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Role of purinergic P2X4 receptors in regulation of dopamine homeostasis in the basal ganglia and associated behaviors

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Role of purinergic P2X4 receptors in regulation of dopamine homeostasis in the basal ganglia and associated behaviors

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Content i

ROLE OF PURINERGIC P2X4 RECEPTORS IN REGULATION OF DOPAMINE
HOMEOSTASIS IN THE BASAL GANGLIA AND ASSOCIATED BEHAVIORS

by

Sheraz Khoja

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 AND TOXICOLOGY)

May 2017

Copyright 2017 Sheraz Khoja

ii

EPIGRAPH

“If I have seen further than others, it is by standing upon the shoulders of giants”

Sir Isaac Newton

iii

ACKNOWLEDGEMENTS

I would like to convey my sincere gratitude to my mentor, Dr. Daryl L.
Davies and my lab colleagues for enriching my research experience. I
would like to thank my mentor, Dr. Daryl L. Davies for giving me the
opportunity to pursue my research goals. He has witnessed me grow and
mature as a research scientist in the past 7 years. Without his
unconditional support, guidance and patience, I would have not achieved
my research goals. He has always aided me in resolving my issues and
provided valuable advice to various tasks related to undertaking of
experiments, writing of fellowships, manuscripts and my PhD dissertation.
He also given me multiple chances in mentoring high school students and
USC undergraduates, which would be instrumental in developing my skills
related to leadership and people management. Additionally, I would also
like to thank Dr. Michael W. Jakowec for allowing me to use his laboratory
space and resources to undertake experiments related to my research
project. His expertise in the field of dopamine system proved to be
extremely helpful for my PhD project. Members of his research team have
also assisted me in performing certain critical experiments of my project. I
would also want to express my deepest appreciation to Dr. Liana Asatryan
for her suggestions in execution of various experiments and drafting of my
first authored manuscripts. I am also grateful to Dr. Kathleen E. Rodgers
iv

for taking the time out in serving on my PhD qualifying and dissertation
committee. Lastly, I would like to thank my lab-mates for performance of
certain experiments as well as creating a conducive environment for me to
perform my research investigations.

Statement of Contributions to works contained in this Dissertation
This dissertation comprises of 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 Sheraz Khoja. The authorship on published manuscripts is
described in greater detail in the following page. Dr. Daryl L. Davies
provided discussion and revisions to manuscripts.

v

AUTHORSHIPS
Published and “under review ” work by the Author incorporated in the
Dissertation
Sheraz Khoja, Vivek Shah, Damaris Garcia, Liana Asatryan, Michael W.
Jakowec, Daryl L. Davies: Role of purinergic P2X4 receptors in regulating striatal
dopamine homeostasis and dependent behaviors: Journal of Neurochemistry
2016; 139 (1): 134-48.

Sheraz Khoja, Nhat Huynh, Liana Asatryan, Michael W. Jakowec, Daryl L.
Davies: Reduced expression of purinergic P2X4 receptors increases voluntary
ethanol intake in C57BL/6 mice: Under Review.

Additional work by the Author relevant to this Dissertation but not forming
part of it

Letisha R. Wyatt, Deborah A. Finn, Sheraz Khoja, Megan Yardley, Liana
Asatryan, , Ronald L, Alkana, Daryl L. Davies. Contribution of P2X4 receptors to
ethanol intake in male C57BL/6 mice: Neurochemical Research 2014; 39 (6):
1127-1139

Liana Asatryan, Megan M.Yardley, Sheraz Khoja, James R.Trudell, Nhat Huynh,
Stan G. Louie, Nicos A.Petasis, Ronald L. Alkana, Daryl L. Davies: Avermectins
differentially affect ethanol intake and receptor function: Implications for
developing new therapeutics for alcohol use disorders: International Journal of
Neuropsychopharmacology 2014; 17(6): 907-916.

Letisha R. Wyatt, Sean C. Godar, Sheraz Khoja, Michael W.Jakowec, Ronald L.
Alkana, Marco Bortolato, Daryl L. Davies. Socio-communicative and
sensorimotor impairments in male P2X4 deficient mice.
Neuropsychopharmacology 2013; 38 (10):1993-2002.

Marco Bortolato, Megan M Yardley, Sheraz Khoja, Sean C.Godar, Liana
Asatryan, Deborah A. Finn, Ronald L. Alkana, Stan G. Louie, Daryl L. Davies.
Pharmacological insights into role of P2X4 receptors in behavior regulation:
lessons from ivermectin. International Journal of Neuropsychopharmacology
2013; 16(5):1059-1070.

Megan M Yardley, Letisha Wyatt, Sheraz Khoja, Liana Asatryan, Marcia
J.Rammaker, Deborah A. Finn, Ronald L. Alkana, Nhat Huynh, Stan G.Louie,
Nicos A.Petasis, Marco Bortolato, Daryl L. Davies . Ivermectin reduces alcohol
intake and preference in mice. Neuropharmacology 2012; 63(2): 190-201.

Research Support
NIAAA/NIH R01 A022448 (D.L.D) and USC School of Pharmacy.
vi

TABLE OF CONTENTS
EPIGRAPH .................................................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................................................. iii
LIST OF FIGURES ....................................................................................................................................... ix
ABSTRACT .................................................................................................................................................. xi
CHAPTER 1 - INTRODUCTION ................................................................................................................... 1
1.1) Purinergic superfamily (P2X) of ligand gated ion channels:- .................................................... 1
1.1.1) General topology and properties of P2X subunits:- .................................................. 1
1.1.2) Pharmacology of P2X subunits:- .............................................................................. 3
1.1.3) Expression of P2X subunits in the central nervous system (CNS):- ........................ 7
1.1.4) Physiological significance of P2XRs in the CNS:- .................................................... 8
1.2) Purinergic P2X4 receptors:- ...................................................................................................... 9
1.2.1) General properties and pharmacology of P2X4Rs:- ................................................. 9
1.2.2) Physiological significance of P2X4Rs in CNS:- ...................................................... 11
1.2.2.1) Synaptic strengthening of neurons in the CA1 region of
hippocampus:- ..................................................................................................... 11
1.2.2.2) Regulation of GABAergic mediated IPSCs in the hypothalamus:- ......... 12
1.2.2.3) Chronic inflammatory and neuropathic pain ........................................... 14
1.3) Behavioral significance of P2X4Rs in the CNS:- .................................................................... 17
1.4) Behavioral deficits induced upon P2X4R manipulation characteristic of DA
dysfunction:- ................................................................................................................................... 23
1.5) Dissertation hypothesis:- ........................................................................................................ 25
CHAPTER 2 - ROLE OF PURINERGIC P2X4 RECEPTORS IN REGULATING STRIATAL
DOPAMINE HOMEOSTASIS AND DEPENDENT BEHAVIORS ............................................................... 28
2.1) ABSTRACT: ............................................................................................................................ 28
2.2) INTRODUCTION: ................................................................................................................... 29
2.3) EXPERIMENTAL PROCEDURES:......................................................................................... 32
2.4) RESULTS: .............................................................................................................................. 42
2.4.1) P2X4R KO mice exhibit alterations in expression of presynaptic DA
markers in striatum:- ......................................................................................................... 42
2.4.2) P2X4R KO mice exhibit significant alterations in expression of DA
receptors and downstream targets:- ................................................................................. 44
2.4.3) IVM significantly affected DARPP-32 and ERK 1/2 phosphorylation, but not
CREB phosphorylation in the dorsal striatum:- ................................................................. 47
vii

2.4.4) IVM significantly affected DARPP-32 phosphorylation, but not ERK 1/2 or
CREB phosphorylation in the ventral striatum:- ................................................................ 49
2.4.5) Pharmacological inhibition of D1Rs and D2Rs significantly enhanced
prepulse inhibition (PPI) of acoustic startle reflex in P2X4R KO mice:- ........................... 51
2.4.6) Pharmacological or genetic manipulation of P2X4R function significantly
influenced L-DOPA induced motor behavior in the 6-OHDA model of DA
depletion:- ......................................................................................................................... 53
2.5) DISCUSSION:......................................................................................................................... 58
CHAPTER 3 - REDUCED EXPRESSION OF PURINERGIC P2X4 RECEPTORS INCREASES
VOLUNTARY ETHANOL INTAKE IN C57BL/6J MICE .............................................................................. 68
3.1) ABSTRACT: ............................................................................................................................ 68
3.2) INTRODUCTION: ................................................................................................................... 69
3.3) MATERIALS AND METHODS: ............................................................................................... 72
3.4) RESULTS: .............................................................................................................................. 79
3.4.1) P2X4R KO mice exhibited increased voluntary ethanol consumption in the
24 hour access drinking paradigm:- .................................................................................. 79
3.4.2) Transfection of BV-2 cells or transfusion in mouse striatum with LV-
shRNA-p2rx4 reduced P2X4R expression:- ..................................................................... 81
3.4.3) LV-shRNA-p2rx4 infused mice exhibited higher ethanol intake and
preference in comparison to naïve mice:- ......................................................................... 83
3.4.4) The LV-shRNA-p2rx4 infused mice exhibited a higher ethanol intake as
compared to mice that only received LV infusion: ............................................................ 84
3.4.5) Infusion of LV alone did not have any significant effect on ethanol intake or
preference in comparison to the naïve mice:- ................................................................... 86
3.5) DISCUSSION:........................................................................................................................ 87
CHAPTER 4 - ROLE OF DOPAMINE RECEPTORS IN IVERMECTIN INDUCED PREPULSE
INHIBITION DEFICITS IN C57BL/6J MICE ................................................................................................ 94
4.1) ABSTRACT: ............................................................................................................................ 94
4.2) INTRODUCTION: ................................................................................................................... 95
4.3) MATERIALS AND METHODS: ............................................................................................... 99
4.4) RESULTS: ............................................................................................................................ 103
4.4.1) Pharmacological blockade of dopamine D1, but not D2 receptors
significantly attenuated IVM-mediated PPI disruption:- .................................................. 103
4.4.2) Dopamine D1 receptor activation did not potentiate the effects of IVM on
PPI function without inducing any alterations in acoustic startle:- .................................. 105
4.5) DISCUSSION:....................................................................................................................... 107
CHAPTER 5 - OVERALL DISCUSSION AND CONCLUSION ................................................................. 116
viii

5.1) Summary of overall findings:- ............................................................................................... 116
5.2) Future directions:- ................................................................................................................. 122
BIBLIOGRAPHY ....................................................................................................................................... 126

ix

LIST OF FIGURES

Figure 2.1 P2X4R KO mice exhibited significant alterations in pre- 43
synaptic and postsynaptic DA markers in dorsal
and ventral striatum.
Figure 2.2 No changes in DA levels in dorsal and ventral striatum 44
of P2X4R KO mice.
Figure 2.3 P2X4R KO mice exhibited dysregulation of DA-dependent 46
signaling pathways in the dorsal and ventral striatum.
Figure 2.4 IVM (5mg/kg) significantly upregulated DARPP-32 and ERK 1/2 48
phosphorylation, but not CREB phosphorylation in the
dorsal striatum.
Figure 2.5 IVM (5mg/kg) significantly affected DARPP-32 phosphorylation 50
but not ERK 1/2 or CREB phosphorylation via P2X4R potentiation
in the ventral striatum
Figure 2.6 SCH 23390 (1mg/kg) and raclopride (3mg/kg) significantly 52
enhanced prepulse inhibition of acoustic startle reflex in
P2X4R KO mice (A) without any changes in startle amplitude (B).
Figure 2.7 Stereotaxic injection of 6-OHDA (4mg/ml) induced destruction 54
of DA neurons in ventral mesencephelon (A) and TH density (B)
in striatum of both WT and P2X4R KO mice to similar extent.
Figure 2.8 IVM (5mg/kg) induced ipsilateral rotations in 6-OHDA WT that were 55
statistically significant from sham WT controls.
Figure 2.9 L-DOPA (5mg/kg) induced rotational behavior is significantly 57
attenuated in P2X4R KO mice. IVM (5mg/kg) significantly
potentiated L-DOPA’s effect on number of contralateral turns in
WT and P2X4R KO mice (A). IVM’s ability to enhance L-DOPA
induced motor behavior was significantly altered in P2X4R KO
mice (B).

Figure 3.1 P2X4R KO mice exhibited significantly higher 10E intake as 80
compared to WT controls (A) and tended to have higher total
fluid intake (D) without any significant changes in 10E preference
(B) or water intake (C).

Figure 3.2 BV-2 cells transinfected with LV-shRNA-p2rx4 reduced P2X4R 82
by 68% and 62% in comparison to non-treated cells (NT) and
LV alone infused cells (A). Microinfusion of LV-shRNA-p2rx4 into
NAc core was verified by detecting ZsGreen1 immunofluorescence
(B).Stereotaxic injection of LV-shRNA-p2rx4 reduced P2X4R
expression by 44% and 39% as compared to naïve mice and LV
alone infused mice after 14 days (C).
x

Figure 3.3 LV-shRNA-p2rx4 infused group exhibited significantly higher 10E 85
intake as compared to naïve mice and mice infused with LV alone
(A). The10E preference of LV-shRNA-p2rx4 group was significantly
higher and lower than that of naïve mice, but not mice infused with
LV alone (B & C). No changes in total fluid intake (D).

Figure 4.1 SCH 23390 (1mg/kg), but not raclopride (3mg/kg) significantly 105
blocked IVM-mediated prepulse inhibition deficits of acoustic
startle reflex.

Figure 4.2 Co-administration of IVM (5mg/kg) and SKF 82958 (0.1mg/kg) 107
did not significantly decrease PPI% in comparison treatment with
either one of the drugs.

xi

ABSTRACT

Purinergic P2X receptors (P2XRs) are cation permeable ionotropic
receptors gated by adenosine-5’-triphosphate (ATP). Until date, seven
subtypes of P2XRs have been identified (P2X1R upto P2X7R). Amongst
the P2X subtypes, P2X4 receptors (P2X4Rs) are expressed in different
types of cells (i.e. neurons and glial cells) in the central and peripheral
nervous system (CNS and PNS). ATP-mediated synaptic transmission via
P2X4Rs has been implicated in regulation of synaptic currents facilitated
by N-Methyl-D-aspartate receptors (NMDARs) and -amino butyric acid
(GABA
A
Rs) at post-synapses and pre-synaptic release of
neurotransmitters including glutamate, GABA and norepinephrine (NE) in
various brain regions. However, the functional significance of P2X4Rs
remains limited due to lack of specific agonists and antagonists. Presently,
P2X4Rs in the hippocampus has been attributed to synaptic plasticity and
P2X4Rs in spinal cord microglia has been linked to pain hypersensitivity.
Investigations from our laboratory group have made significant efforts in
understanding the behavioral role of P2X4Rs. Using pharmacological and
genetic approaches, we suggested a role for P2X4Rs in sensorimotor
gating, social behavior and ethanol drinking behavior. The molecular
mechanisms underlying these behavioral changes are not clearly
understood. To address this issue, I hypothesized that P2X4Rs interact
with the dopamine (DA) system, considering that DA has a significant role
to play in mediation of aforementioned behaviors. Presently, there is very
limited knowledge regarding the interaction of P2X4Rs with the DA
system. Elucidating this interaction could give us important insights into
regulation of certain CNS behaviors including sensorimotor gating, social
behavior and reward behavior as well as relevance of P2X4Rs to
psychiatric disorders characterized by deficits in aforementioned
behaviors. To investigate the gap pertaining to this interaction, the
hypothesis of my dissertation is that P2X4Rs play an important role in
regulation of DA-dependent signaling pathways and associated behaviors.
Chapter 2 investigates the role of P2X4Rs in controlling DA homeostasis
and its relevance to DA-dependent behaviors including motor control and
sensorimotor gating. We used mice deficient in p2rx4 gene [P2X4R
knockout (KO) mice] and ivermectin (IVM), which is a positive modulator
of P2X4Rs, to address the interaction between P2X4Rs and DA
xii

neurotransmission. Chapter 3 makes significant attempts to establish a
direct link between reduced P2X4R expression and increased ethanol
intake. This chapter is based on previous findings wherein, P2X4R KO
mice exhibited transient increase in ethanol intake over short period of
time. In this chapter, we observed the ethanol drinking behavior in P2X4R
KO mice over longer period of time and used the lentivirus-shRNA (LV-
shRNA) strategy to establish a direct role for P2X4Rs in ethanol intake.
Chapter 4 examines the interaction between IVM and DA receptor
agonist/antagonists in regulation of prepulse inhibition (PPI) of acoustic
startle reflex. Taken together, findings from my dissertation provide us with
important insights into role for P2X4Rs in DA neurotransmission and DA-
dependent behaviors such as motor control and sensorimotor gating.
Moreover, the findings indicate that perturbation of this interaction may be
relevant to pathogenesis of neuropsychiatric disorders characterized by
DA dysfunction including schizophrenia, bipolar disorder, Parkinson’s
disease, alcohol use disorder (AUD) and supports P2X4Rs as novel drug
targets for therapeutic intervention for these disorders.

1

CHAPTER 1
INTRODUCTION

1.1) Purinergic superfamily (P2X) of ligand gated ion channels:-

1.1.1) General topology and properties of P2X subunits:-
P2X receptors (P2XRs) represent a superfamily of non-selective cation
permeable ion channels that are gated by ATP (Valera et al. 1994). The
cloning of P2XRs happened nearly 20 years after the inception of the
concept “purinergic transmission” as put forth by Dr. Geoffrey Burnstock,
in which ATP mediated signaling, was characterized in the adrenergic,
cholinergic neurons of the enteric nervous system and subsequently was
identified as a neurotransmitter (Burnstock 1972). Within two years after
the isolation of cDNA encoding P2XRs, the entire family of ionotropic
receptors was elucidated. To date, seven genes encoding P2XRs (P2X1-
P2X7) have been identified. These genes differ significantly in their sizes,
possessing 11-13 exons and having well-conserved intron/exon
boundaries (Urano et al. 1997). The P2X subunits range from 379 to
595 amino acids in length (Brake et al. 1994, North 1996). Each of these
subunits comprises of two hydrophobic membrane spanning regions (TM1
& TM2) that are connected by an ectodomain which has ten conserved
2

cysteine residues that form disulfide bonds (North 1996). The first half of
the ectodomain consist of six cysteine residues encoded by exons 2, 4
and 5; the remaining four cysteine residues are encoded by exons 7 and
8. The ability of these cysteine residues to form disulfide bonds gives rise
to a tertiary structure of the P2XRs (Clyne et al. 2002, Ennion & Evans
2002). Certain regions of ectodomain adjacent to TM1 and TM2 contain
the binding sites for ATP. The intracellular NH and COOH terminals
contain binding motifs for different protein kinases (Khakh et al. 2001,
Nicke et al. 1998). The COOH terminus is highly diverse among the
P2XRs with P2X7Rs having the longest COOH terminus and possessing
an additional hydrophobic domain (North 2002). This topology was later
confirmed by a series of experiments involving site-directed mutagenesis
of asparagine residues in the consensus sequence for N-linked
glycosylation of P2X2Rs (Newbolt et al. 1998). This consensus sequence
for N-linked glycosylation on the extracellular domain is homologous
among species variants, but differs significantly among the receptor
subunits (Newbolt et al. 1998).
P2XRs are capable of forming hetromers or homomers on cell surface,
with each receptor containing atleast three monomers. Most P2XRs can
assemble into homotrimers, with the exception of P2X6Rs (Volonte et al.
2006). Examples of hetrotrimers include P2X1/P2X5 (Haines et al. 1999,
Torres et al. 1998), P2X2/P2X3 (Radford et al. 1997), P2X2/P2X6 (King et
3

al. 2000) and P2X4/P2X6 (Lˆ et al. 1998). Recently P2X7Rs have been
shown to hetromerize with P2X4Rs (Guo et al. 2007).
Interestingly, P2XRs differ with respect to their Ca
2+
permeability ranging
from 1-5 (ratio of Ca
2+
ions to monovalent ions), with P2X1R having the
highest Ca
2+
permeability of 4.8 (Egan et al. 2006). Notably, P2X
hetromers in neuronal species of certain brain regions such as the
somatosensory cortex, have a high Ca
2+
permeability (~10-12), which is
comparable to that of NMDARs (Edwards et al. 1997). Hence, the
presence of P2XRs in the postsynapses of those neurons can be an
important route for Ca
2+
entry into the neurons when NMDARs are inactive
due to presence of Mg
2+
block, leading to activation of signaling pathways
that regulate gene expression and protein expression (Pankratov et al.
2002, Pankratov et al. 2009). Moreover, entry of Ca
2+
ions through P2XRs
on presynapses can trigger release of neurotransmitters into synapses
(MacDermott et al. 1999).
1.1.2) Pharmacology of P2X subunits:-
In comparison to -amino butyric acid (GABA), glutamate and dopamine
(DA) neurotransmitter system, the P2X pharmacology is relatively limited
due in part to the elucidation of X-ray crystal structure of protein not
occurring until lates 2000s (Kawate et al. 2009, Hattori & Gouaux 2012).
Sensitivity of P2XRs to ATP vary considerably with P2X1R and P2X3R
4

having a low EC
50
(0.07 & 0.5 µM) and P2X7Rs requiring a high
concentration of ATP for activation (100 µM) (North 2002, Khakh & North
2012). The classical agonists for P2XRs are 2-MethylthioATP (2-
MeSATP), α, β-methylene ATP (α, β-meATP); whereas classical P2XR
antagonists are suramin, pyridoxal-phosphate-6-azophenyl-2
’
, 4’-disulfonic
acid (PPDAS) and 2
’
, 3’,-O-(2, 4, 6-trinitrophenyl ATP) (TNP-ATP) (Evans
et al. 1995, Virginio et al. 1998, Valera et al. 1994, Bianchi et al. 1999,
Garcia-Guzman et al. 1997, Lewis et al. 1995). P2X2Rs, P2X4Rs and
P2X7Rs are considered insensitive to aforementioned pharmacological
drugs. In the case of P2X7Rs; 2
’
,3
’
-O-(benzoyl-4-benzoyl) –ATP ) (BzATP)
was demonstrated to be more effective than ATP in potentiating P2X7R
function (Bianchi et al. 1999) and drugs such as NF 279 (an analog of
suramin), Brilliant Blue G (BBG) have been shown to antagonize P2X7R
function at nanomalor concentrations (Jiang et al. 2000). Divalent cations
(Zn
2+
, Cu
2+
) or acidification of extracellular pH have been demonstrated to
be helpful in investigating functional properties of P2X2Rs, P2X4Rs and
P2X7Rs (Xiong et al. 1999, King et al. 1997, Wildman et al. 1998, Stoop et
al. 1997). For instance, low concentrations of Cu
2+
, Zn
2+
or protons can
potentiate P2X2Rs (King et al. 1997, Wildman et al. 1998) , whereas Zn
2+
,
but not Cu
2+
(Xiong et al. 1999) can activate P2X4Rs. Additionally,
P2X2Rs and P2X7Rs are sensitive to blockade by divalent cations
5

including Mg
2+
, Mn
2+
, Ca
2+
and Ba
2+
(Ding & Sachs 1999, Ding & Sachs
2000, Virginio et al. 1997).
In regards to specific modulation of P2X4Rs, semi-synthetic macrocyclic
lactone derivatives that belong to avermectin family such as ivermectin
(IVM), abamectin (ABA) and moxidectin (MOX) have been shown to
potentiate P2X4R function and prolong its rate of desensitization
(Asatryan et al. 2010, Asatryan et al. 2014, Priel & Silberberg 2004,
Jelinkova et al. 2008). A unique property of the avermectins is that they
are selective to P2X4Rs and does not potentiate activity of other P2X
subtypes (Khakh et al. 1999). The presence of two carbohydrate moieties
and an allylic hydroxyl group seem to be pertinent to IVM’s effect on
P2X4Rs, as elimination of one of carbohydrate moieties and replacement
of the allylic hydroxyl group by an unsaturated ketoxime resulted in
generation of another avermectin compound, selamectin, which does not
possess any ability to modulate ATP-mediated currents (Asatryan et al.
2014). The one structural difference between ABA and IVM is the
presence of a double bond at C22-23 in abamectin, whereas in the case
of ivermectin, it is single bond.
A few selective P2X4R antagonists have been synthesized and developed
so far (Tian et al. 2014, Hernandez-Olmos et al. 2012). Paroxetine, a
selective serotonin reuptake inhibitor, has been reported to block P2X4R
6

activity and this pharmacological effect may underlie its potential to
alleviate neuropathic pain (Nagata et al. 2009). It was also assumed that
paroxetine may cause downregulation of P2X4R function by interfering
with its lysosomal secretion (Toulme et al. 2010). 5-(3-bromophenyl) 1, 3-
dihydro-2H-benzofuro [3, 2-e] 1, 4-diazepin-2-one (5-BDBD) (Donnelly-
Roberts et al. 2008) and TNP-ATP have been reported to block P2X4R
activity within the micromolar range (Khakh & North 2012). Recently, N-
substituted phenoxazine and carbamazepine derivatives have been
claimed to possess a greater degree of selectivity to human P2X4Rs (Tian
et al. 2014) in relation to other P2X subtypes. N-(Benzyloxycarbonyl)
phenoxazine displayed a high degree of potency towards P2X4Rs in the
micromolar range and had fifty fold selectivity for human P2X4Rs in
comparison to P2X2, P2X3 and P2X7 receptors (Tian et al. 2014). N-(p-
methylphenylsufonyl)-substituted phenoxazine also exhibited antagonistic
effect within micromolar range and has a higher affinity for P2X4Rs in
comparison to other P2X subtypes. 1-(2,6-dibromo-4-isopropyl phenyl)-3-
(pyridyl) urea (BX-430) also displayed a high degree of affinity to human
P2X4Rs and a hundred fold selectivity to P2X4Rs versus other P2X
subtypes (Ase et al. 2015).
7

