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The role of excitatory amino acid receptors in the basal forebrain in the locomotor response produced by psychostimulants and the non-competitive NMDA receptor antagonist MK801

Willins, David Lawrence, Ph.D.

The Ohio State University, 1992

UMI 300 N. ZeebRd. Ann Aibor, MI 48106 THE ROLE OF EXCITATORY AMINO ACID RECEPTORS IN THE BASAL FOREBRAIN IN THE LOCOMOTOR RESPONSE PRODUCED BY PSYCHOSTIMULANTS AND THE NON-COMPETITIVE NMDA RECEPTOR ANTAGONIST MK801

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By David L. Willins, B.S., R.Ph.

•k ie ic ie -k

The Ohio State University 1992

Dissertation Committee: Approved by: Michael C. Gerald, Ph.D. Lane J. Wallace, Ph.D. Advisor Norman J. Uretsky, Ph.D. College of Pharmacy God, where shall I find Thee, Whose glory fills the universe? Behold I find Thee Wherever the mind is free to follow its own bent, Wherever words come out from the depth of truth, Wherever tireless striving stretches its arms towards perfection, Wherever men struggle for freedom and right, Wherever the scientist toils to unbare the secrets of nature, Wherever the poet strings pearls of beauty in lyric lines, Wherever glorious deeds are done. - Prayer Book of the Jewish Reconstructionist Foundation Colette, you cannot know how much your support and love have meant to me during the last five years. You shared with me your courage and confidence when I had all but lost mine. You gave endlessly of your love and your strength at the times when I have needed them the most. I could not have made it without you. For this I am forever in your debt. As a small measure of my love and commitment, I dedicate this dissertation to you. ACKNOWLEDGEMENTS

Firstly, I would like to thank my fellow students. The experience of graduate school would not have been as enjoyable, or as endurable, without your companionship. Some of the best and brightest moments have been because of you. Rose Smith, Cheryl Crooks, Ron Dent, Hazel Benson, Laura Lingham, and Kathy Brooks Thanks for everything you have done to help untangle the web of Ohio State. I would also like to thank the members of my committee, Dr. Lane J. Wallace, Dr. Michael C. Gerald and Dr. Dennis R. Feller. Your guidance and advice during the course of my graduate career has been invaluable. I especially want to thank my advisor, Dr. Norman J. Uretsky. You have a special talent, the ability to be both friend and mentor. You have balanced these two roles remarkably and in so doing have made my years as a graduate student infinitely more productive and enjoyable. I will never forget the lessons you have taught. Finally, I want to thank my parents. Dad, you cannot know, for I did not know myself until the completion of my goal, what an inspiration you have been to me. I have always respected and admired you and I have tried to model my life after yours. I hope someday to make you as proud of me as I have always been of you. Mom, I have always felt your love and caring. From you I have learned to be curious and it is curiosity that drives the scientist. You have helped me to set my priorities and to accomplish one of my major goals. Thank you both.... VITA

July 23, 1963 ...... Born - Radford, Virginia 1986 B.S. Pharmacy, The Ohio State University, Columbus, Ohio 1986-1989 Graduate Teaching Associate, The Ohio State University, Columbus, Ohio 1989-1992 Graduate Research Associate, The Ohio State University, College of Pharmacy, Columbus, Ohio

PUBLICATIONS Willins, D.L., L.J. Wallace, and N.J. Uretsky, The role of excitatory amino acid receptors within the subpallidum in the hypermotility response induced by systemically administered stimulants of locomotor activity. FASEB J., A4233, 1989. Boldry, R.C., D.L. Willins, L.J. Wallace, and N.J. Uretsky, The interactions of AMPA and dopamine in the nucleus accumbens. FASEB J., A4133, 1989. Hill, R., D.D. Miller, L.J. Wallace, D.E. Supko, D.L. Willins, and N.J. Uretsky, Synthesis and SAR for some 2,3-dihydroxy- quinoxalines as glutamate antagonists. American Assoc, of Pharmaceutical Scientists 5th Annual Meeting and Exposition, Pharm. Res. 7, MNPC 5038 drugs. Pharmacologist, 32, 126 (1990). Presented at the Annual Meeting of the American Society for Pharmacology and Experimental Therapeutics, Milwaukee, August 1990.

iv Willins, D.L., D.E. Supko, R . A. Hill, L.J. Wallace D.D. Miller, and N.J. Uretsky, Quisqualate receptor antagonists in the subpallidum decrease the hypermotility response to amphetamine. Soc. Neurosci. 20th annual meeting, Abs: 489.9, 1990. Willins, D.L., P.E. Shreve, L.J. Wallace, and N.J. Uretsky, The role of glutamatergic receptors in the subpallidum in regulating locomotor activity elicited by psychostimulant drugs., The Basal Forebrain: Anatomy to Function, Chicago, 1989. Willins, D.L, L.J. Wallace, and N.J. Uretsky, AMPA/kainate receptor antagonists in the nucleus accumbens selectively decrease the hypermotility response to amphetamine. Soc. Neurosc. 21st annual meeting, Abs: 567.13, 1991. Boldry, R.C., D.L. Willins, L.J. Wallace, and N.J. Uretsky, The role of dopamine in the hypermotility response to intra- accumbens AMPA. Brain Res., 559:100-108, 1991. Willins D.L., L.J. Wallace, D.D. Miller and N.J. Uretsky, AMPA/kainate receptor antagonists in the nucleus accumbens and the subpallidum decrease the hypermotility response to psychostimulant drugs. J. Pharmacol. Exp. Ther. 260:1145-1151, 1992.

FIELDS OF STUDY Major field: Pharmacy Studies in: Neuropharmacology

v TABLE OF CONTENTS

DEDICATION ...... ii ACKNOWLEDGEMENTS ...... iii VITA ...... iv LIST OF TABLES ...... x LIST OF FIGURES ...... xi CHAPTER I INTRODUCTION ...... 1 EXCITATORY AMINO ACIDS ...... 1 EXCITATORY AMINO ACID RECEPTOR SUBTYPES...... 4 THE NMDA RECEPTOR ...... 4 THE TRANSMITTER RECOGNITION SITE ...... 5 THE ION-CHANNEL SITE ...... 6 THE GLYCINE SITE ...... 9 THE ZINC SITE ...... 11 THE POLYAMINE SITE ...... 12 KAINATE AND QUISQUALATE (AMPA) RECEPTORS ...... 12 THE AP4 RECEPTOR ...... 15 THE METABOTROPIC RECEPTOR ...... 17 ANATOMY OF THE LOCOMOTOR CIRCUIT ...... 19 THE VENTRAL TEGMENTAL AREA ..... 2 0 THE NUCLEUS ACCUMBENS ...... 23 THE VENTRAL PALLIDUM ...... 26 STATEMENT OF THE PROBLEM ...... 27 vi CHAPTER II AMPA/KAINATE RECEPTOR ANTAGONISTS IN THE NUCLEUS ACCUMBENS AND VENTRAL PALLIDUM DECREASE THE HYPERMOTILITY RESPONSE TO PSYCHOSTIMULANT DRUGS ...... 29 INTRODUCTION ...... 2 9 METHODS ...... 33 ADMINISTRATION OF DRUGS INTO THE BRAIN ...... 33 MEASUREMENT OF LOCOMOTOR ACTIVITY ...... 34 HISTOLOGY ...... 35 DRUGS ...... 35 STATISTICS ...... 36 RESULTS ...... 37 EFFECT OF THE BILATERAL MICROINJECTION OF DNQX OR GAMS INTO THE NUCLEUS ACCUMBENS ON STIMULANT INDUCED HYPERMOTILITY ... 37 THE EFFECT OF BILATERAL MICROINJECTION OF DNQX OR GAMS INTO THE VENTRAL PALLIDUM ON STIMULANT INDUCED HYPERMOTILITY ... 43 EFFECT OF AFQX INJECTED INTO THE NUCLEUS ACCUMBENS OR SUBPALLIDUM ON THE STIMULATION OF LOCOMOTION PRODUCED BY AMPHETAMINE ...... 44 DISCUSSION ...... 48 CHAPTER III THE ROLE OF DOPAMINE AND AMPA/KAINATE RECEPTORS IN THE NUCLEUS ACCUMBENS IN THE HYPERMOTILITY RESPONSE TO MK801 ...... 55 INTRODUCTION ...... 55 METHODS ...... 58 ADMINISTRATION OF DRUGS INTO THE BRAIN ...... 58 MEASUREMENT OF LOCOMOTOR ACTIVITY ...... 59 HISTOLOGY ...... 59 vii DRUGS ...... 60 STATISTICS ...... 61 RESULTS ...... 61 THE EFFECTS OF MK801 ON LOCOMOTOR ACTIVITY IN NORMAL AND DOPAMINE DEPLETED RATS ...... 61 THE EFFECT OF DOPAMINE RECEPTOR ANTAGONISTS ON LOCOMOTOR ACTIVITY STIMULATED BY MK801 ...... 65 THE EFFECT OF THE BILATERAL ADMINISTRATION AMPA/KAINATE RECEPTOR ANTAGONISTS INTO THE NUCLEUS ACCUMBENS ON THE HYPERMOTILITY PRODUCED BY SYSTEMIC MK801 ...... 70 DISCUSSION ...... 73 CHAPTER IV THE NON-COMPETITIVE NMDA RECEPTOR ANTAGONIST MK801 STIMULATES LOCOMOTOR ACTIVITY FOLLOWING INJECTION INTO THE VENTRAL TEGMENTAL AREA AND THE NUCLEUS ACCUMBENS IN RATS: DEPENDANCE ON CENTRAL DOPAMINERGIC AND GLUTAMATERGIC MECHANISMS ...... 81 INTRODUCTION ...... 81 METHODS ...... 85 ADMINISTRATION OF DRUGS INTO THE B R A I N ...... 85 MEASUREMENT OF LOCOMOTOR ACTIVITY ...... 86 HISTOLOGY ...... 87 DRUGS ...... 87 STATISTICS ...... 88 RESULTS ...... 88 THE ROLE OF DOPAMINE IN THE LOCOMOTOR STIMULATION FOLLOWING THE ADMINISTRATION OF MK801 DIRECTLY INTO THE VENTRAL TEGMENTAL AREA...... 88 THE ROLE OF DOPAMINE IN THE LOCOMOTOR STIMULATION FOLLOWING THE ADMINISTRATION OF MK801 DIRECTLY INTO THE NUCLEUS ACCUMBENS ...... 93 DISCUSSION ...... 97 CHAPTER V SUMMARY AND CONCLUSIONS ...... 101 REFERENCES ...... 112 viii LIST OF TABLES

TABLE PAGE 1. Effect of the administration of DNQX or GAMS into either the nucleus accumbens or the ventral pallidum on locomotor activity ...... 40

ix LIST OF FIGURES

FIGURE PAGE 1. Representative injection sites for the intracranial administration of drugs into the nucleus accumbens and the ventral pallidum ...... 39 2. Effect of DNQX injected into the nucleus accumbens on locomotor activity stimulated by amphetamine, caffeine and scopolamine ... 41 3. Effect of GAMS injected into the nucleus accumbens on locomotor activity stimulated by amphetamine, caffeine and scopolamine ... 42 4. Effect of DNQX injected into the ventral pallidum on locomotor activity stimulated by amphetamine, caffeine and scopolamine ... 45 5. Effect of GAMS injected into the ventral pallidum on locomotor activity stimulated by amphetamine, caffeine and scopolamine ... 46 6. Effect of AFQX injected into the nucleus accumbens or the ventral pallidum on locomotor activity stimulated by amphetamine, caffeine and scopolamine ..... 47 7. The effect of reserpine on the locomotor stimulation induced by MK801 or amphetamine ...... 63 8. The effect of a-methyl-p-tyrosine on the locomotor stimulation induced by MK801 ... 64 9. The effects of systemic administration of dopamine receptor antagonists on MK801 stimulated locomotor activity ...... 66

x 10. The effect of intraaccumbens administration of dopamine receptor antagonists on d-amphetamine or MK801 stimulated locomotor activity ...... 68 11. The effect of intraaccumbens administration of DNQX on MK801 stimulated locomotor activity ...... 71 12. The effect of intraaccumbens administration of GAMS on MK801 stimulated locomotor activity ...... 72 13. The effect of direct bilateral injection of MK801 into the ventral tegmental area ... 90 14. The effects of pretreatment with the dopamine D1 receptor antagonist, SCH233 90 or the D2 receptor antagonist, Eticlopride on locomotor activity stimulated by direct bilateral injection of MK801 into the ventral tegmental area ...... 91 15. The effect of reserpine pretreatment 18 hours prior to the administration of MK801 into the ventral tegmental area ..... 92 16. The effect of direct bilateral injection of MK801 into the nucleus accumbens ...... 94 17. The effects of pretreatment with the dopamine D1 receptor antagonist, SCH233 90 or the D2 receptor antagonist, Eticlopride on locomotor activity stimulated by intra-accumbens MK801 ...... 95 18. The effect of reserpine pretreatment 18 hours prior to the administration of MK801 into the nucleus accumbens ...... 96

xi CHAPTER I INTRODUCTION

EXCITATORY AMINO ACIDS

The dicarboxylic acids, L-glutamate and L-aspartate, are now generally accepted as the primary mediators of fast excitatory transmission in the mammalian central nervous system. These transmitters appear to be involved in many physiological phenomena ranging from the processing of sensory information to control of coordinated movement patterns to cognitive processes including learning and memory. Disfunction of these transmitter systems has been implicated in an increasing number of disease states, including neurological disorders such as epilepsy, stroke induced brain damage, and various neurodegenerative diseases (e.g. Huntington's and Alzheimer's diseases) as well as psychological disorders including affective disorder, schizophrenia and drug addiction. It is evident that a more thorough understanding of the mechanisms involved in the regulation and control of excitatory amino acid function in the central nervous system will be of great value in

1 understanding many of the diseases that affect the human brain.

While the first demonstrations of the ability of amino acids to depolarize neurons took place nearly forty years ago (Hayashi, 1954; Curtis et. al., 1959), only in the past two decades have the excitatory amino acids been accepted as neurotransmitter substances. Much of the early hesitancy in accepting these amino acids as transmitter substances was due to the multiplicity of roles they play in cellular physiology. L-Glutamate is involved in protein structure, the metabolic processing of glucose and fatty acids, and the synthesis of the inhibitory neurotransmitter GABA. L- Aspartate is also a structural element of proteins and is also involved in the Kreb's cycle. In addition L-aspartate is involved in the regulation of ammonia levels via the urea cycle. Due to the ubiquitous nature of these processes, high concentrations of these amino acids in neurons did not immediately suggest a role as neurotransmitters.

With continuing advancement of research techniques it has now been demonstrated that glutamate meets many of the criteria to be considered as a neurotransmitter. Glutamate release has been shown to be calcium dependant (Cotman and Nadler, 1981) and there exists a high affinity uptake mechanism for glutamate into nerve terminals (McGeer et. al., 1987). Radioligand binding studies have demonstrated selective binding sites for glutamate (Honore et.al., 1982; Monoghan et. al., 1984) and autoradiographic evaluation of these binding sites has suggested that high concentrations of binding sites exist in regions believed to receive glutamatergic afferent projections (Cotman et.al., 1987).

The excitatory amino acid receptors were originally classified into three subtypes based upon the selective agonists N-methyl-D-aspartate (NMDA), quisqualate, and kainate (Watkins and Evans, 1981; Davies et.al., 1979; McLennan, 1983). The recent rapid advances in this field have primarily resulted from the identification and radiolabeling of potent and selective agonists and antagonists for these receptors. Currently there are at least five different receptor subtypes including NMDA, quisqualate, and kainate receptors and two additional sites termed the AP-4 receptor (after the amino acid analog D-2- amino-phosphonobutyric acid) and a metabotropic receptor (which has been associated with the turnover of phosphotidyl inositol). Additionally, molecular biological approaches have isolated and identified a number of different receptor proteins that respond to glutamate and exhibit pharmacologic profiles similar to each of the three original excitatory amino acid receptors (Barnard and Henley, 1991) . 4 EXCITATORY AMINO ACID RECEPTOR SUBTYPES

THE NMDA RECEPTOR

The best characterized of the excitatory amino acid receptors is the N-methyl-D-aspartate receptor. The NMDA receptor has been demonstrated to play a role in a variety of physiological functions, including developmental plasticity (Tsumoto et.al., 1987), learning and memory (Morris et.al., 1986; Collingridge and Bliss, 1987) and sensory transmission (Salt, 1986). This receptor has also been implicated in a number of neurological disorders including ischemic brain damage (Meldrum, 1985), hypoglycemia (Weiloch, 1985), and epilepsy (Croucher et .al., 1982).

Recent findings show the existence of at least 6 different pharmacologically distinct sites within the NMDA receptor complex to which agonists and antagonists can bind to modify the response to glutamate. These sites include (a) a transmitter recognition site, (b) an ion channel site (Mg+2) , (c) a PCP site, (d) a strychnine insensitive glycine site, (e) a zinc site, and (f) a polyamine site. 5 THE TRANSMITTER RECOGNITION SITE

The primary binding site, to which NMDA, glutamate and aspartate bind, is located on the outer surface of the receptor protein and operates to facilitate the opening of a cation channel. A large number of competitive antagonists now exist for this site and have been demonstrated to have electrophysiological, biochemical and behavioral activity. Nearly all of these antagonists are D-isomers of longer chain analogs of glutamate (Watkins and Olverman, 1987) . The initial selective and useful NMDA receptor antagonists were D-a-aminoadipate (DAA) and D-a-aminosuberate (DAS). The optimum chain length for these analogs was determined to be 5 and 7 carbons (DAA and DAS respectively). The next major advancement in selectivity and potency came with the development of omega phosphonate analogs of DAA and DAS. The 5 carbon (D-2-amino-phosphonovaleric acid, AP5) and 7 carbon (D-2-amino-phosphonoheptanoic acid, AP7) phosphonic acid analogs of these original antagonists have good selectivity and affinities in the low micromolar range for the transmitter recognition site (Watkins and Olverman, 1987). While these compounds have been of great value in determining many of the physiological roles that NMDA receptors play, they are highly polar and therefore do not penetrate the central nervous system readily following systemic administration. They are therefore not good candidates as therapeutic agents. Several conformationally restricted analogs of AP5 and AP7 have been developed in an attempt to produce selective and potent NMDA antagonists with increased lipophilicity. These antagonists include the rigid piperazine analog 3-(2-carboxy-piperazin-4-yl)propyl- 1-phosphonic acid (CPP, Davies et.al., 1986) derived from AP7 and cis-4-{phosphonomethyl) -2-piperidine-carboxylic acid (CGS19755, Lehmann et.al., 1988) derived fromAP5. Recently an AP5 analog, made more lipophilic through the addition of a double bond and a methyl group, CGP37849 (D,L-(E)-2-amino- 4-methyl-5-phosphono-3-pentanoic acid), was shown to have anticonvulsant activity following oral administration (Schmutz et.al., 1989).

THE ION-CHANNEL SITE

The first agent identified as a blocker of the NMDA receptor gated ion channel was magnesium (Mg*2) . In 1980 Watkins and co-workers demonstrated that Mg*2, in a preparation of spinal cord tissue, selectively inhibited depolarization produced by NMDA while having no effect on that produced by quisqualate or kainate (Ault et.al., 1980) . It has subsequently been shown that Mg*2 ions produced a voltage-dependant blockade of the NMDA ion channel by binding to a site within the channel (Ascher and Nowak, 1988) . One significant feature about this Mg*2 blockade is that it occurs at concentrations well below those normally present in the extracellular fluid. This confers a unique voltage dependency on NMDA mediated responses. At normal resting potential the NMDA receptor is quiescent but as the membrane becomes depolarized, allowing Mg*2 to diffuse out of the ionophore, NMDA receptor mediated responses become apparent. This type of voltage dependency is believed to have a significant role in the physiological processes involving the NMDA receptor such as long term potentiation (Collingridge and Bliss, 1987).

