivo. (Left) coronal section from 7 week
old mouse immediately following injection of methylene blue dye for validation of NAc coordinates.
(Center) Schematic of coronal section (Franklin and Paxinos, 2007) representing area of lentivirus
infection relative to 1.18 mm Bregma. (Right) High magnification image showing transduction efficiency
of lentiviral infection in cells with neuronal morphology.
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24-hr Access, Two-Bottle Ethanol and Tastant Drinking
Mice that received microinjections of shRNA-based lentivirus targeting P2X4 did not
have overt signs of toxicity or immunogenicity from the viral injections. shRNA-injected
mice appeared healthy and without overt signs of toxicity throughout the experiment,
showing consistent increasing weight over the three week drinking period from 21.5 +
0.08 to 22.9 + 0.2 grams. Food intake for WT controls was 4.6 + 0.2 versus 4.9 + 0.3
grams for shRNA-injected mice during the study.
From initial analysis of cumulative ethanol intake for each week, it was apparent that
consumption of a 10% ethanol (10E) solution was increased in shRNA-injected mice
compared to WT controls (Fig. 4.6a). Since this is the first study of our lentiviral vectors
in vivo, intake was analyzed by drinking session for evaluation of daily variability within
the groups and to obtain a reference point for timing of maximal behavioral effect from
shRNA knockdown (Fig. 4.6b). Over all 12 days, two-way repeated measures ANOVA
revealed a significant effect of drinking session [F(11, 77) = 2.27; p = 0.02] and a non
significant trend for the effect of treatment [F(1, 77) = 2.61, p = 0.11] on 10E intake, with
no significant drinking session x genotype interaction [F(11, 77) = 0.63, p = 0.63].
There was no significant effect of treatment or drinking session on water intake and
ethanol preference in shRNA-injected mice compared to controls (Fig. 4.6c-d).
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FIGURE 4.6
Figure 4.6 Ethanol intake is increased in C57BL/6 mice after shRNA-mediated reduction of P2X4Rs
in the nucleus accumbens. (a) Weekly two-bottle choice intake of a 10% ethanol (10E) solution was
increased in mice with lentivirus-mediated reduction of P2X4Rs in the NAc compared to WT controls. (b)
10E intake by drinking session showing maximal behavioral effect in shRNA-injected mice occurring
during week 2 of the study. (c-d) Water intake and ethanol preference did not differ between the groups.
Values represent mean + SEM.
WT and shRNA-injected mice were also tested for quinine and saccharin intake using
an abbreviated (4 day) two-bottle choice procedure, similar to the ethanol study. Intake
and preference for a 0.015 mM quinine solution did not significantly differ between
shRNA-injected and WT controls (Fig. 4.7a-b). Conversely, shRNA-injected mice
consumed significantly more 0.033% saccharin than WT mice (p<0.05) with no
difference between the groups in preference (Fig. 4.7c-d).
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FIGURE 4.7
Figure 4.7 Saccharin but not quinine intake is increased in C57BL/6 mice after shRNA-mediated
reduction of P2X4Rs in the nucleus accumbens. (a-b) Intake and preference for quinine was equivalent
between shRNA-injected mice and WTs while (c) saccharin intake, but not (d) preference, was
significantly higher following P2X4R knockdown in the NAc. Values represent mean + SEM. *p<0.05.
DISCUSSION
In the current study we sought to develop tools for the investigation of the role of
individual P2XR subunits in different brain regions on ethanol intake. We tested the
hypothesis that there is an inverse relationship between P2X4R expression and ethanol
intake and predicted that lentiviral-mediated reduction of P2X4Rs in the NAc would
increase ethanol consumption in mice. In support of the hypothesis, we found that
reducing accumbal P2X4Rs increases ethanol intake in male C57BL/6 mice compared to
controls. These results complement our findings using P2X4R KO mice and extend this
work by providing information regarding the involvement of accumbal P2X4Rs in
ethanol responses.
The successful development of lentiviral vectors for altering expression of functional
P2X4Rs via expression or suppression of p2rx4 is illustrated by the extensive validation
experiments. All vectors employed were preliminarily assessed for their capability to
transduce mammalian cells and primary neuronal cultures. In both HEK293 cells and
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cultured E18 rat hippocampal neurons, we observed high expression of GFP fluorescence
supporting the efficacy of the constructed lentiviral vectors and the reliability of the
protocols used to create and concentrate lentivirus particles in our laboratory.
Furthermore, the lentiviral expression vectors developed for these experiments were
useful for delivery of p2rx4 genetic material to cultured neurons, resulting in detectable
P2X4 protein expression in cultures that otherwise did not express P2X4 as well as
functional P2X4Rs as determined by their electrophysiological properties. Lentiviral
vectors for RNAi applications should also involve additional validation to quantify the
level of protein knockdown. This was accomplished as demonstrated by our in vitro
studies showing variability in shRNA efficiencies. In agreement with previous work
reported by others (Song et al., 2008), we achieved high levels of targeted gene
suppression, which ranged between 80-90% when combining our S-1+S-2 shRNA
lentiviral vectors.