1.1.3) Expression of P2X subunits in the central nervous system
(CNS):-
Multiple immunohistochemical, electron microscopy and in situ
hybridization studies have indicated that P2XRs are expressed in the CNS
(brain and spinal cord) and peripheral nervous system (sympathetic
ganglia, parasympathetic ganglia, enteric nervous system) (Burnstock
2007, North 2002). Expression of P2XRs in the CNS is heterogeneous
(Rubio & Soto 2001, Llewellyn-Smith & Burnstock 1998, Amadio et al.
2007, Xiang et al. 1998, Soto et al. 1996b). Among the P2XRs, P2X2Rs,
P2X4Rs and P2X6Rs are abundantly expressed, whereas P2X1Rs,
P2X5Rs are sparsely distributed in the CNS. Purkinje neurons of the
cerebellar cortex (Balcar et al. 1995, Hervas et al. 2005) and the CA1/CA3
pyramidal neurons of the hippocampus (Rodrigues et al. 2005, Sim et al.
2006) express all the P2XRs. Moderate expression of P2XRs is found on
the medium spiny neurons (MSNs) and interneurons in the caudate
putamen, caudate nucleus and nucleus accumbens (NAc) of the basal
ganglia (Amadio et al. 2007). P2X subunits are also expressed in different
parts of the midbrain including GABA releasing presynaptic terminals of
ventral tegmental area (VTA) (Xiao et al. 2008), substantia nigra (SN)
(Amadio et al. 2007) and the periaqueductal gray area (Worthington et al.
1999). A dense expression of P2X4Rs was found in the arcuate (Arc) and
ventromedial (VMH) nuclei of the hypothalamus (Jo et al. 2011, Xu et al.
8

2016). P2XRs are also expressed in the ventrobasal complex of thalamus
(Bo & Burnstock 1994, Le et al. 1998), the medial habenula (Robertson et
al. 1999) and the locus coeruleus (Collo et al. 1996, Frohlich et al. 1996).
In addition, there are significant levels of P2X4Rs expressed in the
susbtantia gelatinosa of the nucleus caudalis and dorsal horn (DH) of the
spinal cord (Le et al. 1998). Moreover, immunohistochemical studies have
shown expression of P2XRs on soma and dendrites of postsynaptic
neurons and presynaptic terminals (Rubio & Soto 2001). Finally, P2XRs
are also expressed on different types of glial cells including
oligodendrocytes, astrocytes, microglia and Schwann cells (Kirischuk et al.
1995a, Kirischuk et al. 1995b, Moller et al. 2000).
1.1.4) Physiological significance of P2XRs in the CNS:-
ATP-mediated synaptic transmission has been implicated in regulation of
excitatory potential synaptic currents (EPSCs) mediated by NMDARs
(Pankratov et al. 2009, Pankratov et al. 2002), nicotinic acetylcholine
receptors (nAchRs) (Searl & Silinsky 1998, Nakazawa 1994, Nakazawa et
al. 1991, Khakh et al. 2005) and GABA
A
receptor (GABA
A
Rs) mediated
inhibitory potential synaptic currents (IPSCs) (Jo et al. 2011, Xu et al.
2016, Shrivastava et al. 2011) as well as presynaptic release of
neurotransmitters including glutamate (Gu & MacDermott 1997, Li et al.
1998b), GABA (Jo & Schlichter 1999, Hugel & Schlichter 2002) and
9

norepinephrine (NE) (Boehm 1999). ATP-mediated neurotransmission via
P2XRs was first elucidated in the medial habenula (Edwards et al. 1992).
Subsequently, P2XRs have been implicated in excitatory and inhibitory
neurotransmission in hippocampus (Rodrigues et al. 2005, Pankratov et al.
1999, Pankratov et al. 2002, Wieraszko & Ehrlich 1994), hypothalamus (Jo
& Role 2002, Jo et al. 2011, Sorimachi et al. 2001), cortex (Phillis & Wu
1981, Pankratov et al. 2003) and spinal cord (Hugel & Schlichter 2002, Jo
& Schlichter 1999, Bardoni et al. 1997).
1.2) Purinergic P2X4 receptors:-
1.2.1) General properties and pharmacology of P2X4Rs:-
The p2rx4 gene which encodes P2X4Rs is located on chromosome
12q24.32 spanning about 12Kbp with 12 exons (Gu et al. 2010).
Presently, few studies have focused on transcription factors that bind to
the promoter region of p2rx4 gene and initiate its transcription.
Interestingly, it was originally suggested that Sp1 transcription factor
regulated p2rx4 gene expression. This observation was later contested
(Korenaga et al. 2001) when additional studies the same group of
investigators suggested that GATA-2 was the critical promoter that
regulated p2rx4 gene expression (Gu et al. 2010). In addition to GATA-2,
interferon regulatory factor-5 (IRF5) has been recently linked to de novo
expression of P2X4Rs in activated microglia in the spinal cord following
10

peripheral nerve injury (PNI), by binding to a promoter region on p2rx4
gene that was identified as interferon-stimulated response element (ISRE)
(Masuda et al. 2014). This hypothesis was strengthened by the
observation that mice lacking Irf5 did not exhibit upregulation of P2X4Rs in
microglia following PNI and the mice exhibited less allodynic behavior as
compared to wildtype (WT) littermates as revealed by behavioral assays
that induce the hallmark features of neuropathic pain (Masuda et al.
2014).
P2X4Rs have a slow desensitization rate (>20 seconds), similar to that of
P2X2Rs and P2X7Rs (North 2002). ATP-mediated currents at P2X4Rs
typically decline within 5-10 seconds after application of maximal ATP
concentration (100 µM) (North 2002). The activity of P2X4Rs can be
regulated through trafficking. This includes endocytosis, lysosomal
secretion and lateral mobility. P2X4Rs undergo constitutive and regulated
endocytosis via the non-canonical endocytic motif (YXXGL) (Royle et al.
2002, Royle et al. 2005). P2X4Rs are resistant to lysosomal degradation
owing to their heavily glycosylated structure (Qureshi et al. 2007). Notably,
microglial activation has been shown to increase the lateral mobility and
lysosomal secretion of P2X4Rs (Toulme & Khakh 2012).
11

1.2.2) Physiological significance of P2X4Rs in CNS:-
1.2.2.1) Synaptic strengthening of neurons in the CA1 region of
hippocampus:-
NMDAR- mediated LTP in the CA1 region of the hippocampus is one of
the forms of synaptic plasticity, an underlying mechanism for learning and
memory (Luscher & Malenka 2012, Hunt & Castillo 2012). It involves
monitoring the changes in amplitude of AMPAR mediated synaptic
potentials subsequent to tetanic stimulus to the Schaffer collateral
commissure fiber tract. An influx of Ca
2+
through NMDARs results in
increased delivery of AMPAR subunits to synaptic surface and
subsequent induction of LTP (Hayashi et al. 2000, Shi et al. 2001). It was
presumably thought that P2X4Rs may have a role in synaptic
strengthening of CA1 neurons on account of their perisynaptic location on
the postsynaptic membrane in CA1 region of the hippocampus (Rubio &
Soto 2001) as well as their high Ca
2+
permeability (Buell et al. 1996).
Moreover, they have a slow desensitization constant (North 2002), thus
remaining open for significant amount of time to allow Ca
2+
ions to influx
into membrane and causing induction of cellular pathways related to gene
expression and protein translation. Using pharmacological and genetic
approaches, the role of P2X4Rs in synaptic strengthening was deciphered
to some degree.
12

In the event of LTP, a significant rise in AMPAR-related EPSCs is usually
detected following depolarization paired with multiple stimuli to Schaffer
collaterals. This phenomenon of rise in sEPSCs following a train of
depolarization pulses was significantly impaired in P2X4R KO mice
(Baxter et al. 2011), possibly due to low influx of Ca
2+
ions in the P2X4R
KO mice which negatively impacts the delivery of AMPAR subunits to
synaptic surface.
In addition to AMPA component of EPSCs, the facilitation of NMDAR
EPSCs in a Mg
2+
free solution which develops over a period of 10-20
minutes and is usually reduced in presence of 1,2-bis(o-
aminophenoxy)ethane-N,N,N’,N’ –tetraacetic acid (BAPTA) or ifenpodril
(NMDAR antagonist), is absent in P2X4R KO mice (Baxter et al. 2011).
Moreover, facilitation of NMDA EPSCs in P2X4R KO mice was unaffected
by BAPTA or ifenpodril (Baxter et al. 2011). A plausible explanation for
this absence of facilitation of NMDAR EPSCs is inability of NR2B subunits
to be incorporated into synaptic membrane in P2X4R KO mice.
1.2.2.2) Regulation of GABAergic mediated IPSCs in the
hypothalamus:-
In the VMH nucleus of the hypothalamus, P2X4Rs have been reported to
be present in the steroidogenic factor 1 (SF-1) neurons. These receptors
have been implicated in regulating neuronal excitability of SF-1 neurons,
13

as determined by biochemical approaches wherein the endocytosis of
P2X4Rs was blocked by a site specific interference peptide that targets
the endocytic motif of the receptor (Jo et al. 2011). In the same neuronal
population, P2X4Rs were demonstrated to physically interact with α2β3
subunit of GABA
A
Rs via two successive amino acid residues (Tyr 374 and
Val 375) in the C-terminal domain of P2X4Rs (Jo et al. 2011). This
interaction between P2X4Rs and GABA
A
Rs was proposed to be
antagonistic in nature, since co-application of ATP and GABA in a
recombinant cell system such as Xenopus oocytes expressing these two
receptors resulted in currents that were of significantly lower amplitude
than the predicted sum of responses upon separate application of two
ligands (Jo et al. 2011). To gain insights into physiological relevance of
this interaction, experiments involving blockade of P2X4R internalization
by an interference peptide resulted in decrease in frequency and
amplitude of GABAergic mediated sIPSCs in SF-1 neurons, which further
supports the cross-inhibition between the two ionotropic receptors in vivo
(Jo et al. 2011).
In addition to P2X4Rs’s role in modulating GABAergic sIPSCs in the VMH,
using a BAC transgenic mouse model that expresses Td-tomato
fluorescence under the control of p2rx4 gene, P2X4Rs were reported to
be expressed on the agouti-related peptide-neuropeptide Y neurons
(AgRP-NPY), propiomelanocortin (POMC) neurons and tanycytes in the
14

Arc nucleus of the hypothalamus (Xu et al. 2016). ATP-mediated currents
via P2X4Rs were shown to facilitate the presynaptic release of GABA from
AgRP-NPY neurons onto POMC neurons as well as PVN neurons in the
hypothalamus, both of which receive GABAegric input from the AgRP-
NPY neurons (Xu et al. 2016). Moreover, the ability of P2X4Rs to enhance
mIPSCs frequency was diminished in a state of food deprivation and this
phenomenon was reversed by leptin, a satiety hormone that regulates
energy homeostasis, implicating that ATP-mediated GABA release by
P2X4Rs can be altered by metabolic state (Xu et al. 2016). However, the
behavioral relevance of P2X4Rs to regulate GABAergic mIPSCs
frequency is not fully elucidated, since the same investigation reported
that infusion of ATP analogues into Arc nucleus did not have any effect on
food intake and P2X4R KO mice did not exhibit any phenotypes with
respect to feeding behavior (Xu et al. 2016).
1.2.2.3) Chronic inflammatory and neuropathic pain:-
Building evidence supports role of P2X4Rs in pathogenesis of neuropathic
pain (Tsuda et al. 2003, Tsuda et al. 2009a, Ulmann et al. 2010, Ulmann
et al. 2008, Trang et al. 2009, Tsuda et al. 2005). Animal models of
neuropathic pain have demonstrated that development of mechanical
hypersensitivity correlates with the increase in microglial P2X4R
expression (Tsuda et al. 2003). Moreover, hallmark features of
15

neuropathic pain including tactile allodynia and mechanical hyperalgesia
were abolished in mice deficient in p2rx4 gene [i.e. P2X4R knockout
(KO)], implying that blockade of P2X4Rs can alleviate symptoms
associated with neuropathic pain (Tsuda et al. 2009a).
The microglia-neuronal communication in the DH of the spinal cord is
defined as a critical mechanism underlying the nociceptive process
network of the spinal cord (Inoue 2006). BDNF is considered an important
molecule in regulating the microglial-neuronal signaling (Coull et al. 2005).
Interaction of BDNK with its cognate receptor, TrkB, induces
downregulation of K
+
/Cl
-
transporter, leading to accumulation of Cl
-
ions
and inhibition of GABA and glycine mediated function in DH neurons of
spinal cord (Coull et al. 2003, Coull et al. 2005). P2X4Rs have been
suggested to mediate release of BDNF from activated microglia to spinal
laminae Ӏ neurons in the spinal cord (Ulmann et al. 2008). Notably, deficits
in BDNF release from activated microglia and resultant accumulation of
BDNF in DH microglia were observed in P2X4R KO mice, linking the
abnormalities in BDNF-related signaling in microglia with increased
resistance to pain hypersensitivity in P2X4R KO mice (Ulmann et al.
2008). P2X4Rs were demonstrated to regulate release and expression of
BDNF in cultured microglial cells via Ca
2+
influx and activation of p38-
MAPK, an important enzyme involved in pain hypersensitivity (Trang et al.
2009). Apart from impaired release of BDNF from microglial cells that
16

alleviates tactile allydonia in P2X4R KO; these mice also exhibit impaired
production of prostaglandin E2 (PGE2), which is key lipid in processing
inflammatory pain, as well as reduction in ATP-induced activation of
cytosolic phospholipase A2 (cPLA2) and release of arachidonic acid (AA)
(Ulmann et al. 2010). Thus, P2X4Rs can contribute to symptoms of
neuropathic pain by their involvement in BDNF expression, secretion as
well as release of pro-inflammatory lipids such as PGE2.
Further, multiple factors have been linked to upregulation of microglial
P2X4Rs in spinal cord during the state of chronic inflammatory pain. For
instance, CCL21, a microglial chemokine that is released from damaged
neurons results in an upregulation of P2X4Rs on cell surface via
chemokine CC receptor (CCR2) since mice deficient in CCL21or
administration of CCL21 neutralizing antibody resulted in suppression of
tactile allodynia as well as upregulation of microglial P2X4R expression
(Biber et al. 2011). Moreover, injection of CCL21 into P2X4R KO mice had
no effect on tactile allodynia. The cytokine, interferon , has also been
implicated in P2X4R expression (Tsuda et al. 2009b). Further, fibronectin
has also been associated with upregulation of P2X4Rs (Nasu-Tada et al.
2006) through stimulation of tyrosine kinase, Lyn, (Tsuda et al. 2008) and
downstream activation of intracellular signaling enzymes such as
phosphotidylinositol-3 kinase (PI-3K), mitogen activated protein kinase
kinase (MEK) and extracellular signal-regulated kinase (ERK) (Tsuda et
17

al. 2009c), all of which can impact P2X4R expression. PI3K-Akt signaling
can induce degradation of p53, which may modulate P2X4R expression.
MEK-ERK signaling has been linked to activation of eukaryotic translation
initiation factor 4E (eIF4E), which can control the translation of P2X4Rs
(Tsuda et al. 2013).
1.3) Behavioral significance of P2X4Rs in the CNS:-
The role of P2X4Rs in a wide array of behaviors including anxiety or
depressive like reaction in response to acute stressors, working memory,
information processing and reward behavior was recently investigated
using IVM as a pharmacological approach and P2X4R KO mice as a
genetic approach (Bortolato et al. 2013, Wyatt et al. 2013). IVM exhibited
a diverse, multifaceted profile in various anxiety-related paradigms that
focus on different aspects of anxiety-related behavior. For instance, IVM
induced an anxiolytic –like effect in elevated plus maze and marble
burying test, but a thigmotactic response in the open field test (Bortolato et
al. 2013). In the elevated plus maze test, IVM treated mice spent more
time in the open arms as compared to their duration in the closed arms
and in the marble burying paradigm, IVM treated mice buried fewer
marbles and exhibited a reduction in digging behavior in relation to vehicle
treated mice (Bortolato et al. 2013). In contrast to these findings, in the
open field test, IVM treated mice spent more time at the periphery of the
18

compartment, indicating an anxiogenic-like response, without any
significant alterations in locomotor activity or exploratory parameters
(Bortolato et al. 2013). The open field test and marble burying capture
different facets of anxiety-related behavior and anxiety is induced by
diverse factors in these behavioral assays (Njung'e & Handley 1991,
Ramos et al. 1997, Carola et al. 2002, Prut & Belzung 2003). In marble
burying, anxiety is induced upon presence of a novel object, which is
perceived as a sign of threat or repulsiveness by the mouse and burying
the object is considered as a defensive behavior to eliminate that source
of threat (Njung'e & Handley 1991, Poling et al. 1981). In open field test,
anxiety is induced upon placing the mouse in a large, brightly lit,
compartment that is several times larger than its usual habitat and the
mouse in confronted with a situation between exploration and neophobia
(Prut & Belzung 2003). However, these pharmacological effects of IVM
did not seem to be elicited via P2X4R modulation since IVM was able to
induce the same behavioral effects in elevated plus maze and open field
in P2X4R KO mice (Bortolato et al. 2013). Moreover, P2X4R KO mice did
not show any differences in anxiety-like responses under baseline
conditions in these behavioral paradigms (Wyatt et al. 2013), suggesting
that IVM may be mediating these responses via a different target, possibly
GABA
A
Rs (Dawson et al. 2000). This speculation is not unfounded
considering that multiple positive modulators of GABA
A
Rs such as
19

benzodiazepines (diazepam, alprazolam, flurazepam, midazolam) have
been reported to exhibit anxiolytic-like response in open field and elevated
plus maze (Lopez et al. 1988, Nazar et al. 1997, Stefanski et al. 1993,
Crawley 1981, Rodgers et al. 1992, Albrechet-Souza et al. 2005).
IVM also induced depressive-like response in the tail suspension and
forced swim test, wherein in both the studies, IVM significantly increased
the immobility duration in relation to control group (Bortolato et al. 2013).
Both these paradigms focus on creating an inescapable situation, which
eventually precipitates depressive like behavior in mice (Porsolt et al.
1979, Porsolt et al. 1977). However, the role of P2X4Rs in mediation of
IVM’s effects is still undetermined as these behavioral studies have not
been undertaken in P2X4R KO mice. Moreover, we are unaware if P2X4R
KO mice exhibit any differences in depressive-related behavior in these
studies in relation their WT littermates.
IVM also induced deficits in prepulse inhibition (PPI) of acoustic startle
reflex, without causing any alterations in startle amplitude. Notably, IVM
did not induce the same behavioral response in P2X4R KO mice
(Bortolato et al. 2013) and these genetically modified mice also exhibited
reduced PPI function in comparison to their WT counterparts (Wyatt et al.
2013). These findings indicate that P2X4Rs may be involved in the neural
circuitry modulating PPI. PPI is considered as a reliable measure of
20

sensorimotor gating, wherein a subject processes incoming sensory
information and filters or gates irrelevant stimuli from significant ones in a
stimulus-laden environment (Braff & Geyer 1990, Braff & Light 2004).
Deficits in PPI have been associated with impairments in information
processing, leading to an overflow or flooding of sensory stimuli (Karper et
al. 1996, McGhie & Chapman 1961). PPI abnormalities have been linked
to a wide spectrum of neuropsychiatric and developmental disorders
including schizophrenia (Braff & Geyer 1990, Braff et al. 2001), bipolar
disorder (Perry et al. 2001), attention-deficit hyperactivity (ADHD) (Feifel
et al. 2009) and autism-spectrum disorders (Fragile X, Rett syndrome,
Down syndrome) (Perry et al. 2007). Considering that PPI has been
considered as an effective behavioral endophenotype in genetic
investigations of aforementioned disorders, especially schizophrenia, the
findings from IVM and P2X4R KO mouse model, suggests a role for
P2X4Rs in pathogenesis of neuropsychiatric diseases.
In addition to PPI deficits, P2X4R KO mice also exhibited significant
reduction in various patterns of social exploration. P2X4R KO mice
displayed significantly lower frequency and duration of sniffing bouts in
comparison to their WT littermates (Wyatt et al. 2013). Moreover, P2X4R
KO pups displayed significant reduction in maternal separation-induced
ultrasonic vocalization, citing communication deficits (Wyatt et al. 2013).
The aberrant social behavior of P2X4R KO adult mice and pups was
21

reinstated by the observation that P2X4R KO did not display any olfactory
impairment in olfactory discrimination test, which could potentially
confound the results from social interaction and ultrasonic vocalization test
(Wyatt et al. 2013). The social deficits displayed by P2X4R KO mice are
highly reminiscent of behavioral manifestations exhibited in mouse models
of neurodevelopmental diseases (Duncan et al. 2004, Won et al. 2012,
Moy et al. 2008, Bortolato et al. 2012). Considering that P2X4Rs are
expressed in rat brain from postnatal day 1 (Cheung et al. 2005) and that
ATP-mediated synaptic transmission has been attributed to multiple
aspects of neurogenesis (Zimmermann 2006, Del Puerto et al. 2013); the
constitutive deficiency of P2X4Rs may lead to abnormalities in
physiological process of development, ultimately resulting in social and
communication deficits at behavioral level.
The role of P2X4Rs in ethanol drinking behavior has been extensively
investigated (Wyatt et al. 2014, Asatryan et al. 2011, Asatryan et al. 2014,
Franklin et al. 2015). IVM was reported to significantly reduce ethanol
consumption across a wide spectrum of alcohol drinking paradigms that
capture multiple aspects of drinking behavior (Yardley et al. 2012,
Asatryan et al. 2014). IVM showed significant effects in reducing ethanol
intake in the 24 hour two bottle choice, drinking in the dark (DID) and
operant chamber paradigms (Yardley et al. 2012). In addition,
intracerebroventricular (ICV) administration of IVM significantly reduced
22

