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UNIVERSITY OF CINCINNATI

______, 20 _____

I,______, hereby submit this as part of the requirements for the degree of:

______in: ______It is entitled: ______

Approved by: ______

Differential Involvement of Opioid Receptors in Regulating the Behavioral Response to Amphetamine in C57BL/6 Mice.

A dissertation submitted to the

Division of Research and Advanced Studies of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

DOCTORATE OF PHILOSOPHY (Ph.D.)

in the Department of Cell Biology, Neurobiology, and Anatomy of the College of Medicine

2002

Jonathan W. Yates

B.A., Knox College, 1991

Committee Chair: Lei Yu

Abstract:

Drug addiction is recognized as a serious brain disease and with the rapid expansion in neuroscience research over the past few decades, great progress has been made in understanding the causes and effects of drug addiction and its relevance to more basic components of human behavior. It is now accepted that drugs of abuse (DOA) act on the mesolimbic dopamine (DA) brain reward system to mimic the rewards induced by more ‘natural’ stimuli such as food and sex. Acute administrations of DOA, particularly the psychostimulant amphetamine (AMPH), and the opiate heroin, lead to increases in extracellular levels of the neurotransmitter dopamine in regions comprising the brain reward system, including the nucleus accumbens (NAc), the ventral tegmental area (VTA), the striatum, and the dorsal caudate (DC). Changes in DA levels contribute to both the rewarding properties of DOA, as well as behavioral aspects of their use.

As knowledge of the molecular and behavioral effects of DOA has advanced, evidence has accumulated that opioids not only share similar rewarding properties as psychostimulants, but that the endogenous opioid system can directly modulate the mesolimbic dopaminergic system as well. However, the individual contributions of the three opioid receptors subtypes (µ,

κ, and δ) in modulating the brain reward system are still unclear. In an attempt to investigate this, we developed an animal model of acute amphetamine (AMPH) –induced behaviors and studied the effect of antagonizing the individual opioid receptors (ORs) on these behaviors.

Using the induction of hyperlocomotion following a low dose (2mg/kg) of AMPH as a behavioral correlate of DA levels, we found that antagonizing the δOR significantly attenuated behavior, antagonism of κORs failed to effect behavior, and antagonism of µORs had a slight, but non-significant, augmentation of DA-dependent hyperlocomotion. In contrast, utilizing the induction of stereotypic behaviors with a high dose (12mg/kg) of AMPH as our behavioral correlate, antagonism of all three ORs significantly attenuated DA-dependent stereotypy. It is

assumed that these OR-dependent effects are mediated through differential regulation of target

neurons within the mesolimbic reward system and indicate that the endogenous opioid system can regulate behavioral responses to rewarding stimuli.

Table of Contents

Chapter One ______4 Introduction ______4 1.1. Introduction: ______5 1.2. Dopamine and the Brain Reward System ______6 1.3. The Opioid System and Receptors: ______10 1.4. The Dopamine System and Receptors: ______12 1.5. Opioid System Regulation of DA System:______13 1.6. The Goals for this Project: ______20

Chapter Two ______23 Evaluation of Amphetamine-induced Behavior in C57BL/6 Mice______23 2.1. Introduction: ______24 2.2. Materials and Methods: ______27 Subjects: ______27 Locomotor Activity:.______27 Drugs:. ______28 Data Analysis: ______29 2.3. Results:______29 2.3-A: Amphetamine-induced Locomotion:______29 2.3-B: Amphetamine-induced Stereotypy:______33 2.3-C: Comparison of Selected AMPH doses: ______36 2.3-D: Time Course Evaluation: ______45 2.4. Discussion: ______51

Chapter Three ______56 Differential Involvement of Opioid Receptors in Amphetamine-induced Behaviors in C57BL/6 Mice ______56 3.1. Introduction: ______57 3.2. Materials and Methods: ______63 Subjects: ______63 Locomotor Activity: ______63 Drugs: ______64 Data Analysis: ______65 3.3. Results: ______65 3-3-1: Antagonist Dose Responses:______65 3-3-2: Antagonist Pretreatment + Amphetamine:______71 Locomotion Results: ______74 3-3-2A: Naloxone + AMPH: ______74 3-3-2B: β-FNA + AMPH: ______77 3-3-2C: Naltrindole + AMPH: ______80 3-3-2D: nor BNI + AMPH:______83 Stereotypy Results: ______88

1 3-3-3 Stereotypy: ______88 3.4. Discussion: ______93

Chapter Four ______104 Discussion ______104 4.1. Discussion: ______105 4.2. Development of Animal Model: ______107 4.3. Analysis of Amphetamine Response in C57 Mice: ______109 4.4. Amphetamine-induced Locomotion and Stereotypy: ______111 4.5. Identification of Time Interval for Analysis: ______112 4.6. Effects of Opioid Antagonists on Basal Activity in C57 Mice: ______115 4.7. Opioid Antagonists and Amphetamine-induced Behaviors: ______118 4.8. Mechanisms for Opioid Receptor Regulation of Amphetamine-induced Behaviors: ______120 4.9. Summary: ______126 4.10. Future Directions: ______128

Chapter Five ______130 Bibliography ______130

2 Table of Figures

CHAPTER 1 FIGURE 1-1 ------08

CHAPTER 2 FIGURE 2-1------32 FIGURE 2-2------35 FIGURE 2-3A ------38 FIGURE 2-3B ------35 FIGURE 2-3C ------37 FIGURE 2-4------47 FIGURE 2-5------50

CHAPTER 3 FIGURE 3-1 ------58 FIGURE 3-2 ------59 FIGURE 3-3 ------61 FIGURE 3-4 ------63 FIGURE 3-5 ------65 FIGURE 3-6------82 FIGURE 3-7------85 FIGURE 3-8------87 FIGURE 3-9------90 TABLE 1 ------92

Chapter One

Introduction

1.1. Introduction:

Drug addiction is a chronically relapsing condition characterized by an uncontrollable compulsion to take drugs, an inability to stop the intake of these drugs, and the appearance of a withdrawal syndrome when drug use is discontinued (Koob and Bloom, 1998). Drug addiction is now recognized as a serious brain disease which costs society not only in monetary terms for health care, treatment, law enforcement, and the legal system, but also in non-monetary terms such as the emotional impact on the addict and his or her family and friends (White, 2002). With the rapid expansion in basic neuroscience research over the last few decades, great progress has

been made in understanding the causes and effects of drug addiction and the relevance of this

condition to more basic components of human behavior. In fact, it is now generally accepted

that drugs of abuse (DOA) act on the brain reward system, and mimic the rewards induced by

more ‘natural’ stimuli (Kelley and Berridge, 2002). Much of the scientific progress made to date

on addiction, both at the molecular/cellular level and at the behavioral/systems level, has come

through work on a limited number of drug classes or types. In particular, psychostimulants such as cocaine and amphetamine, and opioids such as heroin and morphine have been the compounds of choice for studying drug addiction (Nestler and Aghajanian, 1997). There are a number of reasons for this, including the fact that these classes of drugs are among the most widely abused; these compounds, especially opioids, have considerable use in health care and pain management; and finally because abuse of psychostimulants can result in a psychotic state that in many aspects resembles other brain diseases, such as schizophrenia, and thus can provide insight into these conditions (Goldstein and Deutch, 1992; Nestler and Aghajanian, 1997). For these reasons and

5 many others, furthering our understanding of the actions and functioning of these compounds is

vital.

1.2. Dopamine and the Brain Reward System

The mesocorticolimbic dopamine system is thought to be a primary site of action for the

rewarding and reinforcing effects of natural and artificial stimuli (Everitt and Wolf, 2002). The reinforcement of responses to natural stimuli, such as food and sex, are thought to be evolutionarily important for survival, reproduction, and the overall fitness of a species (Kelley and Berridge, 2002). In contrast, the reinforcement of responses to artificial stimuli, such as drugs of abuse, can lead to the development of drug addiction. In an attempt to understand and explain the brain reward system, researchers have studied the effects of these ‘unnatural’ stimuli on organisms at both the molecular/cellular level and at the behavioral/systems level. Much of this work has focused on identifying changes induced in the mesolimbic dopamine system by drugs of abuse, including psychomotor stimulants, opiates, ethanol, and others. Studies have well established that acute administration of various drugs of abuse, particularly cocaine and amphetamine (AMPH), which act as indirect dopamine agonists, lead to increases in extracellular dopamine (DA) levels in a number of regions comprising the mesolimbic DA system, including the nucleus accumbens (NAc), the ventral tegmental area (VTA), the striatum, and the dorsal caudate (DC) (Heidbreder et al, 1996; Wise, 1981). The mechanisms for this increase in DA concentrations include an inhibition of DA reuptake by the transporter and/or by promoting reverse transport of DA, and increases in DA release (Koob, 1992). It has been theorized that this effect on DA levels within the mesolimbic DA system, especially in the NAc, contributes significantly to not only the rewarding properties of these drugs, but also various

6 behavioral aspects of their use, including modulation of locomotor activity and the development

of stereotypies.

While the involvement of the NAc in the rewarding properties of DOA has been well

established, there is still considerable debate over the specific mechanisms involved (Hoffman

and Lupica, 2001). In a simplified model, shown in Figure 1-1, the critical circuitry of the brain

reward system is thought to be between the NAc and the VTA, as these regions are intrinsically

involved with the rewarding properties of drugs of abuse (Wise, 1982; Koob and Bloom, 1988).

The VTA contains the cell bodies of the mesolimbic DA system which project to the NAc and

frontal cortex, providing dopaminergic input to these structures (Carlezon and Wise, 1996). The

majority of neurons within the NAc are GABAergic medium spiny neurons (MSNs) which

receive this DA input and also receive glutamatergic input from the hippocampus, amygdala, and

prefrontal cortex and which in turn send their inhibitory output projections to several brain

structures, including the VTA (Groenewegen et al, 1991; Steffensen et al, 1998; Christie et al,

1985, 1987). Glutamate and dopamine are thought to have opposite actions on their target

neurons in the NAc – glutamate is believed to excite MSNs and DA inhibits them (Carlezon and

Wise, 1996). Many drugs of abuse, including AMPH and opioids, act to increase DA concentrations within the NAc and also inhibit amino-acid mediated synaptic transmission in this structure (DiChiara and Imperato, 1988; Chieng and Williams, 1998). It is through these effects of DA levels and NAc neuronal activity that the rewarding properties of DOA are manifested

(Hoffman and Lupica, 2001).

Hippocampus Amygdala PFC Glutamatergic Neurons

Rewards/Drugs of Abuse

DA DA Neuron + Ventral Tegmental Area GABAergic MSNs

Nucleus Accumbens GABAergic interneuron

Rewards/Drugs of Abuse Additional Brain regions

8 Figure 1-1

A diagram of the critical components of the brain reward system. Dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) can inhibit

GABAergic neurons within the NAc. Inhibition of GABAergic neurons leads to decreased inhibitory signals to various brain regions, including the VTA. Glutamatergic input from a number of regions into the NAc can excite GABAergic neurons and increase inhibitory signals.

Natural rewards and drugs of abuse act to increase DA levels within the NAc and to inhibit

GABAergic neurons within the VTA and NAc, all contributing to the rewarding properties of these compounds. Figure adapted from Spanagel and Weiss, 1999.

9 1.3. The Opioid System and Receptors:

Opioids are among the most effective drugs used in a clinical setting for the control and

management of severe pain (Hardman et al, 1996). Along with their analgesic properties, opioids are involved in the regulation of other physiological events, including hormone secretion,

neurotransmitter release, food-intake, gastrointestinal motility, and respiratory activity (Paternak,

1988). However, the use of opioids can result in a euphoric effect, thus conferring the

reinforcing properties of these drugs and contributing to opioid dependence. Dependence is the

need for continued drug exposure to avoid a withdrawal syndrome, characterized by physical and

psychological disturbances, when the drug is withdrawn (Nestler et al, 1993). Dependence upon

the opioid narcotic heroin, which is converted to morphine and related metabolites in vivo, is a

major social and medical problem. It is estimated that up to a million people in the United States

are addicted to heroin, with millions more worldwide. Both exogenous opioids, like morphine

and heroin, and endogenous opioid peptides, like ß-endorphin and enkephalins, achieve their

effects by activating membrane-bound receptors of three formal classes: mu (µ), kappa (κ), and

delta (δ) (Pasternak, 1993). The mu opioid receptor (µOR) is of primary clinical interest since

both the majority of medically relevant opioid narcotics and the most commonly abused

narcotics exert their effects via activation of this receptor (Yu, 1996). Drug dependence is a

complex process involving an interaction of both environmental and biological factors.

Although quite a bit is known about the functioning of the opioid receptors at the cellular level,

we are only now beginning to elucidate the biological basis of opioid and other drug addictions.

As members of the G-protein-coupled receptor (GPCR) superfamily, opioid receptors are

structurally composed of an extracellular ligand-binding amino end, seven transmembrane

domains forming three extracellular and three intracellular loops, and an intracellular effector-

10 binding carboxyl end (Pasternak, 1993). The µ, κ, and δ, opioid receptors have significant sequence homology along their length, but are most divergent in their amino and carboxyl termini (Kreek et al, 2002). Opioid receptors couple to inhibitory G-proteins (Gi/Go) and upon

acute exposure to agonists, act to inhibit adenylyl cyclase to decrease cAMP levels and activate

G-protein-coupled, inwardly rectifying potassium channels and inhibit calcium channels to

decrease membrane excitability and inhibit neuronal firing (Yu, 1996; Kreek et al, 2002). Upon

repeated or long term opioid exposure, neurons develop tolerance to these effects with a

subsequent recovery of firing rates, up-regulation of the cAMP system, and increased expression

of Giα/Goα and other signal transduction cascade proteins, including cyclic AMP-dependent

protein kinase A (PKA) (Guitart and Nestler, 1993). It is thought that these cellular adaptations

to long term agonist exposure may parallel the development of tolerance in a person abusing an

opioid narcotic, but they do not themselves cause tolerance and dependence. Once these

adaptations are established, however, the continued presence of agonist is needed, otherwise the

cell quickly becomes depolarized (more excited) due to the up-regulated cAMP system and the elevated electrical excitability (Guitart and Nestler, 1993). Finally, opioid receptors are widely

expressed in the mammalian brain, with varying and unique distribution patterns seen for each

receptor type in different species. Autoradiographic mapping studies of mouse brain coronal

sections indicate that µORs are highly expressed in the caudate putamen, NAc, amygdala,

thalamus, hypothalamus, VTA, and substantia nigra, while δORs are highly expressed in

olfactory bulbs, caudate putamen, NAc, amygdala, and cortex (Kitchen et al, 1997). Lastly,

κORs are highly expressed in NAc, ventral pallidum, preoptic areas, hypothalamus, and

substantia nigra (Kitchen et al, 1997). As can be seen, opioid receptors are expressed in regions

involved in the brain reward pathway, including the VTA, NAc, cortex, amygdala, and thalamus,

11 although there are differences in the type and level of receptor expressed in these regions.

(Mansour et al, 1987, 1988; Tempel and Zukin, 1987; Kitchen et al, 1997; De Vries and

Shippenberg, 2002).

1.4. The Dopamine System and Receptors:

Dopamine is a critical neurotransmitter involved in regulating a number of physiological

functions, including voluntary movement, motivated behaviors, endocrine regulation, learning,

and memory (White, 1989; Robbins, 1992). Similar to opioids, DA also exerts its regulatory and

modulatory effects on neurons through binding to receptors of the GPCR superfamily. However,

unlike the opioid receptor ‘family’ which contains three main subtypes, DA receptors are

subdivided into two classes, D1 and D2, with multiple subtypes within each class. The D1 class

includes D1 and D5 DA receptor subtypes, which stimulate adenylyl cyclase leading to increases in cAMP levels, while the D2 class includes D2, D3, and D4 subtypes and generally inhibits

adenylyl cyclase to decrease cAMP levels (Civelli et al, 1993; Beatty, 1995). These subtypes

differ in regards to their gene structure and by alternative splicing, differing affinities for DA,

differing localization and expression within the brain, and differing effects on effector molecules

(Goldstein and Deutch, 1992; Bunzow et al, 1988; Caron, et al, 1991). For instance, D1

receptors can stimulate adenylyl cyclase leading to increases in cAMP levels, while a D2 variant

can regulate a calcium current, and a D3 form may not even couple to a G-protein (Goldstein and

Deutch, 1992). This complexity among DA receptors has contributed to the difficulty of

assigning specific functions to different members of the two DA receptor classes. However,

12 there is general agreement that DA D1, D2, and D3 receptor subtypes are all involved in drug

reward and drug-induced behavioral responses (Bardo, 1998; Xu et al, 1997). The evidence for

this includes the expression of these receptors subtypes within the brain – D1 and D2 receptors

are highly expressed in the striatum, while D3 receptors are highly expressed in the NAc – two

areas known to be involved in the brain reward pathway and in regulating drug-dependent

behaviors (Xu et al, 1997; Wise, 1982; Koob and Bloom, 1988). Additionally, studies using

receptor-subtype specific agonists and antagonists, knockout and transgenic mice, and neuronal

lesioning experiments indicate these receptors influence the locomotor and stereotypic responses

to psychostimulants, including cocaine and AMPH (Hiroi and White, 1991; Laviola et al, 1994;

Xu et al, 1997; Bardo, 1998). In particular, it is believed that D1 and D2 receptor activation

results in increased locomotion and D3 receptor stimulation results in decreased locomotion,

presumably through the differing actions of DA on the receptor subtypes within various brain

regions (Richtand et al, 2000; Ekman, et al, 1998; Waters et al, 1993).

1.5. Opioid System Regulation of DA System:

With the identification of evolutionarily-conserved molecular components comprising the

brain reward system, including (but not limited to) DA, G proteins, GPCRs, amine transporters,

various effector molecules, and endogenous peptide agonists and antagonists, the similarities and

differences in the reinforcing actions of drugs of abuse have been studied. Additionally, the

crosstalk between different classes of drugs and their effects on the dopaminergic system have

been investigated. There is growing evidence to indicate that opioids not only share many

similar rewarding properties of psychostimulants, but that the endogenous opioid system can

13 modulate the mesolimbic dopaminergic system as well. For instance, opioids have been shown

to induce membrane hyperpolarization and neuronal inhibition in multiple structures that provide

major afferent input to the VTA of the mesocorticolimbic DA system and in this manner are in

position to regulate multiple neurotransmitter systems (De Vries and Shippenberg, 2002). These

structures include the nucleus accumbens (NAc) and ventral pallidum which are primary inhibitory GABA inputs, the prefrontal cortex, amygdala, and mediodorsal thalamus as sources of stimulatory glutamate input, and pedunculopontine tegmental nucleus as a source of acetylcholine input (DeVries and Shippenberg, 2002). These structures are characterized by high densities of opioid receptors and are involved in goal-directed behavior and the rewarding effects of psychostimulants (Mansour et al 1995; Pierce and Kalivas, 1997; Koob et al, 1998).

The individual contributions of the three types of opioid receptors (µ, κ, and δ) to the brain reward system, however, are not as well understood. Much of this work has focused on the

µOR as it is the receptor responsible for the rewarding effects of heroin and morphine. Deletion or blockade of µORs, but not other opioid receptors, has been shown to attenuate opioid self- administration and the conditioned place preference animals exhibit for the contextual cues previously associated with opioid administration (Negaus et al, 1993; Matthes et al, 1996).