Earlier work by our lab and others indicates that P2X4R function is inhibited by
alcohol in vitro (Davies et al., 2002; Davies et al., 2005, Xiong et al., 2005; Popova et al.,
2010; Ostrovskaya et al., 2011) and that modulation of receptor function can alter ethanol
intake in vivo (Yardley et al., 2012). This work coupled with recent evidence that rodents
with a high alcohol drinking phenotype have reduced p2rx4 expression (Kimpel et al.,
2007; Tabakoff et al., 2009), supports the notion that P2X4Rs play a role in the
regulation of ethanol intake. We recently provided the first direct evidence to support
this hypothesis, reporting increased ethanol intake by male P2X4R KO mice (Wyatt et
al., in revision). In the present study, our efforts to target shRNA-based lentivirus to the
NAc increased intake of a 10% ethanol solution in C57BL/6 mice, augmenting initial in
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vivo studies demonstrating increased ethanol intake by P2X4R KO mice. Moreover, the
increase in ethanol intake by mice with shRNA knockdown of P2X4Rs in the NAc
provides additional support for the role of these receptors in ethanol consumption with
brain-regional specificity.
Previous theories in the alcohol field postulated a global effect of alcohol on the brain
as opposed to selective, regional effects on brain function (White et al., 2000). More
recent work by other laboratories investigating ethanol behaviors by using brain-region
selective knockdown strategies against proteins of interest dispels this notion (Baek et al.,
2010; Cozzoli et al., 2012; Lasek et al., 2007). For example, the Szumlinski lab has
shown that alcohol intake can be altered by overexpression and knockdown of accumbal
Homer2, proteins that are responsible for coordinating the function of synaptic glutamate
receptors (Cozzoli et al., 2009; Szumlinski et al., 2008). Our demonstration of a brain-
region selective knockdown of P2X4Rs also supports the latter notion.
Interestingly, in the present study, increased intake by mice injected with shRNA
against p2rx4 in the NAc was not selective for ethanol, as saccharin intake was also
significantly higher in these mice compared to controls. Conversely, intake and
preference for an aversive bitter solution (i.e., quinine) was comparable between shRNA-
injected and WT mice. The NAc has been determined to be the target region of the
mesolimbic reward pathway involved in the regulation of natural reward and the
reinforcement of drugs of abuse (Wise, 2004). Medium spiny neurons (MSNs), the
primary cell type within the NAc, receive dopaminergic input from the VTA and project
GABAergic efferents to the ventral pallidum (Girault, 2012; Pierce et al., 2006).
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Presumably, knockdown of P2X4Rs alters inhibitory function of these MSNs, producing
increased ethanol and saccharin intake phenotypes in mice.
An alternate possibility for this effect may involve the deactivation of microglial
P2X4Rs. Recent studies have implicated a role for microglia in drug reward responses
and plasticity whereby activated microglia inhibit opioid function in the NAc (Inoue,
2006; Kovacs, 2012). Opioid agonists have been shown to increase ingestive behaviors
related hedonic value (Zhang and Kelley, 2002). Though, further studies are needed to
better understand the intricacies of underlying molecular pathways, results from this
experiment suggest that P2X4Rs located in the NAc may contribute to alterations in
general reward processing or consumption of hedonic substances that is likely not due to
alterations in gustatory processes.
In addition to the acquisition of spatial information, lentiviral strategies provide some
temporal resolution for investigation of specific proteins and receptor systems. In the
present study, shRNA-injected mice appear to gain their increased ethanol drinking
phenotype 4-weeks post virus injection. This effect may be associated with differences in
the efficacy of the lentivirus in each animal. In vivo reports of inter-individual variations
of shRNA efficiencies are suspected to be caused by construct integration into active
versus quiescent locations on the chromosome thereby affecting the levels of circulating
shRNA and degree of the knockdown (Prawitt et al., 2004). The Janak lab reports
efficacy of their viral-mediated gene silencing on ethanol intake occurring 10 days post-
injection, peaking about 8 days later, and lasting approximately 2 weeks. Janak and
colleagues attribute this effect to the time needed for processing and protein
90
downregulation and recycling (Nie et al., 2011). In our case, more studies are necessary
to see if this is the case for our current findings.
Notably, ethanol intake differences between both shRNA-injected and P2X4R KO
mice and their respective WT counterparts are more evident by daily drinking patterns
rather than overall intake. Average weekly ethanol intake levels of shRNA-injected mice
appeared to be moderately higher than WTs, however when analyzed for daily intake,
temporal information can be gleaned. Similar to reports by Furay and colleagues (2011),
analysis of the microarchitecture underlying ethanol intake provides more information
regarding differences between the groups. Parameters such as bout size and duration may
magnify subtle P2X4R-mediated differences in ethanol drinking that are seemingly
transient and unobvious from a cumulative analysis.
Several limitations to the study should be acknowledged including the lack of the Scr
control group and the need for assessment of shRNA knockdown in brain tissue.
Although WT controls and the availability of P2X4R KO data provide comparative
groups for the shRNA-injected mice, we cannot rule out the possibility that increased
ethanol intake is not due to an off-target affect mediated by a viral injection into the
brain. Additionally, ethanol intake by shRNA-injected mice was not as robust as
expected. This may be explained by either unilateral elimination of P2X4Rs and/or the
timeframe in which ethanol measurements occurred post-infusion of the shRNA virus. It
will be important to determine the time course for the effect of shRNA-mediated P2X4R
knockdown in brain tissue and how this correlates to ethanol intake. This information
may help define the optimal time for capturing ethanol drinking measurements in
shRNA-injected mice. These experiments are currently underway.
