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Modulation of cortical ACh release by GABAA-dopamine receptor interactions in the basal forebrain

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the

Degree of Doctor of Philosophy in the Graduate School of

The Ohio State University

Holly Moore, B.S.

The Ohio State University

1995

Dissertation Committee: Approved by

John P. Bruno, Ph.D. Martin Sarter, Ph.D. Lane Wallace, Ph.D. Bennett Givens, Ph.D. / Advisor ( Neuroscience Program Department of Psychology UMI Number: 9544646

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, Ml 48103 ACKNOWLEDGEMENTS

I sincerely thank my mentors, Dr. John P. Bruno and Dr. Martin Sarter for their guidance. Being a part of their scientifric collaboration has been an honor an enriching experience. Thanks to Dr. James King and to members of the Neuroscience Graduate Studies Committee for their creative energies and perserverance in continually increasing the quality of the Neuroscience Graduate

Program. I also thank my dissertation committee for their interest in this project and their important suggestions. Special acknowledgements are made to

Lee Ann Holley Miner for her constant generous offering of computer equipment, transportation, moral support and academic enlightenment, to

Kathlene R. Merendo for her insight and resourcefulness, and to Scott

Stuckman for his efforts on this project. Finally, I offer unending gratitude to my family for their patience and support.

ii To Leslie, Marcia, Hope, Virginia and Grace,

my constant sources of inspiration.

iii VITA

December 14, 1962 Born, Urbana, Ohio

1990 B.S. Wright State University; dual degree in Psychology and Chemistry

1990-present The Ohio State University Graduate School Neuroscience Ph.D. Program

RESEARCH PUBLICATIONS

Bruno, J.P., Moore, H., Dudchenko, P., & Sarter, M. (1992). Modulation of frontal cortical acetylcholine release by benzodiazepine receptor ligands: Age-dependent effects and behavioral correlates. In E.M. Meyer, F.T. Crews, & J.W. Simpkins (Eds.), The treatment of dementias: A new generation of progress. NY: Plenum Press, pp. 277- 298.

Moore, H., Dudchenko, P., Bruno, J.P., & Sarter, M. (1992). Towards modelling age-related changes of attentional abilities in rats: simple and choice reaction time tasks and vigilance. Neurohiol Aging. 12., 759-772.

Moore, H., Dudchenko, P., Comer, K.S., Bruno, J.P., & Sarter, M. (1992). Central versus peripheral effects of muscarinic antagonists: the limitations of quaternary ammonium derivatives. Psvchopharmacolog v. 108. 241-243.

Moore, H., Sarter, M., & Bruno, J.P. (1992). Age-dependent modulation of in vivo cortical acetylcholine release by benzodiazepine receptor ligands. Brain Research. 596. 17-29.

iv Tamborski, A., Moore, H., Lucot, J.B., Hennessy, M. (1993). Monoamine activity in anterior hypothalamus of guinea pig pups separated from their mothers. Behavioral Neuroscience. 108, 171-176.

Bruno, J.P., Moore, H., Stuckman, S., Johnson, B. & Sarter, M. (1994). Repeated microdialysis sampling as a valid technique to study cortical and striatal acetylcholine efflux. Current Separations. 15, 1-3.

Moore, H., Sarter, S. & Bruno, J.P. . Bidirectional modulation of cortical acetylcholine release by infusion of benzodiazepine receptor ligands into the basal forebrain. Neuroscience Letters. 189. 31-34

Moore, H., Stuckman, S., Sarter, M., & Bruno, J.P. (1995). Stimulation of cortical acetylcholine efflux by FG 7142 measured with repeated microdialysis. Synapse. 21, 71-76.

PUBLISHED ABSTRACTS

Moore, H., Sarter, M., and Bruno, J.P. (1990). GABAergic mediation of frontal cortical acetylcholine release in awake rats. Current Separations. 10. Abstract #19.

Moore, H., Berntson, G., Sarter, M. & Bruno, J.P. (1991). Age dependent modulation of cortical acetylcholine release by benzodiazepine ligands. Presented at the Society for Neuroscience 21st Annual Meeting. Abstract 632.8.

Quigley, K., Moore, H., Dudchenko, P., Bruno, J.P., & Sarter, M. (1991). Age-related changes in attentional abilities in Fischer-344 rats: Effects of benzodiazepine receptor ligands and amphetamine. Society for Neuroscience 21st Annual Mtg.. Abtract 116.15.

Sarter, M., Dudchenko, P., Moore, H., & Bruno, J.P. (1991). Cognition enhancement based on GABA-cholinergic interactions. Satellite Symposium: Neurotransmitter Interactions and Cognitive Function. Society for Neuroscience 21st Annual Meeting. New Orleans, LA.

Sarter, M., Moore, H., Dudchenko, P., & Bruno. J.P. (1991). Disinhibition of cortical cholinergic activity and attenuation of age-related attentional impairments. American Federation for Aging Research Grantee Conference. NY, NY.

v Moore, H., Berntson, G., Sarter, M. & Bruno, J.P. (1991). Age dependent modulation of cortical acetylcholine release by benzodiazepine ligands. Presented at the Society for Neuroscience 21st Annual Meeting. Abstract 632.8.

Tamborski, A., Lucot, J.B., Moore, H., & Hennessy, M.B. (1991). Catecholamine turnover in anterior hypothalamus of guinea pig pups separated from their mothers in a novel environment. Presented at International Society for Developmental Psychobiology. New Orleans, LA.

Moore, H., Sarter, M., & Bruno, J.P. (1992). Interactions between environmental manipulations and benzodiazepine receptor ligands on cortical acetylcholine release. Society for Neuroscience 22nd Annual Meeting. Abstract 47.2.

Bruno, J.P., Moore, H., and Sarter, M. (1993). FG 7142, a negative modulator at the GABAA/Benzodiazepine receptor, increases cortical acetylcholine efflux: Interactions with dopamine receptor ligands. Society for Neuroscience 23nd Annual Meeting. Abstract 128.11.

Moore, H., Sarter, M., and Bruno, J.P. (1993). Benzodiazepine receptor mediation of cortical acetylcholine efflux: The determining roles of

vi environmental/behavioral manipulations. Curr Separations. 12, Abstract # 47.

Moore, H., Sarter, M. & Bruno, J.P. (1993). Modulation of stimulated cortical acetylcholine efflux by benzodiazepine receptor ligands is mediated in the basal forebrain. Society for Neuroscience 23nd Annual Meeting. Abstract 128.12.

Moore, H., Stuckman, S., Sarter, M. and Bruno, J.P. (1993). Modulation of cortical ACh efflux by GABA/benzodiazepine receptor ligands: Effects of repeated testing and interactions with dopamine receptors. Current Separations. 12, Abstract ft 8.

Sarter, M., Bruno, J.P., Moore, H., McGaughy, J., Dudchenko, P., Holley, L.A. and Turchi, J. (1993). Basal forebrain GABAergic modulation of cortical acetylcholine release and of attentional abilities. British Association for Psvchopharmacologv. Cambridge, U.K.

Stuckman, S., Moore, H., Berntson, G., Sarter, M. and Bruno, J.P. (1993). Effect of repeated microdialysis testing on stimulated cortical acetylcholine efflux. Society for Neuroscience 23nd Annual Meeting. Abstract 128.13.

Moore, H., Stuckman, S., Sarter, M. & Bruno, J.P. (1994). Stimulation of cortical ACh efflux by local ionic changes, but not local muscarinic blockade, is diminished in aged rats. Society for Neuroscience 24th Annual Meeting.

Bruno, J.P., Sarter, M., Moore, H. & Fadel, J. (1995). Trans-synaptic modulation of cortical acetylcholine release: A new approach to restore cholinergic function. Neurodegenerative Disorders: Common Molecular Mechanisms. April, 1995, Jamaica.

Fields of Study

Major Field: Neuroscience

Field of specialization: Behavioral neuroscience

vii TABLE OF CONTENTS

ACKNOWLEDGMENTS...... ii

VITA ...... iv

LIST OF TA BLES...... x

LIST OF FIGURES...... xi

LIST OF P L A T E S ...... xii

CHAPTER PAGE

I. INTRODUCTION 1

A. The behavioral significance of cortical cholinergic transmission ...... 2 B. Functional connectivity among structures of the basal forebrain and c o rte x ...... 5 C. Evidence for control of cortical ACh release by transmission at GABAa/BZ receptors in the SI 10 D. Evidence for the modulation of cortical ACh efflux by dopamine receptor ligands ...... 12 E. The in vivo measurement of neurotransmitter release by microdialysis ...... 16 F. Specific a im s ...... 19

II. GENERAL METHODS...... 23 A. Habituation and training of subjects ...... 23 B. Intracranial surgery ...... 24 C. Microdialysis protocol ...... 25 D. Measurement of neurotransmitters and motor activity 26 E. Statistics...... 29

viii III. STIMULATION OF CORTICAL ACETYLCHOLINE EFFLUX BY THE GABAa/BENZODIAZEPINE RECEPTOR PARTIAL INVERSE AGONIST FG 7142 ASSESSED WITH REPEATED IN VIVQ MICRODIALYSIS SESSIONS 30

A.Background and rationale ...... 33 B. Experimental design and m ethods ...... 32 C. Results ...... 38 D. Discussion ...... 43

IV. MODULATION OF BEHAVIORALLY-STIMULATED CORTICAL ACETYLCHOLINE EFFLUX BY BENZODIAZEPINE RECEPTOR LIGANDS...... 50

A. Background and Rationale ...... 50 B. Experimental Design and Methods ...... 52 C. R esu lts ...... 55 D. Discussion ...... 57