ethanol in high alcohol drinking (HAD-2) female rats (Franklin et al. 2015).
In all of these studies, IVM did not induce any alterations in water intake or
body weight, suggesting that IVM did not have any deleterious effect on
physiology of drinking as well as did not induce any toxicity that could
negatively impact drinking behavior. In further support of P2X4Rs’s role in
ethanol intake, P2X4R KO mice consumed significantly more ethanol,
without any overt changes in water intake, in relation to their WT
littermates in paradigms that mimic social and binge drinking behavior
(Wyatt et al. 2014). Notably, IVM’s anti-alcohol effects were significantly
attenuated in P2X4R KO mice (Wyatt et al. 2014), indicating that IVM is
mediating its effects, in part, via P2X4R modulation. Additionally, lower
mRNA expression of p2rx4 gene was found in inbred ethanol preferring
(iP) strains of rats in relation to ethanol non-preferring (niP) strains of rats
(Tabakoff et al. 2009, Kimpel et al. 2007), indicating an inverse correlation
between p2rx4 expression and ethanol drinking behavior. Furthermore,
lentivirus mediated sh-RNA (LV-shRNA) of p2rx4 in posterior VTA of HAD-
2 female rats significantly reduced ethanol intake (Franklin et al. 2015),
citing a direct relationship between P2X4Rs and ethanol consumption.
Overall, studies from IVM and P2X4R KO mouse model suggest a role for
P2X4Rs in sensorimotor gating, social interaction and ethanol drinking
behavior and that P2X4R dysfunction may contribute to pathogenesis of
23

neuropsychiatric disorders such as schizophrenia, bipolar disorder,
alcohol addiction and autism-spectrum disorders.
1.4) Behavioral deficits induced upon P2X4R manipulation
characteristic of DA dysfunction:-
Presently, there is paucity of information regarding the molecular
mechanisms underlying role of P2X4Rs in CNS behaviors. The deficits in
sensorimotor gating and alterations in ethanol drinking behavior suggests
an interplay between P2X4Rs and various neurotransmitter systems. Until
date, P2X4Rs have been implicated in modulation of NMDAR, AMPAR
and GABA
A
R-mediated synaptic transmission in specific brain sites such
as hippocampus (Sim et al. 2006, Baxter et al. 2011) and hypothalamus
(Xu et al. 2016, Jo et al. 2011). However, the role of P2X4Rs in regulation
of DA neurotransmission is not clearly understood. At present, there is no
direct evidence for P2X4Rs’ role in modulating DA function. However, it is
known that P2XR potentiation on GABA releasing presynaptic terminals in
VTA can enhance GABA release in synapses and induce inhibition of
firing of DA neurons (Xiao et al. 2008). However, these experiments used
antagonists and agonists that are thought to be insensitive to P2X4Rs
(Buell et al. 1996), and it is unlikely that this finding can be attributed to
P2X4Rs. P2X4Rs are expressed on GABAergic MSNs and interneurons
of the caudate putamen as well as DAergic neurons in the ventral
24

midbrain and projections that extend into the striatum (Amadio et al.
2007). Notably, ablation of DA neurons by 6-Hydroxydopamine (6-OHDA)
induced alterations in P2X4R expression on MSNs in the SN of ventral
midbrain and caudate putamen, suggesting an interaction between
P2X4Rs expression and manipulation of DAergic system (Amadio et al.
2007).
The disruptions in PPI function in P2X4R KO mice and upon IVM
treatment are characteristic of DA dysfunction, considering the critical role
of DA neurotransmission in regulation of PPI. Indirect and direct DA
agonists have been reported to induce PPI dysfunction (Ralph-Williams et
al. 2002, Doherty et al. 2008, Ralph & Caine 2005) and DA receptor
antagonists have been shown to alleviate PPI deficits (Swerdlow & Geyer
1993, Swerdlow et al. 1991, Geyer et al. 2001). Animal models that are
characterized by perturbations in DA activity have been shown to exhibit
PPI abnormalities (Ralph et al. 2001, Kinkead et al. 2005, Wolinsky et al.
2007, Ohgake et al. 2009, Vuillermot et al. 2011). Several antipsychotics
including chlorpromazine, haloperidol, risperidone, clozapine, olanzapine,
that have multiple receptor targets in DAergic and serotonergic system
have all been demonstrated to ameliorate PPI deficits in clinical population
(Braff et al. 2001) as well as genetic animal models of psychiatric
disorders (Swerdlow & Geyer 1993, Geyer et al. 2001). In addition, the
increased ethanol consumption in P2X4R KO mice is characteristic of DA
25

hyperactivity. Animal models that are created upon deficiency of Drd1 or
Drd2 gene, which encodes the DA receptors, have been reported to
exhibit abnormalities in ethanol drinking behavior (Delis et al. 2013,
Phillips et al. 1998, Bulwa et al. 2011). Notably, direct knockdown of DA
receptors by viral vectors have also been demonstrated to alter ethanol
drinking patterns, thus establishing a direct link between DA perturbation
and ethanol intake (Bahi & Dreyer 2012). DA receptor antagonists have
been linked to reducing ethanol consumption and preference in wide
spectrum of alcohol drinking paradigms (Bahi & Dreyer 2012, Hauser et al.
2015).
Collectively, findings from our behavioral studies in conjunction with
previous investigations suggests that there is disruption of interaction
between purinergic signaling via P2X4Rs and DA neurotransmission and
that this interaction may be involved in neural circuits regulating behaviors
such as sensorimotor gating and reward behavior.
1.5) Dissertation hypothesis:-
To investigate the gaps pertaining to the interaction between P2X4Rs and
DA system, the overarching hypothesis of my dissertation is that P2X4Rs
play an important role in regulation of DA-dependent signaling pathways
and behaviors. Elucidating the interaction between purinergic P2X4Rs and
26

DA system will aid us in better understanding the behavioral changes in
P2X4R KO mouse model and upon IVM treatment.
In chapter 2 of my dissertation, I test the overarching hypothesis by
investigating the expression levels of different markers of DA
neurotransmission as well as phosphorylation states of different signaling
molecules linked to DA receptor activation using P2X4R KO mouse model
as a genetic approach and IVM as a pharmacological approach. I
measured expression levels of DA markers including tyrosine hydroxylase
(TH), dopamine transporter (DAT) and DA receptors (D1 and D2
receptors) in the dorsal and ventral striatum using Western
immunoblotting. Amongst the signaling molecules, I measured the degree
of phosphorylation of dopamine and cyclic-AMP regulated phosphoprotein
of 32 kDa (DARPP-32), extracellular regulated kinase-1/2 (ERK 1/2) and
cyclic-AMP response element binding protein (CREB) in same brain
regions. In regards to behavioral context, I investigated the interaction
between P2X4Rs and dopaminergic drugs in regulation of motor control
using IVM and P2X4R KO mouse model. The behavioral paradigm used
to test this function was the 6-Hydroxydopamine (6-OHDA) model of DA
depletion, wherein the neurotoxin, 6-OHDA was stereotactically injected
into left medial forebrain bundle (MFB), which is a part of striatonigral
circuitry. In addition, I also tested the response of P2X4R KO mice to D1
27

receptor antagonist, SCH 23390 and D2 receptor antagonist, raclopride in
regulation of PPI.
The investigations in Chapter 3 of my dissertation tested the hypothesis
that reduced expression of P2X4Rs in the NAc core increased voluntary
ethanol intake. This hypothesis was based on previous findings that
deficiency of p2rx4 gene increases ethanol intake and potentiation of
P2X4R function by IVM reduces it. To test this hypothesis, I reduced
P2X4R expression in the NAc core using a lentivirus-mediated shRNA
(LV-shRNA) strategy, followed by subjecting the treatment groups (naïve
controls, LV alone and LV-shRNA-p2rx4) to a 24 hour two bottle choice
paradigm of social drinking.
Over the course of my Chapter 4 experiments, I investigated the
interaction between P2X4Rs and DA receptors in PPI mediation using a
pharmacological approach. I tested the effects of DA receptor antagonists
(SCH 23390 and raclopride) on PPI disruptive effects of IVM.
Furthermore, I tested to see if DA D1 receptor activation by SKF 82958
could potentiate IVM-induced PPI dysfunction.

28

CHAPTER 2
ROLE OF PURINERGIC P2X4 RECEPTORS IN REGULATING
STRIATAL DOPAMINE HOMEOSTASIS AND DEPENDENT BEHAVIORS
2.1) ABSTRACT:-
Purinergic P2X4 receptors (P2X4Rs) belong to the P2X superfamily of ion
channels regulated by ATP. We recently demonstrated that P2X4R
knockout (KO) mice exhibit deficits in sensorimotor gating, social
interaction and ethanol drinking behavior. Dopamine (DA) dysfunction may
underlie these behavioral changes, but there is no direct evidence for
P2X4Rs’ role in DA neurotransmission. To test this hypothesis, we
measured markers of DA function and dependent behaviors in P2X4R KO
mice. P2X4R KO mice exhibited altered density of presynaptic markers
including tyrosine hydroxylase, dopamine transporter; postsynaptic
markers including dopamine receptors and phosphorylation of
downstream targets including dopamine and cyclic-AMP regulated
phosphoprotein of 32kDa (DARPP-32) and cyclic-AMP response element
binding protein (CREB) in different parts of the striatum. Ivermectin, an
allosteric modulator of P2X4Rs, significantly affected DARPP-32 and ERK
1/2 phosphorylation in the striatum. Sensorimotor gating deficits in P2X4R
KO mice were rescued by DA antagonists. Using the 6-Hydroxydopamine
29

(6-OHDA) model of DA depletion, P2X4R KO mice exhibited an
attenuated levodopa (L-DOPA) induced motor behavior, whereas IVM
enhanced this behavior. Collectively, these findings identified an important
role for P2X4Rs in maintaining DA homeostasis and illustrate how this
association is important for CNS functions including motor control and
sensorimotor gating.
2.2) INTRODUCTION:-
Purinergic P2X receptors are cation permeable ion channels gated by
ATP. They form homo- and heterotrimeric channels from seven subunits
namely, P2X1-P2X7 (North 2002, Khakh & North 2012). Amongst them,
P2X4 receptors (P2X4Rs) are abundantly expressed on neurons and glial
(microglia and astrocytes) cells across the CNS as well as the PNS (Li et
al. 1998a). Gene knockout and pharmacological strategies have
implicated P2X4R mediated transmission in hippocampal synaptic
plasticity, inflammatory processes in the spinal cord and neuroendocrine
functions (Sim et al. 2006, Ulmann et al. 2008, Zemkova et al. 2010).
Despite this growing body of evidence, there remains a paucity of
information regarding the functional significance of P2X4Rs in the CNS.
We recently reported that mice deficient in the p2rx4 gene [i.e. P2X4R
knockout (KO)] exhibited deficits in sensorimotor gating, social behavior
30

and ethanol drinking behavior (Wyatt et al. 2013, Wyatt et al. 2014).
However, we did not identify any molecular mechanism that could explain
these behavioral deficits. One plausible mechanism could be a result of
P2X4Rs modulating major neurotransmitter systems including the
glutamate and GABA systems. For instance, P2X4Rs are suggested to
regulate postsynaptic currents mediated by NMDA receptors, AMPA
receptors and GABA
A
receptors as well as presynaptic release of
glutamate and GABA (Baxter et al. 2011, Andries et al. 2007, Jo et al.
2011, Gu & MacDermott 1997, Hugel & Schlichter 2002). Moreover,
P2X4R KO mice exhibited altered subunit expression of multiple
glutamatergic and GABA
A
receptors across multiple brain regions. This
latter finding suggests that P2X4R deficiency disrupts homeostasis of
postsynaptic ionotropic receptors (Wyatt et al. 2014). Notably, disruption
of glutamatergic and GABAergic function has been linked to deficits in
sensorimotor gating, social interaction and ethanol drinking behavior
(Duncan et al. 2004, Du et al. 2012, Blednov et al. 2003). Together, these
findings support the hypothesis that P2X4Rs can interact with other
ionotropic receptors in regulation of multiple CNS functions.
In contrast to the building evidence supporting a role for P2X4Rs in
glutamatergic and GABAergic function, little is known regarding the
interaction of P2X4Rs with dopamine (DA) neurotransmission. Early
evidence suggests that P2X4Rs are indirectly involved in DA
31

neurotransmission (Krugel et al. 2001a, Krügel et al. 2003, Xiao et al.
2008), but the direct role for P2X4Rs in regulating DA homeostasis has not
been demonstrated. Considering that P2X4Rs are expressed on DA
neurons and GABAergic medium spiny neurons (MSNs) of the basal
ganglia (Heine et al. 2007, Amadio et al. 2007) and the behavioral deficits
exhibited by P2X4R KO mice may represent DA dysfunction (Gendreau et
al. 2000, Rodriguiz et al. 2004, Zhou et al. 1995, Ralph et al. 2001), we
hypothesized that P2X4Rs control DA signaling with a relevant impact on
DA associated behaviors.
In the present study, we utilized a P2X4R KO mouse model as a genetic
approach and ivermectin (IVM), a positive allosteric modulator of P2X4Rs
(Priel & Silberberg 2004, Khakh et al. 1999, Jelinkova et al. 2008,
Jelinkova et al. 2006, Hattori & Gouaux 2012), as a pharmacological
approach to test the aforementioned hypothesis. We measured protein
densities of different markers of DA neurotransmission including tyrosine
hydroxylase (TH), dopamine transporter (DAT), dopamine D1 and D2
receptors (D1Rs and D2Rs) and downstream targets integral to DA
signaling including dopamine and cyclic-AMP regulated phosphoprotein of
32 kDa (DARPP-32), extracellular regulated kinase-1/2 (ERK 1/2) and
cyclic-AMP response element binding protein (CREB) in different regions
of the striatum of P2X4R KO and wildtype (WT) male mice. We also
measured the degree of phosphorylation of DARPP-32, ERK 1/2 and
32

CREB isolated from different striatal regions of WT and P2X4R KO mice in
the presence and/or absence of IVM. The interaction between P2X4Rs
and DA system in the regulation of CNS functions was addressed by
employing behavioral pharmacology paradigms. The 6-Hydroxydopamine
model (6-OHDA) of DA depletion was used to link P2X4R function with DA
neurotransmission in modulation of motor control. Finally, using the
prepulse inhibition (PPI) of acoustic startle reflex coupled with DA
antagonists, we evaluated the effects of DA dysregulation as it is
pertained to sensorimotor gating deficits. Overall, the findings support the
hypothesis that P2X4R function plays a role in maintaining DA signaling
with an impact on DA associated behaviors such as motor control and
sensorimotor gating.
2.3) EXPERIMENTAL PROCEDURES:-
Animals:-
Experimentally naïve male WT and P2X4R KO mice were obtained from
our breeding colony at the University of Southern California. The breeding
colony was established from a previous P2X4R KO colony that was
maintained on a C57BL/6 background (Sim et al. 2006). Our overall
breeding scheme for generation of P2X4R KO mice and genotyping has
been described previously (Wyatt et al. 2013, Wyatt et al. 2014). Mice
33

were housed in groups of 5 per cage in rooms maintained at 22
o
C with 12
h/12 h light: dark cycle and ad libitum access to food and water. All
experiments were undertaken in compliance to guidelines established by
National Institute of Health (NIH) and approved by the Institutional Animal
Care and Use Committee (IACUC) of University of Southern California.
2-3 month old P2X4R KO and WT mice were used for biochemical
assays. 2-4 month old P2X4R KO and WT mice were used for the motor
behavior studies. 4-6 month old mice were used for the PPI of acoustic
startle reflex study with DA antagonists.
Materials:-
Levodopa (L-DOPA) (Sinemet; 100 mg L-DOPA, 25 mg Carbidopa per
pill) was diluted in 0.9% saline solution to achieve a concentration of 0.75
mg/ml. IVM (Norbrook, Lenexa, KS) was diluted in 0.9% saline solution, to
achieve a concentration of 0.5 mg/ml and injected at a volume of 0.01 ml/g
of body weight. Propylene glycol (Sigma Aldrich, St.Louis, MO) was used
as the vehicle control for IVM. SCH-23390 HCl and raclopride tartrate
(Sigma-Aldrich, St. Louis, MO) were dissolved in 0.9% saline at
concentrations of 0.2 mg/ml and 0.6 mg/ml respectively. Both drugs were
injected at a volume of 0.005 ml/g of body weight.
Western immunoblotting:-
34

Tissue preparation: The dorsal and ventral striata were dissected from
P2X4R KO and WT mice following euthanasia with CO
2
asphyxiation. For
the experiment that tested the effects of IVM (5 mg/kg, i.p.) on
dopaminergic signaling, the dorsal and ventral striatal regions were
dissected out 8 h post drug administration. The dorsal and ventral striata
were dissected out as per the neuroanatomical landmarks described in the
mouse brain atlas (Franklin & Paxinos 2007). The brain tissues were
homogenized in a buffer containing 50 mM Tris-HCl pH (8.0), 150 mM
NaCl, 1 mM EDTA, 0.1% SDS, 1/100 dilution proteinases inhibitor cocktail
(Millipore,Temecula,CA) and protein content was determined using BCA
assay kit (Thermo Scientific, Rockford, IL). Homogenates were treated
with a cocktail of phosphatase inhibitors (1 mM sodium pyrophosphate, 10
mM sodium fluoride, 0.5 mM sodium orthovandate, 10 mM β-glycerol
phosphate, 1 μM microcysteine LR) (Sigma-Aldrich, St. Louis, MO) for
detection of phosphoproteins.
Immunoblotting procedure: Protein samples of 50 µg ran on 10% SDS
PAGE gels and transferred onto polyvinylidine diflouride membranes using
semi-dry transfer method (Trans turbo blot; BioRad Laboratories,
Hercules, CA). Striatal samples from WT and P2X4R KO mice were made
to run on the same gel and transferred onto the same membrane. Non
specific binding was blocked by incubation in 5% non fat dry milk, followed
by incubation with primary antibodies overnight at 4
o
C. The antibodies
35

used were rabbit anti- TH (1:5000), mouse anti-DAT (1:1000), rabbit anti-
D2 receptor (1:1000), rabbit anti- DARPP-32 (1:1000) (Millipore,
Temecula, CA), rabbit anti -D1 receptor (1:500; SantaCruz biotechnology,
Santacruz, CA), mouse anti-total ERK 1/2 (1:1000), rabbit anti- total
CREB (1:1000) (Cell Signaling Technology, Beverly,MA), mouse anti-β-
actin (1:20,000; Sigma Aldrich, St. Louis, MO), mouse anti- α-tubulin
(1:10,000; Millipore, Temecula, CA). The antibodies for phospho proteins
included rabbit anti-phospho-Thr34- DARPP-32 (1:400; Millipore,
Temecula, CA), rabbit anti-phospho-Ser133- CREB (1:500), rabbit anti-
diphospho- Thr202/Tyr204- ERK 1/2 (1:500) (Cell Signaling Technology,
Beverly, MA). Secondary antibodies included goat anti-mouse and goat
anti-rabbit antibodies (1:10,000; BioRad Laboratories, Hercules, CA).
Bands were visualized using chemiluminescence method (Clarity western
plus ECL substrate; BioRad Laboratories, Hercules, CA) followed by
exposure to HyBlot autoradiography films (Denville Scientific, Metuchen,
NJ). Protein quantification was carried out by optical densitometry using
ImageJ software (NIH, Bethesda, MD). Protein densities were normalized
to β-actin or α-tubulin levels.
HPLC assay:-
Brain tissue was homogenized with 0.5 M perchloric acid, centrifuged at
16,873 x g for 12 mins at 4
o
C and protein was resuspended in 0.5 M
36

NaOH. Protein content was detected by BCA assay. DA concentrations
were determined using electrochemical detection method consisting of a
ESA model Coularray 5600A coupled with a four channel analytical cell at
-175, 50, 220, 300 mV. Samples were injected with ESA autosampler
(Chelmsford, MA) and DA was separated by a 150 x 3.2 mm reverse
phase 3 µm diameter C-18 column regulated at 28
o
C. The mobile phase
MD-TM (ESA) consisted of acetylnitrile in phosphate buffer and a non ion-
pairing reagent delivered at a rate of 0.6 ml/min. The HPLC was integrated
with a Dell GX 280 computer with analytical programs including ESA
Coularray for Windows software.
Acoustic Startle Reflex and PPI of Startle:-
Apparatus: Acoustic startle reflex and PPI were tested as previously
described (Wyatt et al. 2013).The apparatus used for detection of startle
reflex (San Diego Instruments, San Diego, CA) consisted of a standard
cage placed in sound attenuated chambers with fan ventilation. Each cage
consisted of a Plexiglass cylinder of 3 cm diameter, mounted on
piezoelectric accelerometric platform connected to an analog-digital
converter. Background noise and acoustic bursts were conveyed by two
separate speakers, each one oriented appropriately so as to produce a
variation of sound within 1 dB across the startle cage. Both speakers and
startle cages were connected to the main PC, which detected and
37

analyzed all chamber variables with Startle software (San Diego
Instruments, San Diego, CA). Before each testing session, acoustic stimuli
were calibrated via a digital sound level meter.
Startle and PPI session: During the baseline session, mice were exposed
to background noise of 70 dB and after an acclimatization period of 5
mins, were presented with 12 40 ms trials of 115 dB interposed with 3
trials of a 82 dB prestimulus preceding the 115 dB by 100 ms.
Subsequently, treatment groups were established so that the average
startle response and %PPI were equivalent within the WT and P2X4R KO
groups. On the testing day, each mouse was placed in the cage and
exposed to a 5 mins acclimatization period with a 70 dB white noise
background, which continued for remainder of the session. Each session
consisted of three consecutive sequences of trials. Unlike the first and
third session, wherein the mice were exposed to five alone pulse trials of
115 dB, the second period consisted of a pseudorandom sequence of 50
trials, including 12 pulse alone trials, 30 trials of pulse preceded by 73 dB,
76 dB or 82 dB prepulse (respectively, defined as PPI 3, PPI 6 and PPI
12; 10 for each level of prepulse loudness) and 8 no stimulus trials,
wherein the mice were presented with background noise without any
prepulse or pulse stimuli. Inter trial intervals were randomly chosen
between 10 and 15 seconds. Delta PPI (ΔPPI) was calculated as mean
startle amplitude for pulse alone trials – (mean startle amplitude for
38

prepulse trial). DA antagonists, SCH-23390 (1 mg/kg, i.p) and raclopride
(3 mg/kg, i.p) were administered 10 mins prior to testing session.
6-OHDA lesioning in mice and motor behavior testing:-
6-OHDA lesioning surgery: Mice were treated with desipramine
hydrochloride (25 mg/kg, i.p.) (Sigma-Aldrich, St. Louis, MO) 30 mins prior
to surgery to prevent concurrent damage of noradrenergic pathways by 6-
OHDA. Mice were anesthetized with avertin (25 mg/kg, i.p.) and placed in
the stereotaxic apparatus. 2 μl of freshly prepared 6-OHDA bromide salt (4
mg/ml in 0.2% ascorbic acid and 0.9% saline; Sigma-Aldrich, St. Louis,
MO) was unilaterally infused into left median forebrain bundle (from
Bregma point: 1.2 mm posterior, 1.1 mm lateral, 5 mm ventral) (Franklin &
Paxinos 2007) at a rate of 0.5 μl/min using a 10 µl Hamilton syringe and a
microlitre syringe pump. The injection cannula was left in place for 3-5
mins to prevent reflux and ensure complete absorption. Postoperative
procedures involved daily subcutaneous (s.c.) injections with sucrose (5%
w/v in saline) and warming on a heating pad for 2 weeks.
Motor behavior testing: Mice were subjected to behavioral testing 3
weeks after 6-OHDA lesioning surgery. Mice were placed in a plastic
cylinder (318 mm in diameter) with a video camera mounted above it.
Baseline rotational behavior was established by saline injections which
causes the mouse to rotate towards the lesioned side with lesser DA
39

activity (ipsilateral rotations). L-DOPA (5 mg/kg, s.c.) was administered 5
mins prior to behavioral testing and the number of contralateral and
ipsilateral rotations were counted in every ten minute interval for a total
period of 90 mins. After monitoring the behavioral activity with L-DOPA
alone, the same cohort of WT and P2X4R KO mice received a
combination of IVM (5 mg/kg, i.p.) and L-DOPA (5 mg/kg, s.c.) to study the
modulatory effect of IVM on L-DOPA induced motor behavior. IVM and L-
DOPA were administered 8 h and 5 mins respectively prior to behavioral
testing. To monitor effect of IVM alone on rotational behavior, a separate
cohort of 6-OHDA lesioned WT mice received IVM (5 mg/kg, i.p) 8 h prior
to behavioral testing and motor activity was monitored for 2 h. The 8 h
time point was selected since IVM has been shown to achieve maximal
concentration in brain and plasma post 8 h (Yardley et al. 2012).
Peroxidase based immunohistochemistry:-
Collection and processing of brain tissues: Transcardial perfusion was
performed using 0.9 % NaCl/ 4% phosphate buffered paraformaldehyde.
Perfused brains were post fixed overnight followed by storage in 20%
sucrose for 48 h and quickly frozen with 4-Methylbutane on dry ice.
Perfused brains were cut coronally at 25 μm thickness in a cryostat and
stored in a cryoprotective solution containing 30% sucrose in PBS at 4
o
C
until further use.
40

