THE ROLE OF THE VASOPRESSIN 1B RECEPTOR IN THE REGULATION OF SENSORIMOTOR GATING
A thesis submitted to Kent State University in partial fulfillment of the requirements for the degree of Master of Science
By
Monica Dhakar
May 2011
Thesis written by Monica Dhakar M.B.B.S., Byramjee Jeejeebhoy Medical College, 2008 M.S., Kent State University, 2011
Approved by
______Dr. H. Caldwell, Advisor
______Dr. R. Dorman, Director, School of Biomedical Sciences
______Dr. John Stalvey, Dean, College of Arts and Sciences
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TABLE OF CONTENTS
PAGE
LIST OF FIGURES………………………………………………………………vi
LIST OF TABLES……………………………………………………………...viii
ACKNOWLEDGEMENTS………………………………………………………ix
Introduction………………………………………………………………………1
Vasopressin………………………………………………………………..2
Sexual dimorphism in the central vasopressin system…………………….3
Vasopressin and social behavior…………………………………………..3
Vasopressin receptors……………………………………………………..5
The vasopressin 1b receptor………………………………………………6
Vasopressin 1b receptor knockout mice…………………………………..7
Vasopressin, the vasopressin 1b receptor, and neuropsychiatric disorders.8
Sensorimotor gating……………………………………………………...10
Prepulse inhibition of the startle reflex…………………………..11
Cortico-striato-pallido-pontine circuitry…………………………13
Neurotransmitter systems regulating prepulse inhibition of the
startle reflex………………………………………….…………..15
Interactions of vasopressin and the cortico-striato-pallido-pontine
circuitry…………………………………………………………..17
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Objective…………………………………………………………………………17
Methods…………………………………………………………………………..20
General Methods…………………………………………………………………20
Animals…………………………………………………………………..20
Prepulse inhibition the startle reflex……………………………………..20
Drugs……………………………………………………………..20
PPI………………………………………………………………..21
Procedure………………………………………………………...21
Study design……………………………………………………...24
Quantitative real time polymerase chain reaction ……………………….26
Procedure………………………………………………………...26
Individual Experiments…………………………………………………………..26
Experiment 1A and 1B…………………………………………………...26
Experiment 2A and 2B…………………………………………………...27
Experiment 3……………………………………………………………..27
Statistics………………………………………………………………….28
Experiment 4A and 4B…………………………………………………..28
Statistics………………………………………………………………….29
Results……………………………………………………………………………30
Experiment 1A…………………………………………………………...31
Experiment 1B…………………………………………………………...31
Experiment 2A…………………………………………………………...37
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Experiment 2B…………………………………………………………...43
Experiment 3……………………………………………………………..49
Experiment 4A…………………………………………………………...55
Experiment 4B…………………………………………………………...55
Discussion………………………………………………………………………..64
References………………………………………………………………………..74
Appendix I……………………………………………………………………….88
Appendix II………………………………………………………………………93
v
LIST OF FIGURES
PAGE
Introduction
Fig 1: Role of vasopressin in regulation of social behaviors……………...4
Fig 2: Schematic diagram of PPI………………………………………...12
Fig 3: CSPP circuit………………………………………………………14
Methods
Fig 4: Schematic diagram representing the components of SR-LAB……22
Fig 5: Acoustic startle chamber …………………………………………23
Fig 6: Study design for Experiments 1, 2 and 3………………………….25
Results
Fig7: Experiment 1A Habituation of startle in females………………….32
Fig 8: Experiment 1A Baseline PPI percentage in females ……………..34
Fig 9: Experiment 1A PPI percentage following drug treatment in
females…………………………………………………………….35
Fig 10: Experiment 1B Habituation of startle in males …………………38
Fig 11: Experiment 1B Baseline PPI percentage in males………………40
Fig 12: Experiment 1B PPI percentage following drug treatment in
male………………………………………………….…………..41
Fig 13: Experiment 2A Habituation of startle in females…..…………..44
Fig 14: Experiment 2A Baseline PPI percentage in females…………….46
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Fig 15: Experiment 2A PPI percentage following drug treatment in
females…………………………………………………………...47
Fig 16: Experiment 2B Habituation of startle in males………………….50
Fig 17: Experiment 2B Baseline PPI percentage in males………………52
Fig 18: Experiment 2B PPI percentage following drug treatment in
males……………………………………………………………..53
Fig 19: Experiment 3 Habituation of startle in males…………………....56
Fig 20: Experiment 3 Baseline PPI percentage in males………………...58
Fig 21: Experiment 3 PPI percentage following drug treatment in
males…………………………………………………………….59
Fig 22: Experiment 4A Expression of NMDAR1 in males………………62
Fig 23: Experiment 4B Expression of NMDAR2A in males……………..63
Appendix I
Fig 24: PPI percentage in Avpr1b +/− mice following drug treatment….90
Fig 25: PPI percentage following AMP and saline in Avpr1b +/− mice...91
vii
LIST OF TABLES
PAGE
Results
Table 1: Experiment 1A Startle amplitude in females………………...…33
Table 2: Experiment 1A Mean and SEM for PPI in females…………….36
Table 3: Experiment 1B Startle amplitude in males………………...... 39
Table 4: Experiment 1B Mean and SEM for PPI in males………………42
Table 5: Experiment 2A Startle amplitude in females…………………...45
Table 6: Experiment 2A Mean and SEM for PPI in females…………….48
Table 7: Experiment 2B Startle amplitude in males……………………..51
Table 8: Experiment 2B Mean and SEM for PPI in males………………54
Table 9: Experiment 3 Startle amplitude in males……………………….57
Table 10: Experiment 3 Mean and SEM for PPI in males……………….60
Appendix II
Table 11: PPI program…………………………………………………...92
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ACKNOWLEDGEMENTS
I would like to thank my advisor and mentor Dr. Heather Caldwell for giving me an opportunity to work in her lab and her invaluable guidance over the course of three years.
Dr. Caldwell has always provided me constant encouragement and endless support in all my endeavors including research and future career goals. I would also like to thank Dr.
Eric Mintz and Dr. John Johnson for serving on my thesis committee and providing valuable insights and expert advice on my research project.
I am grateful to my colleagues; Erica Stevenson, Megan Rich and Uju Dike for their patience in teaching me all the lab techniques that I know today. Without their support and friendship, this project could not have been completed. I would also like to thank
Shannah Witchey, and all the undergraduate students for their help in conducting some of the experiments.
I would like to extend my gratitude to my friends; Vivek, Priyanka, Sagar and
Harsh for being the family to me for the last three years. A special thanks to my friend,
Charu; for always being there with me during the ups and downs of the graduate school.
Lastly, a heartfelt thanks to the people, whom I owe everything that I have ever achieved; Mom, Dad, Neha, and Dhaval! I cannot thank them all enough for everything that they are to me. I thank each one of them for believing in me and in my dreams.
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Without Dhaval’s love and support, I would not be where I am today. I am grateful to you for being the pillar of strength and encouragement at every step of my life!
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Introduction:
The expression of normal social behavior is crucial for the development and survival of many species (Donaldson and Young, 2008). Broadly, social behavior can be defined as interactions between individuals of the same species and includes affiliation, aggression, reproduction, and communication (Sokolowski, 2010; Insel and Young,
2000). Successful social interactions require animals to observe the actions of others, make predictions about their behavior, and respond appropriately (Skuse and Gallagher,
2009). In humans, aberrant social behavior is characteristic of a variety of neuropsychiatric disorders, such as autism, schizophrenia, obsessive compulsive disorder
(OCD), anxiety, and depression. Across species, the neural substrates and the mechanisms regulating social behavior are highly conserved (Donaldson and Young,
2008).
A number of hormones and neuropeptides are important to the neural regulation of social behavior. In particular, two neurohormones, oxytocin (Oxt) and vasopressin
(Avp), have consistently been implicated in the neural regulation of social behavior (as reviewed in Caldwell et al., 2008; Lee et al., 2009; Ross and Young, 2009).
Evolutionarily, Oxt and Avp and their gene families are largely conserved across species
(Acher et al., 1995). Both peptides are composed of nine amino acids and differ from one another in only two amino acids, i.e., those in the third and eighth positions. Oxt and Avp are hydrophilic in nature and are not thought to readily cross the blood brain barrier.
1
2
Further, both exert their actions through membrane bound G protein coupled receptors.
The effectiveness of Oxt and Avp at regulating behavior is attributed to three characteristics: 1) their localized distribution in the brain 2) their slow and enduring effects, and 3) their plasticity (Insel and Young, 2000).
Vasopressin
Avp is synthesized as a part of a preprohormone, which is then enzymatically cleaved to produce the biologically active nonapeptide, a neurophysin, and a glycopeptide. Once cleaved, Avp is transported along the axons and released (Brownstein et al., 1980; Acher et al., 2002). Avp is synthesized primarily in the magnocellular neurons of the supraoptic (SON) and paraventricular nucleus (PVN) of the hypothalamus that project to the posterior pituitary. Upon appropriate stimulation, Avp is released into the blood stream from the posterior pituitary to produce systemic effects which include the regulation of water balance and maintenance of blood pressure by acting on the blood vessels and the kidneys (Nishimura and Fan, 2003; Bankir, 2001). Centrally, small quantities of Avp are released from the dendrites of the magnocellular neurons to produce local effects. In many species, Avp is also made in small populations of parvocellular neurons within the PVN, the bed nucleus of stria terminalis (BNST), the medial amygdala (MeA), and the suprachiasmatic nucleus (SCN) (Sofroniew, 1983). These neurons provide robust projections to a number of brain areas, including, hippocampus, subiculum, diagonal band of Broca, locus coeruleus, solitary tract nucleus, dorsal motor nucleus of vagus, olfactory tubercle, lateral septum (LS), lateral habenula (LH), ventral 3
tegmental area (VTA), and spinal cord (Buijs, 1978; De Vries and Buijs, 1983; Millan et al., 1984).
Sexual dimorphisms in the central vasopressin system
The distribution of Avp immunoreactive (Avp-ir) neurons and fibers shows a remarkable degree of sexual dimorphism. In most mammals especially rodents, males have a higher density of Avp-ir neurons and fibers in the MeA, BNST, LH and LS, as compared to females (De Vries et al., 1983). However, there are exceptions to these sexual dimorphisms and the distribution varies across species. For example, in primates such as marmosets, macaques, and humans, there is a similar distribution of Avp-ir neurons between males and females (Fliers et al., 1986; Caffe et al., 1989; Wang et al.,
1997). In guinea pigs, there is reverse sexual dimorphism with females having higher
Avp-ir fibers in the inferior colliculus, ventral trapezoid body, and dorsal cochlear nucleus than males (Dubois-Dauphin et al., 1987; Dubois-Dauphin et al., 1989). Since
Avp is involved in the modulation of sex specific behaviors, the distribution of Avp-ir fibers are also sensitive to circulating gonadal hormones (for full review on the interactions of gonadal steroids and vasopressin, please see Dhakar et al., in press ). For
instance, castration reduces Avp-ergic innervations from BNST and MeA in both males
and female rats and administration of testosterone increases the density of Avp fibers in
these areas (de Vries et al., 1984).