V. MODULATION OF FG 7142-STIMULATED CORTICAL ACETYLCHOLINE EFFLUX BY DOPAMINE RECEPTOR SELECTIVE ANTAGONISTS...... 60

A. Background and Rationale ...... 60 B. Experimental Design and Methods ...... 63 C. Results ...... 68 D. Discussion ...... 75

VI. GENERAL DISCUSSION...... 83

APPENDIX ...... 101 TABLES, FIGURES AND PLATES

BIBLIOGRAPHY...... 145 LIST OF TABLES PAGE

1. Description of drugs and doses tested in Chapter V . . . . 101 2. Drug treatment combinations for Chapter V, Exp. 1 . . . 102 3. Drug treatment combinations for Chapter V, Exp. 2 . . . 103 4. Changes in GAB A efflux in the nucleus accumbens during local co-perfusion of DA antagonists ...... 104 5. Summary of the effects of dopamine receptor antagonists on frontal cortical ACh release and accumbal GABA release 105

x LIST OF FIGURES PAGE

1. Schematic time course of a microdialysis session ...... 107 2. Experimental design for assessing repeated microdialysis testing 109 3. Effect of FG 7142 on cortical ACh efflux ...... I l l 4. Effect of FG 7142 on orofacial tremor and locomotor activity . . . 113 5. Effect of repeated dialysis testing on basal and FG 7142-stimulated cortical ACh efflux ...... 115 6. Stimulation of cortical ACh efflux by behavioral activation and systemic administration of the benzodiazepine receptor inverse agonists ...... 117 7. Cortical dialysis probe and basal forebrain drug cannulae placements 119 8. Effects of BZR ligands infused into the basal forebrain on basal and stimulated cortical ACh efflux ...... 121 9. Effects of systemic SCH 23390 on FG 7142-stimulated cortical ACh efflux ...... 123 10. Effects of systemic haloperidol on FG 7142-stimulated cortical ACh efflux ...... 125 11. Effects of systemic clebopride on FG 7142-stimulated cortical ACh efflux...... 127 12. Summary of effects of systemic dopamine receptor antagonists on FG 7142-stimulated cortical ACh efflux ...... 129 13. Schematic diagrams of placements of dialysis probes in the cortex and nucleus accumbens ...... 131 14. Effect of intra-accumbens perfusion of the D1-selective antagonist on FG 7142-stimulated cortical ACh efflux ...... 133 15. Effect of intra-accumbens perfusion of haloperidol on FG 7142-stimulated cortical ACh efflux ...... 135 16. Effects of intra-accumbens perfusion of dopamine receptor antagonists on FG 7142-stimulated cortical ACh efflux 137 17. Summary of effects of BZR ligands on non-stimulated and stimulated cortical ACh efflux ...... 139

xi LIST OF PLATES PAGE

I. Nissl-stained section of cortical probe placement ...... 141

II. Acetylcholinesterase-stained section showing cortical probe . 142

x i i CHAPTER I

INTRODUCTION

A wealth of converging, yet indirect, evidence indicates that cholinergic transmission in the cortex is necessary for normal stimulus processing and learning. The most compelling of this evidence is derived from age-related dementias, especially Alzheimer's type dementia. Alzheimer's Disease is characterized by a profound deterioration of the cholinergic projections that arise from the basal forebrain and innervate all alio- and neocortical structures

(Coyle et al., 1983). The severity of deterioration in these projections is significantly correlated with the cognitive deficits that charaterize the inevitable dementia (Cummings & Benson, 1987). Recently, it has also been postulated that alterations in the activity of the basal forebrain cholinergic system (Sarter,

1994) or its inputs (Grace, 1993) underlie the cognitive deficits associated with other psychiatric disorders, such as schizophrenia. Consistent with this psychopathological and neuropathological evidence, experimental animal models have demonstrated that the basal forebrain cholinergic system and its primary

1 2 inputs are necessary for basic cognitive processes that may underlie learning

(see below). The general purpose of the experiments described in this dissertation was to characterize the control of cortical acetylcholine (ACh) release by neurotransmitter interactions within the basal forebrain and one of its input structures, the nucleus accumbens (nACC). The data provide insights into the dynamic neurochemical processes that regulate the activity of the basal forebrain cholinergic system. The results are discussed in terms of how the regulation of this system may relate to basic cognitive processes.

A. The behavioral significance of cortical cholinergic transmission

The correlation between the reduction of fimction in the cortical cholinergic projection cells and the onset of cognitive impairments in dementia has been modeled experimentally in rodents, primates and normal humans.

These models include assessment of the cognitive effects of muscarinic receptor antagonists or experimental lesions of the basal forebrain cholinergic cells, as well as correlational analyses of cognitive deficits and the integrity

(morphological, histochemical, or neurochemical) of the basal forebrain cholinergic projections/cells in aged rats. In humans, muscarinic blockers, such as scopolamine and atropine, impair performance in a variety of memory tests (i.e. Beatty et al., 1986; Caine et al., 1981; Drachman & Leavitt, 1974; Flicker et al., 1990; Mohs et al.,

1986). However, finer analyses of these effects have indicated that muscarinic blockers primarily disrupt processing of information (e.g. encoding), rather than storage or retrieval (Flicker et al., 1990; Caine et al., 1981). Similarly, in rats, muscarinic blockers disrupt a variety of indices of cognitive performance

(Andrews et al., 1992; Evenden, et al., 1992; Moore et al., 1992a; Soffie &

Lejeune, 1992; Spangler et al., 1988). As with humans, these effects are primarily on acquisition and/or stimulus processing (i.e. Cheal, 1981; Spangler et al., 1988; Hagan et al., 1986; Andrews et al., 1992). Although the cognitive effects of systemic muscarinic blockers have been attributed to central, even cortical, disruption of cholinergic transmission (i.e. Caine et al., 1981; Messer et al., 1990; Barnes, 1990), there is little direct evidence for this. Most attempts to determine the role of the cortical cholinergic projections in learning have used lesions of this system.

Given the neurochemical evidence that cholinergic transmission in the cortex is almost completely dependent on afferents from the basal forebrain, lesions of the basal forebrain are the most common method of depleting 4

neocortical areas of ACh. The cholinergic innervation of the neocortex arises

almost exclusively from the basal forebrain (Eckenstein et al., 1988; Heckers et

al., 1994; Woolf, 1991). Moreover, whearas electrical stimulation of the

substantia innominata/nucleus basalis of Meynert (SI/NBM) increases cortical

ACh efflux (Kurosawa, Sato & Sato, 1989; Rasmusson, Clow & Szerb, 1992),

excitotoxic lesions of this area reduce cortical, but not sub-cortical, ACh efflux by up to 60% (Herrera-Marschitz, et al, 1990). Thus, projections from the basal forebrain provide the most of the presynaptic component of cortical cholinergic transmission.

Scores of studies have shown that excitotoxic lesions of the basal forebrain produce learning impairments (see Everitt et al, 1987) and these impairments have been considered a model of the cognitive deficits of dementia

(Bartus, 1990). As with the systemic muscarinic blocker studies, many of the cognitive effects of SI/NBM lesions are on components of learning considered to be related to stimulus processing or other ‘‘attentional” processes (see

Robbins et al., 1989). A few of these studies provide direct evidence that normal attentional processes depend on the cholinergic projection from the

SI/NBM to the neocortex. For example, Muir et al. (1992a) showed in rats that lesions of the SI disrupted the maintenance of attention to a set of five visual 5 stimuli, and the effect of the lesion was reversed by a ACh-rich graft into the cortex.

B. Functional connectivity among structures of the basal

forebrain and cortex

The area lying ventral to the globus pallidus, extending rostroventrally from the lateral preoptic area to an area ventral to the nucleus accumbens, has been defined, rostrally, as the ventral pallidum (VP) and, caudally, as the sublenticular substantia innominata (SI; see Heimer & Alheid, 1991). This heterogeneous collection of cells has neurochemical and neuroanatomical characteristics aligning it with at least two anatomically-defined circuits: the ventrostriatopallidal system (Groenewegen et al., 1991; Zahm et al., 1987;

Alexander, et al., 1986) and the extended amygdala (Heimer & Alheid, 1991;

Zahm & Brog, 1992). These systems are distinguished by different inputs to and outputs from the nucleus accumbens (nACC).

The part of the nACC most extensively connected with the ventrostriatal pathway is the core (nACCc). The core lies adjacent to the dorsal striatum and its connections are analogous to those of the dorsal striatum (Paxinos and 6

Watson, 1985; Zahm & Brog, 1992). The nACCc (and some of the rostral pole region) receives excitatory inputs from the hippocampus (Kelley et al., 1982), parts of the rostral basolateral amygdala (McDonald, 1991) and the dorsal prefrontal cortex (Berendse et al., 1992; Groenewegen & Berendse, 1994). The majority of the dopaminergic input to the nACCc originates from the medial substantia nigra pars compacta and ventral tegmental area (Nauta et al., 1978;

Zahm, 1991). There is evidence that both excitatory amino acid and dopaminergic inputs synapse onto nACCc projection cells that synthesize gamma-aminobutyric acid (GABA; Onteniente et al., 1987; Smith & Bolam,

1990). Based on histological similarities between the dorsal striatum and nucleus accumbens, it may also be presumed that GABAergic collaterals from striatonigral cells (see below), cortical excitatory input and dopaminergic inputs all converge onto cholinergic and GABAergic intemeurons, which, in turn also synapse onto the GABAergic projection cells (Dimova et al., 1993; Di Chiara &

Morelli, 1993). The nACCc shares reciprocal connections with lateral enkephalin- and calbindin-rich areas of the VP (Grove, 1988a,b; Spooren et al., 1991; Zahm & Brog, 1992). Cells in the VP project to the mediodorsal nucleus of thalamus (MD), which, in turn, projects to medial prefrontal cortex and anterior cingulate cortex (i.e. Alexander et al., 1986; 7

Groenewegen et al., 1991). Other cells in the VP/SI have reciprocal connections with the subthalamic nucleus (STN) and the substantia nigra pars reticulata (Haber et al., 1985), and have projections to the entopeduncular nucleus (EP) in rodents, analogous to the internal pallidal segment (GPi) in primates (Heimer & Alheid, 1991). These ventral pallidal cells, like the

GABAergic cells of the dorsal pallidum, affect the EP/GPi projection to motor thalamic nuclei, and, consequently, motor output from the cortex, either directly, or via the STN. The nACCc also projects to the substantia nigra pars compacta, influencing dopaminergic transmission within the entire striatal complex. Considering all these connections, it appears that in the ventrostriatal pallidal system, the nACCc and VP both subserve and affect limbic cortical structures, similar to the way their dorsal counterparts subserve and affect motor cortical areas. The significance of the ventral circuit is that it provides an anatomical substrate for the control of motor behavior (i.e., via motor thalamus) by cortical structures involved in multimodal sensory and mnemonic processing (i.e. "limbic-motor integration"; Zahm & Brog, 1992; Mogenson &

Yang, 1991; Robbins et al., 1989).