TH staining using peroxidase method of immunohistochemistry in ventral
mesencephelon: Brain sections were heated in 10 mM sodium citrate
buffer (pH 8.5) for 30 mins at 80
o
C followed by permeabilization in TBS +
0.2%Triton-X for 30 mins. Slices were then quenched in solution
containing 10% methanol and 3% H
2
O
2
in TBS for 10 mins and
subsequently blocked in 5% non fat dry milk and in 5% normal goat
serum (each step for 30 mins). Sections were incubated overnight with
rabbit anti-TH (1:5000) (Millipore, Temecula, CA) diluted in TBS +0.2%
Triton-X containing 1% normal goat serum. The avidin-biotin complex
method of detection was used, wherein slices were incubated with
biotinylated goat anti-rabbit antibody (1:500) for 1 h and then with 1%
avidin linked peroxidase complex for 45 mins (Vector Laboratories,
Burlingame, CA). This was followed by treatment with solution containing
0.05% 3,3-diaminobenzidine and 0.015% H
2
O
2
in PBS for 5 mins,
dehydration of slides in a dilution series of ethanol, clearance in xylene
and evaluation under light microscope.
Statistical analyses:-
For the Western blotting analysis, the average of densitometry values of
WT striatal samples was used to arbitrarily normalize WT samples to 1
and the P2X4R KO striatal samples were normalized by dividing each
densitometry value by the average of WT samples. Normalization of the
41

two genotypes was done within the same membrane and presented as
fold change of P2X4R KO versus WT in that membrane. For D1, D2,
DARPP-32, ERK1/2 and CREB immunoblotting; WT and P2X4R KO mice
were generated in separate cohorts. The normalization of P2X4R KO to
WT samples was done within the same cohort followed by combining data
from two cohorts for analyses. The same method of normalization was
used for the study that tested the effects of IVM on dopaminergic signaling
in the dorsal and ventral striatum of WT and P2X4R KO mice. The
average of vehicle treated WT mice was used to arbitrarily normalize the
WT control samples to 1 and the IVM treated WT, vehicle and IVM treated
P2X4R KO mice were normalized by dividing each value by the average
of vehicle treated WT mice. Normalized density of proteins were
expressed as mean ± SEM. Phosphorylation was calculated as ratio of
normalized values of phosphorylated form to total form of protein. DA
levels were expressed as ng/mg of protein content. Unpaired Student t-
test was used for analyzing the differences in protein densities and DA
levels between WT and P2X4R KO groups. Two-way ANOVA with
Bonferroni post hoc test was used to evaluate the effect of IVM on
phosphorylation of various signaling molecules between WT and P2X4R
KO. Pharmacological studies for motor behavior and sensorimotor gating
were analyzed by two way repeated measures ANOVA with time/PPI
intensity and genotype/treatment as within and between subjects
42

variability respectively followed by Bonferroni post hoc test for multiple
comparisons. Significance was set at P<0.05. All data was analyzed using
GraphPad Prism software (San Diego, CA).
2.4) RESULTS:-
2.4.1) P2X4R KO mice exhibit alterations in expression of
presynaptic DA markers in striatum:-
To investigate changes in presynaptic markers of DA neurotransmission,
we compared TH and DAT protein density between P2X4R KO and WT
mice using Western immunoblotting. P2X4R KO mice exhibited a
significant increase in TH protein density in the dorsal striatum by 64%
(p<0.01) but no change in protein density in the ventral striatum [Fig 2.1A
& 2.1B (i)]. P2X4R KO mice exhibited significant increases in DAT protein
density by 106% (p<0.05) and by 98% (p<0.01) in the dorsal and ventral
striatum, respectively [Fig 2.1A &2.1B (ii)].
The significant increase in TH protein density in the dorsal striatum did not
appear to be associated with an increase in DA levels as there were no
significant changes in DA levels in the dorsal striatum (Fig 2.2A).
Moreover, there were no changes in DA levels in the ventral striatum (Fig
2.2B) between P2X4R KO and WT mice.

43

FIGURE 2.1

Figure 2.1: P2X4R KO mice exhibited significant increases in TH protein density in the dorsal
striatum, but no changes in the ventral striatum [A & B (i)]; increased DAT protein density in both
parts of the striatum [A & B (ii)]; increased D1R [A &B (iii)] and D2R [A&B (iv)] protein densities in
the ventral, but no change in the dorsal striatum. The protein levels of DA markers were normalized
to β-actin and expressed as arbitrary units (AU). The average of densitometry value of WT samples
was arbitrarily normalized to 1. P2X4R KO samples were normalized by dividing each value by the
average of WT samples and presented as fold change of P2X4R KO versus WT in that membrane.
Values represent mean ± SEM from 5-8 WT, 7-8 P2X4R KO mice for TH, DAT analyses and 11-12
WT, 12-13 P2X4R KO for D1Rs and D2Rs analyses. 2 representative bands from each genotype
from the same membrane are shown. *P <0.05, ** P <0.01 versus WT controls. Unpaired Student’s
t-test

A.
WT P2X4R KO
Ventral striatum
D2R
D1R
DAT
TH
-actin
-actin
-actin
-actin
WT
P2X4R KO
Dorsal striatum
D2R
D1R
DAT
TH
-actin
-actin
-actin
-actin
B.
i)
TH protein density
(fold change)
Dorsal striatum Ventral striatum
0
1
2
3
4
WT
P2X4R KO
**
ii)
DAT protein density
(fold change)
Dorsal striatum Ventral striatum
0
1
2
3
4
WT
P2X4R KO
**
*
iii)
D1R protein density
(fold change)
Dorsal striatum Ventral striatum
0
1
2
3
4
WT
P2X4R KO
*
iv)
D2R protein density
(fold change)
Dorsal striatum Ventral striatum
0
1
2
3
4
WT
P2X4R KO
*
44

FIGURE 2.2

Figure 2.2: No changes in DA levels in the dorsal (A) and ventral striatum (B) of P2X4R KO mice as
compared to WT controls. Values represent mean ± SEM for 13 WT, 12 P2X4R KO mice in dorsal
striatum and 12 WT, 11 P2X4R KO mice in ventral striatum, Student’s t-test.

2.4.2) P2X4R KO mice exhibit significant alterations in expression of
DA receptors and downstream targets:-
Based on the significant changes in TH and DAT protein densities, we
posit that changes within presynaptic DA environment could impact the
densities of DA receptors and downstream targets. Among the DA
receptor subtypes, D1Rs belong to excitatory G 
s/olf
family of G-coupled
protein receptors (GPCRs), whereas D2Rs belong to inhibitory G 
i/olf
family of GPCRs (Girault 2012). To test this hypothesis, we measured
protein densities of D1Rs, D2Rs and phosphorylation states of major
downstream targets in striatum. P2X4R KO mice exhibited significant
increases in D1R protein density by 159% [p<0.05; Fig 2.1A & 2.1B (iii)]
B. Ventral Striatum
Genotype
DA levels (ng/mg protein)
WT P2X4R KO
0
20
40
60
80
100
A. Dorsal Striatum
Genotype
DA levels (ng/mg protein)
WT P2X4R KO
0
20
40
60
80
100
45

and in D2R protein density by 42% [p<0.05; Fig 2.1A & 2.1B (iv)] in the
ventral striatum as compared to WT mice. There were no changes in
either of protein densities for DA receptors in the dorsal striatum of P2X4R
KO mice.
To investigate downstream pathways regulated by DA receptors, we
measured total and phosphorylated form of DARPP-32, ERK 1/2 and
CREB in the dorsal and ventral striatum using Western immunoblotting.
P2X4R KO mice exhibited a significant increase in DARPP-32
phosphorylation (by 164%, p<0.05) without any changes in total DARPP-
32 protein density in the dorsal striatum as compared to WT counterparts
[Fig 2.3A & 2.3B (i)]. In the ventral striatum, P2X4R KO mice exhibited a
significant decrease in DARPP-32 phosphorylation (by 48%, p<0.05)
without any changes in protein density of total DARPP-32 [Fig 2.3A & 2.3B
(i)]. P2X4R KO mice did not exhibit any significant changes in total ERK
1/2 protein density or ERK 1/2 phosphorylation in the dorsal and ventral
striatum compared to WT mice [Fig 2A & 2B (ii)]. There were no significant
changes in protein density of total CREB or CREB phosphorylation in the
dorsal striatum of P2X4R KO mice [Fig 2.3A & 2.3B (iii)]. On the other
hand, P2X4R KO mice did exhibit a significant decrease in total CREB
density (by 36%, p<0.01) and a corresponding increase in CREB
phosphorylation (by 141%, p<0.001) in the ventral striatum compared to
WT mice [Fig 2.3A & 2.3B (iii)].
46

FIGURE 2.3

Figure 2.3: P2X4R KO mice exhibited increased DARPP-32 phosphorylation in the dorsal striatum,
but a decrease in the ventral striatum [A & B (i)]; no changes in ERK 1/2 phosphorylation in dorsal
or ventral striatum of P2X4R KO mice [A & B(ii)]; increased phosphorylation of CREB in the ventral,
but not in the dorsal striatum [A & B(iii)]. Details of normalization and analyses are presented in
Figure 1. Values represent mean ± SEM from 3-6 WT and 4-8 P2X4R KO mice for DARPP-32
analysis; 6-8 WT & P2X4R KO for ERK 1/2 and CREB analyses. 2 representative bands from each
genotype from the same membrane are shown. *P <0.05, ***P<0.001 versus WT counterparts.
Unpaired Student’s t-test.

A.
WT P2X4R KO
Ventral striatum
CREB
-tubulin
-tubulin
p-CREB
ERK 1/2
-tubulin
-tubulin
p-ERK 1/2
DARPP-32
-actin
-tubulin
p- DARPP-32
CREB
-tubulin
-tubulin
p-CREB
WT
P2X4R KO
ERK /2
-tubulin
-tubulin
p-ERK 1/2
DARPP-32
-actin
p-DARPP-32
Dorsal striatum
-actin
B.
i)
DARPP-32 phosphorylation
(fold change)
Dorsal striatum Ventral striatum
0
1
2
3
4
WT
P2X4R KO
*
*
ii)
ERK1/2 phosphorylation
(fold change)
Dorsal striatum Ventral striatum
0
1
2
3
4
WT
P2X4R KO
iii)
CREB phosphorylation
(fold change)
Dorsal striatum Ventral striatum
0
1
2
3
4
WT
P2X4R KO
***
47

2.4.3) IVM significantly affected DARPP-32 and ERK 1/2
phosphorylation, but not CREB phosphorylation in the dorsal
striatum:-
IVM was used to gain pharmacological insights into the role of P2X4Rs in
dopaminergic signaling in the dorsal striatum. There was no significant
effect of IVM treatment or genotype on total DARPP-32 levels or
phosphorylation but the drug treatment x genotype interaction was
significant for both the total DARPP-32 levels [F(1,18)=5.61, p<0.05] and
DARPP-32 phosphorylation [F(1,18)=5.48, p<0.05]. Bonferroni post hoc
test confirmed significant increase in DARPP-32 phosphorylation upon
IVM treatment in WT (t=2.834, p<0.05), but not in P2X4R KO mice
(t=0.5829, p>0.05) [Fig 2.4A & 2.4B (i)]. There was no significant effect of
IVM treatment or genotype on total ERK 1/2 levels, but the drug treatment
x genotype interaction was significant [F(1,18)=10.00, p<0.01]. There was
a significant effect of IVM treatment [F(1,18)=, p<0.05] but not genotype or
drug treatment x genotype interaction for ERK 1/2 phosphorylation [Fig
2.4A & 2.4B (ii)]. There was no significant effect of IVM treatment,
genotype or drug treatment x genotype interaction for total CREB levels.
There was no significant effect of IVM treatment or drug treatment x
genotype interaction, but the effect of genotype trended towards
significance for CREB phosphorylation [F(1,16)=4.37, p=0.0529] [Fig 2.4A
& 2.4B (iii)].
48

FIGURE 2.4

Figure 2.4: IVM (5 mg/kg) significantly upregulated DARPP-32 phosphorylation [A & B (i)] via
P2X4R potentiation in the dorsal striatum. IVM increased ERK 1/2 phosphorylation independent of
P2X4R function [A& B (ii)]. No effect of IVM on CREB phosphorylation [A& B (iii)]. The average of
densitometry value of vehicle treated WT samples was arbitrarily normalized to 1. The IVM treated
WT mice, vehicle and IVM treated P2X4R KO were normalized by dividing each value by the
average of the vehicle treated WT samples. The data is presented as fold change of IVM treated
WT, P2X4R KO and vehicle treated P2X4R KO samples versus vehicle treated WT samples in that
membrane. Values represent mean ± SEM from 4-6 WT and P2X4R KO mice per treatment group.
2 representative bands from each treatment group are shown. *P<0.05 versus vehicle treated WT
group, Bonferroni post hoc test.

B.
i)
Genotype
DARPP-32 phosphorylation
(fold change)
WT P2X4R KO
0
1
2
3
4
PG
5 mg/kg IVM
*
**
ii)
Genotype
ERK1/2 phosphorylation
(fold change)
WT P2X4R KO
0
1
2
3
4
PG
5 mg/kg IVM
iii)
Genotype
CREB phosphorylation
(fold change)
WT P2X4R KO
0
1
2
3
4
PG
5 mg/kg IVM
WT
CREB
p-CREB
ERK 1/2
p-ERK 1/2
DARPP-32
p-DARPP-32
Vehicle 5mg/kg IVM
-tubulin
-tubulin
-tubulin
P2X4R KO
CREB
p-CREB
ERK 1/2
p-ERK 1/2
DARPP-32
p- DARPP-32
Vehicle 5mg/kg IVM
-tubulin
-tubulin
-tubulin
A.
49

2.4.4) IVM significantly affected DARPP-32 phosphorylation, but not
ERK 1/2 or CREB phosphorylation in the ventral striatum:-
Similar to dorsal striatum, we investigated the effect of IVM on
phosphorylation of the same signaling molecules in the ventral striatum.
There was no significant effect of IVM treatment or genotype on total
DARPP-32 levels or DARPP-32 phosphorylation but the drug treatment x
genotype interaction was significant for both total DARPP-32 levels
[F(1,16)=6.40, p<0.05] and DARPP-32 phosphorylation [F(1,16)=6.20,
p<0.05] [Fig 2.5A & 2.5B (i)]. There was no significant effect of IVM
treatment, genotype or drug treatment x genotype interaction for total ERK
1/2 levels or ERK 1/2 phosphorylation [Fig 2.5A & 2.5B (ii)]. There was no
significant effect of IVM treatment, genotype or drug treatment x genotype
interaction for total CREB levels. Similarly, there was no significant effect
of IVM treatment or genotype on CREB phosphorylation. However, the
drug treatment x genotype interaction trended towards significance for
CREB phosphorylation [F(1,15)=4.43, p=0.0527] [Fig 2.5A& 2.5B (iii)].

50

FIGURE 2.5

Figure 2.5: IVM (5 mg/kg) significantly affected DARPP-32 [A & B (i)] but not, ERK 1/2 [A & B (ii)] or
CREB phosphorylation [A& B (iii)], via P2X4R potentiation in the ventral striatum. Details of
normalization and analyses are presented in Figure 3. Values represent mean ± SEM from 4-6 WT
and P2X4R KO mice per treatment group. 2 representative bands from each treatment group are
shown. *P<0.05 versus vehicle treated WT group, Bonferroni post hoc test.
B.
iii)
Genotype
CREB phosphorylation
(fold change)
WT P2X4R KO
0.0
0.5
1.0
1.5
2.0
PG
5 mg/kg IVM
**
i)
Genotype
DARPP-32 phosphorylation
(fold change)
WT P2X4R KO
0.0
0.5
1.0
1.5
2.0 PG
5 mg/kg IVM
*
ii)
Genotype
ERK1/2 phosphorylation
(fold change)
WT P2X4R KO
0.0
0.5
1.0
1.5
2.0
PG
5 mg/kg IVM
A.
P2X4R KO
CREB
p-CREB
ERK 1/2
p-ERK 1/2
DARPP-32
p- DARPP-32
Vehicle 5mg/kg IVM
-tubulin
-tubulin
-tubulin
WT
CREB
p-CREB
ERK 1/2
p-ERK 1/2
DARPP-32
p-DARPP-32
Vehicle 5mg/kg IVM
-tubulin
-tubulin
-tubulin
51

2.4.5) Pharmacological inhibition of D1Rs and D2Rs significantly
enhanced prepulse inhibition (PPI) of acoustic startle reflex in P2X4R
KO mice:-
We investigated the response of P2X4R KO mice to selective antagonists
for D1Rs (SCH-23390; 1 mg/kg) and D2Rs (raclopride; 3 mg/kg) on PPI
startle response, to link imbalances in DA homeostasis to PPI deficits in
P2X4R KO mice. The doses tested were chosen based on previous
studies that investigated PPI function in C57BL/6 mice (Doherty et al.
2008, Ralph-Williams et al. 2003, Ralph et al. 2001). We found a
significant effect of genotype on PPI function [F(1,26)= 5.50, p<0.05] (Fig
2.6A) which supports our previous findings (Wyatt et al. 2013). SCH-
23390 significantly increased PPI in P2X4R KO mice [F(1,28)=9.33,
p<0.01] with Bonferroni post hoc test identifying a significant increase at
PPI 6 (t=3.294, p<0.01) and PPI 12 (t=3.240, p<0.01) (Fig 2.6A). Similarly,
raclopride significantly enhanced PPI function in P2X4R KO mice
[F(1,28)=11.98, p<0.01] as compared to saline treated P2X4R KO mice
with Bonferroni post hoc test identifying a significant increase at PPI 6
(t=3.748, p<0.001) and PPI 12 (t=3.507, p<0.01) (Fig 2.6A). There were
no significant changes in PPI function in WT mice upon treatment with
SCH-23390 or raclopride (Fig 2.6A). Moreover, neither SCH- 23390 nor
raclopride induced any significant change in startle amplitude in P2X4R
KO mice (Fig 2.6B).
52

FIGURE 2.6

Figure 2.6: SCH-23390 (1 mg/kg) and raclopride (3 mg/kg) significantly increased prepulse
inhibition of acoustic startle reflex in P2X4R KO mice (A) without any changes in startle amplitude
(B). There were no changes in PPI in WT mice upon treatment with both antagonists (A). Values
represent the mean of ΔPPI and mean startle amplitude ± SEM from 14 WT (saline), 15 WT (SCH
23390 and raclopride), 14 P2X4R KO (saline) and 16 P2X4R KO (SCH-23390 and raclopride). *P
<0.05 versus saline treated WT mice, ## P<0.01, ### P<0.001 versus saline treated P2X4R KO
mice, Bonferroni post hoc test.

D
PPI (mV)
0
50
100
150
200
250
300
PPI 3
PPI 6
PPI 12
*
##
##
##
###
A.
B.
Startle amplitude (mV)
0
100
200
300
400
500
53

2.4.6) Pharmacological or genetic manipulation of P2X4R function
significantly influenced L-DOPA induced motor behavior in the 6-
OHDA model of DA depletion:-
Using the 6-OHDA model of DA depletion, we investigated the role of
P2X4Rs in regulation of motor behavior (Schwarting & Huston 1996). As
presented, ablation of DA neurons in ventral mesencephelon in both
genotypes (Fig 2.7A) resulted in reduced TH expression by 95.7% and
96.3% in the striatum of WT (p<0.001) and P2X4R KO mice (p<0.01)
respectively (Fig 2.7B). In the presence of L-DOPA treatment (5 mg/kg),
both genotypes exhibited contralateral rotations, since L-DOPA
metabolizes into DA in the synapses, followed by activation of the
supersensitive postsynaptic DA receptors on lesioned striatum
(Ungerstedt 1971) (Fig 2.9A). In the presence of L-DOPA, we found that
P2X4R KO mice exhibited significantly fewer contralateral turns as
compared to WT mice (Fig 2.9A). There was a significant effect of
genotype [F(1,20)=4.39, p<0.05] and time [F(8,160)=58.46, p<0.001] on L-
DOPA induced rotational behavior in P2X4R KO. There was no significant
time x genotype interaction for L-DOPA induced motor behavior.
Bonferroni post hoc test confirmed significant reduction in L-DOPA
induced motor behavior in P2X4R KO mice during the 45-55 mins interval
(t=3.154, p<0.05) (Fig 2.9A).

54

FIGURE 2.7

Figure 2.7: Stereotaxic injection of 6-OHDA (4mg/ml) induced destruction of DA neurons in the
ventral mesencephelon (A) and TH density in the striatum (B) of both WT and P2X4R KO to similar
extent. U= unlesioned, L=lesioned. Values represent mean ± SEM for 4 mice per genotype. **P
<0.01, *** P <0.001 versus unlesioned side of striatum, Unpaired Student’s t-test.

B.
Normalized density (AU)
Unlesioned Lesioned
0.0
0.2
0.4
0.6
0.8
1.0
1.2
***
Normalized density (AU)
Unlesioned Lesioned
0.0
0.2
0.4
0.6
0.8
1.0
1.2
**
U L
WT
TH
α-tubulin
P2X4RKO
U L
TH
α-tubulin
A.
P2X4RKO
WT
unlesioned VME lesioned VME
unlesioned VME lesioned VME
55

FIGURE 2.8

Figure 2.8: IVM (5mg/kg) induced ipsilateral rotations in 6-OHDA WT mice that were statistically
significant from sham WT controls. Values on the y-axis represent mean of number of ipsilateral
rotations for a period of 2 hrs post IVM treatment ± SEM for 7 6-OHDA WT and 4 sham controls. **
P <0.01, *** P <0.001 versus sham WT controls, Bonferroni post hoc test.

