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The role of excitatory amino acids in the nucleus accumbens and subpallidal region in goal-directed locomotor activity

Shreve, Paul Edward, Ph.D.

The Ohio State University, 1989

UMI 300 N. ZeebRd. Ann Arbor, MI 48106 THE ROLE OF EXCITATORY AMINO ACIDS IN THE

NUCLEUS ACCUMBENS AND SUBPALLIDAL REGION

IN GOAL DIRECTED LOCOMOTOR ACTIVITY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Paul E. Shreve, B.S., R.Ph.

*****

The Ohio State University

1989

Reading Committee: Approved by:

Dr. Norman J. Uretsky

Dr. Allan M. Burkman

Dr. Dennis B. McKay Adviser & College of Pharmacy Dr. Lane J. Wallace DEDICATION

To my wife Sheila,

I have so much to thank you for, and yet, words can not begin to express how much you mean to me. You are more than my loving wife, you are also my best friend whom I can confide in and draw strength from. You have always been there when I needed love and support.

You encouraged and enabled me to achieve one of my major gc'ls in life. I am eternally grateful and indebted. Therefore, as an expression of my immeassurable love for you, I dedicate this dissertation to you.

11 ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to:

Dr. Norman J. Uretsky

for his support, instruction, and guidance throughout my years as a graduate student. I feel priviledged to have worked with such a skilled and knowledgeable scientist. His confidence in me has enabled my graduate career to be a productive endeavor. His friendship and sincere interest in me has made my life as a graduate student an enjoyable experience which I will always remember.

Drs. Burkman, McKay, and Wallace

for their constructive criticism of my work and suggestions in the preparation of this dissertation.

111 VITA

September 23, 1959...... Born - Pomeroy, Ohio, U.S.A.

1982...... B.S. in Pharmacy, College of Pharmacy, University of Cincinnati, Cincinnati, Ohio

1983-1985...... Graduate Teaching Associate The Ohio State University Columbus, Ohio

1986-1989...... Graduate Research Associate The Ohio State University Columbus, Ohio

HONORS

1982...... Eli Lilly Achievement Award

1981...... Rho Chi Honor Society

1981...... Presented paper at Morgantown Undergraduate Research Seminars Morgantown, West Virginia

1988...... ASPET Travel Award

PUBLICATIONS

Kolta, M., P. Shreve, and N.J. Uretsky (1985) Effect of methyl- phenidate pretreatment on the behavioral and biochemical responses to amphetmaine. Eur. J. of Pharmacol. 117:279-282.

Kolta, M.G., P. Shreve, V. De Souza, and N.J. Uretsky (1985) Time course of development of the enhanced behavioral and bio chemical response to amphetamine after amphetamine pretreatment. Neuropharmacology 24:823-829.

Shreve, P.E. and N.J. Uretsky (1988) 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.

iv Shreve, P.E. and N.J Uretsky (1988) Effect of GABAergic trans mission 1n the subpallidum on the hypermotility response to to Intraaccumbens administration of excitatory amino acids and plcrotoxin. Neuropharmacology 27:1271-1278.

Kolta, M.G., P.E. Shreve, and N.J. Uretsky (1989) Effect of pretreatment with amphetamine on the Interaction between amphetamine and dopamine neurons 1n the nucleus accumbens. Neuropharmacology 28:9-14.

Shreve, P.E. and N.J. Uretsky (1989) AMPA, kalnic acid, and N-methyl-D-aspart1c acid stimulate locomotor activity after Injection Into the substantia Innominata / lateral preoptic area. Pharmacol., Blochem., & Behav. (In press).

ABSTRACTS

Grund, V.R., E.C. Hyde, and P.E. Shreve (1981) Stimulation of Insulin secretion 1n dogs with the H2-histamine receptor agonist 1mprom1dine. Diabetes 30:122A.

Grund, V.R., E.C. Hyde, and P.E. Shreve (1981) H2-h1stamine receptors and Insulin secretion. The Pharmacologist 23:161.

Hyde, E.C., P.E. Shreve, and V.R. Grund (1982) Role of free fatty acids 1n histamine-induced insulin secretion in dogs. The Pharmacologist 24:215.

Kolta, M.G., P. Shreve, V. De Souza, and N.J. Uretsky (1984) Time course of development of the enhanced behavioral and bio chemical response to amphetamine after amphetamine pretreatment. The Pharmacologist 26:218.

Kolta, M., P. Shreve, and N.J. Uretsky (1985) Site of action of amphetamine (A) in enhancing locomotor activity (LMA) and stereotyped behavior (STB) in A-pretreated rats. The Pharmacologist 27:124.

Shreve, P.E. and N.J. Uretsky (1987) a-Amino-3-hydroxy-5- methylisoxazole-4-propionate (AMPA)-induced hypermotility after Intraaccumbens injection is antagonized by y-glutamyl- amlnomethlylsulfonate(GAMS). Fed. Proc. 46:710.

Shreve, P.E. and N.J. Uretsky (1988) Kainic acid (KA), a-amino- 3-hydroxy-5-methyl1soxazole-4-prop1onate (AMPA), and N-methyl-D- aspartlc add (NMDA) stimulate locomotor activity (LMA) after injection into the substantia innominata / lateral preoptic area (SI/LPO). Society for Neuroscience Abstracts 14:940.

v Boldry, R.C., P.E. Shreve, and N.J. Uretsky(1988) 6,7-Dinitro- quinoxaline-2,3-dione (DNQX) and y-glutamylaminomethylsulfo nate (GAMS) selectively inhibit the hypermotHity response produced by activation of qulsqualic acid (QA) receptors in the nucleus accumbens (NA). Society for Neuroscience Abstracts 14:940.

Shreve, P.E. and N.J. Uretsky (1988) Role of GABAerglc neurotrans mission in the substantia innominata / lateral preoptic area (SI/ LPO) in the hypermotility responses produced by excitatory amino acids (EAA) Injected into the nucleus accumbens (NA). The Pharmacologist 30:A25.

Field of Study: Pharmacology

vi TABLE OF CONTENTS

PAGE

DEDICATION...... 11

ACKNOWLEDGEMENTS...... 111

VITA...... iv

LIST OF TABLES...... xii

LIST OF FIGURES...... xiii

CHAPTER

I. INTRODUCTION...... 1

Excitatory Amino Acid Pharmacology...... 1

Endogenous Excitatory amino acids...... 1

Excitatory Amino Acid Receptor Classification...... 2

Effects of Excitatory Amino Acids on Neurons...... 6

Excitatory Amino Acids and Long Term Potentiation... 8

Excitatory Amino Acids and Neurotoxicity...... 9

Function and Anatomy of The Nucleus Accumbens...... 11

Function of the Nucleus Accumbens...... 11

Pharmacology of Excitatory Amino Acids in the Nucleus Accumbens ...... 13

Function of the Substantia Innominata / Lateral Preoptic area...... 15

Statement of the Problem...... 18

v i 1 CHAPTER

PAGE

II. Role of Quisqualic Acid Receptors in the Hypermotility Response Produced by the Injection of AMPA into the Nucleus Accumbens ...... 21

Introduction...... 21

Methods...... 24

Surgical Procedure...... 24

Monitoring Locomotor Activity...... 24

Histology...... 24

Radioligand Binding...... 26

Drugs...... 28

Statistics...... 28

Results

Effect of AMPA on Locomotor Activity in the Rat 29

Effect of GAMS on AMPA - Stimulated Locomotor Activity in the Rat...... 29

Effect of GAMS on Quisqualic Acid - Stimulated Locomotor Activity in the Rat...... 30

Effect of GAMS on Kainic Acid - Stimulated Locomotor Activity in the Rat...... 35

Effect of GAMS on N-methyl-D-aspartic Acid - Stimulated Locomotor Activity in the Rat...... 35

Comparison of the Effect of an N-methyl-D-aspartic Acid Receptor Antagonist, D-alpha-Aminoadipic Acid, on AMPA - and N-methyl-D-aspartic acid - Stimulated Locomotor Activity in the Rat...... 39

Effect of AMPA, Quisqualic Acid, and GAMS on Total AMPA Binding ...... 39

D iscussion...... 42

v i 1 i CHAPTER

PAGE

III. Effect of GABAergic Transmission in the Ventral Pallidum on the Hypermotility Response to the Intraaccumbens Administration of Excitatory Amino Acids and Picrotoxin...... 46

Introduction...... 46

Methods...... 49

Surgical Procedure...... 49

Monitoring Locomotor Activity...... 50

Catalepsy ...... 50

Histology...... 51

Drugs...... 51

Statistics...... 52

Results...... 52

Effect of Injection of Picrotoxin and Muscimol into the SI/LPO on the Locomotor Activity in the Rat...... 52

Effect of the Administration of Muscimol into the SI/LPO on the Hypermotility Response to the Intraaccumbens Administration of AMPA...... 53

Effect of the Administration of Muscimol into the SI/LPO on the Hypermotility Responses to the Intraaccumbens Administration of Amphetamine, Picrotoxin, AMPA, Kainic Acid, and N-methyl-D- Aspartlc Acid ...... 54

Effect of the Injection of Muscimol into the Nucleus Accumbens on the Hypermotility Responses Induced by the Intraaccumbens Administration of AMPA or Picrotoxin...... 54

Discussion...... 61

1 x CHAPTER

PAGE

IV. AMPA, Kainic Acid, and N-methyl-D-aspartic Acid Stimulate Locomotor Activity after Injection into Substantia Innominata / Lateral Preoptic Area...... 66

Introduction...... 66

Methods...... 68

Surgical Procedure...... 68

Monitoring Locomotor Activity...... 69

Histology...... 69

Drugs...... 70

Statistics...... 71

Results...... 71

Effect of AMPA, Kainic Acid, and N-methyl-D- Aspartic Acid on Locomotor Activity in the Rat 71

Effect of GAMS on AMPA-, Kainic Acid-, and N-methyl-D-aspartic Acid- Stimulated Locomotor Activity in the Rat...... 72

Effect of DNQX on AMPA-, Kainic Acid, and N-methyl-D-aspartic Acid- Stimulated Locomotor Activity in the Rat...... 80

Effect of D-a-Aminoadip1c Acid on AMPA- and N-methyl-D-aspartic Acid- Stimulated Locomotor Activity in the Rat...... 80

Discussion...... 81

x CHAPTER PAGE

V. Effect of Glutamatergic Transmission in the Subpallidum on the Hypermotility Response to the Intraaccumbens Administration of AMPA and Amphetamine...... 87

Introduction...... 87

Methods...... 89

Surgical Procedure...... 89

Monitoring Locomotor Activity...... 90

Histology...... 91

Drugs...... 91

Statistics...... 93

Results...... 93

Effect of the Administration of GAMS into the SI/LPO on the Hypermoti1ity Responses to the Administration of AMPA or Amphetamine into the Nucleus Accumbens ...... 93

Effect of the Administration of DNQX Into the SI/LPO on the Hypermoti1ity Responses to the Administration of AMPA or Amphetamine into the Nucleus Accumbens ...... 94

Effect of the Administration of DAA into the SI/LPO on the Hypermoti1ity Responses to the Administration of AMPA or Amphetamine into the Nucleus Accumbens ...... 103

Effect of Muscimol on AMPA Stimulated Locomotor Activity in the SI/LPO...... 103

Effect of DNQX, GAMS, or D-a-aminoadipic acid on Picrotoxin Stimulated Locomotor activity in the SI/LPO...... 104

Discussion...... 105

VI. Summary...... Ill

List of References...... 116 LIST OF TABLES

TABLE PAGE

1. Pharmacology of Excitatory Amino Acids...... 6

2. Effect of Different Doses of AMPA on the GAMS - Induced Inhiblton of AMPA - Stimulated Locomotor Activity ...... 33

3. Effect of GAMS on Quisqualic Acid - Stimulated Locomotor Activity after Bilateral Injection into the Nucleus Accumbens ...... 34

4. Effect of D-Alpha-aminoad1pic Acid (DAA) on AMPA and N-methyl-D-aspartic Acid (NMDA) - Stimulated Locomotor Activity after Bilateral Injection into the Nucleus Accumbens ...... 40

x ii LIST OF FIGURES

FIGURE - PAGE

1. Effect of AMPA on locomotor activity after bilateral ■ injection into the nucleus accumbens...... 31

2. Effect of GAMS on AMPA - stimulated locomotor activity after bilateral injection into the nucleus accumbens 32

3. Effect of GAMS on kainic acid - stimulated locomotor activity after bilateral injection into the nucleus accumbens ...... 36

4. Effect of GAMS on N-methyl-D-aspartic acid - stimu lated locomotor activity after bilateral injection into the nucleus accumbens...... 37

5. Effect of GAMS on picrotoxin - stimulated locomotor activity after bilateral injection into the nucleus accumbens ...... 38

6. Effect of AMPA, quisqualic acid, and GAMS on total AMPA binding ...... 41

7. Sites of injection of muscimol 1n the SI/LPO...... 56

8. Effect of muscimol or picrotoxin on locomotor activity after bilateral injection into the SI/LPO 57

9. Locomotor activity response to intraaccumbens AMPA following injection of different doses of muscimol into the SI/LPO...... 58

10. Locomotor activity responses to the intraaccumbens administration of amphetamine, picrotoxin, AMPA, kainic acid, and N-methyl-D-aspartic acid following injection of muscimol into the SI/LPO...... 59

11. Effect of the administration of muscimol into the nucleus accumbens on the hypermoti1ity responses to the intraaccumbens injections of AMPA and picrotoxin...... 60

12. Sites of Injection of AMPA, kainic acid, or N-methyl- D-aspartic acid in the SI/LPO ...... 73

x 111 FIGURE PAGE

13. Effect of AMPA on locomotor activity after bilateral Injection Into the SI/LPO...... 74

14. Effect of kainic acid on locomotor activity after bilateral injection into the SI/LPO...... 75

15. Effect of N-methyl-D-aspartic acid on locomotor activity after bilateral injection into the SI/LPO...... 76

16. Effect of GAMS on AMPA-, kainic acid-, and N-methyl- D-aspartic acid- stimulated locomotor activity after bilateral injection into the SI/LPO...... 77

17. Effect of DNQX on AMPA-, kainic acid-, and N-methyl- D-aspartic acid- stimulated locomotor activity after bilateral injection Into the SI/LPO...... 78

18. Effect of D-o-aminoadipic acid on AMPA- or N-methyl- D-aspartic acid- stimulated locomotor activity after bilateral injection into the SI/LPO...... 79

19. Sites of Injection of muscimol into the SI/LPO...... 92

20. Locomotor activity responses to the intraaccumbens administration of amphetamine or AMPA following injection of GAMS Into the SI/LPO...... 95

21. Locomotor activity responses to the intraaccumbens administration of amphetamine or AMPA following injection of DNQX into the SI/LPO...... 96

22. Locomotor activity responses to the intraaccumbens administration of amphetamine or AMPA following injection of DAA into the SI/LPO...... 97

23. Effect of muscimol on AMPA - stimulated loco motor activity after bilateral injection into the SI/LPO...... 98

24. Effect of GAMS on picrotoxin - stimulated loco motor activity after bilateral injection into the SI/LPO...... 99

25. Effect of DNQX on picrotoxin - stimulated loco motor activity after bilateral injection into the SI/LPO...... 100

x i v FIGURE PAGE

26. Effect of DAA on picrotoxin - stimulated loco motor activity after bilateral injection into the SI/LPO...... 101

27. Effect of DAA and GAMS on picrotoxin - stimulated locomotor activity after bilateral injection into the SI/LPO...... 102

xv CHAPTER I

INTRODUCTION

EXCITATORY AMINO ACID PHARMACOLOGY

ENDOGENOUS EXCITATORY AMINO ACIDS

Glutamate 1s an excitatory amino acid which 1s present 1n great abundance 1n the central nervous system and functions 1n many roles

(McGeer, Eccles, and McGeer, 1987). Glutamate 1s Incorporated Into proteins and peptides which serve as fundamental structural units 1n all phases of chemical and physical activity of cells. Glutamate 1s

Involved 1n fatty acid synthesis, contributes (with glutamine) to the regulation of ammonia levels, and serves as a precursor to GABA and various Intermediates of the Krebs cycle. Glutamate also serves as a chemical constituent of glutathione and folic acid which are

Important cofactors 1n many enzymatic reactions. Thus, glutamate has many different roles 1n the central nervous system.

This multiplicity of functions of glutamate led to the initial skepticism toward considering glutamate a neurotransmitter 1n the central nervous system. However, 1n the years since Hyashl (1954)

Initially observed the excitatory effects of glutamate on cerebral cortical cells, glutamate has now met many of the criteria necessary 1 2

for neurotransmitter status. Thus, glutamate is released from

neurons in a calcium dependent manner in response to elevated

potassium (Cotman and Nadler, 1981). The direct application of

glutamate to neurons has been shown to elicit powerful excitatory

effects (Johnson, 1978; Shank and Aprison, 1988). Glutamate is also

transported with high affinity into the nerve terminals of several

neuronal pathways in the central nervous system (McGeer et.al.,

1987). Additionally, radioligand binding studies have demonstrated

the presence of selective binding sites for glutamate (Honore,

Lauridsen, and Krogsgaard-Larsen, 1982; Monaghan, Yao, Watkins, and

Cotman, 1984; Drejer and Honore, 1988). Although the evidence

supporting glutamate as a neurotransmitter is not as complete as

that for several other compounds, glutamate appears to be the main candidate for an excitatory neurotransmitter in the central nervous

system.

EXCITATORY AMINO ACID RECEPTOR CLASSIFICATION

The consideration of glutamate as a potential excitatory neurotransmitter necessitates the demonstration that glutamate is exerting its excitatory properties by acting at specific receptors.

In the early 1960s, N-methyl-D-aspartic acid was found to elicit a more potent excitatory effect on neurons than glutamate. (Watkins and Olverman, 1987). Since the chemical structure of N-methyl-D- aspartic acid was similar to glutamate, it was thought that the actions of these compounds were mediated by the activation of the 3 same receptor. Much research has ensued in an attempt to find specific antagonists of the excitatory effects of glutamate and its analogs in order to more definitively show that glutamate was acting at specific receptors.

Despite many years of research, the progress in developing both selective agonists and antagonists for the receptors that are activated by glutamate has been slow. There is presently thought to be at least three excitatory amino acid receptor subtypes (Table 1) which have been characterized by the following agonists: N-methyl-D- aspartic acid, kainic acid, and quisqualic acid (Cotman and

Monaghan, 1987). a-Amino-3-hydroxy-5-methyli soxazole-4-propionate

(AMPA) is a glutamate analog which is now utilized as a selective agonist for the quisqualic acid receptor (Honore, et.al., 1982). A fourth receptor subtype which is activated by ibotenic acid and antagonized by L-2-amino-4-phosphonobutyrate (AP4) may also exist

(Cotman and Monaghan, 1987). Although the use of agonists has been useful in delineating this receptor classification, the lack of availability of selective antagonists for all of these receptor subtypes has hindered attempts to confirm this classification scheme.

The N-methyl-D-aspartic acid receptor subtype has been the most extensively studied receptor because of the availability of selective antagonists for this receptor. These antagonists can be classified into competitive and non-competitive N-methyl-D-aspartic acid receptor antagonists. The competitive antagonists produce their inhibitory effects by binding directly to the recognition site of the N-methyl-D-aspartic acid receptor. The most potent competitive N-methyl-D-aspartic acid receptor antagonists are the phosphonocompounds of mono- or dicarboxylic acids which include

D-2-ami no-5-phosphonovalerate (AP5), D-2-ami no-7-phosphonoheptanoate

(AP7), and 3-(2-carboxypiperazam-4-yl)-propyl-l-phosphonate (CPP)

(Drejer and Honore, 1988). Another selective competitive N-methyl-

D-aspartic acid antagonist is D-a-aminoadipic acid. In contrast, the non-competitive N-methyl-D-aspartic acid receptor antagonists

Inhibit the effects of an agonist at the N-methyl-D-aspartic acid receptor by acting at a site different from the agonist recognition site. There are two classes of non-competitive N-methyl-D-aspartic acid receptor antagonists which include MK801 and phencyclidine in the first class, and magnesium, a divalent cation, in the second class (Kemp, Foster, and Wong, 1987). MK801 or phencyclidine, and magnesium, are thought to act at two distinct sites on the ion channel associated with the N-methyl-D-aspartic acid receptor to prevent ion passage. Thus, it has been shown that competitive and non-competitive N-methyl-D-aspartic acid receptor antagonists can selectively inhibit the responses to N-methyl-D-aspartic acid while having no significant effect on those responses elicited by kainic acid or quisqualic acid (Watkins and Olverman, 1987).