The area of the nACC which is more extensively connected with the basal forebrain cholinergic cells is the “shell”, located ventral and, for the most part, medial to the nACC core. The nACC shell (nACCs) has been considered as a major structure in “the extended amygdala”, a circuit which also includes the SI. The most important anatomical features of the extended amygdala circuit are the parallel projections from paralimbic cortices to the amygdala, nACCs and SI, and interconnections among these three structures. The nACCs,

SI and amygdala all receive cortical excitatory inputs from paralimbic cortical structures, such as the medial prefrontal, orbitofrontal, perirhinal and entorhinal cortices (Zaborszky et al., 1991). Moreover, both the SI and the ventromedial nACCs are extensively interconnected with other structures in this network including the amygdala, hypothalamus and bed nucleus of stria terminalis

(Heimer & Alheid, 1991). Excitatory amino acid inputs from the caudal basolateral amygdala (McDonald, 1991) and hippocampus (Sesack & Pickel,

1990) converge with dopaminergic synaptic inputs from the ventral tegmental area onto GABA-containing projection cells of the nACCs (Sesack & Pickel,

1990). These GABAergic cells project to cholinergic and non-cholinergic cells in the VP/SI (Grove et al., 1986), as well as to the lateral hypothalamus and brainstem (Zahm & Heimer, 1993; Zahm & Brog, 1992; Heimer & Alheid,

1991). Cholinergic and some GABAergic cells in the VP/SI project back to the basolateral amygdala (Woolf & Butcher, 1982) and entire neocortex (Rye et al., 1984; Mesulam et al., 1983) in a topographical manner (see below).

Other outputs from the enkephalin-poor portion of the SI are extensively interconnected with the nACCs, hypothalamus, bed nucleus of stria terminalis and other structures within the extended amygdala (Zahm & Heimer, 1993;

Zahm & Brog, 1992; Heimer & Alheid, 1991). The extended amygdala circuit appears to mediate complex somatic and autonomic responses to stimuli based on the significance of those stimuli relative to the internal state

(i.e. temperature, hunger or thirst) of the animal.

The cholinergic corticipetal cells of the VP/SI are in a position to relay information from both the ventrostriatopallidal system and the extended amygdala (Heimer & Alheid, 1991). They receive neurochemically heterogeneous inputs, including GABAergic inputs from the nACC and local interneurons, peptidergic inputs from the accumbens and hypothalamus

(Zaborszky et al.,1991; Bolam et al., 1986), and catecholaminergic, serotonergic, and cholinergic input from mesencephalic and brainstem nuclei

(see Vertes, 1988; Woolf & Butcher, 1986). GABA-synthesizing terminals presumably from the nucleus accumbens synapse onto VP/SI cholinergic cells

(Zaborszky et al., 1986), which possess GABAa/BZRs (Faull & Villiger, 1988;

Sarter & Schneider, 1988; see above). In summary, anatomical evidence 10

suggests that inputs from both ventrostriatopallidal and extended amygdala areas

of the nACC affect activity of corticipetal cholinergic cells of the VP/SI, predominately via GABAergic synapses. Thus, study of DA/GAB A

interactions in the nACC and GABA/cholinergic interactions in the VP/SI will

elucidate how the ventrostriatopallidal and extended amygdala circuits impact on cortical ACh function. Moreover, the most useful paradigm for studying the behavioral significance of these interactions necessarily allows measurement of the dynamics of neurotransmission in these structures and allows correlations with ongoing behavior.

C. Evidence for control of cortical ACh release bv transmission at

GABA^/BZ receptors in the SI

Evidence that the GABAergic input to the SI modulates cortical ACh transmission comes from anatomical, pharmacological and electrophysiological studies (see Sarter et al., 1992, for a review). The SI contains a high density of benzodiazepine binding sites, presumably associated with GABAa receptor complexes (Faull & Villiger, 1988; Penny et al., 1981; Sarter & Schneider,

1988). The GABAa/BZ receptors are possibly located on cholinergic projection 11

neurons, since GABA-containing efferents from the nACC synapse on cholinergic cells in the VP (Zaborszky et al., 1991; but see Henderson, 1995).

As predicted by this anatomical evidence, intra-SI injections of muscimol, a

GABAa receptor agonist, into the SI reduce ACh turnover, high-affmity choline uptake, and in vivo cortical ACh efflux in the cortex (i.e. Casamenti et al.,

1986). Conversely, intra-SI administration of picrotoxin, a blocker of the

GABAa chloride channel, increases cell firing frequency in the SI (Mogenson &

Yang, 1991), and increases in vivo cortical ACh efflux (Bertorelli et al., 1991).

GABA transmission at the GABAa/BZ receptor can be bidirectk nally allosterically modulated by benzodiazepine and beta-carboline compounds

(Haefely, 1989). These compounds, hereafter referred to as benzodiazepine receptor (BZR) ligands, bind to a subunit distinct from the subunit to which

GABA binds. Once bound, BZR inverse agonists decrease, whereas BZR agonists increase, the frequency of the chloride channel opening. Recently, it was shown that cortical ACh efflux can be increased by systemic administration of beta-carboline BZR inverse agonists and decreased by administration of a

BZR agonist (Moore et al., 1992c, 1993a,b). Given the anatomical evidence for GABAa/BZRs in the basal forebrain, and given that the SI has a relatively 12 high affinity for beta-carbolines (Faull & Villiger, 1986; Sarter & Schneider,

1988), the basal forebrain was hypothesized as the major site for the modulation of cortical ACh release by GABAa/BZR ligands. However the ubiquity of

GABAa and BZ receptors and their physiological actions in input structures to the basal forebrain, including the prefrontal cortex (i.e. Oka & Hicks, 1990;

Conners, 1992; Santiago et al., 1993) and nACC (i.e. Zetterstrom & Fillenz,

1990; Finlay et al., 1992; Gruen et al., 1992) raises the possibility that these other sites may also be involved in the regulation of cortical ACh release by

GABAa/BZRs.

D, Evidence for the modulation of cortical ACh release

bv dopamine receptor ligands

A plethora of studies indicate that dopamine transmission in the nACC is an important mechanism underlying motor responses to psychologically and biologically significant stimuli (i.e. Cador et al., 1991; for review see

Salamone, 1994). One mechanism by which DA transmission in the nACC may facilitate preferential processing of psychologically significant stimuli is via disinhibition of cortical cholinergic afferents. For example, presentation of 13 appetitive stimuli, in addition to increasing DA efflux in the nACC (i.e. Phillips et al., 1993; Hernandez & Hoebel, 1991), increases cortical ACh efflux (Moore et al., 1992c). Concomitant increases in accumbal DA release and cortical ACh release is also produced by aversive environmental changes (Imperato et al.,

1992a,b).

There is neurochemical, behavioral and electrophysiological evidence that the nACC and VP/SI are connected, such that increases in DA in the nACC result in decreased GABA release in the VP/SI, and increased ACh release into the cortex. Section II (above) summarizes the anatomical basis such a functional circuit, and available neurochemical experiments support this hypothesis. For example, in addition to evidence that DA agonists increase cortical ACh (see below), it has been shown that BZR agonists and inverse agonists decrease and increase, respectively, both nACC DA (Finlay, Damsma

& Fibiger, 1992; McCullough & Salamone, 1992b) and cortical ACh efflux

(Moore, et al., 1992c, 1993a,b), suggesting that GABA transmission in the

VP/SI has feedback effects on DA in the nACC and feed-forward effects on cortical ACh. More direct, but incomplete, evidence for this functional circuit is provided by the landmark electrophysiological studies by G. Mogenson and colleagues which showed that increased DA transmission in the nACC has 14 complex, but largely negative, effects on the excitation of nACC GABA output neurons by subicular or amygdaloid inputs (Mogenson, Yang & Yim, 1988).

Furthermore, electrical stimulation of the nACC results in a cessation of firing of VP/SI cells, whereas application of DA in the nACC enhanced the firing of these cells, many of which had the firing patterns of cholinergic projection cells

(Mogenson & Yang, 1991; and see Richardson & DeLong, 1990). This relationship between DA transmission in the nACC and GABA transmission in the VP/SI is also revealed by behavioral indices. For example, local infusion of

DA or its agonists into the nACC increases object-directed motor activity

(Salamone, 1994), an effect that can be antagonized with infusion of GABA or muscimol into the VP/SI (Jones & Mogenson, 1980). Conversely, infusion of

DA antagonists into the nACC attenuates locomotion induced by picrotoxin

(which blocks GABAa chloride channels) into the VP (Kalivas et al., 1991).