We reasoned that if genetic disruption of P2X4Rs reduces L-DOPA
response on motor behavior, then pharmacological potentiation of
P2X4Rs should enhance motor behavior. In support of this hypothesis, we
found that IVM (5 mg/kg) significantly increased the number of L-DOPA
induced rotations in WT mice. There was a significant effect of time
[F(8,208)=95.32, p<0.001] and IVM treatment [F(1,26)=33.80, p<0.001] on
L-DOPA induced motor behavior. There was also a significant time x drug
treatment interaction [F(8,208)=10.99, p<0.001] with Bonferroni post hoc
Mins post IVM treatment
Ipsilateral rotations
480 490 560 570
0
5
10
15
20
Sham WT ( 5mg/kg IVM)
6-OHDA WT (5mg/kg IVM)
***
***
**
**
56

test identifying a significant increase in contralateral rotations upon IVM
treatment during 5-15 mins (t=5.509, p<0.001), 15-25 min (t=4.161,
p<0.001), 25-35 min (t=5.491, p<0.001), 35-45 min (t=6.254, p<0.001), 45-
55 min (t=6.484, p<0.001) and 55-65 min (t=4.878, p<0.001) intervals (Fig
5A). IVM alone induced an average of 12 rotations ± 2 rotations (SEM) for
period of 2 hours post drug treatment, although it was still statistically
significant compared to sham controls [F(1,9)=26.64, p<0.001] (Fig 2.8).
There was a significant effect of time [F(8,112)=36.93, p<0.001] and IVM
treatment [F(1,14)=4.80,p<0.05] on L-DOPA induced motor behavior in
P2X4R KO mice (Fig 2.9A). There was also a significant time x drug
treatment interaction [F(8,112)=2.88, p<0.01] with Bonferroni post hoc test
confirming a significant increase in L-DOPA induced motor behavior
during the 5-15 mins interval (t=3.481, p<0.01). Taking into consideration
the lower L-DOPA baseline in P2X4R KO mice, we analyzed the
difference in the number of contralateral rotations between L-DOPA alone
and IVM + L-DOPA in WT and P2X4R KO mice. There was a significant
effect of time [F(8,160)=11.33, p<0.001] and a non-significant trend of
effect of genotype [F(1,20)=3.17, p=0.0903] on contralateral rotations
between L-DOPA and L-DOPA + IVM (Fig 2.9B). There was a significant
time x genotype interaction [F(8,160)=2.07, p<0.05], suggesting that IVM’s
ability to increase L-DOPA induced motor behavior differed between WT
and P2X4R KO mice. Bonferroni post hoc test identified significant
57

differences in L-DOPA induced motor behavior upon IVM treatment
between WT and P2X4R KO mice at 55-65 mins (t=2.948, p<0.05) (Fig
2.9B).
FIGURE 2.9

Figure 2.9: L-DOPA (5 mg/kg) induced rotational behavior is significantly attenuated in P2X4R KO
mice. IVM (5 mg/kg) significantly potentiated L-DOPA’s effect on the number of contralateral turns
in WT and P2X4R KO mice (A). IVM’ ability to enhance L-DOPA induced motor behavior was
significantly altered in P2X4R KO mice (B). Values on the y-axis represent the mean of number of
contralateral turns per 10 minute interval ± SEM from 14 WT, 8 P2X4R KO. *P<0.05, *** P <0.001
versus L-DOPA treated WT mice, ## P<0.01 versus L-DOPA treated P2X4R KO mice for (A),
*P<0.05 versus WT mice for (B), Bonferroni post hoc test.

Time (mins)
Contralateral Rotations
(per ten minute interval)
15 25 35 45 55 65 75 85 95
0
100
200
300
400
500
6-OHDA WT (L-DOPA)
6-OHDAP2X4R KO (L-DOPA)
6-OHDA WT(IVM+L-DOPA)
6-OHDA P2X4R KO(IVM+L-DOPA)
***
***
***
***
***
***
*
##
A.
Time (mins)
Increase in contralateral rotations
from L-DOPA baseline
15 25 35 45 55 65 75 85 95
-50
0
50
100
150
200
250
6-OHDA WT (L-DOPA + IVM)
6-OHDA P2X4R KO (L-DOPA + IVM)
*
B.
58

2.5) DISCUSSION:-
Our current study investigated the role of P2X4Rs in the dopaminergic
system and its impact on DA dependent behaviors. Impairments in DA
neurotransmission were observed with respect to changes in protein
densities of pre- and postsynaptic markers. There was increased TH
protein density in the dorsal, but not the ventral, striatum of P2X4R KO
mice. DAT was significantly increased in both parts of the striatum in
P2X4R KO mice, which is indicative of higher presynaptic reuptake of DA.
In addition to changes in presynaptic markers of DA activity, we also
identified significant changes in protein densities of DA receptors in the
ventral, but not the dorsal, striatum of P2X4R KO mice. The increased DA
receptors’ density in the ventral striatum could be an adaptive response to
the altered synaptic DA availability due to increased DAT expression in
the same brain region of P2X4R KO mice. This interpretation is based on
previous studies that have postulated a positive correlation between DAT
and DA receptor density (Fauchey et al. 2000, Ghisi et al. 2009). The
increased DAT and DA receptor density levels suggest that P2X4R KO
mice may have alterations in DA neurotransmission. However, there were
no significant changes in DA levels in both parts of the striatum of P2X4R
KO mice despite significant alterations in TH density levels in the dorsal
striatum of P2X4R KO mice. There are several other factors that control
DA levels such as storage, release, reuptake, and catabolism (Eells
59

2003). Based on our finding of increased density of DAT and DA
receptors, future studies will be necessary using in vivo microdialysis or
fast scan cyclic voltammetry to help to identify any differences in the
extracellular DA levels between WT and P2X4R KO mice and elucidate
the dopaminergic tone in P2X4R KO mice. Overall, the findings suggest
that p2rx4 deficiency affects DA synthesis and transport that could impact
normal DA neuron function.
In addition to DA receptors, we identified dysregulation of signaling
molecules (i.e. DARPP-32 and CREB) that can be regulated through DA
receptors in different striatal regions of P2X4R KO mice. D1R stimulation
on striatonigral MSNs phosphorylates DARPP-32 at Thr34 via protein
kinase A (PKA) that inhibits PP-1 activity and allows phosphorylation of
ERK1/2 and CREB. D2R stimulation produces the opposite effects in the
striatopallidal MSNs (Girault 2012, Bertran-Gonzalez et al. 2008). Despite
the lack of significant changes in D1Rs protein density, P2X4R KO mice
exhibited a significant increase in DARPP-32 phosphorylation in the dorsal
striatum, which is typically indicative of upregulated D1R mediated
signaling function. On the other hand, we saw a significant decrease in
DARPP-32 phosphorylation in the ventral striatum of P2X4R KO mice that
correlates well with the increased D2R protein density in the same brain
region. But, we saw significant increases in D1R density and CREB
phosphorylation in the same brain region of P2X4R KO mice that did not
60

corroborate with the decreased DARPP-32 phosphorylation. One possible
explanation for these neurochemical differences identified in the P2X4R
KO mice is that there are multiple interactions or involvement of various
neurotransmitter systems besides DA in regulating DARPP-32
phosphorylation such as glutamate, GABA and serotonin (Svenningsson
et al. 2004). Thus, the alterations in signaling molecules including DARPP-
32 and CREB in P2X4R KO mice suggest a complex compensatory
change other than that of DA receptors. Taken together; the increased
density of pre- and postsynaptic markers suggests dysregulation of DA
system in P2X4R KO mice which may partially underlie the behavioral
deficits previously reported in P2X4R KO mice.
In addition to the genetic approach, we used a pharmacological approach
to explore a role for P2X4Rs in regulating DA receptor associated
signaling pathways. Since, there are limited specific antagonists to test
P2X4R related signaling in vivo, we used the P2X4R allosteric modulator,
IVM, to investigate a link between P2X4R function and DA receptor
associated signaling molecules in the dorsal and ventral striatum. As
presented above, there was a significant increase in DARPP-32
phosphorylation upon IVM treatment in the WT, but not in P2X4R KO
mice, suggesting a role for P2X4Rs in regulating DARPP-32
phosphorylation in the dorsal striatum. The changes in DARPP-32
phosphorylation upon P2X4R potentiation by IVM did not fully agree with
61

the result from genetic deletion of P2X4Rs in the dorsal striatum. This
difference in finding may be linked to neurodevelopmental changes in
P2X4R KO mice. This hypothesis is supported by several lines of
evidence: first, IVM did not increase DARPP-32 phosphorylation in P2X4R
KO mice; second, P2X4Rs have been reported to be expressed from
postnatal day 1 (Cheung et al. 2005); third, the P2X4R KO mice exhibit
communication deficits during their pre-adult period (Wyatt et al. 2013).
Since, phospho-Thr34-DARPP-32 can indirectly increase ERK 1/2
phosphorylation in the striatum (Girault 2012), it was not surprising to see
a significant effect of IVM treatment on ERK 1/2 phosphorylation.
However, increase in ERK 1/2 phosphorylation was seen in both the
genotypes, indicating that IVM’s ability to modulate ERK 1/2
phosphorylation is independent of P2X4R function. Interestingly, there
was a significant interaction between IVM treatment and genotype for total
ERK 1/2 levels, indicating that effect of IVM on total ERK 1/2 expression
was dependent upon P2X4R function. Hence, IVM might be increasing
ERK 1/2 phosphorylation in the WT mice by regulating total expression of
the protein upon P2X4R potentiation. Although previous investigations
have used IVM as a pharmacological tool for studying P2X4R function in
vitro and in vivo (Bortolato et al. 2013, Sim et al. 2006, Popova et al. 2013,
Asatryan et al. 2010), IVM does have other protein targets in the CNS
including GABA
A
receptors (Dawson et al. 2000) , nicotinic acetylcholine
62

receptors (Krause et al. 1998) and glycine receptors (Shan et al. 2001).
Hence, the mechanism by which IVM increases ERK 1/2 phosphorylation
in P2X4R KO mice needs further investigation. Similar to the dorsal
striatum, IVM modulated DARPP-32 phosphorylation via P2X4R
potentiation in the ventral striatum. Moreover, IVM had a tendency to
differentially modulate CREB phosphorylation via P2X4R activity in the
same brain region. Taken together, using genetic and pharmacological
approaches, the data suggests that there is a link between P2X4R activity
and DARPP-32 phosphorylation in the striatum and P2X4Rs may have a
role in regulating DA neurotransmission in GABAergic MSNs of the
striatum via modulating DARPP-32 activity.
The significant increases in DA receptor protein density in the ventral
striatum reported herein may underlie the PPI deficits in P2X4R KO mice.
PPI measures reduction in startle reflex that occurs when the eliciting
acoustic burst is immediately preceded by a weak stimulus and is highly
reliable index for measuring sensorimotor gating (Ison & Hoffman 1983).
Multiple findings have reported a critical role for DA receptors in regulation
of PPI function, of which D2Rs have received considerable attention on
basis of findings from pharmacological studies in rats and patient
population (Swerdlow et al. 1991, Abduljawad et al. 1998, Volter et al.
2012, Kumari et al. 1998). However, gene knockout and pharmacological
studies in mice have implicated both D1Rs and D2Rs (Ralph-Williams et
63

al. 2003, Ralph-Williams et al. 2002, Doherty et al. 2008). In the context of
findings from the literature, we used both D1R (SCH-23390) and D2R
(raclopride) antagonists to identify potential contribution of DA receptors to
PPI functioning in P2X4R KO mice. We found that the PPI deficits in
P2X4R KO mice were significantly ameliorated by SCH-23390 and
raclopride, indicating D1Rs and D2Rs as important modulators of PPI
function in mice. The increased density of DA receptors in the ventral
striatum, integral to corticolimbic-striato-pallidal circuitry of PPI (Swerdlow
et al. 2008), of P2X4R KO mice may contribute to PPI dysfunction and
that blocking these receptors can reverse the deficit. The pharmacological
studies provide insights into the functional consequences of altered DA
receptor protein densities on behaviors such as sensorimotor gating in the
P2X4R KO mice. Moreover, we reported IVM mediated PPI disruption in
WT C57BL/6J mice and its attenuated response in P2X4R KO mice, which
further supports a role for P2X4Rs in sensorimotor gating (Bortolato et al.
2013). Overall, these studies identify potential interactions between
P2X4R function and DA neurotransmission in regulating sensorimotor
gating.
The neurochemical and behavioral alterations in P2X4R KO mice could be
relevant to multiple psychiatric disorders such as schizophrenia,
attentional deficit hyperactivity disorder, Obsessive-Compulsive disorder
and bipolar depression. For example, post mortem studies have reported
64

an increase in D2R and DAT expression in psychotic and non-psychotic
disorders (Krause et al. 2000, Brunswick et al. 2003, Pearlson et al. 1995,
Perez et al. 2003). In addition, increased TH expression and presynaptic
DA synthesis has been reported in neuroleptic naïve psychotic patients
(Hietala et al. 1995, Hietala et al. 1999). We observed increased density in
D2Rs in the ventral striatum, DAT in both striatal regions and TH in the
dorsal striatum in P2X4R KO mice. Perhaps, one of the most notable
findings of our study was the identification of increased D2Rs in the
striatum, since D2Rs are considered as an important genetic marker for
susceptibility to neuropsychiatric diseases (Seeman 2013a, Seeman
2013b, Zhan et al. 2011). Additionally, P2X4R KO mice exhibited PPI
deficits, an important behavioral biomarker of neuropsychiatric diseases
(Seeman et al. 2006, Feifel et al. 2009, Perry et al. 2001, Braff 1993).
Interestingly, the upregulation of D2Rs, altered sensitivity to DA receptor
acting drugs and PPI deficits in P2X4R KO mice correlate with findings
from mouse models linked to psychiatric disorders (Wolinsky et al. 2007,
Lipina et al. 2010, Ralph et al. 2001, Kinkead et al. 2005). Taken together,
the altered DA homeostasis and resultant behavioral deficits induced upon
p2rx4 deficiency suggests a role for this receptor in disorders
characterized by DA dysfunction such as schizophrenia, bipolar disorder
and attention deficit hyperactivity.
65

Our findings also suggest that P2X4Rs are involved in other DA
dependent functions of the basal ganglia including motor behavior. To test
this, we used the 6-OHDA animal model in combination with IVM and
P2X4R KO mice, which is a well established model for elucidating DA
interactions with other neurotransmitter systems in motor activity (Fox &
Brotchie 2000, Xiao et al. 2011). Also, this model is used for
understanding the pathogenesis of movement disorders including
Parkinson’s disease and screening of novel therapeutics (Deumens et al.
2002, Schwarting & Huston 1996). We found that the L-DOPA induced
motor behavior was significantly decreased in 6-OHDA lesioned P2X4R
KO mice, indicating that disruption of P2X4R function significantly affected
L-DOPA induced behavioral response. This attenuated response in
P2X4R KO mice may be due to alterations in DA system in striatonigral
circuitry of the basal ganglia. Alternatively, L-DOPA attenuated response
could be linked to its faster clearance or metabolism in P2X4R KO mice.
This hypothesis will be explored in future studies. Conversely, we
demonstrated that pharmacological modulation of P2X4R activity by IVM
significantly enhanced L-DOPA induced rotational behavior. A plausible
mechanism underlying L-DOPA + IVM response is a synergy between
P2X4Rs and D1Rs on MSNs in disinhibiton of neurons projecting from the
substantia nigra pars reticulata (SNR) to the thalamus, superior colliculus
and pendenculopontine nucleus and thereby, producing contralateral
66

rotations. Notably, increased expression of P2X4Rs has been reported in
the MSNs of SNR of 6-OHDA treated rats (Amadio et al. 2007) and so,
these compensatory changes may partially explain the augmented L-
DOPA dependent motor response in presence of IVM. IVM did not
influence motor behavior independently, suggesting that activation of
P2X4Rs alone is not sufficient enough to cause disinhibiton of SNR to
induce such a response. Unlike D1Rs that are present exclusively on
postsynaptic MSNs, P2X4Rs are present both on presynaptic DA neurons
and postsynaptic MSNs. The simultaneous activation of P2X4Rs at the
presynapses and postsynapses would counteract each other, thus
preventing the mice from turning to either side. The lack of effect with IVM
alone supports the notion that IVM has a modulatory effect on L-DOPA’s
motor behavior. In addition to increased behavioral response in WT mice,
IVM also enhanced L-DOPA’s motor behavior in P2X4R KO mice.
However, while comparing the absolute increase in L-DOPA’s motor
behavior in presence of IVM, we saw a significant interaction between
time and genotype, suggesting an altered effect of IVM between WT and
P2X4R KO mice. This finding supports our previous finding that IVM
mediates its behavioral effects partially via action on P2X4Rs (Wyatt et al.
2014, Bortolato et al. 2013). Nevertheless, the increase in L-DOPA
response in P2X4R KO mice suggests that IVM may be modulating L-
DOPA response through a complex network of receptor systems. Overall,
67

the findings suggest that P2X4Rs have a synergistic role in DA modulation
of motor control and can alter behavioral responses to dopaminergic
drugs. As such, P2X4R allosteric modulators may represent potential
adjuvant pharmacotherapies for Parkinson’s disease.
In conclusion, the present investigation supports the hypothesis that there
are signaling interactions between P2X4Rs and DA neurotransmission in
regulation of multiple CNS functions in the basal ganglia. Finally, though at
nascent stage, these findings implicate P2X4Rs in neurobiological
mechanisms of multiple neurological disorders. There is growing interest
in P2XRs as novel drug targets for therapeutic development in psychiatry
disorders (Ortiz et al. 2015). In support of this hypothesis, others report
that the gene for P2X4Rs is located in chromosome 12q24 (Gu et al.
2010) which contains several loci that can alter susceptibility to
schizophrenia, bipolar disorder and attention deficit hyperactivity (Dawson
et al. 1995, Jones et al. 2002, Bailer et al. 2000). Non-synonymous single
nucleotide polymorphisms in p2rx4 gene have been linked to high pulse
pressure and age related macular degeneration (Caseley et al. 2014) but
further investigation is needed before definite conclusions can be drawn
regarding P2X4Rs and psychiatric diseases.

68

CHAPTER 3
REDUCED EXPRESSION OF PURINERGIC P2X4 RECEPTORS
INCREASES VOLUNTARY ETHANOL INTAKE IN C57BL/6J MICE
3.1) ABSTRACT:-
Purinergic P2X4 receptors (P2X4Rs) belong to the P2X superfamily of
ionotropic receptors that are gated by adenosine-5’-triphosphate (ATP).
Accumulating evidence indicates that P2X4Rs play an important role in
regulation of ethanol intake. At the molecular level, ethanol’s inhibitory
effects on P2X4Rs are antagonized by ivermectin (IVM), in part; via action
on P2X4Rs. Behaviorally, male mice deficient in p2rx4 gene [P2X4R
knockout (KO)] have been shown to exhibit a transient increase in ethanol
intake over a period of 4 days as demonstrated by social and binge
drinking paradigms. Furthermore, IVM reduced ethanol consumption in
male and female rodents, whereas, male P2X4R KO mice were less
sensitive to anti-alcohol effects of IVM compared to wildtype (WT), further
supporting a role for P2X4Rs as targets of IVM’s action. The current
investigation extends testing the hypothesis that P2X4Rs play a role in
regulation of ethanol intake. First, we tested the response of P2X4R KO
mice to ethanol for a period of 5 weeks. Second, to gain insights into the
changes in ethanol intake, we employed a lentivirus-shRNA (LV-shRNA)
69

methodology to selectively knockdown P2X4R expression in the nucleus
accumbens (NAc) core in male C57BL/6J mice. In agreement with our
previous study, male P2X4R KO mice exhibited higher ethanol intake than
WT mice. Additionally, reduced expression of P2X4Rs in NAc core
significantly increased ethanol intake and preference. Collectively, the
findings support the hypothesis that P2X4Rs play a role in regulation of
ethanol intake and that P2X4Rs represent a novel drug target for
treatment of alcohol use disorder.
3.2) INTRODUCTION:-
P2X receptors (P2XRs) are becoming a focus of investigation in
neuroscience and ethanol studies (Litten et al. 2012, Burnstock 2008,
Asatryan et al. 2011, Gum et al. 2012, Franklin et al. 2014, Xu et al. 2016).
P2XRs are fast acting cation-permeable ion channels that are gated by
synaptically released extracellular adenosine 5’-triphosphate (ATP) (Chizh
& Illes 2001, Khakh 2001, North 2002). In the CNS, ATP directly mediates
fast excitatory synaptic transmission by acting on P2XRs located on
postsynaptic membranes. In addition, ATP can produce neuromodulator
responses by promoting neurotransmitter release of other major ionotropic
targets (e.g., GABA and glutamate), known to play important roles in
ethanol drinking and other behaviors by acting on P2XRs located on pre-
70

and postsynaptic membranes (Khakh 2001, Jo & Schlichter 1999, Hugel &
Schlichter 2002, Baxter et al. 2011, Xu et al. 2016).
P2X4Rs are the most abundantly expressed P2XR subtype in the CNS
ranging from neurons to microglia (Buell et al. 1996, Soto et al. 1996a)
and are the most sensitive P2XR subtype to ethanol. In vitro studies
report that ethanol concentrations starting at approximately 5 mM
modulate ATP-activated currents in neurons (Li et al. 1994, Li et al. 1998a,
Li et al. 1993, Weight et al. 1999, Xiao et al. 2008) and recombinant
models (Xiong et al. 2000, Xiong et al. 2001, Davies et al. 2002, Davies et
al. 2005, Asatryan et al. 2008, Asatryan et al. 2010). 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 identified as neural substrates of
ethanol [e.g., hippocampus, cerebellum, ventral tegmental area (VTA),
nucleus accumbens (NAc), hypothalamic nuclei including paraventricular
nucleus (PVN) and arcuate nucleus (Arc)] (McCool 2011, Pankratov et al.
2009, Sim et al. 2006, Gonzales et al. 2004, McClintick et al. 2016).
Recent studies implicate P2X4Rs in the regulation of multiple CNS
functions including neuropathic pain (Tsuda et al. 2000, 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
71

addition, P2X4Rs have been recently shown to modulate the function of
other major ionotropic targets, such as GABA
A
Rs (Jo et al. 2011) and
NMDA glutamate (Baxter et al. 2011) receptors. Many of these
physiological and behavioral functions linked to P2XRs are known to be
affected by ethanol.
Building evidence links P2X4Rs to ethanol consumption including
investigations using microarray techniques, which found an inverse
relationship between p2rx4 gene expression and innate rodent intake and
preference for ethanol (Kimpel et al. 2007, Tabakoff et al. 2009). In
agreement with this hypothesis, we recently demonstrated that male P2X4
knockout (KO) mice (i.e., p2rx4 deleted) consumed significantly more
ethanol than wildtype (WT) controls (Wyatt et al. 2014). The present paper
extends the investigation of the role of P2X4Rs in ethanol intake and
addresses two unresolved questions from the recent Wyatt et al paper.
First, we significantly increased the length of time of the ethanol
investigation to gain insights regarding the transient nature of the
increased drinking reported by Wyatt and colleagues (Wyatt et al. 2014).
This was accomplished by testing male P2X4KO mice and WT littermates
for changes in ethanol intake and preference for 5 weeks using a 24 hr
access two-bottle choice paradigm. Second, in that the increase in ethanol
intake previously measured in male P2X4KO mice could partially reflect
compensatory developmental changes, we also utilized a lentiviral-
72

mediated shRNA knockdown strategy (LV-shRNA) to knockdown P2X4R
expression in the NAc core and measured changes in ethanol intake and
preference.
3.3) MATERIALS AND METHODS:-
Animals:-
We used experimentally naïve 2-3 month old male WT and P2X4R KO
mice from our breeding colony at the University of Southern California.
The generation of P2X4R KO mice and the breeding scheme has been
described previously (Sim et al. 2006, Wyatt et al. 2013). For the LV-
shRNA experiments, 2 month old male C57BL/6 mice were obtained from
Jackson laboratories (Bar Harbor, ME). Mice were group housed (i.e. 5
per cage) in the vivarium maintained at 22
0
C and a 12 hr/12 hr light: dark
cycle with free access to food and water. All procedures are carried out in
compliance with the guidelines of National Institute of Health and
approved by the Institutional Animal Care and Use Committee of
University of Southern California.
Drugs:-
The ethanol solution was prepared as a 10% v/v ethanol solution in tap
water from 190 proof USP grade ethanol solution (Koptec, King of Prussia,
PA).
73