In contrast, there are presently no well accepted antagonists for the kainic acid and quisqualic acid receptor subtyes. As a result, these receptors are sometimes collectively refered to as non-N-methyl-D-aspartic acid receptors because the known antagonists for these kainic acid and quisqualic acid receptors often overlap in their spectrum of inhibitory effects. Thus, while these antagonists do not inhibit the excitatory response elicited by N-methyl-D- aspartic acid, they often inhibit the responses produced by either kainic acid or quisqualic acid. Therefore, these non-N-methyl-D- aspartlc acid receptor antagonists have proved useful in distinguishing between N-methyl-D-aspartic acid versus non-N-methyl-

D-aspartic acid receptor mediated responses but have not facilitated the study of the specific responses to kainic acid or quisqualic acid. These non-N-methyl-D-aspartic acid antagonists include N-(p- chlorbenzoyl )piperazine-2,3-dicarboxyl ate (pCB-PzDA), Jf-D-glutamyl ami nomethyl sulfonate (GAMS), Jf-glutamyl taurine, and two recently introduced compounds: 6,7-dinitroquinoxaline-2,3-dione

(DNQX) and 6-cyano-7-dinitro-quinoxaline-2,3-dione (CNQX) (Drejer and Honore, 1988; Honore, Davies, Drejer, Fletcher, Jacobsen, Lodge, and Nielsen, 1988; Stephens, Lee, Boldry, and Uretsky, 1986).

Indeed, GAMS, as well as DNQX and CNQX, have been described as potent non-N-methyl-D-aspartic acid receptor antagonists (Fagg,

1985; Davies, Evans, Jones, Smith, and Watkins, 1982; Honore et.al.,

1988). Preliminary behavioral studies suggest that GAMS and DNQX may selectively inhibit the hypermoti11ty responses elicited by the activation of quisqualic acid receptors in the nucleus accumbens

(Shreve and Uretsky, 1987; Boldry, Shreve, and Uretsky, 1988). Therefore, the evidence to date supports the presence of at

least three excitatory amino acid receptor subtypes refered to as N-

methyl-D-aspartic acid, kainic acid, and quisqualic acid receptors.

Although electrophysiological, binding, and behavioral studies have

contributed to this classification, the search and procurement of

more selective antagonists of the kainic acid and quisqualic acid

receptors will greatly enhance our understanding of the actions

mediated by each of these receptor subtypes.

TABLE 1

PHARMACOLOGY OF EXCITATORY AMINO ACID RECEPTORS

NMDA KA QA

Agonists NMDA KA QA L-glutamate L-glutamate L-glutamate L-aspartate AMPA

Antagonists AP5 GAMS GAMS AP7 DNQX DNQX DAA CNQX CNQX CPP

Abbreviations: NMDA= N-methly-D-aspartic acid, KA= kainic acid, QA= quisqualic acid, AMPA=a-amino-3-hydroxy-5-methylisoxazole- 4-propionate, AP5= D-2-amino-5-phosphonovalerate, AP7= D-2-amino- 7-phosphonoheptanoate, CPP= 3-(2~carboxypiperazam-4-yl)-propyl- 1-phosphonate, DAA= D-a-aminoadipic acid, GAMS ^ -g lu ta myl ami nomethyl sulfonate, DNQX= 6,7-dinitroquinoxaline-2,3-dione, CNQX= 6-cyano-7-dinitro-quinoxaline-2,3-dione.

EFFECTS OF EXCITATORY AMINO ACIDS ON NEURONS.

The use of pharmacological antagonists has provided evidence that the excitatory amino acid receptors can be divided into N- methyl-D-aspartic acid and non-N-methyl-D-aspartic acid receptor

subtypes. Recent electrophysiological studies have provided further

support of this general classification scheme. Thus, evidence

suggests that these excitatory amino acid receptor subtypes can be

distinquised upon the basis of different properties of their

respective ion channels.

While the application of either N-methyl-D-aspartic acid, kainic

acid, or quisqualic acid to a neuron elicits an excitatory

postsynaptlc potential (EPSP), there is a difference in the

characteristics of these EPSPs (MacDermott and Dale, 1987; Ascher

and Nowak, 1987). First, kainic acid and quisqualic acid elicit

fast EPSPs whereas N-methyl-D-aspartic acid elicits a slower and

more prolonged EPSP. Secondly, although the stimulation of all

three receptor subtypes is associated with an increased permeability

to sodium, the stimulation of the N-methyl-D-aspartic receptor is

associated with an increase in the concentration of free calcium

while the stimulation of the kainic acid or quisqualic acid receptor

does not produce an increase in intracellular calcium. Another

difference is that magnesium produces a blockade of the N-methyl-D-

aspartic acid channel in a voltage dependent manner while having no

significant effect on kainic acid or quisqualic acid ion channels.

It has also been shown that glycine can selectively enhance the effects of N-methyl-D-aspartic acid possibly by binding to an allosteric regulatory site on the N-methyl-D-aspartic acid receptor complex. Thus, these differences in the electrophysiology of excitatory amino acid receptors support the pharmacological separation of N-methyl-D-aspartic acid and non-N-methyl-D-aspartic acid receptors.

EXCITATORY AMINO ACIDS AND LONG TERM POTENTIATION

The aforementioned differences in electrophysiological characteristics of N-methyl-D-aspartic acid and non-N-methyl-D- aspartic acid receptors coupled with studies which have utilized N- methyl-D-aspartic acid receptor antagonists has led to the idea that the N-methyl-D-aspartic acid receptor may play an integral role in long term potentiation (Collingridge and Bliss, 1987). Long term potentiation is a long lasting increase in synaptic transmission that follows a brief period of high frequency stimulation of neurons. This increase in synaptic transmission can be maintained for a prolonged period of time, possibly as long as several weeks.

Thus, long term potentiation has been a very intriguing concept to neuroscientists and may provide the physiological basis for infor mation storage in the brain.

Excitatory amino acids are thought to play an important role in long term potentiation. Evidence from hippocampal neurons suggest that the neuron is initially depolarized, possibly by glutamate acting at kainic acid or quisqualic acid receptors. This depolarization would remove the magnesium block of ion channels associated with the N-methyl-D-aspartic acid receptor system allowing calcium to enter the neuron and initiate the sequence of 9

events that result in enhanced synaptic transmission. Thus, it

appears that the N-methyl-D-aspartic acid receptor is involved in

the initiation of the long term potentiation. This hypothesis is

supported by the observation that the potent N-methyl-D-aspartic

acid antagonist, AP5, can inhibit the formation of the long term

potentiation. Although the N-methyl-D-aspart1c acid receptor may

play a pivotal role in the initiation of the long term potentiation,

the mechanism for maintaining the long term potentiation is less

well understood. However, maintenance of the long term potentiation

may involve an Increase presynaptic release of neurotransmitter, an

increase 1n postsynaptic receptors, and / or a morphological change

at the synapse (Col 1ingridge and Bliss, 1987). As more knowledge is

acquired, the role of excitatory amino acids in long term

potentiation should prove useful in the study of learning and

memory.

EXCITATORY AMINO ACIDS AND NEUROTOXICITY

Another important area of pharmacological interest is the

excltotoxic effects of excitatory amino acids on neurons. The

injection of high doses of various excitatory amino acids into discrete brain regions produces a lesion characterized by the destruction of cell bodies while sparing nerve endings and axons of

passage. Additionally, kainic acid has the potential to produce damage in areas remote from the site of injection of the amino acid.

This remote damage appears to depend upon epileptiform activity, 10 possibly causing the release of an endogenous excitatory amino acid from nerve terminals in the remote area. Although the mechanism of the toxic effect of excitatory amino acids is not fully understood, it has been proposed that it may represent an extension of their depolarizing effects (Olney, 1978; McGeer, et.al., 1987). Thus depolarization of the neuron leads to the opening of ion channels resulting in the passage ofsodium and calcium ions across the neuronal membrane into the neurons. When present in high concentrations, the excitatory amino acids are thought to continuously excite the cell, causing excessive intraneuronal accumulation of sodium and calcium ions, leading to a depletion of energy stores and ultimately cell death.

It has been postulated that excitotoxic effects may play a role in brain damage associated with anoxia, stroke, hypoglycemia, epilepsy, and neurodegenerative illnesses such as Huntington's and

Alzheimer's disease (Rothman and Olney, 1987; Stone, 1987).

Glutamate is thought to be the endogenous excitatory amino acid responsible for the neuropathological damage which occurs during ischemia due to anoxia, stroke, or hypoglycemia. Studies suggest that glutamate release is enhanced while glutamate reuptake is diminished, resulting in an accumulation of glutamate in the extracellular space. This accumulation of glutamate then leads to excessive depolarization and neuronal death (Bradford and Peterson,

1988). Supporting this concept is the observation that N-methyl-D- aspartic acid receptor antagonists protect neurons from these 11

pathological insults. The involvement of glutamate in epilepsy is

thought to be twofold. First, glutamate may play a role in the

initiation of the primary abnormal focus of the epileptic seizure.

Secondly, glutamate may also be involved in the spread of this

seizure activity by being released from excitatory glutamatergic

pathways at remote sites (Bradford and Peterson, 1988). Glutamate

may also be involved in Huntington's and Alzheimer's diseases by

producing neurodegeneration of cells in the striatum and the nucleus

basali-s, respectively, although the evidence for this involvement is

not conclusive (McGeer, et.al. 1987; Stone, 1987).

FUNCTION AND ANATOMY OF THE NUCLEUS ACCUMBENS

FUNCTION OF THE NUCLEUS ACCUMBENS

The nucleus accumbens is a forebrain region which appears to be

strategically located between the limbic and motor areas of the

brain (Mogenson, 1984). The anatomical organization of the nucleus

accumbens is such that it receives afferent neural inputs from

limbic structures, such as the hippocampus, amygdala, and the

septum, as well as from the dorsal raphe nucleus, ventral tegmental area, and the frontal cortex (Domeslck, 1981). Biochemical evidence has suggested that the cortical and limbic inputs to the nucleus accumbens utilize glutamate as their neurotransmitter whereas the ventral tegmental area and raphe nucleus inputs utilize dopamine and serotonin respectively (Fonnum and Walaas, 1981). The nucleus accumbens also has important neural outputs which project to the 12 globus pallidus, subpallidal regions (substantia innominata and lateral preotic area), the substantia nigra, and the ventral tegmental area (Fonnum and Walaas, 1981). These neural projections from the nucleus accumbens are thought to use GABA as their neurotransmitter. The nucleus accumbens also contains cholinergic,

GABAergic, and glutamatergic interneurons (Fonnum and Walaas, 1981;

Watkins and Evans, 1981).

These neural connections of the nucleus accumbens have led to the concept that the nucleus accumbens may serve as a functional interface between the limbic and motor systems (Mogenson, Jones, and

Yim, 1980; Mogenson and Yim, 1981). Indeed, the nucleus accumbens has been implicated in such psychomotor disorders as schizophrenia,

Parkinson's disease, and Huntington's disease because of its neural connections with the limbic and motor system (Stevens, 1979; Price,

Forley, and Hornykiewicz, 1978; Hayden, 1981; Bots and Bruyn, 1981).

Limbic structures are thought to initiate skeleto-motor responses as part of complex behavioral acts, such as attack, defense escape reactions, food procurement, and mating behavior. Therefore, it is thought that these limbic structures send pertinent "motivational" information to the nucleus accumbens. The nucleus accumbens would then relay information to the basal ganglia in such a manner as to elicit the appropriate "action" response. In this model, the nucleus accumbens serves as a functional interface between the limbic and motor systems, and in simplest terms, converts motivational stimuli into the necessary action responses. 13

Thus, the nucleus accumbens is thought to play an important role

in the initiation and regulation of normal locomotor activity

(Mogenson, 1984). Indeed, the injection of many pharmacological

agents into the nucleus accumbens will elicit a marked hypermotility

response. Thus it has been shown that the direct administration of

dopamine, carbachol, picrotoxin, and various excitatory amino acids

into the nucleus accumbens can produce a stimulation of locomotor

activity (Pljnenburg, Honig, Van derHayden, and Van Rossum, 1976;

Mogenson and Nielsen, 1983; Morgenstern, Mende, Gold, Lemme, and

Oelssner, 1984; Donzanti and Uretsky, 1983; Austin and Kalivas,

1988). In addition, neural mechanisms in the nucleus accumbens have

been shown to mediate the locomotor activating properties of heroin

and amphetamine (Swerdlow, Vaccarino, Amalric, and Koob, 1986). It

has been proposed that the nucleus accumbens may play a major role

1n initiating and maintaining the positive reinforcing properties of

these drugs and that these properties are related to the locomotor

stimulation produced by these drugs. Therefore, the measurement of

locomotor activity can be used as an experimental model to determine

the involvement of the nucleus accumbens in the initiation and

regulation of locomotor behavior.

PHARMACOLOGY OF EXCITATORY AMINO ACIDS IN THE NUCLEUS ACCUMBENS

The direct injection of the excitatory amino acids, N-methyl-D- aspartic acid, quisqualic acid (or AMPA) or kainic acid, into the the nucleus accumbens has been shown to produce marked dose- 14

dependent increases in locomotor activity (Arnt, 1981; Donzanti and

Uretsky, 1983). At high doses of these excitatory amino acids,

there are subsequent decreases in locomotor activity which are

thought to occur because of the induction of seizure like behavior

by these agents. The hypermotility produced by N-methyl-D-aspartic

acid was selectively inhibited by D-o-aminoadipic acid or magnesium

(Donzanti and Uretsky, 1984a; Donzanti and Uretsky, 1984b).

Although there are presently no selective antagonists available for

both kainic acid and quisqualic acid receptors, GAMS has been shown

to selectively inhibit the hypermoti11ty responses to quisqualic

acid and AMPA while having having no significant effect on kainic

acid or N-methyl-D-aspartic acid stimulated locomotor activity

(Shreve and Uretsky, 1988a). While these studies suggest that the

activation of excitatory amino acid receptors mediate the

hypermotility responses, more selective antagonists are needed to

determine the exact involvement of specific excitatory amino acid

receptors in modulating locomotor activity in the nucleus accumbens.

Drugs which interfere with the function of dopaminergic pathways

in the nucleus accumbens also inhibit excitatory amino acid induced

locomotor activity. Arnt (1981) initially demonstrated that the

locomotor activity produced by the intraaccumbens injection of AMPA was inhibited by flupenthixol, a dopamine receptor antagonist. In agreement with this finding, the injection of another dopamine

receptor antagonist, fluphenazine, into the nucleus accumbens

inhibited kainic acid, quisqualic acid, and N-methyl-D-aspartic acid induced hypermoti1ity (Donzanti and Uretsky, 1983). The systemic

administration of reserpine, which depletes catecholamine stores,

or a-methyl-p-tyrosine, which Inhibits dopamine synthesis, has also

been shown to inhibit the hypermotility responses produced by AMPA,

kainic acid, and N-methyl-D-aspartic acid (Arnt, 1981; Boldry and

Uretsky, 1988; Donzanti and Uretsky, 1983). These results, taken

together, suggest that excitatory amino acids may mediate their

locomotor activating properties by releasing dopamine from nerve

terminals in the nucleus accumbens. However, although an initial

study has shown that 1-glutamate (in high concentrations) is capable

of releasing dopamine (Roberts and Anderson, 1979), a recent study

has demonstrated that N-methyl-D-aspartic acid does not stimulate

endogenous dopamine release from nucleus accumbens slices (Boldry

and Uretsky, 1988). This observation suggests that while the

hypermotility responses produced by excitatory amino acids involve

the activation of dopamine receptors by endogenous dopamine, the

excitatory amino acids do not appear to cause the release of

endogenous dopamine from nerve terminals in this region. Rather,

dopaminergic neurotransmission may play a permissive effect in the

excitatory amino acid induced hypermotility response.

FUNCTION OF THE SUBSTANTIA INNOMINATA / LATERAL PREOPTIC AREA

The substantia innominata / lateral preoptic area (SI/LPO) is a

subpall 1 dal region which appears to be strategically located between the nucleus accumbens and motor areas of the brain (Swanson, Mogenson, Gerfen, and Robinson, 1984; Swerdlow and Koob, 1987). The

anatomical organization of the SI/LPO is such that it receives a

large GABAerglc projection from the nucleus accumbens (Jones and

Mogenson, 1980b; Mogenson and Nielsen, 1983; Mogenson, Swanson, and

Wu, 1983; Nauta, Smith, Faull, and Domesick,1978). Recent evidence

suggests that a glutamatergic (excitatory amino acid) projection

from the amygdala also synapses in the SI/LPO (Fuller, Russchen, and

Price, 1987; Davies, McBean, and Roberts, 1984; Halpain, Wieczorek,

and Rainbow, 1984; Monaghan and Cotman, 1985). The SI/LPO also has

important neural outputs which project to twolocomotor regions, the

pedunculopontine nucleus and the dorsomedial nucleus of the thalamus

(Young, Alheid, and Heimer, 1984; Swerdlow and Koob, 1987; Swanson,

1976; Swanson et.al., 1984; Brudzynski and Mogenson, 1985;

Brudzynski, Wu, and Mogenson, 1988). Although the neurotransmitter

for the projection to the dorsomedial nucleus of the thalamus is

presently not known, acetylcholine appears to be the

neurotransmitter of the projection to the pedunculopontine nucleus

(Brudzynski et.al., 1988). The SI/LPO also sends important

cholinergic efferent fibers to the cortex (Bigl, Woolf, and Butcher,

1982; Casamenti, Deffenu, Abbamondi, and Pepeu, 1986) and contains

nerve cell bodies which cross-react with anti-sera to corticotropin

releasing factor, somatostatin, and substance P (Swanson et.al.,

1984).

Recent evidence suggests that the GABAerglc projection from the nucleus accumbens to the SI/LPO is involved in the hypermoti11ty 17 produced by drugs which act in the nucleus accumbens (Jones and

Mogenson, 1980a; Mogenson and Nielsen, 1983; Mogenson et.al., 1983;

Nauta et.al., 1987). Thus it has been shown that the injection of muscimol or GABA into the SI/LPO inhibited the locomotor activity produced by the activation of dopamine or opioid receptors in the nucleus accumbens (Mogenson and Nielsen, 1983; Swerdlow and Koob,

1984, Swerdlow et.al., 1986). These observations suggest that the hypermotility responses produced by increases in dopaminergic and opioid neurotransmission in the nucleus accumbens may be mediated by a decrease in GABAergic activity in the SI/LPO. Therefore, the injection of muscimol into the SI/LPO would activate GABAergic receptors at this site and decrease these hypermoti11ty responses.

The SI/LPO is thought to mediate the behavioral expression of locomotor activity produced in the nucleus accumbens through efferent projections to two motor areas, the dorsomedial nucleus of the thalamus and the pedunculopontine nucleus, which are believed to directly innervate spinal motor circuitry (Swerdlow, Swanson, and

Koob, 1984; Brudzynski and Mogenson, 1985; Brudzynski, Wu, and

Mogenson, 1988). Thus it has been shown that ibotenic acid - induced lesions of the dorsomedial nucleus of the thalamus inhibited the hypermotility responses elicited by the stimulation of dopamine receptors in the nucleus accumbens or by the injection of picrotoxin into the SI/LPO (Swerdlow and Koob, 1987). The involvement of the pedunculopontine nucleus is not as clear since lesions of this nucleus did not significantly effect these hypermotility resonses 18

(Swerdlow and Koob, 1987). However, another study has shown that

the injection of procaine into the pedunculopontine nucleus

inhibited the locomotor activity produced by the injection of

amphetamine into the nucleus accumbens (Brudzynski and Mogenson,

1985). This suggests that the pedunculopontine nucleus is involved

in ampetamine induced locomotion. The activation of dopamine

receptors in the nucleus accumbens by amphetamine may result in the

activation of cholinergic fibers which project from the SI/LPO to

the pedunculopontine nucleus (Brudzynski et.al., 1988).

Therefore, the SI/LPO appears to play an important role in the

behavioral expression of the hypermotility responses produced by the

activation of dopamine or opioid receptors in the nucleus accumbens.

However, it remains to be shown whether the locomotor activity

elicited by the activation of excitatory amino acid receptors in the

nucleus accumbens is mediated through the SI/LPO. It also remains

to be shown what role the excitatory amino acid receptors in the

SI/LPO play in regards to modulating locomotor activity initiated in

the nucleus accumbens.

STATEMENT OF THE PROBLEM

The nucleus accumbens is a region of forebrain which receives

neuronal input from limbic areas and is involved in the stimulation of locomotor activity. The stimulation of locomotor activity may

involve a major GABAergic projection from the nucleus accumbens to the substantia innominata / lateral preoptic area (SI/LPO). Nerve 19 terminals containing glutamate, believed to be the main excitatory amino acid neurotransmitter, are found in the nucleus accumbens and the SI/LPO. The major objective of this dissertation is to determine the role of excitatory amino acid receptors in the nucleus accumbens and the SI/LPO in the stimulation of locomotor activity.

The specific Aims of this dissertation are:

1. To determine the role of quisqualic acid receptors in the hypermotility response produced by the injection of AMPA into the nucleus accumbens. This was done by studying the effects of the injection of AMPA and quisqualic acid (in the presence and absence of excitatory amino acid antagonists) into the nucleus accumbens on locomotor activity.

2. To determine whether excitatory amino acid induced locomotor activity in the nucleus accumbens is mediated by a decrease in

GABAergic activity in the SI/LPO. This was done by studying the effects of the direct injection of muscimol into the SI/LPO on the hypermotility produced by the intraaccumbens administration of various excitatory amino acids.