Importantly, however, Mogenson and Yang (1991) asserted that although electrophysiological and behavioral findings suggest that "dopamine receptor stimulation reduces accumbens-ventral and subpall idal GABAergic transmission

. . .neurochemical confirmation of a direct association of dopamine inhibiting accumbens-subpallidal output is still needed". It may be added that, in order to understand the neurochemical interactions underlying the selective processing of 15 biologically/psychologically significant stimuli, including selective responding to such stimuli, the relationship between nACC dopamine, VP/SI GABA and the cortically-projecting cholinergic cells must be determined.

At present, the available neurochemical and pharmacological evidence for a relationship among DA transmission in the nACC, GABA transmission in the VP/SI, and cortical ACh release is sparse and indirect. First, systemic administration of the dopamine releaser and reuptake inhibitor, amphetamine

(Cooper, Bloom & Roth, 1991), increases cortical ACh efflux, an effect that is at least partially blocked by antagonists selective for either of the two major DA receptor sub-type families (i.e. D l- or D2-like DA receptor sub-types, see below; Day & Fibiger, 1992). The non-selective, direct dopamine agonist, apomorphine (Day & Fibiger, 1993), and a novel Dl-selective agonist (Acquas, et al., 1994) have also been shown to increase cortical ACh release, and either of these responses can be blocked by a Dl-selective antagonist. While it has been established that the effects of amphetamine on cortical ACh release are not mediated in the frontal cortex (Day & Fibiger, 1993), the DA receptor sub populations and other neurotransmitters mediating the positive relationship between increased DA transmission and increased cortical ACh release have not been elucidated. Thus the "neurochemical confirmation" solicited by Mogenson, specifically as it relates to disinhibition of cortical ACh release, is still needed. This confirmation appears particularly relevant to recent theories about the mediation of the cognitive dysfunctions in schizophrenia and senile dementia by alterations in accumbens DA (i.e. Grace, 1991, 1993) and

GABAergic regulation of basal forebrain cholinergic cells (i.e. Sarter, 1994).

E. The in vivo measurement of neurotransmitter release

bv microdialvsis

Recently, studies aiming to determine the effects of pharmacological and behavioral manipulations on brain neurotransmitter release have made extensive use of the technique of in vivo microdialysis (over 250 citations in 1991 increasing to over 1000 in 1994 - Medline search). With this method, a dialysis membrane tube is implanted into the brain area and perfused with a physiologically inert liquid medium. Neurotransmitters and other diffusable molecules diffuse across the membrane and are taken up by the cross flow of perfusion fluid (Ungerstedt, 1984). The concentric microdialysis probe, the type used in the present studies, is a modification of the push-pull cannula in which the dialysis tube is attached to the outer of two concentric cannulae. The perfusion fluid is driven out of the inner cannula at a constant rate by a delivery

syringe and taken into the outer cannula by the pressure gradient between the

cannulae (Ungerstedt, 1984; Wages, Church and Justice, 1986). The probe is

constructed and the perfusion flow rate is controlled to keep a constant, very

low concentration of the analyte in the perfusion medium. Thus, the major determinant of the concentration gradient across the membrane becomes the extracellular level of the analyte. In this way, the dialysis probe acts similar to a blood capillary in which selectivity is based on molecular weight, and the rate of diffusion of the analyte into the probe is determined only by its concentration in the interstitial space (Ungerstedt, 1984; Benvenista & Huttemeier, 1990).

The transmitter in the perfusion fluid sample (the "dialysate") is usually in femtomolar or picomolar concentrations.

The impact of in vivo microdialysis on the tissue being perfused has not been extensively studied, relative to the extensive use of the technique.

However, several dynamic indices of neuronal function are monitored with microdialysis. Of these, the indicators considered to be most important for most transmitter systems are 1) the stability of the analyte in the ECF under basal conditions and 2) the dependence of changes in the analyte levels on depolarization-dependent neuronal release. The latter criterion is met either by 18

demonstrating a complete suppression of analyte efflux by removal of calcium

ions from the perfusion medium, or addition of the voltage-gated sodium

channel blocker, tetrodotoxin, to the perfusion medium. The "physiological

inertness" of the perfusion medium is achieved by perfusion with a medium that contains physiological levels of the major CSF ions and glucose.

The studies described below use in vivo microdialysis to monitor extracellular levels of ACh and GABA in behaving animals. The limitations of interpreting changes in neurotransmitter uptake by dialysis as changes in neuronal transmitter release have been reviewed (i.e. Benvenista & Huttemeier,

1990). These general issues, as well as the limitations relating specifically 1) to measuring ACh and GABA release and 2) use of repeated dialysis, are addressed in Chapter VI. 19

F. Specific Aims

F .l. To characterize the stimulatory effects of the BZR partial inverse agonist.

FG 7142 on cortical ACh efflux, and to validate an experimental design using repeated in vivo microdialvsis sessions for assessing changes in cortical ACh release

In the first experiment, the effects of the BZR partial inverse agonist,

FG 7142, on cortical ACh efflux were assessed. Pilot experiments had shown that this drug enhances cortical ACh release in resting animals to a significantly greater extent than a previously tested, weaker BZR inverse agonist. Different efficacies of BZR inverse agonists in stimulating cortical ACh efflux potentially allows the their use as tools in examining neurotransmitter interactions underlying the regulation of cortical ACh release. Toward this aim, the efficacy of FG 7142 was characterized.

Secondly, the effects of repeated in vivo microdialysis testing in freely- moving rats were determined. Use of repeated testing with in vivo microdialysis potentially allows evaluation of the effects of multiple pharmacological or behavioral manipulations within an animal, thus potentially increasing statistical and interpretational power of the experimental design. 20

However, an assessment of the validity of this novel method for measuring changes in cortical ACh was required. Thus, the FG 7142 was additionally used to test the effects of repeated dialysis testing on the recovery, neuronal origin and responsivity of cortical ACh. A lack of interaction of repeated testing with an index of neuronal origin or with the ability of cortical ACh efflux to be bidirectionally modulated were considered the most important criteria to be fulfilled in the validation of this experimental design.

F.2. To determine whether the basal forebrain is a maior anatomical site for the modulation of cortical ACh release bv benzodiazepine receptor (BZR) ligands.

The control of cortical ACh efflux by allosteric modulation of GABA in the basal forebrain was assessed. Specifically, the effects of BZR ligands infused into the VP/SI areas of the basal forebrain on cortical ACh efflux in awake, resting animals were compared to their effects on efflux increased by the presentation of an appetitive stimulus. It was determined 1) whether allosteric modulators of GABA act in the VP/SI to modulate cortical ACh release, and

2) whether this modulation is related to the activity level of the cortically- projecting cholinergic neurons. The findings are relevant to the development of

GABAa/BZR negative modulators that might selectively enhance the function of 21 activated cortical cholinergic afferents. Ultimately, this line of research would lead to the determination of the behavioral conditions under which pharmacological stimulation of cortical ACh release would be most therapeutic.

F.3. To determine neurochemical and anatomical sites of action of dopamine receptor ligands and the neuroleptic haloneridol on cortical ACh release.

In these experiments, the effects of dopamine-BZ receptor interactions on cortical ACh release were examined. Based on evidence that increases in

DA release in the nACC are accompanied by functionally-related increases cortical ACh release, it was hypothesized that stimulated cortical ACh release is, in part, regulated by DA transmission in the nACC through the GABAergic projections from the nACC to the VP/SI. First, cortical ACh efflux was stimulated by FG 7142, a BZR partial inverse agonist that also stimulates mesoaccumbal DA release. The ability of selective antagonists at DA receptors to attenuate FG 7142-stimulated cortical ACh efflux was then measured. In addition, because of evidence linking changes in DA transmission in the nACC to the psychopathology of schizophrenia, the effects of the widely-prescribed neuroleptic, haloperidol, were also tested. These findings should add to the current understanding of how neurotransmitters in the nACC-basal forebrain- cortical circuit interact, normally, and in psychopathological states such schizophrenia and senile dementia. CHAPTER II

GENERAL METHODS

A. Habituation and training of subjects

The standard testing apparatus consisted of a clear parabolic bowl (35 cm [ht] x 38 cm [dia]), or a rectangular tub (34 cm [w] x 47 cm [1] x 31 cm

[deep]), each lined with sanitized pine bedding. Dialysis tubing and connecting lines extended down into the chambers from a swivel attached to a counterbalanced arm. Two to three of these chambers were placed in an enclosed, temperature-controlled test room with the microinfusion pump. For at least 7 days prior to cannula implantation, all animals were placed in the testing environment for 4-6 hours. During this time, they were handled extensively about the head and given mock i.p. injections (by holding the animal by the shoulders and poking it in the abdomen). Following this procedure, animals required no restraint during insertion of the probe and typically returned to rest within a few minutes after a systemic injection.

23 24

B. Intracranial surgery

The morning following the last training or habituation session, animals were anesthetized with sodium pentobarbital (52 mg/kg, i.p.), and implanted with cannulae under aseptic conditions. For most cortical probes placements, the following placement was used. The microdialysis guide cannula (0.65 o.d.;

CMA 10, Carnegie Medicin, Acton, MA) was rotated in the stereotaxic apparatus to an angle of 50° angle away from vertical, towards the midline.

The cannula was then inserted, 1 mm beyond the dural surface through a burr hole drilled at 1.8 - 2.7 mm anterior to Bregma and 1.0 mm lateral to the midline. The guide cannula and a small hook were secured to the skull with dental cement and skull screws. In all animals, a small loop which allowed the animal to be tethered during testing was placed in the acrylic as it dried.