Additionally, blockade of µORs in the VTA or NAc via antagonist infusion have also been shown to attenuate heroin self-administration and conditioned approach behavior to cues associated with morphine administration, while infusion of opioid agonists into these regions induce and augment these same behaviors (Shippenberg and Elmer, 1998). There is growing evidence for the involvement of κORs in the reward system as well. The endogenous κOR ligand dynorphin has been shown to be released by medium spiny neurons within the NAc and to produce dysphoria and negative effective states by the activation of NAc opioid receptors and

14 inhibition of DA release (Pfeiffer et al, 1986; Shippenberg and Elmer, 1998). Additionally, elevated expression of dynorphin has been observed after abstinence from repeated opioid administration (Bronstein et al, 1988). It has been postulated that this upregulation of the dynorphin system in the presence of drug diminishes further drug responsiveness (enhanced DA release), but in the absence of drug may contribute to the dysphoria and anhedonia that characterize opioid withdrawal. A similar elevation of dynorphin levels may also contribute to alterations in behavior and DA neurotransmission that occur during abstinence from psychostimulants (Shippenberg et al, 2001). The picture emerging is that there exist tonically active and functionally opposing µ and κ opioid systems that regulate dopamine release in the mesolimbic dopaminergic system (Spanagel et al, 1992).

Because it appears that opioids have many of the same effects on the mesolimbic DA system as psychostimulant administration does, researchers have begun to investigate possible interactions between these two classes of drugs on behaviors associated with their use. The investigation of these interactions within the brain reward system is supported by not only the above findings but also additional data. Neuroanatomically, the three types of opioid receptors have been identified, via binding studies using labeled peptide and non-peptide agonists, on mesolimbic dopaminergic neurons (Spanagel et al, 1992; Mansour et al, 1995). However, the absolute amount and regional distribution of the various receptor types differ. Dense µ receptor binding has been seen in the VTA with little δ or κ receptor binding present, while high

δ and κ opioid receptor binding is seen in the NAc with low to moderate µ-specific binding seen in this region (Mansour et al, 1987, 1988; Tempel and Zukin, 1987). Secondly, at the molecular/cellular level, opioid agonists and antagonists have been shown to modulate extracellular DA levels in the mesolimbic DA system. In fact, there is mounting evidence to

15 indicate that the different opioid receptors have differing regulatory effects on basal DA levels.

Systemic administration of µ and δOR agonists can cause dose-dependent increases in DA levels

in the VTA, NAc, and dorsal caudate, while systemic κOR agonists can cause decreased DA

concentrations within the VTA and NAc (DiChiara and Imperato, 1988; Spanagel et al, 1992;

Devine et al, 1993a). These effects of µ and δ agonists can be blocked by administration of selective receptor antagonists while the κ agonist effects were insensitive to antagonist treatment

(DiChara and Imperato, 1988; Spanagel et al, 1992; Devine et al, 1993a). Direct VTA injections

of either µ or δ-specific peptide agonists can produce significant dose-dependent increases in

ventral striatal DA levels, while injection of the µ peptide antagonist CTOP into the VTA

produced a significant decrease in DA release (Spanagel et al, 1992; Devine et al, 1993b).

Injection of either µ agonist or antagonist into the NAc showed no effect on DA levels (Spanagel

et al, 1992). Conversely, κOR agonists injected directly into the NAc lead to significant

inhibition of DA release, while injection of the κ antagonist nor-BNI into the NAc lead to an

increase in DA release (Takemori et al, 1988; DiChiara and Imperato, 1988; Spangel et al, 1992).

Injections of either κOR agonists or antagonists into the VTA have been shown to have no effect

on DA release (DiChara and Imperato, 1988; Spanagel et al, 1992). The µ-specific antagonist β-

FNA, when injected i.c.v., was shown to decrease the amount of DA released in the NAc and

dorsal caudate in response to morphine, fentanyl, and methadone, but had no effect on DA levels

when given alone (DiChiara and Imperato, 1988). A mechanism proposed for the basal modulation of DA levels within the NAc by opioid receptors suggests activation of µORs on

GABAergic neurons within the VTA leads to a disinhibition of DA neurons projecting to the

NAc and a subsequent increase in DA within the NAc, while activation of κORs located

16 presynaptically within the NAc inhibits DA release to decrease DA levels (DiChiara and

Imperato, 1988; Spanagel et al, 1992).

Studies have also investigated the modulation of DA levels by the opioid system in

response to psychostimulant administration. The non-selective opioid receptor antagonist

naloxone was shown in rats to attenuate the AMPH-dependent increase in extracellular levels of

DA in the NAc and striatum while having no effect on DA levels when administered alone

(Jones et al, 1993; DiChiara and Imperato, 1988). It is assumed that the observed modulation of

DA levels by opioids is mediated via activation or inhibition of their respective receptors in

various parts of the mesolimbic DA system (Spanagel et al, 1992). While µ and δ opioid agonists have been shown to increase DA levels, κ agonists have been shown to either not affect or decrease DA levels depending on the site and method of delivery. In fact, it has been hypothesized that the opposing effects of mu/delta vs. kappa agonists on mesolimbic DA release underlie their opposing effects on motivation and reward (Spanagel et al, 1992)). Generally, µ

and δ agonists, such as morphine and enkephalins, function as positive reinforcers while κ

agonists have negative/aversive effects.

A final line of evidence supporting a modulatory role of the opioid system on the DA

system comes from behavioral/systems research. The administration of AMPH to rodents

generally produces two qualitatively different behaviors. When administered at low doses,

AMPH produces a primary response of hyperlocomotion, while higher doses produce repetitive, stereotyped behaviors such as intense grooming, which disrupt or interfere with locomotion

(Battisti et al, 1999). Both of these behaviors are thought to be due, in large extent, to increases in DA transmission. However, while the AMPH-dependent increase in locomotion is due to increased DA in the NAc, the AMPH-dependent increase in stereotypic behaviors is due to DA

17 increases in the striatum (Creese and Iversen, 1974, Kelly et al, 1975). This inverse relationship

of amphetamine-induced locomotion vs. stereotypy provides two relatively clear behavioral

endpoints with which to study the potential modulating effects of various compounds. Studies

have thus investigated the modulation by opioids of AMPH-induced behaviors in various animal

models. For instance, in Sprague-Dawley (S-D) rats, naloxone has been shown to attenuate the

AMPH-induced increase in locomotor activity (Jones et al, 1993). Similarly, the δOR receptor- selective antagonist naltrindole (NTI) administered i.c.v. has been shown to markedly attenuate the gross motor activity of S-D rats in response to AMPH-administration, while the µ-selective antagonist β-FNA and the κ-selective antagonist nor-BNI both failed to influence the motor response to AMPH (Jones et al, 1993). Lastly, a number of κ agonists have been shown to reduce the spontaneous behavior of rats (DiChiara and Imperato, 1988). A considerable number of studies of AMPH-induced stereotypy have been undertaken in the mouse. It has been shown that, in contrast to rats, particular strains of mice like CF-1 display a limited array of stereotypic behaviors (Karler et al, 1994, 1996). At low doses of AMPH (6-10mg/kg) CF-1 mice exhibit periodic head and paw movements similar to grooming behaviors, which are frequently interrupted by locomotor activity; while at higher AMPH doses (12-20mg/kg), the behavioral response is an animal stationary for long stretches of time exhibiting repetitive head and forelimb movements, similar to grooming. (Karler et al, 1996). However, there is a deficiency of reports investigating the effects of opioids on the AMPH-induced stereotypic behaviors in mice and only a small number of such reports in rats. In fact, the data concerning the effects of opioids on stereotypy in rats is somewhat contradictory and most of this work has been done using the non- selective antagonist naloxone. A number of groups have reported that naloxone has no effect on

AMPH-induced stereotypy in rats (Malick et al, 1977, 1983) while other groups have seen an

18 attenuation of stereotyped behaviors in rats by naloxone (Haber et al, 1978; Hitzman et al, 1982,

Skorupska and Langwinski, 1988). Finally, Adams et al demonstrated a potentiation of AMPH- induced stereotypy by naloxone in the rat (1981). Thus a careful and thorough investigation of the individual contributions each opioid receptor type makes to modulating AMPH-induced locomotion and stereotypy is needed.

The exact mechanism(s) responsible for the opioid system-dependent modulation of

AMPH-induced locomotion and stereotypy are still unclear. It has been established that the

normal response to AMPH requires functioning dopaminergic, glutamatergic, and GABAergic

systems and that these systems are also required for the induction and expression of AMPH-

induced stereotypy (Karler et al, 1994, 1995). Evidence reinforces the hypothesis that AMPH- induced stereotypy is mediated by a disinhibition of an inhibitory influence, likely the

GABAergic system, on an excitatory system, likely the glutamatergic system in the frontal cortex (Everitt and Wolf, 2002). With the realization that multiple neurotransmitter (NT) systems

participate in AMPH-induced behaviors, and given the complexity of neuronal circuits, the

involvement of additional NT systems in regulating AMPH-dependent behaviors seems likely.

As presented above, the opioid system is in a position to directly modulate neurons within the

mesolimbic DA system that contain the NTs glutamate and GABA. Thus it can be assumed that,

at least partly, the effects on AMPH-induced behaviors by the opioid system are likely via

regulation of these NT systems. Additionally, the opioid system participates in the regulation of

the basal DA tone within structures comprising the mesolimbic system and thus likely regulates

AMPH-induced behaviors by directly regulating DA levels as well.

19 1.6. The Goals for this Project:

We therefore wished to study the individual contributions each opioid receptor type

makes in modulating the locomotor-inducing and stereotypy-inducing properties of acute AMPH

administration in mice. We chose to conduct these experiments in mice for a number of reasons.

One being that a characterization of the interaction between the opioid system and the

dopaminergic system as they relate to AMPH-induced behaviors would provide data useful for

further delineating the physiological relationship between these two important neurotransmitter

systems. Second, because of the ability to make knockout and transgenic mice, it is foreseeable

that knowledge of the sensitivity of a particular strain of mouse to drugs of abuse would help

researchers chose the most appropriate stain of mouse for creating such animals (Ralph et al,

2001).

There were a number of steps necessary to facilitate this work. First, we selected three strains of mice to test the locomotor-inducing properties of AMPH administration. We chose to initially study only locomotion using the residential activity chambers (RACs) facility associated with the VA Hospital at the University of Cincinnati because these types of experiments are simpler to perform and the data gathered from them can be analyzed quickly verses stereotypy studies which are much more time and labor intensive. In pilot experiments consisting of groups of 5 mice per AMPH dose, we tested the following strains: BALB/c, CF-1, and C57BL/6.

These strains were chosen based on similar experiments reported in the literature, which gave us an idea of the responses to expect and because, in the case of the C57 strain, it is a common

background strain for creating mutant mice. The results of these pilot experiments confirmed

that the BALB/c strain of mice is insensitive to the locomotion-inducing property of AMPH, and

20 that both CF-1 and C57BL/6 mice respond with increased locomotion at low doses of AMPH and decreased locomotion at high doses (Kitahama and Valatx, 1979; Ralph et al, 2001).

From these results, we chose to pursue further investigation of the interactions between the endogenous opioid system and the DA system using the C57BL/6 strain of mouse. The next step in this work was to determine the optimal doses of acute AMPH needed to induce an increased hyperlocomotive state and a stereotypic state. These experiments and results are presented in the manuscript that comprises Chapter two of this dissertation. The final step for our investigation into opioid modulation of the DA system was to pretreat groups of mice with either a selective or non-selective opioid receptor antagonist followed by administration of either the locomotion-inducing or stereotypy-inducing dose of AMPH. The locomotion and stereotypy data were then recorded and analyzed. We chose to test the non-selective opioid receptor antagonist naloxone, the µOR-selective antagonist β-FNA, the δOR-selective antagonist naltrindole (NTI), and the κOR-selective antagonist nor-BNI and their effects on AMPH-induced behaviors. These experiments and results are detailed in Chapter three of this dissertation.

The results of our experiments confirm that in C57BL/6 mice, the non-selective opioid antagonist naloxone does have an inhibitory effect on AMPH-induced locomotor activity.

However, the contributions of the individual receptor types vary. Specifically, antagonism of the

δOR receptor contributes significantly to the attenuation of AMPH-induced hyperlocomotion, similar to naloxone, while antagonism of the κOR failed to modulate AMPH-induced locomotion in any manner. Interestingly, antagonizing the µOR receptor actually induced a slight, although non-significant, increase in the locomotor-inducing effect of AMPH. These results confirm and expand the belief that δORs are centrally involved with regulating the effects of AMPH on locomotor behaviors in mice while the κORs are not involved and µORs might

21 regulate AMPH-induced locomotion in a manner opposite to the δORs. On the other hand, the results from the experiments investigating the involvement of the opioid receptors in modulating the stereotypy-inducing effects of AMPH in C57 mice indicate that all three receptor types have a similar ability to regulate this behavior. Specifically, antagonism of µ, δ, and κORs can all attenuate the magnitude of stereotypic behaviors induced by acute high dose AMPH administration. These are the first experiments we are aware of that investigated the individual contributions of the opioid receptor subtypes on AMPH-induced stereotypy in mice.

Chapter Two

Evaluation of Amphetamine-induced Behavior in C57BL/6 Mice

23 2.1. Introduction:

There is growing evidence to indicate clear differences in the behavioral responses of different strains of mice to the effects of drugs of abuse. For instance, inbred mouse strains can differ in their response to morphine analgesic tolerance (Lariviere et al, 2001). These authors found that some strains displayed no significant tolerance development, some displayed baseline alterations in tail-withdrawal latencies as a result of chronic morphine treatment and that overall, the magnitude of hyperalgesia and tolerance to morphine were significantly correlated among the strains (Lariviere et al, 2001). The running responses of three inbred strains of mice to several doses of morphine indicate that C57BL/6 mice were most sensitive, BALB/cJ were intermediate, and DBA/2J exhibited little running in response to morphine (Oliverio and Castellano, 1974).

Researchers have also studied strain differences among mice to the effects of the psychostimulant amphetamine (AMPH). The administration of AMPH to rodents generally produces two qualitatively different behaviors. When administered at low doses, AMPH produces a primary response of hyperlocomotion, while higher doses produce repetitive, stereotyped behaviors such as intense grooming, that disrupts or interferes with locomotion

(Battisti et al, 1999). Kitahama and Valatx showed that a low dose of AMPH (1mg/kg) induced hyperactivity in the pigmented strains C57BL/6 and SEC, while inducing hypoactivity or no effect on behavior in the albino strains BALB/c and AKR (1979). In addition to quantitative differences in the amount of AMPH-induced locomotion, differences in the patterns of motor behavior induced by drug administration have been seen. Ralph et al showed that 3mg/kg

AMPH produced both increased activity and a straighter pattern of motor behavior in C57BL/6J mice while a lesser dose of 1mg/kg caused a similar change in amount and pattern of locomotion

24 in 129X1 mice (2001). Fewer studies have analyzed strain differences among mice to the

stereotypy-inducing effects of AMPH administration. The CF-1 strain of mouse has often been

chosen for studying the stereotypical responses to AMPH administration due to its limited

behavioral response, unlike the rat. It has been found that CF-1 mice respond to moderate doses of AMPH (6-10mg/kg) with periodic head and paw movements similar to grooming, which are frequently interrupted by locomotor activity; while at higher doses (12-20mg/kg), the animal is stationary, exhibiting repetitive head and forelimb movements for long stretches of time

(Bedingfield et al, 1996). Additionally, AMPH-induced stereotypy has been demonstrated using two different treatment paradigms – once-daily chronic administration of a low dose of AMPH and once-only administration of a high dose of AMPH (Bedingfield et al, 1996).

The mesocorticolimbic dopamine (DA) system is thought to be a primary site of action for the rewarding and reinforcing effects of drugs of abuse like opioids and amphetamine, and

whose use can lead to the development of drug addiction, a serious disease of modern society

(Everitt and Wolf, 2002; White, 2002). It has been established that both the locomotor-inducing

and stereotypy-inducing properties of AMPH are mediated by increases in DA

neurotransmission, but at different brain sites (Battisti et al, 1999). The locomotor-inducing

effects of AMPH are due to increased DA within the nucleus accumbens, while the stereotypy-

inducing effects are thought to be due to amphetamine’s effect in the striatum (Creese and

Iversen, 1974; Kelly et al, 1975). In addition to providing insights on the mechanisms and

components involved with drug addiction, the study of animal responses to drugs of abuse can

provide information pertinent to other diseases and conditions which affect humans. In

particular, the study of AMPH-induced behaviors can shed light on the complex disease of

schizophrenia. Of interest is the fact that chronic administration of AMPH (in both rodent

25 models and in human drug addicts) can result in a psychotic state that in many aspects resembles

schizophrenia (Goldstein and Deutch, 1992). As mentioned above, chronic low doses or acute

high doses of AMPH can induce significant stereotypic behaviors in rodents. Taken together, the

stereotypic response induced by AMPH administration in rodents has thus served as an animal

model for probing the pathophysiology of schizophrenia and has provided a system for

investigating possible pharmacological agents for the treatment of this condition.

The rationale for the current study was as follows. First, most studies investigating

AMPH-induced behaviors focused primarily on either locomotion or stereotypy, not both. We

wished to systematically characterize and describe both AMPH-induced behaviors. Secondly, we wished to identify one particular strain of animal and carry out this characterization not only

to report our findings in regard to AMPH-induced behaviors, but also to contribute to the growing body of literature describing strain-specific sensitivities to drugs of abuse. Towards this end, we chose the C57BL/6 strain of mouse for these studies as a full characterization of this strain’s behavioral response to AMPH has not been reported and because this strain is commonly chosen as a background strain for knockout and mutant mouse creation (Ralph et al, 2001). In addition to reporting both locomotion and stereotypy data in a single strain of animal, we also chose to investigate a wide dose-response range of AMPH administration and to report both behaviors over an extended period of time (2 hours). Finally, we wished to identify doses of

AMPH that are useful for inducing either a state of prolonged hyperlocomotion or sustained stereotypy which we and other investigators could then use in subsequent studies looking at possible ways to modify or modulate these behaviors. Our data indicate that a dose of 2mg/kg

AMPH s.c. is sufficient for inducing a robust hyperlocomotive state in C57BL/6 mice. A dose of

26 12mg/kg AMPH induces a significant amount of stereotypy, while not completely immobilizing the animal.

2.2. Materials and Methods:

Subjects: Male C57BL/6 mice (Charles River Laboratories, Raleigh, NC) between 5 to 7 weeks

(18-25g) were used in all experiments. The mice were housed 4 to a cage with food and water available ad libitum, and maintained on a 12 hour light: 12 hour dark cycle that corresponded with the day/night cycle. At least 24 hours prior to the initiation of behavioral testing, the mice were moved from the animal care facility to the testing facility. Each mouse was naïve to the drugs used in these experiments and was used only one time.

Locomotor and Stereotypy Activity: Behavioral characterization was carried out in 30 custom- designed residential activity chambers (RACs), modeled after the design of Segal and Kuczenski

(1987). Each chamber consisted of a lighted, ventilated, sound-attenuated cabinet (Cline

Builders, Covington, KY) housing a 16”x16”x15” plexiglass animal enclosure. A fan mounted in the side of the exterior cabinet of each enclosure provided air circulation and constant background noise, and a Sony CCD video camera mounted in the top of each enclosure allowed experimental sessions to be videotaped via an attached VHS VCR. Light inside the chambers was provided by a 15-watt bulb coordinated with the vivarium light cycle, and behavioral testing was performed during the “lights-on” portion of the cycle. Locomotion was monitored with a 16 x 16 photo beam array (San Diego Instruments, San Diego, CA) located ~ 2 cm above the enclosure floor. Locomotor activity was expressed as crossovers, defined as the number of times the animal entered into any of five equally-sized zones (4 ‘corners’ and 1 ‘center’) subdividing

27 the enclosure. The raw photobeam break data was collected by computer using Flex Field

software (San Diego Instruments) for 2 hours and binned into 3-minute time intervals.