3. To determine the function of excitatory amino acids in the SI/LPO on hypermotility responses. This was done by studying the effects of the direct injection of excitatory amino acids in the SI/LPO on hypermotility. I observed that the injection of excitatory amino acids into this subpallidal region elicited a hypermotility response. Thus excitatory amino acids were then injected into the

SI/LPO in the presence and absence of excitatory amino acid 20 antagonists to determine if these hypermotility responses were being mediated by the stimulation of specific excitatory amino acid receptors.

4. To determine whether the initiation of locomotor activity in the nucleus accumbens is mediated by an increase in excitatory amino acid (glutamatergic) neurotransmission in the SI/LPO. This was done by studying the effects of the direct injection of excitatory amino acid antagonists into the SI/LPO on the hypermotility produced by the intraaccumbens administration of AMPA and amphetamine. CHAPTER I I

ROLE OF QUISQUALIC ACID RECEPTORS IN THE

HYPERMOTILITY RESPONSE PRODUCED BY THE

INJECTION OF AMPA INTO ‘THE NUCLEUS ACCUMBENS.

INTRODUCTION

The nucleus accumbens 1s a forebrain region Involved 1n the initiation and regulation of normal locomotor activity (Mogenson and

Y1m, 1980). Biochemical evidence suggests that excitatory amino acids 1n the nucleus accumbens may function as neurotransmitters at the terminals of Interneurons, as well as of neurons derived from the allocortex and the frontal cortex (Watkins and Evans, 1981;

Fonnum and Walaas, 1980). Consistent with this hypothesis, it was found that the Intraaccumbens Injection of the glutamate analogues, kainic acid and quisqualic add, as well as N-methyl-D-aspartic add, an analogue of aspartate, produced a marked hypermotility response which was Inhibited by antagonists of excitatory amino add receptors (Donzanti and Uretsky, 1984; Hamilton, deBelleroche,

Gardiner, and Herberg, 1986). There 1s presently thought to be at least three distinct excitatory amino acid receptors referred to as quisqualic add, kainic acid, and N-methyl-D-aspartic ad d subtypes

(McLennan, 1981; Watkins and Evans, 1981). However, 1t 1s still 21 22

unclear whether the behavioral responses to these compounds are

mediated by specific receptors in the nucleus accumbens because of

the lack of selective antagonists at these receptor subtypes.

a-Am1no-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) is an

excitatory amino acid which on the basis of electrophysiological

(Krogsgaard-Larsen, Honore, Hansen, Curtis, and Lodge, 1980) and

binding studies (Honore, Lauridsen, and Krogsgaard-Larsen, 1982)

appears to selectively activate quisqualic acid receptors. An

autoradiographic study (Monaghan, Yao, and Cotman, 1984) has shown a

significant density of ( 3H)-AMPA binding sites in the nucleus

accumbens suggesting the presence of quisqualic acid receptors in

the nucleus accumbens. Consistent with this hypothesis, AMPA has

been shown to produce a marked dose-dependent increase in locomotor

activity following intraaccumbens injection (Arnt, 1981). However,

glutamic acid diethylester (GDEE), an excitatory amino acid

antagonist, which has been used previously to show the selectivity

of AMPA for quisqualic acid receptors (Hosli, Hosli, Lehmann, and

Eng, 1983; Krogsgaard-Larsen et.al., 1980), was unable to inhibit

this AMPA - induced hypermotility response (Arnt, 1981). This lack of inhibition by GDEE may in part be due to the low dose used (10yg) because a higher dose of GDEE (125yg) did Inhibit quisqualic acid

induced locomotor activity (Donzanti and Uretsky, 1984). But GDEE, at this high dose, was not selective in that it also inhibited the hypermotility response elicited by kainic acid (Donzanti and

Uretsky, 1984). Further evidence that GDEE is not an ideal 23 antagonist to study the interaction of AMPA at quisqualic acid receptors was the lack of GDEE's ability to inhibit ( 3H)-AMPA binding (Honore et. a l. 1982). GDEE also has the potential to hydrolyze in solution (Fagg, Foster, and Ganong, 1986).

Jf-D-gl utamyl ami nomethyl sulphonate (GAMS) is a recently developed excitatory amino acid antagonist which has been described as a potent, but non-selective, antagonist of kainic acid and quisqualic acid receptor subtypes (Fagg, 1985) based upon electrophysiological studies (Davies, Evans, Jones, Smith, and

Watkins, 1982; Jones, Smith, and Watkins, 1984). Studies in the cat spinal cord (Davies and Watkins, 1985) have shown that GAMS selectively antagonized the reponses to excitatory amino acids in the general order kainic acid > quisqualic acid > N-methyl-D- aspartic acid. Consequently, this antagonist would be expected to inhibit the hypermotility response Induced by AMPA.

The purpose of this study was to determine whether the activation of quisqualic acid receptors in the nucleus accumbens produces a hypermotility response. We have, therefore, injected

AMPA directly into the nucleus accumbens and measured the subsequent stimulation of locomotor activity. We have then characterized the ability of GAMS to antagonize this response to AMPA by comparing the doses of GAMS that inhibited the hypermotility response to AMPA with those that Inhibited the responses to other excitatory amino acids and picrotoxin. We also characterized the ability of GAMS to 24 inhibit 3H-AMPA binding. Our results, which indicate that GAMS can selectively antagonize hypermotility responses produced by the activation of quisqualic acid receptors, suggest that activation of quisqualic acid receptors in the nucleus accumbens can produce a hypermotility response. However, the mechanism of this selective effect of GAMS to inhibit AMPA and quisqualic acid induced locomotor activity is currently not known since GAMS was unable to significantly inhibit 3H-AMPA binding.

METHODS

Surgical procedure

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis,

Indiana, U.S.A.) weighing 150-190g, were lightly anesthetized with a halothane/oxygen mixture and placed in a stereotaxic apparatus

(David Kopf Instruments, California, U.S.A.). A midline incision was made in the scalp and holes were drilled on each side of the skull at 9.4mm anterior to the intraaural line and 2.3mm lateral to the saggital suture (Konig and Klippel, 1963). The needle of a lOpd

Hamilton syringe (Hamilton Co., Reno, Nevada, U.S.A.) was then inserted at a 10° angle toward the midline (to avoid puncturing the ventricular system) into the previously drilled holes to a depth of

6.4mm from the surface of the skull. Drugs or vehicle were injected in a 0.5yH volume at a rate of 0.5y)l/m1n. The needle was left in place for an additional 1 min to allow for diffusion of the solution. After the Injection, the needle was removed and the incision was sutured and swabbed with 5% (w/v) lidocaine ointment. 25

Monitoring locomotor activity

After the injections into the nucleus accumbens, the animals recovered from anesthesia within 5 min in all cases. After recovery, the animals were placed in motor activity cages (Opto

Varimex-Minor, Columbus Instruments, Ohio, U.S.A.) and allowed 10 min to adapt to the cages. The motor activity cages contained 12 x

12 Infra-red beams passing a height of 5cm from the bottom of the cage through a ventilated plexiglass box measuring 42cm square and

20cm high. Ambulatory movement was recorded as the number of times two consecutive beams, 3.5cm apart, were interrupted per hour. The data were collected and printed by a Columbus digital counter. The animals were observed for convulsions, rearing, or any other non ambulatory behavior during all recording sessions. All testing was done between 8:00 AM and 4:00 PM in an isolated environmental room, maintained at a temperature of 22 ± 1°C. Prior to the day of the experiment, the animals were housed, four to a cage, in an air- conditioned room kept at 20-21°C with an automatic light-dark cycle

(light on 6:00 AM - 6:00 PM).

Histology

After each experiment, the rats were decapitated and their brains rapidly removed and fixed in a 10% formalin solution for 48 hours. Frozen sections (80y thick) were sliced using a Cryo-Cut

Microtome (American Optical Corp., Buffalo, N.Y., U.S.A.) to check the location of the injection needle. When the tips of the needle 26

tracks were found to be outside of the nucleus accumbens, the

locomotor activity recordings of the animals were not used for the

study.

Radioligand Binding

Crude synaptic membrane preparations were prepared from rat

forebrain by a modification of the procedure of Olsen (1987) and

Honore, Lauridsen, Nielsen, and Krogsgaard-Larsen (1983). Male

Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, Indiana,

U.S.A.) weighing 200-250g, were decapitated and their forebrains

rapidly removed and homogenized in 15 volumes of ice cold 0.32M

sucrose using a teflon glass homogenizer. The homogenate was then

centrifuged at l,000g for 10 min (Sorvall Sr-erspeed RC2-B, DuPont,

Newtown, Conn.) and the supernatant was recentrifuged at 20,000g

for 20 min at 4°C. The resulting pellet was resuspended in ice cold

distilled water using a vortex (Vortex-Genie, Scientific Industries

Inc., Springfield, Mass.) and was centrifuged at 8,000g for 20 min.

The supernatant and buffy upper coat of the resultant pellet were

collected and centrifuged at 48,000g for 20 min. The pellet was

resuspended in ice cold distilled water and centrifuged again at

48,000g for 20 min. This final pellet was resuspended in 1.0ml distilled water and rapidly frozen by dry ice and stored at -20°C

until the day of the experiment.

On the day of the experiment, the frozen pellet was allowed to thaw in an ice water bath, resuspended and centrifuged at 48,000g 27

for 20 min. The pellet was then resuspended in ice cold buffer to

yield a final protein concentration of 0.4-0.8 mg/ml as determined

by the Bio-Rad assay using bovine serum albumin as the standard

(Bradford, 1976). The buffer contained potassium phosphate (lOmM)

and potassium thiocyanate (O.lmM) buffered to pH 7.5 (Olsen, 1987).

For displacement studies, an aliquot (500yd) of the washed crude

synaptic membrane preparation was incubated in duplicate on ice with

20nM (50yd) 3H-AMPA in the presence or absence of various

concentrations (50yd) of AMPA, quisqualic acid, or GAMS for 30 min

at 4°C. Nonspecific binding was determined in the presence of ImM

AMPA. Bound radioactivity was separated from free radioactivity by

centrifugation. The incubations were performed in polycarbonate

high speed centrifuge tubes and the reaction was stopped by

centrifuging at 48,000g for 10 min at 4°C. The tubes were then

placed on ice and the pellets rinsed with ice cold distilled water

(5ml, twice). A small volume (0.5ml) of distilled water was then

added and the pellet was removed from the inside wall of the tube by

gentle vortexing. The resultant solution was decanted into

scintillation vials. Another small volume (0.5ml) of distilled water was added to the tube and gently vortexed to remove any

remaining remnants of the pellet. The pellet was then digested in

tissue solubilizer (1ml Protosol; DuPont-NEN). Scintillation fluid was then added (with 50yd glacial acetic acid 70%) and the

radioactivity quantified with a liquid scintillation counter

(Beckman LS 6800, Beckman Instruments). 28

Drugs

The following compounds were purchased from Sigma Chemical Co.

(St. Louis, MO. U.S.A.): N-methyl-D-aspartic acid, kainic acid, quisqualic acid, picrotoxin, and D-a-aminoadipic acid. 3T-D- glutamylaminomethyl-sulphonate (GAMS) was obtained from Tocris

Chemicals (Essex, England). AMPA was obtained from Research

Biochemicals Inc. (Natick, MA. U.S.A.). 3H-AMPA (specific activity, 26Ci/mmol) was obtained from DuPont-NEN (Boston, MA.).

AMPA was dissolved in phosphate buffer 0.5M (pH 7.4). All other drugs were dissolved in saline and adjusted to pH 7.4 with IN NaOH.

Doses shown refer to the amount injected on each side of the nucleus accumbens. For the studies on the antagonistic actions of GAMS,

AMPA, kainic acid, quisqualic acid, N-methyl-D-aspartic acid, and picrotoxin were administered at doses that produce a similar degree of stimulation of locomotor activity. Control animals were injected with an equal volume (0.5vifc) of saline or vehicle (phosphate buffer).

Statistics

Locomotor activity data were expressed as the mean and standard error of the mean (SEM). Significant differences were evaluated using the two-tailed Mann-Whitney U-test, with a level of p<0.05 being considered significant. The IC50 values for AMPA, quisqualic acid, and GAMS were derived using a log-logit transformation 29

(Bylund, 1980) of specific binding data using 7-17 determinations of

each compound.

RESULTS

Effect of AMPA on locomotor activity in the rat.

Bilateral injection of AMPA (0.5-4nmole) into the nucleus

accumbens produced a dose-dependent increase in locomotor activity

(Fig. 1). At doses of 1, 2, and 4nmole, rats exhibited periods of

prolonged coordinated locomotor activity along with brief

intermittent periods of the "praying" response, which is character

ized by rearing on their hindlimbs with their forepaws extended and

crossed. This behavior is similar to that observed previously for

quisqualic acid (Donzanti and Uretsky, 1983). AMPA, at a dose of

20nmole, induced tremors and labored breathing in all animals, which

seemed to interfere with the hypermotility response and may account

for the reduction in locomotor activity at this dose (Fig. 1). Of

the five animals tested at this dose, one animal died within two

hours after injection.

Effect of GAMS on AMPA - stimulated locomotor activity in the rat.

In order to determine the effect of GAMS, an excitatory amino

acid antagonist (Davies and Watkins, 1985), on AMPA - stimulated

locomotor activity, various doses of GAMS were co-administered with

AMPA (lnmole) Into the nucleus accumbens. GAMS was found to inhibit

the hypermotility response elicited by AMPA; the threshhold inhibitory dose of GAMS being 0.8nmole (Fig. 2). GAMS at doses of

0.8-104nmole produced a 62-86% inhibition of the AMPA - stimulated locomotor activity. The inhibitory effect of GAMS (52nmole) was reversed by administering a higher dose of AMPA (4nmole) along with

GAMS (Table 2). GAMS, administered alone, at doses of 1.6 and

104nmole did not produce a statistically significant effect on locomotor activity (283 ± 112(6) and 460 ± 91(6), respectively) as compared to vehicle alone (232 ± 96(5)).

Effect of GAMS on quisqualic acid - stimulated locomotor activity in the rat.

Quisqualic acid has been shown to stimulate locomotor activity after bilateral injection into the nucleus accumbens. Therefore, the effect of various doses of GAMS on quisqualic acid - induced hypermotility was determined. The hypermotility response to quisqualic acid (5.3nmole) alone was found to vary from day to day.

Consequently, for this study, the experimental results on different days were not pooled; instead the responses of rats to quisqualic acid alone were only compared to those of rats treated on the same day with quisqualic acid and GAMS. The results show that all doses of GAMS tested (1.6-104nmole) inhibited the hypermotility response elicited by quisqualic acid (Table 3). 31

2 5 0 0 0

cr 20000 o=) X \ >- 15000 I- (7) I— o 10000 (5)

5 0 0 0

0 0.5 I 2 4 20 AMPA (nm)

Figure 1. Effect of AMPA on locomotor activity after bilateral Injection Into the nucleus accumbens. AMPA (nm=nanomole) or vehicle was Injected 1n a 0.5yfc volume and the animals were placed 1n motor activity cages for 1 hour. Each point represents the mean ± S.E.M. for the number of observations In parentheses. AMPA 0.5-20nm = 0.125-5.Oyg. * p<0.05 with respect to saline. 32

(7)

5 0 0 0

o: (23) z> o X \ (3) £ 3 0 0 0

( 6 ) (5) ( 4 )

( 6 ) 1000

0.8 6.5 26 52 104 GAMS (nm)

Figure 2. Effect of GAMS on AMPA - stimulated locomotor activity after bilateral injection into the nucleus accumbens. A solution of AMPA (1 nm, nm=nanomole) with or without GAMS (1n increasing doses) was Injected 1n a 0.5yll volume and the animals were placed 1n motor activity cages for 1 hour. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses. GAMS 0.2-104nm = 0.05-25yg and AMPA lnm = 0.25yg. * p<0.05 with respect to AMPA 1 nm alone. 33

TABLE 2

EFFECT OF DIFFERENT DOSES OF AMPA ON THE GAMS-INDUCED INHIBITION OF AMPA-STIMULATED LOCOMOTOR ACTIVITY

Moti1i ty/Hour % Inhibition

AMPA lnm 4136 ± 429 (5) AMPA lnm + GAMS 52nm 510 ± 234 (4)* 88

AMPA 4nm 15478 ± 1807 (5) AMPA 4nm + GAMS 52nm 12837 ± 2055 (5) 17

A solution of GAMS (52nm, nm=nanomole) with AMPA (1 or 4nm) was bilaterally injected into the nucleus accumbens in a 0.5yd volume and the animals were placed in motor activity cages for one hour. Each value represents the mean ± S.E.M. for the number of observations in parentheses. AMPA 1 or 4nm = 0.25 or lyg and GAMS 52nm = 12.5yg. * p<0.05 with respect to AMPA alone. 34

TABLE 3

EFFECT OF GAMS ON QUISQUALIC ACID-STIMULATED LOCOMOTOR ACTIVITY AFTER BILATERAL INJECTION INTO THE NUCLEUS ACCUMBENS

% Inhi Motility/Hour bition

Quisqualic acid 5.3nm 6261 ± 1066 (4) Quisqualic acid 5.3nm 3596 ± 566 (6)* 43 + GAMS 1.6nm

Quisqualic acid 5.3nm 2966 ± 360 (11) Quisqualic acid 5.3nm 1286 ± 263 (11)* 57 + GAMS 6.5nm

Quisqualic acid 5.3nm 2118 ± 466 (15) Quisqualic acid 5.3nm 1075 ± 222 (16)* 49 + GAMS 26nm

Quisqualic acid 5.3nm 6097 ± 1127 (10) Quisqualic acid 5.3nm 1897 ± 542 (10)* 69 + GAMS 52nm

Quisqualic acid 5.3nm 4326 ± 373 (6) Quisqualic add 5.3nm 1206 ± 337 (5)* 72 + GAMS 104nm

A solution of quisqualic acid (5.3nm, nm=nanomole) with and without GAMS (1n Increasing doses) was injected in a 0.5yd volume and the animals were placed in motor activity cages for one hour. Each value represent the mean ± S.E.M. for the number of observations 1n parentheses. Quisqualic acid 5.3nm = lyg and GAMS 1.6-104nm = 0.39-25yg. * p<0.05 with respect to quisqualic acid alone. 35

Effect of GAMS on kainlc acid - stimulated locomotor activity in the rat.

Figure 3 shows that the administration of kainic acid 0.07nmole produced a stimulation of locomotor activity which was similar to that produced by AMPA 1 nmole (Fig. 2). The kainic acid - induced hypermotility was not significantly inhibited by doses of GAMS of

1.6, 6.5, and 26nmole. However, doses of GAMS of 52 and 104nmole did produce a significant 82% and 92% inhibition of kainic acid - stimulated locomotor activity, respectively.

Effect of GAMS on N-methyl-D-aspartic acid - and picrotoxin - stimulated locomotor a c tiv ity in the rat.

N-methyl-D-aspartic acid produced a stimulation of locomotor activity when injected into the nucleus accumbens (Fig. 4).

Similarly, picrotoxin, an inhibitor of endogenous GABA, also produced a stimulation of locomotor activity following intraaccumbens injection (Fig. 5). GAMS at doses of 1.6-52nmole did not significantly inhibit the hypermotility response to either N- methyl-D-aspartic acid (17nmole) or picrotoxin (0.8nmole). However, at a dose of 104nmole, GAMS was able to produce a significant 81% and 52% inhibition of N-methyl-D-aspartic acid and picrotoxin - stimulated locomotor activity, respectively. 36

5000 (25) (4)

(7)

1000

0 1.6 6.5 26 52 104 GAMS (nm)

Figure 3. Effect of GAMS on kainic acid - stimulated locomotor activity after bilateral Injection Into the nucleus accumbens. A solution of kainic acid (0.07 nm, nm=nanomole) with or without GAMS 1n Increasing doses) was Injected 1n a 0.5pfc volume and the animals were placed 1n motor activity cages for 1 hour. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses. Kainic add 0.07nm = 15ng and GAMS 1.6-104nm = 0.39-25pg. * p<0.05 with respect to kainic add 0.07nm alone. 37

5 0 0 0 (4) (5) cc 3 O X \ > 3000 (23)

(9)

1000 (4)

0 1.6 6.5 26 52 104 GAMS (nm)

Figure 4. Effect of GAMS on N-methyl-D-aspartic acid - stimulated locomotor activity after bilateral Injection Into the nucleus accumbens. A solution of N-methyl-D-aspartic acid (17 nm, nm=nanomole) with or without GAMS (1n Increasing doses) was Injected 1n a 0.5yfc volume and the animals were placed 1n motor activity cages for 1 hour. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses. NMDA 17nm = 2.5yg and GAMS 1.6-104nm = 0.39-25pg. * p<0.05 with respect to N-methyl-D-aspart1c add 17 nm alone. 38

(10)

( 6) 5000 ( 2 0 )

cr (4) (9) ID o

>- h - 3000 (II) I— o

1000

0.8 6.5 52 104 GAMS (nm)

Figure 5. Effect of GAMS on picrotoxin - stimulated locomotor activity after bilateral Injection Into the nucleus accumbens. A solution of picrotoxin (0.8 nm, nm=nanomole) with or without GAMS (1n Increasing doses) was Injected 1n a 0.5yd volume and the animals were placed 1n motor activity cages for 1 hour. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses. Picrotoxin 0.8nm = 0.5yg and GAMS 0.8-104nm = 0.2-25yg. * p<0.05 with respect to picrotoxin 0.8 nm alone. 39

Comparison of the effect of an N-methyl-D-aspartic acid receptor

antagonist, D-alpha-aminoadip1c acid, on AMPA- and N-methyl-D-

aspartic acid - stimulated locomotor activity in the rat.