A second site within the NMDA receptor ion channel is a site separate from the magnesium site which binds the dissociative anesthetics phencyclidine (PCP) and ketamine. Lodge first described a non-competitive inhibition of NMDA- induced depolarization by the dissociative anesthetics, PCP and ketamine in 1983 (Anis et.al., 1983). Since that time several other compounds have been identified which interact at this site and which non-competitively antagonize the effects of NMDA. More recently, the dibenzocyclohepteneimine, MK801, has been identified as the most potent NMDA antagonist yet described within this class of compounds (Wong et.al., 1986). The antagonism of NMDA receptor mediated responses by these compounds is voltage dependant and is only apparent at negative intracellular potentials (Honey et.al., 1985). This finding suggests an interaction within the ion channel. Additional evidence suggesting an interaction within the ion channel is that the development of receptor blockade by these non-competitive antagonists shows a strong degree of "use-dependance" (Honey et.al., 1985, Kemp et.al., 1986). The term "use-dependant" means that the blockade of the receptor is increased by the presence of the agonist and recovery from the block is accelerated by repeated applications of the agonist (MacDonald et.al., 1986).

These compounds have been the focus of numerous research reports due to their psychoactive properties. PCP and ketamine have already been mentioned as possessing dissociative properties and PCP is a commonly abused substance. MK801 has been shown to produce a characteristic behavioral pattern including locomotor activity and stereotypy (Clineschmidt et.al., 1982, Trickelbank, et.al., 1989, Benvenga and Spaulding, 1988, Willins et.al., submitted) . MK801 induced behavior and the role of NMDA receptors in behavior have been the focus of a large portion of this dissertation and will be discussed at greater length below (see chapters 3 and 4). 9 THE GLYCINE SITE

Glycine, a classical inhibitory transmitter, was first identified as an endogenous potentiator of NMDA responses in 1987 by Johnson and Ascher (1987). They found that glycine greatly enhanced the electrophysiological responses of NMDA agonists in cultures of mouse brain neurons. Glycine had no effect by itself and the enhancement of NMDA responses was shown to be insensitive to strychnine. Very low concentrations of glycine are required for this effect (EC50: 100-300 nM) . These concentrations are approximately ten times lower than those required to activate the inhibitory, strychnine-sensitive receptor (Wong and Kemp, 1991). Patch- clamp analysis of single NMDA receptor channels indicated that glycine increased the frequency of channel opening and not the current amplitude (Johnson and Ascher, 1987) . These studies were conducted using outside-out patches, suggesting that the effects of glycine do not require a secondary messenger.

In 1985 the demonstration that the pattern of 3H- glycine binding exhibits a distribution that is identical to that of NMDA-sensitive 3H-glutamate binding sites, provided the first concrete evidence that the glycine site may be directly located on the NMDA receptor complex (Monaghan et.al., 1985). In addition to autoradiographic analysis of 10 glycine and NMDA binding, more recent studies have shown that a high affinity glycine recognition site is also present on solubilized preparations of the NMDA receptor (McKernan et.al., 1989). Further evidence has been provided by molecular biology studies in which the NMDA receptor has been expressed in Xenopus oocytes. These studies have demonstrated that responses to NMDA agonists in this preparation are also facilitated by glycine (Kleckner and Dingeldine, 1988). Together, these studies strongly suggest that the glycine recognition site is at least closely associated with the NMDA receptor and in all likelihood is part of the receptor ionophore complex.

Several antagonists have been developed for the glycine site of the NMDA receptor. The non-selective excitatory amino acid receptor antagonist, kynurenic acid, has been shown to competitively inhibit 3H-glycine binding to rat brain membranes (Kemp et.al., 1987), and part of its NMDA receptor blocking effects in tissue slices are reversed by the addition of glycine, supporting an antagonist action at the glycine site (Watson et.al., 1988). The addition of a chlorine atom at the 7 position on kynurenic acid yielded the first selective, high affinity antagonist of the glycine receptor, 7-chloro-kynurenic acid (Kemp et.al., 1988). With the development of the selective antagonists for glycine it was demonstrated that glycine not only facilitates the 11 responses of NMDA receptor agonists, but that it is an absolute requirement for activation of the NMDA receptor (Kemp et.al., 1988). In addition to 7-chloro-kynurenate, HA- 966, a drug that had initially been described as an NMDA antagonist, has also been shown to act selectively at the glycine site to block glycine potentiation of NMDA responses in cultured neurons (Fletcher and Lodge, 1988).

THE ZINC SITE

In addition to the well-defined, voltage-dependent , antagonistic action of Mg*2, increasing evidence indicates that the divalent transition metal ion Zn*2, also produces a selective blockade of NMDA receptor responses. Westbrook and Mayer (1987) described the effects of zinc on dissociated cultures of mouse hippocampal neurons as rapid in onset and reversible. In contrast to Mg*2, this response does not exhibit a voltage-dependency. The differences between the mechanisms of action of Mg*2, and Zn*2 were further highlighted by the observation that, whereas Mg*2 increased the dissociation rate and the binding of 3H-MK801 to rat cortical membranes, Zn*2 had the opposite effect (Reynolds and Miller, 1988) . These results argue for a distinct site of action for the divalent cation, zinc. 12 THE POLYAMINE SITE

The polyandries, spermine and spermidine, are thought to interact with a distinct site on the NMDA receptor complex to enhance responses to glutamate and NMDA agonists. Spermine and spermidine have been shown to positively modulate the binding of 3H-MK801 at the NMDA receptor (Ransom and Stec, 1990) . While this action of the polyamines resembles activation of the receptor by glutamate, spermine and spermidine do not inhibit the binding of 3H-glycine or 3H-CPP, thus arguing that the polyamines act at a site different than the transmitter recognition site.

KAINATE AND QUISQUALATE (AMPA) RECEPTORS

Kainic acid and quisqualic acid are naturally occurring substances derived from the algae Digenea simplex and from the seeds of the terrestrial plant, Quisqualis fructus, respectively. Original studies first described these compounds as potent excitants at the crayfish neuromuscular junction (Takemoto, 1978; Shinozaki and Shibuya, 1974) and in the frog and rat spinal chord (Biscoe et.al., 1975, 1976) . The first demonstration that these compounds acted at different receptors came when it was subsequently demonstrated that the glutamic acid analog, glutamate 13 diethyl ester (GDEE), selectively inhibits quisqualate induced responses, without affecting the responses to NMDA or to kainic acid (Mclennan and Lodge, 1979; Davies and Watkins, 1977) .

A second indication that these agonists are acting at separate sites came with the development in 1980 of a synthetic isoxazole amino acid analog, a-amino-3-hydroxy-5- methyl-isoxazolepropionic acid (AMPA), which was reported to be a potent excitant of spinal neurons and to act at GDEE- sensitive sites (Krosgaard-Larsen et.al., 1980). Subsequent studies with AMPA have demonstrated that it is more selective for the quisqualate site than is quisqualate itself (Krosgaard-Larsen and Honore, 1983; Horne and Simmonds, 1987). AMPA has a higher potency than quisqualate and appears to bind to the receptor site with a higher affinity (Honore et.al., 1982). AMPA appears to be more selective than quisqualate is for the 3H-quisqualate binding site. Thus, in binding studies, unlike quisqualate, AMPA does not inhibit the binding of 3H-kainic acid (Honore et.al., 1982; Murphy et.al., 1987). These findings have prompted a number of researchers to call the site at which AMPA and quisqualate bind the "AMPA" receptor.

Antagonists that are more effective than GDEE in inhibiting AMPA responses have been developed, including y- D-glutamyl-aminomethyl-sulfonate (GAMS) and y-D- glutamyltaurine (GLU-TAU). These agents are broad spectrum antagonists that are less active at NMDA receptors than at AMPA and kainate receptors (Jones et.al., 1984). In 1988, Honore and co-workers developed a series of quinoxalinedione antagonists which have a much greater, selectivity for AMPA and kainate responses than for NMDA mediated responses (Honore et .al., 1988). 6-Cyano-7-nitroguinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) are the two most potent antagonists in this series and displaced 3H- AMPA binding at submicromolar concentrations (IC50 for CNQX: 0.3 |iM) (Honore et.al., 1988). These compounds were 5-fold less potent at displacing 3H-kainate (IC50: 1.5 (JM) and were weak or ineffective at displacing a variety of other neurotransmitter ligands, including the NMDA receptor antagonist, CPP (IC50: 25 |1M) . Functionally, CNQX and DNQX competitively inhibit quisqualate, kainate and to a much lesser degree NMDA stimulated 3H-GABA release from cultured mouse neurons (Drejer and Honore, 1988). To date, no receptor antagonists exist which clearly differentiate between quisqualate (AMPA) and kainate receptor responses. Differences between these receptors have however been identified with respect to distribution and relative agonist potency profiles. In preparations of spinal cord C-fiber afferents, which express a relatively pure population of kainate receptors (Davies et.al., 1979), kainate receptors 15 display a relative agonist potency of: domoic acid > kainic acid > quisqualate » L-glutamate (Davies et.al., 1979). This pattern is the same as that observed for the displacement of 3H-kainate from rat brain membranes (London and Coyle, 1979). In contrast quisqualate receptors, as defined by GDEE sensitivity, display a different rank-order of agonist potency (AMPA > quisqualate > L-glutamate > kainate). Once again this order of agonist potency is repeated in binding studies using 3H-AMPA (Honore et.al., 1982, Murphy and Snowhill, 1987). Autoradiographic analysis of kainate and AMPA receptorbinding shows that these receptors each have a distinct distribution. 3H-Kainate binding sites show highest densities in the CA3 region of the hippocampus and in the striatum, reticular nucleus of the thalamus, and the granule cell layer of the cerebellum (Monaghan and Cotman, 1982) . 3H-AMPA binding shows highest densities in the CA1 region of the hippocampus, the lateral septum and in the molecularlayer of the cerebellum (Monaghan et.al., 1984; Olsen et.al., 1987).

THE AP-4 RECEPTOR

In 1981 Koerner and Cotman (1981) first demonstrated the ability of L-AP4 (L-2-amino-4-phosphonobutyric acid) to suppress responses of dentate gyrus granule cells to stimulation of the lateral perforant path.Subsequent 16 studies have demonstrated that L-AP4 suppresses responses in preparations of cat and immature rat spinal cord (Evans et.al., 1982; Davies and Watkins, 1982), rat olfactory cortex (Hearns et.al., 1986), and mossy fiber-CA3 synapses in guinea pig hippocampal slices (Lanthorn et.al., 1984). While L-AP4 potently blocks synaptic transmission, it is not capable of reversing the effects of co-administered excitatory amino acid agonists, NMDA, kainate, quisqualate, L-glutamate and L-aspartate (Evans et.al., 1982; Davies and Watkins, 1982).

Recent evidence has suggested that the effects of L-AP4 may be mediated at presynaptic receptors. Spontaneous miniature excitatory postsynaptic potentials (MEPSPs) are diminished in the presence of kynurenic acid, a nonselective excitatory amino acid antagonist, indicating a postsynaptic mechanism of action. L-AP4 was unable to block MEPSPs at concentrations which were shown to block EPSPs (Cotman et.al., 1986). This finding supports a role for a presynaptic site of action of L-AP4. Additional evidence supporting a presynaptic action is provided by the finding that AP4 inhibits the release of glutamate from hippocampal synaptosomes (Gannon et.al., 1989). Many of these responses are species specific and the molecular mechanisms underlying the effects of AP4 remain to be determined. 17 THE METABOTROPIC RECEPTOR

In 1985, Sladeczek et.al. (1985) reported that glutamate and quisqualate potently stimulated phosphotidyl inositol (PI) turnover in cultures of striatal neurons. Subsequently this response has been identified in brain slices (Nicoletti et.al., 1986), cultured glial cells (Pearce et.al., 1986), and Xenopus oocytes (Sugiyama et.al., 1987). There are several agonists in addition to quisqualate and glutamate which activate the metabotropic receptor. trans-ACPD (trans-l-aminocyclopentane-1,3-dicarboxylic acid), a conformationally restricted analog of glutamate, has been shown to be a selective agonist for metabotropic receptors (Palmer et.al., 1989). In brain slices, trans-ACPD produces a greater maximal stimulation of PI turnover than quisqualate, glutamate or ibotenic acid (Palmer et.al., 1989; Desai and Conn, 1990) . Finally, a new conformationally restricted analog of glutamate, (2S,3S,4S)-a- (carboxycyclopropyl)-glycine, has recently been reported to be more potent than trans-ACPD in stimulating phosphoinositide hydrolysis in rat hippocampal synaptosomes (Nakagawa et.al., 1990). The selectivity of this compound for the metabotropic receptor is evidenced by its insensitivity to the ionotropic receptor antagonists CPP and CNQX (Nakagawa et.al., 1990). 18 Antagonists also exist for the metabotropic excitatory amino acid receptor. AP4 antagonizes ibotenate or quisqualate stimulated PI turnover (Nicoletti et.al., 1986) but this effect is seen only at very high concentrations of AP4 (greater than 1 mM) . AP3, a S-phosphono-substituted derivative of aspartic acid, displays greater potency and selectivity for the metabotropic receptor than does AP4 (Schoepp and Johnson, 1989).

The highest levels of glutamate activated PI turnover and the highest density of metabotropic receptors, as determined by 3H-glutamate receptor autoradiography, exist in telencephalic regions of the brain including the hippocampus, cortex and striatum (Schoepp and Johnson, 1989; Cha et.al., 1990). This suggests a role for these receptors in the functioning of these higher regions. Glutamate stimulated increase in PI turnover is most active during the early postnatal period in rats suggesting a role in synaptic plasticity and synaptogenesis. This elevation in metabotropic responses to glutamate is also apparent in amygdala and hippocampal slices from kindled rats (Iadorola et.al., 1986). Finally, a role for metabotropic receptors has been proposed in neuronal degeneration. In hippocampal slices of guinea pig cortex, ibotenic acid induced toxicity and enhancement of glutamate release is blocked by AP4 (Jones and Roberts, 1990). In models of brain injury 19 including hypoxic /ischemic brain injury (Seren et.al., 1989) there is enhanced quisqualate-stimulated PI hydrolysis in the damaged tissue.

ANATOMY OF THE LOCOMOTOR CIRCUIT INVOLVED IN PSYCHOSTIMULANT INDUCED LOCOMOTION

The structures of the basal forebrain which have been hypothesized to underlie the locomotor responses to psychomotor stimulant drugs appear to involve a circuit that originates in a midbrain dopaminergic nucleus, the ventral tegmental area. The primary projection site of dopaminergic neurons from the ventral tegmental area is a basal forebrain structure called the nucleus accumbens. In addition to dopaminergic projections from the midbrain, the nucleus accumbens receives a dense glutamatergic projection which originates in higher cortical and limbic regions. A primary projection site of neurons originating in the nucleus accumbens neurons is a region ventral to the globus pallidus called the ventral pallidum. These projections have been shown to be GABAergic in nature and to play a critical role in drug-induced locomotion. Additionally, the nucleus accumbens sends GABAergic projections back to the ventral tegmental area and appears to regulate its own activation. Finally, neurons within the ventral pallidum project to the 20 frontal cortex, and two motor regions, the dorsomedial thalamus and the pedunculopontine nucleus. This circuit, from the ventral tegmental area to the nucleus accumbens to the ventral pallidum, has been the focus of the studies presented in this dissertation and will be discussed in further detail (for extensive reviews see Mogenson et.al., 1980; Heimer and Alheid, 1991; Groenewegen et.al., 1991).

THE VENTRAL TEGMENTAL AREA

Original anatomical studies demonstrated that the dopamine neurons of the mesencephalon form a continuous band extending from the lateral and caudal most extent of the substantia nigra pars lateralis, rostromedially through the pars compacta to the ventral tegmental area, along the midline ventral to the red nucleus and surrounding the interpeduncular nucleus (Carlsson et.al., 1962; Dahlstrom and Fuxe, 1964; Ungerstedt, 1971). Three major divisions of these projections termed A8, A9, and A10 have been made. The A8 projection arises from the substantia nigra pars lateralis and projects to telencephalic structures. The A9 projection arises from the substantia nigra pars compacta and the A10 projection from the ventral tegmental area. The A9 projection, which projects from the substantia nigra pars compacta to the striatum, is also called the nigrostriatal system. The A10 projection originates in the ventral 21 tegmental area of the midbrain and projects to the nucleus accumbens and to structures closely associated with the limbic system and has therefore been named the mesolimbic system.

The A10 dopaminergic neurons have a distinctive electrophysiological signature, as monitored with extracellular single-unit recording, which includes long duration action potentials (greater than 2.5 ms), slow (1-10 Hz) irregular or slow bursting firing patterns and often a distinctive initial segment "notch" (Bunney et.al., 1973a,b). These characteristics have been used to distinguish A10 dopaminergic neurons from non-dopaminergic neurons which also pass through this region.

The A10 dopaminergic projections are under several regulatory controls by both dopaminergic and GABAergic influences. A10 dopamine neurons have been demonstrated to be highly sensitive to the systemic administration of dopaminergic agonists which suppress the spontaneous firing of these neurons (Bunney et.al., 1973b). The iontophoretic administration of dopamine to the cell bodies of A10 dopaminergic neurons also suppresses spontaneous firing of these neurons, suggesting that this action is mediated by somatodendritic dopamine autoreceptors (Aghajanian and Bunney, 1977). These receptors were shown to be of the D2 22 subtype when it was demonstrated that iontophoretic application of lisuride, pergolide, bromocriptine and dopamine ergots, all selective D2 dopamine agonists, suppressed firing of A10 dopamine neurons (White and Wang, 1984) . In contrast, iontophoretic application of the Dl agonist, SKF38393, had no effect on A10 dopamine unit activity (White and Wang, 1984). In addition to autoregulation by dopamine, these neurons also appear to be regulated by GABAergic processes in the form of an inhibitory influence mediated by a long loop feedback regulation from the nucleus accumbens. A GABAergic projection from the nucleus accumbens to the ventral tegmental area has been demonstrated both biochemically (Walaas and Fonnum, 1980) and electrophysiologically (Wolf et.al., 1978). Mogenson and colleagues (Maeda and Mogenson, 1980; Yim and Mogenson, 1980) demonstrated that nearly 50% of the A10 dopamine neurons tested were inhibited by nucleus accumbens stimulation. This response was potentiated by nipecotic acid, an inhibitor of GABA uptake, and inhibited by picrotoxin, which blocks chloride channels associated with the GABA/benzodiazepine receptor complex.

Evidence that a ventral tegmental-nucleus accumbens dopaminergic projection is involved in behavior has been suggested by studies in which the administration of picrotoxin directly into the ventral tegmental area was 23 shown to stimulate locomotor activity. This activity is reversed by the administration of the dopamine receptor antagonist, spiroperidol, into the nucleus accumbens (Mogenson et.al., 1979; Yim and Mogenson, 1980). Additionally, this treatment was also shown to increase dopamine release in the nucleus accumbens (Mogenson and Yim, 1991). These studies argue the involvement of a GABA regulated dopaminergic projection from the ventral tegmental area to the nucleus accumbens in the initiation of locomotor responses.

THE NUCLEUS ACCUMBENS

As mentioned previously, a primary projection site of A10 dopaminergic neurons is the nucleus accumbens. In addition to dopaminergic inputs from the ventral tegmental area the nucleus accumbens receives glutamatergic inputs from a number of limbic and cortical structures. The intersection of limbic and motor inputs in the nucleus accumbens has prompted the suggestion that the accumbens may act as an interface, integrating higher emotional and motivational information with motor behavior (Mogenson et.al., 1980).

A functional role for dopamine in the nucleus accumbens was first suggested by studies that demonstrated that 24 dopaminergic agonists administered directly into the accumbens stimulates locomotor activity (Pijnenburg et.al., 1973). This finding, coupled with the finding that picrotoxin injected into the ventral tegmental area released dopamine in the nucleus accumbens and stimulated locomotor activity (Yim and Mogenson, 1991), suggests that endogenous dopamine plays an integral role in the modulation of locomotor activity. Dopaminergic transmission in the accumbens has also been implicated in the stimulation of locomotor activity produced by drugs of abuse such as amphetamine and cocaine, which act at dopamine nerve terminals to inhibit the uptake of dopamine and, in the case of amphetamine, to displace dopamine from the nerve terminal. Both amphetamine and cocaine have been shown to increase extracellular concentrations of dopamine in the nucleus accumbens (Di Chiara and Imperato, 1988). In addition, the destruction of dopaminergic neurons in the accumbens with 6-hydroxydopamine, a neurotoxin which selectively destroys dopamine neurons, reverses the locomotor effects of these drugs (Kelly and Iversen, 1976) .