Stereotypic behavior was assessed by an observer who was blinded with respect to the treatment

conditions of each mouse. The videotapes made during the testing sessions were analyzed for

stereotypic behavior, as defined as a stationary animal actively engaged in repetitive head and

limb movements, similar to grooming. A stationary animal not engaged in some type of

repetitive movement was not considered to be exhibiting stereotypy. Stereotypy was quantified

by scoring the cumulative time a mouse spent engaged in stereotypic behaviors during the first 2

minutes of every 5 minutes over the 2 hour test period.

The mice were randomly assigned to an individual RAC and allowed to habituate to the

chambers for at least 30 minutes prior to drug treatment. After the habituation period, videotaping of each mouse was begun. Subsequently, the mice were removed one at a time, weighed, and injected with the corresponding drug or vehicle. The mice were then immediately placed back in the RAC and locomotion data for each animal were recorded for 120 minutes. All compounds were injected subcutaneously.

Drugs: d-Amphetamine was obtained from the National Institute on Drug Abuse (NIDA) and dissolved in 0.9% saline solution. The same saline solution was used as the vehicle control for these experiments. The drug weights used were of the salt form and were not corrected for the free base. All injections were in a final volume of 1ml/kg.

28 Data Analysis: Data were subjected to one-way ANOVA with Dunnett's post test performed using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego California

USA, www.graphpad.com).

2.3. Results:

2.3-A: Amphetamine-induced Locomotion: the sensitivity of the C57BL/6 strain of mouse to the locomotion-inducing and stereotypy-inducing properties of acute amphetamine administration.

Numerous studies have established that amphetamine administration causes a dose-dependent bimodal behavioral response. At low doses, (0.5 – 3mg/kg) amphetamine causes an increase in locomotion while at high doses (>10mg/kg), amphetamine administration causes an increase in stereotypic behaviors, as defined by repetitive head and forelimb movements (Bedingfield et al,

1996; Ralph et al, 2001). Additionally, there is a growing body of research on species-specific and strain-specific responses to psychoactive drugs. These studies indicate some mouse strains are more sensitive to amphetamine while other stains are less sensitive or insensitive (Anisman and Kokkinidis, 1975; Kitahama and Valatx, 1979; Logan et al, 1989; Belzung and Barreau,

2000; Ralph et al, 2001;).

For these experiments, groups of mice were injected with either vehicle (saline) or a single dose of amphetamine, and their behaviors recorded for 120 minutes. The doses of amphetamine tested were: 2, 6, 12, and 20mg/kg (s.c.). Looking at the locomotion data first, presented in Figure 2-1, the results confirmed previous studies in this and other strains that the low dose of amphetamine (2mg) caused a long-lasting and pronounced period of significant hyperlocomotion compared to the saline group. This hyperlocomotion was maintained during

29 the first 90 minutes of the observation period and then slowly returned toward control levels

during the final 30 minutes. The increase in activity, as measured by crossovers, reached its

highest significance (p<0.001) compared to the saline group during the middle hour of the 2 hour test period and reached its highest activity levels during the period 30 - 60 minutes post AMPH injection. This is consistent with previous studies, which have established that the peak increase

in locomotion following AMPH administration occurs between ~30 and 60 minutes post

injection (Bedingfield et al, 1997). Conversely, at the two high AMPH doses (12 and 20mg/kg),

there was much less hyperlocomotion induced by drug administration. Following an initial brief

period of increased activity during the first 30 minute period, the two groups displayed locomotion activity similar to vehicle levels. The 12mg group did exhibit a secondary period in activity at the start of the second 30 minute interval, but by 60 minutes, this group’s activity level was approaching control levels. At no time during the 2 hour period did either the 12mg or

20mg group’s activity levels differ significantly (p >0.05) from the control. The middle dose

(6mg/kg) caused a more complex, biphasic, response. There was a short period of increased locomotion during the first 30 minutes after AMPH administration, followed by a decrease in locomotion to levels below that of the 2mg/kg group during the middle ~60 minutes, which was then followed by a second period of increased activity during the final 30 minutes of the 2 hour period. From the locomotion results, it seems clear that a dose of 2mg/kg AMPH is sufficient for the monophasic induction of hyperlocomotion in C57 mice and that from 30 – 60 minutes post drug administration is the period of maximum effect with minimum variability.

200 ** ** 175 ** 150 ** ** ** ** ** 125 ** Saline ** * ** 100 2mg * * ** * 6mg 75 12mg

Crossovers/ 10 min Crossovers/ 50 20mg 25

0 0 30 60 90 120 Minutes

Figure 2-1

Effects of different doses of amphetamine (AMPH) on locomotion in C57BL/6 mice. Saline (n =

13), 2mg/kg AMPH (n = 11), 6mg/kg AMPH (n = 5), 12mg/kg AMPH (n = 11), or 20mg/kg

AMPH (n =5) was injected subcutaneously and locomotion was recorded for 2 hours using the

RACs. Data points represent mean crossovers per 10 minute interval. The X-axis represents

time after AMPH administration. * = p < 0.05, ** = p < 0.001 significant differences compared to saline for the same time point (Dunnett’s multiple comparison post hoc analysis).

32 2.3-B: Amphetamine-induced Stereotypy: Looking at the stereotypy data collected from the

videotape analysis, presented in Figure 2-2, it is clear that AMPH administration caused a dose-

dependent increase in stereotypic behavior in C57BL/6 mice. The 2mg AMPH group displayed

stereotypic behaviors for less than half of the time over the entire 2 hour observation period. The two highest doses (12 and 20mg/kg) displayed elevated levels of stereotypic behavior almost immediately after drug administration and these levels (50 – 90% of the time) were maintained for the remainder of the 2 hour observation period. As mentioned above with regard to the complex locomotion response to the 6mg AMPH dose, the stereotypy response to this middle dose was more complex than the other doses as well. This dose produced stereotypy levels close to the 2mg dose during the first and last 30 minute intervals and stereotypy levels similar to the two highest doses during the middle hour of the observation period. In addition to the dose- dependent increase in overall time spent in stereotypy induced by AMPH administration, the onset of this behavior was also dose-dependent. The 6mg dose took about 45 minutes post injection to reach significant stereotypy levels compared to the 2mg group while the 12 and

20mg doses reached significant levels of stereotypy by 30 minutes post injection vs. 2mg.

100 90 80 70 * *** *** 60 ** *** ** *** 50 40 30

% Time in Stereotypy 2mg/kg 20 6mg/kg 10 12mg/kg 20mg/kg 0 0 30 60 90 120 Minutes

Figure 2-2

Effects of different doses of amphetamine (AMPH) on stereotypy in C57BL/6 mice. 2mg/kg

AMPH (n = 4-8), 6mg/kg AMPH (n = 4), 12mg/kg AMPH (n = 4-7), or 20mg/kg AMPH (n =4-

5) was injected subcutaneously and behavior videotaped for 2 hours. Tapes were analyzed by a blinded observer for stereotypic behaviors for 2 minute periods every 5 minutes. Data points represent mean percent time spent in stereotypy per interval. The X-axis represents time after

AMPH administration. Significant differences (Dunnett’s multiple comparison post hoc analysis) compared to 2mg/kg AMPH for the same time point are as follows: * = p < 0.05 for

6mg/kg AMPH, ** = p <0.05 for 12mg/kg AMPH, and *** = p <0.05 for 20mg/kg AMPH.

Finally, analysis of the 20mg AMPH group appears to indicate that at this dose (and

presumably higher doses) the amount of time spent in stereotypy reaches its maximum level and

in fact these mice likely have their overall motor ability impaired and are thus unable to display

any type of movements, whether gross locomotion or finer stereotypic movements.

2.3-C: Comparison of Selected AMPH doses: The analysis of the locomotion and stereotypy

data indicated that the four doses induced 3 identifiable patterns of behavior. The lowest dose of

2mg induced a pattern dominated by hyperlocomotion. The two highest doses (12 and 20mg/kg)

induced a pattern consisting mostly of stereotypy, and the middle dose (6mg) displayed a

combination of these two behaviors. To further analyze and discuss these three patterns of

behavior, we have presented the locomotion and stereotypy data for the 2mg, 6mg, and 12mg

doses of AMPH on separate graphs, Figures 2-3A, B, and C respectively, for easier comparison.

Looking at the data in this manner clearly demonstrates the inverse relationship between AMPH-

induced hyperlocomotion and stereotypy. With the locomotion-inducing dose of 2mg/kg

AMPH, the comparison graph, Figure 2-3A, shows that when the locomotion activity is at its

highest levels, between 20 and 70 minutes post injection, these mice concomitantly display the

lowest levels of stereotypy. Then during the final 45 minutes of the observation period, as locomotion is on the decline, the amount of stereotypy begins to increase, indicating that the

drug effect is clearly not gone, but that the mice are potentially moving from a state of AMPH-

induced hyperlocomotion to a state of AMPH-induced stereotypy.

175 2mg Locomotion 100 2mg Stereotypy

150 % TimeinStereotypy 75 125

100 50 75

50 Crossovers/ 10 min Crossovers/ 25 25

0 0 0 30 60 90 120 Minutes

Figure 2-3A

Demonstration of relationship between amphetamine-induced locomotion and stereotypy in

C57BL/6 mice. 2mg/kg amphetamine (AMPH) was injected subcutaneously into (n = 11) mice and activity was recorded for 2 hours. Locomotion data points (open circles) represent mean total crossovers +/- SEM per 10 minute interval and are graphed on the left Y-axis. Stereotypy data points (filled circles) represent mean percent time +/- SEM spent in stereotypic behavior during 2 minute intervals and are graphed on the right Y-axis. The X-axis represents time after

AMPH administration.

The comparison graph for the 6mg/kg AMPH dose, Figure 2-3B, clearly demonstrates

this transitioning effect from hyperlocomotion to stereotypy. This group experienced two peaks

of hyperlocomotion, one at 20 minutes post injection and a second peak 100 minutes after

AMPH administration. Separating these two peaks is a period of decreased locomotion with a concurrent single peak in stereotypic behavior. As the hyperlocomotion reaches a minimum between 40 and 70 minutes, the stereotypy observed in these mice reaches its maximum levels at exactly this same time period. This would indicate that the AMPH-induced behaviors of hyperlocomotion and stereotypy are mutually exclusive events and that a mouse will predominately exhibit either one or the other behavior, in response to AMPH administration. We take this pattern of activity to indicate that the 6mg dose is a transitional dose inducing a mix of both hyperlocomotion and stereotypy.

6mg Locomotion 300 6mg Stereotypy 100

250 Stereotypy in % Time 75 200

150 50

100

Crossovers/ 10 min Crossovers/ 25 50

0 0 0 30 60 90 120 Minutes

40 Figure 2-3B

Demonstration of relationship between amphetamine-induced locomotion and stereotypy in

C57BL/6 mice. 6mg/kg amphetamine (AMPH) was injected subcutaneously into (n = 5) mice and activity was recorded for 2 hours. Locomotion data points (open diamonds) represent mean total crossovers +/- SEM per 10 minute interval and are graphed on the left Y-axis. Stereotypy data points (filled diamonds) represent mean percent time +/- SEM spent in stereotypic behavior during 2 minute intervals and are graphed on the right Y-axis. The X-axis represents time after

AMPH administration.

Looking at the comparison graph, Figure 2-3C, for the final pattern of AMPH-induced behavior, we observed that treatment with 12mg/kg caused a clear separation between locomotion and stereotypy almost from the first time point analyzed. This high dose of AMPH initially induced a momentary brief period in hyperlocomotor behavior, but then quickly diminished with a concurrent increase in stereotypy to a level that was maintained over the course of the 2 hour observation period. Finally, this group exhibited a second spike in locomotor behavior, from 20 – 40 minutes post drug injection, while the stereotypy levels were maintained at about 55% during this period.

175 12mg Locomotion 100 12mg Stereotypy

150 % TimeinStereotypy 75 125

100 50 75

50 Crossovers/ 10 min Crossovers/ 25 25

0 0 0 30 60 90 120 Minutes

Figure 2-3C

Demonstration of relationship between amphetamine-induced locomotion and stereotypy in

C57BL/6 mice. 12mg/kg amphetamine (AMPH) was injected subcutaneously into (n = 11) mice

and activity was recorded for 2 hours. Locomotion data points (open squares) represent mean total crossovers +/- SEM per 10 minute interval and are graphed on the left Y-axis. Stereotypy data points (filled squares) represent mean percent time +/- SEM spent in stereotypic behavior during 2 minute intervals and are graphed on the right Y-axis. The X-axis represents time after

AMPH administration.

2.3-D: Time Course Evaluation: A limiting factor in carrying out stereotypy studies is that analysis of the videotapes is time consuming and labor-intensive. For these reasons, the majority of published studies focus on one particular time period or short interval to evaluate and score for stereotypic behaviors. With this in mind, we evaluated both the locomotion and stereotypy data in terms of four 30 minute intervals to determine if a particular period gave a representative indication of the observed behavior over the entire 2 hour period. The second 30 minute interval post injection seems to meet this condition. Looking first at the locomotion data in terms of total crossovers during this 30 minute period, a strong inverse dose-response relationship is apparent.

The lowest dose of 2mg AMPH resulted in the highest amount of crossovers while the highest dose of 20mg AMPH resulted in the fewest total crossovers during this interval. As can be seen in Figure 2-4, total crossovers for the 2mg/kg AMPH group were significantly greater (p <

0.001) than the saline, 12mg, or 20mg AMPH groups during this interval. The 6mg/kg dose did result in a greater number of total crossovers over the entire 2 hour period (data not shown) due to the second peak in locomotor activity, but also a greater variability due to the more complex pattern of activity. Thus, for studies investigating AMPH-induced locomotion, a dose of 2mg/kg analyzed 30 – 60 minutes post drug administration appears to be representative.

800 * * 700

600

500

400

300

Total Crossovers Total 200

100

0 Saline 2mg 6mg 12mg 20mg

46 Figure 2-4

Effects of amphetamine (AMPH) administration on locomotion 30 – 60 minutes post injection in

C57BL/6 mice. Saline (n = 13), 2mg/kg AMPH (n = 11), 6mg/kg AMPH (n = 5), 12mg/kg

AMPH (n = 11), or 20mg/kg AMPH (n =5) was injected subcutaneously and locomotion was recorded for 2 hours using the RACs. The bars represent mean total crossovers +/- SEM during the period 30 – 60 minutes post AMPH administration. * = p < 0.001 significant differences compared to saline for the same time interval (Dunnett’s multiple comparison post hoc analysis).

The stereotypy data from the period 30 – 60 minutes post injection also confirm that this is a good representative time period for analyzing the response to AMPH administration. As can be seen in Figure 2-5, the 2mg AMPH group had mean stereotypy times significantly less (p <

0.001) than the saline group, while the three higher doses of AMPH all induced significantly greater levels of stereotypy (p < 0.001) compared to saline or the 2mg group. The 20mg dose resulted in the highest percentage of time spent in stereotypy for both this period and for the entire 2 hour period (data not shown), but careful analysis of the videotapes seemed to indicate that at this dose, the mouse’s overall ability to exhibit any movements was compromised. Thus, for studies investigating AMPH-induced stereotypy, a dose of 12mg/kg analyzed 30 – 60 minutes post drug administration is sufficient.

70 ** ** 60 **

20 * % Time in Stereotypy in Time %

0 Saline 2mg 6mg 12mg 20mg

49 Figure 2-5

Effects of amphetamine (AMPH) administration on stereotypy 30 – 60 minutes post injection in

C57BL/6 mice. Saline (n = 9), 2mg/kg AMPH (n = 8), 6mg/kg AMPH (n = 5), 12mg/kg AMPH

(n = 7), or 20mg/kg AMPH (n =5) was injected subcutaneously and behavior was videotaped for

2 hours. Tapes were analyzed by a blinded observer for stereotypic behaviors for 2 minute periods every 5 minutes. The bars represent mean total percent time spent in stereotypy +/- SEM during the period 30 – 60 minutes post AMPH administration. * = p < 0.001 significantly different compared to saline; ** = p < 0.001 significantly different compared to 2mg/kg AMPH for the same time interval (Dunnett’s multiple comparison post hoc analysis).

50 2.4. Discussion:

The purpose of the current study was to provide a careful characterization of the C57BL/6 strain

of mouse and its sensitivity to behaviors induced by acute amphetamine administration. A

detailed analysis would serve a number of important functions. First, it would serve as a reference for us and other investigators who are interested in using an animal model to study a

range of issues relating to drugs of abuse and strain differences. Having a good understanding of the sensitivity of a particular strain of mouse to a specific drug of abuse would provide valuable information when choosing a background strain for transgenic or knockout work. Without a careful characterization of the background strain, it is difficult to conclude that observed phenotypes in a subsequent knockout or mutant mouse are due to a specific mutation and not to a particular genetic contribution from one of the parental strains (Ralph et al, 2001). A second objective of this study is identification of suitable doses of AMPH to induce a specific behavior, allowing further investigations into the mechanisms contributing to the behavior and/or into compounds which may modulate these behaviors. Towards this end, we have established that the C57BL/6 strain exhibits the widely observed bimodal response to acute AMPH administration. At lower doses, the primary response to AMPH was an induction of hyperlocomotion, while at higher doses, the hyperlocomotor response was replaced with a significant increase in stereotypic behaviors. We found that a dose of 2mg/kg AMPH is sufficient to induce a pattern of sustained hyperlocomotive behavior in C57BL/6 mice. This dose is consistent with reports from the literature that indicate C57BL/6 mice are quite sensitive to the locomotor-inducing properties of AMPH. Doses between 1 and 5mg/kg AMPH have been shown to induce significant increases in locomotion in C57BL/6 mice under a number of different testing and recording conditions (Davis et al, 1974; Kitahama and Valatx, 1979; Ralph

51 et al, 2001). On the other hand, we found that a dose of 12mg/kg AMPH is sufficient to induce a predominately stereotypic behavioral response while not incapacitating the animal to the point of inhibiting all movement. This is also consistent with reports for the induction of stereotypy in mice. 10mg/kg AMPH has been shown to induce short periods of focal stereotypies in C57BL/6 mice and an acute dose of 12mg/kg induced stereotypy in about 90% of all CF-1 mice treated

(Bedingfield et al, 1997; Ralph et al, 2001). However, this report is the first we are aware of that studied the induction of both AMPH-induced locomotion and stereotypy in a single strain of mouse.

Figures 2-3A, B, and C illustrate the inverse relationship between locomotion and stereotypy induced by acute AMPH administration. A low dose of AMPH induced robust hyperlocomotion for a significant period of time, while a high dose of AMPH induced a predominately stereotypic response for an extended period of time. A middle dose of AMPH produced a mix of both significant hyperlocomotion and significant stereotypy. We have concluded that the 6mg dose is a ‘transitional’ dose which is at or near the threshold for inducing stereotypic behaviors within C57 mice. This complex pattern of locomotion and stereotypy at a middle dose of AMPH is consistent with what has been reported for CF-1 mice, where doses between 6-10mg/kg induce a mixture of the two behaviors while higher doses induce prolonged periods of stereotypy (Bedingfield et al, 1996; Battisti et al, 1999). Additionally, like CF-1 mice, the stereotypic behaviors induced by AMPH administration in C57BL/6 mice are manifested as a stationary animal involved in intense grooming behaviors with repetitive head and paw movements, licking, and gnawing. Finally, the stereotypy data demonstrate that the onset and persistence of stereotypic behaviors are dose-dependent. The higher the dose of AMPH, the earlier the onset of stereotypic behavior and the longer this behavior lasts. Again, the middle

52 dose of 6mg induced a pattern of behavior that was in between the low and high dose groups.