Vasopressin and social behavior 4
VASOPRESSIN
Avpr1 Avpr1 a b
Social Behaviors Aggression Stress
Social Affiliation Memory Anxiety
Figure 1: Schematic diagram showing the role of vasopressin in the regulation of social behaviors
5
Centrally released Avp acts as a neuromodulator for a variety of social behaviors such as, social recognition, social motivation, social memory, aggression, affiliation, and parental behavior (as reviewed in Caldwell et al., 2008; Donaldson et al., 2008; Lee et al.,
2009) (Figure 1). Some evidence which indicates the importance of Avp in the regulation of social behaviors comes from studies in Brattleboro rat (BB-Ho), a naturally occurring
Avp mutant, that lacks the ability to synthesize Avp (Bohus and de, 1998), and thus has diabetes insipidus. BB-Ho rats have deficits in memory, social recognition, social motivation, and attention (Laycock et al., 1983; Williams et al., 1985; Williams et al.,
1983). Exogenous administration of Avp in these rats improves certain social behaviors such as social recognition (Engelmann and Landgraf, 1994). Further, in rodents, intracerebral injection of an Avp analogue facilitates aggression (Ferris et al., 1997;
Caldwell and Albers, 2004), partner preference (Winslow et al., 1993; Cho et al., 1999), and paternal behavior (Wang et al., 1994). In humans, intranasal administration of Avp enhances verbal memory and reaction time in males (Born et al., 1998; Beckwith et al.,
1983).
Vasopressin receptors
Avp exerts it effects via three different receptors: 1) the Avp 1a receptor (Avpr1a)
2) the Avp 1b receptor (Avpr1b) and 3) the Avp 2 receptor (Avpr2). While the Avpr1a and the Avpr1b are found centrally (as well as peripherally), the Avpr2 is only expressed peripherally (Tribollet et al., 1988; Lolait et al., 1992). Across species, the Avpr1a is widely distributed in the central nervous system (CNS) in areas known to be important 6
for neural regulation of social behavior (Johnson et al., 1993; Young et al., 2000;
Tribollet et al., 1997). This thesis will focus specifically on the Avpr1b (for a review of the role of the Avpr in the neural regulation of behavior please see Caldwell et al., 2008;
Bielsky et al., 2004).
The vasopressin 1b receptor
The Avpr1b is a seven transmembrane receptor and belongs to the family of G protein coupled receptors. It is coupled to G αq/11 GTP binding proteins which along with
Gβλ activates a downstream 1,4,5-inositol triphosphate (IP3) second messenger pathway
(Michell et al., 1979; Jard et al., 1987). In the rat brain, in situ hybridization and RT-PCR have localized Avpr1b transcripts to a number of areas that are known to regulate social behaviors; olfactory bulb, LS, cerebral cortex, hippocampus, PVN, SCN, cerebellum, red nucleus, and piriform cortical layer II (Lolait et al., 1995; Vaccari et al., 1998; Saito et al., 1995). However, it should be noted that the probes used in the above mentioned in situ hybridization studies had some identity to the oxytocin receptor and the Avpr1a. A subsequent study which used more specific riboprobes, found a more restricted localization of Avpr1b in the anterior pituitary, and in the CA2 region of the hippocampus in rat, mouse, and human as detected by in situ hybridization and RT-PCR
(Young et al., 2006). So far, the distribution of Avpr1b has not been studied using receptor autoradiography, due to the lack of an available pharmacologically specific radiolabeled ligand, or with immunocytochemistry, due to lack of a specific antibody. Of 7
the species in which the Avpr1b has been sequenced and mapped, there is no evidence of sexual dimorphism in its distribution.
Evidence for the role of the Avpr1b in the regulation of social behaviors such as aggression, social recognition and social motivation comes from studies using an oral
Avpr1b antagonist (SSR149415), Avpr1b agonists and genetically engineered Avpr1b knockout (Avpr1b −/− ) mice (Wersinger et al., 2002; Tanoue et al., 2004).The development of Avpr1b −/− mice, in particular, has been helpful in elucidating the role of the Avrp1b in the neural regulation of social behaviors. The work described in this thesis utilized Avpr1b −/− mice to test our central hypothesis.
Vasopressin 1b receptor knockout mice
Avpr1b −/− mice were first generated in 2002 through a targeted disruption of the
Avpr1b gene (Wersinger et al., 2002). Initial characterization of Avpr1b −/− mice found that the males have deficits in social aggression and social motivation. However, predatory aggression in these mice seems to be normal, indicating that they do not have a global deficit in processing and attacking the stimuli but rather have a deficit in processing the social cues associated with social aggression (Wersinger et al., 2007).
They also have impaired social recognition, despite normal olfactory function (Wersinger et al., 2004; Wersinger et al., 2002). Avpr1b −/− females have deficits in maternal aggression and an abnormal Bruce Effect (Wersinger et al., 2008; Wersinger et al., 2007).
(The Bruce Effect is a pheromonally mediated response wherein the female terminates a pregnancy in response to chemosensory cues from an unfamiliar male). Avpr1b −/− 8
females fail to terminate their pregnancy in presence of unfamiliar male. Given the discreet localization of the Avpr1b within the CA2 region of the hippocampus in mice, these data suggest that Avpr1b might be important for the formation and retrieval of memories about social context (Young et al., 2006). This hypothesis fits well with the phenotypic data in Avpr1b −/− mice demonstrating that they have deficits in social recognition tasks. Avpr1b −/− mice also have an attenuated stress response with suppressed activity of the hypothalamic-pituitary-adrenal (HPA) axis under resting as well as stress condition (Tanoue et al., 2004). Specifically, Avpr1b −/− mice have a blunted adrenocorticotrophic hormone (ACTH) and cortisol response to hypoglycemia and chronic restraint stress (Lolait et al., 2007). Avpr1b −/− mice also have an impaired
stress response following acute immune stress (administration of bacterial
lipopolysaccharide) and alcohol intoxication (Lolait et al., 2007). In summary, these
studies implicate a role for the Avpr1b in the regulation of social behaviors and allow for
the possibility that abnormalities in the Avp system in humans could contribute to
abnormal social behavior.
Vasopressin, the vasopressin 1b receptor, and neuropsychiatric disorders
Alterations in the Avp system have been found in a number of neuropsychiatric
disorders in humans. Compared to controls, patients diagnosed with major depressive
disorder (van et al., 1997), post-traumatic stress disorder (PTSD) (de Kloet et al., 2008),
and OCD (Altemus et al., 1992) all have elevated plasma levels of Avp. In
schizophrenics, baseline levels of plasma Avp are low compared to controls (Elman et al., 9
2003); however, acute psychosis is associated with increased serum levels of Avp
(Raskind et al., 1987). There is evidence that elevations in plasma Avp correlate with central Avp, this is consistent with the effects of Avp on the coordination of physiology and behavior (Dogterom et al., 1978). However, it should be noted that even elevations in
Avp within the CSF may not be reflective of changes in local release.
In depressed patients, Avp mRNA is upregulated within the SON which is thought to be associated with elevated glucocorticoid levels (Meynen et al., 2006; Inder et al., 1997; Chakrabarty et al., 2005). Recently, a study found that a single nucleotide polymorphism (SNP) in the Avpr1b gene seems to be protective against recurrent major depression in adults (van et al., 2004). In children, variations in the Avpr1b gene have been associated with the onset of childhood mood disorders, particularly in females
(Dempster et al., 2007). In adolescents and adults with depression and OCD, treatment with antidepressants such as fluoxetine, a selective serotonin reuptake inhibitor, and clomipramine, a tricyclic antidepressant, decrease CSF levels of Avp (Altemus et al.,
1994; De et al., 1993). In addition, intranasal administration of Avp or its analogue ameliorates some of the negative symptoms of schizophrenia and improves memory
(Brambilla et al., 1986; Brambilla et al., 1989; Iager et al., 1986; Korsgaard et al., 1981).
Studies in animal models also confirm a possible role for Avp and the Avpr1b in psychiatric disorders. SSR149415, which is a specific orally active Avpr1b antagonist, has been used to study the role of Avpr1b. In rodents, the administration of this agent reduces aggression and anxiety-like behaviors, suggesting that the Avpr1b is important 10
for regulation of the circuitry that mediates aggression and anxiety (Griebel et al., 2002;
Blanchard et al., 2005; Stemmelin et al., 2005). Administration of SSR149415 in rodents also produces antidepressant effects in various behavioral tests, such as forced swim in rats (Griebel et al., 2002; Griebel et al., 2003).
In sum, all the above studies suggest that Avp could potentially contribute to the pathogenesis of these neuropsychiatric disorders. However, as Avp is a neuromodulator, it is not likely that it is the cause of neuropsychiatric disorders. Rather, Avp likely interacts with the neurocircuitry and neurotransmitter systems that underlie neuropsychiatric disorders, and in turn affects the susceptibility an individual to a certain psychiatric disorder. Common to many neuropsychiatric disorders, including schizophrenia, are abnormal thought processing, thought fragmentation, and difficulty with attention and cognition. This cognitive fragmentation and disordered thinking is thought to originate in part from deficits in sensorimotor gating.
Sensorimotor gating
Sensorimotor gating is the ability to process and filter incoming sensory and cognitive information and selectively allocate attention to the specific information. In normal awake individuals, sensorimotor gating is constantly active to ‘gate’ any excess or trivial information and selectively process the informational stimuli. Any individual’s gating abilities are viewed as plastic and are known to be shaped by a combination of genetic and environmental factors. Sensorimotor gating is also influenced by changes in the environmental, neurochemical, and hormonal milieu in the body (Swerdlow, 1996). 11
When this ‘gating window’ is pathologically broken, it can lead to abnormalities in orderly processing of information; resulting in sensory flooding, and ultimately thought fragmentation and disruption in cognitive processes (Mcghie and Chapman, 1961;
Venables, 1960). This breakdown of the gating window is thought to be responsible for the cognitive abnormalities which are features of many neuropsychiatric disorders. An operational measure of sensorimotor gating is called prepulse inhibition of the startle reflex (PPI).
Prepulse Inhibition of the startle reflex
The startle reflex is a constellation of responses to sudden, relatively intense
stimuli (auditory, visual or tactile), which is usually defensive in nature (Swerdlow et al.,
2000). The primary mammalian acoustic startle circuit is composed of three synapses
from the auditory nerve to the spinal motor neuron (Davis et al., 1982; Koch and
Schnitzler, 1997). Startle demonstrates several forms of plasticity such as habituation,
and fear potentiation, which are regulated by forebrain neural circuitry that is conserved
across species (Brown et al., 1951; Grillon et al., 1994; Geyer and Braff, 1982).