Glutamate receptors in the nucleus accumbens also appear to play a role in behavior. The direct injection of excitatory amino acids into the nucleus accumbens produces an intense stimulation of locomotor activity (Arnt, 1981) . This response is dose dependant and is limited by the 25 production of seizures at higher doses. The co- administration of selective antagonists has been shown to inhibit the locomotor stimulation produced by NMDA, kainic acid and AMPA (Donzanti and Uretsky, 1984; Shreve and Uretsky, 1988b). The locomotor responses to the excitatory amino acids are also inhibited by drugs which interfere with dopaminergic function. Thus, the stimulation of locomotor activity produced by excitatory amino acids has been shown to be inhibited by the co-administration of fluphenazine (Donzanti and Uretsky, 1983) and also by pretreatment with the tyrosine hydroxylase inhibitor, a-methyl-p-tyrosine (Boldry et.al., 1990). Initially these results suggested that the activation of glutamate receptors may mediate the release of dopamine in the nucleus accumbens, however, other studies have demonstrated that NMDA stimulated only a small amount of 3H-dopamine release from nucleus accumbens slices (Boldry and Uretsky, 1988). In addition, the inhibition of excitatory amino acid stimulated locomotion by cx-methyl-p- tyrosine and reserpine can be reversed by the co­ administration of direct acting dopamine agonists (Boldry et.al., 1991; Willins et.al., unpubl. obs.). This would suggest that a post-synaptic mechanism is involved in the responses to glutamate and in the interaction between glutamate and dopamine mediated behaviors. 26 THE VENTRAL PALLIDUM

A primary projection site of the nucleus accumbens is a region ventral to the globus pallidus that has been called the ventral pallidum. This area receives a dense GABAergic projection from the nucleus accumbens (Jones and Mogenson, 1980; Mogenson and Nielsen, 1983) and glutamatergic afferents from limbic regions (Fuller et.al., 1987). The ventral pallidum also has outputs that project to two locomotor areas, the pedunculopontine nucleus and the dorsomedial thalamus (Swerdlow and Koob, 1987; Brudzynski et.al., 1988). The neurochemical identity of these projections remains to be determined, however, the injection of carbachol into the pedunculopontine nucleus inhibits activity suggesting acetylcholine as a likely candidate (Brudzynski et.al., 1988).

The GABAergic projection from the nucleus accumbens to the ventral pallidum appears to play a role in the locomotor response to drugs which act in the nucleus accumbens (Jones and Mogenson, 1980) . The injection of muscimol into the ventral pallidum inhibits locomotor activity stimulated by the activation of dopamine receptors (Mogenson and Nielsen, 1983, Swerdlow and Koob, 1984) and glutamate receptors (Shreve and Uretsky, 1988a) in the nucleus accumbens. Thus it appears that the ventral pallidum plays an integral role 27 in locomotor behaviors elicited through dopaminergic and glutamatergic mechanisms in the nucleus accumbens.

STATEMENT OF THE PROBLEM

The nucleus accumbens plays a critical role in mediating locomotor behavior elicited by drugs of abuse. Dopaminergic and glutamatergic afferent projections from other areas converge in this region and appear to be involved in translating cognitive and motivational inputs into locomotor responses. More specifically, evidence suggests that the locomotor stimulation induced by drugs acting in the nucleus accumbens may require the concurrent activation of both dopamine and AMPA/kainate receptors. The studies in this dissertation are designed to explore the interaction between glutamatergic and dopaminergic receptors in the nucleus accumbens in the regulation of drug-induced locomotor activity. The specific objectives of the experiments presented in this dissertation was: 1. to determine the role that AMPA/kainate receptors in the nucleus accumbens and the ventral pallidum play in the locomotor responses to the psychostimulant drugs, amphetamine, caffeine and scopolamine. 2. to determine the role of dopaminergic and glutamatergic neurotransmission in the nucleus accumbens in the locomotor response to systemic MK801. 28 3. to determine the neuroanatomical substrates of the locomotor stimulant response to MK801. CHAPTER II

AMPA/KAINATE RECEPTOR ANTAGONISTS IN THE NUCLEUS ACCUMBENS AND VENTRAL PALLIDUM DECREASE THE HYPERMOTILITY RESPONSE TO PSYCHOSTIMULANT DRUGS.

INTRODUCTION

The systemic administration of either amphetamine, caffeine or scopolamine produces a stimulation of locomotor activity (Thornburg and Moore, 1973; Joyce and Koob, 1981). However, the mechanisms by which these drugs produce this effect appear to be different. Thus the effects induced by amphetamine are believed to be mediated indirectly through the enhancement of dopaminergic neurotransmission, while those produced by caffeine and scopolamine are related to the blockade of adenosine and cholinergic muscarinic receptors respectively (Thornburg and Moore, 1973, Snyder et.al. 1981). Intact dopaminergic neurotransmission in the nucleus accumbens appears to be critical for the locomotor stimulation produced by amphetamine because the microinjection of dopaminergic antagonists into the nucleus

29 30 accumbens or lesions of dopaminergic nerve terminals in this region with 6-hydroxydopamine inhibits the amphetamine- induced response (Pijnenberg et.al., 1975; Kelly and Iversen, 1976; Roberts and Fibiger, 1975). In contrast, whereas intraventricular administration of 6-hydroxydopamine has been shown to inhibit the hypermotility produced by caffeine (Erinoff and Snodgrass, 1986), specific lesions of dopaminergic neurons in the nucleus accumbens do not alter the locomotor stimulant responses to either caffeine or scopolamine (Joyce and Koob, 1981). These observations suggest that the hypermotility produced by amphetamine but not caffeine or scopolamine is mediated by enhanced dopaminergic neurotransmission in the nucleus accumbens.

The nucleus accumbens sends a major GABAergic efferent projection to the ventral pallidum (Jones and Mogenson, 1980; Mogenson et.al., 1983; Mogenson and Nielsen, 1983; Nauta et.al., 1978; Zaborsky et.al., 1986). Lesions of the ventral pallidum or bilateral injections of GABA or GABA agonists into this site attenuate the locomotor stimulant actions of compounds that enhance dopaminergic neurotransmission in the nucleus accumbens (Swerdlow and Koob, 1985; Mogenson and Nielson, 1983; Austin and Kalivas, 1988; Shreve and Uretsky, 1988a). These observations support the concept that the inhibition of the GABAergic projection from the nucleus accumbens to the ventral pallidum plays an 31 essential role in the stimulant response to dopaminergic agonists like amphetamine.

Both the nucleus accumbens and the ventral pallidum receive glutamatergic projections from either the neocortex and/or limbic regions (Fuller et.al., 1987; Walaas and Fonnum, 1979; Walaas, 1981; McGeer et.al., 1977) and contain high affinity binding sites for glutamate and excitatory amino acid agonists (Monoghan et.al., 1989). In addition the bilateral administration of excitatory amino acid agonists into either the nucleus accumbens or the ventral pallidum produces a marked stimulation of locomotor activity, which can be inhibited by specific excitatory amino acid antagonists (Donzanti and Uretsky, 1984; Hamilton et.al.,1986; Shreve and Uretsky, 1988b; Shreve and Uretsky, 1989) . Compounds such as a-amino-3-hydroxy-5- methylisoxazole-4-propionic acid (AMPA) and quisqualic acid which activate the receptor now identified as the AMPA receptor, elicit the most consistent and robust hypermotility responses. This suggests that excitatory amino acid mechanisms in the nucleus accumbens and the ventral pallidum may play a role in the regulation of locomotion.

Recently, it has been reported that glutamic acid diethyl ester, (GDEE) , administered systemically or directly into the nucleus accumbens inhibited the locomotor stimulant response to amphetamine (Pulvirente and Koob, 1989; Freed and Cannon-Spoor, 1990). Because GDEE in electrophysiological studies has been classified as a non- NMDA glutamate receptor antagonist (Collingridge and Lester, 1989), these observations suggest that non-NMDA receptors may be involved in the hypermotility response to amphetamine. However the effectiveness and specificity of GDEE as an antagonist for excitatory amino acid receptors has been questioned (Collingridge and Lester, 1989; Hosli, et.al., 1983).

6,7-Dinitroquinoxaline-2,3-dione (DNQX) and y-D- glutamylaminomethyl-sulphonate (GAMS) have been shown to selectively inhibit the electrophysiological effects of AMPA and kainic acid and are therefore classified as AMPA/kainate receptor antagonists (Honore et.al., 1988, Davies and Watkins, 1982; Jones and Watkins, 1984; Fagg, 1985) . In behavioral studies, GAMS, when injected into the nucleus accumbens or ventral pallidum, selectively inhibited the hypermotility response to coinjected AMPA, while DNQX injected into these sites inhibited the responses to AMPA and kainic acid (Shreve and Uretsky, 1988b, Shreve and Uretsky, 1989). The purpose of the present study was to determine the role of AMPA/kainate receptors in the nucleus accumbens and in the ventral pallidum in the locomotor stimulation produced by amphetamine, caffeine and 33 scopolamine. We have therefore investigated the effects of the AMPA/kainate antagonists, DNQX and GAMS, injected bilaterally into either the nucleus accumbens or the ventral pallidum on the behavioral responses to these locomotor stimulant drugs.

METHODS

ADMINISTRATION OF DRUGS INTO THE BRAIN.

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) , weighing 150 to 2 00 grams, were housed 4 animals per cage in a temperature controlled environment (23 ± 1 °C) vivarium with a 12 hour on-off lighting cycle. For direct injection into the brain, the rats were lightly anesthetized with a halothane/oxygen mixture and placed in a stereotaxic frame (David Kopf Instruments, Tajunga, CA). A midline incision was made in the scalp and holes were drilled bilaterally into the skull at the following coordinates: 6.8 mm anterior to the intraaural line and 1.8 mm lateral to the sagittal suture for ventral pallidal injection and 9.4 mm anterior to the intraaural line and 1.2 mm lateral to the sagittal suture for nucleus accumbens injection (Konig & Klippel, 1968) . A 10 |il Hamilton syringe (Hamilton Co., Reno, NE) was then inserted into the holes 34 and lowered to a depth of 7.2 mm (ventral pallidum) or 6.4 mm (nucleus accumbens) as measured from the surface of the skull. Drug solutions or vehicle were infused bilaterally in a volume of 0.5 |il/side at a rate of 0.5 (il/min. The needle was allowed to remain in position for an additional minute to allow for diffusion of the solution away from the needle tip. After removal of the needle, the incision was closed with wound clips and swabbed with 2% (w/v) lidocaine ointment.

MEASUREMENT OF LOCOMOTOR ACTIVITY

After injections into the ventral pallidum, anesthesia was discontinued and the animals were removed from the stereotaxic frame. Following recovery from the anesthetic (5 minutes), animals were placed into motor activity cages (Opto-Varimex Minor, Columbus Instruments, Columbus, OH) and motor activity was monitored. The motor activity cages consisted of a 12 x 12 grid of infrared beams 3.5 cm apart and 5.0 cm from the bottom of the cage in a ventilated plexiglass box measuring 42 cm square and 20 cm high. Ambulatory activity was measured as the number of times 2 consecutive beams were interrupted, and the data were recorded with a digital computer (Columbus Instruments). Experiments in which locomotor activity was monitored were preformed between the hours of 8:00 a.m. and 4:00 p.m. in an 35 isolated environmental room maintained at a temperature of 23°C.

HISTOLOGY

Following each experiment the animals were removed to a chamber containing halothane and anesthetized. Under anesthesia the animals were decapitated and the brains were removed to a 4% formaldehyde solution where they were allowed to fix for 24 hours. Frozen sections, 40 |l in thickness, were cut using a Cryo-Cut microtome (American Optical Corp., Buffalo, NY) to verify the position of the injection cannula. Figure 1 shows representative injection sites for injections into the nucleus accumbens and the ventral pallidum. Data points from animals in which needle tracks were found to terminate outside of the nucleus accumbens or the ventral pallidum were excluded from the study.

DRUGS

d-Amphetamine sulfate and (-)-scopolamine HC1 were purchased from Sigma Chemical Co. (St. Louis, MO.). y-D- Glutamylaminomethyl-sulphonate (GAMS) and 6,7- dinitroquinoxaline-2,3-dione (DNQX) were obtained from Tocris Neuramin (Essex, England). Caffeine was purchased from MCB (Matheson, Coleman and Bell, Norwood, OH.), Halothane U.S.P. from (Halocarbon Laboratories, N. Augusta, S.C.) and 2% Lidocaine ointment was obtained from (Astra Pharmaceuticals, Westborough, MA. ) . 6-amino-7- fluoroquinoxaline-2, 3-dione (AFQX) was provided by Dr. Duane D. Miller (The Ohio State University, College of Pharmacy, Div. of Medicinal Chemistry). DNQX and AFQX were dissolved initially in 0.1 N NaOH and adjusted to the appropriate volume with phosphate buffer 0.5 M (pH 7.4) . All other drugs were dissolved in saline or water and adjusted to pH 7.4 with 1 N NaOH. Doses shown for DNQX, GAMS and AFQX refer to the amount injected on each side into the target brain structure (ventral pallidum or nucleus accumbens). Control animals received injections of equal volumes (0.5 |i.l) of saline or vehicle. d-Amphetamine, caffeine and scopolamine were administered subcutaneously in the doses selected that produced similar degrees of locomotor activity.

STATISTICS

Data were expressed as the mean and standard error of the mean (SEM) . Significant differences were evaluated using the Dunnett's Test, with a level of P < 0.05 being considered significant. 37 RESULTS

EFFECT OF THE BILATERAL MICROINJECTION OF DNQX OR GAMS INTO THE NUCLEUS ACCUMBENS ON STIMULANT INDUCED HYPERMOTILITY

Rats were injected bilaterally into the nucleus accumbens with either DNQX, (1 |ig/0.5 Jil), GAMS, (5 ng/0.5 |ll), or vehicle and then injected with amphetamine (0.5 mg/kg, s.c.). These doses of DNQX and GAMS were chosen because they were shown previously to antagonize the locomotor stimulant effects of AMPA. Locomotor activity was recorded for 1 hour. DNQX and GAMS inhibited amphetamine- stimulated locomotion by 55% and 67% respectively (Figs. 2 and 3) . Neither DNQX nor GAMS, when injected into the nucleus accumbens, significantly inhibited the locomotor activity of animals that were not injected with amphetamine (Table 1).

Systemic administration of either caffeine (20 gm/kg, s.c.) or scopolamine (0.5 mg/kg, s.c.), produced approximately the same intensity of locomotor stimulation as 0.5 mg/kg amphetamine during a 1 hour test period. In contrast to the inhibitory effects of DNQX (1 |ig) and GAMS (5 |ig) on the locomotor stimulation produced by amphetamine, neither excitatory amino acid antagonist when injected into the nucleus accumbens significantly inhibited the locomotor 38 stimulation produced by caffeine or scopolamine (Figs. 2 and 3). In fact, the locomotor response to scopolamine was significantly enhanced in the DNQX-injected animals (Fig.

2 ) . Figure 1. Representative injection sites for the intracranial administration of drugs into (A) the ventral pallidum and (B) the nucleus accumbens. The sites represent the tips of the needle tracks. Closed circles refer to sites which were accepted as being within the bounds of the target structure. Open squares represent injections in which the injection cannulae terminated outside of the target regions. Locomotor activity data from animals in which either target structure was missed were excluded from the study. Adapted from KOnig and Klippel (1963) . 40

Table 1. Effect of the administration of DNQX or GAMS into either the nucleus accumbens or the ventral pallidum on locomotor activity.

Nucleus Ventral Treatment Accumbens Pallidum

Vehicle 632 ± 155 (4) 766 ± 216 (4) DNQX 913 ± 186 (4) 693 ± 177 (4) Vehicle 566 ± 209 (4) 894 ± 64 (4) DNQX 517 ± 177 (4) 662 ± 130 (6)

DNQX (1 |ig) , GAMS (5 |lg) or the appropriate vehicle was injected intracranially in a volume of 0.5 |xl. Values represent the mean locomotor activity accumulated over a 1 hr. period ± S.E.M. for the number of observations in parenthesis. None of the values were significantly different from the respective vehicle-treated controls. 41

X 5000 HU Vehicle D ESS DNQX O X \ CO 4000 (6) I— (4) Z Z) O o 3000

> I— 2000 o < tr o h- 1000 o 2 o o o 0- Amphetamine Caffeine Scopolamine

Figure 2. Effect of injection of DNQX (lfig/Q.5|il) bilaterally into the nucleus accumbens immediately before the s.c. administration of amphetamine, caffeine and scopolamine locomotor activity. Systemic drug solutions were injected at lml/kg in doses of 0.5, 20, and 0.5 mg/kg, respectively. Vehicle, in a volume of 0.5 jil, was injected bilaterally into the nucleus accumbens of control animals. The animals were placed in motor activity cages and locomotor activity was recorded for 1 hour. Each bar represents the mean ± S.E.M. for the number of determinations in parenthesis. ”P < 0.01 with respect to vehicle controls. 42

Od 5000 I I Vehicle ZD O r a GAMS X \ in 4 0 0 0 - (10) i— z> 8 3000 (5) (7) £ (3) > (5) i— 2000 o < o 1000

o o o Amphetamine Caffeine Scopolamine

Figure 3. Effect of injection of GAMS (5(ig/0. 5pl) bilaterally into the nucleus accumbens immediately before the s.c. administration of amphetamine, caffeine 1 and scopolamine on locomotor activity. Systemic drug solutions were injected at 1 ml/kg in doses of 0.5, 20 and 0.5 mg/kg, respectively. Vehicle, in a volume of 0.5 pi., was injected bilaterally into the nucleus accumbens of each control animal. The animals were placed in motor activity cages and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of determinations in parenthesis. ”P < 0.01 with respect to vehicle controls. 43 THE EFFECT OF BILATERAL MICROINJECTION OF DNQX OR GAMS INTO THE VENTRAL PALLIDAL REGION ON STIMULANT INDUCED HYPER­ MOTILITY

Rats were injected bilaterally into the ventral pallidum with either vehicle or various doses of DNQX, or GAMS and then were injected with amphetamine (0.5 mg/kg, s.c.), caffeine (20 mg/kg, s.c.), or scopolamine (0.5 mg/kg, s.c.). Locomotor activity was recorded for 1 hour. The stimulation of locomotion produced by amphetamine was inhibited by 78% when the dose of DNQX injected into the ventral pallidum was 1 Jig (Fig. 4). This dose of DNQX did not significantly alter the activity of animals that were not injected with amphetamine (Table 1). Lower doses of DNQX did not produce a significant inhibition of amphetamine-induced locomotor stimulation (Fig. 4) .

As was shown for amphetamine, DNQX, at a dose of 1 Jig, also inhibited the stimulation of locomotor activity induced by either caffeine (79% decrease) or scopolamine (49% decrease) (Fig. 4) . Lower doses of DNQX did not produce a significant change in the locomotor stimulation produced by these drugs.

The administration of GAMS 5 jig bilaterally into the ventral pallidum significantly inhibited the stimulation of 44 locomotor activity produced by either amphetamine (73% dec.), caffeine (70% dec.), or scopolamine (74% dec.) (Fig. 5). However, this dose of GAMS did not significantly change the locomotor activity of animals that were not injected with amphetamine (Table 1).