Following surgery, all animals were injected with antibiotic (s.c.) and local antibiotic and analgesic were applied to the suture. Animals were given 3 days to recover and be re-habituated or trained in the testing environment.

During this time, they were closely monitored and rehydrated with 5% glucose in isotonic saline if necessary. Animals lost less than 5% of their pre-surgery 25 body weight as a result of the surgery, and, in most cases, recovered to their pre-surgery weight within a few days after surgery.

C. Microdialysis protocol

The standard protocol is illustrated in Figure 1. After the 3-day recovery/retraining period, animals were tested on four sessions, with a drug- free day (about 36 hrs) between each session. Drug combinations werecounterbalanced across session. During each session, animals were allowed to habituate to the chamber for at least 30 min, after which a microdialysis probe (0.5 dia.) was inserted into the cannula. After probe insertion, animals were perfused (1.3-2.0 /d/min) with an artificial CSF (pH =

7.0 ± 0.1) containing (in mM) NaCl 126.5, NaHC03 27.5, KC1 2.4, Na2S04

0.5, KH2P04 0.5, CaCl2 1.1, MgCl2 0.8, glucose 4.9, and neostigmine bromide

0.5 /xM. Animals were then dialyzed for 3 hours before baseline samples were collected. We have shown that this delay is sufficient to allow extracellular

ACh levels to stabilize and become dependent on axonal depolarization (Moore et al., 1992c). Thereafter, samples were collected every 15 min for one hour before the experimental manipulation (see below). 26

Following the 4th dialysis session, animals were sacrificed with an overdose of pentobarbital and perfused with formalin (10%). Probe placements were verified by examination of Nissl- and/or acetylcholinesterase-stained (Tago etal., 1986) sections.

P. Measurement of neurotransmitters and motor activity

D .l. Acetylcholine and GABA

For ACh, cortical dialysates (30 ul) and aliquots (30 ul) of standard solutions were placed on dry ice and stored at - 80 °C until analyzed. It has been established in our laboratory that ACh standards with concentrations as low as 40 fM and in vivo dialysates of similar concentration can be stored in this manner without significant degradation of ACh. A volume of 10-20 /zl of each sample was injected onto a carbon polymer-packed 100 mm column.

Following separation on the column, ACh and choline was catalyzed to choline and H202, respectively on a post-column reactor containing acetylcholinesterase and choline oxidase. The mobile phase (pH = 8.5) consisted of NaH2P04 (35 mM) and either a drop of toluene or 20 ppm of the microbicide, ProClin300.

Hydrogen peroxide corresponding to ACh or Ch was quantified using an 27 electrochemical detector (LC 4C, BAS) with a platinum electrode as the anode referenced to a Ag/AgCl electrode at a potential of +0.5 V (Potter et al.,

1983; Tyrefors & Carlsson, 1990). For the experiments in Chapter V, an additional electrode set-up used a Unijet (BAS) 3 mm glassy carbon electrode coated with an osmium polyvinylpyridine solution containing horseradish peroxidase (BAS). The coated glassy carbon electrode was used as the cathode, referenced to a Ag/AgCl electrode at a potential of 0 mV. With either electrode set-up, LC 4C detector was coupled to an amplifier/A-D converter

(BAS), and interfaced with 386SX computer. ACh was quantified by integrating the area under the corresponding peak using INJECT software

(BAS), and comparing the area to a 3-point external standard curve which bounds the range of expected ACh values.

For GABA, dialysates from the nACC were collected and stored at -

80°C until analyzed. GABA was quantified by a modification of the methods of

Kehr & Ungerstedt (1988). Dialysates and standards were first mixed with a reagent (amounts per 10 ml reagent) containing o-phthaldialdehyde (OPA; 27 mg) and t-butylthiol (45 /xl) in carbonate buffer (pH = 9.6) with 50% methanol.

Using an autosampler, 10.0 /ul of reagent was added to 13 jal of dialysate (or standard) and mixed for 3 min. This mixture (21 n1) was then injected onto a 28

C18 column over which was pumped a 0.1 M sodium acetate buffer, with 50% acetonitrile (pH 4.96). The GABA derivative was detected with an LC 4C electrochemical detector (BAS) with a glassy carbon working electrode referenced to Ag/AgCl at a potential of 0.6 V. The change in current (peak) associated with the thiol derivative of GABA had been confirmed by the addition of nipocotic acid to the aCSF in a previous in vivo microdialysis experiment in this laboratory (McCone-Bymes, 1995). The GABA-specific peak was quantified with the computer-interfaced apparatus, and GABA was quantified using an external standard curve, as described above for ACh.

D.2. Motor activity

During each collection period of all test sessions, locomotor activity was scored with the following scale: 0 = no movement, 1 = head movement only,

2 = movement of head and forelimbs, 3 = movement of 3-4 limbs not resulting in a change in quadrant in the test chamber, 4 = quadrupedal movement resulting in greater than 180° change in position or change in quadrant. During the observation, it was also recorded whether the following behaviors were ongoing: sniffing, grooming, burrowing, chewing, and vacuous chewing. For animals trained with the darkness/cereal procedure, the latency to approach the 29 cereal after the onset of darkness was also recorded. Orofacial tremor, defined as a rapid movement around the vibrissae not correlated with sniffing, was also quantified in experiments using FG 7142. Eight minutes after the beginning of each collection period, the animal was observed for 3 min, during which the number of bouts of tremor was recorded.

E, Statistics

In most experiments, efflux rates were corrected for the in vitro recovery of the probe (9.0 - 14.0%) and a median baseline efflux was determined from the four baseline collection periods. In analyses of effects of between-subjects factors or repeated-sessions on basal efflux, the median baseline was used. Otherwise, efflux values were expressed as percent change from median baseline, and percent change values were subjected to statistical analysis. CHAPTER III

STIMULATION OF CORTICAL ACETYLCHOLINE EFFLUX

BY THE GABAa/BENZODIAZEPINE RECEPTOR

PARTIAL INVERSE AGONIST FG 7142

AS ASSESSED WITH REPEATED IN VIVO MICRODIALYSIS

SESSIONS

A. Background and rationale

Methyl-p-carboline-3-carboxyamide (FG 7142) is a

GABAA/benzodiazepine (BZR) partial inverse agonist which, in vitro, non- competatively inhibits GABA binding and reduces GABA-stimulated chloride conductance (i.e. Braestrup et al., 1984; Ito et al., 1994). Its biochemical efficacy and proconvulsive properties are weaker than those of "full" inverse agonists, such as DMCM, but greater than other BZR ligands, such as

30 31

ZK 93 426 (i.e. Braestrup, et al., 1984; Marescaux et al., 1987; Lambert et al.,

1988). The effects of systemic administration of FG 7142 (i.e. proconvulsive, proconflict, hypothermic, and anorectic effects), as well as its stimulatory effects on central dopamine and norepinephrine, are blocked by the centrally- selective BZR antagonist RO 15-1788 (Corda et al., 1987; File et al., 1987;

Jackson et al., 1991; Cooper, 1985; Bradberry et al., 1991; Giorgi et al., 1988;

Ida et al., 1991). This evidence indicates that in vivo. FG 7142 acts selectively at GABAa/BZRs (see Lambert et al., 1988; Richards, et al., 1982).

Recently, it was demonstrated that cortical ACh efflux in freely-moving rats is increased following systemic administration of the BZR "selective" inverse agonist, ZK 93 426 (Moore et al., 1992c, 1993a). The ZK 93 426- induced increase in basal ACh efflux was small, in contrast to its more potent enhancement of behaviorally-activated efflux. This effect, as well as the ability of the GABAa/BZR agonist, chlordiazepoxide, to decrease activated cortical

ACh efflux, has been reproduced by infusion of BZR ligands directly into the substantia innominata (SI; see Chapter IV). These data converge with evidence for GABAergic terminals and a high density of benzodiazepine binding sites on or near cholinergic cells in the SI (Zaborzsky et al., 1986; Sarter et al., 1988) to support the hypothesis that BZR ligands act in the basal forebrain to 32 bidirectionally modulate cortical ACh release (Sarter et al., 1990).

The first aim of the present study was to determine the effects of FG

7142 on cortical ACh efflux. Since other GABAa/BZR negative modulators have been shown to increase ex vivo (Miller et al., 1990) and in vivo (see above) indices of cortical ACh release, it was predicted that FG 7142 would enhance cortical ACh efflux. Furthermore, whereas administration of

ZK 93 426 had a modest effect on basal efflux, presumably via its negative modulation of GABA in the basal forebrain (see above), FG 7142, because of its greater intrinsic activity at the GABAa/BZR receptor, was expected to more effectively stimulate basal cortical ACh efflux.

The second aim of this study was to use FG 7142-stimulated cortical

ACh efflux to verify the validity of the within-subjects design for assessing changes in cortical ACh with in vivo microdialysis. Within-subjects designs reduce between-subject variability and can provide greater statistical power than between-subjects designs (Winer, 1971). Furthermore, such designs allow trained animals to be retested, which makes possible in vivo animal models for correlating neurochemical changes with learning. On the other hand detrimental effects of repeated or extended microdialysis sampling (i.e. progressive gliosis and changes in vascularization) could potentially disrupt local modulation of 33 neurotransmitter release. The evidence that such changes limit the utility of multiple dialysis designs, is not uniform. For example, Robinson and Camp

(1991) showed that repeated dialysis can significantly reduce amphetamine- stimulated striatal dopamine efflux. In contrast, Westerink and Tuinte (1986) found amphetamine-stimulated striatal dopamine efflux to be relatively stable over several days of dialysis. Moreover, repeated dialysis does not appear to change the responsiveness of striatal dopamine to cocaine (Robinson & Camp,

1991; Kalivas et al., 1990) nor the ability of dopamine receptor antagonists to increase or decrease striatal ACh efflux (Johnson & Bruno, 1995). The heterogeneity of these data underscores the necessity for verifying within each neuroanatomical area the validity of changes in neurotransmitter efflux detected with repeated microdialysis. Thus, effects of repeated insertion and perfusion of the microdialysis probe on basal and FG 7142-stimulated cortical ACh efflux were determined.