While the stereotypy studies we found in the literature indicated the doses of AMPH used and the time periods studied for behavioral analysis, none reported the actual time course of the behavior over an extended observation period nor did they address the kinetics of the response post drug injection. For these reasons, we feel this extended analysis of AMPH- induced stereotypy and locomotion is of note as reference for future work.

It’s clear that all doses of AMPH tested initially induced a period of hyperlocomotion.

This is seen with the elevated numbers of crossovers, compared to saline, induced by AMPH treatment at the first 10 minute time point graphed. Additionally, it is evident that the first 30 minutes post drug injection is the most dynamic period of change for both locomotion, and to a lesser extent, stereotypy. This is seen with the continuous changes in crossovers exhibited by the various treatment groups. The stereotypy data also indicate the first 30 minute interval is a period of ongoing change in behavior levels, with the exception of the 2mg AMPH group, which remained fairly stable in the level of observed stereotypy during this period. The 6mg, 12mg, and 20mg AMPH groups all exhibited, to differing degrees, increasing stereotypy levels during this initial 30 minute period. This period of rapid changes in behavior is consistent with the time needed for the AMPH to enter the mouse’s system and induce a behavioral state of either hyperlocomotion or stereotypy.

Because the analysis of stereotypy data from a long time course experiment (such as two hours) is very time and labor intensive, we wished to determine if a particular interval of the two hour period would provide a suitable window for evaluating both locomotion and stereotypy data and serve as a representative of the entire observation period. As mentioned above, while the first 30 minute interval post-AMPH administration was a period of ongoing changes in behavior

53 levels and intensities, the second 30 minute interval (30-60 minutes post injection) seemed to be

a good candidate period for analysis. As Figure 2-4 indicates, looking at total crossovers during

the second 30 minute interval as a measure of locomotor activity, there is a clear inverse dose-

response relationship seen. The lowest dose of 2mg AMPH lead to the greatest number of crossovers (681); the highest dose of 20mg AMPH caused the least amount of crossovers (165) during this period. The stereotypy data for the second 30 minute interval also supports this period as a good candidate period for analysis. The three highest doses of AMPH all had similar levels of stereotypy (55 – 70% of time spent in stereotypy) during the observed time intervals scored for this 30 minute period, while the 2mg group displayed its lowest level of stereotypy

(20% of the time spent in stereotypy). Thus, we feel that the 30 – 60 minute post AMPH administration time period is a useful interval for analyzing both locomotion and stereotypy data for subsequent experiments.

As stated above, one goal of the present study was to identify doses of AMPH that consistently induce a pattern of either increased locomotor activity or increased stereotypic behavior in C57BL/6 mice. The reason for this was to provide us and other investigators with appropriate doses to use for further study of the AMPH-induced behaviors of hyperlocomotion and stereotypy. By identifying doses which induce a single predominate behavior, while minimizing drug-induced behavioral ‘side effects’, it is hoped that this will lead to an increased ability to design and study modulatory and modifying effects on these behaviors. By using minimum amounts of a drug to induce the desired behavior, the sensitivity of pretreatment or co- treatment paradigms with compounds of interest will be maximized and hopefully increase the ability to detect alterations in the behavior being studied. Conversely, the 6mg/kg dose seemed to induce a transitional state of behaviors where both hyperlocomotion and stereotypy were

54 exhibited to significant levels during the observation period. While we feel this dose is not suitable for studying either one of the AMPH-induced behaviors individually, the possibility remains for the investigation of compounds and treatments along with this dose of AMPH to determine if co-treatment pushes the predominate behavior toward locomotion or stereotypy. In this manner, administration of specific compounds which modulate the dopaminergic, glutamatergic, GABAergic, or other neurotransmitter systems can be investigated and their involvement in AMPH-induced behaviors studied.

Chapter Three

Differential Involvement of Opioid Receptors in Amphetamine-

induced Behaviors in C57BL/6 Mice

56 3.1. Introduction:

The mesocorticolimbic dopamine system is thought to be a primary site of action for the rewarding and reinforcing effects of natural and artificial stimuli (Everitt and Wolf, 2002). The reinforcement of responses to natural stimuli, such as food and sex, are thought to be evolutionarily important for survival, reproduction, and the overall fitness of a species (Kelley and Berridge, 2002). In contrast, the reinforcement of responses to artificial stimuli, such as drugs of abuse (DOA), can lead to the development of drug addiction, a serious disease of modern society (White, 2002). In an attempt to understand and explain the brain reward system, researchers have studied the effects of these ‘unnatural’ stimuli on organisms at both the molecular/cellular level and at the behavioral/systems level. Much of this work has focused on identifying changes induced in the mesolimbic dopamine system by drugs of abuse, including psychomotor stimulants, opiates, ethanol, and others. Studies have well established that acute administration of various drugs of abuse, particularly the psychostimulants cocaine and amphetamine (AMPH), lead to increases in extracellular dopamine (DA) levels in a number of regions comprising the mesolimbic DA system, including the nucleus accumbens (NA), the ventral tegmental area (VTA), the striatum, and the dorsal caudate (DC) (Heidbreder et al, 1996;

Wise, 1981, 1982). The mechanisms by which psychostimulants increase DA concentrations involve an inhibition of DA reuptake by the transporter and/or by promoting reverse transport of

DA (Koob and Bloom, 1988). It has been theorized that this effect on DA levels within the mesolimbic DA system, especially within the NAc, contributes significantly to not only the rewarding properties of these drugs, but also various behavioral aspects of their use, including modulation of locomotor activity and the development of stereotypies (DiChiara and Imperato,

57 1988; Koob and Bloom, 1988) The investigation of drug-induced stereotypies is thought to have

relevance not only to drug abuse and addiction but also to the development of psychoses in

humans (Lieberman, et al, 1997).

However, while the involvement of the NAc in the rewarding properties of DOA has

been well established, there is still considerable debate over the specific mechanisms involved

(Hoffman and Lupica, 2001). In a simplified model, the critical circuitry of the brain reward

system is thought to be between the NAc and the VTA, as these regions are intrinsically involved

with the rewarding properties of drugs of abuse (Wise, 1982; Koob and Bloom, 1988). The VTA

contains the cell bodies of the mesolimbic DA system which project to the NAc and frontal

cortex, providing dopaminergic input to these structures (Carlezon and Wise, 1996). The

majority of neurons within the NAc are GABAergic medium spiny neurons (MSNs) which

receive this DA input and also receive glutamatergic input from the hippocampus, amygdala, and

prefrontal cortex and which in turn send their inhibitory output projections to several brain

structures, including the VTA (Groenewegen et al, 1991; Steffensen et al, 1998; Christie et al,

1985, 1987). Glutamate and dopamine are thought to have opposite actions on their target

neurons in the NAc – glutamate is believed to excite MSNs and DA inhibits them (Carlezon and

(Hoffman and Lupica, 2001).

The identification of evolutionarily-conserved molecular components comprising the brain reward system, including (but not limited to) DA, glutamate, GABA, G proteins, G protein

58 coupled receptors, amine transporters, various effector molecules, and endogenous peptide

agonists, has enabled the similarities and differences in the reinforcing actions of drugs of abuse

to be studied. Additionally, the crosstalk between different classes of rewarding stimuli and their effects on the dopaminergic system have also been investigated. There is growing evidence to indicate that opioids not only share many similar rewarding properties of psychostimulants, but

that the endogenous opioid system can modulate the mesolimbic dopaminergic system as well.

The evidence for this is three-fold. (i.) Neuroanatomically, the three types of opioid receptors (µ,

κ, and δ) have been identified, via binding studies, on mesolimbic dopaminergic neurons

(Spanagel et al, 1992; Mansour et al, 1995). However, the absolute amount and regional

distribution of the various receptor types differ. Dense µ receptor binding has been seen in the

VTA with little δ or κ opioid receptor binding present, while high δ and κ receptor binding is

seen in the NAc with low to moderate µ-specific binding seen in this region (Mansour et al,

1987, 1988; Tempel and Zukin, 1987).

(ii.) Secondly, at the molecular/cellular level, opioid agonists and antagonists have been

shown to modulate extracellular DA levels in the mesolimbic DA system. There is mounting

evidence that the different opioid receptor subtypes have differing effects on the regulation of

basal dopamine levels, depending on method of delivery and region studied. Systemic

administration of µ and δOR agonists can cause dose-dependent increases in DA levels in the

VTA, NAc, and dorsal caudate, while systemic κOR agonists cause decreased DA

concentrations within the VTA and NAc (DiChiara and Imperato, 1988; Spanagel et al, 1992;

Devine et al, 1993a). These effects by µ and δ agonists can be blocked by administration of selective receptor antagonists while the κOR effects were insensitive to antagonist treatment

(DiChara and Imperato, 1988; Spanagel et al, 1992; Devine et al, 1993a). Injections of either the

59 µ agonist DAMGO or δ agonist DPDPE directly into the VTA were shown to cause dose-

dependent ventral striatal DA increases, with the δ agonist being significantly more effective

than the µ agonist (Devine et al, 1993a). Injections of µOR agonists or antagonists directly into

the NAc showed no effect on DA levels within this structure (Spanagel et al, 1992). Conversely,

κOR agonists injected into the NAc lead to significant inhibition of DA release within the NAc,

while injection of the κ antagonist nor BNI into the NAc lead to an increase in DA release

(DiChiara and Imperato, 1988; Spanagel et al, 1992). Injections of either κOR agonist or

antagonist into the VTA have been shown to have no effect on DA release (DiChiara and

Imperato, 1988; Spanagel et al, 1992; Devine et al, 1993a). Studies have also investigated the

modulation of DA levels by the opioid system in response to psychostimulant administration.

The non-selective opioid receptor antagonist naloxone, at 5mg/kg s.c., was shown in rats to attenuate the AMPH-dependent increase in extracellular levels of DA in the NAc and striatum while having no effect on DA levels when administered alone (Jones and Holtzman, 1992;

Hooks et al, 1992). It is assumed that the observed modulation of DA levels by opioids is mediated via activation or inhibition of their respective receptors in various parts of the mesolimbic DA system, particularly the NAc and VTA. It has been hypothesized that the opposing effects of µ/δ vs. κ agonists on mesolimbic DA release underlie their opposing effects on motivation and reward (DiChiara and Imperato, 1988; Spanagel et al, 1992). Generally, mu and delta agonists, such as morphine and enkephalins, function as positive reinforcers while kappa agonists have aversive effects. From these findings, it is clear that activation of opioid receptors can modulate the DA tone within specific brain regions that participate in the reward pathway and it has been suggested that tonic activation of µ and κORs is required for the maintenance of basal DA levels within the NAc (Spanagel et al, 1992).

60 (iii.) The third area of evidence supporting a modulatory role of the opioid system on the

DA system comes from behavioral/systems research. The administration of AMPH to rodents

generally produces two qualitatively different behaviors. When administered at low doses,

AMPH produces a primary response of hyperlocomotion, while higher doses produce repetitive, stereotyped behaviors such as intense grooming, that disrupts or interferes with locomotion

(Battisti et al, 1999). Both of these behaviors are thought to be due, in large extent, to increases

in DA transmission. However, while the AMPH-dependent increase in locomotion is due to

increased DA in the NA, the AMPH-dependent increase in stereotypic behaviors is due to DA

increases in the striatum (Creese and Iversen, 1974; Kelly et al, 1975). This inverse relationship

of amphetamine-induced locomotion vs. stereotypy provides two relatively clear behavioral

endpoints with which to study the potential modulating effects of various compounds. Studies

have thus investigated the modulation by opioids of AMPH-induced behaviors in various animal

models. It has been shown in rats that naloxone can attenuate the AMPH-induced increase in

locomotor activity (Jones and Holtzman, 1992; Hitzman et al, 1982; Skorupska and Langwinski,

1989). Similarly, the δOR receptor-selective antagonist naltrindole (NTI) administered i.c.v. has been shown to markedly attenuate the gross motor activity of rats in response to AMPH- administration, while the µ-selective antagonist β-FNA and the κ-selective antagonist nor-BNI

both failed to influence the gross motor response to AMPH (Jones and Holtzman, 1992; Jones et

al, 1993). The exact mechanisms responsible for the opioid system-dependent modulation of

AMPH-induced locomotor activity are still unclear. Additionally, there are few reports

investigating the contributions of the three opioid receptor subtypes in modulating both AMPH-

induced hyperlocomotion and stereotypy.

61 We therefore wished to study the individual contribution each opioid receptor type makes

in modulating the locomotor-inducing and stereotypy-inducing properties of acute AMPH

administration in the C57BL/6 strain of mice. Our previous work studying the sensitivity of

C57BL/6 mice to AMPH administration indicated that for inducing a prolonged hyperlocomotive

state, an acute dose of 2mg/kg AMPH is sufficient, and for inducing a robust stereotypic

response, 12mg/kg AMPH is sufficient. These doses are consistent with those used in reports

from the literature in this and other strains (Davis et al, 1974; Kitahama and Valatx, 1979;

Bedingfield et al, 1996; Ralph et al, 2001).

The results of our experiments confirm that in C57BL/6 mice, the non-selective opioid

antagonist naloxone does have an inhibitory effect on AMPH-induced locomotor activity.

However, the contributions of the individual receptor types vary. Specifically, antagonism of the

δOR significantly attenuated the AMPH-induced increase in locomotion, similar to naloxone, while antagonizing the κOR failed to significantly alter the locomotor-enhancing activity of

AMPH in C57 mice. However, we did see a slight augmentation of locomotor activity, as

measured by total crossovers, induced by pretreatment with the µOR antagonist β-FNA compared to AMPH alone during the period 30 – 60 minutes post AMPH administration.

Analyzing this same time interval for changes in stereotypic behaviors following AMPH administration, we found that pretreatment with one non-specific opioid antagonist and three receptor-specific antagonists all significantly reduced the level of stereotypic behaviors exhibited by these mice compared to the 12mg/kg AMPH-treatment alone. Thus, while the δOR seems to be involved with inhibiting AMPH-induced locomotion, the µOR might potentially augment

AMPH-induced locomotion, and the κOR does not alter AMPH-induced locomotor behavior. In contrast, it would appear that antagonism of all three opioid receptors can attenuate AMPH-

62 induced stereotypy. This is the first report that we are aware of that systematically investigated the modulatory effects of the individual opioid receptor subtypes on both AMPH-induced hyperlocomotion and stereotypy in mice.

3.2. Materials and Methods:

Subjects: Male C57BL/6 mice (Charles River Laboratories, Raleigh, NC) between 5 to 7 weeks

Locomotor and Stereotypy Activity: Behavioral characterization was carried out in 30 custom- designed residential activity chambers (RACs), modeled after the design of Segal and Kuczenski

(1987). Each chamber consisted of a lighted, ventilated, sound-attenuated cabinet (Cline

Builders, Covington, KY) housing a 16”x16”x15” plexiglass animal enclosure. A fan mounted in the side of the exterior cabinet of each enclosure provided air circulation and constant background noise, and a Sony CCD videocamera mounted in the top of each enclosure allowed experimental sessions to be videotaped via an attached VHS VCR. Light inside the chambers was provided by a 15-watt bulb coordinated with the vivarium light cycle, and behavioral testing was performed during the “lights-on” portion of the cycle. Locomotion was monitored with a 16 x 16 photo beam array (San Diego Instruments, San Diego, CA) located ~ 2 cm above the

63 enclosure floor. Locomotor activity was expressed as crossovers, defined as the number of times

the animal entered into any of five equally-sized zones (4 ‘corners’ and 1 ‘center’) subdividing

the enclosure. The raw photobeam break data was collected by computer using Flex Field

software (San Diego Instruments) for 2 hours and binned into 3-minute time intervals.

Stereotypic behavior was assessed by an observer who was blinded with respect to the treatment

conditions of each mouse. The videotapes made during the testing sessions were analyzed for

stereotypic behavior, as defined as a stationary animal actively engaged in repetitive head and

limb movements, similar to grooming. A stationary animal not engaged in some type of

repetitive movement was not considered to be exhibiting stereotypy. Stereotypy was quantified

by scoring the cumulative time a mouse spent in stereotypic behaviors during the first 2 minutes

of every 5 minutes over the 2 hour test period.

The mice were randomly assigned to an individual RAC and allowed to habituate to the

chambers for at least 30 minutes prior to drug treatment. After the habituation period, the mice were removed one at a time, weighed, and injected with the corresponding drug or vehicle. The mice were then immediately placed back in the RAC and locomotion data were recorded for 120 minutes and each session was videotaped. For the experiments involving pretreatment with opioid antagonists, the mice were initially weighed and injected with antagonist, then placed in a

RAC. Thirty minutes following the antagonist injection, the mice were removed, injected with amphetamine, placed back in the RAC, and locomotor and video activity recorded for 120 minutes. All compounds were injected subcutaneously.

Drugs: Naltrindole hydrochloride (NTI), nor-binaltorphamine (nor-BNI), and β-funaltrexamine

(β-FNA) were purchased from Tocris (Ellisville, Missouri). d-Amphetamine and naloxone were

64 obtained from the National Institute on Drug Abuse (NIDA). All drugs were dissolved in 0.9%

saline solution. The same saline solution was used as the vehicle control for these experiments.

The drug weights used were of the salt forms and were not corrected for the free base. All

injections were in a final volume of 1ml/kg.

Data Analysis: Data were subjected to one-way ANOVA with Dunnett's post test performed using GraphPad Prism version 3.02 for Windows (GraphPad Software, San Diego California

USA, www.graphpad.com). For purposes of presentation, the locomotion data are reported as

mean crossovers per 10 minute interval recorded over two hours or total mean crossovers

recorded during a 30 minute interval. The stereotypy data are presented as mean percent time spent in stereotypic behavior totaled from the scoring of the first two minutes of each five minute interval from 30 – 60 minutes post drug administration.

3.3. Results:

3-3-1: Antagonist Dose Responses: We first wished to determine the basal sensitivity of the

C57BL/6 strain of mice to the opioid receptor antagonists we would be using in combination

with AMPH in the second set of experiments. We chose to test one non-selective receptor

antagonist (naloxone) and three receptor-selective antagonists. The µOR antagonist chosen was

β-FNA. The δOR antagonist was naltrindole (NTI), and the κOR antagonist tested was nor-BNI.

For these experiments, three or four doses of each drug were injected, s.c., into groups of

between 4 and 11 mice. The doses of the µOR and δOR receptor-selective antagonists were

5mg/kg, 20mg/kg, and 50mg/kg. The doses for the κOR-selective antagonist was 5mg/kg,

65 10mg/kg, 20mg/kg, and 50mg/kg and the doses of the non-selective antagonist tested were

2mg/kg, 5mg/kg, and 10mg/kg. The doses tested were selected based on the literature to ensure coverage of the range for each antagonist that had been shown to block or reverse the effects of their respective receptor agonists.

Summarizing the antagonist dose-response locomotion data are Figures 3-1 and 3-2.