D-o-aminoadip1c acid has been characterized as an antagonist at

the N-methyl-D-aspartic acid subtype of excitatory amino acid

receptor (Davies Watkins, 1979; McLennan and Lodge, 1979). The

purpose of this experiment was to determine the effect of

D-a-am1noad1p1c acid (62nmole) on AMPA and N-methyl-D-aspartic acid

- stimulated locomotor activity. Both AMPA (Inmole) and N-methyl-D-

aspartic acid (17nmole) elicited a marked hypermotility response

(Table 4). As shown previously (Donzanti and Uretsky, 1984),

D-a-aminoadipic acid produced a significant (47%) inhibition of N-

methyl-D-aspartic acid - stimulated locomotor activity (Table 4).

However, D-a-aminoadipic acid did not significantly inhibit the

hypermotility response induced by AMPA.

Effect of AMPA, quisqualic acid, and GAMS on total 3H-AMPA binding.

Figure 6 shows that AMPA and quisqualic acid produced

significant inhibitions of total 3H-AMPA binding while GAMS was

unable to significantly inhibit total 3H-AMPA binding. The concentrations of AMPA and quisqualic acid which produced a 50%

inhibition of specific 3H-AMPA binding was 312 ± 34 nM and 127 ± 78 nM respectively. The concentration of GAMS necessary to produce a

50% inhibition of specific 3H-AMPA binding was in excess of lOmM. 40

TABLE 4

EFFECT OF D-ALPHA-AMINOADIPIC ACID (DAA) ON AMPA AND N-METHYL-D-ASPARTIC ACID (NMDA) - STIMULATED LOCOMOTOR ACTIVITY AFTER BILATERAL INJECTION INTO THE NUCLEUS ACCUMBENS

Moti1ity/Hour % Inhibition

AMPA lnm 8240 ± 1617 (7) AMPA lnm + DAA 62nm 6688 ± 1724 (7) 19

NMDA17nm 3493 ± 689 (10) NMDA17nm + DAA 62nm 1853 ± 719 (8)* 47

A solution of AMPA (lnm, nm=nanomole), NMDA (17nm), vehicle (phosphate buffer 0.5M, pH=7.4), or saline with and without DAA (62nm) was injected in a 0.5yd volume and the animals were place in motor activity cages for one hour. The motility / hour of saline treated (control for NMDA study) and vehicle treated (control for AMPA study) animals were 250 ± 172 (4) and 315 ± 99 (5), respectively. The motility / hour produced by DAA, alone, in saline and vehicle was 564 ± 187 (4) and 272 ± 162 (6), respectively. Each value represents the mean ± S.E.M. for the number of observations in parentheses. AMPA lnm = 0.25yg., NMDA 17nm = 2.5yg., and DAA 62nm = lOyg. * p^*i.05 with respect to NMDA alone. 41

100 90 * o c 80 D o CD 70 s 60 < 50 I n rxKL to 40 * a AMPA 30 •— • 0UIS o o GAMS — i- 20 10

-8 -5 -4 10 10 10 10 10 10 (M)

Figure 6. Effect of AMPA, quisqualic acid, and GAMS on total *H-AMPA binding. Crude synaptic membrane preparations were prepared and Incubated 1n the presence of Increasing concentrations of AMPA, quisqualic acid, and GAMS as described 1n the methods. Bound radioactivity was separated from free radioactivity by centrifugation. Each point represents the mean ± S.E.M. for 3-5 experimental observations. 42

DISCUSSION

AMPA 1s an excitatory amino acid which on the basis of electrophyslologlcal (Krogsgaard-Larsen et.al., 1980) and binding studies (Honore et.al., 1982) appears to be a selective agonist for quisqualic acid receptors. The results of this study show that

AMPA, after injection into the nucleus accumbens, produced a dose- dependent stimulation of locomotor activity. This hypermotility response was Inhibited by GAMS, an excitatory amino acid antagonist, at doses which were also able to inhibit the hypermotility response to quisqualic acid but not the responses to other excitatory amino acids, kainic acid and N-methyl-D-aspartic acid. In addition, these low doses of GAMS did not inhibit the hypermotility response to picrotoxin, an Inhibitor of GABA-mediated effects. Thus, it appears that in the nucleus accumbens, GAMS can selectively Inhibit the effects of drugs that activate quisqualic acid receptors. The results of these studies suggest that the activation of quisqualic acid receptors in the nucleus accumbens produces a stimulation of locomotor activity.

In order to determine the role of quisqualic acid receptors in the nucleus accumbens in stimulating locomotor activity, it would be necessary to find a selective antagonist of this receptor. However, antagonists which can discriminate between quisqualic acid and kainic add receptors have not been available. It has been suggested on the basis of electrophysiological studies that glutamic 43

acid diethylester (GDEE) is a weak but selective antagonist of

quisqualic acid - induced responses (Davies and Watkins, 1979;

McLennan and Lodge, 1979). This assumption has been questioned because the results from binding studies have demonstrated that GDEE was unable to inhibit the binding of radiolabeled AMPA (Honore et.al., 1982). In addition, GDEE has the tendency to hydrolyze in

solution and become inactive (Fagg et.al., 1986). In previous

studies on the effects of the intraaccumbens administration of drugs, a high dose of GDEE significantly decreased the hypermotility responses produced by both quisqualic acid and kainic acid (Donzanti and Uretsky, 1984). Therefore, it was not possible in these behavioral studies to determine the effects on locomotor activity of activating quisqualic acid receptors in the nucleus accumbens.

The results from microiontophoretic studies have shown that GAMS can antagonize the effects produced by both kainic acid and quisqualic acid, while exerting a weaker antagonistic action on N- methyl-D-aspartic acid - induced effects. In contrast to these electrophysiological studies, in the present study the intraaccumbens administration of GAMS selectively inhibited the responses to AMPA and quisqualic acid at doses that were ineffective in Inhibiting the responses to kainic acid. The GAMS - induced antagonism of the effects of AMPA was reversed by raising the dose of AMPA, suggesting that the antagonism is competitive. However, the Inability of GAMS to inhibit 3H-AMPA binding, which is in agreement with another study (Murphy, Snowhill, and Williams, 1987), 44

suggests that this selective antagonism is not mediated by a

competitive antagonism at the quisqualic acid receptor. At present,

it is not clear whether the ability of GAMS to discriminate between

the effects of AMPA and quisqualic acid on one hand and kainic acid

on the other is related to differences in the receptor for these two

compounds, to an action of GAMS on a second messenger system coupled

to the quisqualic acid receptor complex, or to some other mechanism.

However, regardless of which explanation is correct, the selective

effects of GAMS in the nucleus accumbens suggest that the

hypermotility responses to AMPA and quisqualic acid is mediated by a

different mechanism than the hypermotility response to kainic acid.

Studies were also performed to determine if AMPA - induced

hypermotility was mediated by an interaction with the N-methyl-D-

aspartic acid receptor. D-a-aminoadipic acid is an excitatory amino

acid antagonist which has been shown in electrophysiological studies

to be selective for the N-methyl-D-aspartic acid receptor (Davies

and Watkins, 1979; McLennan and Lodge, 1979). In addition, it has

been shown that D-a-aminoadipic acid is inactive as an inhibitor of

( 3H)-AMPA binding (Honore et.al., 1982). Supporting these observations is the previous report that the intraaccumbens

injection of D-a-aminoadipic acid antagonized the hypermotility response produced by N-methyl-D-aspartic acid but not that produced by quisqualic acid (Donzanti and Uretsky, 1984). In the present study, D-a-aminoadipic acid, in a dose that produced a significant

(47%) inhibition of N-methyl-D-aspartic acid - stimulated locomotor *

45 activity, did not significantly Inhibit the hypermotility produced by AMPA (Table 3). Additionally a high dose of GAMS (104 nmoles) was required to inhibit N-methyl-D-aspartic acid - stimulated locomotor activity (Fig. 4). This threshhold inhibitory dose of

GAMS was 130 times higher than that required to inhibit AMPA - stimulated locomotor activity. These results suggest that the AMPA

- induced hypermotility response is not mediated by the activation of N-methyl-D-aspartic acid receptors.

In summary, these results indicate that GAMS, which has been classified as a kainic acid / quisqualic acid receptor antagonist, can selectively inhibit AMPA - induced and quisqualic acid - induced hypermotility responses after injection into the nucleus accumbens.

Thus, these observations suggest that the activation of quisqualic acid receptors in the nucleus accumbens produces a stimulation of locomotor activity. CHAPTER I I I

EFFECT OF GABAERGIC TRANSMISSION IN THE VENTRAL PALLIDUM

ON THE HYPERMOTILITY RESPONSE TO THE INTRAACCUMBENS

ADMINISTRATION OF EXCITATORY AMINO ACIDS AND PICROTOXIN.

INTRODUCTION

The nucleus accumbens is a region of forebrain that has been postulated to be involved in the integration of motivational behavior, associated with the limbic system, and motor behavior, associated with the basal ganglia (Mogenson, Jones, and Y1m, 1980;

Mogenson and Yim, 1980). Neuronal mechanisms 1n the nucleus accumbens have been shown to mediate the locomotor activating properties of certain psychostimulant drugs, such as heroin and amphetamine (Swerdlow, Vaccarlno, Amalrlc, and Koob, 1986). In addition, hypermotility can also be produced by the direct

Intraaccumbens administration of dopamine, picrotoxin, carbachol, and various excitatory amino acids (Pijnenburg, Honlg, Van derHeyden, and Van Rossum, 1976; Mogenson and Nielson, 1983;

Morgenstern, Mende, Gold, Lemme, and Oelssner, 1984; Donzantl and

Uretsky, 1983; Austin and Kalivas, 1988).

46 47

Recent evidence suggests that a main GABAergic efferent projection from the nucleus accumbens to the substantia innominata / lateral preoptic area (SI/LPO), located in the ventral pallidum, is involved in the hypermotility produced by drugs which act in the nucleus accumbens (Jones and Mogenson, 1980a; Mogenson, Swanson, and

Wu, 1983; Mogenson and Nielson, 1983; Nauta, Smith, Faull, and

Domesick, 1978). Thus it has been shown that injection of GABA or muscimol into the SI/LPO attenuated the locomotor responses produced by the direct injection of dopamine into the nucleus accumbens

(Mogenson & Nielson, 1983), the systemic administration of apomorphine after 6-hydroxydopamine (6-OHDA) induced denervation of the nucleus accumbens (Swerdlow & Koob, 1984), the peripheral administration of amphetamine (Swerdlow et.al., 1986), and the intraaaccumbens administration of carbachol which is thought to act by stimulating endogenous dopamine release (Austin and Kalivas,

1988). In addition, the stimulation of locomotor activity induced by the systemic administration of heroin, which appears to be due in part to to the activation of opioid receptors in the nucleus accumbens, is inhibited by the direct injection of muscimol into the

SI/LPO (Swerdlow et.al., 1986). These observations suggest that locomotor activation produced by increases in dopaminergic and opioid neurotransmission in the nucleus accumbens may be mediated by a decrease 1n GABAergic activity in the SI/LPO. Therefore, the injection of muscimol into the SI/LPO would activate GABAergic receptors at this site and decrease the hypermotility response. However, the GABAergic pathway from the nucleus accumbens to the

SI/LPO does not seem to be involved in the actions of all drugs that stimulate locomotor activity (Swerdlow et.al., 1986). Thus, the stimulation of locomotor activity induced by the systemic administration of caffeine and corticotropin releasing factor is not antagonized by the injection of muscimol into this site. These observations suggest that caffeine and corticotropin releasing factor produce their activating effects through a mechanism not involving a decrease in GABAergic neuronal activity in the SI/LPO

(Swerdlow and Koob, 1985).

Excitatory amino acid containing neurons (presumably glutamatergic) in the nucleus accumbens may be involved in the stimulation of locomotor activity. These neurons project from the hippocampus and cerebral cortex, and in addition, excitatory amino acid interneurons may exist (Walaas and Fonnum, 1979a; Carter, 1980;

Walaas, 1981). The direct injection of the excitatory amino acids,

N-methyl-D-aspartic acid, kainic acid, quisqualic acid, and a-amino-3-hydroxy-5-methyl1soxazole-4-propionate (AMPA), a quisqualic acid agonist, produce an intense stimulation of locomotor activity (Arnt, 1981; Donzantl and Uretsky, 1983; Shreve and

Uretsky, 1988). The effects of these compounds may be mediated by different neuronal mechanisms, since they can be differentially antagonized by drugs which have been shown to antagonize the electrophysiologlcal effects of excitatory amino acids (Donzanti and

Uretsky, 1984; Shreve and Uretsky, 1988). It is not known whether 49 the hypermotility responses to excitatory amino acids are mediated by the inhibition of the GABAergic projection from the nucleus accumbens to the SI/LPO. To investigate this possibility, we have determined the effect of muscimol injected into the SI/LPO on the stimulation of locomotor activity produced by the intraaccumbens

Injection of various excitatory amino acids. In addition, since the intraaccumbens injection of picrotoxin also stimulates locomotor activity, we have determined whether muscimol inhibits the effects of picrotoxin.

METHODS

Surgical procedure

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis,

Indiana, U.S.A.) weighing 150-190g, were lightly anesthetized with a halothane/oxygen mixture and placed in a stereotaxic apparatus

The coordinates for the nucleus accumbens were 9.4mm anterior to the intraaural line and 2.3mm lateral to the saggital suture (Konig and

Klippel, 1963). The needle of a 10yH Hamilton syringe (Hamilton

skull. The coordinates for the SI/LPO were 6.8mm anterior to the

intraaural line, 1.8mm lateral to the saggital suture, and 2.0mm

above the intraaural line (Konig and Klippel, 1963). Drugs or

vehicle were injected in a 0.5pfc volume at a rate of 0.5pd/min. The

needle was left in place for an additional 1 min to allow for

diffusion of the solution. After the injection, the needle was

removed and the Incision was sutured and swabbed with 5% (w/v)

lidocaine ointment.

Monitoring locomotor activity

After the injections into the nucleus accumbens and/or SI/LPO,

the anesthesia was turned off and the animals recovered from

anesthesia within 5 min. After recovery, the animals were placed in

motor activity cages (Opto Varimex-Minor, Columbus Instruments,

Ohio, U.S.A.) and allowed 10 min to adapt to the cages. The motor

activity was monitored and recorded as described in chapter II.

Catalepsy

Rats that were injected with muscimol Into the SI/LPO were

tested for catalepsy. The catalepsy tests involved placing the rats

on a table with both forepaws on a wooden block (5 X 5cm) with a

height of 3cm. Only rats remaining Immobile on the block for 10 sec were considered to be cataleptic. Additionally, the forepaws of the

rats were placed on a suspended horizontal rod (2cm diameter). Rats

hanging immobile with stretched forelimbs for 6 sec were considered

to be cataleptic (Vrijmoed-de Vries, Tonissen, and Cools, 1987). 51

Hi stology

After each experiment, the rats were decapitated and their brains rapidly removed and fixed in a 10% formalin solution for 48 houfs. Frozen sections (80y thick) were sliced using a Cryo-Cut

Microtome (American Optical Corp., Buffalo, N.Y., U.S.A.) to check the location of the injection needle. When the tips of the needle tracks were found to be outside of the nucleus accumbens and/or

SI/LPO, the locomotor activity recordings of the animals were not used for the study. Figure 7 shows the injection sites for muscimol in the SI/LPO that were effective and ineffective in inhibiting hypermotility produced by injection of drugs into the nucleus accumbens. Muscimol was found to be ineffective when at least one of the two injection sites in an animal was outside the SI/LPO.

Drugs

The following compounds were purchased from Sigma Chemical Co.

(St. Louis, MO. U.S.A.): N-methyl-D-aspartic acid, kainic acid, picrotoxin, muscimol, and D-amphetamine. a-Amino-3-hydroxy-5-methyl i soxazole-4-prorionate (AMPA) was purchased from Research

Biochemicals Inc. (Natick, MA. U.S.A.).

All drugs were dissolved in saline and adjusted to pH 7.4 with

IN NaOH. The doses shown refer to the amount injected on each side of the nucleus accumbens and or SI/LPO. Control animals were injected with an equal volume (0.5yll) of saline. 52

Statistics

Data were expressed as the mean and standard error of the mean

(SEM). The effects of drugs and saline treatment were evaluated

statistically using the nonparametrlc Kruskal-Wal1is one-way

analysis of variance followed by the one-tailed Mann-Whitney U-test,

with a level of p<0.05 being considered significant.

RESULTS

Effect of injection of picrotoxin and muscimol into the SI/LPO on

the locomotor activity in the rat.

Bilateral injection of picrotoxin (0.5yg) into the SI/LPO

produced a 24 fold increase in locomotor activity as compared to

saline treated animals (fig. 8). This observation is in agreement

with previous reports on the effect of picrotoxin injected into this

site (Mogenson and Nielsen, 1983; Swerdlow and Koob, 1987). In

contrast, the bilateral injection of muscimol (25ng) into the SI/LPO

did not significantly affect locomotor activity which is consistent

with the finding that GABA injected into this region also had no

significant effect on locomotor stimulation (Mogenson and Nielsen,

1983). The stimulation of locomotor activity by picrotoxin but not muscimol suggests that the site of injection in the ventral pallidum

in our studies was the same as that reported by Mogenson and Nielson

(1983), and Swerdlow and Koob (1987), but different from that

reported by Scheel-Kruger (1983), in which muscimol but not 53

picrotoxin stimulated locomotor activity. Rats Injected with 25ng

of muscimol Into the SI/LPO were also tested for catalepsy by two

different methods. None of these rats exhibited catalepsy, which is

consistent with the observation that muscimol at this dose did not

produce a significant Inhibition of locomotor activity.

Effect of the administration of muscimol into the SI/LPO on the

hypermotility response to intraaccumbens administration of AMPA.

In order to determine the effect of muscimol, injected into the

SI/LPO, on AMPA - induced hypermotility, various doses of muscimol

were bilaterally injected into the SI/LPO following bilateral

injections of AMPA (0.5yg) into the nucleus accumbens. Muscimol was

found to inhibit the hypermotility response elicited by AMPA in a

dose dependent manner; the threshold inhibitory dose of muscimol was 2.5ng, and a dose of 25ng produced a 96% inhibition of the AMPA

- stimulated locomotor activity (fig. 9). Muscimol administered

into the SI/LPO at a dose of 25ng had no significant effect on

locomotor activity when saline was injected into the nucleus accumbens. 54

Effect of the administration of muscimol into the SI/LPO on the

hypermotility responses to the intraaccumbens administration of

amphetamine, picrotoxin, AMPA, kainic acid, and N-methyl-D-aspartic

acid.

In order to determine the effect of muscimol, injected into the

SI/LPO, on hypermotility elicited by amphetamine, picrotoxin, AMPA,

kainic acid, and N-methyl-D-aspartic acid, muscimol (25ng) was

bilaterally Injected into the SI/LPO following bilateral injections

of the above agents into the nucleus accumbens. Amphetamine (25yg),

picrotoxin (0.5yg), and AMPA (0.5yg) produced the largest increases

in locomotor activity whereas the doses of kainic acid (15ng) and N-

methyl-D-aspartic acid (2.5yg) employed produced smaller but

significant increases in locomotor activity (fig. 10). In all

cases, muscimol (25ng) produced a statistically significant 82, 86,

95, 83, and 95% inhibition of the hypermotility responses elicited

by amphetamine, picrotoxin, AMPA, kainic acid, and N-methyl-D-

aspartic acid respectively.

Effect of the injection of muscimol into the nucleus accumbens on

the hypermotility responses induced by the intraaccumbens

administration of AMPA or picrotoxin.