Short hairpin RNA (shRNA) constructs and LV production:-
cDNA encoding two shRNA sequences targeting different regions of
P2X4 mRNA (S1 and S2) were subcloned into the Clontech biscistronic
pLVX-shRNA2 vector where the shRNA expression was driven by human
U6 promoter, located just upstream of the MCS (Mountain View, CA). The
vector also expressed ZsGreen1 reporter, a human codon optimized
variant of the coral reef green fluorescent protein (GFP), under CMV
promoter control. The shRNA sequences were 5’-
CCACAAATACTCAGGGTTG-3’ and 5’-CTCAGATGGGCTTCAGATA-3’. We
have observed that the simultaneous use of both the sequences resulted
in higher extent inhibition of P2X4R expression. LV was produced by
mixing both shRNA constructs with psPAX2 and pMD2.G packaging
vectors obtained from Addgene (Cambridge, MA) and transfected HEK
293T cells. Virus-containing supernatant was collected, concentrated and
resultant viral titers were determined via the ELISA method. Concentrator
and titration kits were obtained from Clontech Laboratories (Mountain
View, CA).
Stereotaxic surgery and microinjection procedure:-
Mice were anesthetized with a ketamine/xylazine cocktail and 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
74

were measured to ensure an even plane and a small area of dura
removed in the area for microinjection. A ten-microliter syringe (Hamilton,
Reno, NV) was used to deliver 1µL of LV (4.1 x 10
7
to 5.3 x 10
9
IU/mL) to
each NAc (bregma coordinates: anteriorposterior 1.2 mm; mediolateral 1.0
mm; dorsoventral 4.5 mm) at a rate of 0.1 µL/min. After infusion, the
syringe was left in place for a further 5 min. Mice were then allowed to
recover from surgery in their home cages which were placed on heating
pads for 2 days, then transported to the vivarium. Mice had ad libitum
access to food and water during their resting period and were allowed to
acclimate to the vivarium conditions for 1 week prior to start of the ethanol
drinking experiments.
Anatomical verification of LV microinfusion into the NAc core by
fluorescence microscopy:-
Following the stereotaxic surgery and microinjection of LV, transcardial
perfusion was performed on mice using 0.9% NaCl followed by 4%
phosphate buffered paraformaldehyde. Brains were post fixed in 4%
phosphate buffered paraformaldehyde overnight followed by storage in
20% sucrose for 48 hr and frozen in 4-Methylbutane on dry ice. Striatal
sections were cut coronally at 25 µm thickness in a cryostat and later
stored in a cryoprotective solution containing 30% sucrose in PBS at 4
0
C
until further use. The striatal sections were then examined for GFP
75

immunofluorescence under a fluorescent microscope (Olympus BX61
microscope, Shinjuku, Tokyo, Japan).
Verification of LV-shRNA mediated knockdown of P2X4Rs in vitro
and in vivo using Western immunoblotting:-
BV-2 transduction: Knockdown of P2X4Rs by LV-shRNA strategy in vitro
was verified through transduction of mouse microglial BV-2 cells, which
have a high endogenous P2X4R expression. BV-2 cells were cultured in
6-well plates in DMEM/ F12 medium supplemented with
penicillin/streptomycin and fetal bovine serum until they reached
approximately 80% confluence. Cells were transduced with 10
6
infectious
units/ml of shRNA-based LV. Confirmation of virus expression was
visualized by GFP fluorescence after 48 hr.
Microinjection into NAc core: P2X4R knockdown in mouse nucleus
accumbens core by LV-shRNA methodology was verified at 14 days post
infusion of LV-shRNA. 2 and 3 mice were stereotactically injected with the
LV alone and LV-shRNA-p2rx4 respectively. A separate cohort of mice
that have never undergone surgery (will be described as naïve mice) was
used as a positive control for this study. After surgery, mice were allowed
to rest for period of 1 week. At the end of the recovery period, the mice
remained in their cages for period of 14 days during which they had ad
libitum access to food and water. At their respective time points, mice
76

were euthanized using CO
2
asphyxiation and striatum was dissected out
as per landmarks described in the mouse brain atlas (Franklin & Paxinos
2007).
BV-cell lysate or striatal tissue homogenate preparation: Cells or striatal
tissues were treated with lysis buffer containing 50mM tris-HCl pH (7.4),
150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton-X-100, 0.1% SDS,
1% proteinase inhibitor cocktail (Millipore, Temecula,CA). BV-2 cell
lysates were spun at 13,000 rpm for 10 min at 4
0
C and the protein-
containing supernatant was collected. Protein content for BV-2 cells and
ventral striatum was measured by using BCA protein assay (Thermo
Scientific, Rockford, IL).
Immunoblotting procedure: Striatal homogenates or cell lysates (
respectively of 50 µg and 10 µg per lane) ran on 10% SDS PAGE gels
and transferred onto polyvinylidine fluoride membranes using a semi-dry
transfer method (Trans turbo blot, BioRad, Hercules, CA). Non-specific
binding was blocked using 5% non fat dry milk (BioRad, Hercules, CA)
followed by incubation with rabbit anti-P2X4 receptor antibody (Alomone
Labs, Jerusalem, Israel) overnight at 4
0
C. Membranes were then
incubated with goat anti-rabbit secondary antibody for 1 hr at room
temperature. In between incubation steps for antibodies and blocking,
membranes were washed 3 times, 5 min each time, with TBS containing
77

0.05% Tween-20. After secondary antibody incubation, membranes were
incubated with ECL substrate, (BioRad, Hercules, CA) and bands were
visualized using chemilumenescent method (ChemiDoc system, BioRad,
Hercules, CA).
24 hr access two-bottle choice paradigm:-
The 24 hr access two bottle choice paradigm is a model that mimics social
drinking and is used to investigate differences in ethanol intake in
genetically modified animals or upon pharmacological treatment
(Yoneyama et al. 2008, Middaugh & Kelley 1999, Belknap et al. 1993). We
used the procedure previously described (Wyatt et al. 2014, Yardley et al.
2012, Asatryan et al. 2014). Briefly, mice had 24 hr access to 2 inverted
graduated tubes (25 mL) with metal sippers positioned on stainless steel
cage tops. Food was evenly distributed on the cage tops to avoid
association with either of the tubes. Post acclimation, mice had access to
tubes containing water only for the first week. In the second week, one of
the tubes contained water and the other with 10% ethanol solution (10E).
10E and water intake was recorded by measuring the lower meniscus. It
was ensured that positions of the tubes were switched every alternate day
to avoid side preferences. Mice were given fresh solution of 10E and
water once a week. Body weights were measured and used to calculate
the g/kg/24hr ethanol intake. Percent ethanol preference was determined
78

by multiplying the ratio of volume of 10E intake (mL) over total fluid intake
(10E + water) by 100.
For the LV-shRNA-p2rx4 experiment, mice underwent a baseline drinking
sessions post acclimation during which their voluntary consumption of
ethanol, water, total fluid intake and ethanol preference was measured.
Upon stable baseline drinking, mice were randomly assigned to one of the
three treatment options: 1) LV-shRNA-p2rx4, LV alone or naïve mice (i.e.
mice that have never undergone the surgery). ANOVA was used to ensure
that the ethanol intake, preference, water or total fluid intake did not
significantly differ between the three treatment groups. Post surgery, the
mice were transported back to the vivarium and allowed to rest for 1 week
prior to start of ethanol drinking studies.
Statistical Analyses:-
Repeated measures two way ANOVA (genotype x week) was used to
investigate differences in 10E intake, 10E preference, water and total fluid
intake between WT and P2X4R KO mice, followed by Bonferroni post hoc
test for multiple comparisons. Two way ANOVA followed by Bonferroni
post hoc test was used to compare drinking parameters between different
treatment groups for the LV-shRNA-p2rx4 drinking studies. One way
ANOVA with Tukey’s post hoc test was also used to compare the
efficiency of LV-shRNA-p2rx4 transfusion and transfection on P2X4R
79

knockdown in mouse striatum and BV-2 cells. Significance was set at
P<0.05. All data was analyzed using Graph Pad software (Prism, San
Diego, CA).
3.4) RESULTS:-
3.4.1) P2X4R KO mice exhibited increased voluntary ethanol
consumption in the 24 hour access drinking paradigm:-
We tested the effects of global knockout of p2rx4 gene on ethanol intake
using a 24 hr two bottle choice paradigm (10E versus water). As illustrated
in Fig 3.1A, there was a significant effect of genotype [F(1,16)=4.88,
p<0.05], but not week or genotype x week interaction for 10E intake.
There was no significant effect of genotype, week or genotype x week
interaction for 10E preference or water intake between WT and P2X4R
KO mice (Fig 3.1 B & C). There was a non-significant trend towards effect
of genotype [F(1,16)=3.28, p=0.0891] but not week or genotype x week
interaction for total fluid intake. (Fig 3.1D). Considering that there were
changes in 10E intake, but not preference, we evaluated the effect of
genotype and week on body weight. There was a significant effect of
genotype [F(1,16)=4.68, p<0.05] and week [F(4,64)=59.08, p<0.001]
without any significant interaction between the two factors on body weight
between WT and P2X4R KO mice.
80

FIGURE 3.1

Figure 3.1: P2X4R KO mice exhibited significantly higher 10E intake compared to WTcontrols (A)
and tended to have higher total fluid intake (D) without any significant changes in 10E preference
(B) or water intake (C). For each week, the 10E intake, preference and water intake was measured
as an average of 5 days. Values represent mean ± SEM for a duration of 5 days each week for 8
WT and 10 P2X4R KO mice. * P <0.05 versus WT mice, two way-ANOVA.

Week
10E preference (%)
1 2 3 4 5
0
20
40
60
80
100
WT
P2X4R KO
B.
Week
Water Intake (mL)
1 2 3 4 5
0
1
2
3
C.
Week
Total Fluid Intake (mL)
1 2 3 4 5
0
2
4
6
8
D.
A.
Week
10E intake (g/kg/24hr)
1 2 3 4 5
0
5
10
15
*
81

3.4.2) Transfection of BV-2 cells or transfusion in mouse striatum
with LV-shRNA-p2rx4 reduced P2X4R expression:-
We first investigated the efficiency of knockdown of P2X4Rs using a LV-
shRNA strategy in BV-2 cells. As depicted in Fig 3.2A, LV-shRNA
treatment significantly reduced P2X4R expression [F(2,4)=27.88, p<0.01]
in BV-2 cells. Tukey’s post hoc test confirmed that LV-shRNA treatment
significantly reduced P2X4R expression as compared to untreated cells
(q=9.875, p<0.01) and cells treated with LV alone (q=7.377, p<0.05).
We next tested the efficiency of LV-shRNA-p2rx4 infusion on P2X4R
knockdown in the mouse striatum. As illustrated in Fig 3.2C, LV-shRNA
reduced P2X4R expression in the striatum by 45% as compared to naive
mice and 35% as compared to mice that only received the LV infusion
after a period of 14 days. There were no significant differences between
these three groups for this time period.

82

FIGURE 3.2

Figure 3.2: BV-2 cells transinfected with LV-shRNA-p2rx4 reduced P2X4R expression by
68% and 62% as compared to non-treated cells (NT) and LV alone treated cells
respectively (A). Microinfusion of LV into the NAc core was verified by detecting
ZsGreen1 immunofluorescence (B). Stereotaxic injection of LV-shRNA-p2rx4 in NAc core
reduced P2X4R expression by 44% and 39% as compared to naïve mice and LV alone
infused mice respectively after 14 days (C) . Values represent mean ± SEM for 2-3 mice
per treatment group for (C), **P<0.01 v/s non-treated cells,# P<0.05 v/s LV alone treated
cells, Tukey’s post hoc test.

NT LV LV-shRNA-p2rx4
P2X4
-actin
Normalized density (AU)
NT LV LV-shRNA-p2rx4
0.0
0.5
1.0
1.5
**
# **
#
A.
C.
P2X4
-actin
Naïve
LV LV-shRNA-p2rx4
Normalized density (AU)
Naïve LV LV-shRNA-p2rx4
0.0
0.5
1.0
1.5
14 days post infusion
83

3.4.3) LV-shRNA-p2rx4 infused mice exhibited higher ethanol intake
and preference in comparison to naïve mice:-
There was a significant effect of treatment [F(1,25)=6.67, p<0.05],but not
week or week x treatment interaction for 10E intake in mice that received
LV-shRNA-p2rx4 infusion as compared to naïve mice (Fig 3.3A). In
addition, mice receiving LV- shRNA-p2rx4 treatment exhibited significantly
higher 10E preference [F(1,25)=12.15, p<0.01] without any significant
effects of week or week x treatment interaction (Fig 3.3B). On account of
higher preference for 10E, LV-shRNA-p2rx4 group exhibited significantly
lower water intake [F(1,25)=10.93, p<0.01], without any significant effects
of week or week x treatment interaction (Fig 3.3C). There was a significant
effect of week [F(4,100)=9.12, p<0.001] without any significant effects of
treatment or week x treatment interaction for total fluid intake between
two groups (Fig 3.3D). In contrast to P2X4R KO mice, the LV-shRNA-
p2rx4 infused mice did not weigh significantly more than the naïve mice,
although there was significant effect of week on body weight
[F(4,100)=9.47, p<0.001] without any significant week x treatment
interaction.

84

3.4.4) The LV-shRNA-p2rx4 infused mice exhibited a higher ethanol
intake as compared to mice that only received LV infusion:-
There was a significant effect of week [F(4,80)=3.87, p<0.01] and
treatment [F(1,20)=6.08, p<0.05] on 10E intake in the LV-shRNA-p2rx4
group as compared to the group that received LV infusion , without any
significant week x treatment interaction (Fig 3.3A). There was a significant
effect of week [F(4,80)=3.96, p<0.01] but not treatment or week x
treatment interaction for 10E preference between the two groups (Fig
3.3B). Similarly, there was a significant effect of week [F(4,80)=5.57,
p<0.001] but not treatment or week x treatment interaction for water intake
(Fig 3.3C). There was no significant effect of week, treatment or week x
treatment interaction for total fluid intake (Fig 3.3D). There was a
significant effect of week [F(4,80)=16.60, p<0.001] and a significant week
x treatment interaction [F(4,80)=3.39, p<0.05] for body weight between the
two groups. The LV-shRNA-p2x4 infused mice did not weigh significantly
more than the LV alone infused mice.

85

FIGURE 3.3

Figure 3.3: The LV-shRNA-p2rx4 group exhibited significantly higher 10E intake as compared to
naïve mice and mice infused with LV alone (A). The 10E preference and water intake of LV-
shRNA-p2rx4 group was significantly higher and lower respectively than that of naive mice, but not
mice infused with LV alone (B & C). No changes in total fluid intake between the groups (D). Values
represent mean ± SEM for a duration of 5 days for each week for 13 naïve mice, 10 mice infused
with LV alone and 14 mice infused with LV-shRNA-p2rx4. *P<0.05, **P < 0.01 versus naïve mice,
# P<0.05 versus LV alone infused mice, Bonferroni post hoc test

Naïve mice
LV
LV-shRNA-p2rx4
A.
Week
10E intake (g/kg/24h)
1 2 3 4 5
0
5
10
15
*
#
B.
Week
10E preference (%)
1 2 3 4 5
0
20
40
60
80
100
**
C.
Week
Water Intake (mL)
1 2 3 4 5
0
1
2
3
**
D.
Week
Total Fluid Intake (mL)
1 2 3 4 5
0
2
4
6
8
86

3.4.5) Infusion of LV alone did not have any significant effect on
ethanol intake or preference in comparison to the naïve mice:-
There was non-significant trend towards effect of week [F(4,76)=2.35,
p=0.0618] without any significant effect of treatment or week x treatment
interaction for 10E intake in mice infused with LV alone relative to naïve
mice (Fig 3.3A). Similarly, in the context of 10E preference, there was a
non-significant trend towards effect of week [F(4,76)=2.03, p=0.0982]
without any significant effect of treatment or week x treatment interaction
(Fig 3.3B). There was a significant effect of week [F(4,76)=2.59, p<0.05]
but not treatment and the week x treatment interaction trended towards
significance [F(4,76)=2.19, p=0.0782] for water intake between the two
groups (Fig 3.3C). There was a non-significant trend towards effect of
week [F(4,76)=2.21, p=0.0753] and treatment [F(1,19)=3.86, p=0.0642] on
total fluid intake between the two groups of mice. However, there was a
significant week x treatment interaction [F(4,76)=2.75, p<0.05] with
Bonferroni post hoc test indicating reduced total fluid intake in mice
receiving LV infusion relative to naïve mice at week 5 (t=3.329, p<0.01)
(Fig 3.3D). Finally, there was a non-significant trend towards effect of
week on body weight [F(4,76)=2.49, p=0.0503] without any significant
effect of treatment or week x treatment interaction between LV alone
infused mice and naïve mice.
87

3.5) DISCUSSION:-
The current study investigated the role of P2X4Rs in regulation of ethanol
drinking behavior. Overall, the findings support the hypothesis that
P2X4Rs play an important role in the regulation of ethanol intake by
demonstrating that reduced P2X4R expression results in changes in
ethanol drinking behavior. Using a global knockout strategy, we
demonstrated that P2X4R KO mice exhibited significantly increased
ethanol intake. The increased body weights of P2X4R KO mice may
partially contribute to their increased ethanol intake as suggested by lack
of significant change in ethanol preference. On the other hand, there was
no significant change in water intake suggesting that p2rx4 deficiency
affects mechanism of ethanol drinking, without perturbing the physiolgy of
drinking per se. These results are in good agreement with our previous
study wherein we showed that male P2X4R KO mice exhibited higher
ethanol intake over a period of 4 days without changes in ethanol
preference or water intake as compared to their WT littermates (Wyatt et
al. 2014).
Taking into consideration the complex compensatory changes that occur
in a knockout mouse model, the constitutive deficiency of P2X4Rs does
not necessarily represent the full pharmacological blockade of the
receptor. At present, we do not have any selective P2X4R antagonists
88

that can be used to provide a direct link between P2X4R antagonism and
increased ethanol consumption. As a complementary strategy, we
employed a LV-shRNA methodology to address this issue. In the present
investigation, we targeted the NAc core as the site for the LV-shRNA
injection since this is a critical site of the dopamine (DA) mesolimbic
circuitry for various drugs of abuse including ethanol to induce their
reinforcing and rewarding effects (Di Chiara & Imperato 1988, Gonzales et
al. 2004). Moreover, P2X4Rs are expressed in the striatum (Amadio et al.
2007) and endogenous ATP (possibly via activation of P2XRs) has been
implicated in modulation of DA neurotransmission in various regions of the
mesolimbic circuitry including the VTA and NAc (Xiao et al. 2008, Krugel
et al. 2001b). In agreement with our previous and current findings from the
male P2X4R KO study, we found that mice with reduced P2X4R
expression ( via LV-sh-RNA-p2rx4 infusion) exhibited greater ethanol
consumption relative to naïve mice and mice infused with LV alone. There
were no significant changes in ethanol intake upon infusion of LV alone in
relation to naïve mice indicating that the increased ethanol intake in mice
with reduced P2X4R expression is due to shRNA mediated knockdown of
P2X4Rs and not infusion of the virus alone. Moreover, findings from LV-
shRNA studies suggest that the increased ethanol intake observed in
male P2X4R KO mice was a functional consequence upon deletion of
p2rx4 gene and not due to compensatory changes in other receptors such
89

as NMDARs, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptors (AMPARs) or DA receptors, all of which have been previously
reported to be altered in expression levels in different brain regions of
P2X4R KO mice (Khoja et al. 2016). Additionally, shRNA mediated
knockdown of P2X4Rs significantly increased ethanol preference relative
to naïve mice but not mice infused with LV alone. The increased ethanol
preference in LV-shRNA-p2rx4 infused mice may account for the
increased ethanol intake relative to naïve mice since there were no
significant differences in body weights between these group. Unlike in the
LV-shRNA methodology where there is knockdown of a particular gene at
the adult stage, the P2X4R KO mice may exhibit neurodevelopmental
adaptations to compensate for constitutive deficiency of p2rx4 gene, and
such adaptations could nullify the effect of p2rx4 knockout on ethanol
preference. Moreover, the p2rx4 knockdown is in the NAc core which is a
key brain region for expression of ethanol reinforcement and has an
important role in acquisition and maintenance of ethanol seeking behavior
. Thus, possible neurodevelopmental changes in this brain region of
P2X4R KO mice that could significantly interfere with motivational
behavior towards seeking ethanol. The somewhat differing data with
respect to ethanol preference from P2X4R KO and LV-shRNA-p2rx4
infused mice suggests the need for additional investigations using
operant chamber technique to monitor self-administration or conditioned
90

place preference in P2X4R KO mice or LV-shRNA-p2rx4 infused mice to
better understand the role of P2X4Rs in ethanol seeking behavior.
The findings from LV-shRNA-p2rx4 and P2X4R KO experiments are in
agreement with mutiple studies that have reported an inverse correlation
between P2X4R expression and ethanol consumption. For example,
Kimpel and colleagues compared gene expression in brain areas
associated with reward in inbred alcohol preferring (Ip) v/s non-preferring
(Inp) rat lines and found that functional p2rx4 expression was significantly
reduced in Ip rats (Kimpel et al. 2007). Similarly, Tabakoff and colleagues
found lower levels (i.e., inverse relationship) 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 phenotye (Tabakoff et al.
2009). On the other hand, McBride and colleagues reported that p2rx4
gene expression was significantly increased in high alcohol drinking
female (HAD2) rats, relative to their low-alcohol drinking (LAD2)
counterparts (McBride et al. 2012). In addition, increased expression of
P2X4Rs was detected in the periaqueductal gray (PAG), a region
associated with fear and anxiety (Behbehani 1995), in adolescent male P
rats (McClintick et al. 2016). To further validate the correlation of
increased P2X4R expression with increased ethanol intake, LV-shRNA
mediated knockdown of P2X4R expression in the posterior VTA was
recently shown to significantly decrease ethanol intake in female HAD2
91

rats (Franklin et al. 2015). Although there are differences regarding the
direction of change in drinking behavior in these reports , a common
theme emerging from these investigations is that manipulation of p2rx4
expression is associated with significant changes in ethanol consumption.
Multiple investigations from our laboratory as well as others have
supported the hypothesis that inhibition of P2X4R activity increases
ethanol drinking behavior and potentiation of P2X4Rs reduces ethanol
intake as well as the propensity to seek ethanol Based on recent work,
we suggested that ethanol acts as an open channel blocker of P2X4Rs
(Ostrovskaya et al. 2011, Popova et al. 2010) and that positive modulation
of P2X4Rs by ivermectin (IVM) antagonized ethanol induced inhibition of
P2X4Rs (Asatryan et al. 2010). Positive modulation of P2X4Rs by IVM
can reduce ethanol drinking behavior as illustrated in previous reports
showing that IVM administration in C57BL/6J mice reduced ethanol intake
and preference using variety of paradigms that mimic social drinking and
motivation to seek ethanol (Asatryan et al. 2014, Wyatt et al. 2014,
Yardley et al. 2012).The anti-alcohol effects of IVM can be, in part, linked
to P2X4R activity in that the degree of reduction of ethanol intake was
decreased in male P2X4R KO mice (Wyatt et al. 2014). Similar findngs
were obtained from Franklin and colleagues, where IVM significantly
reduced ethanol intake in male and female HAD-1 and HAD-2 rats.
Furthermore, intracerebroventricular (ICV) administration of IVM
92

significantly reduced ethanol intake in female HAD2 rats (Franklin et al.
2015). The physiological significance of ethanol induced inhibition of
P2X4Rs within the mesolimbic circuitry is unknown and currently under
investigation in our laboratory. However, based on previous reports, it is
thought that ethanol induced inhibition of P2XRs on the GABA releasing
terminals in the VTA is linked to disinhibition of VTA DA neurons as
suggested previously (Xiao et al. 2008). Therefore, the study by Xiao and
colleagues provides indirect evidence for P2X4Rs’s role in modulation of
firing of DA neurons in VTA. One possible mechanism for P2X4Rs in
modulation of firing of DA neurons is via its localization in the Arc region of
the hypothalamus. P2X4Rs have been reported to be involved in
regulating presynaptic GABA release onto proopiomelanocortin (POMC)
neurons in the Arc region (Xu et al. 2016) and GABAergic activity in this
region has been associated with an inhibitory influence on the VTA DA
neurons (Tabakoff et al. 2009). However, additional electrophysiological
studies would be needed to test this hypothesis before definitive
conclusions can be drawn. Overall, we do not have sufficient data to
conclusively state that there is a direct link between P2X4R
potentiation/inhibition and firing of DA neurons in the VTA. This is an area
of work that is ongoing. Moreover, elucidating the GABAergic tone or firing
of VTA DA neurons in P2X4R KO mice or mice that received LV-shRNA-
93

p2rx4 infusion would also be important to help provide novel insights into
the mechanism leading to increased ethanol intake in these mice.
In conclusion, the results from our previous work using P2X4R KO mouse
model coupled with current LV-shRNA studies which corroborate
findings from others, supports r the hypothesis that P2X4Rs play an
important role in regulation of ethanol intake. Moreover, the results
support the discovery and development of P2X4R allosteric modulators
as novel therapeutic agents for treatment of AUD.