Figure 3-1 displays the continuous locomotor behavior, as measured by crossovers from one

‘zone’ to another ‘zone’, for selected doses of each antagonist over the course of the two hour observation period. Figure 3-2 displays the total crossovers recorded during the time period of

30 – 60 minutes post drug administration. We have shown that for analyzing locomotion and stereotypy data, this time interval is a good representation of the longer two hour observation period. As can be seen in Figure 3-2, three of the 4 antagonists caused a slight decrease in total crossovers, while β-FNA caused a slight increase in total number of crossovers. However, none of these changes in locomotor activity induced by antagonist administration were significantly different from saline (p >0.05) via ANOVA and Dunnett’s multiple comparison post hoc test.

Saline 40 5mg FNA 5mg NTI 2mg Naloxone 10mg nBNI 30

20 Crossovers/ minutes 10 10

0 0 30 60 90 120 Minutes

67 Figure 3-1

Effects of opioid receptor antagonist administration on locomotion in C57BL/6 mice. Saline (n

= 12), 2mg/kg naloxone (n = 11), 5mg/kg β-FNA (n = 4), 5mg/kg NTI (n = 4), or 10mg/kg nor-

BNI (n = 9) was injected subcutaneously and locomotion was recorded for 2 hours using the

RACs. Data points represent mean crossovers per 10 minute interval. The X-axis represents time after drug administration.

Interval 2

150

125

100

50 Crossovers/ 30 30 minutes Crossovers/ 25

0 Saline 5mg FNA 5mg NTI 2mg Naloxone 10mg nBNI

69 Figure 3-2

Effects of opioid antagonist administration on locomotion during the second 30 minute interval post injection in C57BL/6 mice. Groups and treatments are the same as in Figure 3-1. Bars represent mean total crossovers +/- SEM during the period 30 – 60 minutes post drug administration. None of the drug groups differed significantly compared to saline.

Figure 3-3 summarizes the levels of stereotypy induced by each opioid antagonist during the period of 30 – 60 minutes post administration. As can be seen, acute administration of

2mg/kg naloxone and 5mg/kg NTI both failed to significantly alter stereotypic behaviors (p >

0.05) compared to saline, while 5mg/kg β-FNA caused a significant increase (p < 0.05) in stereotypy compared to saline. Conversely, treatment with the κOR antagonist nor-BNI at

10mg/kg significantly decreased (p < 0.001) stereotypy levels compared to saline. Based on the locomotion and stereotypy results from these and the other doses tested (data not shown), the following doses of each antagonist were chosen for the next set of experiments: β-FNA and nor-

BNI, 10mg/kg; naltrindole, 5mg/kg; and naloxone, 2mg/kg.

3-3-2: Opioid Antagonist Pretreatment + Amphetamine:

We next wished to investigate the contribution of each opioid receptor subtype to

AMPH-induced locomotion and stereotypy in C57BL/6 mice. For these experiments, groups of mice were pretreated with an individual opioid antagonist at the dose determined from part 3-3-

1, followed 30 minutes later by administration of 2mg/kg AMPH for locomotion analysis and

12mg/kg AMPH for stereotypy analysis. Locomotion data were then recorded for two hours and each animal was videotaped as well for subsequent stereotypy scoring.

Saline 2mg Nalox 5mg FNA 5mg NTI

60 10mg nBNI *

30 **

20 % Time% Stereotypy

0 Saline 2mg Nalox 5mg FNA 5mg NTI 10mg nBNI

72 Figure 3-3

Stereotypy induced by opioid antagonist treatment in C57BL/6 mice measured during the period

30 – 60 minutes post drug administration. Saline (n = 9), 2mg naloxone (n = 8), 5mg/kg β-FNA

(n = 4), 5mg/kg NTI (n = 3), or 10mg/kg nor-BNI (n = 5) mice were injected subcutaneously, and stereotypy behavior was analyzed for the interval 30 – 60 minutes post drug administration.

Data bars represent mean percentage of time +/- SEM spent in stereotypy for the first two minutes of every 5 minutes during this time interval. * = p < 0.05 significantly different compared to saline for the same time interval; ** = p < 0.001 significantly different compared to saline for the same time interval (ANOVA and Dunnett’s multiple comparison post hoc analysis).

73 Locomotion Results:

3-3-2A: Naloxone + AMPH: The locomotion data for pretreatment with the non-selective opioid receptor antagonist naloxone followed by administration of 2mg/kg AMPH are presented in Figure 3-4. Two mg/kg naloxone pretreatment caused a decrease in locomotion throughout the 2- hour period vs. 2mg/kg AMPH-alone. The total crossovers (see Figure 3-8) recorded for the naloxone + AMPH group during the interval 30 – 60 minutes post AMPH administration were increased compared to the saline group but failed to reach statistical significance. The activity of the naloxone-alone group did not differ significantly from the control group over the

120-minute period. On a 10-minute point by point comparison, naloxone pretreatment caused significantly less hyperlocomotion (p < 0.05) vs. AMPH-alone treatment at 20, 30, and 90 minutes post AMPH administration.

175

Saline 150 2mg/kg AMPH 2mg/kg Naloxone 125 2mg AMPH + 2mg Naloxone

100 * 75 * 50 * Crossovers/ 10 minutes 10 Crossovers/ 25

0 0 30 60 90 120 Minutes

75 Figure 3-4

The effect of naloxone pretreatment (2mg/kg) on AMPH-induced hyperlocomotion in C57BL/6 mice. Saline (n = 13), 2mg naloxone (n = 11), 2mg/kg AMPH (n = 11), or 2mg/kg naloxone +

2mg/kg AMPH (n = 13) mice were injected subcutaneously and locomotion activity was recorded for 2 hours using the RACs. Data points represent mean +/- SEM crossovers per 10- minute interval. The X-axis represents time after AMPH administration; naloxone pretreatment was 30 minutes prior to AMPH administration. * = p < 0.05 significantly different compared to

2mg/kg AMPH for the same time point (Dunnett’s multiple comparison post hoc analysis).

3-3-2B: β-FNA + AMPH: The locomotion data for pretreatment with the µOR-selective

antagonist β-FNA followed by administration of AMPH are presented in Figure 3-5. Thirty minute pretreatment with 10mg/kg of β-FNA followed by administration of 2mg/kg AMPH did not lead to a significant increase or decrease in hyperlocomotion compared to AMPH treatment

alone. The number of crossovers for the β-FNA pretreatment group and the 2mg/kg AMPH

group mirror each other quite closely during the first 90 minutes of the observation period and it

is not until the final 30 minute interval that the β-FNA group falls below the AMPH-alone group.

The β-FNA pretreatment group did have significantly greater levels of locomotion (p< 0.001)

compared to saline-alone during the period 30 – 60 minutes post AMPH administration, as

shown in Figure 3-8. Additionally, we saw a slight augmentation of locomotion following β-

FNA pretreatment compared to 2mg/kg AMPH during this same period, but this increase did not

reach statistical significance. This slight increase is consistent with the small augmentation of

total crossovers seen by administration of β-FNA by itself in the dose-response experiments

presented above. There were no statistical differences in activity between the saline-alone and β-

FNA alone groups at any point during the 2 hour period.

175 Saline 2mg/kg AMPH 150 5mg/kg FNA 2mg AMPH + 10mg FNA 125

100

50 Crossovers/ 10 minutes 25

0 0 30 60 90 120 Minutes

78 Figure 3-5

The effect of β-FNA pretreatment (10mg/kg) on AMPH-induced hyperlocomotion in C57BL/6 mice. Saline (n = 13), 5mg/kg β-FNA (n = 4), 2mg/kg AMPH (n = 11), or 10mg/kg β-FNA +

2mg/kg AMPH (n = 14) mice were injected subcutaneously and locomotion activity was recorded for 2 hours using the RACs. Data points represent mean crossovers +/- SEM per 10- minute interval. The X-axis represents time after AMPH administration; β-FNA pretreatment was 30 minutes prior to AMPH administration.

3-3-2C: Naltrindole + AMPH: The locomotion data for pretreatment with the δOR-selective antagonist NTI followed by administration of AMPH are presented in Figure 3-6. Thirty minute pretreatment with 5mg/kg NTI followed by 2mg/kg AMPH administration lead to a significant decrease (p< 0.001) in locomotor activity vs. AMPH-alone that was maintained for the majority of the 2 hour observation period. This decrease in hyperlocomotion was significant at each ten minute time point starting at 20 minutes post AMPH administration and continuing until 110 minutes post AMPH administration, and was most significant during the second 30 minute interval (Figure 3-8). This attenuation of AMPH-induced hyperlocomotion by NTI was greater than that caused by naloxone pretreatment. Additionally, unlike β-FNA pretreatment, the NTI +

AMPH group did not have significantly more activity vs. saline-alone at any point during the observation period. Thus, antagonism of the δOR in C57BL/6 mice seemed to completely block the locomotor-activating effect of AMPH.

175

150 Saline 2mg/kg AMPH 5mg/kg NTI 125 2mg AMPH + 5mg NTI

100

75 * * 50 * * * * * Crossovers/ 10 minutes Crossovers/ * * * 25

0 0 30 60 90 120 Minutes

81 Figure 3-6

The effect of naltrindole (NTI) pretreatment (5mg/kg) on AMPH-induced hyperlocomotion in

C57BL/6 mice. Saline (n = 13), 5mg/kg NTI (n = 4), 2mg/kg AMPH (n = 11), or 5mg/kg NTI +

2mg/kg AMPH (n = 12) mice were injected subcutaneously and locomotion activity was recorded for 2 hours using the RACs. Data points represent mean crossovers +/- SEM per 10- minute interval. The X-axis represents time after AMPH administration; NTI pretreatment was

30 minutes prior to AMPH administration. * = p < 0.001 significantly different compared to

2mg/kg AMPH for the same time point (Dunnett’s multiple comparison post hoc analysis).

3-3-2D: nor-BNI + AMPH: The locomotion data for pretreatment with the κOR-selective

antagonist nor-BNI followed by administration of AMPH are presented in Figure 3-7.

Pretreatment with 10mg/kg nor-BNI followed by administration of 2mg/kg AMPH failed to modulate the locomotor response to AMPH in C57BL/6 mice. Similar to β-FNA pretreatment,

locomotor activity of the nor-BNI + AMPH group was significantly greater (p <0.001) than the

saline group during the second 30 minute interval (Figure 3-8). But unlike β-FNA pretreatment,

nor-BNI did not cause any increase in total crossovers compared to 2mg/kg AMPH at any time

point measured.

175 Saline 150 2mg/kg AMPH 10mg/kg nBNI 125 2mg AMPH +10mg nBNI

100

50 Crossovers/ 10 minutes Crossovers/ 25

0 0 30 60 90 120 Minutes

84 Figure 3-7

The effect of nor-BNI pretreatment (10mg/kg) on AMPH-induced hyperlocomotion in C57BL/6 mice. Saline (n = 13), 10mg/kg nor-BNI (n = 9), 2mg/kg AMPH (n = 11), or 10mg/kg nor-BNI

+ 2mg/kg AMPH (n = 13) mice were injected subcutaneously and locomotion activity was recorded for 2 hours using the RACs. Data points represent mean crossovers +/- SEM per 10- minute interval. The X-axis represents time after AMPH administration; nor-BNI pretreatment was 30 minutes prior to AMPH administration.

Saline 2mg AMPH 2mg Nalox + 2mg AMPH 10mg FNA + 2mg AMPH 5mg NTI + 2mg AMPH 10mg nBNI + 2mg AMPH 1250 * 1000 * * 750

500 ** 250

Total Crossovers ** 0 Saline 2mg AMPHNalox + 2mg FNA + 2mg NTI + 2mg BNI + 2mg

86 Figure 3-8

The effect of opioid antagonist pretreatment on AMPH-induced locomotion in C57BL/6 mice during the period 30 – 60 minutes post AMPH administration. Saline ( n = 13), 2mg/kg AMPH

(n = 11), 2mg/kg naloxone + 2mg/kg AMPH (n = 13), 10mg/kg β-FNA + 2mg/kg AMPH (n =

14), 5mg/kg NTI + 2mg/kg AMPH (n = 12), or 10mg/kg nor-BNI + 2mg/kg AMPH (n = 13) mice were injected subcutaneously and locomotion was recorded for 2 hours using the RACs.

The bars represent mean total crossovers +/- SEM during the period 30 – 60 minutes post AMPH administration. * = p < 0.001 significantly different compared to saline treatment; ** = p < 0.001 significantly different compared to 2mg/kg AMPH treatment (ANOVA analysis and Dunnett’s multiple comparison post hoc test)

87 Stereotypy Results:

3-3-3 Stereotypy: The second half of the experiments we performed involved investigating the

effects of pretreatment with the various opioid antagonists had on the stereotypic response of

C57BL/6 mice to a high dose of AMPH. For these experiments, we again used a 30-minute

pretreatment paradigm with each antagonist followed by administration of 12mg/kg AMPH. As

we have established, this dose of acute AMPH induces a sustained period of significantly

increased stereotypy compared to saline or 2mg/kg AMPH treatments. Alterations in the

amount of stereotypy induced by antagonist pretreatment were determined by measuring the total percentage of time spent in stereotypic behavior for each pretreatment group compared to

12mg/kg AMPH during the first two minutes of every five minute interval recorded from 30 – 60

minutes post AMPH administration. An observer who was blinded to the treatment of each group of mice assessed stereotypy. We have not presented the locomotion data for this set of experiments, as we did with the 2mg/kg AMPH treatment (above), because we have found that the 12mg dose of AMPH does not induce locomotor activity significantly different than that induced by saline over the two hour observation period. Additionally, pretreatment with the various antagonists did not lead to either significant increases or decreases in locomotion or obvious changes in the pattern of activity over the two hour period compared to AMPH-alone.

The stereotypy data for the pretreatment experiments are graphically summarized in

Figure 3-9 and the statistical analysis is presented in Table 1. The doses for each opioid antagonist were as follows: naloxone, 2mg/kg; β-FNA, 10mg/kg; NTI, 5mg/kg; and nor-BNI,

10mg/kg.

Saline

60 12mg AMPH Nalox + 12mg FNA + 12mg NTI + 12mg 50 * * BNI + 12mg * 40 *

20 % Time% Stereotypy

0 Saline 12mg AMPH Nalox + 12mg FNA + 12mg NTI + 12mg BNI + 12mg

89 Figure 3-9

The effect of opioid antagonist pretreatment on AMPH-induced stereotypy in C57BL/6 mice during the period 30 – 60 minutes post AMPH administration. Saline (n = 9), 12mg/kg AMPH

(n = 7), 2mg naloxone + 12mg AMPH (n = 6), 10mg/kg β-FNA + 12mg AMPH (n = 7), 5mg/kg

NTI + 12mg/kg AMPH (n = 9), or 10mg/kg nor-BNI + 12mg/kg AMPH (n = 5) mice were injected subcutaneously, and stereotypy behavior was analyzed for the interval 30 – 60 minutes post AMPH administration. Data bars represent mean percentage of time +/- SEM spent in stereotypy for the first two minutes of every 5 minutes during this time interval. Antagonist pretreatment was 30 minutes prior to AMPH administration. * = p < 0.001 significantly different compared to 12mg/kg AMPH (ANOVA and Dunnett’s multiple comparison post hoc analysis).

From Figure 3-9, it can be seen that pretreatment with all four opioid antagonists significantly reduced the magnitude of stereotypic behaviors induced by 12mg/kg AMPH during the interval 30 – 60 minutes post AMPH administration. 12mg/kg AMPH induced stereotypic behaviors 58.0% of the scored time during this interval, which was significantly greater compared to saline (p < 0.001, 41.4%). Compared to the 12mg AMPH group, naloxone- (43.3% of time) and β-FNA- (44.6% of time) pretreated mice both displayed significantly less stereotypic behavior (p < 0.001). Pretreatment with NTI or nor-BNI induced even larger attenuations of AMPH-induced stereotypy. NTI pretreated mice exhibited stereotypy 38.2% of the scored time (p < 0.001), while nor-BNI pretreated mice exhibited stereotypic behaviors

34.6% of the time (p < 0.001). Pretreatment with any of the antagonists followed by AMPH administration did not lead to significantly different stereotypy levels compared to the saline

group. Recall from the initial dose-response experiments presented in section 3-3-1 that

treatment with 5mg/kg β-FNA significantly increased stereotypy compared to saline, while

10mg/kg nor BNI by itself significantly reduced the percentage of time the mice spent in stereotypy compared to saline. Thus, while antagonism of the µ and κORs by themselves induced opposite effects on baseline stereotypy levels, antagonism of all three opioid receptor subtypes decrease the magnitude of stereotypy induced by acute AMPH administration. Table 1 summarizes the statistical analyses of the stereotypy data for the antagonist dose-response and pretreatment experiments.

Table 1 Effects of Opioid Receptor Antagonist Pretreatment on AMPH-induced Stereotypy during the period 30 – 60 minutes post drug administration

Antagonist (mg/kg) Saline 12mg/kg AMPH Naloxone 2mg n.s. p < 0.001 2mg + 12mg AMPH n.s. p < 0.001 β- FNA 5mg p < 0.05 n.s. (increased) 10mg + 12mg AMPH n.s. p < 0.001 Naltrindole (NTI) 5mg n.s. n.s. 5mg + 12mg AMPH n.s p < 0.001 nor BNI 10mg p < 0.001 p < 0.001 (decreased) 10mg + 12mg AMPH n.s. p < 0.001

Indicated significance is antagonist treatment or pretreatment compared to vehicle and 12mg

AMPH administration by ANOVA and Dunnett’s multiple comparison post hoc analysis. n.s. = not significantly different compared to saline or 12mg/kg AMPH

92 3.4. Discussion:

There is growing evidence that the endogenous opioid system can modulate the

dopaminergic (DA) system. A common behavioral assay used to measure changes in the DA

system is the increase in locomotor activity seen after administration of low doses of the

psychostimulant amphetamine (AMPH) and increases in stereotypic behaviors following

administration of high doses. A number of studies have been undertaken in rats looking at the

interaction between the opioid system and the DA system and the effects of opioid agonists and antagonists on AMPH-induced behavior. Less thorough are such studies in mice. Thus, we wished to study the interaction between the endogenous opioid system and the DA system in

C57BL/6 mice, a commonly used background strain for the generation of transgenic and knockout mice (Ralph et al, 2001). For this characterization, we pretreated groups of mice with one of a number of opioid antagonists followed by either a low (2mg/kg) or high (12mg/kg) dose of AMPH and then recorded and analyzed both locomotor activity and stereotypic behaviors.

Our results from the locomotion studies indicate that naloxone (non-selective opioid receptor antagonist) and NTI (δOR antagonist) both significantly attenuated the increase in locomotion induced by 2mg/kg AMPH administration, while pretreatment with the κOR antagonist nor-BNI

failed to alter the locomotor activity in any way, and pretreatment with the µOR antagonist β-

FNA induced a slight, though non-significant, augmentation of locomotor activity. The results from the stereotypy data indicate that pretreatment with each of the four opioid antagonists followed by administration of the high 12mg/kg dose of AMPH significantly reduced the levels of stereotypic behaviors exhibited by C57BL/6 mice compared to the high AMPH-alone animals.

This is the first report we are aware of investigating the individual contributions of all the opioid receptor subtypes in modulating AMPH-induced stereotypic behaviors in C57BL/6.