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P2X4Rs are located throughout the NAc and so future studies can investigate whether
sub-regional changes in P2X4R expression can exert changes in ethanol intake. The
distinction between NAc core and shell has been described in previous alcohol and drug-
related studies but is not completely understood. Though both the shell and core have
been implicated as mediators of ethanol intake and reinforcement, the shell is considered
to have a more prominent role (Everitt et al., 2008). Virus-mediated knockdown of
proteins in the shell versus core reportedly cause differential ethanol consumption
(Cozzoli et al., 2012). Studies by Nie et al. (2011) demonstrate differences between gene
silencing in the lateral versus medial NAc shell on ethanol behaviors. Moreover,
consideration of additional brain regions, particularly those that are part of the
mesolimbic dopamine system, will continue to aid our understanding of the role of these
receptors in addictive behaviors.
CONCLUSION
The present work utilized lentiviral vectors for site-specific, long-lasting suppression of
P2X4Rs to begin investigating the contribution of accumbal P2X4Rs in ethanol
consumption in C57BL/6 male mice. The increase in ethanol intake by shRNA-injected
mice extend our previous work with P2X4R KO mice by demonstrating that changes in
ethanol intake are linked to spatial and temporal changes in p2rx4 gene expression.
These studies, where P2X4R expression was reduced in the NAc, suggest a role for
P2X4Rs in mediating the rewarding effects of ethanol and provide additional support for
increased ethanol intake by P2X4R KO mice. Continued investigation with brain-
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regional knockdown of P2X4Rs will provide insight to the role of P2X4Rs in other areas
of the brain. Such studies may aid the development of novel therapeutics for the
treatment of AUDs.
MATERIALS AND METHODS
Animals
Male C57Bl/6 mice used for these studies were obtained from our colonies maintained at
the University of Southern California. Mice were genotyped by Transnetyx (Cordova,
TN) using ear biopsies probing for P2X4 and LacZ. After weaning, mice were housed 4-
5 per cage in a temperature and humidity controlled room on a 12/12 h light-dark cycle
(lights off at 19:00 h). Mice were 5-6 weeks old at the time of stereotaxic microinjection
so that all mice were between 8-12 weeks at the start of the drinking studies.
Construction of Lentiviral Vectors & Lentivirus production
Lentivirus was produced according to previously published methods (Ostrovskaya et al.,
2011). Briefly, cDNA encoding rat WT P2X4R was subcloned into the pLVX-AcGFP-
N1 lentiviral expression vector. In addition, cDNA encoding two shRNA sequences
targeting different regions of P2X4 mRNA (called S-1 and S-2) or a scramble sequence
were subcloned into the pLVX-shRNA2 vector. Both viral vectors were obtained from
Clontech Laboratories (Mountain View, CA) and contained genetic material for
expression of green fluorescent protein for tracking transduced cells. The P2X4
expression vector produced a GFP fusion protein (e.g., P2X4-GFP) while GFP expression
93
was under bicistronic control in the shRNA vector. From here forward, “shRNA” will be
used to describe gene sequences targeting P2X4 mRNA or “scramble (Scr)” for gene
sequences that do not target P2X4 mRNA. All lentiviral vector constructs were verified
by enzymatic digestion and gel electrophoresis followed by DNA sequencing
(USC/Norris DNA Core Facility). Lentivirus was produced by mixing constructs with
psPAX2 and pMD2.G packaging vectors obtained from Addgene (Cambridge, MA) and
used to transfect 293T cells. Virus-containing supernatant was collected and
concentrated with Lenti-X Concentrator. Viral titers were determined via the Elisa
method using the Lenti-X p24 Rapid Titer Kit Elisa. Concentrator and titration kits were
obtained from Clontech Laboratories (Mountain View, CA).
Validation of Lentivirus-Based P2X4R Expression in HEK293 Cells and Neurons
HEK 293 cells and primary hippocampal neurons were utilized to validate virus
expression. Primary neurons were isolated from the hippocampi of E18 pups obtained
from timed pregnant Sprague-Dawley rats (Harlan, Indianapolis, IN). Dissected
hippocampi were enzymatically digested in 0.02% trypsin and titrated through a series of
Pasteur pipettes with diameters that were progressively smaller. Cells were plated on
poly-D-lysine coated plates and maintained in neurobasal medium supplemented with
primocin, B-27, and 25 um glutamate for the first 3-4 days and then media with
supplements and no glutamate thereafter. Neurons were allowed to mature for at least 5
days prior to lentiviral transduction. All cells were infected between days in vitro 5 to 7
with 10
6
infectious units/ml. After 48-hrs incubation with viral particles, transduction
efficiency of virus expression was visualized by GFP fluorescence using a fluorescence
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microscope (Zeiss, Thornwood, New York). A portion of non-transduced and transduced
primary neurons were homogenized in ice-cold hypotonic lysis buffer (10 mM Tris-HCl,
pH 7.4, 1 mM MgCl
2
) containing protease inhibitor (1:100 dilution). Lysates were spun
at 13,000 rpm for ten minutes at 4°C and the protein-containing supernatant was
collected and stored at -80 degrees C until used for western immunoblotting.