EFFECT OF AFQX INJECTED INTO THE NUCLEUS ACCUMBENS OR VENTRAL PALLIDUM ON THE STIMULATION OF LOCOMOTION PRODUCED BY AMPHETAMINE

In order to evaluate the specificity of the effects of DNQX, the effects of AFQX, a chemical analogue of DNQX prepared in our laboratory, were determined. Previous studies have shown that while DNQX inhibited the high affinity binding of [3H] -AMPA to brain membranes AFQX at concentrations as high as 10~4 M did not produce even a 50% inhibition of [3H]-AMPA binding. The bilateral administration of AFQX into either the nucleus accumbens or the ventral pallidum in a dose of l(J,g/0.5(0.1/side did not significantly inhibit the locomotor stimulation produced by amphetamine (Fig. 6). 45

Cd □ d-AMPHEJAMINE 0.5 mg/kg Z) 4 0 0 0 -p ES CAFFEINE 20 mg/kg o X ESI SCOPOLAMINE 0.5 mg/kg \cn (5) x (6) Z> 3 0 0 0 -• (16) (11) o o

= 2000 -* 0) o < Cd o I— 1000 -- (a) o 2 o o o DNQX: 0.0 0.1 0-3 1.0 0.0 0.1 O J 1.0 0.0 0.1 0 J 1.0

Figure 4. Effect of injection of DNQX bilaterally into the ventral pallidum immediately prior to the s.c. administration of amphetamine, caffeine and scopolamine on locomotor activity. Systemic drug solutions were injected at 1 ml/kg in doses of 0.5, 20 and 0.5 mg/kg, respectively. Vehicle, in a volume of 0.5 |il., was injected bilaterally into the ventral pallidum of each control animal. The animals were placed in motor activity cages and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of determinations in parenthesis. ’P < 0.05, *’P < 0.01 with respect to vehicle controls. 46

(ZD GAMS 0.0 fig . 5000 ESI GAMS 2.5 fig . ESI GAMS 5.0 fig . z (10) r> 4 0 0 0 - o o £ 3 0 0 0 - > i— (10) o < 2000 - QL I-O o •* s ** o 1000 - o —Io

d—AMPHETAMINE CAFFEINE SCOPOLAMINE

Figure 5. Effect of injection of GAMS bilaterally into the ventral pallidum immediately prior to the s.c. administration of amphetamine, caffeine and scopolamine on locomotor activity. Systemic drug solutions were injected at 1 ml/kg in doses of 0.5, 2 0 and 0.5 mg/kg, respectively. Vehicle, in a volume of 0.5 111., was injected bilaterally into the ventral pallidum of each control animal. The animals were placed in motor activity cages and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of determinations in parenthesis. 'P < 0.05, **P < 0.01 with respect to vehicle controls. 47

a: 4000 t □ d-AMPHETAMINE 0.5 mg/kg Z> o KS d-AMPHETAMINE + AFQX 1/ig X \ in t- z 3 0 0 0 - (4) Z> o o (6)

£ 2000 - o < C£ 0 1--- o 1000- o o o

0 NUCLEUS VENTRAL ACCUMBENS PALLIDUM

Figure 6. Effects of AFQX on locomotor activity stimulated by the s.c. administration of d-amphetamine (0.5 mg/kg). AFQX (1 |ig) was injected in a 0.5 |ll volume into the nucleus accumbens or the ventral pallidum immediately before the s.c. administration of d-amphetamine. Vehicle/ in a volume of 0.5 Hi., was injected bilaterally into the nucleus accumbens or the ventral pallidum of each control animal. The animals were placed in motor activity cages and locomotor activity was recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of determinations in parenthesis. 48 DISCUSSION

The purpose of this study was to determine whether the AMPA/kainate excitatory amino acid receptors in the nucleus accumbens and the ventral pallidum play a role in the motor activating effects of the three stimulant drugs, caffeine, scopolamine and amphetamine. The present study shows that DNQX and GAMS, two AMPA/kainate excitatory amino acid receptor antagonists that were administered directly into the nucleus accumbens and the ventral pallidum, can attenuate drug-induced stimulation of locomotor activity but that the selectivity of the effects varies between the different regions. Thus, GAMS and DNQX, when injected into the nucleus accumbens in doses that have been shown previously to inhibit the locomotor stimulation produced by the excitatory amino acid AMPA (Shreve and Uretsky, 1988b; Boldry et.al., 1988) antagonized the locomotor stimulation produced by amphetamine but not caffeine or scopolamine (Figs. 2 and 3). The administration of these two antagonists into the ventral pallidum inhibited the stimulation of locomotion produced by either amphetamine, caffeine or scopolamine (Figs. 4 and 5). These results suggest that the activation of AMPA/kainate excitatory amino acid receptors in the nucleus accumbens is important in the stimulation of locomotion produced by amphetamine, while activation of these receptors in the ventral pallidum is involved in the hypermotility response to all three drugs. 49 In contrast to their effects on drug-stimulated locomotion, the administration of GAMS and DNQX into either the nucleus accumbens or the ventral pallidum did not significantly inhibit the locomotor activity of control animals that did not receive the locomotor-stimulant drugs (Table 1) . This observation suggests that activation of AMPA/kainate receptors in the nucleus accumbens or the ventral pallidum is not required for the regulation of normal unstimulated locomotor activity.

The neural pathways mediating the locomotor stimulant effects of amphetamine are believed to be different from the pathways that mediate the stimulant effects of caffeine and scopolamine (Swerdlow et.al., 1986). This conclusion is supported by the observations that 1.) 6-hydroxydopamine- induced lesions of the nucleus accumbens and the administration of dopamine receptor blocking agents inhibit the locomotor stimulation produced by amphetamine but not caffeine or scopolamine (Joyce and Koob, 1981) and 2.) the administration of GDEE systemically or directly into the nucleus accumbens inhibited the hypermotility response to amphetamine (Pulvirenti et.al., 1989; Freed and Cannon-Spoor, 1990), but not to caffeine (Pulvirenti et.al., 1989). The present studies, showing that the inhibitory effects of GAMS and DNQX administered into the nucleus accumbens, antagonized selectively amphetamine-stimulated locomotion provide further support for this hypothesis. Because amphetamine, scopolamine and caffeine at the doses administered produced a similar intensity of locomotor stimulation, the selective inhibitory effects of the excitatory amino acid antagonists on the locomotor-stimulation induced by amphetamine cannot be explained by a rate dependency effect of the inhibition. These results are consistent with the concept that locomotor stimulation induced by an increase in dopaminergic neurotransmission in thenucleus accumbens may require activation of AMPA/kainate receptors by endogenous excitatory amino acids, presumably glutamic acid.

In contrast to their effects in the nucleus accumbens, the injection of DNQX and GAMS into the ventral pallidum inhibited the locomotor stimulation produced by caffeine and scopolamine as well as amphetamine (Figs. 4 and 5). Inasmuch as the inhibition of the locomotor stimulation induced by all three compounds is compatible with a nonselective action of the antagonists at this site, this possibility was investigated further by determining the effects of the administration of the compound, AFQX, into the ventral pallidum on the locomotor stimulation induced by amphetamine. AFQX is a chemical analog of DNQX that does not inhibit the high affinity binding of [3H]-AMPA to its receptor in brain membranes (Supko et.al., 1990). The administration of AFQX into the ventral pallidum at a dose comparable to the 51 effective inhibitory dose of DNQX had virtually no effect on the amphetamine-induced locomotor stimulation (Fig. 6) . Thus, the relative effectiveness of DNQX and AFQX in inhibiting stimulant induced locomotion correlates with their respective ability to inhibit [3H]-AMPA binding, suggesting that binding at the receptor is the critical step in inhibiting drug stimulated locomotion. In addition, the observation that the ventral pallidal administration of DNQX and GAMS did not depress the locomotor activity of control animals that did not receive stimulant drugs also argues against a nonspecific depressant action of the antagonists. These results suggest that activation of AMPA/kainate receptors in the ventral pallidum may mediate the locomotor stimulation produced by all three stimulant drugs tested. This suggests that the neural pathways involved in the hypermotility response to all three stimulants may converge at a common site in the ventral pallidum, in which AMPA/kainate receptors are activated. Alternatively, the neural pathways mediating the hypermotility responses to the three stimulant drugs may pass through the ventral pallidum but remain separate. In this case injected antagonists would diffuse throughout the ventral pallidum and inhibit different AMPA/kainate receptors associated with each independent pathway. The injection sites in the ventral pallidum were close to the medial forebrain bundle, which contains the ascending dopaminergic neurons that project to various areas of the forebrain such as the cerebral cortex and 52 the striatum as well as the nucleus accumbens. Consequently, the possibility cannot be excluded that DNQX and GAMS interact with dopaminergic projections of the medial forebrain bundle to nonselectively inhibit the locomotor stimulation produced by the different stimulant drugs.

Whereas DNQX has been shown to inhibit the high affinity binding of radiolabeled AMPA and kainic acid, it has also been shown to inhibit the binding of glycine to a site associated with the NMDA receptor and to antagonize NMDA-induced responses (Birch et.al., 1988; Lester et.al., 1989). It is therefore possible that the inhibitory effects of DNQX observed on drug-stimulated locomotion are mediated by the inhibition of glycine receptors, which would lead to the inhibition of NMDA receptor function. However, DNQX, when injected into the nucleus accumbens or ventral pallidum in doses that inhibited the hypermotility produced by AMPA and kainic acid, did not inhibit the response to NMDA (Shreve and Uretsky, 1989; Boldry et.al., 1988). It is, therefore, unlikely that the inhibitory effects of DNQX on drug-induced locomotor stimulation are related to its ability to functionally antagonize the responses to NMDA.

The inhibitory effect of GAMS on drug-stimulated locomotion also argues for the importance of non-NMDA receptors in the actions of the locomotor stimulant drugs. It 53 has been shown previously that GAMS can selectively inhibit the electrophysiological responses of single neurons to kainic acid and quisqualic acid (Jones et.al., 1984; Davies and Watkins, 1985). In addition, the coadministration of GAMS with excitatory amino acid agonists into either the nucleus accumbens or the ventral pallidum inhibited the locomotor stimulation produced by AMPA but not that produced by either kainic acid or NMDA (Shreve and Uretsky, 1988b; 1989) . This latter observation suggests that the inhibition of drug- induced locomotor stimulation by GAMS may reside in its ability to functionally inhibit the AMPA receptor.

In conclusion, the results of this study indicate that AMPA/kainate excitatory amino acid antagonists can inhibit the locomotor stimulant responses to amphetamine, caffeine and scopolamine. When the antagonists were injected into the nucleus accumbens, they selectively inhibited the stimulant effects of amphetamine, and when they were administered into the ventral pallidum, they inhibited the stimulant responses to all three drugs. However, the antagonists had no effect on locomotor activity that was not drug-induced. These results suggest that the locomotor stimulation produced by amphetamine involves the activation of AMPA/kainate excitatory amino acid receptors in both the nucleus accumbens and the ventral pallidum, while the locomotor stimulation produced by scopolamine and caffeine involves the activation AMPA/kainate receptors only in the ventral pallidum. CHAPTER III

THE ROLE OF DOPAMINE AND AMPA/KAINATE RECEPTORS IN THE NUCLEUS ACCUMBENS IN THE HYPERMOTILITY RESPONSE TO MK801.

INTRODUCTION

MK801 ((+)-5-methyl-10,ll-dihydro-5H-dibenzo[a,d]cyclo- hepten-5,10-imine maleate) is a noncompetitive antagonist of the NMDA receptor that binds to the cation channel associated with the NMDA-receptor complex (Wong et.al., 1986). Recently there has been a great deal of interest in MK-801 as a potential anticonvulsant and neuroprotective agent (Clineschmidt et.al., 1982; Gill et.al., 1987; Sonsalla et.al., 1988). The administration of MK801 to rats produces a characteristic behavioral pattern which includes hyperloc­ omotion at lower doses and lateral head weaving and ataxia at higher doses (Benvenga and Spaulding, 1988; Clineschmidt et.al., 1982a; 1982b; 1982c; Tricklebank et.al., 1989). It has been suggested that some of these behavioral effects may be mediated by an enhancement in central dopaminergic transmission. Thus, the dopamine receptor antagonist,

55 56 haloperidol, has been shown to inhibit the hyperlocomotion induced by MK801 (Clineschmidt et.al., 1982b). Additionally, the motor activating effects of dopamine receptor agonists, such as L-DOPA and apomorphine, are potentiated by MK801 (Carlsson and Carlsson, 1990; Klockgether and Turski, 1976). These behavioral observations are consistent with the finding that MK801 produces an increase in dopamine turnover in various brain regions (Hiramatsu et.al., 1989; Loscher et.al., 1991; Rao et.al., 1990) and an increase in the firing rate of dopaminergic neurons originating in the ventral tegmental area (French and Ceci, 1990). It, therefore, appears that the behavioral activation elicited by MK801 may be mediated, in part, by dopaminergic mechanisms in the brain.

The results of other studies do not support this hypothesis and suggest that the behavioral effects produced by MK801 are not mediated by endogenous dopamine. Thus, MK801 stimulates locomotion in mice depleted of dopamine by pretreatment with a combination of reserpine and a-methyl-p- tyrosine (Carlsson and Carlsson, 1989; Carlsson and Svensson, 1990) . In addition, the direct administration of MK801 into the nucleus accumbens stimulates an intense locomotor response, which is not antagonized by haloperidol (Raffa et.al., 1989). These studies suggest that the behavioral activation produced by MK801 is mediated by a mechanism which, at least in part, is independent of brain dopamine. 57 In the present study we report additional experiments aimed at resolving the divergent views regarding the role of dopamine in the hypermotility response to MK801. We first reexamined the importance of endogenous dopamine in mediating the hypermotility response elicited by a dose of MK801 that does not produce ataxia by determining its effects in animals pretreated with reserpine, a-methyl-p-tyrosine, and dopamine receptor antagonists. Our results support the hypothesis that the locomotor stimulation produced by MK801 is mediated by endogenous dopamine. Previous studies have shown that dopamine receptors in the nucleus accumbens are critical for the behavioral responses to drugs, such as amphetamine and cocaine, whose effects are mediated by endogenous dopamine. Since the stimulation of activity induced by MK801 appears to be mediated by endogenous dopamine, we extended these studies by determining the role of D1 and D2 dopamine receptors in the nucleus accumbens in this response. In addition, activation of AMPA/kainate receptors as well as dopamine receptors in the nucleus accumbens may be required for the expression of the hypermotility elicited by dopaminergic agonists (Willins et.al., 1992) . Therefore, we also determined the involvement of these receptors in the nucleus accumbens in the hyper­ motility response to MK801. 58 METHODS

ADMINISTRATION OF DRUGS INTO THE BRAIN

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN), weighing 200 to 300 grains, were housed 4 animals per cage in a temperature controlled environment (23 ± 1°C) with a 12 hour on-off lighting cycle. For direct injection into the brain, the rats were lightly anesthetized with a halothane/oxygen mixture and placed in a stereotaxic frame (David Kopf Instruments, Tajunga, CA) . A midline incision was made in the scalp, and holes were drilled bilaterally into the skull at the following coordinates: 10.2 mm anterior to the intraaural line and 1.2 mm lateral to the sagittal suture. (Paxinos and Watson, 1982) . A 10 [ll Hamilton syringe (Hamilton Co., Reno, NE) was then inserted into the holes and lowered to a position 0.2 mm above the intraaural line. Drug solutions or vehicle were infused bilaterally in a volume of 0.5 (ll/side at a rate of 0.5 (4,1/min. The needle was allowed to remain in position for an additional minute to allow for diffusion of the solution away from the needle tip. After removal of the needle, the incision was closed with wound clips and swabbed with 2% (w/v) lidocaine ointment. 59 MEASUREMENT OF LOCOMOTOR ACTIVITY

Following direct injections into the nucleus accumbens, anesthesia was discontinued, and the animals were removed from the stereotaxic frame. Following recovery from the anesthetic (5 minutes), animals were placed into motor activity cages (Opto-Varimex Minor, Columbus Instruments, Columbus, OH), and motor activity was monitored. The motor activity cages consisted of a 12 x 12 grid of infrared beams 3.5 cm apart and 5.0 cm from the bottom of the cage in a ventilated plexiglass box measuring 42 cm square and 20 cm high. Ambulatory activity was measured as the number of times 2 consecutive beams were interrupted, and the data were recorded with a digital computer (Columbus Instruments). Experiments in which locomotor activity was monitored were performed between the hours of 8:00 a.m. and 4:00 p.m. in an isolated environmental room maintained at a temperature of 23 ± 1°C.

HISTOLOGY

Following each experiment the animals were removed to a chamber containing halothane and anesthetized. Under anesthesia the animals were decapitated, and the brains were removed to a solution of 10% formalin where they were allowed to fix for 24 hours. Frozen sections, 40 microns in thickness, were cut using a Cryo-Cut microtome (American 60 Optical Corp., Buffalo, NY) to verify the position of the injection cannula. Data points from animals in which needle tracks were found to terminate outside of the nucleus accumbens were excluded from the study.

DRUGS

d-Amphetamine sulfate, reserpine HC1 and a-methyl-p- tyrosine methyl ester were purchased from Sigma Chemical Co. (St. Louis, MO.). Y-D-Glutamylaminomethyl-sulphonate (GAMS) and 6, 7-dinitroquinoxaline-2, 3-dione (DNQX) were obtained from Tocris Neuramin (Essex, England). Eticlopride HCl and SCH23390 HCl were purchased from Research Biochemicals Inc. (Natick, MA.). Halothane U.S.P. was obtained from Halocarbon Laboratories (N. Augusta, S.C.), and 2% Lidocaine ointment was obtained from Astra Pharmaceuticals (Westborough, MA.). DNQX was dissolved initially in 0.1 N NaOH and adjusted to the appropriate volume with phosphate buffer 0.5 M (pH 7.4). Reserpine was initially dissolved in a minimum amount of glacial acetic acid and adjusted to the appropriate volume with water. All other drugs were dissolved in saline or water and adjusted to pH 7.4 with 1 N NaOH. Doses shown for DNQX and GAMS refer to the amount injected on each side into the target brain structure. Control animals received injections of equal volumes (0.5 jj.1) of saline or vehicle. 61 STATISTICS

Data were expressed as the mean and standard error of the mean (SEM). In experiments involving one control group and one treatment group, data were evaluated using Student's t- test, with a level of p < 0.05 being considered significant. In experiments where multiple groups were compared to the same control group significant differences were evaluated using the Dunnett's Test, with a level of p < 0.05 being considered significant.

RESULTS

THE EFFECTS OF MK801 ON LOCOMOTOR ACTIVITY IN NORMAL AND DOPAMINE DEPLETED RATS

The administration of MK801 (0.1 mg/kg, sc) produced a marked stimulation of locomotor activity (Fig. 7) . Locomotion produced by this dose of MK801 was observed to be coordinated and controlled. Animals retained the ability to proceed around obstacles placed in their path and did not run into the cage walls. Doses of MK801 higher than 0.1 mg/kg produced a lower intensity stimulation of locomotor activity and were associated with repetitive stereotyped behavior that interfered with locomotion (data not shown). Still higher 62 doses are known to produce ataxia and, therefore, were not used in the present study (Amalric et.al., 1991; Loscher and Honak, 1992).

In order to investigate the role of vesicular dopamine in the locomotor stimulation produced by MK801, rats were pretreated with reserpine (5 mg/kg, i.p.) administered 18 hours prior to MK801 to deplete dopamine stores. This pretreatment completely abolished the hypermotility response produced by MK801 (Fig. 7) . For comparison, the effect of reserpine pretreatment on amphetamine-induced locomotor activity was evaluated. Reserpine did not inhibit the locomotor stimulatory response to amphetamine (2 mg/kg, s.c.) (Fig. 7).