93 We initially performed limited dose-response experiments with the four opioid

antagonists to determine the basal sensitivity of C57BL/6 mice to these compounds in regards to

locomotor and stereotypic behaviors. Figures 3-1 and 3-2 summarize the locomotion results for a selected dose of each antagonist while Figure 3-3 summarizes the stereotypy data for the same

doses of each compound. Looking first at the locomotion data in Figures 3-1 and 3-2, the dose

of 5mg/kg β-FNA did not significantly alter locomotor behavior in C57BL/6 mice compared to

vehicle over the two hour observation period or during the second 30 minute period post

administration. Similar to β-FNA, there was no significant effect on locomotion either over the

entire 2 hour period or the second 30 minute interval by treatment with the lowest dose of the

δOR specific antagonist naltrindole (NTI). Based on this data and the data from the 20mg and

50mg/kg doses (not shown), we chose doses of 10mg/kg β-FNA and 5mg/kg NTI for the

pretreatment experiments. For the κOR receptor-selective antagonist, we tested nor-BNI at four

doses and the locomotion data for the 10mg/kg dose are presented. Again, there was no

significant alteration of locomotor activity induced by this dose of κOR antagonist. The non-

selective antagonist we tested was naloxone and again as presented, the lowest dose of 2mg/kg

did not significantly alter locomotor behavior either over the course of the two hour observation

period or during the interval 30 – 60 minutes post drug administration. The lack of effects on

locomotor behavior by opioid antagonists alone is consistent with reports in the literature (Jones

and Holtzman, 1992; Jones et al, 1993), suggesting that opioid receptors have little, if any, effect

on the basal motor activity of mice. The stereotypy data from the antagonist dose-response

experiments are presented in Figure 3-3. Again, the same individual dose of each compound as

presented for the locomotion data are summarized for the second 30 minute interval post drug

administration. It can be seen that treatment with 2mg/kg naloxone or 5mg/kg NTI by

94 themselves did not significantly alter stereotypy levels compared to saline-treated mice.

However, treatment with 10mg/kg of the κOR antagonist nor-BNI significantly decreased the percentage of time C57 mice spent in stereotypic behaviors compared to saline while treatment with 5mg/kg β-FNA significantly increased stereotypic behaviors compared to saline. There are limited reports of the effects of opioid antagonists on stereotypic behaviors in unstimulated rats.

For instance, NTI injected i.c. was shown to cause a reduction in fine motor movements (a partial measure of stereotypy) compared to saline, while naloxone, nor-BNI and β-FNA injected i.c. had no effects on fine motor movements compared to saline injections (Jones and Holtzman,

1992; Jones et al, 1993).

The antagonist pretreatment experiments lead to a number of interesting findings regarding AMPH-induced locomotion. Consistent with previous reports in rats and mice

(Skorupska and Langwinski, 1989; Chatterjie, et al 1998; Jones and Holtzman, 1992; Hitzman et al, 1982), the non-selective antagonist naloxone attenuated the AMPH-induced increase in locomotor activity in C57 mice. Naloxone pretreatment decreased crossovers by 43% compared to AMPH-alone during interval 2. Investigating the individual contributions of each opioid receptor subtype to the modulation of AMPH-induced locomotion provided three findings. First, pretreatment with the delta receptor antagonist NTI significantly reduced the AMPH-induced increase in locomotor activity. This decrease was most apparent during the second 30 minute interval with NTI causing a 69% reduction in crossovers compared to 2mg/kg AMPH administration. Again, this finding is in line with previous reports of the inhibitory action of delta antagonists on psychostimulant-induced locomotor activity in rats and mice (Jones et al,

1993, Skorupska and Langwinski, 1989; Hitzman et al, 1982). Further, this finding would seem to confirm that in the C57 strain of mouse, the delta opioid receptor plays a similar role in

95 modulating AMPH-induced locomotion as in rats. There are reports in the literature that some

strains of mice have aberrant or dysfunctional endogenous opioid systems and thus do not exhibit the expected responses to opioid agonist or antagonist treatment. For instance, 129 mouse strains are considered to perform poorly on a number of behavioral paradigms used to measure reward to opioids and other drugs of abuse compared to C57 mice (Dockstader and van der Kooy, 2001).

Additionally, there are reports of differences in the expression of the opioid receptor subtypes in the brains of different mouse strains and the analgesic properties of morphine-like opioids and naloxone among different strains as well (Shuster et al, 1975; Reith et al, 1981; Vaccarino et al,

1988; Lariviere et al, 2001). The more interesting finding from our studies involves the slight augmentation of AMPH-induced locomotion by pretreatment with the µOR antagonist β-FNA.

While this increase in locomotor activity did not reach statistical significance via ANOVA

analysis, the trend was clearly present, with the maximum effect of a 28% increase in total

crossovers recorded during the second 30 minute interval. Literature searches have failed to find

a similar report of opioid antagonist-dependent augmentation of AMPH-induced locomotor

activity, and in fact conflicts with the findings of Jones et al who showed that β-FNA had no

effect on AMPH-induced locomotion in the rat (1993). We are currently investigating this

finding further. Lastly, pretreatment with the kappa receptor antagonist nor-BNI failed to

modulate AMPH-induced locomotor activity in C57 mice. Again this finding is consistent with

previous reports in the literature (Jones et al, 1993).

The stereotypy results from the opioid antagonist pretreatment experiments indicate that

all three opioid receptor subtypes can attenuate AMPH-induced stereotypic behaviors in C57

mice. This is seen by the reduction in the mean percentage of time the pretreated mice spent in

stereotypy during the second 30 minute interval compared to the 12mg/kg AMPH group. β-FNA

96 pretreatment reduced AMPH-induced stereotypy by 14%. NTI pretreatment reduced stereotypy by 20% and nor-BNI reduced stereotypy by about 25% of the AMPH-induced levels. There are only limited studies investigating the effects of opioid antagonists on AMPH-induced stereotypic behaviors. Additionally, the data concerning these effects are somewhat contradictory and most of this work has been done in rats using the non-selective antagonist naloxone. A number of studies have reported that naloxone had no effect on AMPH-induced stereotypy in rats (Malick et al, 1977, 1983) while other groups have seen an attenuation of stereotyped behaviors by naloxone (Haber et al, 1978; Hitzman et al, 1982; Skorupska and Langwinski, 1988), and one group demonstrated a potentiation of AMPH-induced stereotypy by naloxone in the rat (Adams et al, 1981). Finally, Jones et al (1993) demonstrated that pretreatment with NTI had no effect on AMPH-induced fine motor movements but did attenuate gross motor movements in rats. One possible explanation for these contradictory results is that rats often display a wide and varied stereotypical response to AMPH administration while mice have been shown to exhibit a much more limited behavioral response (Battisti et al, 1999; Bedingfield et al, 1996). The need to score a wider range of behaviors or to focus on one particular behavioral response to AMPH when using rats could obscure the actual effects of a test compound and thus lead to incorrect or inconsistent conclusions. By using mice and measuring AMPH-induced stereotypy as a stationary animal exhibiting repetitive head and forelimb movements for long stretches of time, we clearly see an attenuation of the magnitude of stereotypic behavior induced by acute administration of AMPH following pretreatment with any one of the opioid antagonists.

From these results, it would seem that the opioid receptor subtypes contribute differentially to regulating AMPH-induced behaviors in C57 mice. While µ and δOR agonists are thought to have similar or overlapping effects, including promoting an increase in DA

97 concentrations within the mesolimbic dopaminergic system, in the context of AMPH-induced locomotion, antagonizing these receptors has opposing effects. Additionally, antagonism of all three receptor subtypes attenuated the AMPH-induced stereotypy exhibited by C57BL/6 mice.

What could be the mechanism(s) for these differing actions of the opioid receptors? It is likely that differences in the localization and activity of the various receptor subtypes within VTA,

NAc, and striatum contribute to the behavioral effects seen following AMPH administration.

µORs are highly expressed within the VTA while δ and κORs are highly expressed in NAc

(Mansour et al, 1987, 1988; Tempel and Zukin, 1987). VTA contains neurons which project to

NAc, providing dopaminergic input; NAc contains GABAergic medium spiny neurons (MSNs) which project to several brain regions, including VTA, providing inhibitory input (Carlezon and

Wise, 1996; Groenewegen et al 1991; Steffensen et al, 1988; Christie et al, 1985, 1987).

Additionally, there are GABAergic interneurons within the VTA providing inhibitory input to the dopaminergic neurons within this structure (Devine et al, 1993b; Spanagel et al, 1992).

Many drugs of abuse, including AMPH and opioids, act to increase DA concentrations within

NAc and can also inhibit GABA- and glutamate-mediated synaptic transmission (DiChiara and

Imperato, 1988; Chieng and Williams, 1998; Jones et al, 1993). It is believed that endogenous or exogenous µ agonists can act upon µORs within the VTA to hyperpolarize both GABAergic interneurons and GABAergic neurons presynaptic to dopaminergic neurons projecting to NAc

(Devine et al, 1993a,b). This hyperpolarization would inhibit these GABAergic neurons, resulting in a decrease of inhibition on the NAc-projecting DA neurons and result in increases in

DA levels within the NAc (Devine et al, 1993b; Spanagel et al, 1992). Antagonism of VTA µ receptors would reduce the inhibition on these GABAergic neurons, resulting in an increase of

DA neuron inhibition and a net decrease in NAc DA levels. In contrast, activation of δORs has

98 been shown to decrease the activity of GABAergic neurons within NAc and striatum, but not in

VTA (Jiang and North, 1992). A decrease in GABA signaling from neurons projecting from

NAc to VTA could result in decreased inhibition on target neurons within VTA and potentially lead to subsequent increases in DA levels within NAc – a kind of positive feed-forward circuit.

Antagonism of the NAc δORs would increase GABA signaling from NAc to VTA, increase inhibition of target DA neurons and result in decreased DA levels within NAc. In this manner,

both µ and δOR agonists can increase extracellular DA concentrations within NAc, consistent

with other rewarding and abused drugs and compounds.

Behaviorally, increases in NAc DA levels are measured as an increase in locomotion, and

decreases in DA levels are measured as a decrease in locomotion. The results from our studies

support the above involvement of δORs in regulating DA levels within NAc and AMPH-induced

locomotion. Pretreatment with NTI significantly attenuated the AMPH-induced

hyperlocomotion, which would be consistent with a reduction in DA levels within the NAc,

possibly mediated via an increase in GABA signaling from NAc to VTA. On the other hand, we

failed to see a significant decrease in AMPH-induced locomotion following β-FNA pretreatment,

and in fact we saw a potential augmentation of locomotor activity during the peak effect time

following AMPH administration. Interestingly, Devine and coworkers, using microdialysis

studies, found that VTA injections of the µOR antagonists CTOP or β-FNA actually elevated

DA levels in the VTA (1993b). Increases in DA within VTA could further inhibit GABA

interneurons within the VTA that synapse onto the NAc-projecting DA neurons, resulting in

greater dopaminergic input to NAc. This increased DA signaling to NAc, coupled with the

AMPH-dependent increase in DA levels could combine to increase locomotor behavior, as we

saw with a slight increase in total crossovers in the β-FNA pretreated mice compared to the

99 AMPH-alone treated mice. This effect within VTA might also help explain the slight increase in locomotor activity we observed in the β-FNA treated group compared to saline in the antagonist dose-response experiments. The failure of κOR antagonism to significantly alter AMPH- induced locomotion is consistent with previous reports (Jones and Holtzman, 1992; Jones et al,

1993), even though nor-BNI has been shown to increase DA levels following injection into NAc

(Spanagel et al, 1992). Despite the ability of κ antagonists to increase DA levels within NAc, the apparent lack of effect on AMPH-induced locomotion by systemic nor-BNI has been postulated to indicate that κORs are not involved with tonic regulation of DA function within brain regions

(or neurons?) that control motor activity (Manzanares et al, 1991; Jones and Holtzman, 1992).

Spanagel et al (1992) postulated that tonic activation of µ and κORs are required for the maintenance of basal DA levels within NAc, but our results and the results of others seem to indicate that this κOR regulation of DA levels is not involved with regulating the AMPH- induced DA increases within NAc and/or AMPH-induced hyperlocomotion.

A possible explanation for the observed effects of opioid antagonist pretreatment on

AMPH-induced stereotypy likely involves alterations of DA levels within the striatum.

Increases in DA levels within the striatum have been identified as a primary mechanism underlying AMPH-induced stereotypic behaviors (Creese and Iversen, 1974; Costall and Naylor,

1974). δOR activation has been shown to decrease the activity of GABAergic neurons in the striatum, similar to their effects within NAc (Jiang and North, 1992). Additionally, δ receptors on DA neurons within the striatum have been shown to mediate a receptor-induced release of newly synthesized DA, and it is this same pool of DA that is preferentially released by AMPH

(Kuczenski and Segal, 1989; Dourmap et al, 1992; Trovero et al, 1990). It has been postulated that AMPH may activate endogenous opioids within the DA system, resulting in a reduction of

100 GABA-mediated inhibition of DA neurons. Decreases in GABAergic neuronal activity within

the striatum induced by δ agonists would result in increases in both DA levels and dopaminergic neuronal activity (Jones et al, 1993). Blockade of δORs (and other opioid receptor subtypes?) would be expected to prevent this loss of GABA-mediated inhibition, thereby decreasing the amount of DA released by AMPH administration. Systemic naloxone administration has been shown to do just this, with an attenuation of the AMPH-induced increase in extracellular DA levels. Naloxone presumably antagonizes striatal δORs and disinhibits GABAergic neurons, resulting in an increase in inhibitory signals to target DA neurons and a decrease in DA levels within the striatum. Thus, in the context of AMPH administration, naloxone (and δ antagonist) pretreatment would lead to a net reduction in DA levels within the striatum and a subsequent decrease in stereotypic behaviors. This is exactly what we observed, not only by pretreatment with naloxone, but also by pretreatment with all three opioid receptor-specific antagonists. It is therefore possible that µ and κ opioid receptors are functioning within the striatum in a similar manner as δORs to modulate GABAergic neuronal activity and thus regulate DA levels in response to AMPH administration.

A potential issue to consider whenever employing the use of antagonists in experiments is the specificity and affinity of the compounds used. β-FNA is one of the most thoroughly characterized irreversible opioid antagonists studied and achieves its antagonistic effects almost entirely via the µOR (Broadbear et al, 2000; Takemori et al, 1981). A potential confounding effect of this antagonist is a reversible κOR-agonistic activity that initially coincides with its µ- selective antagonist effects (Takemori et al, 1981). However, as demonstrated in rats, κ agonists have been shown to decrease both DA release in the NA and spontaneous activity (DiChiara and

Imperato, 1988; Spanagel et al, 1992). Thus, it would seem plausible that the κOR agonistic

101 activity of β-FNA would either have no effect or a potential inhibitory effect on AMPH-induced

locomotor activity. Our finding that the κOR antagonist nor-BNI failed to modulate AMPH-

induced hyperlocomotion in C57 mice points to a lack of involvement of kappa receptors in our

behavioral assay, and would support the view that β-FNA is exerting its effect predominantly via

the µOR. A method for circumventing any possible κ agonistic properties of β-FNA on AMPH-

induced locomotion would be to increase the pretreatment period from 30 minutes to 24 hours.

This pretreatment paradigm has been shown to sufficiently isolate the µOR antagonistic property

of β-FNA (Broadbear et al, 2000). Alternatively, the mice could be pretreated with a κ

antagonist (nor-BNI?) followed by β-FNA treatment before AMPH administration. A final point

to consider is that β-FNA acts to antagonize the µOR by alkylation of a subset of µ receptors, up

to 50% of total receptors, and thus its effects may be due to modification of only a particular

subtype or population of µORs present (Broadbear et al, 2000). This selective modification

could somehow target those µORs that are in a position to decrease DA levels and lead to

alterations in AMPH-induced behaviors. Finally, NTI is the prototypic non-peptide δOR antagonist (Portoghese et al, 1988) and is thought to be highly selective for the delta receptor.

NTI at doses of 0.5-1mg/kg has been shown to selectively antagonize δOR-mediated behaviors in the rat (Heidbreder et al, 1996). However, a number of reports suggest that at higher doses

(2mg/kg and higher), NTI is able to block the µOR as well. For example, NTI at 2.0mg/kg was able to antagonize the release of corticosterone induced by the µOR selective agonist fentanyl

(Kitchen and Kennedy, 1990). Thus there exists the potential that the dose of NTI used in our studies (5mg/kg) could have antagonized µORs as well as δORs. However, the significant attenuation of locomotor activity by NTI pretreatment and the slight increase in hyperlocomotion

102 by β-FNA following AMPH administration would seem to indicate that NTI is functioning

predominately through δORs.

In summary, the findings from our studies indicate that of the three opioid receptor

subtypes, Antagonism of δORs is clearly involved with the attenuation of locomotion following acute administration of 2mg/kg amphetamine in C57BL/6 mice. κORs do not appear to be involved in modulating AMPH-induced locomotion, while antagonism of µORs might potentially augment the locomotor-stimulating effects of AMPH. In contrast to the differing effects of opioid receptors on AMPH-induced locomotion, antagonism of all three receptor subtypes can attenuate the stereotypic response induced by 12mg/kg AMPH in these mice. It is assumed that these receptor-dependent effects are mediated through differential regulation of neurons, presumably GABAergic and dopaminergic, within the VTA, NAc, and striatum of the mesolimbic dopaminergic system.

103

Chapter Four

Discussion

104 4.1. Discussion:

There were a number of aims for these studies, all related to investigating and expanding the current literature on interactions between the endogenous opioid system and AMPH-induced behaviors. The reinforcing, and thus addictive, effects of both opioids like morphine and heroin and psychostimulants like amphetamine and cocaine are thought to be due to actions on the brain reward system (Kelley and Berridge, 2002). Studies have well established that acute administration of these drugs lead to increases in extracellular dopamine (DA) levels in a number of brain regions comprising the mesolimbic DA system, including the nucleus accumbens (NAc), the ventral tegmental area (VTA), the striatum, and the dorsal caudate (DC); and it is these same regions that in part comprise the brain reward system (Heidbreder et al,1996; Wise, 1981). The mechanisms for this increase in DA concentrations include an inhibition of DA reuptake by the dopamine transporter, promotion of reverse transport of DA, and increased release of DA (Koob,

1992). It has been theorized that this effect on DA levels within the mesolimbic DA system significantly contributes to both the rewarding properties of these drugs and various behavioral aspects of their use, including modulation of locomotor activity and the development of drug- induced stereotypies.

However, while the involvement of the NAc in the rewarding properties of drugs of abuse (DOA) has been well established, there is still considerable debate over the specific mechanisms involved (Hoffman and Lupica, 2001). In a simplified model (see Figure 1-1), the critical circuitry of the brain reward system is thought to be between the NAc and the VTA, as these regions are intimately involved with the rewarding properties of drugs of abuse (Wise,

1982; Koob and Bloom, 1988). VTA contains the cell bodies of the mesolimbic DA system

105 which project to NAc and frontal cortex, providing dopaminergic input to these structures

(Carlezon and Wise, 1996). The majority of neurons within the NAc are GABAergic medium

spiny neurons (MSNs) which receive this DA input and also receive glutamatergic input from the

hippocampus, amygdala, and prefrontal cortex and which in turn send their inhibitory output

projections to several brain structures, including the VTA (Groenewegen et al, 1991; Steffensen

et al, 1998; Christie et al, 1985, 1987). Glutamate and dopamine are thought to have opposite

actions on their target neurons in the NAc – glutamate is believed to excite MSNs and DA

inhibits them (Carlezon and Wise, 1996). Many DOA, including AMPH and opioids, have been

shown to increase DA concentrations within the NAc and also inhibit amino-acid mediated

synaptic transmission in this structure (DiChiara and Imperato, 1988; Chieng and Williams,

1998). It is through these effects of DA levels and NAc neuronal activity that the rewarding

properties of DOA may be manifested (Hoffman and Lupica, 2001).