In order to determine the effect of muscimol in the nucleus

accumbens on AMPA- and picrotoxin- stimulated locomotor activity,

various doses of muscimol (2.5 and 25ng) were co-administered with

AMPA (0.5yg) or picrotoxin (0.5yg) respectively. These doses of muscimol were selected because they produced a statistically 55 significant inhibition of AMPA- and picrotoxin- elicited hypermotility when Injected into the SI/LPO (figs. 9 & 10 respectively). In contrast to the 75% inhibition of AMPA- stlmulated locomotor activity produced by the injection of 2.5ng of muscimol into the SI/LPO (fig. 9), the injection of this dose of muscimol directly into the nucleus accumbens had no significant effect on AMPA- mediated hypermotility (fig. 11). The dose of muscimol (25ng) which produced a near complete (95%) inhibition of

AMPA stimulated locomotor activity when injected into the SI/LPO

(fig. 9), produced a smaller but significant 75% inhibition when injected directly into the nucleus accumbens. Additionally, muscimol, at a dose of (25ng), administered with picrotoxin into the nucleus accumbens did not significantly inhibit locomotor activity

(fig 11.) while this dose when injected into the SI/LPO produced a significant 86% inhibition of picrotoxin- stimulated locomotor activity (fig. 10). This data shows that doses of muscimol that inhibited hypermotility when injected into the SI/LPO were either without effect or less effective when injected into the nucleus accumbens. 56

Figure 7. Sites of Injection of muscimol In the SI/LPO. These sites represent the tips of the needle tracks. Closed circles refer to sites that produced an Inhibition of the hypermotility response to drugs that were Injected Into the nucleus accumbens; open circles were sites that did not produce a significant Inhibition of the hypermotility response. Abbreviations: AC, anterior commissures; GP, globus palHdus; OC, optic chiasm; ST, striatum. 57

( 6 ) 50 0 0 QC 3 O 4 0 0 0 X \ >- 3 0 0 0 h-

oh" 2000

1000

{5)

SAL MUS PTX 25ng 0.5/zg

Figure 8. Effect of muscimol (MUS) or picrotoxin (PTX) on locomotor activity after bilateral Injection Into the SI/LPO. A solution of MUS (25ng), PTX (0.5yg), or saline was Injected 1n a 0.5yd volume and the animals were placed 1n motor activity and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses: Kruskal-Wal11s analysis of variance H=10.49, p<0.05. * p<0.05 with respect to saline (Mann- Whltney U-test). 58

(12) 16000 r

14000

12000 CC ZD O 10000 X (5) \ >- 8000 h-

6000 * 4000 (4) T I 200 0 * (4) (4) (4) r^i N. Acc. SAL SAL AMPA 0.5ug SI/LP0(ng) 0 25 1.0 2.5 25

Figure 9. Locomotor activity response to 1ntra-accumbens AMPA following Injection of different doses of muscimol Into the SI/LPO. A solution of AMPA (0.5yg) or saline was bilaterally injected Into the nucleus accumbens, followed by the bilateral Injection of a solution of muscimol (1n increasing doses) or saline Into the SI/LPO. The animals were then placed 1n motor activity cages and locomotor activity recorded for 1 hour. All Injections were administered in a 0.5y£, volume. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses: Kruskal- Wallis analysis of variance for the AMPA experiment H=14.18, p<0.05. *p<0.05 with respect to AMPA alone (Mann-Wh1tney U-test). 59

12000

10000 - (5) (5) 8000

I— 6000 (4)

(4)

2000 (4) (4) (5) (5)

AMPH PTX AMPA KAIN NMDA 25 fig 0.5fig 0.5/ig I5ng 2.5fig

Figure 10. Locomotor activity responses to the Intraaccumbens administration of amphetamine (AMPH), plcrotoxln (PTX), AMPA, kalnic acid (KAIN), and N-methyl-D-aspart1c acid (NMDA) following Injection of muscimol or saline Into the SI/LPO. A solution of either AMPH (25yg), PTX (0.5yg), AMPA (0.5yg), KAIN (15ng), or NMDA (2.5yg) was bilaterally Injected Into the nucleus accumbens followed by muscimol (hatched bars) or saline (open bars) bilaterally Injected Into the SI/LPO. The animals were then placed 1n motor activity cages and locomotor activity recorded for 1 hour. All Injections were administered 1n a 0.5yft volume. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses. * p<0.05 with respect to saline treatment 1n the SI/LPO. 60

F I SALINE

MUSCIMOL 2.5ng

MUSCIMOL 25.0ng ( 8) 12000 (r (4) ZD o 10000 X (4) \ jz 8000 □ (4) I- 6000 O 2 * 4000 (4)

2000 (4) (7) (4) Vx r + i F l I AMPA 0.5(iq PTX 0.5Mg

Figure 11. Effect of the administration of muscimol Into the nucleus accumbens on the hypermotl11ty responses to the Intraaccumbens Injections of AMPA and plcrotoxln (PTX). Rats were Injected bilaterally Into the nucleus accumbens with saline or muscimol (2.5ng and 25ng) either alone or with AMPA (0.5yg) or PTX (0.5yg). The animals were then placed 1n the motor activity cages and locomotor activity recorded for 1 hour. Each value represents the mean ± S.E.M. for the number of observations 1n parentheses: Kruskal-Wal 11 s analysis of variance H=0.88, p>0.05 for the control experiment, H=7.87, p<0.05 for the AMPA experiment. * p<0.05 with respect to saline control (Mann-Wh1tney U-test). 61

DISCUSSION

The major finding of this study is that the hypermotility responses induced by the intraaccumbens injections of the excitatory amino acids, AMPA, kainic acid, and N-methyl-D-aspartic acid, which are thought to act by different receptor mechanisms, are antagonized by the direct injection of muscimol into the SI/LPO. In addition, muscimol injection into this region inhibited the stimulation of locomotor activity induced by the intraaccumbens administration of picrotoxin, which antagonizes GABA-A receptor mediated responses.

Previous studies have presented evidence in support of a major

GABAergic pathway that originates in the nucleus accumbens and synapses in the SI/LPO (Jones and Mogenson, 1980; Walaas and Fonnum,

1979b). The results of the present study are consistent with the concept that the hypermotility responses induced by the intraaccumbens injection of various excitatory amino acids and picrotoxin are mediated by the inhibition of the GABAergic pathway originating in the nucleus accumbens and projecting to the SI/LPO, resulting in a decrease in GABAergic neurotransmission in the

SI/LPO.

In agreement with the results of Mogenson and Nielson (1983) and

Swerdlow and Koob (1987), these results show that picrotoxin can stimulate locomotor activity when injected into the ventral pallidum at sites in the SI/LPO, while muscimol at these sites did not significantly alter locomotor activity. In contrast to these 62 observations, Scheel-Kruger (1983) has reported that the administration of muscimol, but not picrotoxin, into ventral pallidal sites can stimulate locomotor activity. While we have not systematically studied the effects of muscimol and picrotoxin in different regions of the ventral pallidum, the location of the sites

1n the present study appear to be more caudal and lateral to those described by Scheel-Kruger (1983). Taken together, these different observations on the effects of muscimol and picrotoxin suggest that there is more than one type of GABAergic synapse in the ventral pallidum that can influence locomotor activity.

Previous studies have suggested that the injection of either muscimol or GABA into the SI/LPO can inhibit the stimulation of locomotor activity produced by drugs that activate dopaminergic and opioid receptors in the nucleus accumbens (Jones and Mogenson,

1980a; Mogenson et.al., 1983; Mogenson and Nielson, 1983; Swerdlow et.al., 1986). Thus, the stimulation of locomotor activity produced by the systemic administration of amphetamine or apomorphine (in rats pretreated with 6-hydroxydopamine) was inhibited by muscimol injected into the SI/LPO. Similarly, muscimol injected into this site inhibited the stimulation of locomotor activity produced by heroin. The hypermotility responses to heroin or apomorphine and amphetamine appear to be mediated through their action in the nucleus accumbens as these responses are inhibited by the intraaccumbens injection of opioid or dopaminergic antagonists respectively (Amalric and Koob, 1985; Pijnenburg, Honig, and Van 63

Rossum, 1975). In addition, the administration of muscimol or GABA

into the SI/LPO has been shown to inhibit the stimulation of loco

motor activity induced by the direct administration into the nucleus

accumbens of dopamine and carbachol, which appears to act by

releasing endogenous dopamine (Mogenson and Nielson, 1983; Austin

and Kalivas, 1988). In contrast, the hypermotility produced by’the

systemic administration of caffeine or the intraventricular

administration of corticotropin releasing factor are not antagonized

by the injection of muscimol into the SI/LPO (Swerdlow and Koob,

1985). The reason for the inability of muscimol to inhibit the

effect of these latter compounds is unclear but it is possible that

the nucleus accumbens is not a critical substratefor their

locomotor stimulating effects (Swerdlow and Koob, 1985). According

to this hypothesis, the activation of GABAergic receptors in the

SI/LPO may only inhibit the stimulation of locomotor activity

produced by drugs whose stimulatory effects are mediated by

mechanisms within the nucleus accumbens. This hypothesis is

supported by the observations of the present study that muscimol

injected into the SI/LPO can inhibit the hypermotility responses to

the intraaccumbens administration of various excitatory amino acids, picrotoxin, and amphetamine.

The injection of muscimol into the SI/LPO produced an almost complete inhibition of the hypermotility responses produced by the intraaccumbens administration of amphetamine, the various excitatory amino acids, and picrotoxin (fig. 10). Therefore, the GABAergic pathway from the nucleus accumbens to the SI/LPO may be the main

efferent pathway from the nucleus accumbens that mediates

hypermotility responses to these compounds. The results of the

present study differ from that of Swerdlow and Koob (1986), who

reported that the injection of muscimol into the SI/LPO produced

only a partial inhibition of the hypermotility responses to heroin,

amphetamine, and apomorphine. While the latter results could

indicate that the SI/LPO GABAergic synapses play only a partial role

in mediating the hypermotility induced by drugs in the nucleus

accumbens, the greater inhibitory effects of muscimol observed in

the present study could be attributed to the higher dose of muscimol

injected, which would produce a greater activation of the GABAergic

receptors in the SI/LPO. Alternatively, it is possible that the

higher dose of muscimol could have resulted in the diffusion of the

drug to GABAergic receptors at sites outside the SI/LPO which could

also antagonize the hypermotility responses. The activation of

GABAergic receptors in the nucleus accumbens has been shown

previously to antagonize the hypermotility responses to the

intraaccumbens administration of dopamine and folic acid (Pycock and

Horton, 1979; Stephens and Uretsky, 1986). However, it is unlikely that muscimol injected into the SI/LPO inhibited the stimulation of

locomotor activity by diffusing to GABAergic receptors in the nucleus accumbens, since doses of muscimol which inhibited the hypermotility responses to AMPA and picrotoxin when injected into the SI/LPO were less effective when co-injected with these drugs 65

into the nucleus accumbens. Thus muscimol appears to be a less

potent inhibitor of locomotor activity when injected into the

nucleus accumbens as compared to the SI/LPO.

In summary, these studies show that the hypermotility responses

to the Intraacumbens injection of three prototype excitatory amino

acid agonists, AMPA, kainic acid, and N-methyl-D-aspartic acid, as

well as to picrotoxin are almost completely inhibited by the

bilateral injection of muscimol into the SI/LPO. The effects are,

therefore, similar to those induced by the activation of

dopaminergic and opioid receptors in the nucleus accumbens, which

are also antagonized by the administration of muscimol into the

SI/LPO. These results support the concept that a GABAergic neuronal

projection from the nucleus accumbens to the SI/LPO represents a

major efferent pathway from the nucleus accumbens and that these

neurons are inhibited by drugs that act in the nucleus accumbens to

stimulate locomotor activity. It has been suggested that the

hypermotility induced by an increase in dopaminergic neuro

transmission in the nucleus accumbens may reflect goal oriented behavior. Evidence has been presented that the nucleus accumbens contains glutamatergic terminals, which can release glutamate to activate excitatory amino acid receptors. It is, therefore, possible that an increase in glutamatergic neurotransmission in the nucleus accumbens may, like dopaminergic neurotransmission, play a role in goal oriented behavior. CHAPTER IV

AMPA, KAINIC ACID, AND N-METHYL-D-ASPARTIC ACID STIMULATE

LOCOMOTOR ACTIVITY AFTER INJECTION INTO THE

SUBSTANTIA INNOMINATA / LATERAL PREOPTIC AREA.

INTRODUCTION

The substantia Innominata / lateral preoptic area (SI/LPO) 1s a subpalHdal region which receives a large GABAergic projection from the nucleus accumbens (Jones and Mogenson, 1980b; Mogenson and

Nielsen, 1983; Mogenson, Swanson, and Wu, 1983; Nauta, Smith, Faull, and Domeslck, 1978), a region of the forebrain that has been postulated to be Involved in the Integration of motivational and motor behavior (Mogenson, Jones, and Y1m, 1980; Mogenson and Yim,

1980). The Inhibition of this GABAergic pathway appears to be involved in the stimulation of locomotor activity produced by the actions of various drugs 1n the nucleus accumbens (Jones and

Mogenson, 1980a). Thus it has been shown that the stimulation of locomotor activity produced by the activation of opioid (Swerdlow,

Vaccarlno, Amalrlc, and Koob, 1986), dopamine (Mogenson and Nielsen,

1983; Swerdlow and Koob, 1984; Swerdlow, Swanson, and Koob, 1984;

Swerdlow et.al., 1986), and excitatory amino acid (Shreve and

Uretsky, 1988b) receptors 1n the nucleus accumbens can be Inhibited

66 67

by the injection of GABA or muscimol into the SI/LPO. In addition

to the effects of muscimol, the injection of picrotoxin, an

inhibitor of endogenous GABA, into the SI/LPO has been shown to

stimulate locomotor activity (Mogenson and Nielsen, 1983; Shreve and

Uretsky, 1988b; Swerdlow and Koob, 1987). Therefore, GABAergic

synapses in this subpallidal region appear to regulate locomotor

activity stimulated by mechanisms in the nucleus accumbens.

In addition to the presence of GABA, an inhibitory neuro

transmitter, glutamate, an excitatory neurotransmitter, is also

found in the SI/LPO. Thus, autoradiographic studies have shown that

this region contains a high concentration of excitatory amino acid

binding sites that are thought to represent receptors for glutamic

acid (Monaghan and Cotman, 1985; Halpain, Weiczorek, and Rainbow,

1984). Furthermore, synaptosomes prepared from the SI/LPO possess

high-affinity uptake sites for L-glutamate and D-aspartate and can

release glutamate in a calcium dependent manner in response to

depolarization (Davies, McBean, and Roberts, 1984). Based on a

recent neuroanatomical study which utilized 3H-D-aspartate as a

selective retrograde tracer, the cells of origin of these glutamate

terminals appear in part to be located in the amygdala, a region of

the limbic system (Fuller, Russchen, and Price, 1987). While the

functional effects of glutamate 1n the SI/LPO are not known, it is

possible that glutamate, an excitatory amino acid, may serve to directly or indirectly oppose the Inhibitory actions of GABA in this

subpallidal region on locomotor activity. 68

The purpose of the present study was to determine the actions of the excitatory amino acids, a-amino-3-hydroxy-5-methyl-isoxazole-4- propionate (AMPA), kainic acid, and N-methyl-D-aspartic acid, on locomotor activity following their direct injection into the SI/LPO.

These amino acids were studied because they have been shown to be agonists at different excitatory amino acid receptors. Our results show that AMPA, kainic acid, and N-methyl-D-aspartic acid elicited marked stimulations of locomotor activity and the effect of each compound could be distinguished by administering excitatory amino acid antagonists.

METHODS

Surgical procedure

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis,

Indiana, U.S.A.) weighing 150-190g, were lightly anesthetized with a halothane/oxygen mixture and placed in a stereotaxic apparatus

(David Kopf Instruments, California, U.S.A.). A midline incision was made in the scalp and holes were drilled on each side of the skull to facilitate bilateral injections into the substantia innominata / lateral preoptic area (SI/LPO). The coordinates for the SI/LPO were 6.8mm anterior to the intraaural line, 1.8mm lateral to the saggital suture, and 2.0mm above the intraaural line (Konig and Klippel, 1963). The needle of a 10yd Hamilton syringe (Hamilton

Co., Reno, Nevada, U.S.A.) was then inserted into the previously drilled holes. Drugs or vehicle were injected in a 0.5yd volume at 69 a rate of 0.5yll/m1n. The needle was left In place for an additional

1 min to allow for diffusion of the solution. After the injection, the needle was removed and the incision was sutured and swabbed with

5% (w/v) lidocaine ointment.

Monitoring locomotor activity

After the injections into the SI/LPO, the anesthesia was turned off and the animals recovered from anesthesia within 5 min. After recovery, the animals were placed in motor activity cages (Opto

Varimex-Minor, Columbus Instruments, Ohio, U.S.A.) and allowed 10 min to adapt to the cages. The motor activity was monitored and recorded as described in chapter II.

Histology

After each experiment, the rats were decapitated and their brains rapidly removed and fixed in a 10% formalin solution for 48 hours. Frozen sections (80y thick) were sliced using a Cryo-Cut

Microtome (American Optical Corp., Buffalo, N.Y., U.S.A.) to check the location of the injection needle. When the tips of the needle tracks were found to be outside of the SI/LPO, the locomotor activity recordings of the animals were not used for the study.

Figure 12 shows the injection sites for AMPA, kainic acid, or N- methyl-D-aspartic acid in the SI/LPO that were effective and ineffective 1n stimulating locomotor activity. AMPA, kainic acid, or N-methyl-D-aspartic acid were found to be ineffective when at 70

least one of the two injection sites in an animal were outside the

SI/LPO.

Drugs

The following compounds were purchased from Sigma Chemical Co.

St. Louis, MO. U.S.A.): N-methyl-D-aspartic acid, kainic acid, and

D-o-aminoadipic acid. a-Amino-3-hydroxy-5-methyl-isoxazole -4-

propionate (AMPA) was purchased from Research Biochemicals Inc.

(Natick, MA. U.S.A.). 6,7-Dinitro- quinoxaline-2,3-dione (DNQX) and

y-glutamyl-aminomethyl-sulfonate (GAMS) were obtained from Tocris

Chemicals (Essex, England).

N-methyl-D-aspartic acid was dissolved in saline and adjusted to

pH 7.4 with IN NaOH. All other drugs were dissolved in phosphate

buffer (pH=7.5). The doses shown refer to the amount injected on

each side of the SI/LPO. For the studies on the antagonistic action

of GAMS and D-o-aminoadipic acid, AMPA and kainic acid were

administered at doses that produced a similar degree of locomotor activity. Although this level of locomotor activity could not be achieved with N-methyl-D-aspart1c acid, the highest dose which did not elicit seizure activity was utilized for these studies. Control animals were Injected with an equal volume (0.5yd) of saline or phosphate buffer. 71

Statisties

Data were expressed as the mean and standard error of the mean

(SEM). The effects of drugs and saline treatment were evaluated statistically using the nonparametric Kruskal-Wallis one-way analysis of variance followed by the one-tailed Mann-Whitney U-test, with a level of p<0.05 being considered significant.

RESULTS

Effect of AMPA, kainic acid, and N-methyl-D-aspartic acid on locomotor activity in the rat.

The bilateral injection of either AMPA (0.1-lyg), kainic acid

(15-60ng), or N-methyl-D-aspartic acid (1-2.5yg) into the SI/LPO produced a dose dependent increase in locomotor activity (figs

13,14, and 15 respectively). At these doses, rats exhibited periods of intense prolonged coordinated locomotor activity similar to that observed when these agents were injected into the nucleus accumbens.

While the movements of the rats were vigorous, they were also controlled. Thus, the rats avoided obstacles placed in their path and never ran into the cage walls. AMPA (lyg) produced the greatest hypermotility response which was 65 times greater than the response of saline treated animals. Kainic acid (60ng) and N-methyl-D- aspartic acid (2.5yg) were less effective, producing maximum hypermotility responses which were only 25 and 12 times greater than the reponses of control animals respectively. Higher doses of AMPA 72

(3yg), kainic acid (120ng), and N-methyl-D-aspartic acid (5yg) induced tremors and labored breathing which appeared to interfere with the hypermotility response and may account for the reduction in locomotor activity at these doses.

Effect of GAMS on AMPA-, kainic acid-, and N-methyl-D- aspartic acid- stimulated locomotor activity in the rat.

Jf-Glutamyl ami nomethyl sulfonate (GAMS) has been characterized as a selective antagonist of the effects of quisqualic acid in the nucleus accumbens (Shreve and Uretsky, 1988a). The purpose of this experiment was to determine the effect of GAMS on AMPA-, kainic acid-, and N-methyl-D-aspartic acid- stimulated locomotor activity after bilateral injection into the SI/LPO. GAMS (1 and 25yg) was co-administered with AMPA (0.5yg), kainic acid (30ng), and N-methyl-

D-aspartic acid (2.5yg) into the SI/LPO. For this study AMPA and kainic acid produced comparable levels of locomotor activity while the hypermotility response elicited by N-methyl-D-aspartic acid was only one third of that produced by AMPA and kainic acid (fig 16).