Western Immunoblotting
Lysates from cultures of primary hippocampal neurons and BV-2 cells that were non-
transduced or infected with lentivirus were used for analysis of P2X4 protein. Briefly,
samples were run on 10% SDS-PAGE and transferred to PVDF membranes. Membranes
were incubated with a rabbit anti-P2X4 primary antibody (1:2000 dilution) (Millipore,
Temecula, CA) and a secondary anti-rabbit antibody (1:10000 dilution) prior to
visualizing the P2X4 bands using enhanced chemiluminescence (Pierce Biotechnology).
Whole-Cell Patch-Clamp Electrophysiology
Whole-cell patch-clamp recordings were performed for validation of expression of
functional P2X4Rs as described in Ostrovskaya et al. (2011). In brief, primary
hippocampal neurons that were non-transduced (control) and transduced with lentivirus
for P2X4R expression were voltage-clamped at -50 mV and ATP-induced currents were
captured with an Axopatch 200B amplifier, Digidata 1320 interface, and pClamp 9.0
software (Molecular Devices, Sunnyvale, CA). External solutions contained 135 mM
NaCl, 5.4 mM KCl,1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose
with pH of 7.4 adjusted with NaOH and the internal solution contained 140 mM KCl, 2
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mM MgCl2, 2.5 mM EGTA, 2 mM TEA-Cl, 4 mM K2ATP, and 10 mM HEPES, pH
adjusted to 7.25 with KOH. Fast drug applications were performed through a three-barrel
flowpipe using a Warner SB-77B Fast Perfusion apparatus and VC-6 Valve Controller
(Warner Instruments, Hamden, CT).
Validation of shRNA-based Lentiviral Knockdown of P2X4Rs in Mouse Microglial
(BV-2) Cells
shRNA-mediated knockdown of P2X4Rs was validated through transduction of mouse
microglial cells (BV-2 cells), which contain high endogenous expression of P2X4
protein. BV-2 cells were cultured in 6-well plates in DMEM F12 media supplemented
with penicillin/streptomycin and fetal bovine serum until they reached 70-80%
confluency. Once confluent, cells were transduced with 10
6
infectious units/ml of
shRNA-based lentivirus. Confirmation of virus expression was visualized by GFP
fluorescence 24-48 hours later. Cells were then lysed with lysis buffer containing
protease inhibitor (1:100 dilution). Lysates were spun at 13,000 rpm for ten minutes at
4°C and the protein-containing supernatant was collected and stored at -20°C until used
for western immunoblotting.
Stereotaxic Microinjection Surgeries & Viral Gene Transfer
Male mice were anesthetized with a ketamine (100 mg/kg) and xylazine (10 mg/kg)
cocktail then placed in a mouse stereotaxic frame (David Kopf Instruments, Tujunga,
CA). A small incision was made to the skin exposing the skull. Bregma and lambda were
measured to ensure an even plane and a small area of dura bilaterally removed in the area
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for microinjection. A ten-microliter syringe (Hamilton, Reno, NV) was used to deliver
0.25 microliters of shRNA or Scr lentivirus bilaterally to the NAc (bregma coordinates:
anteriorposterior 1.3 mm; mediolateral, 1.0 mm; dorsoventral, 4.5 mm) at a rate of 0.1
microliters/min.
24-hr Access, Two-Bottle Choice Drinking
Mice were tested in a 24-hr, two bottle-choice procedure as previously described by
others (see (Yardley et al., 2012; Yoneyama et al., 2008). The three experimental groups
were WT C57Bl/6 mice (no surgery) (n=6), mice receiving Scr virus (n=5), and mice
receiving shRNA virus (n=5). Mice that underwent viral injections began ethanol
drinking 3 weeks-post surgery and continued drinking for a period of 12 days (or 3
consecutive weeks). During ethanol intake test periods, mice had 24-hr access to a 25
mL bottle containing a solution of 10% ethanol (v/v, 10E) and another containing tap
water. Following ethanol testing and using the same two-bottle choice procedure, mice
received 0.015 mM quinine then 0.033% saccharin versus water measured for 4
consecutive days with a 48-hr (water only) period between all three tastants. Bottle
volumes were measured at the same hour each morning with positions switched every
other day to avoid side preferences. Body weights were recorded to calculate the intake
level for each tastant. The percent preference for each tastant was calculated as the
volume consumed of the tastant divided by the total fluid volume consumed x 100.
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Tissue Processing for Validation of Targeting and Virus Expression
Prior to initiating surgeries for the ethanol drinking experiment, a subset of mice received
microinjections of methylene blue dye to assess stereotaxic coordinates targeting the
NAc. These mice were transcardially perfused with ice-cold saline followed by 4%
paraformaldehyde (PFA). Brains were removed, post-fixed in PFA, and cryoprotected in
a 20% sucrose solution. A cryostat was used to make 25 micron sections which were
mounted on subbed slides and coverslipped. Sections used to validate methylene dye
targeting were lightly stained with crysl violet. Mice that received microinjections of
shRNA or Scr virus were also transcardially perfused at the end of the study. To assess
the location of the virus via GFP fluorescence, brains from these mice were sectioned
coronally at 25 microns, mounted on subbed slides, treated with 95% ethanol and
xylenes.