As our understanding of the individual components involved with opioid and psychostimulant effects at the cellular, molecular, and behavioral levels has grown, the study of interactions between these different classes of drugs and their effects on the brain reward system has likewise grown. Not only has it been shown that the endogenous opioid system can regulate and modulate DA levels within the mesolimbic system, but opioids are also known to modulate the effects of AMPH-induced changes in DA levels and behavior (Heidbreder et al 1996; Wise,

1981; Jones et al, 1993; DiChiara and Imperato, 1988). However, the mechanisms for the modulation of the dopaminergic system by opioids and their effects on AMPH-induced behaviors are still not completely clear. In particular, the contributions of the three types of opioid receptors (µ, κ, and δ) to both the brain reward system and to AMPH-induced behaviors are only now being carefully investigated. Therefore, the purpose of this work was to establish

106 an animal model system of the two main behavioral effects of acute AMPH administration -

hyperlocomotion and stereotypy, and to study what effects manipulation of the individual opioid receptors has on these behaviors.

4.2. Development of Animal Model:

The first step for this investigation was to establish an animal model of the AMPH-

induced behaviors. Acute administration of AMPH to rodents, such as rats and mice, generally

produces two qualitatively different behaviors. When administered at low doses, AMPH

produces a primary response of hyperlocomotion, while higher doses produce repetitive,

stereotyped behaviors such as intense grooming, that disrupts or interferes with locomotion

(Battisti et al, 1999). After our initial pilot experiments measuring the hyperlocomotion induced

by acute AMPH administration in three strains of mice, the C57BL/6 strain of mouse was chosen

for further analysis. The next step was to undertake a careful characterization of the behavioral

sensitivity of this strain of mouse to acute amphetamine administration (Chapter 2). For this,

dose-response experiments were carried out with 4 doses of AMPH (2, 6, 12, and 20mg/kg) and

locomotor and stereotypy data were recorded for 2 hours using the RACs and subsequently

analyzed. This type of detailed analysis would serve a number of important functions. First, it

would provide a reference for us and other investigators who are interested in using an animal

model to study a range of issues relating to drugs of abuse, strain differences, and drug-induced

behaviors. Having a good understanding of the sensitivity of a particular strain of mouse to a

specific drug of abuse provides valuable information when choosing a background strain for

transgenic or knockout work. Without a careful characterization of the background strain, it is

107 difficult to conclude that observed phenotypes in a subsequent knockout or mutant mouse are due to a specific mutation and not to a particular genetic contribution from one of the parental

strains (Ralph et al, 2001) A second goal for this characterization was the identification of appropriate doses of AMPH to use to induce a specific behavior, either hyperlocomotion or stereotypy, allowing further investigations into the mechanisms contributing to the behavior and/or into compounds or systems which may modulate these behaviors. A third goal of establishing an animal model for AMPH-induced behaviors is that the study of behaviors induced by acute or chronic administration of drugs of abuse can shed light on complex mental conditions or diseases seen in humans. Acute high doses or chronic use of drugs of abuse, in both rodents and humans, can produce behaviors ranging from sedation, mental disturbances, euphoria, narcolepsy-catalepsy, hallucinations, and stereotypies (Segal et al, 1979; Griffith et al

1968; Angrist and Gershon, 1970; Skorupska and Langwinski, 1989). In particular, chronic administration of AMPH (in both rodent models and in human drug addicts) can result in a psychotic state that in many aspects resembles schizophrenia (Goldstein and Deutch, 1992;

Lieberman et al, 1997). Although a precise etiology and pathophysiology of this heterogeneous disease have thus far eluded researchers, one favored potential mechanism - “the dopamine hypothesis” - has gained substantial support. This hypothesis posits that there exist functional alterations in central dopaminergic systems in the brain of schizophrenics that can result in a pathologic condition of neurochemical sensitization similar to a drug-induced behavioral sensitization (Goldstein and Deutch, 1992; Lieberman et al, 1997). Thus, developing an animal model for studying AMPH-induced behaviors is critical for furthering our ability to study both drug addiction and other mental diseases.

108

4.3. Analysis of Amphetamine Response in C57 Mice:

Toward this end, we established that the C57BL/6 strain exhibits the widely observed

bimodal response to acute AMPH administration. At lower doses, the primary response to

AMPH was an induction of hyperlocomotion, while at higher doses, the hyperlocomotor

response was replaced with a significant increase in stereotypic behaviors. We found that a dose

of 2mg/kg AMPH is sufficient to induce a pattern of sustained hyperlocomotive behavior in

C57BL/6 mice. This dose is consistent with reports from the literature that indicate C57BL/6 mice are quite sensitive to the locomotor-inducing properties of AMPH. Doses between 1 and

5mg/kg AMPH have been shown to induce significant increases in locomotion in C57BL/6 mice under a number of different testing and recording conditions (Davis et al, 1974; Kitahama and

Valatx, 1979; Ralph et al, 2001). Likewise, we found that a dose of 12mg/kg AMPH is sufficient to induce a predominately stereotypic behavioral response while not incapacitating the animal to the point of inhibiting all movements. This is also consistent with reports from the literature for the induction of stereotypy in mice. 10mg/kg AMPH has been shown to induce short periods of focal stereotypies in C57BL/6 mice and an acute dose of 12mg/kg induced stereotypy in about 90% of all CF-1 mice treated (Bedingfield et al, 1997; Ralph et al, 2001).

The inverse relationship between locomotion and stereotypy induced by AMPH administration in C57BL/6 mice is clearly demonstrated in the dose response experiments in

Chapter 2, Figures 2-3A - C. The lowest dose (2mg) induced a steady increase in hyperlocomotion that was maintained for a significant period of time and then slowly decreased toward control levels. As the 2mg AMPH-induced hyperlocomotion began to decline during the

109 final hour of the observation period, the extent of stereotypy began to increase. The higher dose

of 12mg AMPH induced an initial brief period of locomotor activity which then gave way to an

extended period of hypolocomotion, with a concurrent induction of an extended period of

stereotypy. The middle dose (6mg) produced a complex mix of hyper- and hypolocomotion,

with two periods of hyperlocomotion separated by a single peak of robust stereotypy. We have

concluded that the 6mg dose is a ‘transitional’ dose which is at or near the threshold for inducing

robust stereotypic behaviors within C57 mice. This complex pattern of locomotion and

stereotypy at a middle dose of AMPH is consistent with what has been reported for CF-1 mice, where doses between 6-10mg/kg induce a mixture of the two behaviors while higher doses induce prolonged periods of stereotypy (Bedingfield et al, 1996; Battisti et al, 1999).

Additionally, like CF-1 mice, the stereotypic behaviors induced by AMPH administration in

C57BL/6 mice are manifested mainly as a stationary animal involved in intense grooming behaviors with repetitive head and paw movements, licking, and gnawing. Finally, the stereotypy data demonstrate that the onset and persistence of stereotypic behaviors are dose- dependent. The higher the dose of AMPH, the earlier the onset of stereotypic behavior and the longer this behavior lasts. Again, the middle dose of 6mg induced a pattern of behavior that was in between the low and high dose groups. While the stereotypy studies we found in the literature indicated the doses of AMPH used and the time periods studied for behavioral analysis, few have reported the actual time course of the behavior over an extended observation period (2 hours) nor did they address the kinetics of the response post drug injection. For these reasons, we feel our extended analysis of AMPH-induced stereotypy and locomotion in characterizing our animal model is of note as reference for future work.

110 4.4. Amphetamine-induced Locomotion and Stereotypy:

The mechanisms underlying the transition from a predominately hyperlocomotive response to a more stereotypic response, while containing a clear AMPH dose-dependent component, are not fully known. Most likely it involves differences in the absolute DA concentrations in specific regions of the mesolimbic system induced by AMPH administration.

One mechanism potentially involved with this transition is the phenomenon of sensitization.

Sensitization is the enhancement of a drugs effects following repeated and intermittent

administration of the compound (Heidbreder et al, 1996; Kalivas and Barnes, 1993). One well-

studied form of sensitization thought to have relevance to psychiatric disorders such as

schizophrenia, bipolar disorder, and drug addiction is behavioral sensitization (Richtand et al,

2000; Goldstein and Deutch, 1992; Jimerson, 1987). Behavioral sensitization is the progressive

and long-lasting increase in specific behaviors following the repeated administration of various

drugs of abuse, commonly psychostimulants such as amphetamine or cocaine (Richtand et al,

2000). Behavioral sensitization is associated with increased basal DA release in the NAc and

striatum, enhanced DA overflow in response to subsequent drug challenge, and an increase in

DA receptor sensitivity (Weiss et al, 1992; Heidbreder and Shippenberg, 1994; Kalivas and

Duffy, 1990, 1993; Hummel and Unterwald, 2002). AMPH-induced increases in locomotion are

thought to be due to increased DA in the NAc, while the AMPH-induced increases in stereotypic

behaviors are due to DA increases in the striatum (Creese and Iversen, 1974, Kelly et al, 1975).

Additionally, it has been shown that, at least in mice, three neurotransmitter systems, dopamine,

glutamate, and GABA, participate in the acute stereotypy response to AMPH and cocaine

(Karler et al, 1994, 1995). It is believed that one effect of AMPH administration is to induce the

111 release of newly synthesized DA within the striatum (Kuczenski and Segal, 1989; Dourmap et al,

1992; Trovero et al, 1990). With this mechanism, AMPH may then function to regulate

endogenous signaling systems, particularly the GABA system, resulting in a reduction of

GABA-mediated inhibition of DA neurons. Decreases in GABAergic neuronal activity within

the striatum would result in further increases in DA levels and additional dopaminergic neuronal

activity (Jones et al, 1993). As our experimental paradigm used acute administration of AMPH

and not a multiple or chronic dosing scheme, it appears that there exists a threshold dose of the

psychostimulant which induces a behavioral switch from hyperlocomotion to stereotypy. And

this behavioral switch may reflect changes in DA concentrations within the NAc compared to the

striatum and potentially other brain regions. However, the exact mechanism that acts as the

trigger for this switch remains to be investigated.

4.5. Identification of Time Interval for Analysis:

It is interesting to note that all doses of AMPH tested initially induced a period of

hyperlocomotion. This is seen with the elevated numbers of crossovers, compared to saline,

induced by AMPH treatment at the first 10 minute time point graphed. Additionally, it is evident

that the first 30 minutes post drug injection is the most dynamic period of change for both

locomotion, and to a lesser extent, stereotypy. Looking at locomotion during this period, the

crossovers per interval for the 2mg AMPH group steadily increased to about its maximum value by the end of the first 30 minutes. The 6mg AMPH group exhibited its initial period of

hyperlocomotion during the first 30 minutes, with a peak at 20 minutes, and then a steep decline

to sub-2mg levels by 40 minutes. Finally, the 12 and 20mg AMPH groups experienced their

112 maximum levels of locomotion at 10 minutes post injection and then rapidly dropped to near

control levels by 20 minutes post-AMPH administration. The stereotypy data also indicate the

first 30 minute interval is a period of ongoing change in behavior levels, with the exception of

the 2mg AMPH group, which maintained a fairly low level of stereotypy of about 20%. The

6mg AMPH group had steadily increasing stereotypy levels during this initial 30 minute period,

which continued to increase to a peak of ~70% by 50 minutes post injection, followed by a slow

decline in magnitude over the remainder of the observation period. The 12 and 20mg AMPH

groups had steeply increasing levels of stereotypy during the first 30 minutes post injection, with

peaks at 20 minutes, to levels which were then maintained over the next 40-50 minutes. Over

the last hour, the 20mg AMPH group actually exhibited an additional increase to its highest level

of stereotypy - 85.8% at 70 and 80 minutes post injection. Thus, there is a time component

involved in the change from a hyperlocomotive state to a stereotypic state in response to AMPH

administration. Part of this is likely the pharmacokinetics involved with AMPH administration

and the time needed to enter the mouse’s central nervous system, alter DA levels once there, and thus induce a behavioral state of either hyperlocomotion or stereotypy.

The analysis of stereotypy data from a long time course experiment (such as two hours in our studies) is very time and labor intensive, so we therefore wished to determine if a particular interval of the two hour period would provide a suitable window for evaluating both locomotion and stereotypy data and serve as a representative of the entire observation period. Identification of such an interval would lead to an expedited analysis of future behavioral experiments. As mentioned above, while the first 30 minute interval post-AMPH administration was a period of

ongoing changes in behavior levels and intensities, the second 30 minute interval (30-60 minutes

post injection) seemed to be a good candidate period for analysis. Looking at total crossovers

113 during the second 30 minute interval as a measure of locomotor activity, there is a clear inverse

dose-response relationship seen. The lowest dose of 2mg AMPH lead to the greatest number of

crossovers (681); the highest dose of 20mg AMPH caused the least amount of crossovers (165)

during this period. The stereotypy data for the second 30 minute interval also supports this

period as a good candidate period for analysis. The three highest doses of AMPH all had similar

levels of stereotypy (55 – 70% of time spent in stereotypy) during the observed time intervals

scored for this 30 minute period, while the 2mg group displayed its lowest level of stereotypy

(20% of the time spent in stereotypy). Thus, we feel that the 30 – 60 minute post AMPH

administration time period is a good representative interval for analyzing both locomotion and

stereotypy data for subsequent experiments. This time period is consistent with reports from the

literature. Karler et al (1998) scored stereotypy during a 5 minute interval 30 minutes after

AMPH administration, which they identified as the approximate peak-effect time for stimulant

action. Similarly, Battisti et al (1999) scored stereotypy over 1 minute intervals every 10

minutes at 30, 40, 50, and 60 minutes post drug administration and used the highest percentage

of animals exhibiting stereotyped behavior at one of these time points as the maximum response

to AMPH.

As stated above, one goal of the present study was to identify doses of AMPH that

consistently induce a pattern of either increased locomotor activity or increased stereotypic behavior in C57BL/6 mice. The reason for this was to provide us and other investigators with appropriate doses to use for further study of the AMPH-induced behaviors of hyperlocomotion and stereotypy. By identifying doses which induce a single predominate behavior, while

minimizing drug-induced behavioral ‘side effects’, it is hoped that this will lead to an increased

ability to design and study modulatory and modifying effects on these behaviors. By using

114 suitable amounts of a drug to induce the desired behavior, the sensitivity of pretreatment or co- treatment paradigms with compounds of interest will be maximized and hopefully increase the ability to detect alterations in the behavior being studied. Conversely, the 6mg/kg dose seemed to induce a transitional state of behaviors where both hyperlocomotion and stereotypy were exhibited to significant levels during the observation period. While we feel this dose is not suitable for studying either one of the AMPH-induced behaviors individually, the possibility remains for the investigation of compounds and treatments along with this dose of AMPH to determine if co-treatment pushes the predominate behavior toward locomotion or stereotypy. In this manner, administration of specific compounds that modulate the dopaminergic, glutamatergic, GABAergic, or other neurotransmitter systems, can be investigated and their involvement in AMPH-induced behaviors studied further.

4.6. Effects of Opioid Antagonists on Basal Activity in C57 Mice:

The second major aim of this study was to investigate the effects of manipulation of the endogenous opioid system on AMPH-induced behaviors. Once we had established the mice model for AMPH-induced behaviors, including the optimum doses for inducing hyperlocomotion vs. stereotypy, we were ready to proceed to the experiments looking at the contributions the individual opioid receptors have on these behaviors. As presented in the

Introduction, and elsewhere, there is growing evidence that the endogenous opioid system can modulate the dopaminergic (DA) system. To further explore these interactions, we pretreated groups of mice with one of a number of opioid antagonists followed by either a low (2mg/kg) or high (12mg/kg) dose of AMPH and then recorded and analyzed both locomotor activity and

115 stereotypy behavior for two hours post AMPH administration. We initially performed limited

dose-response experiments with the four opioid antagonists to determine the basal sensitivity of

C57BL/6 mice to these compounds in regards to locomotor and stereotypic behaviors. Figures

3-1 and 3-2 in Chapter 3 summarize the results from these experiments. For the µOR-selective antagonist β-FNA, we tested three doses - 5mg/kg, 20mg/kg, and 50mg/kg. Figures 3-1 and 3-2 indicate that the 5mg/kg dose of β-FNA did not significantly alter locomotor behavior in

C57BL/6 mice compared to vehicle over the two hour observation period or during the second

30 minute period post administration, respectively. For the δOR-selective antagonist naltrindole

(NTI), we again tested three doses - 5mg/kg, 20mg/kg, and 50mg/kg and the locomotor data for the lowest dose of 5mg/kg are displayed in Figures 3-1 and 3-2. Similar to β-FNA, there was no significant effect on locomotion observed. For the third receptor-specific antagonist, we tested the κ-selective antagonist nor-BNI at the doses of 5mg, 10mg, 20mg, and 50mg/kg. The

locomotion data for the 10mg/kg dose of nor-BNI are shown in Figures 3-1 and 3-2. Again, this

dose did not seem to significantly alter locomotor activity. Interestingly, treatment with the various opioid receptor antagonists, by themselves, did have some significant effects on basal

stereotypy behaviors displayed by C57BL/6 mice. From Figure 3-3 in Chapter 3, it can be seen

that treatment with 2mg/kg naloxone or 5mg/kg NTI by themselves did not significantly alter

stereotypy levels compared to saline-treated mice. However, treatment with 10mg/kg nor-BNI

significantly decreased the percentage of time C57 mice spent in stereotypic behaviors compared

to saline, while treatment with 5mg/kg β-FNA significantly increased stereotypic behaviors compared to saline. There are limited reports of the effects of opioid antagonists on stereotypic behaviors in unstimulated rats. For instance, NTI injected i.c.v. was shown to cause a reduction in fine motor movements (a measure of stereotypy) compared to saline, while naloxone, nor-BNI

116 and β-FNA injected i.c.v. had no effects on fine motor movements compared to saline injections

(Jones and Holtzman, 1992).

There are a number of possible explanations for the conflicting results of our dose- response studies to the limited reports in the literature. One likely possibility is that the work done by Jones and Holtzman (1992) was performed in rats while our studies were conducted in mice. As mentioned, stereotypy studies in rats are complicated by the divergent range of behaviors these animals display in response to AMPH administration compared to mice (Battisti et al, 1999; Bedingfield et al, 1996). The scoring of a wider range of behaviors or the need to focus on one particular behavioral response to AMPH in rats could obscure the actual effects of a test compound and potentially lead to erroneous conclusions. A second explanation for our findings could be the method of delivery of the opioid antagonists. We utilized a systemic delivery via subcutaneous injections of all compounds, while Jones and Holtzman used i.c.v. injections directly into the CNS. While the mechanism for the alterations in basal stereotypy induced by opioid antagonist administration is not fully known, one possibility comes from limited clinical results testing the effects of opioid antagonist administration on mentally normal human subjects. It has been found that naloxone treatment in such subjects can produce irritation, anger, hostility, anxiety, fear, and in general, effect overall mood (Jones and Herning,

1979; Pickar et al, 1982). It is possible that these mental effects may be manifested in mice as alterations in stereotypic behaviors as intense grooming, gnawing, and head and forelimb movements. It is interesting to note that at the doses analyzed, administration of opioid antagonists did not result in a similar biphasic change in behavior seen following AMPH administration. That is, β-FNA treatment induced increases, to varying degrees, in both basal

117 stereotypy levels and locomotion, while nor-BNI treatment induced a significant decrease in basal stereotypy levels, but failed to induce a corresponding increase in locomotion.