PPI is the profound decrease in the startle magnitude when the startling pulse is
preceded by a prepulse of weaker intensity by a very brief time window (Graham, 1975;
Hoffman and Searle, 1968; Ison et al., 1973) (Figure 2). The ‘gating’ period is
determined to be approximately 30-500 msec and serves to protect the sensory
information within the weak stimulus so that it is processed without being disrupted by
subsequent stronger stimuli (Hoffman and Searle, 1968; Ison et al., 1973; Swerdlow, 12
Figure 2: Schematic diagram showing the phenomenon of prepulse inhibition (PPI). PPI is the decrease in startle magnitude when a weaker prepulse precedes the startling pulse. (Modified and adapted from Swerdlow et al., 2000)
13
1996). PPI is a normal phenomenon that occurs virtually in all mammals, including primates, and does not exhibit habituation or extinction with multiple trials.
PPI is one form of startle plasticity that shows striking similarities across species
from rodents to humans, and is easily quantified (Swerdlow et al., 1999). It is a robust
experimental phenomenon that occurs irrespective of the sensory modality of the startling
stimulus and the prepulse. In humans, PPI is measured by using electromyography of the
orbicularis oculi muscle which is a part of the blink reflex of the startle component (Braff
et al., 1992). In rats and mice, the whole body reflexive flinch to startle stimulus can be
measured by using a stabilimeter chamber (Swerdlow et al., 2000).
Deficits in PPI can be seen in a number of disorders with thought fragmentation
such as schizophrenia (Bolino et al., 1994; Braff et al., 1992; Braff et al., 1978; Braff et
al., 1999; Grillon et al., 1992; Kumari et al., 1999; Weike et al., 2000), OCD (Swerdlow
et al., 1993; Schall et al., 1996), Huntington’s disease (Swerdlow et al., 1995), Tourette
syndrome (Castellanos et al., 1996), nocturnal enuresis, and attention deficit hyperactive
disorder (ADHD) (Ornitz et al., 1992). Deficits in PPI are not unique to a particular
disease but are characteristic of various disorders with pathological disruption of circuitry
regulating it called the cortico-striato-pallido-pontine (CSPP) circuit (Swerdlow et al.,
1992).
Cortico-striato-pallido-pontine circuitry
Studies in rats have identified the neural substrates that regulate PPI. This serial
and parallel neural circuitry is called the CSPP circuit (Figure 3). It connects the limbic 14
Ventral tegmental area
Hippocampus mPFC
Amygdala Nucleus accumbens
Ventral pallidum CSPP Circuit
Pedunculopontine tegmental nucleus
S R
Cochlear Caudal pontine Motor nucleus reticular nucleus neuron
Acoustic stimulus Response Primary acoustic startle circuit
Figure 3: Schematic diagram showing the main components of the CSPP circuit and its regulatory control over startle reflex. The acoustic stimulus S produces a motor response R through the simple pontine circuit. Prepulse effects on R are mediated via the pedunculopontine tegmental nucleus, which is under the control of serial and parallel descending projections from the forebrain. (Modified and adapted from Koch and Schnitzler et al., 1997; Swerdlow et al., 2001) (Abbreviations: mPFC, medial prefrontal cortex) 15
cortex with the ventral striatum, ventral pallidum, pontine tegmentum and converges with the primary startle circuit at the level of caudal pons (Koch and Schnitzler, 1997;
Swerdlow and Geyer, 1998; Swerdlow et al., 2001; Koch et al., 1993). Though the main inhibitory effect of prepulse on startle is exerted at the level of pons, the CSPP circuit regulates the quality of the inhibitory activity and thus the degree of inhibition of the response to stimuli when preceded by a prepulse (Swerdlow et al., 2001). The CSPP circuit is known to be regulated by a variety of neurotransmitter systems in humans and rodents, including the dopaminergic, serotonergic, and glutamatergic systems.
Neurotransmitter systems regulating prepulse inhibition of the startle reflex
Abnormalities in the dopamine, the glutamate, and the serotonin system have been consistently implicated in pathogenesis of a number of neuropsychiatric disorders such as schizophrenia, major depression, panic disorders and OCD (Carlsson et al.,
1997). Since these neurotransmitters are also reported to act on various neural substrates in the CSPP circuitry, pharmacological manipulation of these neurotransmitter systems using various psychotomimetics, can be used to produce deficits in PPI in rodents.
The dopamine system has been studied extensively both in humans and rodents.
In healthy human subjects, direct dopamine agonists like bromocriptine (a D2 agonist) and indirect dopamine agonists like amphetamine (AMP) have been shown to disrupt PPI
(Abduljawad et al., 1998; Abduljawad et al., 1999; Kumari et al., 1998; Hutchison and
Swift, 1999). Similarly, in many preclinical studies in rats and mice, apomorphine
(APO), which is a direct dopamine agonist, and AMP produce robust decreases in PPI 16
(Mansbach et al., 1988; Swerdlow et al., 2000; Dulawa and Geyer, 1996; Ralph et al.,
1999; Ralph et al., 2001). These PPI disrupting effects of dopamine agonists can be effectively blocked by pretreatment with typical antipsychotics such as haloperidol
(which primarily acts on D2 receptors) suggesting that dopamine and its receptors are important regulators of PPI (Abduljawad et al., 1998; Abduljawad et al., 1999). In humans, dopamine hyperactivity has been implicated in the pathogenesis of schizophrenia and deficits in sensorimotor gating. Few studies report that the PPI deficits seen in schizophrenic patients can be successfully reversed by administration of typical and atypical antipsychotics (which have primary actions on dopamine receptors), suggesting the role of dopamine in the regulation of PPI (Kumari et al., 1999; Kumari et al., 2000; Weike et al., 2000).
Another important neurotransmitter implicated in the regulation of PPI is
glutamate. Dysregulation of the glutamatergic system is thought to be involved in the
etiology of schizophrenia, OCD, and mania; all disorders with abnormal thought
processing (Chakrabarty et al., 2005; Javitt, 1987). In humans, schizophrenia-like
symptoms, including PPI deficits, can be produced by administering NMDA receptor
antagonists, like ketamine. In rodents and non-human primates, deficits in PPI can be
modeled using other NMDA receptor antagonists, like phencyclidine (PCP) or
dizocilpine (MK-801) (Curzon and Decker, 1998; Mansbach and Geyer, 1989; Furuya et
al., 1999; Linn et al., 1999). Further, the effects of NMDA receptor antagonists on PPI
can be reversed using atypical antipsychotics like clozapine, olanzapine, and quetiapine,
which have actions on multiple receptors (Swerdlow et al., 1996; Swerdlow et al., 1998; 17
Yamada et al., 1999). The reversal of PPI with atypical antipsychotics suggests that the regulation of PPI by glutamate might not be due to a single receptor interaction, but might involve many downstream receptors, synapses, and other neurotransmitter systems such as serotonin (Olney et al., 1999).
Interactions of vasopressin and the cortico-striato-pallido-pontine circuitry
Avp is a neuromodulator that is known to interact with the dopamine, glutamate and serotonin systems in various brain areas (Skuse and Gallagher, 2009; Syed et al.,
2007). Abnormalities of the Avp system have been implicated in a variety of neuropsychiatric disorders which are all characterized by a dysfunction of these neurotransmitters. The Avp mutant, BB-Ho rats have also been found to have abnormalities in several neurotransmitter systems including dopamine and serotonin
(Feenstra et al., 1990). They also exhibit profound deficits in PPI at baseline, which can be successfully reversed by using both typical and atypical antipsychotics (Feifel and
Priebe, 2001; Feifel et al., 2007), which suggests that Avp does play some role in regulating PPI and might interact with more than one neurotransmitter system. Exactly how Avp interacts with these neurotransmitter systems still needs to be explored further.
Moreover, whether this deficit in PPI is mediated via Avpr1a or Avpr1b has yet to be investigated. The work described in this thesis set out to determine if the Avpr1b interacts with the neural circuitry that regulates PPI.
Objective 18
Our central hypothesis is that Avp acting via the Avpr1b contributes to the regulation of PPI through its interaction with either the glutamatergic or dopaminergic systems. This hypothesis is based on several pieces of evidence suggesting a role of Avp in regulation of PPI. First, BB-Ho rats, which are genetically deficient in the production of Avp, have been shown to have deficits in baseline PPI. Second, these deficits in PPI can be reversed by infusion of atypical antipsychotics such as clozapine and risperidone
(Feifel et al., 2007). We tested our central hypothesis by treating Avpr1b +/+ and Avpr1b
−/− mice with psychotomimetics and measuring their effects on PPI. The psychotomimetics used in this study were dopamine agonists (APO and AMP) and
NMDA receptor antagonist (MK-801 and PCP), since these are the drugs most commonly used to disrupt PPI.
Experiment 1, was performed to examine the effects of APO, AMP, and MK-801
on PPI in Avpr1b −/− and Avpr1b +/+ mice. It was hypothesized that the Avpr1b −/− mice would be more susceptible to the PPI disruptive effects of psychotics. Experiment 2, was performed to confirm the results of Experiments 1 by comparing the effects of a single drug MK-801 to saline. It was hypothesized that Avpr1b −/− mice treated with
MK-801 would have more disrupted PPI as compared to Avpr1b +/+ mice. Experiment 3, was performed to compare the effects of two different NMDA receptor antagonists MK-
801 and PCP on PPI in Avpr1b −/− and Avpr1b +/+ mice. It was hypothesized that a
stronger disruption of PPI will be seen following treatment with PCP in Avpr1b −/− mice
as compared to MK-801, since PCP augments the effects of glutamate through sigma 19
receptors. Experiment 4 was designed to find out where in the brain the Avp system interacts with glutamatergic system to affect PPI. Therefore, in Experiment 4, quantitative real time PCR was performed to quantify the expression of NMDA receptor mRNA (NMDAR1 and NMDAR2A ) in cortex, striatum and hippocampus in male Avpr1b
−/− and Avpr1b +/+ mice. It was hypothesized that developmental lack of Avpr1b in
Avpr1b −/− mice would cause significant changes in expression of NMDA receptor
mRNA as compared to Avpr1b +/+ mice.
Methods :
General Methods
Animals
This line was originally generated at the National Institute of Mental Health
(NIMH) by W. Scott Young, III (Wersinger et al., 2002). These mice were fully backcrossed into a C57BL/6J background and shipped to Kent State University in 2007 where they have been bred in the vivarium in Cunningham Hall ever since. All the animals used in the study (Avpr1b −/− and Avpr1b +/+ male and females) were offspring from heterozygous breeding pairs. After weaning, animals were reared in the same-sex sibling groups. At the time of weaning (21 days), tails were clipped in order to extract the
DNA, and PCR was performed using the specific primers for Avpr1b for genotyping the animals (Wersinger et al., 2002). For each Experiment, the animals were adults between three and six months of age. Food and water were available ad libitum throughout the studies. All behavior testing was done during the light phase of the 12:12 light dark cycle.
Experiments were conducted in accordance with the protocol approved by the Kent State
University Institutional Animal Care and Use Committee.
Prepulse Inhibition of the Startle Reflex
Drugs
20
21
The following doses of APO, AMP, MK-801 and PCP were used and were chosen based on the literature (Caldwell et al., 2009; Yee et al., 2004) and pilot experiments (Appendix I); APO (10mg/kg), AMP (12mg/kg), MK-801 (0.7mg/kg) and
PCP (8mg/kg). All the drugs were dissolved in 0.9% saline. APO and AMP were diluted to 1.5mg/ml, MK-801 to 10.5mg/ml and PCP to 1.2mg/ml. Saline was given in the same volume as APO and AMP and served as control.