Statistical Analysis
Experiments were conducted using a between subjects design. All data were analyzed
with GraphPad Prism (San Diego, CA) software and are presented as mean + SEM for
each experiment. Densitometric analysis of western blot bands and ATP-induced
currents in transduced neurons were analyzed by one-way ANOVA. For the ethanol
drinking study, two-way repeated measures ANOVA was used to analyze the effect of
treatment (e.g., shRNA-injected or control) and week or drinking session on ethanol
(g/kg) and water (ml) intake and ethanol preference. Quinine and saccharin intake and
preference were analyzed by two-tailed, student’s t-tests. Statistical significance was set
at p < 0.05.
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CHAPTER 5:
OVERALL DISCUSSION AND CONCLUSIONS
Summary of overall findings
The findings presented in this dissertation include the initial behavioral characterization
and ethanol drinking phenotype of P2X4R knockout (KO) mice. This work substantially
adds to our knowledge regarding the involvement of the p2rx4 gene and P2X4Rs in
regulating aspects of general behavior and alcohol use disorders (AUDs). Using a multi-
pronged approach, my studies found that 1) P2X4R deficiency causes social behavioral
and communication deficits in addition to altering sensory function; 2) P2X4Rs regulate
ethanol intake; and 3) p2rx4 gene expression in the nucleus accumbens (NAc) is involved
with regulation of ethanol intake and possibly other non-ethanol tastants. Furthermore,
during the course of my studies, I begin to investigate mechanisms that may underlie the
behavioral changes resulting from alterations in expression of P2X4Rs. Taken together,
findings from my dissertation investigations identify important roles for the p2rx4 gene in
mediating neurodevelopmental processes and the propensity to consume alcohol.
As presented in Chapter 1, alcohol misuse is a pervasive socio-economic problem in
the United States. Alcohol and neuroscience research communities have identified brain
regions considered to be neural targets of alcohol. However there remains a paucity of
information regarding the initial site(s) and mechanism(s) of ethanol action. To date, the
majority of alcohol studies have focused on GABA and glutamate neurotransmitters and
their respective receptor systems. More recently, studies of recombinant P2X4Rs in vitro
and genetic studies in rodents have indirectly linked the regulation of ethanol intake to
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P2X4R expression and function. These investigations set the stage for my current
research dissertation project.
The studies in Chapter 2 were the first to characterize the behavioral phenotype of
male P2X4R KO mice. Previous work has suggested a role for P2X4Rs in hippocampal
plasticity (Sim et al., 2006), regulation of neuropathic pain (Tsuda et al., 2003), and
neuroendocrine function (Zemkova et al., 2010). The current Chapter 2 investigations
extended into new potential roles for P2X4Rs. This was accomplished by investigating
the behavioral responses of P2X4R heterozygous (HZ), and KO mice in a variety of
testing paradigms designed to assess complementary aspects of sensory function,
emotional reactivity, and cognitive organization.
The p2rx4 gene, encoding P2X4Rs, is ubiquitously expressed throughout the central
nervous system and homozygous deletion of this gene is not lethal, thus allowing for
testing of P2X4R KO mice to gain insights into the behavioral role(s) of P2X4Rs.
Behavioral characterization of male P2X4R KO mice revealed perturbations in select
sensory functions and social-communicative behavior. While this strain of mice appears
normal compared to their wildtype (WT) counterparts for spontaneous locomotor
activity, emotionality, and cognition, interestingly they displayed deficits in auditory
function, communication, social behavior, and a hypersensitivity to tactile stimuli (Wyatt
et al., in press).
Furthermore, our studies show that subunits of both NMDA and AMPA glutamate
receptors are differentially expressed in the prefrontal cortex, hippocampus, and
cerebellum of P2X4R KO mice compared to controls. The molecular changes occurred
in brain regions which can be linked to developmental deficits like autism-spectrum
100
disorder (ASD) as well as alcohol use disorders (AUDs). Taken together, these findings
represent neurodevelopmental deficits that resemble endophenotypes of ASD. Overall,
the findings highlighted a putative role of P2X4Rs in the regulation of perceptual and
socio-communicative functions and suggested that P2X4Rs may represent a new
therapeutic target for disturbances associated with neurodevelopmental disorders.
Extending the use of P2X4R KO mice, the work presented in Chapter 3 provided the
first direct evidence for the regulation of ethanol intake by P2X4Rs. Over the years,
investigations from our lab and others have implicated a role for P2X4Rs in ethanol-
induced behaviors. Support for this notion comes from in vitro reports of ethanol
inhibition of P2X4Rs (Davies et al., 2005; Ostrovskaya et al., 2011; Popova et al., 2010)
and ivermectin (IVM) antagonism of ethanol inhibition of P2X4Rs (Asatryan et al.,
2010). In addition, two independent genetic microarray studies reported a reduced
expression of p2rx4 in rodents with high alcohol-drinking phenotypes (Kimpel et al.,
2007; Tabakoff et al., 2009), thus providing support for the importance of P2X4Rs in
alcohol intake. Most recently, our group reported that IVM can reduce ethanol intake in
C57Bl/6J mice (Yardley et al., 2012). Follow-on studies linked at least a portion of
IVM’s anti-alcohol pharmacological effects to actions on P2X4Rs (Bortolato et al.,
2012b). The availability of P2X4R KO mice allowed for the first direct in vivo validation
of the hypothesis that P2X4Rs regulate ethanol intake, whereby in agreement with the
aforementioned genetic studies, mice deficient in P2X4Rs consumed significantly more
ethanol than WT controls in two ethanol drinking paradigms (Wyatt et al., in revision).