4.7. Opioid Antagonists and Amphetamine-induced Behaviors:

The antagonist pretreatment experiments lead to a number of interesting findings.

Looking first at the locomotion studies, consistent with previous reports in rats and mice

(Skorupska and Langwinski, 1989; Chatterjie et al, 1998; Jones and Holtzman, 1992; Hitzman et al, 1982), the non-selective antagonist naloxone attenuated the AMPH-induced increase in locomotor activity in C57 mice. Naloxone pretreatment decreased crossovers by 43% vs.

AMPH-alone during the second 30 minute interval post AMPH administration. Investigating the individual contributions of each opioid receptor subtype to the modulation of AMPH-induced locomotion provided three important findings. First, pretreatment with the δOR antagonist NTI significantly reduced the AMPH-induced increase in locomotor activity. This decrease was most apparent during the second 30 minute interval with NTI causing a 69% reduction in crossovers vs. AMPH. Again, this finding is in line with previous reports of the inhibitory action of δ antagonists on psychostimulant-induced activity in rat (Jones et al, 1993). Further, this finding would seem to confirm that in the C57 strain of mouse, the delta opioid receptor plays a similar role in modulating AMPH-induced locomotion as in rats. There are reports in the literature of strain differences among mice in regards to their endogenous opioid systems and their responses to opioid agonist or antagonist treatment. For instance, 129 mouse strains are considered to perform poorly on a number of behavioral paradigms used to measure reward to opioids and other drugs of abuse compared to C57 mice (Dockstader and van der Kooy, 2001). Additionally,

118 there are reports of differences in the expression of the opioid receptor subtypes in the brains of

different mouse strains and the analgesic properties of morphine-like opioids and naloxone

among different strains as well (Shuster et al, 1975; Reith et al, 1981; Vaccarino et al, 1988;

Lariviere et al, 2001). The more interesting finding from our locomotion studies involves the augmentation of AMPH-induced locomotion by pretreatment with the µOR antagonist β-FNA.

While this increase in locomotor activity did not reach statistical significance via ANOVA

analysis, the trend was clearly present, with the maximum effect of a 28% increase in total

crossovers compared to the AMPH group observed during the period 30 – 60 minutes post

AMPH administration. Lastly, pretreatment with the κOR antagonist nor-BNI failed to modulate

AMPH-induced locomotor activity in C57 mice. Again this finding is consistent with previous

reports in the literature (Jones et al, 1993).

The stereotypy results from the opioid antagonist pretreatment experiments indicate that

all three opioid receptor subtypes can attenuate AMPH-induced stereotypic behaviors in C57

mice. This is seen by the reduction in the mean percentage of time the pretreated mice spent in

stereotypy during the second 30 minute interval compared to the 12mg/kg AMPH group. β-FNA

pretreatment reduced AMPH-induced stereotypy by 14%. NTI pretreatment reduced stereotypy

by 20% and nor-BNI reduced stereotypy by about 25% of the AMPH-induced levels. There are

only limited studies investigating the effects of opioid antagonists on AMPH-induced stereotypic

behaviors in animal models. Additionally, the data concerning these effects are somewhat

contradictory and most of this work has been done in rats using the non-selective antagonist

naloxone. A number of groups have reported that naloxone had no effect on AMPH-induced

stereotypy in rats (Malick et al, 1977, 1983) while other groups have seen an attenuation of

stereotyped behaviors by naloxone (Haber et al, 1978; Hitzman et al, 1982; Skorupska and

119 Langwinski, 1988) and one group demonstrated a potentiation of AMPH-induced stereotypy by

naloxone in the rat (Adams et al, 1981). Finally, Jones and Holtzman (1992) demonstrated that

pretreatment with NTI had no effect on AMPH-induced fine motor movements but did attenuate

gross motor movements in rats. Again, as mentioned above, a possible explanation for these

contradictory results could be that rats often display a wider range of stereotypical responses to

AMPH administration compared to mice, and thus changes in the magnitude of stereotypy by

pretreatment paradigms could be obscured. By using mice and considering AMPH-induced

stereotypy as a stationary animal exhibiting repetitive head and forelimb movements for long

stretches of time, we clearly see an attenuation of the magnitude of stereotypic behavior induced

by acute administration of AMPH following pretreatment with any one of the opioid antagonists.

4.8. Mechanisms for Opioid Receptor Regulation of Amphetamine-induced Behaviors:

From these results, it would seem that the opioid receptor subtypes contribute

differentially to regulating AMPH-induced behaviors in C57 mice. While µ and δOR agonists are thought to have similar or overlapping effects, including promoting an increase in DA concentrations within the mesolimbic dopaminergic system, in the context of AMPH-induced locomotion, we found that antagonizing these receptors has opposing effects. Additionally, antagonism of all three receptor subtypes attenuated the AMPH-induced stereotypy exhibited by

C57BL/6 mice. What could be the mechanisms and physiological significance for these differing actions of the opioid receptors? Mechanistically, it is likely that differences in the localization and activity of the various receptor subtypes within the VTA, NAc, and striatum contribute to the behavioral effects seen following AMPH administration. µORs are highly

120 expressed within the VTA while δ and κORs are highly expressed in the NAc (Mansour et al,

1987, 1988; Tempel and Zukin, 1987). The VTA contains neurons which project to the NAc,

providing dopaminergic input and the NAc contains GABAergic medium spiny neurons (MSNs)

which project to several brain regions, including the VTA, providing inhibitory input (Carlezon

and Wise, 1996; Groenewegen et al 1991; Steffensen et al, 1988; Christie et al, 1985, 1987).

Additionally, there are GABAergic interneurons within the VTA providing inhibitory input to the dopaminergic neurons within this structure (Devine et al, 1993b; Spanagel et al, 1992).

Many drugs of abuse, including AMPH and opioids, act to increase DA concentrations within the

NAc and can also inhibit GABA- and glutamate-mediated synaptic transmission (DiChiara and

Imperato, 1988; Chieng and Williams, 1998; Jones et al, 1993). It is believed that endogenous or exogenous µ agonists can act upon µORs within VTA to hyperpolarize both GABAergic interneurons and GABAergic neurons presynaptic to dopaminergic neurons projecting to NAc

(Devine et al, 1993a, b). This hyperpolarization would inhibit these GABAergic neurons, resulting in a decrease of inhibition on NAc-projecting DA neurons and result in increases in DA levels within the NAc (Devine et al, 1993b; Spanagel et al, 1992). Antagonism of VTA µ receptors would reduce the inhibition on these GABAergic neurons, resulting in an increase of

DA neuron inhibition and a net decrease in NAc DA levels. In contrast, activation of δORs has been shown to decrease the activity of GABAergic neurons within NAc and striatum, but not in

VTA (Jiang and North, 1992). A decrease in GABA signaling from neurons projecting from

NAc to VTA could result in decreased inhibition on target neurons within the VTA and potentially lead to subsequent increases in DA levels within the NAc – a kind of positive feed- forward circuit. Antagonism of NAc δORs would increase GABA signaling from NAc to VTA, increase inhibition of target DA neurons and result in decreased DA levels within the NAc. In

121 this manner, both µ and δOR agonists can increase extracellular DA concentrations within the

NAc, consistent with other rewarding and abused drugs and compounds.

Behaviorally, increases in NAc DA levels are measured as an increase in locomotion, and decreases in DA levels are measured as a decrease in locomotion. The results from our studies support the above involvement of δORs in regulating both DA levels within the NAc and

AMPH-induced locomotion. Pretreatment with NTI significantly attenuated the AMPH-induced hyperlocomotion, which would be consistent with a reduction in DA levels within the NAc, possibly mediated via an increase in GABA signaling from NAc to VTA. On the other hand, we failed to see a significant decrease in AMPH-induced locomotion following β-FNA pretreatment, and in fact we saw a potential augmentation of locomotor activity during the peak effect time following AMPH administration. Interestingly, Devine and coworkers, using microdialysis studies, found that VTA injections of the µOR antagonists CTOP or β-FNA actually elevated

DA levels in the VTA (1993b). Increases in DA within VTA could further inhibit GABA interneurons within the VTA that synapse onto NAc-projecting DA neurons, resulting in greater dopaminergic input to the NAc. This increased DA signaling to the NAc, coupled with the

AMPH-dependent increase in DA levels could combine to increase locomotor behavior, as we saw with a slight increase in total crossovers in the β-FNA pretreated mice compared to the

AMPH-alone treated mice. This effect within the VTA might also help explain the slight increase in locomotor activity we observed in the β-FNA treated group compared to saline in the antagonist dose-response experiments. The failure of κOR antagonism to significantly alter

AMPH-induced locomotion is consistent with previous reports (Jones and Holtzman, 1992;

Jones et al, 1993), even though nor-BNI has been shown to increase DA levels following injection into the NAc (Spanagel et al, 1992). Despite the ability of κ antagonists to increase DA

122 levels within the NAc, the apparent lack of effect on AMPH-induced locomotion by systemic

nor-BNI has been postulated to indicate that κORs are not involved with tonic regulation of DA

function within brain regions (or neurons?) that control motor activity (Manzanares et al, 1991;

Jones and Holtzman, 1992). Spanagel and coworkers (1992) postulated that tonic activation of µ and κORs are required for the maintenance of basal DA levels within the NAc, but our results and the results of others seem to indicate that this κOR regulation of DA levels is not involved with regulating the AMPH-induced DA increases within the NAc.

A possible explanation for the observed effects of opioid antagonist pretreatment on

AMPH-induced stereotypy likely involves alterations of DA levels within the striatum.

Increases in DA levels within the striatum have been identified as a primary mechanism underlying AMPH-induced stereotypic behaviors (Creese and Iversen, 1974; Costall and Naylor,

1974). δOR activation has been shown to decrease the activity of GABAergic neurons in the striatum, similar to their effects within the NAc (Jiang and North, 1992). Additionally, δ receptors on DA neurons within the striatum have been shown to mediate a receptor-induced release of newly synthesized DA, and it is this same pool of DA that is preferentially released by

AMPH (Kuczenski and Segal, 1989; Dourmap et al, 1992; Trovero et al, 1990). It has been postulated that AMPH may cause the release of endogenous opioids within the DA system, resulting in a reduction of GABA-mediated inhibition of DA neurons. Decreases in GABAergic neuronal activity within the striatum induced by δ agonists would result in increases in both DA levels and dopaminergic neuronal activity (Jones et al, 1993). Blockade of δORs (and other opioid receptor subtypes?) would be expected to prevent this loss of GABA-mediated inhibition, thereby decreasing the amount of DA released by AMPH administration. Systemic naloxone administration has been shown to do just this, with an attenuation of the AMPH-induced increase

123 in extracellular DA levels. Naloxone presumably antagonizes striatal δORs and disinhibits

GABAergic neurons, resulting in an increase in inhibitory signals to target DA neurons and a decrease in DA levels within the striatum. Thus, in the context of AMPH administration, naloxone (and δ antagonist) pretreatment would lead to a net reduction in DA levels within the striatum and a subsequent decrease in stereotypic behaviors. This is exactly what we observed, not only by pretreatment with naloxone, but also by pretreatment with all three opioid receptor-

selective antagonists. It is therefore possible that µ and κ opioid receptors are functioning within

the striatum in a similar manner as δORs to modulate GABAergic neuronal activity and thus regulate DA levels in response to AMPH administration.

A potential issue to consider whenever employing the use of antagonists in experiments is the specificity and affinity of the compounds used. β-FNA is one of the most thoroughly

characterized irreversible opioid antagonists studied and achieves its antagonistic effects almost

entirely via the µOR (Broadbear et al, 2000; Takemori et al, 1981). A potential confounding

effect of this antagonist is a reversible κOR-agonistic activity that initially coincides with its µ-

selective antagonist effects (Takemori et al, 1981). However, as demonstrated in rats, κ agonists

have been shown to decrease both DA release in the NAc and spontaneous activity (Di Chiara

and Imperato, 1988; Spanagel et al, 1992). Thus, it would seem plausible that the κOR agonistic

activity of β-FNA would either have no effect or a potential inhibitory effect on AMPH-induced

locomotor activity. Our finding that the κOR antagonist nor-BNI failed to modulate AMPH-

induced hyperlocomotion in C57 mice points to a lack of involvement of kappa receptors in our

behavioral assay and would support the view that β-FNA is exerting its effect predominantly via

the µOR. A method for circumventing any possible κ agonistic properties of β-FNA on AMPH-

induced locomotion would be to increase the pretreatment period from 30 minutes to 24 hours.

124 This pretreatment paradigm has been shown to sufficiently isolate the µOR antagonistic property

of β-FNA (Broadbear et al, 2000). Alternatively, the mice could be pretreated with a κ

antagonist such as nor-BNI followed by β-FNA treatment before AMPH administration. A final

point to consider is that β-FNA acts to antagonize the µOR by alkylation of a subset of µ

receptors, up to 50% of total receptors, and thus its effects may be due to modification of only a

particular subtype or subpopulation of µORs present (Broadbear et al, 2000). This selective

modification might somehow target those µORs that are in position to decrease DA levels and

lead to alterations in AMPH-induced behaviors. Finally, NTI is the prototypic non-peptide δOR

antagonist (Portoghese et al, 1988) and is thought to be highly selective for the delta receptor.

NTI at doses of 0.5-1mg/kg has been shown to selectively antagonize δOR-mediated behaviors in the rat (Heidbreder et al, 1996). However, a number of reports suggest that at higher doses

(2mg/kg and higher), NTI is able to block the µOR as well. For example, NTI at 2.0mg/kg was able to antagonize the release of corticosterone induced by the µOR-selective agonist fentanyl

125 4.9. Summary:

In summary, the findings from the characterization of a mouse model of AMPH-induced behaviors indicate that the C57BL/6 strain of mice exhibit the expected bimodal behavioral response to acute AMPH administration. A low 2mg/kg dose of AMPH induced robust and prolonged hyperlocomotion with little induction of stereotypy while administration of high doses of AMPH (12 and 20mg/kg) induced robust stereotypy with little hyperlocomotion. A middle dose of 6mg/kg AMPH induced a pattern of behavioral responses lying in between the low and high dose groups, with periods of substantial hyperlocomotion surrounding a period of robust stereotypy. We determined that the time interval 30 – 60 minutes post AMPH administration is a reasonable time period, as a single index number, for the analysis of both locomotion and stereotypy data. This time period was found to contain the most significant changes in both locomotion and stereotypic behavior induced by drug administration and as such acted as a good representative of the behavioral responses seen throughout the longer two-hour observation period. Finally we found that an acute dose of 2mg/kg AMPH was sufficient for inducing a predominately hyperlocomotive response in C57 mice and that 12mg/kg AMPH was most useful

for inducing a predominately stereotypic response in these mice.

Using these predetermined AMPH doses and representative time interval for behavioral

recording and analysis, we then utilized this animal model of AMPH-induced behaviors to study

the involvement of the endogenous opioid system in regulating locomotion and stereotypy

following acute AMPH administration. The results from these studies indicate that of the three

opioid receptor subtypes, antagonism of δORs is involved with the attenuation of locomotion

following acute administration of 2mg/kg amphetamine in C57BL/6 mice. κORs do not appear

126 to be involved in modulating AMPH-induced locomotion, and antagonism of µORs might act to potentially augment the locomotor-stimulating effects of AMPH. In contrast to the differing effects of opioid receptors on AMPH-induced locomotion, antagonism of all three receptor subtypes can attenuate the stereotypic response induced by 12mg/kg AMPH in these mice. It is assumed that these receptor-dependent effects are mediated through differential regulation of neurons, presumably GABAergic and dopaminergic, within the VTA, NAc, and striatum of the mesolimbic dopaminergic system.

In the context of physiological significance, why would the endogenous opioid system regulate the dopaminergic system? As presented in the Introduction in Chapter 1, natural rewards such as food and sex elicit pleasurable responses within organisms and as such are thought to be reinforced via largely dopamine-dependent changes within the mesocorticolimbic system (Everitt and Wolf, 2002; Kelley and Berridge, 2002). From our opioid antagonist dose- response studies, and reports from the literature, it is clear that the opioid system can regulate the brain reward system. Treatment of normal human subjects with the opioid antagonist naloxone can cause irritation, fear, and anger (Jones and Herning, 1979; Pickar et al, 1982), which would imply that endogenous opioids such as β-endorphin and enkephalin (and others) are functioning to regulate brain systems involved in mood and behavior. By being able to regulate mood and behavior, the opioid system can act to further reinforce or diminish the responding of an organism to a wide variety of stimuli. The reinforcement of stimuli that positively regulate the opioid system and lead to increases in DA levels, would lead to an increase in perceived reward and a subsequent increase in the likelihood that those stimuli will be sought out in the future.

A second potential outcome of crosstalk between the opioid system and the DA system is that a greater diversity of physiological responses and processes can be regulated in response to

127 various stimuli. For example, there is increasing evidence that many of the same molecular and

biochemical changes seen with long term use or exposure to drugs of abuse mimic changes seen

in learning and memory. Thus, just as drug addiction can be viewed as a form of ‘learned’

behavior, stimuli that impact on the endogenous opioid system can induce neurochemical

changes, such as long term potentiation (LTP), within critical brain regions like the

hippocampus, that are important for the process of learning and the generation of memory. In

this manner, not only can a pleasurable stimulus be made even more so by the interaction of the

opioid system and the DA system, but the organism will begin to associate and ‘remember’ this stimulus as rewarding. Conversely, stimuli that are harmful or unrewarding to an organism may impact the opioid and DA systems in an equal but opposite manner leading to a negative memory of a particular stimulus. This generation of both positive and negative memories would likely contribute to the overall success and survival of the organism.

4.10. Future Directions:

There are a number of interesting findings from these studies that would be worth

following up with additional and expanded investigations. The first concerns the complex mix

of behaviors induced in C57 mice following an intermediate dose (6mg/kg) of AMPH. We

found that after administration of this dose, the mice exhibited periods of both robust

hyperlocomotion and stereotypy. Because, as we and others have shown, multiple

neurotransmitter systems participate in regulating AMPH-induced behaviors, the effect on these

behaviors following pretreatment with agonists and antagonists for the GABA, glutamate, and/or

128 opioid systems followed by 6mg/kg AMPH could be investigated. These types of studies could

provide additional insight into the possible trigger mechanism that ultimately determines whether

an animal will display drug-induced hyperactivity or stereotypy. A second finding that warrants

further investigation is the slight augmentation of AMPH-induced hyperlocomotion following

pretreatment with the µOR antagonist β-FNA. For this, additional groups of mice need to be

tested with similar doses of both the antagonist and AMPH, along with mice pretreated with

differing doses of β-FNA followed by AMPH, to study if this effect is dose-dependent on the antagonist in any way. If this finding is confirmed, neurochemical studies could be performed to

identify if β-FNA treatment is in fact increasing VTA DA levels and in this way upregulating the

overall DA system, as proposed by Devine et al (1993b). The last findings that would be

valuable to investigate further are the effects on basal stereotypy induced by treatment with the µ

and κOR-specific antagonists by themselves. There have been a few clinical studies reported

over the years investigating the use of various opioid antagonists in treating a range of human

conditions, including heroin and alcohol addiction and schizophrenia. As the stereotypic

response to AMPH can, in some ways, mimic aspects of schizophrenia, further investigations,

both behaviorally and neurochemically, into the effects of opioid antagonists on basal and

AMPH-induced stereotypic behaviors could lead to new avenues of research into potential

treatments for these conditions.

129

Chapter Five

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