PPI
Two startle chambers were used for all the experiments (SR-LAB; San Diego instruments, San Diego, CA, USA) (Figure 4 and 5). The startle chamber consisted of a small plexiglass cylinder (8 cm diameter, 16cm long) which is mounted on a platform inside a chamber. The loudspeaker inside the chamber generated the background noise as well as all the prepulse and pulse alone tones. The startle response was measured from the whole body reflexive flinch of the animals to acoustic startle stimuli. Vibrations of the animal were sensed and transduced by the piezoelectric unit (motor sensor) attached to the bottom of the platform which is then converted into arbitrary units by the potentiometer and stored in the computer.
Procedure
Each testing session consisted of a 5 min acclimation period followed by 60 trials for 20 min. During each session, background noise of 74dB was generated by the loudspeaker. Sessions consisted of NO PULSE, PULSE ALONE and PREPULSE +
PULSE trials. During NO PULSE trails, no tone was presented. The PULSE ALONE 22
Figure 4: Schematic diagrams representing the components of the SR-LAB system.
23
Figure 5: Acoustic startle chamber (SR LAB, San Diego, CA)
24
trial consisted of 120dB pulse of 40ms duration. During the PREPULSE + PULSE trial, three prepulse tones each 20ms long, of 77dB (3dB above the background), 80dB (6dB above the background) and 86dB (12dB above the background) were presented 100 msec prior to the PULSE tone. Each session began and ended with 5 trials of pulse alone tones.
In between the pulse alone trials, each session consisted of 10 blocks of 5 trials, during which different stimuli were presented in a random order.
Study Design
Prior to drug testing, each animal was tested for baseline PPI without any drug
treatment. Following baseline testing, repeated measures testing with drugs was done
with each animal. APO, AMP, MK-801, PCP and saline (0.9%) were administered
intraperitoneally (i.p.) to animals in a counterbalanced manner, though which drug was
administered depended on the Experiment. A minimum of 1 week between testing was
observed in order to avoid any residual drug effects. PPI percentage was calculated using
the following equation: (1-(startle amplitude of the PREPULSE + PULSE/startle
amplitude of the PULSE ALONE)) × 100. Habituation of startle was calculated using the
average responses from the first 5 PULSE ALONE trials (Pulse 1) and last 5 PULSE
ALONE (Pulse 2) trials. Magnitude of the startle was calculated from the average of all
the PULSE ALONE trials during the entire session excluding the first and last blocks of
PULSE ALONE trials (Figure 6).
25
10 Blocks of 5 Trials 5 Pulse 5 Pulse Alone Trials NO PULSE Alone Trials
PREPULSE + PULSE (3X) PULSE 1 PULSE 2
PULSE ALONE
Startle amplitude was Habituation of startle PPI percentage was was measured from calculated from the calculated from the average responses to the average responses average response PULSE ALONE of the first 5 (PULSE from the 10 blocks of trials. Comparisons 1) and last 5 pulse 5 trials each using
were made between alone (PULSE 2) the equation: (1- drug treatments as trials and (startle amplitude of compared to saline. comparisons were the prepulse + pulse made within drug trial / startle treatment. amplitude of pulse
alone trial)) × 100.
Comparisons were made between genotypes and drug treatment with respect to saline.
Figure 6: Study design used to analyze startle amplitude, habituation of startle, and PPI percentage.
26
Quantitative Real Time Polymerase Chain Reaction
Procedure
Brains were collected from male Avpr1b −/− and Avpr1b +/+ mice and frozen on dry ice. Tissue punches of 1.00 mm diameter were collected from a 400 µm slab cut on
cryostat, from the striatum, hippocampus and frontal cortex. Samples were homogenized
using homogenizer and RNA was extracted using the RiboPure kit (Ambion, The RNA
Company, Austin, TX). The purity of the sample was established by using the Nanodrop
Spectrophotometer ND-100 (Thermo-Scientific) and 1 µl of extract. Samples having a
ratio of 260/280 between 1.8-2.0 were included in the assay. Purified RNA was reverse
transcribed into cDNA using a ThermoScript RT- PCR system (Invitrogen, Carlsbad, CA,
USA). Specific Taqman primers for NMDAR1 and NMDAR2A were purchased from
Applied Biosystems. Quantitative real time PCR was performed using Stratagene QPCR
System (Agilent Technologies, Foster City, CA, USA). The genes quantified were
NMDAR1 and NMDAR2A within the brain areas mentioned using specific primers.
Triplicates of 20 µl reaction with Taqman Universal master mix and 2 µl of cDNA in
each well were used to perform the assay. GAPDH was used as the control for normalization.
Individual Experiments
Experiment 1: Assessment of prepulse inhibition of the startle reflex in A) female
Avpr1b +/++/++/+ and Avpr1b −/−−/−−/− mice and B) male Avpr1b +/++/++/+ and Avpr1b −/−−/−−/− mice following treatment with APO, AMP, MK-801, and saline 27
In Experiment 1A, 13 Avpr1b +/+ and 10 Avpr1b −/− female mice were used as subjects. In Experiment 1B, 11 Avpr1b +/+ and 12 Avpr1b −/− male mice were used. The animals were run in two different cohorts at two different times. Each cohort consisted of a mix of males and females. However, for analysis purposes, males and females were considered separately. Each animal was initially tested for baseline PPI measurement without any drugs. For the drug studies, APO, AMP, MK-801 and saline (0.9%) were administered i.p. 10 min prior to testing in a counterbalanced manner. Habituation, startle and PPI were measured as described under statistics section.
Experiment 2: Assessment of the effects of MK-801 and saline on prepulse inhibition
of the startle reflex in A) female Avpr1b +/++/++/+ and Avpr1b −/−−/−−/− mice and B) male
Avpr1b +/++/++/+ and Avpr1b −/− mice
In Experiment 2A, subjects were 9 Avpr1b +/+ and 7 Avpr1b −/− female mice. In
Experiment 2B, 9Avpr1b +/+ and 10 Avpr1b −/− male mice were used. Males and
females were run at the same time; however analyzed separately. MK-801 and saline
(0.9%) were given i.p. 10 min prior to testing. Data were analyzed as mentioned under
statistics section.
Experiment 3: Assessment of prepulse inhibition of startle reflex in response to MK-
801, PCP, and saline in Avpr1b +/++/++/+ and Avpr1b −/−−/−−/− male mice
Only 10 Avpr1b −/− and 7 Avpr1b +/+ male mice were used as study subjects
since a robust effect of MK-801 on PPI was seen only in males in the Experiment 1. MK- 28
801, PCP and saline (0.9%) were administered i.p. in a counterbalanced manner and data were analyzed as described under statistics section.
Statistics
For Experiments 1, 2, and 3, baseline PPI percentage was analyzed across genotypes using repeated measure of analysis of variance (ANOVA) comparing the main effects of genotype, sex and prepulse intensity as well as their interactions. For the drug studies, PPI percentage was determined using repeated ANOVA and main effects of genotype, drug treatment, prepulse intensity and their interactions were compared.
Effects in both the sexes were analyzed independently. Animals with PPI percentage within two standard deviations of the mean were included in statistical analysis. Since we were interested in habituation across the test session; effect of drugs on habituation was measured using repeated ANOVA with weight as a covariate. To correct for multiple comparisons, the α was adjusted to p<0.013. Main effects of the first five PULSE
ALONE trials were compared to last five PULSE ALONE trials as well as those of genotype and any interactions. Startle was compared using a repeated measures ANOVA.
Main effects of drug treatment, genotype, and their interactions were examined.
Experiment 4: Assessment of gene expression of A) NMDAR1 and B) NMDAR2A in male Avr1b +/++/++/+ and Avpr1b −/−−/−−/− mice
Subjects were 5 Avpr1b −/− and 5 Avpr1b +/+ male mice. Animals were killed by cervical dislocation and brains removed. Brains were immediately frozen on dry ice and 29
stored at -80C until tissue collection and quantitative real time PCR procedure were performed as described under quantitative real time PCR methods section.
Statistics
For Experiment 4, the Ct values from the samples were normalized against
GAPDH . The expression values were then calculated using the equation: 2 -∆Ct .
Comparisons were then made between genotypes by using t-test.
Results:
Experiment 1A: Assessment of PPI in Avpr1b +/++/++/+ and Avprb1 −/−−/−−/− female mice
following treatment with saline, APO, AMP and MK-801.
Habituation of startle
For all the drug treatments, there were no main effects of the drugs, genotype or any
interactions on habituation of startle. There was no habituation to startle between Pulse 1
and Pulse 2 trial sessions within any of the drugs tested (Figure 7).
Startle amplitude
There was a main effect of the drugs on startle amplitude (F 3,54 = 16.042, p<0.05), with
APO and AMP decreasing the startle amplitude, whereas MK-801 increased the startle
amplitude. There was no main effect of genotype, or any interactions on the startle (Table
1).
PPI Percentage
Baseline : There were no genotypic differences in the baseline PPI percentage. There were
the expected main effects of prepulse intensity (F 2,36 = 25.073, p<0.05), resulting in
increased PPI percentage with increasing prepulse tone (Figure 8).
30
31
Drug treatment : There was a main effect of the drug treatment (F3,54 = 11.636, p<0.05) with APO, AMP and MK-801 all resulting in disruption of PPI as compared to saline.
There was also a main effect of prepulse intensity (F 2,36 = 60.785, p<0.05). However,
there were no genotypic differences in the females on PPI percentage or any other
interactions (Figure 9) and (Table 2).
Experiment 1B: Assessment of PPI in male Avpr1b +/++/++/+ and Avprb1 −/−−/−−/− mice
following treatment with saline, APO, AMP and MK-801
Habituation of startle
There were no main effects of drug treatments, genotype or any interactions on the
habituation of startle across Pulse 1 and Pulse 2 sessions in male Avpr1b +/+ and Avpr1b
−/− mice (Figure 10).
Startle amplitude
There was a main effect of the drugs on startle amplitude (F 3,60 = 34.775, p<0.05), with
APO decreasing the startle amplitude whereas AMP and MK-801 increased the startle
amplitude. There was no main effect of genotype, or any interactions on the startle (Table
3).