GAMS (lyg) produced a significant 82% inhibition of AMPA- stimulated locomotor activity whereas this dose of GAMS did not produce a significant inhibition of either kainic acid (16%) or N-methyl-D- aspartic acid (25%). A further Increase in the dose of GAMS to 25yg with kainic acid and N-methyl-D-aspartic acid produced no inhibition and a significant 62% inhibition respectively. GAMS, administered alone at these doses, did not produce a statistically significant effect on locomotor activity. 73

Figure 12. Sites of Injection of AMPA, kainic acid, or N-methyl-D- aspartic acid 1n the SI/LPO. These sites represent the tips of the needle tracks. Closed circles, triangles, and squares refer to sites that produced a significant stimulation of locomotor activity 1n response to AMPA, kainic acid, or N-methyl-D-aspartic acid Injected into the SI/LPO respectively; open circles were sites that did not produce a significant hypermotility response. Adapted from reference 13. Abbreviations: AC, anterior commissures; GP, globus palHdus; OC, optic chiasm; ST, striatum. 74

* (4) 16000 14000 12000 13 O X 10000 80 0 0 (5) 60 0 0 (5) 4000 (4) (5) 2000

0.1 0.5 1.0 3.0 A M P A (^g)

Figure 13. Effect of AMPA on locomotor activity after bilateral Injection Into the SI/LPO. A solution of AMPA or vehicle was Injected 1n a 0.5y£ volume and the animals were placed 1n motor activity cages, and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations in parentheses: Kruskal-Wal11s analysis of variance H=17.62, p<0.05. * p<0.05 with respect to vehicle (Mann-Wh1tney U-test). 75

* 8000 ( 6) (4) 7000 - 6000 1 3 ® 5000 4000 ^ 3000 - (5) 2000 (3)

1000 - ( 6) j f i 15 30 60 120 Kainic Acid (ng)

Figure 14. Effect of kainic acid on locomotor activity after bilateral Injection Into the SI/LPO. A solution of kainic acid or vehicle was Injected 1n a 0.5yt volume and the animals were placed 1n motor activity cages and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations in parentheses: Kruskal-Wal11s analysis of variance H=18.98, p<0.05. * p<0.05 with respect to vehicle (Mann-Wh1tney U- test). 76

5 0 0 0

(5) 4 0 0 0 3 X 3000 ( 6) \

= 2000 o (5) 1000 ( 6)

1.0 2.5 5.0 N M D A (^ g )

Figure 15. Effect of N-methyl-D-aspartic acid on locomotor activity after bilateral Injection Into the SI/LPO. A solution of N-methyl- D-aspartic acid or vehicle was Injected 1n a 0.5yH volume and the animals were placed 1n motor activity cages and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses: Kruskal-Wal11s analysis of variance H=9.86, p<0.05. * p<0.05 with respect to vehicle (Mann- Whltney U-test). 77

□ - Vehicle 0-GAMS 1/xg ■ - GAMS 25Mg 14000 (5) (4) 12000 3 O X 10000 (7) N (4) >N 8000 6000 // * ( 8) 4000 * (7) ( 6) 2000 (5) (4) ( 6) (4) _cSl ■ AMPA 0.5fig KAIN 30ng NMDA 2.5fig

Figure 16. Effect of GAMS on AMPA-, kainic acid-, and N-methyl-D- aspartic acid- stimulated locomotor activity after bilateral injection Into the SI/LPO. A solution of AMPA (0.5yg), kainic acid (30ng), or N-methyl-D-aspartic add (2.5yg) with or without GAMS (l-25yg) was injected in a 0.5y£ volume and the animals were placed 1n motor activity cages and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses: Kruskal-Wal1is analysis of variance H=2.38, p>0.05 for the control experiment, H=1.8 p>0.05 for the kainic add experiment, H=4.53, p=0.05 for the N-methyl-D-aspartic add experiment. *p<0.05 with respect to vehicle controls (Mann- Whitney U-test). 78

□ -Vehicle

12000 DNQX 1Mg 2 10000 8000 6000 4000 2000 (4) (4)

AMPA 0.5/xg KA 30ng

Figure 17. Effect of DNQX on AMPA-, kainic acid-, or N-methyl-D- aspartic acid- stimulated locomotor activity after bilateral Injection Into the SI/LPO. A solution of AMPA (0.5yg), kainic add (30ng), or N-methyl-D-aspartic add (2.5yg) with or without DNQX (lyg) was injected In a 0.5y£ volume and the animals were placed in motor activity cages and locomotor activity recorded for 1 hour. Each point represents the mean ±S.E.M. for the number of observations in parentheses. * p<0.05 with respect to vehicle control s. 79

-DA A 10/xg 14000 — ( 6) 12000—

10000— ( 6)

8000— '/ '/ 6000 —

4000 — 1 (6) * 2000— (4) (4) (4) (4) [*] <|> 1 * 1 | W | 21 r ^ i r4 i 1 I m PB AMPA 0.5/ig SAL NMDA 2.5^g

Figure 18. Effect of D-a-aminoadipic acid on AMPA- or N-methyl-D- aspartic acid- stimulated locomotor activity after bilateral Injection Into the SI/LPO. A solution of AMPA (0.5yg) or N-methyl- D-aspartic acid (2.5yg) with or without DAA 10yg was Injected 1n a 0.5yd volume and the animals were placed in motor activity cages and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses. *p<0.05 with respect to vehicle controls. 80

Effect of DNQX on AMPA-, kainic acid-, and N-methyl-D-aspart1c acid- stimulated locomotor activity 1n the rat.

6,7-Dinitroqu1noxaline-2,3-dione (DNQX) has recently been characterized as a selective qulsqualic acid receptor antagonist on the basis of JH-AMPA binding studies (Honore, Davies, Drejer,

Fletcher, Jacobsen, Lodge, and Nielsen, 1988). The purpose of this experiment was to determine the effect of DNQX on AMPA-, kainic acid-, and N-methyl-D-aspartic acid- stimulated locomotor activity after bilateral injection into the SI/LPO. DNQX (lyg) was coadministered with either AMPA (0.5yg), kainic acid (30ng), or N- methyl-D-aspartic acid (2.5yg) into the SI/LPO. AMPA alone produced the greatest hypermotility response which was two and five times greater than kainic acid and N-methyl-D-aspartic acid respectively

(fig 17). DNQX (lyg) produced a significant 64% and 84% inhibition of AMPA- and kainic acid- stimulated locomotor activity respectively. However, this dose of DNQX did not significantly affect the hypermotility response elicited by N-methyl-D-aspartic acid. The administration of DNQX alone did not produce a significant effect on locomotor activity.

Effect of D-a-aminoadipic acid on AMPA- and N-methyl-D aspartic acid- stimulated locomotor activity 1n the rat.

D-a-am1noadip1c acid, an antagonist of the N-methyl-D-aspartic acid subtype of excitatory amino acid receptors, has been shown to selectively inhibit the hypermotility responses to N-methyl-D- 81 aspartic acid in the nucleus accumbens (Donzanti and Uretsky,

1984a). In this study, D-a-aminoadipic acid (10yg) was coadministered with AMPA (0.5yg) and N-methyl-D-aspartic acid

(2.5yg) into the SI/LPO. AMPA and N-methyl-D-aspartic acid produced significant increases in locomotor activity as compared to vehicle controls although the hypermotility response to AMPA was over four times greater than the response to N-methyl-D-aspartic acid (fig

18). D-a-aminoadipic acid produced a significant 58% inhibition of

N-methyl-D-aspartic acid stimulated locomotor activity but did not produce a statistically significant inhibition of AMPA stimulated locomotor activity. D-a-aminoadipic acid, administered alone, did not produce a statistically significant effect on locomotor activity.

DISCUSSION

The major finding of this study is that the direct injection of the excitatory amino acids, AMPA, kainic acid, and N-methyl-D- aspartic acid, into the SI/LPO produced marked dose - dependent increases in locomotor activity in the rat. These hypermotility responses to AMPA and N-methyl-D-aspartic acid were selectively inhibited by excitatory amino acid receptor antagonists suggesting that the responses are mediated by the activation of specific excitatory amino acid receptors. These results suggest that excitatory amino acid neurotransmisslon in the SI/LPO 1s involved in the stimulation of coordinated locomotor activity. 82

Studies were designed to determine the involvement of non-N-

methyl-D-aspartic acid receptors in the SI/LPO in stimulating

locomotor activity. DNQX has recently been described as a potent

competitive non-N-methyl-D-aspart1c acid receptor antagonist based

on its ability to selectively inhibit 3H-AMPA and 3H-kainic acid

binding to the quisqualic acid and kainic acid receptors

respectively, while being a much weaker inhibitor of 3H-CPP binding

to the N-methyl-D-aspartic acid receptor (Honore, et.al., 1988). In

the present study DNQX (lyg) produced a significant 64% and 84%

inhibition of AMPA- and kainic acid- stimulated locomotor activity

while producing no significant effect on N-methyl-D-aspartic acid-

stimulated locomotor activity. These results, which are in

agreement with the inhibitory effects of DNQX on the responses to

AMPA and kainic acid in electrophysiological studies (Honore et.al.,

1988), suggest that the activation of non-N-methyl-D-aspartic acid

receptors in the SI/LPO can produce a stimulation of locomotor

•activity.

The effects of excitatory amino acids in the SI/LPO were also

determined in the presence of GAMS which has been described as a

potent, but non-selective, antagonist of kainic acid and quisqualic

acid receptor subtypes (Fagg, 1985) based upon electrophysiological

studies (Davies, Evans, Jones, Smith, and Watkins, 1982; Jones,

Smith, and Watkins, 1984). Thus, iontophoretic studies on neurons of the cat spinal cord have shown that GAMS selectively antagonized

the responses to excitatory amino acids in the general order kainic 83

acid > quisqualic acid » N-methyl-D-aspartic acid (Davies and

Watkins, 1985). However, despite its ability to inhibit the

electrophysiological effects of AMPA and kainic acid, recent binding

studies have shown that GAMS is a very weak inhibitor of both 3H-

AMPA and 3H-ka1nic acid binding (Honore et.al., 1988

Murphy, Snowhill, and Williams, 1987) suggesting that GAMS does not

inhibit the effects of AMPA and kainic acid by blocking their

respective receptors. In the present study, GAMS at a dose of lyg

produced a marked (82%) inhibition of AMPA- stimulated locomotor

activity while having no significant effect on kainic acid- and N-

methyl-D-aspartic acid- stimulated locomotor activity. The

selective effect of GAMS in inhibiting AMPA-induced responses is in

agreement with the results of a previous study which showed that

GAMS selectively Inhibited the hypermotility responses to AMPA and

quisqualic acid in the nucleus accumbens while being much less

effective in inhibiting the responses to kainic acid and N-methyl-D-

aspartic acid (Shreve and Uretsky, 1988a). Although the mechanism

for the selectivity of GAMS in the SI/LPO and nucleus accumbens is

currently not known, these results suggest that the hypermotility

response to AMPA in the SI/LPO is mediated by a different mechanism

than that produced by kainic acid or N-methyl-D-aspartic acid and are consistent with the actions of these compounds at separate

receptors.

Similarly, the hypermotility response produced by the direct

injection of N-methyl-D-aspartic acid into the SI/LPO seems to be selectively mediated by the activation of N-methyl-D-aspartic acid

receptors. D-ot-ami noadipic acid is an excitatory amino acid

antagonist, which has been shown in electrophysiological (Davies and

Watkins, 1979; McLennan and Lodge, 1979), binding (Olverman, Jones,

and Watkins, 1988), and behavioral (Donzanti and Uretsky, 1984a;

Shreve and Uretsky, 1988a), studies to be selective for the N-

methyl-D-aspart1c acid receptor. In the present study

D-a-am1noadipic acid, at a dose that produced a significant 58%

inhibition of N-methyl-D-aspartic acid- induced locomotor activity,

did not significantly inhibit AMPA- induced locomotor activity.

Although this result is consistent with a selective inhibition of

the effects of N-methyl-D-aspartic acid, the stimulation of

locomotor activity produced by the maximally effective dose of N-

methyl-D-aspartic acid (2.5yg) was three to four times less than

that produced by a low dose of AMPA (0.5yg). It is therefore

possible that the locomotor activity produced by AMPA may not be as

easily antagonized as that produced by N-methyl-D-aspartic acid.

Although this possibility can not be excluded, the greater

hypermotility response to AMPA was found to be selectively inhibited

by GAMS and DNQX at doses that had no significant effect on the

smaller response to N-methyl-D-aspartic acid. Furthermore, we have

found in a previous study (Donzanti and Uretsky, 1988a) that the dose of D-a-aminoadipic acid used in the present study selectivity

inhibited the hypermotilty response elicited by N-methyl-D-aspartic acid but had no significant effect on that produced by AMPA in the 85 nucleus accumbens. Therefore these results suggest that activation of N-methyl-D-aspartic acid receptors in the SI/LPO leads to the stimulation of locomotor activity.

The SI/LPO has previously been shown to play an important role in modulating locomotor activity inititated 1n the nucleus accumbens

(Jones and Mogenson, 1980a). Thus, it has been shown that the locomotor activity produced by the stimulation of opioid, dopamine, and excitatory amino acid receptors in the nucleus accumbens can be inhibited by the injection of GABA or muscimol into the SI/LPO

(Mogenson and Nielsen, 1983; Shreve and Uretsky, 1988b; Swerdlow and

Koob, 1984; Swerdlow et.al., 1984; Swerdlow et.al., 1987). These observations suggest that the stimulation of locomotor activity produced by drugs acting in the nucleus accumbens is mediated by a decrease in GABAergic neurotransmission in the SI/LPO. This hypothesis is supported by the observation that the injection of picrotoxin, an inhibitor of the effects of GABA, into the SI/LPO can stimulate locomotor activity (Mogenson and Nielsen, 1983; Swerdlow and Koob, 1987). These observations taken together with the present observations suggest that both the inhibition of GABAergic neurotransmission and the stimulation of glutamatergic (excitatory amino acid) neurotransmission in the SI/LPO can produce a stimulation of coordinated locomotor activity.

The SI/LPO has previously been shown to play an important role in modulating locomotor activity initiated by the injection of drugs 86 into the nucleus accumbens. Thus the stimulation of locomotor activity induced by the injection of drugs into the nucleus accumbens is thought to be mediated by the inhibition of a GABAergic projection from the nucleus accumbens to the SI/LPO. These present results indicate that excitatory amino acids when injected into the

SI/LPO stimulate locomotor activity, suggesting that excitatory amino acids may either directly or indirectly oppose the actions of

GABA in this region. Therefore, since neuronal mechanisms in the nucleus accumbens have been implicated in goal-oriented behavior, excitatory amino acid as well as GABAergic mechanisms in the SI/LPO may function to regulate this behavior. CHAPTER V

Effect of glutamatergic transmission 1n the subpalHdum

on the hypermotl11ty response to the Intraaccumbens

administration of AMPA and amphetamine.

INTRODUCTION

The nucleus accumbens 1s a region of forebrain which 1s thought to be Involved in the Initiation and regulation of normal locomotor activity (Mogenson, Jones, and Yim, 1980; Mogenson and Y1m, 1980).

Thus 1t has been shown that amphetamine and heroin produce hypermotl11ty responses through neuronal mechanisms 1n the nucleus accumbens (Swerdlow, Vaccarlno, Amalrlc, and Koob, 1986). The direct injection of dopamine, carbachol, and various excitatory amino acids into the nucleus accumbens also elicit marked increases

1n locomotor activity (Pijnenberg, Honig, Van derHeyden, and Van

Rossum, 1976; Mogenson and Nielsen, 1983; Morgenstern, Mende, Gold,

Lemme, and Oelssner, 1984; Arnt, 1981; Donzanti and Uretsky, 1983;

Austin and Kalivas, 1988).

Previous studies have shown that an Inhibitory GABAergic projection from the nucleus accumbens to the substantia innominata /

lateral preoptic area (SI/LPO) may mediate the hypermoti1ity produced by drugs which act in the nucleus accumbens (Jones and

87 Mogenson, 1980a; Nauta, Smith, Fail'll, and Domesick, 1978; Mogenson and Nielsen, 1983; Mogenson, Swanson, and Wu, 1983). Thus it has been shown that the injection of muscimol or GABA into the SI/LPO inhibited the locomotor activity produced by the activation of opioid (Swerdlow et.al., 1986), dopamine (Mogenson and Nielsen,

1983; Swerdlow and Koob, 1984), and excitatory amino acid receptors

(Shreve and Uretsky, 1988b) in the the nucleus accumbens.

Therefore, GABAergic mechanisms in the SI/LPO appear to regulate locomotor activity initiated in the nucleus accumbens.

Recent evidence suggests that the SI/LPO also contains excitatory amino acid neuronal synapses. Thus, autoradiographic studies in this subpalli da 1 region have shown the presence of excitatory amino acid binding sites that are thought to represent receptors for glutamate (Monaghan and Cotman, 1985; Halpain,

Wieczorek, and Rainbow, 1984). In addition, synaptosomes prepared from the SI/LPO can release endogenous glutamate in a calcium dependent manner 1n response to depolarization and possess high affinity uptake sites for L-glutamate and D-aspartate (Davies,

McBean, and Roberts, 1984). The location of the cells of origin of these glutamatergic nerve terminals have not been completely defined but recent evidence suggests that they may originate in the amygdala, a region of the limbic system (Fuller, Russchen, and

Price, 1987). These findings are consistent with the concept that glutamate, an excitatory amino acid, may act at excitatory amino acid receptors in the SI/LPO. 89

We have found that the direct injection of the excitatory amino

acids, AMPA, kainic acid, and N-methyl-D-aspartic acid, into the

SI/LPO elicits marked dose dependent increases in locomotor activity

(unpublished observations). These hypermotility responses were

selectively inhibited by the co-administration of excitatory amino

acid antagonists suggesting that these responses were mediated by

the activation of specific excitatory amino acid receptors. This

observation taken with previous observations, of the effects of

GABAergic mechanisms, suggest that, in the SI/LPO, activation of excitatory amino acid receptors can stimulate while GABA receptor activation can inhibit locomotor activity. However, it is unclear whether excitatory amino acid receptors in the SI/LPO play a role in the hypermotility responses produced by the administration of drugs into the nucleus accumbens. To investigate this possibility, we have determined the effect of excitatory amino acid antagonists injected into the SI/LPO on the stimulation of locomotor activity produced by the intraaccumbens injection of AMPA and amphetamine.

METHODS

Surgical procedure

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis,

Indiana, U.S.A.) weighing 150—190g, were lightly anesthetized with a halothane/oxygen mixture and placed in a stereotaxic apparatus

(David Kopf Instruments, California, U.S.A.). A midline incision was made in the scalp and holes were drilled on each side of the 90 skull to facilitate bilateral injections into the nucleus accumbens and/or the substantia innominata / lateral preoptic area (SI/LPO).

The coordinates for the nucleus accumbens were 9.4mm anterior to the intraaural line and 2.3mm lateral to the saggital suture (Konig and

Klippel, 1963). The needle of a 10yd Hamilton syringe (Hamilton

Co., Reno, Nevada, U.S.A.) was then inserted at a 10° angle toward the midline (to avoid puncturing the ventricular system) into the previously drilled holes to a depth of 6.4mm from the surface of the skull. The coordinates for the SI/LPO were 6.8mm anterior to the intraaural line,. 1.8mm lateral to the saggital suture, and 2.0mm above the intraaural line (Konig and Klippel, 1963). Drugs or vehicle were injected in a 0.5yd volume at a rate of 0.5yd/min. The needle was left in place for an additional 1 min to allow for diffusion of the solution. After the injection, the needle was removed and the incision was sutured and swabbed with 5% (w/v) lidocaine ointment.

Monitoring locomotor activity

After the Injections into the nucleus accumbens and/or SI/LPO, the anesthesia was turned off and the animals recovered from anesthesia within 5 min. After recovery, the animals were placed in motor activity cages (Opto Varimex-Minor, Columbus Instruments,

Ohio, U.S.A.) and allowed 10 min to adapt to the cages. The motor activity was monitored and recorded as described in chapter II. 91

Hi stology

After each experiment, the rats were decapitated and their brains rapidly removed and fixed in a 10% formalin solution for 48 hours. Frozen sections (80y thick) were sliced using a Cryo-Cut

Microtome (American Optical Corp., Buffalo, N.Y., U.S.A.) to check the location of the injection needle. When the tips of the needle tracks were found to be outside of the nucleus accumbens and/or

SI/LPO, the locomotor activity recordings of the animals were not used for the study. Figure 19 shows the injection sites for excitatory amino acid antagonists in the SI/LPO that were effective and ineffective in inhibiting hypermotility produced by injection of drugs into the nucleus accumbens. Animals were excluded when at least one of the two injection sites in an animal was outside the

SI/LPO.

Drugs

The following compounds were purchased from Sigma Chemical Co.

(St. Louis, MO. U.S.A.): N-methyl-D-aspartic acid, kainic acid, D- amphetamine, and D-a-aminoadipic acid. a-Amino-3-hydroxy-5

-methylisoxazole-4- propionate (AMPA) was purchased from Research

Biochemicals Inc. (Natick, MA. U.S.A.).