B. Experimental design and methods

B .l. Animals and surgery

Young adult (4-5 months) BNNia/F344 male rats (NIA colony at Charles 34

Rivers), were maintained in a temperature- and humidity-controlled environment with food and water available ad libitum. Following at least one week of handling and habituation to the testing environment, stainless steel guide cannulae were implanted into the frontoparietal cortex as described in the

General Methods. The cannula was directed at a 50° angle through a hole drilled in the skull 1.5 mm anterior to bregma and 1.5 mm left of the midline.

The cannula was then inserted 1 mm beyond the dural surface and permanently fixed to the skull with stainless steel screws and dental acrylic. All placements were verified histologically as described in the General Methods.

B.2. Experimental design

Following recovery from surgery (see Chapter II), subjects were divided into three groups and dialyzed on every other day for four test sessions. The general design for determining the effects of repeated dialysis sessions were assessed in Groups 1 and 2. This design is illustrated in Figure 2. Group 1

(n=5) was injected with vehicle (10% Cremephor EL in isotonic saline [CEL];

BASF, Ludwigshafe, FRG) on

Session 1, and injected with FG 7142 (8 mg/kg, i.p.) on Session 4. Group 2

(n=6) was injected with FG 7142 (8.0 mg/kg) on both Sessions 1 and 4. Group 3 (n=6) was injected with FG 7142, 4.0 or 16.0 mg/kg (doses counterbalanced), on Sessions 1 and 4. Animals in all groups received vehicle injections on Sessions 2 and 3.

B.3. Microdialvsis protocol

On each test day, the animals were placed in the bowls 30 min before insertion of the concentric dialysis probes (0.5 mm o.d., membrane extending

2.0 mm beyond the guide cannula; CMA 10, Carnegie Medicin, Stockholm).

After probe insertion, the animals were dialyzed (2.0 jd/min) with the standard artificial CSF (see General Methods).

After the 3-hr equilibration period, dialysates were collected every 15 min. Four baseline samples were collected and then vehicle (CEL) or FG 7142 was injected (i.p.). Following injection, samples were collected every 15 min for 2 more hours. Behavioral observations were recorded during the middle 5 minutes of each collection period. Samples were analyzed for ACh as outlined in the General Methods. All values of ACh efflux were corrected for the in vitro recovery of the probe based on a 0.2 fiM ACh/choline standard (average probe recovery = 11.7 ±. 0.5%). 36

B.4. Determination of the dependency of efflux on axonal ■depolarization

In a separate set of animals, on Session 1 (n=l) or on Session 4 (n=l) tetrodotoxin (TTX; 1.0 fM ) was co-infused beginning on the 5th collection interval. Three more collections were taken, then FG 7142 (8.0 mg/kg, i.p.) was injected and three more collections were taken. The ability of TTX to suppress baseline efflux and block the ability of FG-7142 to increase efflux was considered to reflect the extent to which cortical ACh efflux was derived from depolarization of cholinergic axon terminals (see Westerink et al., 1987).

B.5. Statistical analyses

Effect ofFG 7142 on cortical ACh. Efflux values, expressed as percent change from median baseline (see above), were analyzed using a "split plot" design (see Winer, 1971). With this design, the 16.0 and 4.0 mg/kg doses were tested in a separate group of animals (Group 3; see above) than the vehicle and

8.0 mg/kg dose. Separate analyses were used to assess the effects of each dose relative to vehicle. The 16.0 and 4.0 mg/kg doses were each compared to vehicle with separate DOSE (2 levels; between subjects) x COLLECTIONS

PERIOD (9 levels; within-subjects mixed ANOVAs. The 8.0 mg/kg dose was compared to vehicle with a DOSE (2 levels) x COLLECTION PERIOD (9 37 levels) within-subjects ANOVA. Significant effects of DOSE or significant interactions of DOSE x COLLECTION PERIOD were further analyzed by 2 methods: 1) comparing each dose of FG 7142 to vehicle at individual collection periods, and 2) by comparing post-injection collection periods to baseline (i.e. last pre-injection collection, see above) for each dose. Appropriate independent or paired t-tests, using the pooled variance (across COLLECTION PERIOD) as the error term (Winer, 1971) were applied in post-hoc analyses.

Effects of repeated microdialysis sessions. To determine the effect of repeated dialysis sessions on basal cortical ACh efflux, the median baseline efflux values from Session 1 and 4 from all drug-naive animals were compared using a paired t-test. To reveal effects of repeated testing on FG 7142 (8.0 mg/kg)-stimulated ACh efflux, efflux values, expressed as percent change from median baseline (see above), from Group 2, Session 1 and Group 1, Session 4, were compared using a SESSION (2 levels; between-subjects) x COLLECTION

PERIOD (9 levels; within-subjets) mixed ANOVA. Effects of COLLECTION

PERIOD and COLLECTION PERIOD x SESSION interactions were further assessed with within-SESSION paired t-tests comparing post-injection collection periods to the last baseline collection period. For all planned comparisons, the pooled error term was used in order to correct individual tests for the average 38

variance of the overall analysis (Winer, 1971). The criterion for significance

(a) for all statistical tests was set at 0.05.

C. Results

C.1. Probe placement

All probes terminated 1.8 ± 0.25 mm (mean ± S.E.M.) anterior to bregma (estimated based on the atlas of Paxinos and Watson [1985]), lying completely within the cortex with the tips extending to 4.6 ± 0.23 mm lateral from the midline. As shown by the example given in Plate I, Nissl-stained sections from brains of animals receiving four sessions showed a moderate density of microglia along the border of and invading the tract of the guide cannula. Less gliosis was observed in the tract of the membrane tip. These observations were nearly identical to those previously-reported (Moore et al.,

1992c, 1993a,b). In addition, as shown in Plate II, the density of AChE- positive fibers surrounding the probe tract appeared unaltered, relative to adjacent tissue and the intact contralateral hemisphere. 39

C.2. Effect of FG 7142 on cortical ACh efflux and behavior

The mean baseline efflux rate collapsed over all Groups and both

Sessions was 0.15 ± 0.04 pmol/min. As shown in Figure 3, FG 7142 increased cortical ACh efflux to a maximum of 154 (± 68), 280 (±64) and

470 (± 185) percent above baseline for 4.0, 8.0 and 16.0 mg/kg, respectively.

The highest dose (16 mg/kg) increased ACh relative to vehicle, as indicated by a significant DOSE effect (F[l,9] = 9.23, p < .02). Planned t-tests revealed that following the 16.0 mg/kg dose, efflux was significantly increased over baseline for 105 min (all paired t[5]'s > 2.0, p's < .05), and was significantly increased relative to vehicle-treatment at 30, 45 and 75 min

(all independent t[9]'s > 2.1, p's < .05). The 8.0 mg/kg dose produced a shorter-duration increase, as reflected by the significant DOSE x

COLLECTION PERIOD interaction (F[8,72] = 2.61; p < .02). Planned comparisons revealed that at 15 and 30 min following this dose, ACh efflux was significantly increased relative to baseline (paired t[5]'s > 2.5, p < .05) and relative to vehicle treatment (independent t[9]'s > 2.3; p's < .05). Analysis of the lowest dose (4.0 mg/kg) revealed only a main effect of COLLECTION

PERIOD (F [l,7] = 3.57; p < .01). Planned comparisons revealed a significant difference between baseline and post-FG 7142 ACh efflux at 15 min 40

(t[5] = 1.9, p < .05) and a difference between vhicle and drug effects at

30 min (t[9] = 1.8; p < .05). An independent analysis of the vehicle treatment showed that following vehicle injection, ACh efflux was never significantly different than baseline efflux.

The data in Figure 3 and comparisons of each dose with vehicle (see above) revealed dose-dependent differences in the duration of the FG 7142- induced increase in cortical ACh efflux. Thus, as a more specific test for effects of DOSE on FG 7142-stimulated ACh efflux, a statistical analysis was performed on DURATION, defined a minutes post-injection that efflux remained at least two standard deviations above the median baseline efflux

(standard deviations based on the 4 pre-injection baseline collections). This analysis revealed that the increase in cortical ACh efflux was significantly prolonged with the higher dose of FG 7142. DURATION values were

(minutes, mean ± S.E.M.) 39 ± 18, 57 ± 8, and 90 ± 16 following 4.0, 8.0, and 16.0 mg/kg, respectively. The DURATION of the highest dose was significantly longer than that of 8.0 mg/kg (t[14] = 2.03, p < 0.05) and

4.0 mg/kg (t[4] = 2.07, p = 0.05).

The behavioral changes following injection of FG 7142 included the appearance of an orofacial tremor consisting of tremulous movements of the masseter and adjacent facial muscles, moving caudally to recruit muscles around

eyes (i.e. Koene et al., 1993). Typically the tremor lasted 7 to 20 seconds and

was often terminated with a few seconds of grooming about the head or vacuous mouth movements. Locomotor activity was also stimulated by FG 7142.

Figure 4 shows the dose-dependency of the probability for a bout of orofacial tremor within each collection period (top) and the level of locomotor activity

(bottom). Both the probability for a bout of orofacial tremor and the level of locomotor activity remained increased for a duration dependent on the dose of

FG 7142. Aside from the first 15 min following injection of FG 7142, the locomotor activity score range (1.5-2.5) indicates forepaw and head movement, including grooming of the head, behaviors that appeared in many cases to be in reaction to the tremor (see above).