PPI Percentage 32
Saline APO
300 300 * Pulse 1 Pulse 1 250 Pulse 2 250 Pulse 2
200 200
150 150 Startle Amplitude 100 Startle Amplitude 100
50 50
0 0 Avpr1b +/+ Avpr1b −/− Avpr1b +/+ Avpr1b −/−
AMP MK-801
300 300 * Pulse 1 Pulse 1 250 Pulse 2 250 Pulse 2
200 200
150 150 Startle Amplitude 100 Amplitude Startle 100
50 50
0 0 Avpr1b +/+ Avpr1b −/− Avpr1b +/+ Avpr1b −/−
Figure 7: Habituation of startle in female Avpr1b +/+ and Avpr1b −/− mice. There was no habituation of startle to drugs; Saline (0.9%), APO (10mg/kg), AMP (12mg/kg) and MK-801 (0.7mg/kg), in Avpr1b +/+ and Avpr1b −/− female mice. Data are expressed as means + standard error of the mean (Abbreviations: APO, apomorphine; AMP, amphetamine; MK-801, dizocilpine). 33
Mean ± Standard Error of the Mean (Startle amplitude)
Drugs Avpr1b +/++/++/+ Avpr1b −/−−/−−/−
Saline 126.36 ± 18.25 111.83 ± 12.75
APO* 56.91 ± 9.70 47.88 ± 7.95
AMP* 82.50 ± 8.72 93.33 ± 10.01
MK-801* 127.97 ± 27.23 159.25 ± 9.21
Table 1: Startle amplitude in female Avpr1b +/+ and Avpr1b −/− mice. There was a main effect of the drug treatment (p<0.05), with APO and AMP decreasing the startle amplitude whereas MK-801 increased the startle amplitude in both the genotypes. There was no genotypic difference between the Avpr1b +/+ and Avpr1b −/− mice. Startle was calculated from the averages responses from the first and last pulse alone (120dB) trials. (*p < 0.05 indicating a main effect of drug compared to saline) (Abbreviations: APO, apomorphine; AMP, amphetamine; MK-801, dizocilpine).
34
Avpr1b +/+ 100 −/− * Avpr1b * 80
60
40 % Prepulse Inhibition %
20
0 3dB 6dB 12dB
Figure 8: Baseline PPI percentage in female Avpr1b +/+ and Avprb1 −/− mice. There was no genotypic difference in baseline PPI percentage but there was a main effect of prepulse intensity with the higher decibel prepulse tone resulting in greater PPI percentage (* indicates p<0.05). Data are expressed as means + standard error of the mean.
35
Avpr1b +/+
100 3dB 6dB 12dB
80
60
40 % Prepulse Inhibtion Prepulse %
20
0 SALINE APO AMP MK801
Avpr1b −/−
100 3dB 6dB 12dB
80
60
40 % Prepulse% Inhibtion
20
0 SALINE APO AMP MK801
Figure 9: PPI percentage in female Avpr1b +/+ and Avpr1b −/− mice following drug treatment. Treatment with all the three drugs, APO, AMP, and MK-801 resulted in disruption of PPI as compared to saline (p<0.05). There were however no genotypic differences between Avpr1b +/+ and Avpr1b −/− female mice. Data are expressed as means + standard error of the mean. (Abbreviations: APO, apomorphine; AMP, amphetamine; MK-801, dizocilpine). 36
Mean ± Standard Error of the Mean (PPI percentage) Drugs and Prepulse Avpr1b +/++/++/+ Avpr1b −/−−/−−/− Levels
Saline 77 63.40 ± 4.56 66.61 ± 3.53
Saline 80 73.42 ± 3.19 74.14 ± 3.52
Saline 86 78.68 ± 2.21 82.47 ± 1.74
APO 77 48.91 ± 4.02 38.60 ± 9.87
APO 80 57.35 ± 3.62 52.87 ± 6.67
APO 86 58.46 ± 3.02 55.39 ± 10.41
AMP 77 46.51 ± 4.76 38.92 ± 7.58
AMP 80 55.60 ± 5.01 55.75 ± 2.71
AMP 86 63.22 ± 4.14 62.53 ± 3.51
MK-801 77 31.49 ± 9.29 40.22 ± 4.28
MK-801 80 47.78 ± 5.99 42.98 ± 6.62
MK-801 86 63.31 ± 5.62 68.43 ± 4.37
Table 2: Mean and standard error of the mean for drug treatment in female Avpr1b +/+ and Avpr1b −/− mice. (Abbreviations: APO, apomorphine; AMP, amphetamine; MK- 801, dizocilpine)
37
Baseline: There was a main effect of the prepulse intensity (F 2,40 = 38.869, p<0.05), with
greater PPI percentage with higher intensity tone. However there were no genotypic
differences in baseline PPI percentage in Avpr1b +/+ and Avpr1b −/− males (Figure 11).
Drug treatment: There was a main effect of drug treatment (F 3,60 = 28.954, p<0.05) and
prepulse intensity (F 2,40 = 121.057, p<0.05). APO, AMP and MK-801 disrupted the PPI
as compared to saline in both Avpr1b +/+ and Avpr1b −/− male mice but there was no
main effect of genotype. However, there was a genotype × drug interaction (F 3,60 = 6.018, p<0.05). Post hoc tests with repeated ANOVA revealed that treatment with MK-801 resulted in more disruption of PPI in Avpr1b −/− mice as compared to Avpr1b +/+ mice
(p<0.05). There were no other interactions of genotype with any other drug treatment
(Figure 12) and (Table 4).
Experiment 2A: Assessment of prepulse inhibition of startle reflex in female
Avpr1b +/++/++/+ and Avpr1b −/−−/−−/− mice following administration of MK-801
Habituation of Startle
There was no habituation to startle reflex in female Avpr1b +/+ and Avpr1b −/− mice with saline or MK-801 (Figure 13). There was no main effect of genotype, drug or any other interaction.
Startle amplitude 38
Saline APO
300 300 * * Pulse 1 Pulse 1 250 Pulse 2 250 Pulse 2
200 200
150 150
Startle Amplitude Startle 100 Startle Amplitude Startle 100
50 50
0 0 Avpr1b +/+ Avpr1b −/− Avpr1b +/+ Avpr1b −/−
AMP MK-801
300 300 * * Pulse 1 Pulse 1 250 Pulse 2 250 Pulse 2
200 200
150 150
Startle Startle Amplitude 100 Startle Amplitude Startle 100
50 50
0 +/+ −/− 0 Avpr1b Avpr1b Avpr1b +/+ Avpr1b −/−
Figure 10: Habituation of startle in male Avpr1b +/+ and Avpr1b −/− mice. There were no main effects of the drugs; Saline (0.9%), APO (10mg/kg), AMP (12mg/kg) and MK- 801 (0.7mg/kg) on habituation of startle on Avpr1b +/+ and Avpr1b −/− mice. Data are expressed as mean + standard error of the mean. (Abbreviations: APO, apomorphine; AMP, amphetamine; MK-801, dizocilpine)
39
Mean ± Standard Error of the Mean (Startle amplitude)
Drugs Avpr1b +/++/++/+ Avpr1b −/−−/−−/−
Saline 137.12 ± 18.95 116.91 ± 13.95
APO* 76.19 ± 11.65 96.99 ± 11.38
AMP* 142.35 ± 13.80 117.23 ± 14.56
MK-801* 243.21 ± 29.89 232.95 ± 32.07
Table 3: Startle amplitude in male Avpr1b +/+ and Avpr1b −/− mice. There was a main effect of the drug treatment on startle amplitude (p<0.05), with APO decreasing the startle amplitude whereas AMP and MK-801 increased the startle amplitude in both genotypes. There was no genotypic difference between the Avpr1b +/+ and Avpr1b −/− mice. Startle was calculated from the averages responses from the first and last pulse alone (120dB) trials. (*p < 0.05 indicating a main effect of drug compared to saline) (Abbreviations: APO, apomorphine; AMP, amphetamine; MK-801, dizocilpine).
40
Avpr1b +/+ 100 −/− * Avpr1b * 80
60
40 % Prepulse Inhibition Prepulse %
20
0 3dB 6dB 12dB
Figure 11: Baseline PPI percentage in male Avpr1b +/+ and Avprb1 −/− mice. There was no genotypic difference in baseline PPI percentage but there was a main effect of prepulse intensity with the higher decibel prepulse tone resulting in greater PPI percentage (* indicates p<0.05). Data are expressed as mean + standard error of the mean. 41
Avpr1b +/+
100 3dB 6dB 12dB
80
60
40 % PrepulseInhibtion %
20
0 SALINE APO AMP MK801
Avpr1b −/−
100 3dB 6dB 12dB
80
*
60 *
40 % Inhibtion Prepulse
20
0 SALINE APO AMP MK801
Figure 12: PPI percentage following drug treatment in males. Treatment with all the three drugs, APO, AMP, and MK-801 resulted in disruption of PPI as compared to saline (p<0.05). There was no main effect of the genotype; however there was a genotype × drug interaction (p<0.05). Specifically, treatment with MK-801 resulted in greater disruption of PPI in male Avpr1b −/− mice as compared to Avpr1b +/+ mice (* indicates p<0.05). Data are expressed as mean + standard error of the mean. (Abbreviations: APO, apomorphine; AMP, amphetamine; MK-801, dizocilpine) 42
Mean ± Standard Error of the Mean (PPI percentage) Drugs and Prepulse Avpr1b +/++/++/+ Avpr1b −/−−/−−/− Levels
Saline 77 67.33 ± 3.62 57.16 ± 3.89
Saline 80 73.25 ± 3.63 71.90 ± 2.08
Saline 86 78.06 ± 2.47 80.03 ± 1.79
APO 77 51.56 ± 6.30 50.17 ± 5.53
APO 80 66.23 ± 4.03 62.22 ± 4.41
APO 86 65.91 ± 4.11 65.54 ± 4.66
AMP 77 30.11 ± 2.26 39.46 ± 3.98
AMP 80 46.88 ± 5.10 54.62 ± 4.83
AMP 86 54.28 ± 3.62 62.29 ± 3.29
MK-801 77 45.37 ± 3.86 34.62 ± 5.09
MK-801 80 57.80 ± 6.35 42.88 ± 6.20
MK-801 86 73.44 ± 3.55 54.57 ± 7.72
Table 4: Mean and standard error of the mean for drug treatments on male Avpr1b +/+ and Avpr1b −/− mice. (Abbreviations: APO, apomorphine; AMP, amphetamine; MK- 801, dizocilpine)
43
There was a main effect of the drug on startle amplitude (F 1,14 = 13.842, p<0.05), with
MK-801 increasing the startle amplitude. There was no main effect of genotype, or any
interactions on the startle amplitude (Table 5).
PPI Percentage
Baseline : There was no genotypic difference between Avpr1b +/+ and Avpr1b −/− female
mice in baseline PPI percentage. However, there was a main effect of prepulse intensity
(F 2,26 = 14.393, p< 0.05), with higher prepulse tone resulting in greater PPI percentage
(Figure 14).
Drug treatment : There was a main effect of the drug treatment (F 1,14 = 13.095, p<0.05)
and prepulse intensity (F 2,28 = 34.016, p<0.05). There was no genotypic effect or any
interactions in Avpr1b +/+ and Avpr1b −/− female mice (Figure 15) and (Table 6).
Experiment 2B: Assessment of prepulse inhibition of startle reflex in Avpr1b
+/+ and Avpr1b −/−−/−−/− male mice following administration of MK-801
Habituation of startle reflex
Male Avpr1b +/+ and Avpr1b −/− mice did not show habituation to startle reflex across the test sessions with either saline or MK-801 (Figure 16).