P2X4R KO did not significantly affect water intake or saccharin intake, suggesting that
the effects did not generalize to tastants other than ethanol.
101
Chapter 3 findings also began to provide insight to the mechanism(s) underlying the
increase in ethanol intake by P2X4R KO mice. This was achieved by comparing the
hypnotic effect of ethanol on P2X4R KO mice and WT controls. To this end, the loss of
righting reflex (LORR) behavioral test and blood ethanol concentrations when mice
regained their righting reflex (BECRR) were analyzed to assess differences in brain
sensitivity to ethanol. The results from this study suggested altered ethanol
pharmacokinetics in P2X4R KO mice. We also observed differences between WT and
P2X4R KO mice in the expression of GABA
A
Rs whereby P2X4R KO mice had greater
cerebellar expression of the 1 subunit compared to controls. This finding implicates a
role for P2X4Rs in altering GABAergic signaling pathways and provides new evidence
supporting P2X4R regulation of ethanol intake.
Findings presented in Chapter 4 serve as an extension to the investigations from
Chapter 3 by exploring the role of P2X4Rs localized in the NAc. The goal of this
investigation was to further explore the mechanisms for increased ethanol intake by
P2X4R KO mice. These studies involved lentivirus-mediated knockdown of the p2rx4
gene in the NAc of C57Bl/6 mice. The spatial (site-specific) and temporal control over
gene expression as enabled by lentiviral vector technology, provided experimental
conditions that may avoid some of the issues related with developmental compensation
by global gene deletion.
For these studies, intake of ethanol and other tastants was measured using a 24-hr,
two-bottle choice paradigm, similar to test conditions for P2X4R KO mice. We found
that knockdown of accumbal P2X4Rs caused increased ethanol intake in mice compared
to WT controls. Interestingly, saccharin intake was also significantly increased in these
102
mice, suggesting the involvement of P2X4Rs in the NAc in mediating general reward
processing or ingestion of hedonic substances. Moreover, it is apparent from these
experiments that a thorough study is needed on the time course of action for shRNA-
mediated knockdown of P2X4Rs and how this correlates to the effect on drinking
behavior.
Neurobiological Prospective
As demonstrated in Chapters 2 and 3, P2X4R KO mice exhibit alterations in NMDA and
AMPA glutamate receptor subunit expression as well as GABA
A
R 1 expression.
Considering the importance of excitatory glutamate and inhibitory GABA
neurotransmitter across a broad range of behavioral responses, it is of interest to relate the
behavioral and drinking phenotypes of P2X4R-null mice in light of these neurobiological
findings. P2X4R KO mice have altered glutamate subunit expression that mediate
changes in sensory function, social behavior, and communication, and it is likely that
aberrant glutamatergic signaling has some effect on the ethanol drinking phenotype of
these mice.
It is widely accepted that excitatory neurotransmission is depressed by ethanol (Tsai et
al., 1998) and that this action elicits the increase of inhibitory GABAergic responses
(Tsai et al., 1998). Accordingly, reduced NMDA glutamate receptor activity might
contribute to the sedative/depressive effects of alcohol intoxication (Tsai et al., 1998).
Alternatively, glutamatergic control over GABA interneurons is partially responsible for
the inhibition of dopamine release in certain regions of the brain. In the case of P2X4R
KO mice that consume more ethanol than their WT counterparts, it seems reasonable to
suspect that reduced NMDA function results in disinhibition, leading to increased
103
dopamine release or alcohol reinforcement. In support of this notion, I observed an
increased expression of GABA
A
R 1 subunit in P2X4R KO mice. Though baseline
behavioral measures related to disinhibition that may co-occur with ASD (i.e.,
impulsivity, hyperactivity, etc.) were not tested in these animals, it is possible that
P2X4R KO mice would react differently compared to controls in such tests due to their
enhanced GABA
A
R expression.
Limitations of the Study
The studies in this dissertation project sought to understand the role of P2X4Rs in the
regulation of ethanol intake and behaviors related to AUDs. To this end, the intrinsic and
ethanol drinking phenotypes of P2X4R KO mice were characterized. One limitation to
the investigations of both the behavioral and drinking phenotypes is the lack of
experimentation involving female mice. The findings in Chapter 2 suggest that P2X4R
deficiency elicits behaviors that resemble ASD. Notably, in the human condition, ASD
predominately affects males. Therefore, it would be of great benefit to perform these
behavioral assays with female P2X4R KO mice to determine whether a similar deficiency
has more relevance in male P2X4R KO mice than females.
An additional limitation within the ethanol drinking studies in experimental Chapters
3 and 4 involve the testing of liquid tastants saccharin and/or quinine to assess the
selectivity of increased ethanol consumption by P2X4R KO mice. Similar studies
reported in the literature typically use low and high concentrations of 0.033% and
0.066% for saccharin and 0.015 mM and 0.03 mM for quinine. In our studies with
P2X4R KO mice, we observed no differences in saccharin consumption for the 24-hr,
104
two-bottle choice (low) and intermittent, limited access (high) ethanol intake paradigms.