C.3. Effect of repeated dialysis sessions on basal and FG 7142-stimulated efflux

Effects of repeated testing on baseline cortical ACh efflux are shown in

Figure 5, top panel. The paired t-test (collapsed across Group; performed on natural log values to correct for non-normal distribution) comparing Session 1 and 4 revealed that repeated dialysis sessions resulted in a significant decrease in baseline efflux (t[15] = 2.45, p < 0.05). This decrease in baseline efflux 42

over sessions was not correlated with the amplitude or duration or the FG 7142 effect, since Pearson correlation coefficients between median baseline efflux and the percent increase in efflux in all post-injection collection periods were non significant (r's < 0.57; p > 0.1).

The effects of repeated testing on FG 7142-stimulated ACh efflux are shown in Figure 5, bottom panel. The ANOVA revealed significant differences in efflux over COLLECTION PERIOD (F[8,72] = 5.32, p < 0.001) but no effect of SESSION (F[l ,9] = 1.85, P > 0.2) and no interaction

(F[8,72J = 1.34, p > 0.2). Post-hoc analyses confirmed that the basis for the

COLLECTION PERIOD effect was the expected significant increase in ACh efflux (see 8.0 mg/kg results above) at 15 (t[10j = 4.72, p < 0.001),

30 (t[10] = 3.00, p < 0.01) and 45 min (t[10] = 2.24, p < 0.05). At

120 min post-injection, efflux fell to significantly below baseline

(t[10] = -1.86, p < 0.05).

As shown in Figure 5 (bottom panel, open symbols), TTX (1.0 /xM) suppressed both basal and FG 7142-stimulated cortical ACh efflux. Moreover, while only a single animal was tested at each session, this TTX-dependency appeared to be unaffected by repeated dialysis sessions. Within 30 min after the addition of TTX to the perfusion medium, basal ACh efflux was suppressed by 43

90%. With continuous co-perfusion with TTX, post-FG 7142 (8.0 mg/kg) efflux increased to only 50% of basal efflux in the first 15 min, then returned to below 10% of basal values by 30 min post-injection.

D. Discussion

Two hypotheses were tested in these experiments: 1) that systemic administration of FG 7142 increases basal cortical ACh efflux, and 2) that repeated insertion and perfusion of the microdialysis probe do not alter the responsivity of cortical ACh efflux. FG 7142 produced a substantial increase in cortical ACh efflux that lasted up to 90 min, depending on the dose. Responses of cortical ACh to FG 7142 and TTX did not differ between drug-naive rats tested on the first versus the fourth microdialysis session, indicating that changes cortical ACh can be validly measured for up to four microdialysis sessions. These results are discussed below in terms of possible mechanisms underlying the stimulatory effect of FG 7142 on cortical ACh efflux, and evidence for the validity of the within-subjects design for cortical microdialysis experiments. 44

P.l. FG 7142 enhancement of basal cortical ACh efflux

The effects of FG 7142 in this study were seen on "basal" ACh efflux, defined as efflux from well-habituated animals at rest prior to drug administration. As expected, based on previous results with ZK 93 426

(Moore et al., 1992c), FG 7142 effectively stimulated basal efflux. The principal expression of dose-responivity in the effects of FG 7142 was in the duration of the stimulation of ACh efflux (see Figure 3). Interestingly, the assessments of the motoric effects similarly reflected a dose-dependent increase in duration of effect. FG 7142 induced an orofacial tremor and increased locomotor activity. Behavioral observations suggested that the dose of FG 7142 was positively related to the number of collection periods in which an animal would display orofacial tremor and/or increased locomotor activity (See Figure

4). Importantly, the dose-response relationship of the amplitude of the increase in cortical ACh appears robust (see Figure 3), and the dose-related differences in duration may be secondary to the dose-dependency of the proportion of available receptors occupied.

Unlike the weak inverse agonist, ZK 93 426 (Moore et al., 1993a),

FG 7142 effectively increased cortical ACh efflux in resting animals. The increased efficacy of FG 7142, relative to ZK 93 426, on basal cortical ACh 45 efflux is illustrated Figure 6. As shown, ZK 93 426 (5.0 mg/kg, i.p.) minimally stimulates basal efflux, and is effective only in enhancing the ACh efflux during behavioral activation (i.e. activation following darkness/cereal presentation, see Chapter IV). FG 7142, on the other hand increases basal efflux.

General pharmacokinetics probably do not contribute significantly to the apparent difference in efficacy in ZK 93 426 and FG 7142. The greater efficacy of FG 7142 is more likely due to its greater intrinsic activity at the

GABAa receptor. ZK 93426 has a very low activity in gating of GABAa/BZR- gated chloride conductance in in vitro preparations (i.e. Malatynska et al.,

1989), and lacks many of the behavioral effects of other central BZR inverse agonists (Jensen et al., 1984). In contrast, FG 7142 has partial efficacy at

BZRs and has behavioral and neurochemical effects characteristic of BZR inverse agonists (Corda et al., 1987; File et al., 1987; Jackson et al., 1991;

Cooper, 1985; Bradberry et al., 1991; Giorgi et al., 1988; Ida et al., 1991).

Thus, it is possible that the apparently greater effect of systemic FG 7142 on cortical ACh is entirely due to its greater intrinsic activity at the central BZR.

Given the extensive anatomical and vivo neurochemical evidence for

GABAergic modulation of the cortical cholinergic afferents from the basal 46

forebrain (see above), the primary mechanism contributing to FG 7142-induced

increases in cortical ACh is likely to be disinhibition of these cells within the basal forebrain (Sarter et al., 1990). Furthermore, infusion of BZR full inverse

agonist, P-CCM, into the SI is sufficient for stimulation of cortical ACh efflux

(see Chapter IV). Thus, FG 7142, like other negative modulators of the

GABAa/BZR, may reduce GABA-mediated inhibition of cholinergic neurons in the SI, thereby increasing cortical ACh release.

Another, perhaps parallel, mechanism for FG 7142-stimulated cortical

ACh efflux may be via stimulation of meso-accumbal dopamine release (see

McCullough et al., 1992b). Electrophysiological and behavioral evidence indicate that FG 7142-induced increases in dopamine release in nucleus accumbens could lead to a decrease in GABA transmission in the SI (see

Mogenson et al, 1991). The actions of FG 7142 in the nucleus accumbens and the SI could be parallel, reducing both GABA levels and the binding and activity of the remaining GABA. It is therefore probable that the ability of

FG 7142 to enhance mesoaccumbal DA contributes to its relatively potent enhancement of basal cortical ACh efflux. The parallel stimulatory actions of

FG 7142 on mesolimbic DA and basal forebrain cholinergic neurons may make this drug a useful tool in modelling conditions under which both of these 47

systems are activated (see above). FG 7142 is used in this context in the experiments described in Chapter V.

12,2. The validity of repeated microdialysis sessions for measuring

cortical ACh efflux

Comparisons between groups undergoing insertion and perfusion of a concentric microdialysis probe on the fourth versus first dialysis session indicated that capacity of cortical ACh efflux to be stimulated by BZR ligands is not altered by repeated probe insertion and dialysis. This was concluded from 1) the nearly identical responses of Groups 1 and 2 to the 8.0 mg/kg dose of FG 7142, and 2) the similar time courses and levels of suppression of efflux by TTX on Session 1 versus Session 4 (see Figure 5, bottom panel).

Furthermore, differences in the amplitude or time course of the FG 7142- stimulated cortical ACh were not observed with the two other doses (data not shown).

The experiment also revealed, however, that repeated microdialysis testing is associated with a reduction in basal cortical ACh efflux (Figure 5, top panel) and with possible differences in time to return to basal efflux levels following FG 7142 (see Figure 5, bottom panel). Reduced basal efflux as a 48 function of multiple dialysis sessions has been previouly observed (i.e. Moore, et al., 1992b, 1993a), and possibly reflects a reduction in the in vivo diffusion coefficient of the neurotransmitter as a result of increased tissue disruption (see

Benvenista & Huttemeier, 1990). It is not associated with differences in within- session dynamics in basal or stimulated ACh efflux, since basal and stimulated levels of efflux were not correlated. This lack of correlation between baseline efflux and the effect of FG 7142 corroborates previous findings indicating that pharmacologically- or behaviorally-induced changes in cortical ACh efflux are not sensitive to reductions in in vivo recovery as a result of repeated testing

(i.e. Moore et al., 1992b,c, 1993a). The origin for the apparent prologation of the FG 7142 effect in Session 4 animals is unknown. However, because it would be reflected in a reduced level of suppression by TTX, an increase in extracellular ACh derived from damaged cholinergic terminals does notunderlie these apparent differences between sessions.

The conclusion that the protocol of repeated probe insertion and perfusion leaves pharmacological modulation of cortical ACh release unaltered is supported by previous findings. For example, robust enhancement of cortical

ACh by the addition of potassium ion (100 mM) or atropine to the perfusion fluid (Moore et al., 1995), or following systemic scopolamine (Moore et al., 1992b), has been demonstrated for up to five sessions. Also, similar to its effects on FG 7142-stimulated efflux, local TTX effectively suppresses basal and scopolamine-stimulated ACh efflux over multiple sessions

(Moore, et al. 1992b; Moore unpublished observations). Moreover, a similar experimental design has been shown to be valid for measuring modulation of striatal ACh (Johnson & Bruno, 1995) and GABA (McCone-Byrnes, 1995).