Reserpine pretreatment affects the storage and release of all monoamine transmitters. In order to further investigate the specific role of dopamine in MK801-induced locomotion, rats were pretreated with a-methyl-p-tyrosine (250 mg/kg, i.p.) four hours and one hour prior to systemic administration of MK801 (0.1 mg/kg, s.c.). a-Methyl-p-tyrosine pretreatment caused a 60 % reduction in MK801 stimulated locomotor activity (Fig. 8). 63

O3 j: 5000 I I Vehicle m □20 Reserpine 3c o 4000

<

^ 2000 --

s

0.1 mg/kg 2.0 mg/kg

MK-801 d-AMPHETAMINE

Figure 7. The effect of reserpine on the locomotor stimulation induced by MK801 or amphetamine. Rats were pre­ treated with reserpine (5 mg/kg, s.c.) 18 hours prior to the administration of MK801 (0.1 mg/kg, s.c.) or d-amphetamine (2 mg/kg, s.c.). Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0 . 01. 64

(6) 8000 3 JZO \ 7000 CZI Vehicle CO EZl a-MPT pretreatment § 6000

^ 5000 + * (6) | 4000 < g 3000 o 2 O 2000 o o 100 0

Figure 8. The effect of a-methyl-p-tyrosine on the locomotor stimulation induced by MK801. Rats were pre-treated with a- methyl-p-tyrosine (250 mg/kg, i.p.) 4 hours and 1 hour prior to the administration of MK801 (0.1 mg/kg, s.c.). Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0.01. 65 THE EFFECT OF DOPAMINE RECEPTOR ANTAGONISTS ON LOCOMOTOR ACTIVITY STIMULATED BY MK801

In order to investigate the role of dopamine receptor subtypes in the locomotor stimulation induced by MK801, rats were pretreated with selective dopamine receptor antagonists 20 minutes prior to MK801 (0.1 mg/kg, sc). The D1 dopamine receptor antagonist, SCH23390, was administered subcutaneously at a dose of 0.03 mg/kg, which is in a dose range (0.01-0.05 mg/kg) that has been previously demonstrated to inhibit functional dopaminergic effects (Amalric et.al., 1986). This dose had no significant affect on MK801-induced locomotion (Fig. 9) . However, pretreatment with the D2 receptor selective antagonist, eticlopride (0.03 mg/kg, SC), a dose which is one tenth the dose that has been previously reported to inhibit amphetamine-stimulated locomotor activity (Boldry et.al., 1991), inhibited locomotor activity stimulated by MK801 by approximately 70% (Fig. 9). The combination of the D1 and D2 antagonists (at the doses previously given) produced a 97% decrease in MK801-stimulated locomotor activity (Fig. 9) . Rats that received either SCH23390, eticlopride or the drugs in combination maintained the ability to respond to external stimulation, indicating that the dopamine receptor antagonists did not produce catalepsy at these doses. 66

C=3 Vehicle 7 0 0 0 - 3 ES SCH23390 O -C \ 6 0 0 0 - EZ2 Eticlopride m (7) ■ 1 SCH23390 -r Eticlopride u0 5 0 0 0 - 1 4000 rh O < 3 0 0 0 - (8)

* o 2000 - 2 O o (5) o 1000 - * 0 I

Figure 9. The effects of systemic administration of dopamine receptor antagonists on MK801 stimulated locomotor activity. MK801 was administered subcutaneously at a dose of 0.1 mg/kg. SCH23390 (0.03 mg/kg, sc) or Eticlopride (0.03 mg/kg,sc) or the combination of the two drugs was administered 20 minutes prior to MK801. Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0 .01. 67 To determine if the activation of dopamine receptors in the nucleus accumbens is required for the stimulation of locomotor activity produced by MK801, dopamine receptor antagonists were administered bilaterally into the nucleus accumbens of rats. This was followed immediately (within 2 minutes of the initial injection of antagonist) by the subcutaneous administration of either d-amphetamine (0.5 mg/kg, sc) or MK801 (0.1 mg/kg, sc). Locomotor activity was recorded for 1 hour. The direct administration of either SCH- 23390 or eticlopride into the nucleus accumbens produced a dose-dependent inhibition of activity stimulated by either d- amphetamine or MK801. The amphetamine-induced stimulation of locomotor activity was more sensitive to eticlopride pretreatment than was the response to MK801. While the intra- accumbens administration 0.03 Jig eticlopride inhibited the locomotor stimulation produced by d-amphetamine by 68%, it did not significantly change the locomotor response to MK801 (Fig. 10A and 10B) . However, when this dose of eticlopride was given concomitantly with 0.03 (ig SCH23390, a dose which also did not significantly inhibit the response to MK801, the combination produced an 87% decrease in the hypermotility response to MK801 (Fig. 10B). 68

A 3000 S' 3 = 2500 C H S C H 2 3 3 9 0 N *2 EZ3 ETICLOPRIDE z 3 2000 o | 1500- ai— < 1000 -

o 500-- 3

ANTAG. C0NC. (jig): 0.0 0.03 0.3 0.0 0.03 0.3

Figure 10. The effect of intraaccumbens administration of dopamine receptor antagonists on (A) d-amphetamine (0.5 mg/kg, s.c.) stimulated locomotor activity and (B) MK801 (0.1 mg/kg, s.c.) stimulated locomotor activity. Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0.01. 69

Figure 10 (continued)

B CD SCH23390 ZZ3 ETICLOPRIDE § 8000 E 3 SCH + ETIC I . 7000 t/i z D 6000 - O o 4-, 5000 1 £ 2 4000 a * cr 3000-- £ rh M o -- 2 2000 o O 1000 -- &

0.0 0.03 + 0.0 0.03 0.3 1.0 0.0 0.03 0J 1.0 ANTAG. C0NC. Cug): 0.03 70 THE EFFECT OF THE BILATERAL ADMINISTRATION AMPA/KAINATE RECEPTOR ANTAGONISTS INTO THE NUCLEUS ACCUMBENS ON THE HYPERMOTILITY PRODUCED BY SYSTEMIC MK801

In order to investigate whether AMPA/kainate glutamate receptors in the nucleus accumbens are involved in the locomotor response to systemic MK801, rats were injected bilaterally into the nucleus accumbens with either DNQX, (1 fig/0.5 (ll), GAMS, (5 (ig/0.5 jul) , or vehicle and then immediately injected subcutaneously with MK801 (0.1 mg/kg). These doses of DNQX and GAMS were chosen because they have been demonstrated to markedly inhibit the locomotor stimulation produced by the intra-accumbens administration of the excitatory amino acid, AMPA (Boldry et.al., 1991), or by the systemic administration of d-amphetamine and cocaine (Kaddis et.al., 1991; Willins et.al., 1992). Both DNQX and GAMS were found to inhibit MK801 stimulated locomotion by 68% and 56% respectively (Figs. 11 and 12). At these doses, neither AMPA/kainate antagonist reduced the amount of locomotor activity seen in rats treated with saline alone (1 ml/kg, sc) (Willins et.al., 1992). 71

3u JZO \ CZ] Vehicle V) ESZ DNQXc o £ > g 2000- CC I g 1000- o 3

Figure 11. The effect of intraaccumbens administration of DNQX (1 jig) on MK801 (0.1 mg/kg, s.c.) stimulated locomotor activity. Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0 .01. 72

M

3O 9 0 0 0 - -C CZJ Vehicle

| 7500' ESZI GAMS 5 /ig. O3 6 0 0 0 - £ > 4 5 0 0 - 1 d: 3 0 0 0 - 21 O o Q 1 5 0 0 -

Figure 12. The effect of intraaccumbens administration of GAMS (5 jig) on MK801 (0.1 mg/kg, s.c.) stimulated locomotor activity. Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0 .01. 73 DISCUSSION

The results of the present study suggest that the stimulation of locomotor activity produced by the non­ competitive NMDA receptor antagonist, MK801, is dependent upon central dopaminergic neurotransmission. Disruption of the dopaminergic system by the administration of drugs which interfere with the synthesis and storage of dopamine or block dopamine receptors markedly inhibited the hypermotility elicited by MK801. The dopamine receptors that mediate this response appear to be located in the nucleus accumbens, since the direct administration of dopamine receptor antagonists into this region inhibited the MK801-induced locomotor stimulation. Further evidence in support of the hypothesis that dopamine is involved in the locomotor response to MK801 comes from the finding that intra-accumbens administration of the AMPA/kainate receptor antagonists, DNQX and GAMS, also inhibited MK801 stimulated locomotion. Previous studies have demonstrated that drugs which depend on the activation of dopamine receptors in the nucleus accumbens to elicit a locomotor stimulatory response also require intact glutamate receptors within this region (Freed and Cannon-Spoor, 1990; Kaddis et.al., 1991; Willins et.al., 1992) . These observations demonstrate that the locomotor stimulation elicited by MK801 is dependent upon the activation of both AMPA/Kainate and dopamine receptors in the nucleus accumbens. 74 The results observed with dopamine-depleting drugs strongly suggest that vesicular dopamine is involved in the hypermotility elicited by MK801. Reserpine, which inhibits the storage and release of monoamines by impairing synaptic vesicle function, did not inhibit the locomotor stimulation produced by amphetamine (Fig. 7, see also Hiroi and White, 1990 and Stolk and Rech, 1967). This shows that animals that received reserpine were capable of responding to locomotor stimulation and is consistent with the hypothesis that the effects of amphetamine are produced by the release of dopamine from a cytoplasmic pool rather than from a vesicular storage pool in dopamine nerve terminals. Reserpine, however, almost completely inhibited the stimulation of locomotor activity produced by MK801, suggesting the involvement of stored monoamines in this effect. This hypothesis is consistent with the results of other studies showing that MK801 administered to rats with unilateral 6-hydroxydopamine lesions of the striatum, produces ipsilateral circling (Clineschmidt et.al., 1982b). This suggests that intact dopaminergic terminals are involved in the behavioral response to MK801.

Pretreatment of rats with the selective tyrosine hydroxylase inhibitor, a-methyl-p-tyrosine, also reduced the stimulation of locomotor activity elicited by MK801 (Fig. 8). This observation also supports a role for catecholamines in the MK801-induced locomotor response. While the locomotor 75 stimulation elicited by MK801 was reduced by a-methyl-p- tyrosine, it was not entirely eliminated. This may indicate that sufficient endogenous catecholamine stores remain available after a-methyl-p-tyrosine pretreatment to mediate some degree of locomotor stimulation.

Dopamine receptor antagonists were administered with MK801 to evaluate the role of dopaminergic receptors in the locomotor stimulant response to this drug. In agreement with a recent report by Amalric et.al. (1991), the systemic administration of a low dose of SCH23390 (0.03 mg/kg), the Dl receptor antagonist, did not significantly inhibit the locomotor stimulation induced by MK801. In contrast, the D2 dopamine receptor antagonist, eticlopride (0.03 mg/kg), produced a greater than 50% reduction in MK801-stimulated locomotor activity. While these data alone would seem to suggest that Dl dopamine receptors are not involved, co­ administration of SCH23390 and eticlopride had a greater inhibitory effect on MK801-induced locomotion than did eticlopride alone (Fig. 9). Thus it appears that while the availability of D2 dopamine receptors alone may be sufficient to maintain activity, Dl receptors are involved in this response and may serve to modulate the effects produced by the activation of D2 receptors. 76 The dopamine receptors that are necessary for the locomotor stimulation produced by MK801 appear to be located in the nucleus accumbens. The administration of either SCH23390 or eticlopride into the nucleus accumbens produced an inhibition of the locomotor response to MK801 as well as to amphetamine, which stimulates locomotor activity by enhancing dopamine neurotransmission in the nucleus accumbens (Kelly and Iversen, 1976; Pijnenberg et.al., 1975). The D2 antagonist was able to block amphetamine stimulated locomotion at doses which were ineffective in inhibiting the locomotor response to MR801 (Figs. 10A and 10B) . One possible explanation for this can be derived from the finding that reserpine completely inhibited the locomotor response to MK801. This suggests that the locomotor activity produced by MK801 is dependent upon the depolarization-induced release of dopamine. The blockade of presynaptic D2 receptors may, therefore, facilitate dopaminergic transmission by reducing the effects of autoinhibition in the accumbens. This would result in an increase in the synaptic concentration of dopamine in response to MK801 which in turn may counteract the effect of a competitive antagonist of postsynaptic D2 receptors. The combination of Dl and D2 antagonists, when administered into the nucleus accumbens at doses that individually had little effect on MK801-stimulated activity, produced a marked decrease (85%) in MK801-stimulated activity (Fig. 10B). These studies show that MK801 stimulated locomotor activity requires 77 the activation of both Dl and D2 dopamine receptors in the nucleus accumbens.

In contrast to the present findings, it has been reported that MK801 was able to stimulate locomotor activity in mice pretreated with a combination of reserpine and a-methyl-p- tyrosine (Carlsson and Carlsson, 1989). There are many differences between the present study and the one by Carlsson and Carlsson (1989). Firstly, the species used were different in the two studies. Secondly, the conditions under which locomotor activity was evaluated were different. While in the present study, each rat was tested in an individual activity monitoring cage, in the study by Carlsson and Carlsson (1989) the activity of three mice was evaluated in a single cage. Thus, it is possible that social interaction may have had a significant effect on locomotor activity. Thirdly, the type of locomotor stimulation produced by MK801 in animals pretreated with reserpine and a-methyl-p-tyrosine appears to be qualitatively different from that of the animals used in the present study. Mice that were given MK801 following reserpine and a-methyl-p-tyrosine were reported to exhibit locomotion in a forward direction only and to stop moving completely if presented with an obstacle, such as the cage wall (Carlsson and Engberg, 1992). In the present study, rats receiving MK801 ran around the perimeter of the cages, and turned to avoid running into the walls of the activity cages and 78 obstacles in their path. Finally, the dose of MK801 used in the studies of Carlsson and Carlsson (1989) was 10 to 40 times higher than the doses used in the present studies. Such high doses of MK801 may stimulate locomotor activity in mice by mechanisms that are independent of dopamine.

The finding that the stimulation of locomotor activity produced by the intra-accumbens administration of MK801 was not reversed by the co-administration of haloperidol (Raffa et.al., 1989) suggests that the effects produced by MK801 at this site is independent of dopaminergic neurotransmission. However, blockade of dopamine receptors in the nucleus accumbens inhibited the stimulation of locomotion produced by the systemic administration of MK801 (Fig. 10B). Therefore the site where MK801 is acting, following systemic administration, must be outside the nucleus accumbens. It has been shown that systemic administration of NMDA antagonists, PCP and MK801, markedly increase the firing rate of dopaminergic neurons in the ventral tegmental area, the site of the dopaminergic perikarya that project to the nucleus accumbens (French and Ceci, 1990). This is consistent with the finding that NMDA antagonists administered into the ventral tegmental area produce a hypermotility response (Dawbarn and Pycock, 1981). These observations suggest that the locomotor stimulation produced by the systemic administration of MK801 may be due to an action of this drug 79 in the ventral tegmental area, leading to an increased firing of dopaminergic neurons and an enhanced dopaminergic neurotransmission in the nucleus accumbens.

In addition to dopaminergic afferents from the ventral tegmental area, the nucleus accumbens receives afferent glutamatergic projections from the hippocampus, the amygdala and the medial prefrontal cortex. It has been suggested that these projections play a role in the regulation of locomotor activity (Mogenson et.al., 1980). In support of this concept is the observation that infusion of excitatory amino acid agonists into the nucleus accumbens produced a hypermotility response, which can be inhibited by specific excitatory amino acid antagonists (Donzanti and Uretsky, 1984; Hamilton et.al., 1986; Shreve and Uretsky, 1988). In addition antagonists of AMPA/kainate receptors infused into the nucleus accumbens inhibit the stimulation of locomotor activity produced by the indirectly acting dopaminergic agonists, amphetamine and cocaine, and directly acting dopaminergic agonists (Kaddis et.al., 1991; Willins et.al., 1992). The finding that the locomotor stimulation produced by MK801 is inhibited by antagonists of both AMPA/kainate receptors (Figs. 11 and 12) and dopaminergic receptors supports the hypothesis that the stimulation of locomotion produced by drugs that enhance dopaminergic neurotransmission in the nucleus accumbens 80 require the activation of both AMPA/kainate receptors and dopaminergic receptors at this site. CHAPTER IV

THE NON-COMPETITIVE NMDA RECEPTOR ANTAGONIST MK801 STIMU­ LATES LOCOMOTOR ACTIVITY FOLLOWING INJECTION INTO THE VEN­ TRAL TEGMENTAL AREA AND THE NUCLEUS ACCUMBENS IN RATS: DEPENDANCE ON CENTRAL DOPAMINERGIC AND GLUTAMATERGIC MECHANISMS

INTRODUCTION

The noncompetitive NMDA receptor antagonist, MK801 has been shown to stimulate a number of characteristic behavioral changes in rats. Following systemic administration of low doses of MK801 rats exhibit an increase in ambulatory locomotion. This locomotor stimulation appears to be depen­ dant upon central dopaminergic mechanisms (Clineschmidt et.al., 1982, Rao et.al., 1990, Loscher et.al., 1991; Loscher and Honack, 1992; Willins et.al., submitted). Electrophysiol- ogic studies, that have attempted to describe regional changes in neuronal function in response to MK801, have shown that neurons originating in a midbrain dopaminergic nucleus called the ventral tegmental area increase their firing rate in re-

81 82 sponse to intravenous MK801 (French and Ceci, 1990) . While the mechanism of the enhancement of dopamine neurotransmission is not known, this finding is significant because the ventral tegmental area is a primary source of dopaminergic innervation for several structures which are believed to be involved in locomotor function, including the striatum and the nucleus accumbens. The fact that MK801 activates neurons projecting from this region further strengthens the suggestion that locomotor stimulation in response to MK801 involves dopamine.

One of the primary projection sites of the ventral tegmental area is the nucleus accumbens. The release of dopamine and subsequent activation of dopamine receptors in the accumbens has been suggested to be a critical step in mediating the locomotor stimulant responses to many drugs (Swerdlow et.al., 1985; 1986). This hypothesis is supported by the finding that lesions of this region with the selective dopaminergic neurotoxin 6-hydroxydopamine as well as the direct administration of dopamine receptor antagonists inhibits the locomotor stimulation produced by psychostimulants (Joyce and Koob, 1981; Kelly and Iversen, 1976; Pijnenburg et.al., 1975) . The systemic administration of low doses of MK801 also stimulates locomotor activity. Evidence has been presented which suggests that dopaminergic neurotransmission in the nucleus accumbens is involved in the locomotor response to MK801. Thus pretreatment with reserpine, 83 or dopamine receptor antagonists (both systemically and injected directly into the accumbens) , have been shown to inhibit this response (see Chapter III). The direct adminis­ tration of MK801 into the nucleus accumbens also stimulates locomotor activity (Raffa et.al., 1989). While dopaminergic transmission in the nucleus accumbens appears to be critical for the stimulation of locomotor activity produced by systemically administered MK801, the locomotor activity produced by the direct administration of MK801 into the accumbens appears to be independent of accumbens dopamine. This is supported by the observation that haloperidol, when co-administered with MK801 into the nucleus accumbens, did not effect the locomotor activity produced by MK801 (Raffa et.al., 1989) .

Taken together, the results of these studies may indicate that the behavioral effects of MK801 are produced through actions of the drug at more than one site. Both the ventral tegmental area and the nucleus accumbens receive glutamatergic projections and contain glutamate binding sites (Walaas, 1981; McGeer et.al., 1977) at which MK801 could act to modulate locomotor activity. Each of these structures is believed to be involved in a circuit which may subserve locomotor activity (Mogenson et.al., 1980). It is possible therefore, that MK801 may act at either or both of these areas to produce locomotion. 84 In the present study MK801 injected directly into the ventral tegmental area produced a significant degree of locomotor activity. This activity was blocked by dopamine receptor antagonists and also by depletion of catecholamines with reserpine. MK801 injected directly into the nucleus accumbens also produced an increase in locomotor activity. This locomotor response was blocked by the D-l receptor antagonist, SCH23390 but not by the D-2 receptor antagonist, eticlopride. Reserpine pretreatment did significantly reduce the degree of locomotor activity produced by intra-accumbens MK801, however the degree of inhibition was not as great as that following intra-ventral tegmental MK801. Thus, it appears that intra-ventral tegmental administration of MK801 produces locomotor activity which is critically dependant upon dopaminergic transmission. The pharmacologic profile of intra- ventral tegmental MK801, with respect to dopaminergic transmission, exhibits many of the characteristics previously demonstrated for systemic MK801 (Willins et.al., submitted). This suggests that a primary site of action of systemically administered MK801 might be the ventral tegmental area. 85 METHODS

ADMINISTRATION OF DRUGS INTO THE BRAIN

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN), weighing 180 to 320 grams, were housed 4 animals per cage in a temperature controlled environment (23 ± 1°C) with a 12 hour on-off lighting cycle. For direct injection into the brain, the rats were lightly anesthetized with a halothane/oxygen mixture and placed in a stereotaxic frame (David Kopf Instruments, Tajunga, CA) . A midline incision was made in the scalp and holes were drilled bilaterally into the skull at the following coordinates: (nucleus accumbens): 10.2 mm anterior to the intraaural line and 1.2 mm lateral to the sagittal suture, (ventral tegmental area): 4.8 mm anterior to the intraaural line and 2.3 mm lateral to the sagittal suture (Paxinos and Watson, 1982) . A 10 |i.l Hamilton syringe (Hamilton Co., Reno, NE) was then inserted into the holes. For intra-accumbens injections the needle was then lowered to a position 0.2 mm above the intra­ aural line. For injections into the ventral tegmental area, the needle was positioned at the surface of the skull at a 10 degree angle and then lowered to a position 8.6 mm below the skull surface. Drug solutions or vehicle were infused bilaterally in a volume of 0.5 |il/side at a rate of 0.5 Hl/min. The needle was allowed to remain in position for an 86 additional minute to allow for diffusion of the solution away from the needle tip. After removal of the needle, the incision was closed with wound clips and swabbed with 2% (w/v) lidocaine ointment.