However, following lentiviral-mediated knockdown of P2X4Rs in the NAc of C57Bl/6
mice, we found a significant increase of saccharin intake compared to controls.
Therefore, the high levels of saccharin used in the behavioral studies for both the test and
control groups, may be linked to a ceiling effect.
In a similar manner, I also found that the level of quinine consumed by lentivirus-
injected mice was similar to controls. This result may not fully reflect the entire story as
the preference for a bitter tastant that the animals are expected to avoid should at least be
below 50%. Since the preference level for the control group was slightly greater than
50%, a higher concentration of quinine (e.g., 0.03 mM) would be useful in this test. As
such, in the case of both tastants, future studies would provide more insight if the levels
of saccharin and quinine in the study were first calibrated to reflect optimized baseline
preference levels for controls. This might bring about group differences in tastant intake
where they were not previously identified.
Future Directions
The studies presented in my dissertation provided the first direct evidence supporting a
role for P2X4Rs in the regulation of alcohol intake. This is a new area of investigation
and many avenues can continue to be explored regarding the role of P2X4Rs in both
general behavior and alcoholism pathology. Addressing the limitations outlined above is
of major importance in future investigations. For instance, the work presented in this
dissertation does not include experimentation with female P2X4R KO mice. The
prevalence of ASD has been described to be lower in females, but the topic is considered
105
to be controversial, prompting those in the field to investigate why this may be true.
Some studies report a possible gender bias in diagnosing ASD in young children
(Dworzynski et al., 2012) while others believe that females do better at functioning with
ASD symptoms (Baron-Cohen and Wheelwright, 2004). Interestingly, specific
behavioral differences have been detected in autistic males and females with males
displaying more difficulties with communication and attention, while both sexes have
similar deficits in social behavior (May et al., 2012). Characterizing the behavior of
female P2X4R KO mice would be of interest in future studies, particularly to determine
whether the receptor has similar regulatory roles in males and females and if deficits
produce endophenotypes of ASD in female P2X4R KO mice.
As for ethanol studies, cohorts of female mice were tested when available and the
drinking phenotype of these mice was consistent with our hypotheses that P2X4Rs
regulate ethanol intake and that there is an inverse relationship between ethanol intake
and p2rx4 expression. That is, our preliminary findings suggested that female P2X4R
KO mice consume significantly more ethanol than their WT counterparts (unpublished
data). This effect was primarily observed in the high-level (intermittent, limited access)
ethanol drinking paradigm.
When tested in the LORR behavioral paradigm, female P2X4R KO exhibited
significantly reduced LORR duration and had similar BECRR compared to WT controls.
These data suggest that female P2X4R KO mice metabolize ethanol faster than WT
controls but this hypothesis has yet to be tested. Interestingly, the effect of global p2rx4
elimination may be somewhat sex dependent. As reported in Chapter 3, we found that
knocking out P2X4Rs in male mice caused a transient increase in ethanol intake whereas
106
the increase in alcohol intake in female P2X4R KO mice appeared to be more persistent.
In addition, the data taken from the LORR studies, looking at both males and females,
suggested that there is more influence of P2X4R KO on metabolism in female than male
mice. The reasons for this are currently unknown. One plausible explanation may be
related to the interactions of P2X4Rs and circulating hormones or differences in body
composition. We know such gender differences have been observed in humans where
females have faster ethanol clearance rates and are less sensitive to the aversive effects of
ethanol (Mumenthaler et al., 1999; Norberg et al., 2003).
Another direction for future studies is a more in-depth analysis of compensatory
changes arising from a deficit in the p2rx4 gene. We have demonstrated alterations in
glutamate and GABA receptor expression in P2X4R KO mice, likely occurring from the
activation of compensatory mechanisms and may also occur over time during
experimentation with animals that receive lentiviral gene silencing. Such studies may
provide some clues about changes in ethanol-related behaviors and thus, it would also be
of interest to measure compensatory changes in these receptors pre- and post-ethanol
exposure in both P2X4R KO and WT mice.
Lentivirus-mediated gene delivery is a useful alternative strategy for investigating the
role of P2X4Rs in ethanol behaviors. Lentiviral techniques are particularly useful with
regard to concerns about compensation that may occur during development. Thus, our
shRNA studies helped avoid some of the issues related to developmental compensation.
Though initial investigation here utilized shRNA knockdown of P2X4R expression,
interfering RNA technology may have confounds related to variability in knockdown
efficiencies making data difficult to interpret. It is imperative that shRNA vectors are
107
validated and quantified for success in reducing protein expression both in vitro and in
vivo as individual differences between animals can cause variability that may mask the
behavioral effect of interest. Furthermore, certain genes may have expression level
thresholds that activate compensatory mechanisms making the need for similar levels of
knockdown in test animals all the more important. Gene rescue experiments are another
method for study of P2X4Rs in ethanol behaviors. Coupling the p2rx4 overexpressing
vector developed in Chapter 4 with P2X4R KO mice would complement these studies.