Startle amplitude
There was a main effect of the drug (MK-801) on startle amplitude, (F 1,15 = 5.587,
p<0.05), with MK-801 increasing the amplitude of the startle reflex. There was no 44
Saline MK-801
300 300 * * Pulse 1 Pulse 1 250 Pulse 2 250 Pulse 2
200 200
150 150 Startle Amplitude Startle 100 Amplitude Startle 100
50 50
0 0 Avpr1b +/+ Avpr1b −/− Avpr1b +/+ Avpr1b −/−
Figure 13: Habituation of startle in Avpr1b +/+ and Avpr1b −/− female mice. There was no habituation of startle between PULSE 1 and PULSE 2 trials with saline (0.9%) and MK-801 (0.7mg/kg) in both genotypes. Data are expressed as mean + standard error of the mean. (Abbreviations: MK-801, dizocilpine)
45
Mean ± Standard Error of the Mean (Startle amplitude)
Drugs Avpr1b +/++/++/+ Avpr1b −/−−/−−/−
Saline 49.94 ± 6.60 56.84 ± 11.46
MK-801* 95.06 ± 22.44 93.43 ± 18.18
Table 5: Startle amplitude in female mice following drug treatment. There was a main effect of the drug on startle amplitude (p<0.05), with MK-801 increasing the startle amplitude in both the genotypes. There was no genotypic difference between the Avpr1b +/+ and Avpr1b −/− mice. Startle was calculated from the averages responses from the first and last pulse alone (120dB) trials. (*p < 0.05 indicating a main effect of drug compared to saline) (Abbreviations: MK-801, dizocilpine)
46
Avpr1b +/+ 100 Avpr1b −/− * * 80
60
40 % Prepulse Inhibition %
20
0 3dB 6dB 12dB Prepulse Intensities dB above background A
Figure 14: Baseline PPI percentage in females. There was no genotypic difference between Avpr1b +/+ and Avpr1b −/− females, but an effect of prepulse intensity was present (p<0.05), with higher prepulse tone resulting in greater PPI percentage. (* indicates p<0.05). Data are expressed as mean + standard error of the mean.
47
Avpr1b +/+
100 3dB 6dB 12dB 80
60
40 % Prepulse% Inhibtion
20
0 SALINE MK801
Avpr1b −/−
100 3dB 6dB 12dB
80
60
40 % Prepulse Inhibtion Prepulse %
20
0 SALINE MK801
Figure 15: PPI percentage following drug treatment in females. There was a main effect of the drug (p<0.05) with MK-801 disrupting the PPI in both Avpr1b +/+ and Avpr1b −/− female mice. However, there was no genotypic difference in PPI between Avpr1b +/+ and Avpr1b −/− females. Data are expressed as mean + standard error of the mean. (Abbreviations: MK-801, dizocilpine)
48
Mean ± Standard Error of the Mean (PPI Percentage) Drugs and Prepulse Avpr1b +/++/++/+ Avpr1b −/−−/−−/− Levels
Saline 77 64.24 ± 6.57 67.29 ± 4.05
Saline 80 71.46 ± 4.33 72.62 ± 3.25
Saline 86 78.86 ± 2.49 78.43 ± 2.98
MK-801 77 34.72 ± 7.63 46.68 ± 6.31
MK-801 80 51.19 ± 7.54 58.20 ± 6.63
MK-801 86 60.58 ± 4.53 65.38 ± 6.63
Table 6: Mean and standard error of the mean for drug treatment in female Avpr1b +/+ and Avpr1b −/− mice. (Abbreviations: MK-801, dizocilpine)
49
genotypic difference between Avpr1b +/+ and Avpr1b −/− male mice in startle amplitude
(Table 7).
PPI Percentage
Baseline : As expected there was a main effect of the prepulse intensity (F 2,32 = 17.968, p<0.05), but there was no genotypic difference (Figure 17).
Drug Treatment : There was a main effect of the drug treatment (F1,17 = 20.883, p<0.05)
with MK-801 disrupting the PPI as compared to saline in both Avpr1b +/+ and Avpr1b
−/− male mice. There was also a main effect of the prepulse intensity (F 2, 34 = 71.671,
p<0.05), but there was no main effect of the genotype or any other interactions (Figure
18) and (Table 8).
Experiment 3: Assessment of prepulse inhibition of startle reflex in male Avpr1b +/++/++/+ and Avpr1b −/−−/−−/− mice following administration of MK-801, PCP and saline
Habituation of startle
There was no habituation to startle reflex across the test trials PULSE 1 and PULSE 2 in
both Avpr1b +/+ and Avpr1b −/− males (Figure 19).
Startle amplitude
There was a main effect of the drug on startle amplitude (F 2,28 = 10.803, p<0.05), with
MK-801 increasing the startle amplitude and PCP decreasing the startle amplitude. 50
Saline MK-801
300 300 * * Pulse 1 Pulse 1 250 Pulse 2 250 Pulse 2
200 200
150 150 Startle Amplitude Startle 100 Amplitude Startle 100
50 50
0 0 Avpr1b +/+ Avpr1b −/− Avpr1b +/+ Avpr1b −/−
Figure 16: Habituation of startle reflex in males. There was no main effect of saline (0.9%) and MK-801 (0.7mg/kg) on habituation of startle across test trials PULSE 1 and PULSE 2 on male Avpr1b +/+ and Avpr1b −/− mice. Data are expressed as mean + standard error of the mean. (Abbreviations: MK-801, dizocilpine)
51
Mean ± Standard Error of the Mean (Startle Amplitude)
Drugs Avpr1b +/++/++/+ Avpr1b −/−−/−−/−
Saline 58.00 ± 4.02 73.01 ± 8.09
MK-801* 110.33 ± 21.32 109.30 ± 20.55
Table 7: Effects of drugs on startle amplitude on males. Treatment with MK-801 resulted in an increase in the startle amplitude as compared to saline in both Avpr1b +/+ and Avpr1b −/− male mice (p<0.05). There was no genotypic difference between the Avpr1b +/+ and Avpr1b −/− male mice. Startle was calculated from the averages responses from the first and last pulse alone (120dB) trials. (*p < 0.05 indicating a main effect of drug compared to saline) (Abbreviations: MK-801, dizocilpine)
52
Avpr1b +/+ 100 * Avpr1b −/− *
80
60
40 % Prepulse Inhibition Prepulse %
20
0 3dB 6dB 12dB
Figure 17: Baseline PPI percentage in males. There was a main effect of the prepulse intensity (p<0.05); with increased PPI percentage with higher decibel prepulse tone. However, there was no effect of the genotype on baseline PPI percentage in male Avpr1b +/+ and Avpr1b −/− mice. (* indicates p<0.05). Data are expressed as mean + standard error of the mean.
53
Avpr1b +/+
100 3dB 6dB 12dB
80
60
40 % Prepulse Prepulse Inhibtion %
20
0 SALINE MK801
Avpr1b −/−
100 3dB 6dB 12dB
80
60
40 % Prepulse Inhibtion Prepulse %
20
0 SALINE MK801
Figure 18: PPI percentage following drug treatment in males. There was a main effect of the drug (p<0.05), with MK-801 disrupting the PPI in both Avpr1b +/+ and Avpr1b −/− male mice. However, there was no genotypic difference in PPI percentage between Avpr1b +/+ and Avpr1b −/− males. Data are expressed as mean + standard error of the mean. (Abbreviations: MK-801, dizocilpine)
54
Mean ± Standard Error of the Mean (PPI Percentage)
Drugs Avpr1b +/++/++/+ Avpr1b −/−−/−−/−
Saline 77 72.11 ± 5.88 58.29 ± 7.90
Saline 80 77.75 ± 3.78 64.66 ± 8.37
Saline 86 86.59 ± 1.58 77.40 ± 4.11
MK-801 77 48.53 ± 4.47 30.29 ± 6.78
MK-801 80 63.12 ± 5.46 51.21 ± 6.07
MK-801 86 72.03 ± 4.19 56.27 ± 6.40
Table 8: Mean and standard error of the mean of PPI percentage following drug treatment in Avpr1b +/+ and Avpr1b −/− males. (Abbreviations: MK-801, dizocilpine)
55
However there was no main effect of genotype or any other interactions on Avpr1b +/+
and Avpr1b −/− mice (Table 9).
PPI Percentage
Baseline: There was a main effect of prepulse intensity (F 2,28 = 24.079, p<0.05), with greater PPI percentage with higher decibel prepulse tone. There was no main effect of the genotype on baseline PPI percentage (Figure 20).
Drug Treatment: There was a main effect of drug treatment (F 2,28 = 4.659, p<0.05) with both MK-801 and PCP disrupting the PPI in Avpr1b +/+ and Avpr1b −/− mice as compared to saline. There was also a main effect of the prepulse intensity (F 2,28 =
105.553, p<0.05). However there was no genotypic effect or any other interactions
(Figure 21) (Table 10).
Experiment 4A: Gene expression of NMDAR1 in Avpr1b +/++/++/+ and Avpr1b −/−−/−−/− male
mice
In this experiment, expression of NMDAR1 was examined in cortex, striatum and hippocampus in Avpr1b +/+ and Avpr1b −/− male mice. There were no genotypic differences in mRNA levels of NMDAR1 receptors in cortex (p=0.365), striatum
(p=0.083) or hippocampus (p=0.137) (Figure 22).
Experiment 4B: Gene expression of NMDAR2A in Avpr1b +/++/++/+ and Avpr1b −/−−/−−/− male
mice 56
Saline
300 * Pulse 1 250 Pulse 2
200
150
Startle Startle Amplitude 100
50
0 Avpr1b +/+ Avpr1b −/−
MK-801 PCP
300 300 * * Pulse 1 Pulse 1 250 Pulse 2 250 Pulse 2
200 200
150 150 Startle Amplitude Startle 100 Startle Amplitude 100
50 50
0 0 Avpr1b +/+ Avpr1b −/− Avpr1b +/+ Avpr1b −/−
Figure 19: Habituation of startle in males. There was no habituation of startle between PULSE 1 and PULSE 2 in both Avpr1b +/+ and Avpr1b −/− males on treatment with saline (0.9%), MK-801 (0.7mg/kg) and PCP (1.2mg/kg). Data are expressed as mean + standard error of the mean. (Abbreviations: MK-801, dizocilpine; PCP, phencyclidine)
57
Mean ± Standard Error of the Mean (Startle amplitude)
Drugs Avpr1b +/++/++/+ Avpr1b −/−−/−−/−
Saline 90.83 ± 7.11 68.51 ± 8.31
MK-801* 103.83 ± 16.36 143.71 ± 17.83
PCP* 85.91 ± 13.20 96.14 ± 11.53
Table 9: Effects of drugs on startle amplitude on males. There was a main effect of the drugs on startle amplitude (p<0.05) with MK-801 increasing the startle amplitude and PCP decreasing the startle amplitude. There was no genotypic difference between the Avpr1b +/+ and Avpr1b −/− mice. Startle was calculated from the averages responses from the first and last pulse alone (120dB) trials. (*p < 0.05 indicating a main effect of drug compared to saline) (Abbreviations: MK-801, dizocilpine; PCP, phencyclidine)
58
Avpr1b +/+ 100 Avpr1b −/− * 80 *
60
40 % PrepulseInhibition %
20
0 3dB 6dB 12dB
Figure 20: Baseline PPI percentage in males. There was no genotypic difference between male Avpr1b +/+ and Avpr1b −/− mice in baseline PPI percentage. There was the expected main effect of prepulse intensity with greater PPI percentage with higher decibel prepulse tone (* indicates p<0.05). Data are expressed as mean and standard error of the mean.