MEASUREMENT OF LOCOMOTOR ACTIVITY

Following direct injections into the brain, anesthesia was discontinued, and the animals were removed from the stereotaxic frame. Following recovery from the anesthetic (5 minutes) , animals were placed into motor activity cages (Opto- Varimex Minor, Columbus Instruments, Columbus, OH), and motor activity was monitored. The motor activity cages consisted of a 12 x 12 grid of infrared beams 3.5 cm apart and 5.0 cm from the bottom of the cage in a ventilated plexiglass box measuring 42 cm square and 20 cm high. Ambulatory activity was measured as the number of times 2 consecutive beams were interrupted, and the data were recorded with a digital computer (Columbus Instruments). Experiments in which locomotor activity was monitored were performed between the hours of 8:00 a.m. and 4:00 p.m. in an isolated environmental room maintained at a temperature of 23 ± 1°C. 87 HISTOLOGY

Following each experiment the animals were removed to a chamber containing halothane and anesthetized. Under anesthesia the animals were decapitated, and the brains were removed to a solution of 10% formalin where they were allowed to fix for 24 hours. Frozen sections, 40 microns in thickness, were cut using a Cryo-Cut microtome (American Optical Corp., Buffalo, NY) to verify the position of the injection cannula. Data points from animals in which needle tracks were found to terminate outside of the target regions were excluded from the study.

DRUGS

d-Amphetamine sulfate, reserpine HC1 and a-methyl-p- tyrosine methyl ester were purchased from Sigma Chemical Co. (St. Louis, MO.). Eticlopride HC1 and SCH23390 HC1 were pur­ chased from Research Biochemicals Inc. (Natick, MA.). Halothane U.S.P. was obtained from Halocarbon Laboratories (N. Augusta, S.C.), and 2% Lidocaine ointment was obtained from Astra Pharmaceuticals (Westborough, MA.). Reserpine was initially dissolved in a minimum amount of glacial acetic acid and adjusted to the appropriate volume with water. All other drugs were dissolved in saline or water and adjusted to pH 7.4 with 1 N NaOH. 88 STATISTICS

Data were expressed as the mean and standard error of the mean (SEM). In experiments involving one control group and one treatment group, data were evaluated using Student's t- test, with a level of p < 0.05 being considered significant. In experiments where multiple groups were compared to the same control group significant differences were evaluated using the Dunnett's Test, with a level of p < 0.05 being considered significant.

RESULTS

THE ROLE OF DOPAMINE IN THE LOCOMOTOR STIMULATION FOLLOWING THE ADMINISTRATION OF MK801 DIRECTLY INTO THE VENTRAL TEGMENTAL AREA

In order to determine the role of the ventral tegmental area in MK801 stimulated locomotor activity we first injected MK801 directly into this region in rats. The results of this study show that intra-tegmental administration of MK801 produced a large and significant increase in locomotor activity (Fig. 13) . To test the hypothesis that this increase in activity may involve the activation of dopamine receptors, rats were pretreated (20 minutes prior to injection of MK801) with either the dopamine Dl receptor antagonist, SCH23390 or the D2 receptor antagonist, eticlopride. SCH23390, at a dose of 0.1 mg/kg (sc) was able to reduce the response to MK801, relative to vehicle pretreated control rats, by more than 90% (Fig. 14) . Eticlopride (0.03 mg/kg, sc) also had an effect on locomotor activity stimulated by MK801, causing a 71% decrease in activity (Fig. 14). To determine whether vesicular dopamine is involved in the behavioral response to MK801, rats were pretreated with reserpine (5 mg/kg, sc) 18 hours prior to ventral tegmental administration of MK801. As shown in Figure 15, pretreatment of rats with reserpine caused a 96% decrease in locomotor activity with respect to control animals that were treated with MK801 but not reserpine. 90

* (4) 14- !=□ SALINE 12' ___j I MK801 > o 10 & xxxxxxx < 31_ o 8- e JC o (0 6- O° 35 O. O o 4-

2 - (4)

0-

Figure 13. The effect of direct bilateral injection of MK801 (10 |ig/0.5 (ig) into the ventral tegmental area. Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0.01. 91

(5)

14- CZ3 MK801 ES MK801 + SCH23390 12' e ? ESS MK801 + ETICLOPRIDE > ° lO- S X < L.* ^ 2 fi- ° 4 (6) -2§ > ,+j 6- O C O 3 3.8 4- (4)

2 -

0- *

Figure 14. The effects of pretreatment with the dopamine D1 receptor antagonist, SCH23390 (0.1 mg/kg, s.c.) or the D2 receptor antagonist, Eticlopride (0.03 mg/kg, s.c.) on locomotor activity stimulated by direct bilateral injection of MK801 (10 jig/ 0.5 jil) into the ventral tegmental area. Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0.01. 92

10 ' (4) I I Vehicle fc 7 Reserpine > o 8- o < 3 6 - £T o O JZ H- \ O CO 2 •4- > o c 4- o oZJ o o (4)

2 - *

0- -EvViSffia.

Figure 15. The effect of reserpine pretreatment (5 mg/kg, s.c.) 18 hours prior to the administration of MK801 (10 |ig) into the ventral tegmental area. Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0.01. 93 THE ROLE OF DOPAMINE IN THE LOCOMOTOR STIMULATION FOLLOWING THE ADMINISTRATION OF MK801 DIRECTLY INTO THE NUCLEUS ACCUMBENS

As shown previously by Raffa (1986), direct injection of MK801 into the nucleus accumbens of rats stimulates locomotor activity (Fig. 16). In order to evaluate the role of specific dopamine receptors in the hyperlocomotor response produced by the direct injection of MK801 into the nucleus accumbens, rats were pretreated systemically with SCH23390 (0.1 mg/kg, sc) or eticlopride (0.03 mg/kg, sc) 20 minutes prior to the intra- accumbens administration of MK801 (10 |ig). SCH23390 produced a 70% inhibition of locomotor activity relative to saline pretreated controls (Fig. 17). In contrast, the D2 receptor antagonist, eticlopride, had no inhibitory effect on MK801- induced locomotor activity (Fig. 17) . In fact, although it was not significant, the average locomotor activity over one hour for the eticlopride pretreated group was 33% higher than the control group. To determine the role of vesicular dopamine in the response to intra-accumbens MK801, rats were pretreated with reserpine (5 mg/kg, sc, 18 hours) prior to the injection of MK801 (10 (ig) into the nucleus accumbens. This pre­ treatment reduced the amount of activity produced by MK801 by 62% relative to non-reserpinized controls (Fig. 18). 94

12' (4) CZD SALINE

IOI 10' MK801 > 0 x 8- < i_T oz. n W V V i V VVA%W r r A V e | 6 - v Xt v vK v 1 5 #VlV*r4 O c3 4- o, o ° (4) 2 -

oJ

Figure 16. The effect of direct bilateral injection of MK801 (10 (ig/0.5 |lg) into the nucleus accumbens. Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0.01. 95

(5) □ MK801 12 E S MK801 + SCH23390 ■ ro 10 (6) > o MK801 O + ETICLOPRIDE < 8 cc. 3 o o -C 6- \ -4-

2 -

0-

Figure 17. The effects of pretreatment with the dopamine D1 receptor antagonist, SCH23390 (0.1 mg/kg, s.c.) or the D2 receptor antagonist, Eticlopride (0.03 mg/kg, s.c.) on locomotor activity stimulated by intra-accumbens MK801 (10 |ig) . Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0.01. 96

8 -- (4) d H Vehicle £ 01 o tWi Reserpine > r— 6 -- o X < iS cr n o o i— jr o \w ■ *c - > o n o o o u _l 2 -

Figure 18. The effect of reserpine pretreatment (5 mg/kg, s.c.) 18 hours prior to the administration of MK801 (10 jig) into the nucleus accumbens. Data are expressed as mean ± S.E.M. of counts registered in a one hour test period. Numbers in parenthesis indicate the number of animals used in each experiment. * p < 0.01. 97 DISCUSSION

The results of the present study show that the noncompetitive NMDA receptor antagonist, MK801, stimulates locomotor activity after direct injection into the ventral tegmental area. This stimulation appears to be dependant on dopamine in a manner which resembles that previously demonstrated for systemically administered MK801 (Chapter III) . Pretreatment of rats with reserpine, which depletes vesicular stores of dopamine, completely inhibited the locomotor responses to intra-ventral tegmental MK801. Additionally, this response was inhibited by pretreatment of rats with either SCH23390, a D1 receptor antagonist, or eticlopride, a D2 receptor antagonist. This evidence strongly suggests that dopamine is involved in the locomotor response to intra-tegmental MK801.

Dopaminergic neurons from the ventral tegmental area project to a number of sites within the central nervous system, including the nucleus accumbens (Dahlstrom and Fuxe, 1964; Ungerstedt, 1971; Hockfelt et.al., 1974). MK801 injected directly into the nucleus accumbens also stimulated an increase in locomotor activity. While this stimulation of locomotor activity was reduced by reserpine pretreatment, it was not completely inhibited as was the case for the locomotor response to intra-ventral tegmental MK801. This may indicate 98 that the locomotor response to intra-accumbens MK801 is only partially dependant on vesicular dopamine stores. The pretreatment of rats with dopamine receptor antagonists also demonstrated a partial response. Pretreatment of rats with SCH23390 produced a 70% decrease in the amount of locomotor activity as compared to saline pretreated controls, and eticlopride pretreatment did not decrease the locomotor response to intra-accumbens MK801. This is consistent with the findings of Raffa et.al. (1989) showing that co-administration of haloperidol with MK801 into the nucleus accumbens did not inhibit locomotion produced by MK801. These results seem to indicate that the stimulation of locomotor activity produced by MK801 injected into the nucleus accumbens is only partially dependant upon intact dopaminergic transmission. The results from the dopamine receptor antagonist studies suggest that D1 dopamine receptors may be critically involved in the stimulation of locomotor activity by intra-accumbens MK801, while the activation of D2 receptors is not apparently necessary for this response.

The present study indicates that the ventral tegmental area may be the site at which MK801 acts, following systemic administration, to stimulate locomotor activity. While it has been shown that MK801 increases the firing rate of dopaminergic neurons in the ventral tegmental area, the mechanism of this effect is not known. Dopaminergic 99 projections from the ventral tegmental area are under regulation by GABAergic projections from the nucleus accumbens. Thus, electrical stimulation of the nucleus accumbens inhibits the firing of AlO dopaminergic neurons in the ventral tegmental area and this response was demonstrated to be potentiated by nipecotic acid (Maeda and Mogenson, 1980, Yim and Mogenson, 1980) . One possible mechanism through which MK801 could stimulate locomotion through an action in the ventral tegmental area is via an interaction with these GABAergic neurons. It is possible that presynaptic NMDA receptors on GABAergic neurons within the ventral tegmental area may regulate the firing of these neurons, thereby regulating the firing of AlO dopaminergic neurons. MK801 may inhibit the effects of NMDA on these neurons and may therefore facilitate the firing of AlO dopamine neurons.

Previous studies have shown that the intravenous adminis­ tration of MK801 increases the firing rate of dopaminergic neurons in this region (French and Ceci, 1990) . The intra- ventral tegmental administration of MK801 may therefore increase the firing rate of these neurons, producing a subsequent increase in extracellular dopamine levels within the nucleus accumbens. Increases in extracellular dopamine in the nucleus accumbens have been associated with increases in locomotor activity (Mogenson and Yim, 1991). Together these findings support the hypothesis that the increase in locomotor activity induced by MK801 injected into the ventral tegmental area may be mediated by the release of vesicular dopamine from terminals within the nucleus accumbens. CHAPTER V

SUMMARY AND CONCLUSIONS

The overall hypothesis of this dissertation is that both dopaminergic and glutamatergic receptor activation is required for the expression of behavior in response to psychostimulant drugs such as amphetamine and cocaine. Thus, the major goal of this work is the characterization of glutamate/dopamine interactions in brain regions critical for regulation of responses to psychoactive drugs. The mesolimbic dopamine system appears to play a critical role in the stimulation of locomotor activity and the modulation of reinforced behaviors produced by drugs of abuse, such as amphetamine and cocaine. In addition to its role in drug reward, the meso-accumbens dopamine system appears to play a role in schizophrenia. The pathophysiologic changes which underlie this disorder have been hypothesized to involve the over-activity of the meso­ limbic dopamine system. This is based on the fact that most drugs currently used in the management of this disorder are dopamine receptor antagonists. Thus, it is apparent that the ability to modulate the mesolimbic dopamine system has

101 1 0 2

important implications in the understanding and treatment of

addiction and schizophrenia. Most studies of the modulation of

mesolimbic dopaminergic function have utilized drugs that

directly interact with dopaminergic receptors, however, a

variety of other neuronal pathways interact with this system.

For example, a tonically active inhibitory GAB pathway,

innervating the dopaminergic cells in the ventral tegmental

area regulates the firing of dopaminergic neurons projecting

from this region. Activation of these dopaminergic projections

is thought to decrease the firing of GABAergic neurons

originating in the nucleus accumbens and projecting to the

ventral pallidum. Finally, several neuronal pathways in

addition to the mesolimbic dopamine projections converge in

the nucleus accumbens. At least three of the converging pathways utilize glutamate as a neurotransmitter. These

anatomical connections provide much of the neurobiological

support for the hypotheses investigated in the present work.

The initial studies described in this dissertation

(Chapter II) have demonstrated that locomotor activity which

is produced by amphetamine, a drug which is known to act through accumbens dopamine, reguires the activation of glutamate receptors in this region. These studies have further demonstrated that the receptors involved appear to be of the

AMPA and/or kainate subtype. While the ability of the NMDA receptor antagonists to diminish the response to amphetamine 103

was not tested, it appears unlikely that these agents would

block amphetamine-stimulated locomotor activity as they have

been shown to stimulate locomotion themselves. One possible

hypothesis which is consistent with these results is that two

distinct glutamatergic pathways terminating in the nucleus

accumbens have a role in regulating locomotor activity. The

first of these pathways activates AMPA receptors and is

involved with dopamine receptor activation to mediate

increases in locomotor activity. The second glutamatergic

pathway activates NMDA receptors which may tonically inhibit motility. Drugs, such as caffeine and scopolamine, which

stimulate locomotor activity via a mechanism which does not require intact dopaminergic transmission within the nucleus accumbens were shown to be independent of accumbens glutamate

as well.

Efferent projections from the nucleus accumbens include

GABAergic and enkephalinergic projections which terminate in a region ventral to the globus pallidus called the ventral pallidum. This region appears to play a critical role in locomotor stimulation produced by glutamatergic and dopaminergic drugs injected directly into the nucleus accumbens. Thus, GABAergic agonists injected into the ventral pallidum inhibit the stimulation produced by these drugs. In addition to the GABAergic projection from the nucleus accumbens, the ventral pallidum receives glutamatergic projections from the neocortex and from limbic regions. The

direct injection of excitatory amino acids into the ventral pallidum stimulates locomotor activity and high affinity binding sites exist for glutamate in this region. This suggests that excitatory amino acid receptors in this region are involved in the mediation of locomotor responses. In the present series of experiments it was shown that amphetamine stimulated locomotor activity is inhibited by AMPA/kainate receptor antagonists injected directly into the ventral pallidum. This strengthens the suggestion that glutamate receptors in this area are critical for the stimulation of locomotor activity produced by psychoactive drugs.

Anatomically this may suggest that a tonically active glutamate input to the ventral pallidum exists which is under regulation by GABAergic projections from the nucleus accumbens. The activation of dopamine receptors within the nucleus accumbens would inhibit the firing of these GABAergic projections and subsequently would relieve the inhibitory influence of these neurons on ventral pallidal glutamatergic afferents.Increases in the activity of glutamatergic projections in the ventral pallidum may subsequently mediate expression of locomotor activity. Interestingly, caffeine and scopolamine, which stimulate locomotion via a mechanism which appears to be independent of dopaminergic transmission in the nucleus accumbens, are also sensitive to AMPA kainate receptor antagonists administered into this region. This suggests that 105

the stimulation of locomotion by caffeine and scopolamine

involves an alternative pathway which does not depend upon

dopaminergic or glutamatergic mechanisms in the nucleus

accumbens, but is dependent upon the activation of

AMPA/kainate receptors in the ventral pallidum.

The role of dopamine and glutamate in the locomotor response stimulated by the non-competitive NMDA receptor antagonist, MK801, has also been the subject of a number of recent studies. The purpose of the present series of experiments (Chapter III) was to evaluate the role of endogenous dopamine in the hypermotility response to MK801.

Pretreatment of rats with reserpine or a-methyl-p-tyrosine markedly inhibited the locomotor response to MK801, suggesting that a vesicular pool of endogenous dopamine is involved in this response. Similarly, pretreatment with the D2 antagonist, eticlopride (0.03 mg/kg, sc), produced a 70% decrease in the hypermotility response to MK801 while the D1 antagonist,

SCH23390 (0.03 mg/kg, sc) had no effect. However, the combination of this dose of eticlopride, and SCH23390 completely inhibited this response (97% decrease). This supports the suggestion that the activation of both D1 and D2 receptors is involved in the response to MK801.

The role of dopamine receptors in the nucleus accumbens in the locomotor response to MK801 was then evaluated by examining the effects of direct injection of the dopamine

receptor antagonists into the accumbens. The administration of

SCH23390 into the nucleus accumbens reversed the locomotor

response to MK801 at a threshold dose of 0,3 fj, g, while eticlopride reversed this response at a dose of 1 fig. However, the combination of SCH23390 and eticlopride at doses that did not significantly inhibit the response to MK801 when administered alone had a synergistic effect. This suggests that D1 and D2 receptors in the nucleus accumbens are involved in the response to MK801. Finally, the intra-accumbens administration of the AMPA/kainate receptor antagonists, DNQX or GAMS, also inhibited the locomotor response produced by

MK801. This finding provided evidence that glutamate receptors, other than the NMDA receptor, are involved in the response to MK801. Additionally, this response may reflect the involvement of dopamine in the response to MK801, as drugs such as amphetamine and cocaine, which are also dependant on dopamine, are also blocked by AMPA/kainate receptor antagonists injected into the nucleus accumbens.

In order to determine the site where MK801 is acting to stimulate locomotor activity, we injected MK801 into either the nucleus accumbens or the ventral tegmental area and evaluated the locomotor response in the presence and absence of drugs that impair dopaminergic transmission. We have determined (Chapter IV) that MK801 administered directly into the accumbens stimulates a locomotor response which is

partially inhibited by reserpine pre-treatment. Rats

pretreated with the D1 dopamine receptor antagonist, SCH23390

demonstrated a 70% decrease in the locomotor response to

MK801. This suggests that D1 receptors are involved in

locomotor activity stimulated by intra-accumbens MK801. The D2

receptor antagonist, eticlopride, at a dose that markedly

inhibited the stimulatory response to the administration of

MK801 directly into the ventral tegmental area or

systemically, had no effect on the locomotor response after

intra-accumbens administration of MK801. Early studies which

suggested that intra-accumbens MK801 stimulates behavior in a manner which is independent of dopamine receptor activation were preformed with the dopamine receptor antagonist, haloperidol. Haloperidol is primarily a D2 receptor antagonist and the finding that eticlopride (which is also selective for

D2 receptors) did not block MK801 stimulated activity agrees with these studies. In contrast, the fact that SCH23390 inhibits this response suggests that a dopaminergic component mediated by the activation of D1 receptors may be involved in the responses to intra-accumbens MK801.