Clinical Implications
The findings from my dissertation studies support a role for P2X4RS in the regulation of
ethanol intake and other important behavioral domains. Compared to WT controls
P2X4R KO mice display behavioral disturbances that resemble endophenotypes
associated with neurodevelopmental disorders. Specifically, these include significant
reductions in social interaction and communication as well as altered sensory function, all
of which are considered symptoms of ASD. P2X4R KO mice also consume significantly
more ethanol than their WT counterparts. Initially, it may seem peculiar that P2X4R-
deficient mice would have these two distinct behavioral profiles; however, a search in the
literature supports a connection between mental disorders and substance use disorders.
For example, a four-year longitudinal study monitoring young adults to adulthood found
that social phobia and panic disorders may act as a predictor for future alcohol disorders
(Zimmermann et al., 2003). In addition, mounting evidence demonstrates genetic
overlap in alcoholism and mental disorders like autism (Schumann et al., 2011).
Notably, a recent study of familial neuropsychiatric disorders in families with autistic
individuals uncovered a higher incidence of alcoholism in these families (Miles et al.,
108
2003). The authors suggest that the higher incidence of alcoholism is distinct for families
with autism since of the control families in this study were those having children with
Down syndrome. Despite their being some similarity between these two
neurodevelopmental disorders, families of children with Down syndrome did not have
increased prevalence of alcoholism. Of particular importance was that none of the
autistic children, whose families were evaluated, met the criteria for fetal alcohol
syndrome (FAS). Although, autism behavioral phenotypes were not assessed for the
children in this study, two main differences were identified between those from families
with high alcoholism versus low alcoholism. These included 1) that autistic children
were more likely to be macroencephalic and 2) to have regressive autism where the onset
of the disorder was accompanied by a loss of language. Notably, the authors suggest an
association between the latter finding and maternal alcoholism that is not related to FAS.
Clinical subjects such as those referenced from the Miles study would be great
candidates for genetic investigations to determine whether there were alterations of any
type to the sequence, localization, or expression of the p2rx4 gene. A deeper
understanding of the overlap of genetic alterations mediating the two may greatly impact
the treatment of ASD and AUDs. Many symptoms and co-occurring disorders related to
alcoholism are associated with ASD. For example, inherent impulsiveness is considered
to be a factor in an individual’s likelihood to have a problem with alcohol and deficits in
inhibitory control are also co-morbid with ASD (Aragues et al., 2011; Christ et al.,
2007). Recognition of P2X4Rs as a regulator of ethanol intake and aspects of ASD may
provide a novel target for treatment of one or both disorders. Kessler et al. (2005) reports
that observations in the clinic indicate that “patients with both mental and substance
109
abuse disorders are more persistent, severe, and treatment-resistant than patients with
pure disorders”. It would be an ideal situation to have a pharmacological treatment for
AUDs that could also address symptoms of other co-occurring disorders in some
individuals.
In summary, the work presented in this dissertation substantially advances our
knowledge regarding the involvement of P2X4Rs in the regulation of ethanol intake.
These studies provide the first direct evidence that alteration of P2X4R expression in vivo
causes significant behavioral changes in ethanol intake. Additionally, our investigation
into the native behavior of P2X4R KO mice demonstrates a role for the p2rx4 gene over a
broader domain of behaviors. Findings from these studies suggest a role for P2X4Rs in
behavioral organization, including social behavior, communication, sensory function and
alcohol consumption. Collectively, the findings put forth in this dissertation, support
future investigations toward modulation of P2X4R expression and/or function as a
putative target for the development of new therapies to treat AUDs and other associated
disorders.
110
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Abstract (if available)
Abstract
P2X receptors (P2XRs) are a family of cation-permeable, ligand-gated ion channels gated by synaptically released extracellular adenosine-5'-triphosphate (ATP). Of the seven P2XR subtypes (P2X1-P2X7), P2X4 is the most abundantly expressed subtype in the central nervous system and to date is the most ethanol sensitive when measured in recombinant expression systems. Previous work demonstrates that ethanol inhibits ATP-activated currents in rat and mouse neurons, suggesting a role for P2X4Rs in ethanol-related behaviors. A recent in vivo study identified p2rx4 as a candidate gene linked to ethanol intake and/or preference in rodents. Despite these reports, the lack of specific agonists and antagonists has hampered our ability to directly determine the role that P2X4Rs play in regulating ethanol-induced behaviors. The availability of p2rx4 null (i.e., knockout
Linked assets
University of Southern California Dissertations and Theses
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Asset Metadata
Creator
Wyatt, Letisha Renee
(author)
Core Title
Regulation of ethanol intake by purinergic P2X4 receptors
School
School of Pharmacy
Degree
Doctor of Philosophy
Degree Program
Molecular Pharmacology and Toxicology
Publication Date
04/29/2013
Defense Date
01/31/2013
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
Alcohol,behavior,Mice,Neuroscience,OAI-PMH Harvest,P2X receptor,pharmacology
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Davies, Daryl L. (
committee chair
), Alkana, Ronald L. (
committee member
), Bortolato, Marco (
committee member
), Wood, Ruth I. (
committee member
)
Creator Email
lrwyatt@gmail.com,lwyatt@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c3-248523
Unique identifier
UC11293560
Identifier
etd-WyattLetis-1624.pdf (filename),usctheses-c3-248523 (legacy record id)
Legacy Identifier
etd-WyattLetis-1624.pdf
Dmrecord
248523
Document Type
Dissertation
Format
application/pdf (imt)
Rights
Wyatt, Letisha Renee
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
Tags
behavior
P2X receptor