94

CHAPTER 4
ROLE OF DOPAMINE RECEPTORS IN IVERMECTIN INDUCED
PREPULSE INHIBITION DEFICITS IN C57BL/6J MICE
4.1) ABSTRACT:-
Purinergic P2X4 receptors (P2X4Rs) are cation –permeable channels
gated by adenosine 5’-triphosphate (ATP). Previous investigations have
explored the role of P2X4Rs in regulation of sensorimotor gating.
Ivermectin (IVM), a positive modulator of P2X4Rs, induces disruptions in
prepulse inhibition (PPI) of acoustic startle reflex, which is an operational
measure for sensorimotor gating. The PPI disruptive effects of IVM were
significantly blunted in mice deficient in p2rx4 gene [i.e. P2X4R knockout
(KO)], suggesting that IVM, in part, is mediating its effects through P2X4R
modulation. Moreover, P2X4R KO mice exhibit PPI deficits that were
rescued by dopamine (DA) receptor antagonists, SCH 23390 (for DA D1
receptors) and raclopride (for DA D2 receptors), demonstrating that p2rx4
gene deficiency induces abnormalities in DA neurotransmission, leading to
PPI deficits. Based on potential interactions between P2X4Rs and DA
receptors in PPI regulation using P2X4R KO mouse model, we
hypothesized that the PPI disruptive effects of IVM are dependent upon
this interaction. To test this hypothesis, we investigated the effects of IVM
95

on PPI in presence of DA receptor antagonists, SCH 23390, raclopride
and DA D1 receptor agonist, SKF 82958 in mediation of PPI function.
SCH 23390, but not raclopride, significantly attenuated IVM –induced PPI
dysfunction, indicating a role for DA D1 receptors in IVM’s mechanism of
action. Surprisingly, there was no synergistic interaction between SKF
82958 and IVM in PPI regulation. Since, SCH 23390 has pharmacological
targets in serotonin 5-HT system (it is a 5HT
2C
agonist), future studies
warrant the use of anti-psychotics with targets in DA and 5-HT system in
elucidating IVM’s mechanism of action. Overall, these behavioral studies
would identify the role of P2X4Rs in PPI regulation, which is a critical
endophenotype for multiple psychiatric disorders.
4.2) INTRODUCTION:-
Purinergic P2X4 receptors (P2X4Rs) belong to the P2X superfamily of
cation permeable ligand gated ion channels (LGICs) gated by extracellular
adenosine-5-triphosphate (ATP) (North 2002, Khakh & North 2012, North
& Verkhratsky 2006). Until date, seven subtypes of P2XRs have been
identified, out of which P2X4Rs are abundantly expressed on different
types of cells including neurons and glial cells (astrocytes and microglial)
in the central nervous system (CNS) (Khakh & North 2012, Abbracchio et
al. 2009). P2X4Rs have been directly or indirectly reported to be involved
in presynaptic neurotransmitter release such as glutamate , GABA (Jo &
96

Schlichter 1999, Hugel & Schlichter 2002), dopamine (DA); modulation of
synaptic currents mediated by postsynaptic receptors including glutamate
N-methyl-D-aspartate receptors (NMDARs) (Baxter et al. 2011, Sim et al.
2006) and -aminobutyric acid receptors (GABARs) (Jo et al. 2011, Xu et
al. 2016) in brain regions linked to learning, memory, reward as well as
regulation of inflammatory signaling cascades in the spinal cord (Ulmann
et al. 2010, Ulmann et al. 2008). These physiological roles of P2X4Rs
have been linked to CNS functions including synaptic plasticity,
neuropathic pain and neuroendocrine functions. Pharmacological and
genetic evidence from our laboratory have suggested a role for P2X4Rs in
regulation of other CNS behaviors including sensorimotor gating, socio-
communication and drug reward (Wyatt et al. 2013, Wyatt et al. 2014,
Yardley et al. 2012, Bortolato et al. 2013).
Amongst these behaviors, we were interested in the role of P2X4Rs in
modulation of sensorimotor gating. Prepulse inhibition (PPI) of acoustic
startle reflex was used as an operational measure of sensorimotor gating
and is considered as a reliable index for this behavior. Ivermectin (IVM), a
positive modulator of P2X4Rs, disrupted PPI functioning in male
C57BL/6J mice and this effect was attenuated in mice deficient in the
p2rx4 gene [i.e. P2X4R knockout (KO) mice] (Bortolato et al. 2013)
suggesting that IVM is inducing deficits in PPI function via modulation of
P2X4Rs. Moreover, male P2X4R KO mice exhibited deficits in PPI
97

function in comparison to their WT littermates (Wyatt et al. 2013) . To
provide a molecular basis for this behavioral deficit, we investigated the
homeostasis of striatal DA neurotransmission in P2X4R KO mice, since
DA plays a major role in regulation of PPI. We found increased expression
of dopamine D1 and D2 receptors in the ventral striatum of P2X4R KO
mice (Khoja et al. 2016). Since the ventral striatum is part of the cortico-
pallido-thalamic circuitry involved in PPI mediation (Swerdlow et al. 2008,
Swerdlow et al. 2011), the altered expression of DA receptors in this brain
region may partially underlie the PPI deficits in P2X4R KO mice. In
agreement with our explanation, previous investigations using knockout
mouse models have cited a role for increased expression of either of DA
receptors in PPI deficits (Lipina et al. 2010, Wolinsky et al. 2007). To
further strengthen our argument, treatment with dopamine D1 receptor
antagonist, SCH 23390 or dopamine D2 receptor antagonist, raclopride,
significantly ameliorated the PPI deficits in P2X4R KO mice, providing a
direct link between inherent perturbations in DA homeostasis and PPI
dysfunction in P2X4R KO mice (Khoja et al. 2016).
On the basis of our findings that DA dysregulation does have a role to pay
in PPI deficits in P2X4R KO mice, we hypothesized that IVM is causing
PPI disruption via activation of DA neurotransmission. Our basis for this
hypothesis stems from previous findings where IVM has been shown to
modulate DA dependent behaviors and signaling pathways. For instance,
98

IVM enhanced levodopa (L-DOPA) induced motor behavior in the 6-
Hydroxydopamine (6-OHDA) model of DA depletion via P2X4R
potentiation, supporting a role for IVM in modulation of motor control
(Khoja et al. 2016). In addition, IVM significantly modulated
phosphorylation of dopamine and cyclic AMP regulated phosphoprotein of
32kDa (DARPP-32) in the dorsal and ventral parts of the striatum via
P2X4Rs, which is a critical downstream target regulated by DA receptor
agonists and antagonists (Khoja et al. 2016). Notably, the ability of IVM to
reduce PPI function in C57BL/6J mice is reminiscent of non-selective DA
agonists including amphetamine, methamphetamine cocaine,
apomorphine, all of which have been shown to disrupt PPI function in
rodents (Doherty et al. 2008, Ralph-Williams et al. 2002, Ralph-Williams et
al. 2003).
To investigate this hypothesis, we tested the effects of IVM (10 mg/kg, i.p.)
on PPI function in the presence of DA receptor antagonists, SCH 23390
and raclopride. Considering the critical role for D1 receptors in regulation
of PPI function in C57BL/6J mice, we also tested the effects of IVM (5
mg/kg, i.p.) in combination with a selective D1 receptor agonist, SKF
82958.

99

4.3) MATERIALS AND METHODS:-
Animals:-
Two separate cohorts of C57BL/6J mice were used for the behavioral and
immunoblotting studies with IVM and dopaminergic drugs. The first cohort
comprised of 37 male mice aged 3-5 month old from Jackson Laboratories
(Bar Harbor, ME). The second cohort comprised of 40 male mice aged
2.5-4 months old, out of which, 27 mice were purchased from Jackson
Laboratories and 13 mice were wildtype (WT) littermates from our P2X4R
KO breeding colony at University of Southern California (USC). The
method for establishing P2X4R KO breeding colony has been described
elsewhere. The first and second cohort was used for the behavioral
studies involving IVM and DA receptor antagonists. The second cohort
was used for behavioral studies involving IVM and selective D1 receptor
agonist. We did not find any statistical significant differences in acoustic
startle or PPI% between C57BL/6J mice from Jackson Labs and our
breeding colony during the baseline studies. Mice from second cohort
were later sacrificed after the behavioral studies for Western
immunoblotting study with IVM and dopaminergic drugs. Mice were group
housed in cages of 5 in a vivarium maintained at 22
0
C and 12h/12h light:
dark cycle. All experiments were undertaken as per guidelines established
100

by National Institute of Health (NIH) and approved by Institutional Animal
Care and Use Committee (IACUC) at USC.
Materials:-
IVM (5 mg/kg, 10 mg/kg, i.p.) (Norbrook, Lenexa, KS) was diluted in 0.9%
saline at a concentration that would allow for injection volume of 0.01Ml/g
of body weight. Propylene glycol (Sigma –Aldrich, St. Louis, MO) was
used as vehicle control for IVM. SCH 23390 (1mg/kg, i.p.), raclopride (3
mg/kg, i.p.) and SKF 82958 (0.1 mg/kg, i.p.) (Sigma-Aldrich, St. Louis,
MO) were diluted in 0.9% saline to achieve concentrations of 0.2 mg/Ml,
0.6 mg/Ml and 0.02 mg/Ml respectively. All three drugs were injected at
volume of 0.005Ml/g of body weight. IVM or its vehicle was injected 8
hours prior to behavioral testing. The DA receptor antagonists and D1
agonist were injected 10 minutes prior to behavioral testing.
Acoustic Startle and PPI of acoustic startle reflex:-
Apparatus: Acoustic Startle reflex and PPI session were tested as
described previously (Wyatt et al. 2013, Khoja et al. 2016). The apparatus
used for detection of acoustic startle and PPI (San Diego Instruments, San
Diego, CA) consisted of a standard cage placed in sound attenuated
chambers with fan ventilation. Each cage consisted of a Plexiglass
cylinder mounted on a piezoelectric accelerometric platform connected to
an analog-digital converter. Background noises and acoustic bursts were
101

conveyed by two separate speakers, each one oriented so as to produce
a variation of 1Db across startle cage. Both speakers and startle cages
were connected to the main PC, which detected all the chamber variables.
Before baseline or testing session, the machine was calibrated using
digital sound level meter.
Startle and PPI session: In the baseline session, mice were exposed to
background noise of 70 Db and after an acclimation period of 5 minutes,
were presented with 12 ms of 40 trials of 115 Db interposed with 3 trials of
a 82 Db prestimulus preceding the 115 Db by 100 ms. Treatment groups
were established so that the acoustic startle magnitude and PPI % are
equivalent across all the groups. On the testing day, each mouse was
exposed to an acclimation period of 5 mins which comprised of
background sound 70 Db, which continued for remainder of session. Each
session consisted of 3 consecutive blocks of trials. During the first and
third block, the mice were exposed to five pulse alone trails of 115 Db. In
the second block, the mice were exposed to pseudorandom sequence of
50 trials, which consisted of 12 pulse alone trials, 30 trials of pulse
preceded by 73 Db, 76 Db or 82 Db (defined as PPI 3, PPI 6 and PPI 12
respectively; 10 for each level of prepulse loudness) and 8 no stimulus
trials, wherein there was only background sound without any prepulse or
pulse stimuli. Inter trial intervals were chosen between 10 and 15 seconds.
102

PPI% was calculated as 100-[(mean startle amplitude for pre-pulse pulse
trials / mean startle amplitude for pulse alone trials) x 100].
Statistical Analyses:-
The PPI% and acoustic startle data from cohorts 1 & 2 were combined to
investigate the effect of DA receptor antagonists on IVM-induced PPI
deficits. It was ensured that there were no statistical significant differences
in baseline acoustic startle magnitude or PPI% for any particular treatment
group between both the cohorts. Moreover, upon combining the cohorts,
there were no statistical significant differences in either of the baseline
parameters between the treatment groups. One way ANOVA followed by
Tukey’s post hoc test was used to analyze these parameters. For the
behavioral experiment involving IVM and SKF 82958, PPI % and acoustic
startle data from only the second cohort was used. Repeated measures
two way ANOVA using PPI intensity and drug treatment as repeated and
independent variables respectively was used to evaluate effects of
dopaminergic drugs on IVM-mediated PPI regulation, followed by
Bonferroni post hoc test for multiple comparisons. Non-repeated
measures two way ANOVA followed by Bonferroni post hoc test was used
to analyze interactions between IVM and dopaminergic drugs in regulation
of acoustic startle magnitude.
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4.4) RESULTS:-
4.4.1) Pharmacological blockade of dopamine D1, but not D2
receptors significantly attenuated IVM-mediated PPI disruption:-
There was a significant effect of IVM treatment [F(1,34)=20.38, p<0.001]
and prepulse intensity [(F2,68)=18.96, p<0.001] on PPI% relative to
control group (Fig 4.1). There was also a significant treatment x prepulse
intensity interaction [F(2,68)=4.00, p<0.05] with Bonferroni post hoc test
indicating that IVM significantly reduced PPI % at PPI 6 (t=2.881, p<0.05)
and PPI 12 (t=4.939, p<0.001). Lack of any significant effect of SCH
23390 alone on PPI function, indicates the inability of SCH 23390 to
modulate PPI independently (Fig 4.1). However, co-administration of IVM
and SCH 23390 significantly increased PPI% in comparison to mice
treated with IVM alone [F(1,32)=7.43, p<0.05] along with a significant
effect of prepulse intensity [F(2,64)=31.91, p<0.001] and a significant
treatment x prepulse intensity interaction [F(2,64)=7.23, p<0.01] (Fig 4.1).
Bonferroni post hoc test confirmed significant increase in PPI function
upon co-administration of IVM and SCH 23390 at PPI 6 (t=2.668, p<0.05)
and PPI 12 (t=3.862, p<0.001). The PPI function of mice treated with
combination of IVM and SCH 23390 was comparable to that of control
group [F(1,34)=3.32, p=0.0771], suggesting that SCH 23390 partly
negated the PPI-disruptive effects of IVM (Fig 4.1). In addition, there was
104

a significant effect of IVM [F(1,61)=10.86, p<0.01], but not SCH 23390, on
acoustic startle magnitude. There was no significant IVM x SCH 23390
treatment interaction for acoustic startle magnitude.
Similar to SCH 23390, there was no significant effect of raclopride alone
on PPI function. However, in contrast to SCH 23390, there were no
significant changes in PPI function upon co-administration of IVM and
raclopride in comparison to IVM group. On the other hand, raclopride was
unable to abolish the PPI disruptive effects of IVM, since the PPI % of
mice treated with both IVM and raclopride was significantly lower than that
of control group [F(1,26)=6.15, p<0.05]. In contrast to SCH 23390, there
was a significant effect of raclopride [F(1,50)=4.37, p<0.05] on acoustic
startle magnitude, without any significant IVM x raclopride treatment
interaction.

105

FIGURE 4.1

Figure 4.1: SCH-23390 (1 mg/kg), but not raclopride (3 mg/kg) significantly blocked IVM-mediated
prepulse inhibition deficits of acoustic startle reflex. Administration of SCH 23390 in combination
with IVM significantly increased PPI% in relation to that of mice treated with IVM alone and PPI% of
IVM + SCH 23390 group was comparable to that of control group. On the other hand, co-treatment
raclopride and IVM did not significantly change PPI% in relation to that of mice treated with IVM
alone. PPI% of mice treated with IVM and raclopride was still significantly lower than that of control
group. Values represent the mean PPI% ± SEM for each decibel level from 10-18 WT mice per
treatment group. *P <0.05, ***P <0.001 versus control group, # P<0.05 versus IVM treated mice,
Bonferroni post hoc test.

4.4.2) Dopamine D1 receptor activation did not potentiate the effects
of IVM on PPI function without inducing any alterations in acoustic
startle:-
Similar to a high dose of IVM (10 mg/kg), a lower dose of IVM treatment (5
mg/kg) did significantly disrupt PPI function [F(1,17)=4.47, p<0.05]
alongwith a significant effect of prepulse intensity [F(2,34)=22.60,
IVM
SCH 23390
raclopride
+
+
+
+
+ +
+ -
-
-
-
-
-
-
-
-
-
-
PPI 3
PPI 6
PPI 12
PPI %
0
10
20
30
40
50
#
***
*
106

p<0.001] in comparison to control group (Fig 4.2). There was a non-
significant trend towards effect of SKF 82958 (0.1 mg/kg) [F(1,15)=3.35,
p=0.0871] and a significant effect of prepulse intensity [F(2,30)=11.79,
p<0.001] on PPI function in comparison to vehicle-treated mice (Fig 4.2).
Similarly, there was a non-significant trend towards effect of co-treatment
of IVM + SKF 82958 [F(1,17)=3.62, p=0.0740] and a significant effect of
prepulse intensity [F(2,34)=8.48, p<0.01] on PPI% in comparison to the
control group (Fig 4.2). However, the co-administration of IVM and SKF
82958 did not significantly reduce PPI function in comparison to mice
treated with IVM or SKF 82958 alone (Fig 4.2). Similar to effects induced
by 10mg/kg dose, there was a significant effect of 5 mg/kg dose of IVM
[F(1,35)=10.76, p<0.01] on acoustic startle magnitude. In addition, there
was a significant effect of SKF 82958 treatment [F(1,35)=4.77, p<0.05] on
acoustic startle magnitude. There was no significant IVM x SKF 82958
treatment interaction for acoustic startle magnitude.

107

FIGURE 4.2

Figure 4.2: Co-administration of IVM (5 mg/kg) and SKF 82958 (0.1 mg/kg) did not significantly
decrease PPI% in comparison to treatment with either one of the drugs. IVM caused a significant
reduction in prepulse inhibition of acoustic startle reflex. Administration of SKF 82958 alone tended
to reduce PPI% in relation to that of control group. Similarly, co-treatment IVM and SKF 82958
showed a non-significant trend towards decreasing PPI% in relation to that of control group. Values
represent the mean PPI% ± SEM for each decibel level from 8-11 mice per treatment group. *P
<0.05 versus control group, Bonferroni post hoc test.
4.5) DISCUSSION:-
The current study investigates the mechanisms underlying IVM induced
PPI disruption in C57BL/6J mice. The PPI deficits induced upon P2X4R
potentiation by IVM were attenuated via antagonism of dopamine D1, but
not D2, receptors, implicating a role for D1 receptors in IVM’s mechanism
of action. Unfortunately, we did not observe a synergistic interaction
between SKF 82958 and IVM in PPI regulation since IVM alone was able
to disrupt PPI function and SKF 82958 tended to have a similar effect on
IVM
SKF 82958
+
+
+
+
-
- -
-
PPI 6
PPI 3
PPI 12
PPI %
0
10
20
30
40
*
108

its own. The findings from the DA receptor antagonist study are in
agreement with multiple studies that have previously reported a more
important role for D1 receptors than D2 receptors in PPI regulation in mice
(Ralph-Williams et al. 2003, Ralph-Williams et al. 2002, Ralph & Caine
2005) and certain strains of rats (Mosher et al. 2016). For instance, non-
selective DA agonists such as apomorphine, cocaine or pergolide have
been reported to disrupt PPI function via D1 receptors as their
pharmacological effects were attenuated either in mice deficient in Drd1
gene [i.e. D1 receptor knockout (KO)] (Ralph-Williams et al. 2002) or in
the presence of D1 receptor blockade (Ralph-Williams et al. 2003, Doherty
et al. 2008). These behavioral effects of apomorphine in C57BL/6J mice
are in contrast to what was observed in Sprague-Dawley rats since
raclopride, but not SCH 23390, blocked apomorphine mediated PPI
dysfunction (Swerdlow et al. 1991). In further support of D1 receptors’ role
in PPI regulation, selective D1 receptor agonists such as SKF 82958 and
dihydrexidine have also been shown to disrupt PPI functioning in
C57BL/6J and 129S6 mice (Ralph-Williams et al. 2003) at doses lower
than those required to exhibit similar behavioral effects in rats (Swerdlow
et al. 2000, Wan et al. 1996) . In the context of genetic mouse models,
mice deficient in nitric oxide synthase (NOS KO) exhibited increased D1
receptor mediated signaling and were hypersensitive to PPI disruptive
effects of D1 receptor selective agonist, SKF 81297 in comparison to
109

wildtype (WT) littermates (Tanda et al. 2009). On the other hand, mice
deficient in neurotensin (NT KO) were resistant to PPI disruptive effects of
SKF 82958, relative to their WT counterparts (Chastain et al. 2015). In
addition to mice, D1 receptors may also have a prominent role in PPI
functioning in certain strains of rats. SKF 82958 was able to disrupt PPI in
Long-Evans rats and this effect was blocked by SCH 23390 but not D2
receptor antagonist, L741626 (Mosher et al. 2016). Such an interaction
was not seen in other rat strains including Sprague –Dawley and Wistar
rats as SKF 82958’s effects were not D1 receptor mediated in these
strains (Mosher et al. 2016). Additionally, D1 receptor activation by SKF
8393 potentiated dizocilpine-mediated PPI disruption in Sprague-Dawley
rats and this interaction was blocked by SCH 23390, but not by
haloperidol or clozapine, indicating an interaction between NMDARs and
D1 receptors in PPI functioning (Bortolato et al. 2005). Overall, in
agreement with findings from previous studies, the ability of SCH 23390 to
block IVM’s disruptive effects on PPI in our current study implies an
interaction between P2X4Rs and D1 receptors in PPI regulation in mice.
While there is sufficient evidence to demonstrate the importance of D1
receptors in PPI regulation, the role of D2 receptors cannot be
disregarded. For instance, indirect DA agonists such as amphetamine and
cocaine disrupt PPI function via D2 receptor activation since their effects
were blunted in mice deficient in Drd2 gene (Ralph-Williams et al. 2002) or
110

in the presence of raclopride (Doherty et al. 2008). In addition, there are
several genetic mouse models such as mice deficient in dopamine
transporter (DAT) (Ralph et al. 2001), disrupted in schizophrenia complex
(DISC) (Lipina et al. 2010) and trace amine receptor-1 (TA1) (Wolinsky et
al. 2007) that exhibit exacerbated D2 receptor functioning and their PPI
deficits were rescued in presence of D2 receptor blockade. In coherence
with evidence from pharmacological and genetic studies, the PPI deficits
in P2X4R KO mice were reversed by raclopride in addition to SCH 23390
and there was a significant interaction between treatment and genotype
for both the drugs, indicating that D2 receptors can be involved in PPI
dysfunction in P2X4R KO mice (Khoja et al. 2016).
The molecular mechanism by which IVM disrupts PPI function is still
unknown. Previously, we have shown that IVM can modulate DA receptor
associated signaling cascades in various parts of the striatum including
caudate putamen (CPu) and ventral striatum [consisting of nucleus
accumbens (NAc)]. For example, we have evidence that IVM (5mg/kg
dose) reduces DARPP-32 phosphorylation but tended to modulate CREB
phosphorylation in the ventral striatum via P2X4R potentiation (Khoja et al.
2016). The ventral striatum which comprises of nucleus accumbens core
(NAc) is part of the cortico-pallido-striatal-thalamic circuitry that is involved
in regulation of PPI (Swerdlow et al. 2011, Swerdlow et al. 2008, Wan &
Swerdlow 1996, Wan et al. 1995). Considering that DA receptor agonists
111