59
Avpr1b +/+
100 3dB 6dB 12dB
80
60
40 % Prepulse Inhibtion %
20
0 SALINE MK-801 PCP
Avpr1b −/−
100 3dB 6dB 12dB
80
60
40 % Prepulse % Inhibtion
20
0 SALINE MK-801 PCP
Figure 21: PPI percentage following drug treatment in males. Treatment with MK-80 and PCP resulted in disruption of PPI as compared to saline in both Avpr1b +/+ and Avpr1b −/− mice (p<0.05). However there was no genotypic difference in PPI percentage. Data are expressed as mean + standard error of the mean. (Abbreviations: MK-801, dizocilpine; PCP, phencyclidine) 60
Mean ± Standard Error of the Mean Drugs and Prepulse Avpr1b +/++/++/+ Avpr1b −/−−/−−/− Levels
Saline 77 57.89 ± 6.61 51.75 ± 6.45
Saline 80 70.53 ± 4.36 63.39 ± 4.59
Saline 86 78.39 ± 2.50 73.78 ± 5.38
MK-801 77 51.97 ± 6.27 35.92 ± 9.39
MK-801 80 63.81 ± 6.03 47.16 ± 7.83
MK-801 86 69.41 ± 7.37 60.94 ± 7.48
PCP 77 41.95 ± 6.00 39.72 ± 8.23
PCP 80 50.63 ± 6.00 53.48 ± 6.96
PCP 86 67.80 ± 4.93 62.27 ± 7.62
Table 10: Mean and stand error of the mean for drug treatment on PPI percentage in male Avpr1b +/+ and Avpr1b −/− mice. (Abbreviations: MK-801, dizocilpine; PCP, phencyclidine)
61
In this experiment, expression of NMDAR2A was evaluated in cortex, striatum and hippocampus of Avpr1b +/+ and Avpr1b −/− male mice. Avpr1b −/− mice had significantly higher mRNA levels of NMDAR2A receptors as compared to Avpr1b +/+ mice in the hippocampus (p= 0.003). No differences in NMDAR2A levels were found in cortex (p=0.897) and striatum (p=0.527) (Figure 23).
62
200 p=0.14
Avpr1b +/+ Avpr1b −/−
150
p=0.08
100 Relative Expression Relative 50
0 Cortex Striatum Hippocampus
Figure 22: Expression of NMDAR1 in male Avpr1b +/+ and Avpr1b −/− mice. There were no genotypic differences in mRNA levels of NMDAR1 in cortex, striatum and hippocampus. Data are expressed as mean + standard error of the mean.
63
** 200
Avpr1b +/+ Avpr1b −/− 150
100 Relative Expression Relative 50
0 Cortex Striatum Hippocampus
Figure 23: Expression of NMDAR2A in male Avpr1b +/+ and Avpr1b −/− mice. Avpr1b −/− males had significantly higher mRNA levels of NMDAR2A expression in the hippocampus as compared to Avpr1b +/+ mice (** indicates p<0.01). There were no genotypic differences in NMDAR2A expression in cortex and striatum. Data are expressed as mean + standard error the mean.
Discussion
The work described in this thesis has helped elucidate the role of the Avpr1b in the neural circuitry that underlies sensorimotor gating. In Experiments 1A and 2A, we examined the effect of various PPI-disrupting drugs on PPI and habituation in female
Avpr1b +/+ and Avpr1b −/− mice. It was found that treatment with APO, AMP, MK-801, all resulted in disruption of PPI in both Avpr1b +/+ and Avpr1b −/− females. However, no genotype or genotype × drug differences were observed between Avpr1b +/+ and
Avpr1b −/− females. Our results are consistent with many other studies where no genotypic differences have been found in PPI in females despite its presence in males.
Further, given the lack of effect in females, we did not include them in Experiment 3.
In Experiment 1B, we found that treatment with MK-801, but not APO and AMP, resulted in greater disruption of PPI in male Avpr1b −/− mice as compared to male
Avpr1b +/+ mice, indicating that male Avpr1b −/− mice are more susceptible to the PPI- disrupting effects of MK-801. As MK-801 is a selective NMDA receptor antagonist
(Wong et al., 1986), these data suggest that male Avpr1b −/− mice have an impairment in their glutamatergic system.
Experiment 2B was performed to verify the results of Experiment 1B, though, only two drugs were used, MK-801 and saline. We found that in male Avpr1b +/+ and
Avpr1b −/− mice, there were no genotypic differences in PPI percentage following
64
65
treatment with MK-801. Although the differences in PPI percentage between the genotypes were not statistically significant, they showed a similar trend as in Experiment
1B; with male Avpr1b −/− mice having an inclination for greater PPI disruption by MK-
801 as compared to male Avpr1b +/+ mice (p=0.19).
In Experiment 3, we wanted to repeat aspects of Experiment 1 and 2 and add another NMDA antagonist, PCP, which has a slightly different pharmacology than MK-
801. PCP is the most commonly used NMDA receptor antagonist to study PPI (Geyer et al., 2001 and the references within). However, PCP binds to receptors other than NMDA receptors, such as sigma opioid receptors (Contreras et al., 1988; Largent et al., 1986;
Sonders et al., 1988),which are known to further facilitate glutamate transmission (Su and
Hayashi, 2003). It was found that PCP produced a stronger disruption of PPI than MK-
801 in both Avpr1b +/+ and Avpr1b −/− mice. However, contrary to our hypothesis, there were no drug specific genotypic differences.
Based on the finding from Experiment 1B, we decided to explore possible changes in the glutamatergic system in male Avpr1b −/− mice in Experiment 4, using quantitative real time PCR to quantify mRNA levels of the NMDA receptors subunits
NMDAR1 and NMDAR2A. Interestingly, we found that the NMDAR2A subunit is increased in the hippocampus of Avpr1b −/− mice as compared to Avpr1b +/+ mice.
The lack of differences in PPI between Avpr1b +/+ and Avpr1b −/− females in
Experiment 1A and 2A was not surprising since there are other studies which have found a differential response of females to these drugs as compared to males. The lack of a 66
genotypic effect in females may be due to the variability in the hormonal milieu throughout the estrous cycle. This idea is supported by one study in humans, which found that there are sex differences in PPI, with women having lower PPI than males
(Swerdlow et al., 1993). Further, there is evidence that cyclic changes in gonadal steroids, in particular estrogens, can affect PPI across the menstrual cycle (Swerdlow et al., 1997).
These data from humans are consistent with data in female rats, that show that PPI varies across the estrous cycle (Aurebach et al., 1997, (Koch, 1998). In a number of other studies, estrogens have been reported to influence monoamines in brain regions which are important for regulation of PPI (Becker, 1990; Joyce and van, 1984; Thompson and
Moss, 1997; Van and Joyce, 1986). These hormonal effects of estrogens on PPI in females make it difficult to interpret the results seen in our study. It would be interesting to investigate PPI in ovarectomized female mice treated with estradiol.
The data in Experiment 2B and 3 did not support our findings from Experiment
1B, where a genotypic difference was found in Avpr1b males following treatment with
MK-801. The lack of genotypic difference in PPI percentage with MK-801 and PCP seen in Experiment 2B and 3, could be due to a number of reasons. First, in Experiment 2B, by using just two drugs instead of four as in Experiment 1B, we might have eliminated the possible drug interactions which could have resulted in a significant difference. Second, a careful analysis of the PPI data in Experiment 2B revealed that saline treatment resulted in lower PPI in Avpr1b −/− mice as compared to Avpr1b +/+ mice. Since the control
itself resulted in differences between Avpr1b +/+ and Avpr1b −/− males, it is possible that the disruption of PPI that was due to MK-801 did not appear as robust. This 67
genotypic difference in PPI following saline treatment between Avpr1b +/+ and Avpr1b
−/− mice has not been observed in any previous experiments and we are unclear as to
why this is happening. We are hopeful that a repeat of this study will shed some light
onto these aspects. In Experiment 3, we are fairly confident that our lack of effect in
Avpr1b −/− mice is due to a decrease in the potency and efficacy of drugs in disrupting
PPI. The drugs used in this Experiment were at least a year old and it is likely that there
was a decrease in their effects due to their breakdown over time. This hypothesis is
supported by the fact that the disruptions of PPI by MK-801 and PCP were not as
profound as that observed in previous Experiments. Another reason that there were not
drug-specific genotypic differences in PPI may be due to the smaller numbers of animals
that were tested. It is likely that a repeat of this study with a larger sample size and using
freshly prepared drugs would reproduce the results of Experiment 1B.
With regards to startle magnitude, in Experiment 1B, the genotypic difference in
PPI percentage does not seem to be due to changes in the startle magnitude. So far, the
relationship between startle and PPI is not well understood. One study compared the
prepulse and startle magnitude in 12 different inbreed mouse strains and found no
association between the two; while another study with 20 different strains found a
positive correlation between the two (Paylor and Crawley, 1997; Logue et al., 1997). Our
present findings suggest that the Avpr1b plays a role in regulation of PPI by interacting
with the glutamatergic system. 68
Data suggesting a link between glutamate and PPI has been established by a number of studies. Across species, treatment with PCP and MK-801 has been shown to disrupt PPI through antagonism of NMDA receptors (Caldwell et al., 2009; Jerram et al.,
1996; Yee et al., 2004). The lack of genotypic difference in PPI following treatment with
AMP and APO in Experiment 1B suggests that the dopaminergic system is not involved in the regulation of PPI in Avpr1b −/− male mice. However, it is naïve to think that an altered glutamatergic system is solely responsible for the deficient sensorimotor gating observed in Avpr1b −/− mice following treatment with MK-801. In a number of studies, it has been shown that there is a great degree of crosstalk between the various neurotransmitters- glutamate, dopamine and serotonin (Carlsson et al., 2000; Carlsson et al., 1999). It has been reported that drugs that block NMDA receptors not only increase the release of glutamate, but also dopamine and serotonin (margos-Bosch et al., 2006;
Martin et al., 1998; Mathe et al., 1999; Moghaddam et al., 1997; Schmidt and Fadayel,
1996). Further in rats and mice, PPI deficits induced by PCP and MK-801, which are specific NMDA antagonists, can be effectively reversed by pretreatment with atypical antipsychotics such as clozapine and olanzapine, which act on many different receptors
(Bubenikova et al., 2005). Additionally, there are few reports which show that the release of serotonin and dopamine, due to administration of MK-801 or PCP, can be effectively suppressed by atypical neuroleptics (Lopez-Gil et al., 2007). Hence it is possible that altered functioning of glutamate receptors in Avpr1b −/− mice may lead to deficits in PPI through some indirect mechanism involving other neurotransmitters, such as by 69
increasing the levels of dopamine. Further studies will be required to explore the relationship of different neurotransmitters and their effects on PPI.