All drugs were dissolved in saline and adjusted to pH 7.4 with

IN NaOH. The doses shown refer to the amount injected on each side of the nucleus accumbens and or SI/LPO. Control animals were injected with an equal volume (0.5y£) of saline. 92

A I GP ; AC II *

"oc

o f : jf » 'I* 0 * B be

!/ o V» c be

Figure 19. Sites of injection of excitatory amino acid antagonists in the SI/LPO. A = GAMS, B = DNQX, and C = DAA. These sites represent the tips of the needle tracks. Closed circles refer to sites that produced an inhibition of the hypermotl1ity response to drugs that were Injected into the nucleus accumbens; open circles were sites that were located outside of the SI/LPO and were excluded from the study. Abbreviations: AC, anterior commissures; GP, globus pallidus; OC, optic chiasm; ST, striatum. 93

Stati sties

Data were expressed as the mean and standard error of the mean

(SEM). The effects of drugs and saline treatment were evaluated statistically using the nonparametric Kruskal-Wal1is one-way analysis of variance followed by the one-tailed Mann-Whitney U-test, with a level of p<0.05 being considered significant.

RESULTS

Effect of the administration of GAMS into the

SI/LPO on the hypermoti1ity responses to the administration of AMPA or amphetamine into the nucleus accumbens.

GAMS has been characterized as a selective antagonist of the effects of quisqualic acid in the nucleus accumbens (Shreve and

Uretsky, 1988a) and SI/LPO (unpublished observations). The purpose of this experiment was to determine the effects of GAMS, injected into the SI/LPO, on the hypermoti 1 ity produced by the injection of

AMPA or amphetamine into the nucleus accumbens. Various doses of

GAMS or vehicle were bilaterally injected into the SI/LPO after bilateral injection of AMPA (0.25yg), amphetamine (lOyg), or vehicle into the nucleus accumbens. GAMS was found to inhibit the hypermotility responses to AMPA and amphetamine (Fig. 20). GAMS (5yg) has been shown to inhibit the hypermotility response to the

Injection of AMPA in the SI/LPO but not kainic acid or N-methyl-D- aspartic acid (Data not shown). This dose of GAMS produced a 94 significant 83% and 77% inhibition of the locomotor activity stimulated by AMPA and amphetamine, respectively. In contrast, GAMS did not produce a significant change in the locomotor activity of rats which received vehicle injections into the nucleus accumbens.

Effect of the administration of DNQX into the SI/LPO on the hypermotility responses to the administration of AMPA or amphetamine into the nucleus accumbens.

DNQX has recently been described as a selective quisqualic acid receptor antagonist on the basis of 3H-AMPA binding studies

(Honore, Davies, Drejer, Fletcher, Jacobsen, Lodge, and Nielsen,

1988). The purpose of this experiment was to determine the effect of DNQX, injected into the SI/LPO, on the hypermotility produced by the injection of AMPA or amphetamine into the nucleus accumbens.

DNQX (lyg) or vehicle was bilaterally injected into the SI/LPO following the bilateral injection of AMPA (0.5yg), amphetamine

(lOyg), or vehicle into the nucleus accumbens. DNQX was found to produce a significant 75% and 74% inhibition of AMPA and amphetamine stimulated locomotor activity, respectively (Fig. 21). DNQX did not significantly effect the locomotor activity of rats which received vehicle injections into the nucleus accumbens. 95

SI/LPO Injection □ - Vehicle

12000 -GAMS v 9 V. (6) 3 -GAMS 5f±q o 10000 X \ 8000 >> ."tz 6000 (7)

o 4000 (4) * -* ( 6) 2000 (4) (4) (4) (4) ^ A . 1 ■ i N. Acc. Vehicle AMPA 0.25/ig AMPHET 10/ig

Figure 20. Locomotor activity responses to the Intraaccumbens administration of amphetamine (AMPHET), AMPA, or vehicle following injection of GAMS or vehicle Into the SI/LPO. A solution of either AMPHET (lOyg), AMPA (0.25yg), or vehicle was bilaterally Injected into the nucleus accumbens followed by GAMS (lyg - hatched bars, 5yg - solid bars) or vehicle (open bars) bilaterally injected into the SI/LPO. The animals were then placed 1n motor activity cages and locomotor activity recorded for 1 hour. All Injections were administered 1n a 0.5y£ volume. Each point represents the mean ± S.E.M. for the number of observations in parentheses. Kruskal- WalHs analyls of variance H=9.08, p<0.05. * p<0.05 with respect to vehicle treatment in the SI/LPO. 96

SI/LPO Injection □ - Vehicle

12000 -DNQX 1/Lig

3 10000 8 0 0 0 6 0 0 0 4 0 0 0 2000

N. Acc. Vehicle AMPA 0.25 pQ AMPHET lO^g

Figure 21. Locomotor activity responses to the Intraaccumbens administration of amphetamine (AMPHET), AMPA, or vehicle following Injection of DNQX or vehicle Into the SI/LPO. A solution of either AMPHET (lOyg), AMPA(0.25yg), or vehicle was bilaterally Injected Into the nucleus accumbens followed by DNQX (lyg - hatched bars) or vehicle (open bars) bilaterally Injected Into the SI/LPO. The animals were then placed 1n motor activity cages and locomotor activity recorded for 1 hour. All Injections were administered 1n a 0.5yd volume. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses. * p<0.05 with respect to vehicle treatment 1n the SI/LPO. 97

SI/LPO Injection □ - Vehicle

12000 z> 10000 8000 6000 400 0 2000

N. Acc. Vehicle AMPA 0 . 2 5 ^ AMPHET 10^g

Figure 22. Locomotor activity responses to the Intraaccumbens administration of amphetamine (AMPHET), AMPA, or vehicle following Injection of DAA or vehicle Into the SI/LPO. A solution of either AMPHET (lOyg), AMPA (0.25yg), or vehicle was bilaterally Injected into the nucleus accumbens followed by DAA (lOyg - hatched bars) or vehicle (open bars) bilaterally Injected Into the SI/LPO. The animals were then placed 1n motor activity cages and locomotor activity recorded for 1 hour. All Injections were administered in a 0.5yH volume. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses. * p<0.05 with respect to vehicle treatment in the SI/LPO. 98

□ - Vehicle ^-Muscimol 25ng

14000 ( 6) 12000 3 O “T 10000 s 8000 = 6000 o 4000 * (7) 2000 (4) (4) i ^ i Vehicle AMPA 0.5/tg

Figure 23. Effect of muscimol on AMPA - stimulated locomotor activity after bilateral Injection Into the SI/LPO. A solution of AMPA (0.5yg) or vehicle with or without muscimol (25ng) was Injected 1n a 0.5y£ volume and the animals were placed in motor activity cages and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations in parentheses. *p<0.05 with respect to vehicle controls. 99

|~l - Vehicle -GAMS 5fj.g -GAMS 25p.g

(14) 6000 ( 8) 3 5000 o ^ 4000 :]= 3000 o 2000 * 1000 ( 6) ( 8) (4) (4) r* ! * i Vehicle PTX 0.5^g

Figure 24. Effect of GAMS on plcrotoxln (PTX) - stimulated locomotor activity after bilateral Injection into the SI/LPO. A solution of PTX (0.5yg) or vehicle with or without GAMS (5 or 25yg) was injected 1n a 0.5yH volume and the animals were placed in motor activity cages and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations in parentheses. Kruskal-Wal1is analysis of variance H=13.25, p<0.05. *p<0.05 with respect to vehicle controls. 100

Q-Vehicle -DNQX1^g

5000 — 1 3 O ( 8) X 4000 —

3000 —

2000 — -X- ( 8) 1000 — (4) (4) i^i 1 Vehicle PTX 0.5/xg

Figure 25. Effect of DNQX on plcrotoxln (PTX) - stimulated locomotor activity after bilateral Injection Into the SI/LPO. A solution of PTX (0.5yg) or vehicle with or without DNQX (lyg) was Injected 1n a 0.5y£ volume and the animals were placed 1n motor activity cages and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations in parentheses. *p<0.05 with respect to vehicle controls. 101

□ -Vehicle □ - DAA 10^g

(5) (5) 7000 >- 6000 x 5000 >. 4000 ~ 3000 O ^ 2000 1000 a(4) (4) Vehicle PTX 0.5/i.g

Figure 26. Effect of DAA on plcrotoxin (PTX) - stimulated locomotor activity after bilateral Injection Into the SI/LPO. A solution of PTX (0.5yg) or vehicle with or without DAA (lOyg) was Injected 1n a 0.5y£ volume and the animals were placed 1n motor activity cages and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses. *p<0.05 with respect to vehicle controls. 102

DAA 10/xg + 6AMS 5/j.q

(6) 8000 7000 6000 5000 4000 * 3000 ( 6 ) 2000

1000 (4) (4 ) J3_ Vehicle PTX 0.5/xg

Figure 27. Effect of DAA and GAMS on plcrotoxln (PTX) - stimulated locomotor activity after bilateral injection Into the SI/LPO. A solution of PTX (0.5yg) or vehicle with or without DAA (lOyg) + GAMS (5yg) was Injected 1n a 0.5yH volume and the animals were placed 1n motor activity cages and locomotor activity recorded for 1 hour. Each point represents the mean ± S.E.M. for the number of observations 1n parentheses. *p<0.05 with respect to vehicle control s. 103

Effect of the administration of DAA Into the SI/LPO on the hypermotlI1ty responses to the administration of AMPA or amphetamine into the nucleus arcumbens.

D-a-aminoadipic acid has been characterized as a selective receptor antagonist of the N-methyl-D-aspartic acid receptor on the basis of electrophysiological, binding, and behavioral studies

(Davies and Watkins, 1979; McLennan and Lodge, 1979; Olverman,

Jones, and Watkins, 1988; Donzanti and Uretsky, 1984a; Shreve and

Uretsky, 1988a). In order to determine the effect of

D-a-aminoadipic acid, injected into the SI/LPO, on the hypermotility produced by the injection of AMPA or amphetamine into the nucleus accumbens, D-a-aminoadipic acid (lOyg) was bilaterally injected into the SI/LPO following the bilateral injection of AMPA (0.5yg), amphetamine (lOyg), or vehicle into the nucleusaccumbens.

D-a-aminoadipic acid did not significantly inhibit the locomotor activity produced by the injection of AMPA, amphetamine, or vehicle into the nucleus accumbens (Fig. 22).

Effect of muscimol on AMPA stimulated locomotor activity in the SI/LPO.

The purpose of this experiment was to determine the effect of muscimol, a GABA-A receptor agonist, on the hypermoti1ity produced by the injection of AMPA into the SI/LPO. Muscimol (25ng) was co administered with AMPA (0.5pg) into the SI/LPO. The bilateral injection of AMPA (0.5pg) into the SI/LPO elicited a marked 104 stimulation of coordinated locomotor activity. The Injection of muscimol (25ng) produced a significant 82% inhibition of this AMPA stimulated hypermotility while having no effect on normal locomotor activity (Fig. 23).

Effect of DNQX, GAMS, or D-a-aminoadipic acid on picrotoxin stimulated locomotor activity in the SI/LPO.

The purpose of this experiment was to determine the effect of various excitatory amino acid antagonists on the hypermotility produced by the injection of picrotoxin, an inhibitor of the effects of GABA, into the SI/LPO. Picrotoxin (0.5yg) was bilaterally injected into the SI/LPO in the presence and absence of the excitatory amino acid antagonists, GAMS, DNQX, or D-a-aminoadipic acid. DNQX (lyg) produced a significant 64% inhibition of picrotoxin stimulated locomotor activity (Fig. 25). GAMS at a dose of 5yg, which has been shown to selectively antagonize the hypermoti1ity elicited by AMPA, did not produce a significant inhibition of picrotoxin induced locomotor activity (Fig. 24). A further increase in the dose of GAMS (25yg) produced a significant

86% inhibition of the hypermotility produced by picrotoxin.

D-a-aminoadipic acid (lOyg) did not produce a significant effect on the locomotor activity elicited by picrotoxin (Fig. 26). The combination of GAMS (5yg) and D-a-aminoadipic acid (lOyg), at doses which were ineffective in inhibiting picrotoxin when administered alone, produced a significant 66% inhibition of picrotoxin induced 105 hypermoti1ity (Fig. 27). In these experiments, the injection of these excitatory amino acid antagonists, alone, into the SI/LPO did not produce changes in locomotor activity as compared to vehicle control s.

DISCUSSION

The major finding of this study is that the injection of excitatory amino acid antagonists into the SI/LPO inhibited the stimulation of locomotor activity produced by the injection of AMPA or amphetamine Into the nucleus accumbens. Thus, the bilateral injection of GAMS or DNQX into the SI/LPO produced a significant inhibition of the hypermotility elicited by the bilateral injection of AMPA or amphetamine into the nucleus accumbens, while the

Injection of D-a-aminoadipic acid into the SI/LPO did not produce a significant inhibition of the AMPA or amphetamine induced locomotor activity. Since GAMS and DNQX have been shown to antagonize responses mediated by the activation of non-N-methyl-D-aspartic acid receptors, these results suggest that quisqualic acid and kainic acid receptor mediated mechanisms in the SI/LPO may be involved in the modulation of locomotor activity initiated in the nucleus accumbens.

Studies were performed to determine whether the inhibition of non-N-methyl-D-aspartic acid receptors in the SI/LPO are involved in the regulation of locomotor activity produced by the injection of

AMPA or amphetamine into the nucleus accumbens. DNQX has recently 106

been characterized as a competitive non-N-methyl-D-aspartic acid

receptor antagonist on the basis of binding and electrophysiological

studies. DNQX (lpg) has also been shown to selectively inhibit the

hypermotility responses to kainic acid or AMPA, but not to N-methyl-

D-aspartic acid, when co-injected with these excitatory amino acid agonists into the SI/LPO (unpublished observations). In the present

study, the injection of DNQX (lyg) into the SI/LPO produced a

significant Inhibition of the hypermotility produced by the

injection of AMPA or amphetamine into the nucleus accumbens. These results suggest that the inhibition of quisqualic acid and kainic acid receptors in the SI/LPO can inhibit locomotor activity initiated in the nucleus accumbens.

The effect of GAMS, injected into the SI/LPO, on the hypermotility produced by the injection of AMPA or amphetamine into the nucleus accumbens was also determined. GAMS has been described as a potent but non-selective antagonist of kainic acid and quisqualic acid receptor subtypes based upon electrophysiological studies. Recent behavioral studies have shown that GAMS inhibited the hypermotility effects produced by AMPA and quisqualic acid when co-injected with these excitatory amino acids into either the the nucleus accumbens or the SI/LPO. In contrast, GAMS was much less effective 1n inhibiting the responses to kainic acid and N-methyl-D- aspartic acid. Despite this apparent selectivity for the quisqualic acid receptor, GAMS has been shown to be a very weak inhibitor of JH-AMPA binding suggesting that GAMS does not inhibit the 107 responses to AMPA by blocking the quisqualic acid receptor. Thus, the mechanism of the antagonism of AMPA effects by GAMS is not clear. In the present study, the injection of GAMS (5]ig) into the

SI/LPO, produced a significant inhibition of the locomotor activity elicited by the injection of AMPA or amphetamine into the nucleus accumbens. These results suggest that the inhibition of quisqualic acid receptor mediated mechanisms in the SI/LPO can inhibit locomotor activity initiated in the nucleus accumbens.

In contrast, the inhibition of N-methyl-D-aspartic acid receptors in the SI/LPO does not appear to be involved in modulating locomotor activity produced by the Injection of AMPA or amphetamine into the nucleus accumbens. D-a-aminoadipic acid has been characterized as a competitive N-methyl-D-aspartic acid receptor antagonist on the basis of electrophysiological, binding, and behavioral studies. The injection of N-methyl-D-aspartic acid into the SI/LPO produced a marked stimulation of co-ordinated locomotor activity which was selectively antagonized by the co-administration of D-a-aminoad1pic acid (unpublished observations). In the present study, the Injection of D-a-aminoadipic acid (lOyg) into the SI/LPO did not significantly inhibit the hypermotility produced by the injection of AMPA or amphetamine into the nucleus accumbens. Thus, while the activation of N-methyl-D-aspartic acid receptors in the

SI/LPO produces a stimulation of locomotor activity, the present results suggest that the inhibition of these N-methyl-D-aspartic acid receptors in this region does not play a role in modulating locomotor activity initiated in the nucleus accumbens. Previous studies have shown that the stimulation of excitatory amino acid (glutamatergic) receptors, as well as the antagonism of

GABAergic receptor mediated responses in the SI/LPO can produce a stimulation of co-ordinated locomotor activity. Thus, the injection of the excitatory amino acids, AMPA, kainic acid, and N-methyl-D- aspartlc acid, into the SI/LPO produced marked stimulations of co ordinated locomotor activity which were selectively antagonized by excitatory amino acid antagonists. Additionally, it has been shown that the injection of picrotoxin,an inhibitor of the effects of

GABA, into the SI/LPO stimulated locomotor activity. Indeed, it has been suggested that GABAergic synapses in the SI/LPO are involved in the stimulation of locomotor activity initiated by the injection of drugs into the nucleus accumbens. This hypothesis is based on the observation that the injection of GABA or muscimol into the SI/LPO inhibited the hypermotility responses produced by the stimulation of opioid, dopamine, and excitatory amino acid receptors in the nucleus accumbens, suggesting that these responses are mediated by a decrease in GABAergic neurotransmission in the SI/LPO. The present results suggest that an increase in glutamatergic neurotransmission in the SI/LPO is involved in the stimulation of locomotor activity initiated 1n the nucleus accumbens, since the injection of GAMS or

DNQX into the SI/LPO significantly inhibited the hypermotility produced by the injection of AMPA or amphetamine into the nucleus accumbens. Therefore, it appears that the GABAergic and glutamatergic

neural components in the SI/LPO may interact in some manner to produce a stimulation of locomotor activity. This possibility was examined in the present study by first determining the effect of muscimol on the locomotor activity produced by the injection of AMPA into the SI/LPO. In the present study, the hypermotility response elicited by the stimulation of quisqualic acid receptors in the

SI/LPO was significantly inhibited by the coadministration of muscimol, a GABA-A receptor agonist. These results initially suggested that the stimulation of excitatory amino acid receptors in the SI/LPO may produce locomotor activity by either directly or indirectly opposing the effects of GABA in this region.

We also examined the effect of excitatory amino acid antagonists on the locomotor activity produced by the injection of picrotoxin, an inhibitor of the effects of GABA, into the SI/LPO. In the present study, DNQX, at a dose which selectively inhibited the hypermotility responses to kainic acid and AMPA (but not to N- methyl-D-aspartic acid) in the SI/LPO, significantly inhibited this picrotoxin induced locomotor activity. GAMS also produced a significant inhibition of the hypermotility elicited by picrotoxin, but only at a higher dose which has been shown to antagonize the effects of both AMPA and N-methyl-D-aspart1c acid, but not to kainic acid. However, D-a-aminoadipic acid or a lower dose of GAMS, which have been shown to selectively inhibit the hypermoti1ity responses to N-methyl-D-aspartic acid or AMPA respectively, did not produce an 110 effect on the locomotor activity elicted by picrotoxin. Thus, these results suggest that the hypermotility elicited by picrotoxin can only be antagonized by the inhibition of more than one population of excitatory amino acid receptors. This concept is further supported by the present results which show that the coadministration of

D-a-aminoadipic acid and the lower dose of GAMS significantly inhibited the hypermotility response to picrotoxin. CHAPTER VI

SUMMARY

The nucleus accumbens is thought to play an important role 1n the initiation and regulation of normal locomotor activity. Studies have shown that the Injection of dopamine, carbachol, picrotoxin, and various excitatory amino acids Into the nucleus accumbens will produce a stimulation of locomotor activity. The neural connections of the nucleus accumbens with limbic and motor areas of the brain has led to the concept that the nucleus accumbens may serve as a functional interface between these two regions to modulate locomotor activity.

The direct intraaccumbens injection of the excitatory amino acids, AMPA, qulsqualic acid, kainic acid, and N-methyl-D-aspartic acid, has been shown to produce marked increases In locomotor activity. However, the unavailability of selective excitatory amino acid antagonists has hindered the complete characterization of these agents to elicit hypermotility by acting at specific excitatory amino add receptor subtypes. In the present study, GAMS, an excitatory amino add antagonist, was utilized to characterize the ability of AMPA (or quisqualic acid), kainic add, or N-methyl-D-

111 112 aspartic acid to stimulate locomotion by activating specific receptors in the nucleus accumbens. GAMS has previously been described as a potent, but non-selective, antagonist of quisqualic acid and kainic acid receptors. Despite this classification of

GAMS, the direct intraaccumbens injection of GAMS selectively inhibited the AMPA- and quisqualic acid- induced stimulation of locomotor activity. The mechanism of this selectivity of GAMS for the quisqualic acid receptor is not presently known since GAMS was unable to inhibit the binding of 3H-AMPA. However, these observations suggest that the activation of quisqualic acid receptors in the nucleus accumbens produces a stimulation of locomotor activity.

Recent evidence suggests that a GABAergic projection from the nucleus accumbens to the substantia innominata / lateral preoptic area (SI/LPO) is involved in the hypermotility produced by drugs which act in the nucleus accumbens. Studies have shown that the injection of GABA or muscimol into the SI/LPO inhibited the locomotor activity elicited by drugs which activate dopamine and opioid receptors in the nucleus accumbens. These observations suggest that hypermotility produced by increases in dopaminergic and opioid neurotransmission in the nucleus accumbens may be mediated by a decrease 1n GABAergic activity in the SI/LPO.