The administration of MK801 directly into the ventral tegmental area produced an intense stimulation of locomotor activity. This response, like the response to MK801 after systemic administration, was severely inhibited by reserpine pre-treatment and the administration of dopamine receptor

antagonists. The ventral tegmental area contains the cells for

dopaminergic afferents to the nucleus accumbens and therefore

is likely to play an integral role in the locomotor circuit.

The fact that the NMDA receptor antagonist stimulates

locomotor activity following injection directly into the ventral tegmental area implies that glutamatergic afferents to

this region provide some sort of tonic inhibitory modulation

of activity. The further finding that this response is

sensitive to inhibition of dopaminergic transmission, suggests that NMDA receptors in this region may regulate locomotor activity by modulating the activity of dopaminergic projections to the accumbens. One potential mechanism by which

NMDA receptors could modulate this response is through an interaction with GABAergic terminals in the ventral tegmental area. As described previously, GABAergic processes do terminate on neurons identified as AlO dopaminergic cells and activation of these receptors regulates the firing of dopaminergic afferents projecting from this region. Glutamate release in the ventral tegmental area and subsequent activation of NMDA receptors on the GABAergic cells in this area would serve to increase the firing of these projections causing an increase in the release of GABA and a subsequent inhibition of the firing of AlO dopamine projections. MK801 may inhibit the effects of glutamate at these NMDA receptors and in so doing would decrease the activity of GABAergic 109

cells. This would remove the inhibitory regulation of the

dopaminergic cells and increase the firing rate of these

neurons.

Dopaminergic neurotransmission in the nucleus accumbens

has been implicated in goal oriented and reward behavior and

is thought to be involved in the initiation and regulation of

locomotor activity. Drugs of abuse, such as amphetamine, are

thought to by-pass the normal reward circuitry, and to

selectively activate dopaminergic mechanisms within the nucleus accumbens. Thus circuitry originally set up to

reinforce behaviors which have "positive" affects on the organism, when activated by drugs of abuse, begins to reinforce behaviors which lead to obtaining these drugs. In the present series of experiments it has been shown that excitatory amino acid receptor mediated mechanisms in the nucleus accumbens and the ventral pallidum are involved in the locomotor responses to amphetamine. In separate studies in our laboratory it has been subsequently shown that the

AMPA/kainate receptor antagonist, DNQX, inhibits the development of a conditioned place preference to amphetamine.

Together these studies strongly suggest that excitatory amino acid receptors of the AMPA (or kainate) subtype may play a role in the rewarding effects of drugs such as amphetamine which activate dopaminergic mechanisms in the nucleus accumbens. This may indicate that the development of drugs 1 1 0 which inhibit AMPA/kainate receptor function could be of some use in treatment of drug addiction.

The locomotor responses to the non-competitive NMDA receptor antagonist, MK801, were demonstrated to involve the activation of dopamine D1 and D2 receptors in the nucleus accumbens and in the ventral tegmental area. MK801 stimulated locomotion was also shown to be dependant upon the availability of AMPA/kainate glutamate receptors in the nucleus accumbens as are other drugs which depend on the activation of dopamine receptors in this region. MK801-induced locomotion was also demonstrated to involve processes in the ventral tegmental area which may serve to regulate dopaminergic function in the nucleus accumbens. MK801 has behavioral actions that appear to be very similar to those of the commonly abused drug, phencyclidine (PCP). MK801 and PCP both stimulate locomotor activity in rats and are postulated to act at the same site on the NMDA receptor ion-channel.

MK801 has recently been demonstrated to elicit a conditioned place preference in rats, suggesting that it may also be rewarding. MK801, therefore, appears to be a potential drug of abuse. Studies involving MK801 and PCP suggest that the NMDA receptor may be an additional site at which abused drugs could act in the brain and at which drugs could be targeted in the treatment of drug addiction. REFERENCES

Aghajanian, G.K. and Bunney, B.S. Dopamine autoreceptors: pharmacological characterization by microiontophoretic single cell recording studies. Naunyn-Schmiedeberg's Arch. Pharmacol. 297:1-7, 1977.

Amalric, M.; Koob, G.F.; Creese, I. and Swerdlow, N. "Selective" D-l and D-2 receptor antagonists fail to differen­ tially alter supersensitive locomotor behavior in the rat. Life Sci. 39:1985-1993; 1986.

Amalric, M.; Ouagazzal, A. and Nieoullon, A. Glutamate- dopamine functional interaction within the striatum and the nucleus accumbens. Soc. Neurosci. Abst. 17:503, A198.20; 1991.

Anis, N.A. ; Berry, S.C.; Burton, N.R. and Lodge, D. The dissociative anesthetics ketamine and phencyclidine, selectively reduce excitation of central mammalian neurons by N-methyl-aspartate. Br. J. Pharmacol. 79:565-575, 1983.

Arnt, J. Hyperactivity following injection of a glutamate agonist and 6,7-ADTN into the rat nucleus accumbens and its inhibition by THIP Life Sci. 28:1597-1603, 1981.

Ascher, P. and Nowak, L. The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture. J. Physiol. 399:247-266, 1988.

Ault, B., Evans, R.H., Francis, A.A., Oaks, D.J. and Watkins, J.C. Selective depression of excitatory amino acid induced depolarizations by magnesium ions in isolated spinal chord preparations. J. Physiol. 307:413-428, 1980.

Austin, M.C. and Kalivas, P.W. The effect of cholinergic stimulation in the nucleus accumbens on locomotor behavior. Brain Res. 441: 209-214, 1988.

Barnard, E. and Henley, J.M. The non-NMDA receptors: types, protein structure and molecular biology, in: The Excitatory Amino Receptors, Trends Pharmacol. Sci. (Special Report), 82- 89, 1991.

Ill 1 1 2

Benvenga, M.J. and Spaulding, T.C. Amnesic effect of the novel anticonvulsant, MK801. Pharmacol. Biochem. Behav. 30:205-207; 1988.

Birch, P.J.; Grossman, C.J. and Hayes, A.G. 6,7-Dinitro- quinoxaline-2,3-dion and 6-nitro,7-cyano-quinoxaline-2,3-dion antagonise responses to NMDA in the rat spinal cord via an action at the strychnine-insensitive glycine receptor. Eur. J. Pharmacol. 156: 177-180, 1988.

Biscoe, T.J., Evans, R.H., Headley, P.M., Martin, M.R. and Watkins, J.C. Domoic and quisqualic acids as potent amino acid excitants of frog and rat spinal neurones. Nature (Lond.) 255:166-167, 1975.

Biscoe, T.J., Evans, R.H., Headley, P.M., Martin, M.R. and Watkins, J.C. Structure-activity relationships of excitatory amino acids on frog and rat spinal neurones. Br. J. Pharmacol. 58:373-382, 1976.

Boldry ,R.C. and Uretsky, N.J. The importance of dopaminergic neurotransmission in the hypermotility response produced by the administration of N-methyl-D-aspartatic acid into the nucleus accumbens. Neuropharmacol. 27:569-577, 1987.

Boldry, R.A.; Shreve, P.E. and Uretsky, N.J. 6,7- Dinitroquinoxaline-2,3-dione (DNQX) and y-aminomethyl- sulphonate (GAMS) selectively inhibit the hypermotility response produced by activation of quisqualic acid (QA) receptors in the nucleus accumbens., Soc. Neurosci. Abst. 14: 380.13, 1988.

Boldry, R.C.; Willins, D.L.; Wallace, L.J. and Uretsky, N.J. The role of endogenous dopamine in the hypermotility response to intra-accumbens AMPA. Br. Res. 559:100-108; 1991.

Brudzynski, S.M., Wu, M. and Mogenson, G.F. Modulation of locomotor activity induced by injections of carbachol into the tegmental pedunculopontine nucleus and adjacent areas in the rat. Brain Res. 451:119-125, 1988.

Bunney, B.S.; Walters, J.; Roth R.H. and Aghajanian, G.K. Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single-cell activity. J. Pharmacol. Exp. Therap. 185:560-571, 1973a.

Bunney, B.S.; Aghajanian, G.K. and Roth, R.H. Comparison of effects of L-DOPA, amphetamine and apomorphine on firing rate of rat dopaminergic neurons. Nat. New Biol. 245:123-125, 1973b. 113

Carlsson, A.; Falck, B. and Hillarp, N.A. Cellular localization of brain monoamines. Acta Physiologica Scandinavica 196:1-27 (suppl.), 1962.

Carlsson, M. and Carlsson, A. The NMDA antagonist MK801 causes marked locomotor stimulation in monoamine-depleted mice. J. Neural Transm. 75:221-226; 1989.

Carlsson, M. and Carlsson, A. Interactions between glutamat- ergic and monoaminergic systems within the basal ganglia - implications for schizophrenia and Parkinson's disease. Trends Neurosci. 13:272-276; 1990.

Carlsson, M. and Svensson, A. Interfering with glutamatergic neurotransmission by means of NMDA antagonist administration discloses the locomotor stimulatory potential of other transmitter systems. Pharmacol. Biochem. Behav. 36:45-50; 1990.

Carlsson, M. and Engberg, G. Ethanol behaves as an NMDA antagonist with respect to locomotor stimulation in monoamine- depleted mice. J. Neural Trans. 87:155-160; 1992.

Cha, J.J., Makowiec, R.L., Penney, J.B. and Young, A.B. Soc. Neurosci. Abstr. 16:548, 1990.

Clineschmidt, B.V.; Martin, G.E. and Bunting, P.R. Anticonvul­ sant activity of (±)-5-methyl-10,ll-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK801), a substance with potent anticonvulsant, central sympathomimetic, and apparent anxiolytic properties. Drug Dev. Res. 2:123-134; 1982a.

Clineschmidt, B.V.; Martin, G.E.; Bunting, P.R.; Papp, N.L. Central sympathomimetic activity of (±)-5-methyl-10,11- dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK801), a substance with potent anticonvulsant, central sympathomimetic, and apparent anxiolytic properties. Drug Dev. Res. 2:134-145; 1982b.

Clineschmidt, B.V.; Williams, M.; Witoslawsky, J.J.; Bunting, P.R.; Risley, E.A. and Totaro, J.A. Restoration of shock- suppressed behavior by treatment with (±)-5-methyl-10,ll- dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK801), a substance with potent anticonvulsant, central sympathomimetic, and apparent anxiolytic properties. Drug Dev. Res. 2:147-163; 1982c.

Collingridge, G.L. and Bliss, T.V.P. NMDA-receptors-Their role in long-term potentiation. Trends Neurosci.10:288-293, 1987. 114

Collingridge, G.L. and Lester, R.A.J. Excitatory amino acid receptors in the vertebrate central nervous system. Pharmacol. Rev. 40: 143-210, 1989.

Cotman, C.W. and Nadler, J.V. In: Glutamate: Transmitter in the Central Nervous System, Ed. Roberts, P.J.;Storm-Mathisen, J. and Johnson, G.A.R., John Wiley and Sons, London, pp.117- 154, 1981.

Cotman, C.W., Flatman, J.A., Ganong, A.H. and Perkins, M.N. Effects of excitatory amino acid antagonists on evoked and spontaneous excitatory potentials in guinea pig hippocampus. J. Physiol. 378:403-415, 1986.

Cotman, C.W. and Iversen L.L. Excitatory amino acids in the brain- focus on NMDA receptors. Trends Neurosci. 10:2 63-265, 1987.

Cotman, C.W., Monaghan, D.T., Ottersen, O.P. and Storm- Mathisen, J. Anatomical organization of excitatory amino acid receptors and their pathways. Trends Neurosci. 10:273-280, 1987.

Croucher, M.J.; Collins, J.F. and Meldrum, B.S. Anticonvulsant action of excitatory amino acid antagonists. Science 216:899-901, 1982.

Curtis, D.R., Phillis, J.W. and Watkins, J.C.: Chemical excitation of spinal neurons. Nature (Lond.) 183: 611-612, 1959.

Curtis, D.R. and Watkins, J.C. The excitation and depression of spinal neurones by structurally related amino acids. J Neurochem 6:117-141, 1960.

Curtis, D.R. and Watkins, J.C. Acidic amino acids with strong excitatory actions on mammalian neurones. J Physiol 166:1-14, 1963.

Curtis, D.R. The actions of amino acids upon mammalian neurones. In: Curtis, D.R. and McIntyre, A.K. (eds), New York, Springer, pp.34-42, 1965.

Dahlstrom, A. and Fuxe, K. Evidence for the existence of mono­ amine containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta Physiologica Scandinavica 232:1-55 (suppl. 62), 1964.

Davies, J. and Watkins, J.C. Selective antagonism of amino acid induced and synaptic excitation in cat spinal chord. J. Physiol. (Lond.) 297:621-635, 1977. 115

Davies, J and Watkins JC Selective antagonism of amino acid- induced and synaptic excitation in the cat spinal chord. J Physiol 297: 621-635, 1979.

Davies, J., Evans, R.H., Francis, A.A. and Watkins, J.C. Excitatory amino acid receptors and synaptic excitation in the mammalian central nervous system. J. Physiol. 75:641-654, 1979.

Davies, J, Francis AA, Jones AW, and Watkins JC 2-Amino- phosphonovalerate (2APV), a potent and selective antagonist of amino acid-induced and synaptic excitation. Neurosci Let 21:77-81, 1981.

Davies, J.; Evans, R.H.; Jones, A.W.; Smith, D.A.S. and Watkins, J.C. Differential activation and blockade of excitatory amino acid receptors in the mammalian and amphibian central nervous system. Comp. Biochem. Physiol. 72C: 211-224, 1982.

Davies, J. and Watkins, J.C. Actions of D- and L- forms of 2- amino-5-phosphonovalerate and 2-amino-4-phosphonobutyrate in the cat spinal cord. Brain Res. 235:378-386, 1982.

Davies, J. and Watkins, J.C. Depressant action of y-D- glutamylaminomethyl sulfonate (GAMS) on amino acid-induced and synaptic excitation in the cat spinal cord. Brain Res. 327: 113-120, 1985.

Davies, J., Evans, R.H., Harrling, P., Jones, A.W., Olverman, H.J. CPP, a new potent and selective NMDA antagonist. Depression of central neuron responses, affinity for 3H-D-AP5 binding sites on brain membranes and anticonvulsant activity. Brain Res. 383:169-173, 1986.

Dawbarn, D. and Pycock, C.J. Motor effects following application of putative excitatory amino acid antagonists to the region of the mesencephalic dopamine cell bodies in the rat. Naunyn Schmiedeberg's Arch. Pharmacol. 318:100-104; 1981.

Desai, M.A. and Conn, P.J. Neurosci. Lett. 109:157-162, 1989.

Di Chiara, G. and Imperato, A. Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc. Nat. Acad. Sci. U.S.A. 85:5274-5278, 1988.

Donzanti, B.A. and Uretsky, N.J. Effects of excitatory amino acids on locomotor activity after bilateral microinjection into the rat nucleus accumbens: possibile dependence on dopaminergic mechanisms. Neuropharmacology 22:971-981, 1983. 116

Donzanti, B.A. and Uretsky, N.J. Antagonism of the hypermotility response induced by excitatory amino acids in the rat nucleus accumbens. Naunyn Schmiedeberg's Arch. Pharmacol. 325:1-7; 1984.

Drejer, J. and Honore, T. New quinoxalinediones show potent antagonism of quisqualate responses in cultured mouse cortical neurons. Neurosci. Lett. 87:104-108, 1988.

Erinoff, L. and Snodgrass, S.R. Effects of adult or neonatal treatment with 6-hydroxydopamine or 5,7-Dihydroxytryptamine on locomotor activity, monoamine levels and response to caffeine. 24: 1039-1045, 1986.

Evans, R.H., Francis, A.A., Jones, A.W., Smith, D.A.S. and Watkins, J.C. The effects of a series of -phosphonic - carboxylic amino acids induced responses in isolated spinal cord preparations. Br. J. Pharmacol. 75:65-75, 1982.

Fagg, G.E. L-Glutamate, excitatory amino acid receptors and brain function. Trends Neurosci. 8: 207-210, 1985.

Fletcher, E.J. and Lodge, D. Glycine reverses antagonism of N- methyl-D-aspartate (NMDA) by l-hydroxy-3aminopyrrolidone-2 (HA-966) but not D-2-amino phosphonovalerate (D-AP5) on rat cortical slices. Eur. J. Pharmacol. 151:161-162, 1988.

Freed, W.J. and Cannon-Spoor, H.E. A possible role of AA2 excitatory amino acid receptors in the expression of stimulant drug effects. Psychopharm., 101:456-464, 1990.

French, E.D. and Ceci, A. Non-competitive N-methyl-D-aspartate antagonists are potent activators of ventral tegmental A10 dopamine neurons. Neurosci. Let. 119:159-162; 1990.

Fuller, T.A.; Russchen, F.T. and Price, D.L. Sources of presumptive glutamatergic/aspartergic afferents to the rat ventral striatopallidal region. J. Comp. Neurol. 258: 317-338, 1987.

Gannon, R.L., Baty, L.T. and Terrian, D.M. Brain Res. 495:151- 155, 1989.

Gill, R. ; Foster, A.C. and Woodruff, G.N. Systemic administra­ tion of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil. J. Neurosci. 7:3343-3349; 1987.

Groenewegen, H.J.; Berendese, H.W.; Meredith, G.E.; Haber, S.N.; Voorn, P.; Wolters, J.G. and Lohman, A.H.M. Functional anatomy of the ventral, limbic system-innervated striatum, in: The Mesolimbic Dopamine System: From Motivation to Action. 117

Eds. P. Willner and J. Scheel-Kruger, pp. 33-59, John Wiley and Sons Ltd., New York, 1991.

Haldeman S, and McLennan H, (1972) The antagonistic action of glutamic acid diethylester towards amino acid induced and synaptic excitations of central neurones. Brain Res 45: 393- 400.

Hall, J.G.; McLennan, H. and Wheal, H.V. The actions of certain amino acids as neuronal excitants. J. Physiol. 272: 52-53, 1977.

Hall, J.G.; Hicks, T.P.; McLennan, H.; Richardson, T.H. and Wheal, H.V. The excitation of mammalian central neurones by amino acids. J. Physiol. 286: 29-39., 1979.

Hamilton, M.H.; Debelleroche, J.S.; Gardiner, I.M. and Heberg, L.J. Stimulatory effect of N-methyl aspartate on locomotor activity and transmitter release from rat nucleus accumbens. Pharmacol. Biochem. Behav. 25:943-948; 1986.

Hayashi, T. (1954) Effects of sodium glutamate on the nervous system. Keio J Med 3: 183-192.

Hayashi, T. Effects of sodium glutamate on the nervous system. Keio J. Med. 3:183-192, 1954.

Hearns, T. , Ganong, A.H. and Cotman, C.W. Antagonism of lateral olfactory tract synaptic potentials in rat pre- pyriform cortex slices. Brain Res. 379:372-376, 1986.

Heimer, L. and Alheid, G.F. Piecing together the puzzle of basal forebrain anatomy, in: The Basal Forebrain: Anatomy to Function. Eds. P. Kalivas, and Hanin, I., Plenum Press, New York, pp.1-42, 1991.

Hiramatsu, M.; Cho, A.K. and Nabeshima, T. Comparison of the behavioral and biochemical effects of the NMDA receptor antagonists MK801 and phencyclidine. Eur. J. Pharmacol. 166:359-366; 1989.

Hiroi, N. and White, N. The reserpine-sensitive dopamine pool mediates (+)-amphetamine-conditioned reward in the place preference paradigm. Brain Res. 510:33-42; 1990.

Hockfelt, T., Ljungdahl, A., Fuxe, K. and Johansson, O. Dopamine nerve terminals in the limbic cortex: aspects of the dopamine hypothesis of schizophrenia. Science 184:177-179, 1974.

Honey, C.R. , Miljkovic, Z. and MacDonald, J.F. Neoursci. Lett. 61:135-139, 1985. 118

Honore, T., Laurisden, J. and Krosgaard-Larsen, P. The binding of [3H]-AMPA, a structural analog of glutamic acid, to rat brain membranes. J. Neurochem. 38:173-178, 1982.