Our findings from Experiment 4 support our hypothesis that the glutamatergic system is altered in male Avpr1b −/− mice. The increases in the NMDA receptor subunit
NMDAR2A within the hippocampus are consistent with Avpr1b localization in the brain, with the highest density of Avpr1b in the CA2 region of hippocampus (Young et al.,
2006). Thus, the developmental lack of Avpr1b could alter the PPI neurocircuitry by changing the levels of expression of NMDA receptor subunits in this area. Although the levels of NMDAR1 did not differ significantly between Avpr1b +/+ and Avpr1b −/− mice, the level of NMDAR1 was decreased in the striatum in Avpr1b −/− mice as compared to Avpr1b +/+ (P=0.08). We are currently quantifying these same subunits
(NMDAR1 and NMDAR2A) in Avpr1b +/+ and Avpr1b −/− mice by western blot analysis to determine whether these observed changes in mRNA result in changes in protein levels as well.
Currently, hypo-functioning of the NMDA receptor is believed to partly
contribute to the PPI deficits seen in patients with schizophrenia or mood disorders.
However, in individuals with psychiatric disorders characterized by PPI deficits, the data
regarding NMDA transcripts within the brain are highly inconsistent. In one study by
Gao and colleagues (2000), they reported an increase in NMDAR2A and decrease in
NMDAR1 transcripts in the hippocampus of schizophrenic patients as compared to
healthy controls, with no changes in protein levels. Conversely, other studies of NMDA 70
receptors in individuals diagnosed with either bipolar disorder or schizophrenia have shown decreases in NMDAR1 and NMDAR2A transcripts in hippocampus
(McCullumsmith et al., 2007). As NMDA receptors are tetrameric and composed of two obligatory NMDAR1 subunits and either two facultative NMDAR2 (A-D) or NMDAR3
(A-B) subunits (Moriyoshi et al., 1991; Sugihara et al., 1992; Paoletti and Neyton, 2007), it is likely that absolute numbers may not be particularly telling. Rather, the ratios of subunits within the receptors are more likely to be altered in these diseased states which in turn would result in altered functioning. Therefore, further studies are required to confirm these results and explore their functional significance.
While the role of glutamate in PPI is well known, the interaction of Avp and glutamate is still under investigation. In a couple of studies, it has been found that MK-
801 abolishes Avp-induced increases in memory, suggesting that Avp does interact with
NMDA receptors in the process of memory formation (Artemowicz and Wisniewski,
1998; Wisniewski et al., 1996) . A recent study also reported that Avp increases glutamate release in the astrocytes of the hippocampus and cortex (Syed et al., 2007).
This effect on hippocampal astrocytes was shown to be specifically mediated via Avpr1b receptors. Given all these interactions between Avp and glutamate, it would be interesting to examine the levels of glutamate within the hippocampus of Avpr1b −/− mice by
microdialysis. It can be speculated that the similar to the mRNA levels of NMDA
receptors, the amount of glutamate would also be different between Avpr1b +/+ and
Avpr1b −/− mice. 71
There is another line of Avpr1b −/− mice that were engineered by another research group. Contrary to the work from this thesis, in this line there are reported baseline differences in PPI between Avpr1b +/+ and Avpr1b −/− mice (Egashira et al.,
2005). There are several reasons why these inconsistencies may exist. First, the mice used in the above study were hybrids between 129Sv and C57BL/6J from F3 to F5 generations, whereas the mice used our study were fully backcrossed into the C57BL/6J background. This is significant because there are reports which indicate that PPI differs between the different inbred mouse strains (Logue et al., 1997). Thus, it is possible that the differences in the background could have resulted in differences in the results seen with PPI. A second difference between the two studies is the technique of creating the
Avpr1b −/− mice (Tanoue et al., 2004; Wersinger et al., 2002). It can be speculated that the differences in generation of knockout could have differently altered other systems as well which resulted in discrepancy in the results.
Nevertheless, the study with two different Avpr1b −/− mice as well as BB-Ho rats suggests that Avp and Avpr1b do play a role in the regulation of sensorimotor gating.
Interestingly, PPI deficits in Avpr1b −/− mice and BB-Ho rats are effectively reversed by atypical antipsychotics (Feifel et al., 2004; Egashira et al., 2005). These deficits might be sensitive to some particular property of these atypical neuroleptics. It would be interesting to see if we can reverse the deficits with atypical antipsychotics.
One of caveat of our study is that Avpr1b −/− mice completely lack the Avpr1b since the time of conception. This developmental lack of Avpr1b could have resulted in 72
some compensatory changes in these animals. It is also possible that all the results seen are due to some compensatory changes due to developmental lack of Avpr1b and not primarily due to its absence. The same reason also makes it difficult to extrapolate the results for humans in the pathogenesis of neuropsychiatric disorders. Hence, while our studies strongly suggest the role of Avpr1b in regulating sensorimotor gating; its translational implications for humans need to be explored further.
Overall conclusions
In summary, our studies have provided evidence for the role of Avpr1b in the regulation of sensorimotor gating. However, lack of Avpr1b by itself is not sufficient to cause deficits in sensorimotor gating. We have demonstrated that the glutamatergic system is altered in Avpr1b −/− mice, which might be contributing to the PPI deficits found in Experiment 1B. Thus, we hypothesize that under altered glutamatergic signaling following treatment with psychotomimetics, the lack of the Avpr1b increases the susceptibility to develop deficits in sensorimotor gating. However, since glutamate and
Avp are both known to interact with other neurotransmitters systems, it is likely these may also be altered in these mice. Therefore, further research will be required to explore how and where Avpr1b is interacting with these neurotransmitter systems to regulate PPI.
In order to further explore the relationship of glutamate and Avp, it would be interesting to examine the changes in NMDA receptors after acute and chronic administration of MK-801 and PCP in Avpr1b +/+ and Avpr1b −/− mice. We could also examine other aspects of glutamatergic system such as measuring the glutamate levels in 73
specific brain regions at baseline and following administration of psychotics to look for changes.
In conclusion, our study further strengthened the evidence for the role of central
Avp system in pathogenesis of neuropsychiatric disorders. With more evidence emerging implicating Avp in regulation of social behavior and its alteration in many diseases states, further research could open new avenues for development of new treatment options.
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APPENDIX I
PILOT STUDY
Objective: To determine the optimum dose of drug to disrupt the PPI in both male and female mice
Methods: 6 female and 6 male heterozygous (Avpr1b +/−) animals of ages 3-6 months were used for the study. All the testing was conducted in the light phase of the 12:12 light dark cycle. APO, AMP and MK-801 were in the following doses; APO (10mg/kg), AMP
(10mg/kg) and (12mg/kg), MK-801 (0.7mg/kg). All the drugs were dissolved in 0.9% saline. Saline was given in the same volume as APO and AMP and served as control.
Animals were tested as per the protocol mentioned in the methods section. PPI percentage was calculated using the following equation: (1 −(startle amplitude of the
PREPULSE + PULSE/startle amplitude of the PULSE ALONE)) × 100. PPI percentage was determined using repeated ANOVA and main effects of drug treatment, prepulse intensity and their interactions were compared. Data from both males and females were collapsed and analyzed together.
Results:
PPI percentage : There was a main effect of the drug treatment with APO (10mg/kg) (F 1,9
= 6.005, P<0.05) and MK-801(0.7mg/kg) (F 1,9 = 16.384, P<0.05) all resulting in
88
89
disruption of PPI as compared to saline. There was also a main effect of prepulse intensity (F 2,18 = 58.048, P<0.001) with higher decibel tone resulting in increased PPI percentage. However, treatment with AMP (10mg/kg) did not result in significant disruption of PPI. Hence the experiment was repeated with an increased dose of AMP-
12mg/kg. Treatment with AMP (12mg/kg) resulted in significant disruption of PPI (F 1,9 =
11.220, P<0.05) as compared to saline (P<0.05).
Conclusions:
APO (10mg/kg), AMP (12mg/kg) and MK-801 (0.7mg/kg) resulted in significant
disruption of PPI as compared to saline in Avpr1b +/− mice. The above mentioned doses
were selected for conducting experiments 1, 2, and 3.
90
100 3dB 6dB 12dB a b 80
60 3DB 6DB 12 DB Col 8 40 % Prepulse Inhibtion Prepulse %
20
0 SALINE APO AMP MK801 0.9% 10mg/kg 10mg/kg 0.7mg/kg
Figure 24: PPI percentage in Avpr1b +/− mice following drug treatment: Treatment with APO (a), and MK-801 (b) resulted in disruption of PPI as compared to saline (P<0.05). There was also a main effect of prepulse intensity, with higher decibel tone resulting in increased PPI percentage. Treatment with AMP at 10mg/kg did not result in significant disruption of PPI as compared to saline. Data are expressed as means and standard error of means. (Abbreviations: APO, apomorphine; AMP, amphetamine; MK-801, dizocilpine).
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100 * 3dB 6dB 12dB
80
60 3DB 6DB 12 DB Col 8 40 % Prepulse Inhibtion Prepulse %
20
0 SALINE AMP 0.9% 12mg/kg
Figure 25: PPI percentage in Avpr1b +/− mice following treatment with AMP: Treatment with AMP (12mg/kg) resulted in disruption of PPI as compared to saline (P<0.05). Data are expressed as means and standard error of means. (Abbreviations: AMP, amphetamine)
APPENDIX II
PPI Program used in Experiment 1, 2, and 3
Trial Decibel 1 pulse120 Parameters 2 pulse120 PULSE 1 3 pulse120 5 min acclimation 4 pulse120 Background 74dB 5 pulse120 6 pulse120 Pulse 120- 40 msec duration 7 PPI77 Block 1 8 PPI80 After variable ITI- 12-30 sec 9 PPI86 10 nopulse Prepulse 77- 20 msec duration, 11 PPI80 100 msec wait and pulse 120 12 pulse120 Prepulse 80- 20 msec duration, Block 2 13 nopulse 100 msec wait and pulse 120 14 PPI77
15 PPI86 Prepulse 86- 20 msec duration, 16 PPI77 100 msec wait and pulse 120 17 Nopulse Block 3 18 PPI86 19 pulse120 20 PPI80 21 Nopulse 22 PPI86 Block 4 23 PPI80 24 PPI77 25 pulse120 26 PPI86 27 PPI80 Block 5 28 pulse120 29 nopulse 30 PPI77 31 pulse120 32 PPI86 Block 6 33 PPI77 92
93
34 nopulse 35 PPI80 36 PPI77 37 pulse120 Block 7 38 nopulse 39 PPI86 40 PPI80 41 pulse120 42 PPI80 Block 8 43 PPI86 44 nopulse 45 PPI77 46 PPI80 47 nopulse Block 9 48 PPI86 49 PPI77 50 pulse120 51 nopulse 52 PPI86 Block 10 53 PPI77 54 PPI80 55 pulse120 56 pulse120 57 pulse120 PULSE 2 58 pulse120 59 pulse120 60 pulse120