induce PPI dysfunction and DA receptor antagonists reverse PPI deficits,
it is presumably thought that increased DAergic function in the NAc
(Swerdlow et al. 2001a), leads to inhibition of GABAergic neurons
projecting towards the ventral pallidum (VP), which has a tonic control
over the pedunculopontine nucleus (PPTg), which mediates PPI function
(Fendt et al. 2001). The ability of IVM to reduce DARPP-32
phosphorylation is reminiscent of PPI modulating agents that are known to
regulate DARPP-32 phosphorylation in the striatopallidal neurons [i.e.
GABAergic medium spiny neurons (MSNs) exclusively expressing D2
receptors] within the mesolimbic circuitry. This includes PPI disruptive
agents like quinpirole or sumanirole (selective D2 receptor agonists) that
can reduce DARPP-32 phosphorylation in the striatopallidal neurons and
pharmacological agents known to reverse psychomimetic-induced PPI
deficits such as haloperidol, clozapine, olanzapine (D2,D4 and 5-HT
2A

receptor antagonists) (Svenningsson et al. 2000), rolipram
[phosphodiasterase-4 (PDE-4) inhibitor] (Nishi et al. 2008) and CGS-2680
(adenosine A2A receptor agonist), all of which can increase DARPP-32
phosphorylation in striatopallidal neurons (Svenningsson et al. 1998).
While the localization of D2 receptors (Alexander & Crutcher 1990, Yung
et al. 1995), PDE-4 (Nishi et al. 2008) or A2A (Fink et al. 1992, Schiffmann
et al. 1991) receptors has been elucidated in neuronal sub-populations in
the basal ganglia, the precise neuronal localization of P2X4Rs within the
112

basal ganglia is yet to be investigated. Until date, P2X4Rs have been
reported to be expressed in the DA neurons of the ventral tegmental area/
substantia nigra region (VTA/SN) in the ventral mesencephelon and DA
projections that innervate the CPu and the NAc (Amadio et al. 2007, Heine
et al. 2007). In addition to its presence on DA neurons, P2X4Rs are also
expressed on GABAergic MSNs, GABA interneurons (Amadio et al. 2007)
in the striatum as well as GABA releasing synaptic terminals that project
into ventral mesencephalon (Xiao et al. 2008). Considering that we saw
attenuation of behavioral effects of IVM by SCH 23390 and not by
raclopride, it is logical to assume that P2X4Rs are expressed on the
striatonigral neurons that exclusively express D1 receptors. However,
there is a sub-population of MSNs in the nucleus accumbens (a critical
region regulating PPI) that expresses both D1 and D2 receptors (Perreault
et al. 2011, Gangarossa et al. 2013). Moreover, the high dose of SCH
23390 used in this study may involve other receptor targets of SCH 23390
such as 5-HT2C receptor (Millan et al. 2001, Briggs et al. 1991). Notably,
5-HT2C receptor agonists can rescue PPI deficits induced by
psychomimetics (Marquis et al. 2007). Thus, the delineation of P2X4Rs in
neuronal sub-population within the basal ganglia circuitry would be critical
to understanding the anatomical site for IVM mechanism of action.
PPI is defined as an operational measure of sensorimotor gating, which
refers to filtering irrelevant or insignificant information in a stimulus-laden
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environment and accordingly, generates a motor output that is
proportional to a significant sensory stimulus (Powell et al. 2012, Geyer et
al. 2001, Geyer & Braff 1987, Braff & Light 2004). Deficits in PPI can be
correlated to disruptions in pre-attentional information processing,
ultimately leading to overflow of sensory information and obliteration of
cognition (McGhie & Chapman 1961). Deficits in sensorimotor gating has
been linked to a wide spectrum of “perceptual” disorders including
schizophrenia (Braff 1993, Braff et al. 2001), bipolar disorder (Perry et al.
2001), obsessive-compulsive disorder (Swerdlow et al. 1993), attention-
deficit hyperactivity (Feifel et al. 2009) and Tourette’s syndrome
(Swerdlow et al. 2001b) as well as to autism spectrum disorders such as
autism (Perry et al. 2007), Fragile X syndrome (Frankland et al. 2004).
Mutant mouse models for susceptible genes that have been suggested to
be involved in the pathophysiology of aforementioned diseases exhibit PPI
dysfunction (Vuillermot et al. 2011, Ralph et al. 2001, Lipina et al. 2010,
Willi et al. 2010, Ohgake et al. 2009, Tanaka et al. 2006, Duncan et al.
2004, Kinkead et al. 2005), making PPI a reliable endophenotype in the
genetic studies of neuropsychiatric disorders (Braff et al. 2007, Powell et
al. 2012). Moreover, several of the clinical typical and atypical anti-
psychotics such as haloperidol, risperidone, clozapine, olanzapine,
quetiapine have been demonstrated to ameliorate PPI deficits in genetic
and pharmacological models of such diseases (Swerdlow & Geyer 1993,
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Geyer et al. 2001, Bast et al. 2001, Levin et al. 2007, Kumari & Sharma
2002). Thus, PPI has been elucidated as a useful behavioral assay in
investigating molecular mechanisms of psychiatric diseases as well as
screening of potential antipsychotic drugs. The ability of IVM to disrupt PPI
function via P2X4R potentiation (Bortolato et al. 2013) as well as the
reduced PPI function in the P2X4R KO mouse model (Wyatt et al. 2013)
indicates a role for P2X4Rs in the normal physiological process of
sensorimotor gating. Additionally, evidence from our previous and current
study indicates an interesting interaction between P2X4Rs and DA
receptors in striatal signaling pathways and this association may partially
contribute to sensorimotor gating. Since, the link between dysregulated
dopaminergic function and PPI abnormalities in psychiatric diseases
(Swerdlow et al. 2001a, Braff et al. 2001, Braff & Geyer 1990, Swerdlow et
al. 1986) has been well consolidated over past decades, our preclinical
studies suggests a role for P2X4Rs in pathophysiology of psychiatric
diseases. However, further investigations at the clinical level would be
warranted to link p2rx4 gene mutations (leading to abberations in
functioning) with neuropsychiatric disorders in order to ascertain such a
role for P2X4Rs in patients diagnosed with psychiatric illnesses. Until
date, single nucleotide polymorphisms in human p2rx4 gene have been
only linked to hypertension and age-related macular degeneration
(Caseley et al. 2014).
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Further studies using viral vectors which would specifically knockdown
P2X4R expression in a brain region critical for PPI regulation [such as
NAc or medial prefrontal cortex (mPFC) or VP] would be necessary to
establish a direct link between P2X4R function and sensorimotor gating.
More importantly, the anatomical site for P2X4Rs’ effect on PPI could be
characterized using viral vectors, since sensorimotor gating operates at
several functional levels. Furthermore, it would be also be helpful to
knockdown signaling molecules such as DARPP-32 or CREB or use
genetic mouse models deficient in those genes, to correlate the molecular
effects of IVM in the ventral striatum with its behavioral effect. Overall, the
current investigation indicates a potential interaction between P2X4Rs and
DA receptors in modulation of PPI and the development of P2X4R
antagonists as potential anti-psychotics for treatment of diseases linked to
DA perturbations.

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CHAPTER 5
OVERALL DISCUSSION AND CONCLUSION
5.1) Summary of overall findings:-
The overall findings from my dissertation signify an interesting interaction
between P2X4Rs and DA system in regulation of striatal signaling
pathways and CNS functions such as motor behavior and sensorimotor
gating. In chapter 2, I found that observed that deficiency in the p2rx4
gene induced disruptions in homeostasis of DA system and that positive
modulation of P2X4Rs by IVM directly impacts the DA receptor associated
signaling pathways in the dorsal and ventral striatum. The observed
neurochemical changes in P2X4R KO suggests a reduction in
dopaminergic tone (hypodopaminergia). Further investigations monitoring
the extracellular levels of DA in synapses would add support to this
conclusion. In that, changes in DA neurotransmission identified using a
P2X4R KO mouse model suggests a role for P2X4Rs in may underlie the
sensorimotor gating, social deficits and increased ethanol drinking
behavior in P2X4R KO mice. From the pharmacological aspect, using
IVM, I demonstrated a role for P2X4Rs linked to modulation of DARPP-32
phosphorylation in striatal regions. A significant interaction between IVM
treatment and P2X4R KO genotype, provided mechanistic explanation for
IVM’s molecular mechanism of action. Furthermore, IVM-mediated
117

enhancement of DARPP-32 and ERK 1/2 phosphorylation is reminiscent
of mechanistic actions of DA agonists including cocaine and
amphetamines (Bertran-Gonzalez et al. 2008, Valjent et al. 2005, Pascoli
et al. 2014, Svenningsson et al. 2000). IVM-mediated reduction in
DARPP-32 phosphorylation in ventral striatum is similar to that of D2
receptor antagonists (haloperidol, risperidone) (Bertran-Gonzalez et al.
2008, Svenningsson et al. 2000) that are used as anti-psychotics for
treatment of psychiatric illnesses. Overall, the findings suggest that
P2X4Rs play a role homeostasis of DA system and that P2X4R
dysregulation can lead to behavioral manifestations that are relevant to
disorders characterized by DA dysfunction.
To investigate a role for P2X4Rs in DA-dependent behaviors, the impact
of P2X4R manipulation on motor behavior was undertaken. Using the 6-
OHDA mouse model of DA depletion, P2X4R KO mice exhibited a
reduction in L-DOPA induced motor behavior and conversely, IVM
potentiated L-DOPA induced motor behavior. Moreover, I observed that
IVM mediated this pharmacological effect, in part, via P2X4R modulation
as IVM’s effect on L-DOPA induced motor behavior tended to attenuate in
P2X4R KO mice. The ability of IVM to potentiate L-DOPA’s
pharmacological effect represents a novel adjunct therapy for treatment of
neurodegenerative disorders characterized by DA hypoactivity including
Parkinson’s disease and Huntington’s disease, since 6-OHDA mouse
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model is extensively used to screen novel pharmacological agents for
these disease states.
As mentioned previously, the changes in DA receptor expression may
account for the PPI abnormalities previously reported in P2X4R KO mice.
PPI is considered as a reliable index of sensorimotor gating (Ison &
Hoffman 1983) and these deficits in P2X4R KO mice suggests that loss of
p2rx4 function reduces the ability to filter insignificant stimuli from salient
ones in a stimulus-laden environment, resulting in inundation of incoming
sensory information and obliteration of integral cognitive functioning. This
hypothesis was tested using SCH 23390 and raclopride (DA receptor
antagonists), wherein the P2X4R KO mice were hypersensitive to
pharmacological effects of DA receptor antagonists, since DA receptor
blockade in P2X4R KO significantly alleviated PPI dysfunction in P2X4R
KO without altering PPI function in WT mice. These findings are similar to
findings using other genetic mouse models of neuropsychiatric diseases,
which exhibit PPI dysfunction and is ratified upon DA receptor antagonism
(Lipina et al. 2010, Ralph et al. 2001, Kinkead et al. 2005). Furthermore,
PPI is commonly used as a predictor endophenotype for various
neuropsychiatric disorders (Powell et al. 2012, Swerdlow et al. 2008) and
multiple antipsychotics that are used in pharmacological intervention can
rescue PPI deficits in clinical population (Braff et al. 2001). Hence, the
role of P2X4Rs in regulation of sensorimotor gating may help to bridge an
119

association between P2X4Rs and pathophysiology of neuropsychiatric
diseases.
While chapter 2 focused on interaction between P2X4Rs and DA
receptors in PPI regulation using the genetic approach, chapter 4
investigated a direct relationship between these two receptor systems
using a pharmacological approach. The demonstration that IVM reduced
PPI function and that the effects were partially abolished in P2X4R KO
mice, adds support to role of P2X4Rs in PPI. As presented, the disruptive
effects of IVM were diminished in presence of SCH 23390, but not
raclopride, indicating that DA D1 receptors and not D2 receptors have a
role in IVM’s behavioral effects. Notably, these results corroborate the
present notion that D1 receptors may be an important contributor to PPI
regulation, atleast in C57BL/6J mice (Ralph-Williams et al. 2003, Ralph-
Williams et al. 2002, Ralph & Caine 2005). Interestingly, I did not observe
a synergistic interaction between IVM and SKF 82958 in PPI regulation,
but this maybe due to ‘floor’ effects since both drugs appeared to
modulate PPI independently. In addition, SCH 23390 positively modulated
5-HT
2C
receptors (Millan et al. 2001, Ramos et al. 2005) and so, a high
dose of SCH 23390 used in my PPI studies may reflect a complex network
of various receptors. Notably, 5-HT
2C
agonism can alleviate PPI
aberrations (Marquis et al. 2007), suggesting an alternative mechanism for
SCH 23390-mediated blockade of IVM-induced PPI deficits. Hence, future
120

studies warrant testing the effects of clinical anti-psychotics such as
risperidone, clozapine or olanzapine that possess binding affinities for
both DA receptors and serotonin 5-HT
2
receptors on IVM-mediated PPI
disruption. These studies could provide interesting mechanistic
information considering the role of 5-HT

receptors in regulation of PPI
(Mansbach et al. 1989, Martinez & Geyer 1997, Nakaya et al. 2011, van
den Buuse & Gogos 2007) and their interaction with DA receptors in
pharmacological effects of anti-psychotics (Meltzer & Massey 2011,
Meltzer & Huang 2008). Additionally, testing the impact of haloperidol or
risperidone on IVM-induced effects could provide insights into interaction
between P2X4Rs, DA and 5-HT
2
receptors in regulation of sensorimotor
gating. Finally, the IVM findings reported herein suggested that
antagonism of P2X4Rs could potentially increase PPI function and
improve PPI deficits induced by psychomimetics such as cocaine,
methamphetamine or amphetamine. Collectively, these findings suggest
that P2X4R antagonists could be used in discovery of novel
pharmacotherapies for treatment of psychiatric disorders.
Chapter 3 investigated the direct relationship between P2X4R expression
and ethanol intake using LV-shRNA methodology. By selectively knocking
down P2X4R expression in the NAc core, I found a significant increase in
ethanol intake without any changes in ethanol preference or any
perturbations in physiology of drinking behavior. Injection of LV-shRNA-
121

p2rx4 construct did not significantly alter body weight. The LV-shRNA
findings were in good agreement with previous studies using P2X4R KO
model, wherein P2X4R KO consumed significantly more ethanol than WT
counterparts without any changes in ethanol preference or water intake
(Wyatt et al. 2014). Interestingly, P2X4R KO mice did weigh significantly
more than WT mice, which could partially underlie their increased ethanol
consumption, since we did not observe any changes in ethanol
preference. The findings from LV-shRNA study further reinforces the
notion that reduced P2X4R expression is correlated with high ethanol
drinking behavior. This is supported by genomic studies wherein ethanol
preferring strains of rats had a lower mRNA expression of p2rx4 in relation
to that of low ethanol preferring rats (Tabakoff et al. 2009, Kimpel et al.
2007). Conversely, we also successfully demonstrated that potentiation of
P2X4R function by IVM significantly reduces ethanol intake (Yardley et al.
2012, Asatryan et al. 2014) and that this phenomenon is mediated, in part,
via P2X4Rs (Wyatt et al. 2014). The mechanism underlying P2X4Rs’ role
in ethanol intake is not clearly understood, but there are reports that have
suggested that P2XR activation in VTA leads to GABA release, resulting
in inhibition of DA neurons (Xiao et al. 2008). This could be a reasonable
explanation for the reduction in ethanol intake in presence of IVM and
increased ethanol intake upon loss of P2X4R function by knockout or LV-
shRNA strategy, but future investigations using selective P2X4 agonists
122

and antagonists would be required to support this hypothesis. Overall,
chapter 3 studies reinforce the hypothesis that P2X4Rs in the mesolimbic
circuitry play an important role in regulation of ethanol intake and that
P2X4R positive modulators represent a novel pharmacotherapy for
treatment of AUD.
5.2) Future directions:-
The findings from my dissertation provide initial insights into role of DA in
behavioral functions including motor activity and sensorimotor gating
mediated by P2X4Rs. In addition, the alterations in different markers of
DA neurotransmission mediated by P2X4R manipulation provide new
insights into molecular mechanisms underlying behavioral deficits in
P2X4R KO mice and/or IVM-mediated behavioral effects. These are
preliminary steps into investigating the state of DA homeostasis in P2X4R
KO mice and/or in presence of IVM. Nevertheless, future investigations
related to elucidating the DAergic tone in P2X4R KO mice as well as the
effect of IVM on modulating DA release in different brain sites of the basal
ganglia circuitry would be required to understand the physiological
importance of P2X4Rs in different basal ganglia circuitries controlling
motor activity, sensorimotor gating and drug reward behavior. The
DAergic tone in P2X4R KO mice or in mice treated with IVM can be
characterized by using in vivo microdialysis or cyclic scan voltametry.
123

Considering I found changes in phosphorylation states of signaling
molecules such as DARPP-32 in P2X4R KO, it would be necessary to
investigate the functional activity of DA receptors in various sites in the
striatum using radioligand binding assay with DA receptor antagonists.
The radioligand binding assay would give us insights into the sensitivity of
DA receptors to DA receptor acting drugs as well as the number of DA
receptors present on neuronal membrane in P2X4R KO mice. Additionally,
undertaking this assay would provide us with a plausible explanation for
upregulation of DARPP-32 phosphorylation in the dorsal striatum of
P2X4R KO mice, since we observed no changes in DA receptor
expression as well as resolve the discrepancy between changes in DA
receptor expression and reduced DARPP-32 phosphorylation in the
ventral striatum of P2X4R KO mice.
At present, the anatomical localization of P2X4Rs in the basal ganglia
circuitry is not clearly elucidated. While, there is preliminary evidence
regarding presence of P2X4Rs in GABAergic MSNs in striatum (Amadio et
al. 2007), the precise localization in different neuronal sub-populations is
not delineated, that is presence of P2X4Rs in striatonigral neurons ( D1
receptor expressing) or striatopallidal neurons (D2 expressing).
Investigating the distribution of P2X4Rs in the direct and indirect pathway
of basal ganglia in different sites within striatum (caudate putamen, NAc
core, NAc shell) will provide knowledge regarding the regulation of DA
124

receptor associated signaling pathways in P2X4R KO mice and in
presence of IVM. Additional biochemical methods could be used to help in
determining the localization of P2X4Rs on MSNs. For example, a study
using double immunofluorescence (i.e. antibody for P2X4R and markers
for MSNs) would give us some insights in this regard. In addition,
fluorescence activated cell sorting (FACS) methodology to segregate
different populations of MSNs from BAC transgenic mouse models (for
example, drd1a-EGFP mice where D1 neurons are labeled and drd2-
EGFP mice were D2 neurons are labeled).
While there have been efforts to design and develop selective P2X4R
antagonists in vitro (Tian et al. 2014, Hernandez-Olmos et al. 2012), the in
vivo potential of those drugs is yet to be determined. However, IVM has
given us some valuable insights into P2X4Rs-mediated striatal signaling
pathways as well as behaviors and some of those significant effects were
confirmed in P2X4R KO mice. IVM does have other pharmacological
targets including the nAchRs (Krause et al. 1998), GABA
A
Rs (Dawson et
al. 2000) and glycine receptors (GlyRs) (Shan et al. 2001) in the CNS.
Considering the effects of IVM on PPI, the effect of P2X4R antagonists on
DA agonists that are known to disrupt PPI function could be tested and
this class of drugs could represent a novel therapeutic strategy for
treatment of neuropsychiatric disorders characterized by gating deficits.
To address the issue of lack of specific antagonists, the LV-shRNA
125

methodology could be used as an effective technique to help establish a
direct link between reduced P2X4R expression and certain behaviors such
as ethanol drinking behavior. Future studies would involve elucidating the
role of P2X4Rs in sensorimotor gating and L-DOPA induced motor
behavior using LV-shRNA methodology.

126

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Abstract (if available)

Abstract Purinergic P2X receptors (P2XRs) are cation permeable ionotropic receptors gated by adenosine-5’-triphosphate (ATP). Until date, seven subtypes of P2XRs have been identified (P2X1R upto P2X7R). Amongst the P2X subtypes, P2X4 receptors (P2X4Rs) are expressed in different types of cells (i.e. neurons and glial cells) in the central and peripheral nervous system (CNS and PNS). ATP-mediated synaptic transmission via P2X4Rs has been implicated in regulation of synaptic currents facilitated by N-Methyl-D-aspartate receptors (NMDARs) and gamma-amino butyric acid (GABAARs) at post-synapses and pre-synaptic release of neurotransmitters including glutamate, GABA and norepinephrine (NE) in various brain regions. However, the functional significance of P2X4Rs remains limited due to lack of specific agonists and antagonists. Presently, P2X4Rs in the hippocampus has been attributed to synaptic plasticity and P2X4Rs in spinal cord microglia has been linked to pain hypersensitivity. Investigations from our laboratory group have made significant efforts in understanding the behavioral role of P2X4Rs. Using pharmacological and genetic approaches, we suggested a role for P2X4Rs in sensorimotor gating, social behavior and ethanol drinking behavior. The molecular mechanisms underlying these behavioral changes are not clearly understood. To address this issue, I hypothesized that P2X4Rs interact with the dopamine (DA) system, considering that DA has a significant role to play in mediation of aforementioned behaviors. Presently, there is very limited knowledge regarding the interaction of P2X4Rs with the DA system. Elucidating this interaction could give us important insights into regulation of certain CNS behaviors including sensorimotor gating, social behavior and reward behavior as well as relevance of P2X4Rs to psychiatric disorders characterized by deficits in aforementioned behaviors. To investigate the gap pertaining to this interaction, the hypothesis of my dissertation is that P2X4Rs play an important role in regulation of DA-dependent signaling pathways and associated behaviors. Chapter 2 investigates the role of P2X4Rs in controlling DA homeostasis and its relevance to DA-dependent behaviors including motor control and sensorimotor gating. We used mice deficient in p2rx4 gene [P2X4R knockout (KO) mice] and ivermectin (IVM), which is a positive modulator of P2X4Rs, to address the interaction between P2X4Rs and DA neurotransmission. Chapter 3 makes significant attempts to establish a direct link between reduced P2X4R expression and increased ethanol intake. This chapter is based on previous findings wherein, P2X4R KO mice exhibited transient increase in ethanol intake over short period of time. In this chapter, we observed the ethanol drinking behavior in P2X4R KO mice over longer period of time and used the lentivirus-shRNA (LV-shRNA) strategy to establish a direct role for P2X4Rs in ethanol intake. Chapter 4 examines the interaction between IVM and DA receptor agonist/antagonists in regulation of prepulse inhibition (PPI) of acoustic startle reflex. Taken together, findings from my dissertation provide us with important insights into role for P2X4Rs in DA neurotransmission and DA-dependent behaviors such as motor control and sensorimotor gating. Moreover, the findings indicate that perturbation of this interaction may be relevant to pathogenesis of neuropsychiatric disorders characterized by DA dysfunction including schizophrenia, bipolar disorder, Parkinson’s disease, alcohol use disorder (AUD) and supports P2X4Rs as novel drug targets for therapeutic intervention for these disorders.

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Asset Metadata

Creator Khoja, Sheraz (author)

Core Title Role of purinergic P2X4 receptors in regulation of dopamine homeostasis in the basal ganglia and associated behaviors

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology and Toxicology

Publication Date 05/02/2017

Defense Date 03/20/2017

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

Tag alcohol use disorder,dopamine receptors,ivermectin,OAI-PMH Harvest,P2X4 receptors,Parkinson's disease,schizophrenia

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Davies, Daryl L. (committee chair), Jakowec, Michael W. (committee member), Rodgers, Kathleen E. (committee member)

Creator Email sheraz.khoja@gmail.com,skhoja@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-368167

Unique identifier UC11255848

Identifier etd-KhojaShera-5302.pdf (filename),usctheses-c40-368167 (legacy record id)

Legacy Identifier etd-KhojaShera-5302.pdf

Dmrecord 368167

Document Type Dissertation

Rights Khoja, Sheraz

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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

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