Honore, T., Davies, S.N., Drejer, J., Fletcher, E.J,, Jacobsen, P., Lodge, D. and Nielsen, F.E. Quinoxalinediones: potent competitive non-N-methyl-D-aspartate glutamate receptor antagonists. Science (Wash. D.C.) 241:701-703, 1988.

Horne, A.L. and Simmonds, M.A. Comparison of the excitatory amino acids quisqualate and AMPA on slices of rat cerebral cortex. Br. J. Pharmacol. 92:543P, 1987.

Hosli, L.; Hosli, E.; Lehmann, R. and Eng, P. Effect of the glutamate analogue AMPA and its interaction with antagonists on cultured rat spinal and brain stem neurons. Neurosci. Let. 36: 59-62, 1983.

Iadorola, M.J., Nicoletti, F., Naranjo, J.R., Putnam, F. and Costa, E. Brain Res. 374:174-178, 1986.

Jones, D.I. and Mogenson G.J. Nucleus accumbens to globus pallidus GABA projections subserving ambulatory activity. Am. J. Physiol. 283: R63-R69, 1980.

Jones, A.W., Smith, D.A.S. and Watkins, J.C. Structure- activity relationships of dipeptide antagonists of excitatory amino acids. Neurosci. 13:573-581, 1984.

Jones, P.G. and Roberts, P.J. Neurosci. Lett. 111:228-232, 1990.

Joyce, E.M. and Koob, G.F. Amphetamine-, scopolamine- and caffeine-induced locomotor activity following 6- hydroxydopamine lesions of the mesolimbic dopamine system. Psychopharmacology 73:311-313, 1981.

Kaddis, F.G.; Wallace, L.J. and Uretsky, N.J. Role of glutamatergic transmission in mediating the locomotor stimulation produced by cocaine and dopamine receptor agonists. Soc. Neurosci. Abst. 17:886, A348.4; 1991.

Kelly, P.H. and Iversen, S.D. Selective 6-OHDA-induced destruction of mesolimbic dopamine neurons: Abolition of psychostimulant induced locomotor activity in rats. Eur. J. Pharmacol. 40:45-56, 1976.

Kemp, J.A.; Priestly, T. and Woodruff, G.N. Br. J. Pharmacol. 89:539P, 1986. 119

Kemp, J.A.; Grimwood, S. and Foster, A.C. Characterisation of the antagonism of excitatory amino acid receptors in rat cortex by kynurenic acid. Br. J. Pharmacol. 91:314P, 1987.

Kleckner, N.W. and Dingeldine, R. Requirement of glycine in activation of NMDA-receptors expressed in Xenopus oocytes. Science 241:835-837, 1988.

Klockgether, T. and Turski, L. NMDA antagonists potentiate antiparkinsonian actions of L-DOPA in monoamine-depleted rats. Ann. Neurol. 28:539-546; 1990.

Konig, J.R.F. and Klippel, R.A. The rat brain: A stereotaxic atlas of the forebrain and lower parts of the brainstem. New York: Krieger, 1963.

Krosgaard-Larsen, P., Honore, T., Hansen, J.J., Curtis, D.R. and Lodge, D. New class of glutamate agonist structurally related to ibotenic acid. Nature (Lond.) 284:64-66, 1980.

Krosgaard-Larsen, P. and Honore, T. Glutamate receptors and new glutamate agonists. Trends Pharmacol. Sci. 4:31-33, 1983.

Lanthorne, T.H., Ganong, A.H. and Cotman, C.W. 2-Amino-4- phosphonobutyrate selectively blocks mossy fiber-CA3 responses in guinea pig but not rat hippocampus. Brain Res. 290:174-178, 1984.

Lehmann, J., Chapman, A.G., Meldrum, B.S.,Hutchinson, A., Tsai, C. and Wood, P.L. CGS19755 is a potent and competitive antagonist at NMDA receptors. Eur. J. Pharmacol. 154:89-93, 1988.

Lester, R.A.J.; Quarum, M.L.; Parker, J.D.? Weber, E. and Jahr, C.E. Interaction of 6-cyano-7-nitroquinoxaline-2,3,- dione with the N-methyl-D-aspartate receptor-associated glycine binding site. Mol. Pharmacol. 35: 565-570, 1989.

Lodge, D. ; Headley, P.M. and Curtis, D.R. Selective antagonism by D-alpha-aminoadipate of amino acid and synaptic excitation of cat spinal neurons. Brain Res. 152: 603-608, 1978.

London, E.D. and Coyle, J.T. Specific binding of 3H-kainic acid to receptor sites in rat brain. Mol. Pharmacol. 15:492- 505, 1979.

Loscher, W.; Annies, R. and Honack, D. The N-methyl-D-aspar- tate receptor antagonist MK-801 induces increases in dopamine and serotonin metabolism in several brain regions of rats. Neurosci. Let. 128:191-194; 1991. 1 2 0

Loscher, W.; Honack, D. The behavioural effects of MK-801 in rats: involvement of dopaminergic, serotonergic and nor­ adrenergic systems. Eur. J. Pharmacol. 215:199-208; 1992.

MacDonald, J.F., Schneiderman, J.H. and Miljkovic, Z. in Advances in Experimental Biology and Medicine (Vol. 203: Excitatory Amino Acids and Epilepsy) (Schwartz, R. and Ben- Ari, Y., eds.), pp.425-437, 1986, Plenum Press.

Maeda, H. and Mogenson, G.J. An electrophysiological study of inputs to neurons of the ventral tegmental area from the nucleus accumbens and medial preoptic-anterior hypothalamic areas. Brain Res. 197:365-377, 1980.

McGeer, P.L., McGeer, E.G., Scherer, U. and Singh, K. A glutamatergic corticostriatal path? Brain Res. 128:393-373, 1977.

McGeer, P.L.; Eccles, J.C. and McGeer Putative excitatory neurons: glutamate and aspartate. In: Glutamine and Glutamate in Mammals Vol. II., Ed. E. Kvamme. CRC Press Inc. Boca Raton, Florida, 1987.

McKernan, R.M., Castro, S., Paot, J. and Wong, E.H.F. Soluabilization of the N-methyl-D-aspartate receptor channel complex from rat and porcine brain. J. Neurochem. 52:777-785, 1989.

McLennan, H.; Marshall, K.C. and Huffman R.D. The antagonism of glutamate action at central neurones. Experentia 27: 1116, 1971.

McLennan, H. and Lodge, D. The antagonism of amino acid- induced excitation of spinal neurones in the cat. Brain Res. 169:83-90, 1979.

McLennan, H, and Liu JR The actions of six antagonists of the excitatory amino acids on neurones of the rat spinal cord. Exp Brain Res 45:151-156, 1982.

McLennan, H. Receptors for the excitatory amino acids in the mammalian central nervous system. Prog. Neurobiol. 20:251-271, 1983.

Meldrum, B.S. Possible therapeutic application of antagonists of excitatory amino acid neurotransmitters. Clin. Sci. 68:113- 121, 1985.

Mogenson, G.J., Wu, M. and Manchanda, S.K. Locomotor activity initiated by microinfusion of picrotoxin into the ventral tegmental area. Brain Res. 161:311-319, 1979. 1 2 1

Mogenson, G.J.; Jones, D.L. and Yim, C.Y. From motivation to action: Functional interface between the limbic system and the motor system. Prog. In Neurobiol., 14:69-97; 1980.

Mogenson, G.J. and Nielsen, M.A. Evidence that an accumbens to subpallidal GABAergic projection contributes to locomotor activity. Brn. Res. Bull. 11: 309-314, 1983.

Mogenson, G.J.; Swanson, L.W. and Wu, M. Neural projections from nucleus accumbens to globus pallidus, substantia innominata, and lateral preoptic-lateral hypothalamic area: An anatomical and electrophysiological investigation in the rat. J. Neurosci. 3: 189-202, 1983.

Mogenson, G.J. and Yim, C.C. Neuromodulatory functions of the mesolimbic dopamine system: electrophysiological and behavioural studies, pp.105-130 in The Mesolimbic Dopamine System: From Motivation to Action. Eds. Willner, P. and Scheel-Kruger, J., John Wiley & Sons, Chichester, 1991.

Monoghan, D.T. and Cotman, C.W. Distribution of 3H-kainic acid binding sites in rat CNS as determined by autoradiography. Brain Res. 252:91-100, 1982.

Monoghan, D.T., Yao, D. and Cotman, C.W. Distribution of 3H- AMPA binding sites in rat brain as determined by quantitative autoradiography. Brain Res. 324:160-164, 1984.

Monoghan, D.T.; Yao, D. and Cotman, C.W. Distribution of NMDA- sensitive L-3H-glutamate binding sites in rat brain as determined by quantitative autoradiography. J. Neurosci. 5:2909-2919, 1985.

Monoghan, D.T.; Bridges, R.J. and Cotman, C.W. The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system. Ann. Rev. Pharmacol. Toxicol. 29: 365-402, 1989

Morris, R.G.M., Anderson, E., Lynch, G.S. and Baudry, M. Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP-5. Nature 319:774-776, 1986.

Murphy, D.E., Snowhill, E.W. and Williams, M. Characterization of quisqualate recognition sites in rat brain tissue using D, L-[3H]-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay. Neurochem. Res. 12:775-782, 1987.

Nakagawa, Y.; Saitoh, K., Ishihara, T.; Ishida, M. and Shinozaki, H. Eur. J. Pharmacol. 184:205-206, 1990. 1 2 2

Nauta, W.J.H.; Smith, G.P.; Faull, R.L.M. and Domesick, V.B. Efferent connections and nigral afferents of the nucleus accumbens septi in the rat. Neurosci. 3: 385-410, 1978.

Nicoletti, F., Meek, J.L., Iadorola, M.J., Chung, D.M., Roth, B.L. and Costa, E. J. Neurochem. 46:40-46, 1986.

Olsen, R.W., Szamraj, O. and Houser, C.R. 3H-AMPA binding to glutamate receptor subpopulations in rat brain. Brain Res. 402:243-254, 1987.

Palmer, E., Monaghan, D.T. and Cotman, C.W. Eur. J. Pharmacol. 166:585-587, 1989.

Paxinos, G. and Watson, C. The Rat Brain in Stereotaxic Coordinates. Academic Press, Australia; 1982.

Pearce, B., Albrecht, J., Morrow, C. and Murphy, S. Neurosci. Lett. 72:335-340, 1986.

Pijnenberg, A.J.J.; Honig, W.M.M. and VanRossum, J.M. Inhibi­ tion of d-amphetamine-induced locomotor activity by injection of haloperidol into the nucleus accumbens of the rat. Psycho­ pharmacology (Berl.) 41:87-95; 1975.

Pulvirenti, L.; Swerdlow, N.R. and Koob, G.F. Microinjection of a glutamate antagonist into the nucleus accumbens reduces psychostimulant locomotion in rats. Neurosci. Let. 103: 213- 218, 1989.

Raffa, R.; Ortegon, M.E.; Robisch, D.M. and Martin, G.E. In vivo demonstration of the enhancement of MK801 by L-glutamate. Life Sci. 44:1593-1599; 1989.

Rao, T.S.; Kim, H.S.; Lehmann, J.; Martin, L.L. and Wood, P.L. Interactions of phencyclidine receptor agonist MK801 with dopaminergic system: Regional studies in the rat. J. Neurochem. 54:1157-1162; 1990.

Reynolds, I.J. and Miller, R.J. 3H-MK801binding to the NMDA receptor/ionophore complex is regulated by divalent cations: evidence for multiple regulatory sites. Eur. J. Pharmacol. 151:103-112, 1988.

Roberts, D.C.S.; Zis, A. P. and Fibiger, H.C. Ascending catecholaminergic pathways and amphetamine induced locomotion: Importance of dopamine and apparent non-involvement of norepinephrine. Brain Res. 93: 441-445, 1975.

Salt, T.E. Mediation of thalamic sensory input by both NMDA receptors and non-NMDA receptors. Nature 322:263-265, 1986. 123

Schmutz, M., Klebs,K., Olpe, H.R., Allgeir, H. and Heckebdorn, R. CGP37849: competitive receptor antagonist with potent oral anticonvulsant activity. Br. J. Pharmacol. 97:581P, 1989.

Schoepp, D.D. and Johnson, B.G. J. Neurochem. 53:1865-1870, 1989.

Schoepp, D.D. and Johnson, B.G. J. Neurochem. 53:273-278, 1989. [both of these references are given for AP4 antag. of metabo recept, which is correct?]

Seren, M.S., Aldinio, C., Zanoni, R., Leon, A. and Nicoletti, F. J. Neurochem. 53:1700-1705, 1989.

Shinozaki, H. and Shibuya, I. A new potent excitant, quisqualic acid: effects on crayfish neuromuscular junction. Neuropharmacol. 13:665-672, 1974.

Shreve, P.E. and Uretsky, N.J. Effect of GABAergic transmission in the subpallidal region on the hypermotility response to the administration of excitatory amino acids and picrotoxin into the nucleus accumbens. Neuropharm. 27: 1271- 1277, 1988a.

Shreve, P.E. and Uretsky, N.J. Role of quisqualic acid receptors in the hypermotility response produced by the injection of AMPA into the nucleus accumbens. Pharmacol. Biochem. Behav. 30:379-384; 1988b.

Shreve, P.E. and Uretsky, N.J. AMPA, kainic acid, and N- methyl-D-aspartic acid stimulate locomotor activity after injection into the substantia innominata/lateral preoptic area. Pharmacol. Biochem. Behav. 34: 101-106, 1989.

Sladeczek, F., Pin, J-P., Recasens, M., Bockaert, J. and Weiss, S. Nature 317:717-719, 1985.

Snyder S.H.; Katims, J.J.; Annau, Z.; Burns, R.F. and Daly, J.W. Adenosine receptors and behavioral actions of methylxanthines. Proc. Nat. Acad. Sci. USA 78: 3260-3264, 1981.

Sonsalla, P.K. ; Nicklas, W.J. and Heikkila, R.E. Role for excitatory amino acids in methamphetamine-induced nigrostriatal dopaminergic toxicity. Science 243:398-400; 1988.

Stolk, J.M. and Rech, R.H. Enhanced stimulant effects of d- amphetamine on the spontaneous locomotor activity of rats treated with reserpine. J. Pharmacol. Exp. Therap. 158:140- 149; 1967. 124

Sugiyama, H., Ito, I. and Hirono, C. Nature 325:531-533, 1987.

Supko, D.E.; Hill, R.A.; Uretsky, N.J.; Miller, D.D. and Wallace, L.J. Substituted O-tyrosines and quinoxaline-2,3- diones as potential quisqualate analogs. FASEB J., 4: A603, 1990.

Swerdlow, N.R. and Koob, G.F. The neural substrates of apomorphine stimulated locomotor activity following denervation of the nucleu accumbens. Life Sci. 35:2537-2544, 1984.

Swerdlow, N.R. and Koob, G.F. Separate neural substrates of the locomotor activating properties of amphetamine, heroin, caffeine and corticotropin releasing factor (CRF) in the rat. Pharmacol. Biochem. Behav. 23: 303-307, 1985.

Swerdlow, N.R., Vaccarino, F.J., Amalric, M. and Koob, G.F. Seperate neural substrates for the motor-activating properties of psychostimulants: A review of recent findings. Pharmacol. Biochem. Behav. 25:233-248, 1986.

Swerdlow, N.R. and Koob, G.F. Lesions of the dorsomedial thalamus, medial prefrontal cortex, and pedunculopontine nucleus: effects on locomotor activity mediated by nucleus accumbens - ventral pallidal circuitry. Brain Res. 412:233- 243, 1987.

Takemoto, T. Isolation and structural identification of naturally occuring excitatory amino-acids. In: Kainic Acid as a Tool in Neurobiology, ed. McGeer, E.C., Olney, J.W. and McGeer, P.L., pp.1-15, New York: Raven, 1978.

Thornburg, J.E. and Moore, K.E. Inhibition of anticholinergic drug induced locomotor stimulation in mice by methyltyrosine. Neuropharm. 12: 1179-1185, 1973.

Tricklebank, M.D.; Singh, L. ; Oles, R.J.; Preston, C. and Iversen, S.D. The behavioural effects of MK801: A comparison with antagonists acting non-competitively and competitively at the NMDA receptor. Eur. J. Pharmacol. 167:127-135; 1989.

Tsumoto, T., Hahihara, K., Sato, H. and Hata, Y. NMDA receptors in the visual cortex of young kittens are more effective than those of adult cats. Nature 327:513-514, 1987.

Ungerstedt, U. Stereotaxic mapping of the monoamine pathways in the rat brain. Acta Physiologica Scandinavica 367:1-48 (suppl), 1971. 125

Waelsch, H. Metabolism of glutamic acid and glutamine. In Elliot KAC, Page IH, Queastel JH (eds) "Neurochemistry: The Chemical Dynamics of Brain and Nerve", Illinois: C.C.Thomas, pp.173-203, 1955.

Walaas, I. Biochemical evidence for overlapping neocortical and allocortical glutamate projections to the nucleus accumbens and rostral caudatoputamen in the rat brain. Neuroscience 6:399-405, 1981.

Walaas, I. and Fonnum F. The effects of surgical and chemical lesions on neurotransmitter candidates in the nucleus accumbens in the rat. Neurosci. 4: 209-216, 1979.

Walaas, I, and Fonnum, F. Biochemical evidence for gamma- aminobutyrate containing fibers from the nucleus accumbens to the substantia nigra and ventral tegmental area in the rat. Neuroscience 5:63-72, 1980.

Watkins, J.C. and Evans, R.H. Excitatory amino acid transmitters. Annu. Rev. Pharmacol. Toxicol. 21: 165-204, 1981.

Watkins, J.C. and Olverman, H.J. Agonists and antagonists for excitatory amin acid receptors. Trends Neurosci. 10:265-272, 1987.

Watson, G.B.; Hood, W.F., Monahan, J.B. and Lanthorn, T.H. Kynurenate antagonizes actions of N-methyl-D-aspartate through a glycine sensitive receptor. Neurosci. Res. Commun. 2:169- 174, 1988.

Weiloch, T. Hypoglycaemia-induced neuronal dammage prevented by an N-methyl-D-aspartate antagonist. Science 230:681-683, 1985.

Westbrook, G.L. and Mayer, L.M. Micromolar concentrations of Zn+2 antagonize NMDA and GABA responses of hippocampal neurons. J. Neurosci. 328:640-643, 1987.

White, F.J. and Wang, R.Y. Pharmacological characterization of dopamine autoreceptors in the rat ventral tegmental area: microiontophoretic studies. J. Pharmacol. Exp. Therap. 231:275-280, 1984.

Willins, D.L.; Wallace, L.J.; Miller, D.D. and Uretsky, N.J. a-Amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor antagonists in the nucleus accumbens and ventral pallidum decrease the hypermotility response to psychostimulant drugs. J. Pharmacol. Exp. Therap. 2 60:1145- 1151; 1992. 126

Willins, D.L. ; Wallace, L.J. and Uretsky, N.J. The role of dopamine and ampa/kainate receptors in the nucleus accumbens in the hypermotility response to MK801., Pharmacol. Biochem. Behav., submitted.

Wolf, P. ; Olpe, H.R.; Avrith, D. and Haas, H.L. GABAergic inhibition of neurons in the ventral tegmental area. Experentia 34:73-74, 1978.

Wong, E.H.F.; Kemp, J.A.; Priestly, T.; Knight, A.R.; Woodruff, G.N.; Iversen, L.L. The anticonvulsant MK801 is a potent N-methyl-D-aspartate antagonist. Proc. Nat. Acad. Sci. USA 83:7104-7108; 1986.

Wong, E.H.F. and Kemp, J.A. Sites for antagonism on the N- methyl-D-aspartate receptor channel complex. 31:401-425, 1991.

Yim, C.Y. and Mogenson, G.J. Effect of picrotoxin and nipecotic acid on inhibitory response of dopaminergic neurons in the ventral tegmental area to stimulation of the nucleus accumbens. Brain Res. 199:466-472, 1980.

Zaborsky, L. ; Heimer, L. ; Eckenstein, F. and LERANTH, C. GABAergic input to cholinergic basal forebrain neurones: an ultrastructural study using retrograde tracing of HRP and double immunolabelling. J. Comp. Neurol. 250: 282-295, 1986.