In the present study, we Investigated whether excitatory amino acid- induced stimulation of locomotor activity in the nucleus 113 accumbens is mediated by a decrease in GABAergic activity in the

SI/LPO. The injection of muscimol into the SI/LPO produced a significant inhibition of the hypermotility response produced by the intraaccumbens administration of AMPA, kainic acid, and N-methyl-D- aspartic acid. This inhibitory effect appears to be selective for this brain region since muscimol injected outside of the SI/LPO did not inhibit the hypermotility produced by the activation of excitatory amino acid receptors in the nucleus accumbens. These results suggest that the locomotor activity produced by increases in glutamatergic neurotransmission in the nucleus accumbens is mediated by a decrease in GABAergic activity in the SI/LPO.

In addition to the presence of GABA, an inhibitory neurotransmitter, glutamate, an excitatory neurotransmitter, is also found in the SI/LPO. Autoradiographic studies have shown that the

SI/LPO contains excitatory amino acid binding sites that are thought to represent receptors for glutamate. In the present study, we studied the role of excitatory amino acids in the SI/LPO on hypermotility responses. The injection of the excitatory amino acids, AMPA, kainic acid, or N-methyl-D-aspartic acid, into the

SI/LPO elicited marked dose - dependent increases in locomotor activity. The hypermotility responses produced by AMPA and N- methyl-D-aspartic acid were selectively Inhibited by GAMS and

D-a-aminoadipic acid, respectively. DNQX produced a significant inhibiton of AMPA- and kainic acid- stimulated locomotor activity but did not inhibit N-methyl-D-aspartic acid- stimulated locomotor 114 activity. These results suggest that the activation of specific excitatory amino acid receptors in the SI/LPO mediates the locomotor activating properties of these excitatory amino acids.

The SI/LPO has previously been shown to play an important role in modulating locomotor activity initiated in the nucleus accumbens by a decrease in GABAergic neurotransmission in this subpallidal region. In the present study, we have shown that the injection of excitatory amino acids into this subpallidal region produces significant increases in locomotor activity. Therefore, we performed experiments to ascertain whether the hypermotility responses produced by the intraaccumbens adminstration of amphetamine or AMPA are mediated by an increase in glutamatergic activity in the SI/LPO. The injection of GAMS or DNQX into the

SI/LPO produced a significant inhibition of the locomotor activity elicited by the intraaccumbens Injection of amphetamine or AMPA.

However, the injection of D-a-am1noadipic acid into the SI/LPO did not inhibit the locomotor activity produced by the injection of amphetamine or AMPA into the nucleus accumbens. These results suggest that the inhibition of quisqualic acid and kainic acid receptor mediated mechanisms in the SI/LPO can inhibit hypermotility responses Initiated in the nucleus accumbens.

Therefore, the locomotor activity produced by neuronal mechanisms 1n the nucleus accumbens may be mediated by either a decrease in GABAergic and / or an increase in glutamatergic 115 neurotransmission in the SI/LPO. In the present study, we examined the possibility that these GABAergic and glutamatergic neural components may Interact in some manner to mediate these hypermotl1i 1ty responses. We observed that the AMPA- stimulated locomotor activity, following injection into the SI/LPO, was inhibited by the co-administration of muscimol. We also observed that the locomotor activity produced by the injection of picrotoxin, an inhibitor of the effects of GABA, into the SI/LPO was inhibited by the co-administration of various excitatory amino acid antagonists. Although the mechanism of this interaction between

GABA and glutamate in the SI/LPO is presently not known, these results provide preliminary evidence to suggest that these two neurotransmitters may either directly or indirectly oppose each other to produce locomotor activity.

Neuronal mechanisms in the nucleus accumbens have been implicated in goal oriented behavior. In the present study, we have used the measurement of locomotor activity as an experimental model to determine the involvement of the nucleus accumbens in the initiation and regulation of locomotor behavior. The stimulation of locomotor activity by the nucleus accumbens appears to be mediated by neuronal mechanisms in the SI/LPO. We have shown that excitatory amino acid receptor mediated mechanisms in the nucleus accumbens and the SI/LPO can initiate and regulate locomotor activity. Therefore, glutamatergic neurotransmission in the nucleus accumbens and the

SI/LPO appears to play an important role in goal directed behavior. LIST OF REFERENCES

1. Amalric, M. and G.F. Koob (1985) Low doses of methylnalox- onium 1n the nucleus accumbens antagonize hyperactivity Induced by heroin 1n the rat. Pharmacol. Blochem. and Behavior 23:411-415.

2. Arnt, J. (1981) Hyperactivity following Injection of a glutamate agonist and 6,7-ADTN Into rat nucleus accumbens and its Inhibition by THIP. Life Sci 28:1597-1603.

3. Ascher, P. and L. Nowak (1987) Electrophyslologlcal studies of NMDA receptors. Trends 1n Neuroscl. 10:284-288.

4. Austin, M.C. and P.W. Kallvas (1988) The effect of cholinergic stimulation 1n the nucleus accumbens on locomotor behavior. Brain Res. 441:209-214.

5. B1gl, V., Woolf, N.J., and L.L. Butcher (1982) Cholinergic projections from the basal forebrain to frontal, parietal, temporal, occipital, and clngulate cortices: A combined fluorescent and acetylcholinesterase analysis. Brain Res. Bull. 8:727-750.

6. Boldry, R.C. and N.J. Uretsky (1988) The Importance of dopa minergic neurotransmlsslon 1n the hypermotility response produced by the administration of N-methyl-D-aspartic add into the nucleus accumbes. Neuropharm. 27:569-577.

7. Boldry, R., P. Shreve, and N.J. Uretsky (1988) 6,7-Di- nltroqulnoxaline-2,3-dione (DNQX) and y-glutamyl- amlnomethlysulfonate (GAMS) selectively Inhibit the hypermotility response produced by activation of quisqualic add (QA) receptors 1n the nucleus accumbens. Society for Neuroscience Abstracts 14(2):940.

116 117

8. Bots, G.Th.A.M. and G.W. Bruyn (1981) Neuropathological changes of the nucleus accumbens in Huntington's chorea. Acta Neuropath. 55:21-22.

9. Bradford, H.F. and D.W. Peterson (1988) Excitatory amino acids in epilepsy: In: Glutamate and Glutamine in Mammals Vol. II. Edited by E. Kvamme. CRC Press. Boca Raton, Florida.

10. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of pro tein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

11. Brudzynski, S.M. and G.J. Mogenson (1985) Association of the mesencephalic locomotor region with locomotor activity induced by the injections of amphetamine into the nucleus accumbens. Brain Res. 334:77-85

12. Brudzynski, S.M., Wu, M., and G.J. Mogenson (1988) 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.

13. Bylund, D.B. (1980) Analysis of receptor binding data. In: Receptor Binding Technigues. Society for Neuroscience, pp. 70-99.

14. Carter, C.J. (1980) Glutamatergic pathways from the medial prefrontal cortex to the anterior striatum, nucleus accumbens, and the substantia nigra. Br. J. Pharmacol. 70:50P-51P.

15. Casamenti, F., G. Deffenu, A.L. Abbamondi, and G. Pepeu (1986) Changes in cortical acetylcholine output induced by modulation of the nucleus basalis. Brain Res. Bull. 16:689-695.

16. Col 1 inridge, G.L. and T.V.P. Bliss (1987) NMDA receptors - their role in long-term potentiation. Trends in Neurosci. 10:288-293. 17. Cotman, C.W. and D.T. Monoghan (1987) Chemistry and anatomy of excitatory amino acid systems. In: Psychopharamacoiogy: The Third Generation of Progress. Edited by H.Y. Meltzer. Raven Press, New York.

18. Cotman, C.W. and J.V. Nadler (1981) In: Glutamate: Transmitter in the Central Nervous System, Edited by P.J. Roberts, J. Storm-Mathisen, andG.A.R. Johnston. John Wiley & Sons, London, pp. 117-154.

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

20. Davies, S.W., G.J. McBean and P.J. Roberts (1984) A glutamatergic innervation of the nucleus basalis / substantia innominata. Neuroscience Letters 45:105-110.

21. Davies, J., and J.C. Watkins (1979) Selective antago nism of amino acid-induced and synaptic excitation in the cat spinal cord. J Physiol (London) 297:621-635.

22. Davies, J. and J.C. Watkins (1985) Depressant actions of 2f-D-glutamyl ami nomethyl sulfonate (GAMS) on amino acid- induced and synaptic excitation in the cat spinal cord. Brain Res. 327:113-120.

23. Domesick, V.B. (1981) Further observations on the anatomy of the nucleus accumbens and caudatoputamen in the rat: similarities and contrasts. In Neurobiology of the Nucleus Accumbens. Edited by R.B. Chronister, J.F. DeFrance, Haer Institute, pp. 7-39.

24. Donzanti, B.A. and N.J. Uretsky (1983) Effects of excit- tory amino acids on locomotor activity after bilateral microinjection into the rat nucleus accumbens: possible dependence on dopaminergic mechanisms. Neuropharmacology 22(8):971-981. 25. Donzanti, B.A. and N.J. Uretsky (1984a) Antagonism of the hypermotility response induced by excitatory amino acids in the rat nucleus accumbens. Naunyn-Schmiedeberg1s Arch Pharmacol 325:1-7.

26. Donzanti, B.A. and N.J. Uretsky (1984b) Magnesium selectively inhibits N-methyl-D-aspartic acid-induced hypermotility after intraaccumbens injection. Pharm. Biochem. & Behav. 20:243-246.

27. Drejer, J. and T. Honore (1988) Excitatory amino acid receptors: In: Glutamate and Glutamine in Mammals Vol. II. Edited by E. Kvamme. CRC Press, Boca Raton, Florida.

28. Fagg, G.E. (1985) L-Glutamate, excitatory amino acid receptors and brain function. Trends in Neuroscience 8:207-210.

29. Fagg, G.E., A.C. Foster, and A.H. Ganong (1986) Excitatory amino acid synaptic mechanisms and neurological function. Trends in Pharmacological Science 7(9):357-363.

30. Fonnum, F. and I. Walaas (1980) Localization of neuro- transmiiters in the nucleus accumbens. In: The Neurobiol ogy of the Nucleus Accumbens, Edited by R.B. Chronister and J.F. DeFrance. Brunswick: Haer Inst., pp. 259-272.

31. Fuller, T.A., F.T. Russchen, and J.L. Price (1987) Sources of presumptive glutamatergic / aspartergic afferents to the rat ventral striatopal11da 1 region. J. Compar. Neurology 258:317-338.

32. Halpain, S., C,M. Wieczorek, and T.C. Rainbow (1984). Localization of L-glutamate receptors in rat brain by quantitative autoradiography. J. Neurosci. 4:2247-2258.

33. Hamilton, M.H., J.S. deBelleroche, I.M. Gardiner, and L.J. Herberg (1986) Stimulatory effect of N-methyl-D-aspartate on locomotor activity and transmitter release from rat nucleus accumbens. Pharmacol Biochem Behav 25:943-948. 34. Hayden, M.R. (1981) Huntington's Chorea. Springer- Verlag. New York. pp. 161-167.

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

36. Honore, T., J. Lauridsen, and P. Krogsgaard-Larson (1982) The binding of ( 3H)AMPA, a structural analogue of glutamic acid, to rat membrane. J Neurochem 38:173-178.

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

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

39. Johnson, J.L. (1978) The excitant amino acids, glutamic and aspartic acid, as -transmitter candidates in the verte brate central nervous system. Prog. Neurobiol. 10:155-162.

40. Jones, A.W., D.A.S. Smith, and J.C. Watkins (1984) Structure activity relations of dipeptide antagonists of excitatory amino acids. Neuroscience 13:573-581.

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

42. Jones, D.I. and G.J. Mogenson (1980b) Nucleus accumbens to globus pallidus GABA projection: electrophysiological and iontophoretic investigations. Brain Res. 188:93-105.

43. Kemp, J.A., A.C. Foster, and E.H.F. Wong (1987) Non competitive antagonists of excitatory amino acid receptors. Trends in Neurosci. 10:294-298. 44. Konig, J.R.F., and R.A. Klippel (1963) The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. New York: Krieger..

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

46. McDermott, A.B. and N. Dale (1987) Receptors, ion channels, and synaptic potentials underlying the integrative actions of excitatory amino acids. Trends In Neurosci. 10:280-284.

47. McGeer, P.L., J.C. Eccles, and E.G. McGeer (1987) Putative excitatory neurons: glutamate and aspartate. In: Glutamine and Glutamate in Mammals Vol. II., Edited by E. Kvamme. CRC Press Inc. Boca Raton, Florida.

48. McLennan, H. (1981) On the nature of the receptors for various excitatory amino acids in the mammalian central nervous system. Adv Biochem Psychopharmacol 27:253-262.

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

50. Mogenson, G.J. (1984) Limbic-motor integration with emphasis on initiation of exploratory and goal-directed locomotion: In: Modulation of Sensorimotor Activity. Alan R. Liss Inc. New York, New York. pp. 121-137.

51. Mogenson, G.J., D.L. Jones, and C.Y. Yim (1980) From motivation to action: Functional interface between the limbic system and the motor system. Progress in Neurobiol. 14:69-97. 122

52. Mogenson, G.J. and M.A. Nielsen (1983) Evidence that an accumbens to subpallidal GABAergic projection contributes to locomotor activity. Brain Res. Bulletin 11:309-314.

53. Mogenson, G.J., L.W. Swanson, and M. Wu (1983) Neural projections from the 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.

54. Mogenson, G.J. and C.Y. Yim (1980) Electrophysiological and neuropharmacological-behavioral studies of the nucleus accumbens: implications for its role as a limbic- motor interface. In: The Neurobiology of the Nucleus Accumbens, Edited by R.B. Chronister and J.F. DeFrance. Brunswick: Haer Inst. pp. 210-229.

55. Monaghan, D.T. and C.W. Cotman (1985) Distribution of N-methyl-D-aspartate sensitive L3H-glutamate binding sites in rat brain. J. Neurosci. 5:2909-2919.

56. Monaghan, D.T., D. Yao, and C.W. Cotman (1984) Distribution of ( 3H)AMPA binding sites in rat brain as determined by quantitative autoradiography. Brain Res 324(1): 160-164.

57. Monaghan, D.T., D. Yao, H.J. Olverman, J.C. Watkins, and C.W. Cotman (1984) Autoradiography of D-(3-H)-2-amino-phosphonopentanoate binding sites in the rat brain. Neurosci. Lett. 52:253-260.

58. Morgenstern, R., T. Mende, R. Gold, P. Lemme, and W. Oelssner (1984) Drug induced modulation of locomotor hyperactivity induced by picrotoxin in nucleus accumbens. Pharmacol. Biochem. and Behavior 21:501-506.

59. Murphy, D.E., E.W. Snowhill, and M. Williams (1987) Characterization of quisqualate recognition sites in rat brain tissue using DL3H-a-am1no-3- hydroxy-5-methylisoxazole-4-propion1c acid (AMPA) and a filtration assay. Neurochemical Res. 12: 775-782. 123

60. Nauta, G.P. Smith, R.L.M. Faull, and V.B. Domesick (1978) Efferent connections and nigral afferents of the nucleus accumbens septi in the rat. Neuroscience 3:385-410.

61. Olney, J.W. (1978) Neurotoxicity of excitatory amino acids. In: Kainic acid as a tool in Neurobilogy. Edited by McGeer, E.G., J.W. Olney, and P.L. McGeer. Raven Press, New York. pp. 95-121.

62. Olverman, H.J., A.W. Jones, and J.C. Watkins (1988) {3H}-D-2-amino-5-phosphonopentanoate as a ligand for N-methyl-D-aspartate receptors in the mammalian central nervous system. Neuroscience 26(1):1-15.

63. Pijnenberg, A.J.J., W.M.M. Honig, J.A.M. VanDerHeyden, and J.M. VanRossum (1976) Effects of chemical stimula tion of the mesolimbic dopamine system upon locomotor activity. Eur. J. Pharmacol. 35:45-58.

64. Pijnenberg, A.J.J., W.M.M. Honig, and J.M. VanRossum (1975) Inhibition of d-amphetamine - induced locomotor activity by injection of haloperldol into the nucleus accumbens ofthe rat. Psychopharmacol. (Berl) 41:87-95.

65. Price, K.S., I.J. Forley, and M. Hornykiewicz (1978) Neurochemistry of Parkinson's disease: relation between striatal and limbic dopamine. In: Advances in Biochemistry and Psychopharmacology Vol. 19. Edited by P.J. Roberts, G.N. Woodruff, and LL. Iverson. Raven Press, New York. pp. 293-300.

66. Pycock, C.J. and R.W. Horton (1976) Possible GABA - mediated control of dopamine - dependent behavioral effects from the nucleus accumbens of the rat. Psychopharmacol. (Berl) 49:173-178.

67. Roberts, P.J. and S.D. Anderson (1979) Stimulatory effect on L-glutamate and related amino acids on ( 3H) dopamine release from rat striatum: an 1nv1tro model for glutamate actions. J. Neurochem. 32:1539-1545. 68. Rothman, S.M., and J.W. Olney (1987) Excitotoxicity and the NMDA receptor. Trends in Neurosci. 10:299-301.

69. Scheel-Kruger, J. (1983) The GABA receptor and animal behavior. In The GABA Receptor, Edited by S.J. Enna. The Humana Press. Clifton, New Jersey.

70. Shank, R.P. and M.H. Aprison (1988) Glutamate as a neurotransmitter; In: Glutamine and Glutamate in Mammals Vol. II. Edited by E. Kvamme, CRC Press Inc. Boca Raton, Florida.

71. Shreve, P. and N.J. Uretsky (1987) a-Amino-3-hydroxy- 5-methylisoxazole-4-propionate (AMPA)-induced hypermotility after intraaccumbens injection is antagonized by Jr-glutamylaminomethylsulfonate(GAMS). Fed. Proc. 46(3):710.

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

73. Shreve, P.E. and N.J. Uretsky (1988) Effect of GABAergic transmission in the subpallidum on the hyper motility response to the intraaccumbens administration of excitatory amino acids and picrotoxin. Neuropharmacology 27:1271-1277.

74. Stephens, J.R. (1979) Schizophrenia and dopamine regulation in the mesolimbic system. Trends in Neuro. Scien. 2:102-105.

75. Stephens, R.L., T. Lee, R. Boldry, and N.J. Uretsky (1986) Activation of excitatory amino acid receptors may mediate the folate-induced stimulation of loco motor activity in thenucleus accumbens. J. Pharmacol. Exp. Ther. 239:627-633. 125

76. Stephens, R.L. and N.J. Uretsky (1986) Folate induced - hypermotility response after bilateral injection into the nucleus accumbens of the rat: Possible mediation through dopaminergic mechanisms. Neuropharmacology 25:887-896.

77. T.W. Stone (1987) Excitatory amino acid neurotransmitters and anticonvulsant drug action. ISI Atlas of Science: Pharmacology pp.71-73.

78. Swanson, L.W. (1976) An autoradiographic study of the afferent connections of the preoptic region in the rat. J. Comp. Neurol. 167:227-256.

79. Swanson, L.W., G.J. Mogenson, C.R. Gerfen, and P. Robinson (1984) Evidence for a projection from the lateral preoptic area and substantia innominata to the 'mesencephalic locomotor region' in the rat. Brain Res. 295:161-178.

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

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

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

83. Swerdlow, N.R., L.W. Swanson, and G.F. Koob (1984) Substantia innominata: Critical link in the behavioral expression of mesolimbic dopamine stimulation in the rat. Neurosci. Letters 50: 19-24. 84. Swerdlow, N.R., F.J. Vaccarino, M. Amalric, and G.F. Koob (1986) The neural substrates for the motor - activating properties of psychostimulants: A review of recent findings. Pharmacol. Biochem. and Behavior. 25:233-248.

85. Vrijmoed-de Vries, M.C., H. Tonissen, and A.R. Cools (1987) The relationship between hindlimb disturbances, forelimb disturbances and catalepsy after increasing doses of muscimol injected into the striatal- pallidal complex. Psychopharmac. 92:73-77.

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

87. Walaas, I. and F. Fonnum (1979a) The effects of surgical and chemical lesions on neurotransmitter candidates in the nucleus accumbens of the rat. Neuroscience 4:209-16.

88. Walaas, I. and F. Fonnum (1979b) The distribution and origin of glutamate decarboxylase and choline acetyltransferase in ventral pallidum and other basal forebrain regions. Brain Res. 177:325-336.

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

90. Watkins, J.C. and H.J. Olverman (1987) Agonists and antagonists for excitatory amino acid receptors. Trends in Neuroscience 10: 265-272.

91. Young, W.S., G.F. Alheid, and L. Heimer (1984) The ventral pallidal projection to the mediodorsal thala mus: a study with fluorescent retrograde tracers and immunohistofluorescence. J. Neurosci. 4: 1626-1638.