In conclusion, despite its limitations which will be discussed further in the General Discussion (below), the repeated dialysis experimental design is valid for measuring changes in cortical ACh efflux and was thus employed in the experiments described below. CHAPTER IV

MODULATION OF STIMULATED CORTICAL

ACETYLCHOLINE RELEASE BY INFUSION OF BENZODIAZEPINE

RECEPTOR LIGANDS INTO THE BASAL FOREBRAIN

A. Background and rationale

In rat, as in humans, projections from the basal forebrain provide the majority of cholinergic innervation to neocortical structures (see Chapter I).

Furthermore, these projections provide essentially all of the ACh released into the cortex, and, consequently, pharmacological manipulations within the basal forebrain can influence cortical ACh release (see Chapter I). I previously found that benzodiazepine receptor (BZR) ligands, when administered systemically, could bidirectionally modulate cortical ACh release (Moore et al., 1992c,

1993a). Specifically, the BZR selective inverse agonist ZK 93 426 (Jensen et al., 1984) increased, while the BZR agonist chlordiazepoxide (CDP;

50 Haefely, 1989) decreased, ACh efflux measured in vivo with microdialysis.

Importantly, the efficacy of the BZR ligands appeared to be dependent on the

initial level of cortical ACh efflux. While these ligands were minimally

effective on ACh efflux in resting animals (Moore et al., 1992c), they

significantly affected efflux that had been stimulated by the presentation of a multimodal appetitive stimulus (Moore et al., 1993a; Sarter & Bruno, 1994) .

Based on anatomical and pharmacological evidence for regulation of basal forebrain cholinergic cell activity by GABA transmission in the basal forebrain (see Chapter I), it was postulated that the bidirectional modulation of stimulated cortical ACh efflux by BZR ligands was through the actions of these ligands on receptors within the basal forebrain. Thus the present study was designed to determine whether the effects of systemically administered BZR ligands on cortical ACh release could be accounted for by their actions in the basal forebrain. Accordingly, the effects of a BZR agonist and a BZR inverse agonist infused into the basal forebrain on cortical ACh efflux in behaviorally- activated rats were assessed. The BZR ligands tested were the full agonist CDP and a water-soluble BZR inverse agonist P-CCM. 52

It. Experimental design and methods

F344/BNNia FI rats (National Institute of Aging at Charles Rivers,

Wilmington, Mass.), age 4-7 months, were trained to associate presentation of palatable food (a piece of fruit cereal) with onset of darkness in the testing chamber. This procedure reliably increases cortical ACh efflux (Moore et al.,

1993a). Following 7 days of training, using the general surgical procedures described in Chapter II, animals were surgically implanted with bilateral drug infusion cannulae into the basal forebrain and a microdialysis guide cannula into the frontal cortex. The drug infusion cannulae (28 ga.) were implanted into the ventral pallidum/substantia innominata (relative to Bregma [in mm]: AP -0.8,

DV 8.0, L 2.5; Paxinos & Watson, 1985) Once these cannulae were secured to skull screws with dental acrylic, the microdialysis guide cannula was placed in the frontal cortex as described in Chapter II and fixed to the skull with additional acrylic.

For 3 days following surgery, animals were allowed to recover and continued to be trained. Animals were then tested every other day for 4 microdialysis test sessions. On three of these sessions, the effects of intracranial infusion of chlordiazepoxide (40.0 /^g/hemisphere), P-CCM 53

(3.0 Mg/hemisphere), or vehicle were tested. The vehicle was adjusted with

acetic acid to the osmolarity and pH of the CDP solution. The fourth session

was used to test additional doses of CDP (10.0 ^g/hemisphere) and P-CCM (9.0

Mg/hemisphere). All drug/dose conditions were counterbalanced across the four sessions. Comparable doses of these drugs infused into the basal forebrain have been shown to have behavioral effects as early as 5 to 15 min, lasting up to an hour following infusion (Dudchenko & Sarter, 1992; Mayo et al., 1992;

Stackman & Walsh, 1992). In addition, preliminary studies in our laboratory indicated that 3.0 Mg/hemisphere of P-CCM and 40.0 Mg/hemisphere of CDP were effective on stimulated cortical ACh efflux, but not on basal efflux.

During each dialysis session, the dialysis probe was inserted through the cortical cannula, and the subject was perfused (2.0 ML/min) with the standard artificial CSF (see General Methods); collection began after 3 hours (see

Chapter II). Following the collection of four 15-min baseline samples, the drug-infusion needles (30 ga., flush with guide cannulae) were inserted into the basal forebrain. Two more 15-min samples were then collected in the resting animals. During the middle 5 min of the 2nd of these collection periods, the drug was infused (0.5 nL at 0.1 ^L/min). This infusion volume and rate are within parameters reported by Routtenberg (1972) for complete diffusion of 54 drug from the cannula tip with the volume of diffusion limited to less than

1 mm3. Five min after infusion of the drug ended, a new collection was begun, the lights in the testing room were extinguished and the food was presented.

Collection continued for an additional 15 min. Samples were stored at -80°C until analyzed for ACh by high pressure liquid chromatography according to methods described above.

Previously, it was observed that in the absence of drug pretreatment, the darkness/cereal stimulus increased ACh efflux for up to 15 min after its presentation, after which efflux approached baseline levels (Moore et al.,

1993a). Accordingly, statistics were limited to efflux during the last pre darkness period (during drug infusion) and the first post-darkness period in order to test the effects of BZR ligands on basal and stimulated efflux, respectively. Efflux values from those two collection periods were expressed as percent change from the median of the 4 collections prior to needle insertion

(see above). The percent change values were subjected to paired t-tests in which CDP and P-CCM were compared to vehicle. 55

C. Results

Due to analytical equipment failure, dialysates from the P-CCM

(3.0 /ig/hemisphere) session were lost for 4 of the 12 animals tested, and data from the CDP (40 jug/hemisphere) session were lost for 4 others. Therefore, each drug was analyzed separately (with DOSE as the within-subjects factor) using data from 8 subjects in the analysis of each drug.

As shown in Figure 7, all microdialysis cannulae lay at approximately a

50° angle within the frontal or frontoparietal cortex, within a range of 2.2 to

4.2 mm anterior to Bregma (Paxinos & Watson, 1985). All drug infusion cannulae (including those contralateral to the dialysis probe; placements not shown) were placed within 0.2 to 1.8 mm posterior to Bregma, between

2.4 - 3.0 mm lateral to the midline, and 7.5 to 8.0 mm below the dural surface.

No differences in basal or stimulated efflux as a function of dialysis probe or infusion cannula position were detected.

For the 8 animals included in the study, average baseline efflux

(pmol/min; see above) was 0.34 (± 0.08), 0.26 (± 0.04) and 0.20 (± 0.06) for vehicle, CDP, and P-CCM, respectively; it did not differ significantly between vehicle and either drug. Figure 8, top panel, shows changes in cortical ACh efflux as a function of collection period. The time courses of changes in

ACh efflux following drug administration and the presentation of the darkness/cereal stimulus were similar to what had been previously observed with systemic BZR ligand administration. Following vehicle infusion, the darkness/cereal stimulus produced an increased in cortical ACh efflux that lasted for up to 15 min (one collection period). As with systemic administration, infusion of BZR ligands into the basal forebrain appeared to bidirectionally modulate the stimulation of ACh efflux in the first collection period following darkness/cereal presentation.

The modulation of behaviorally-stimulated cortical ACh efflux is illustrated in Figure 8, bottom panel. As this figure shows, efflux prior to darkness/cereal presentation (i.e. 5-10 min following drug infusion; left panel) was not significantly affected by either P-CCM or CDP (compared to vehicle, t[7] < 1.5, p's > 0.3). In contrast, efflux following darkness/cereal presentation was significantly greater with p-CCM treatment than with vehicle

(t[7] = 3.44, p < .01), and significantly lower with CDP than with vehicle

(t[7] = -1.94, p < .05). In order to confirm a bidirectional modulation of cortical ACh release by the BZR ligands, the effects of the BZR inverse and full agonist on stimulated ACh efflux were assessed within animals. Accordingly, 57 an ANOVA was performed on the subset of data used in the above analyses from the 4 animals that had received all three drug treatments. This analysis revealed a significant effect of drug (F [2,6] = 11.7, p < 0.01) that was produced by a significant augmentation with p-CCM (t[3] = 2.72, p < 0.02) and a significant reduction with CDP (t[3] = -1.63, p = 0.05).

Behaviorally, prior to needle insertion and during drug infusion, all animals were at rest, with infrequent movement limited to the head and forepaws. During presentation of the cereal, all animals exhibited rearing and quadrupedal locomotion, regardless of drug (or vehicle) treatment.

D. Discussion

Replicating results previously produced with systemic administration

(Moore et al., 1993a), the present findings demonstrate that BZR ligands, infused into the basal forebrain, bidirectionally modulate cortical ACh efflux.

Specifically, a BZR inverse agonist (P-CCM) significantly enhanced, while the full agonist (CDP) suppressed, the stimulation of cortical ACh efflux by a behaviorally-activating stimulus. Although the actions of BZR ligands in alternate neuroanatomical sites may contribute to their systemic effects, the 58

present results indicate that their action in the basal forebrain is sufficient to produce bidirectional modulation of stimulated cortical ACh release.

The cortical ACh efflux measured in the present study represents ACh

release that has been enhanced by a multimodal stimulus which predicted an unconditioned appetitive stimulus (fruit cereal). Moreover, this stimulated cortical ACh release, as opposed to basal release, was modulated by the action of BZR ligands in the basal forebrain. These results are consistent with evidence that electrophysiological activity within the basal forebrain is stimulated by similar sensory/behavioral events. For example, it has been shown in primates that electrophysiological activity within the basal forebrain

(Richardson & DeLong, 1991; Wilson & Rolls, 1990) and cortical ACh efflux

(Smith, 1994) are stimulated during the presentation of operantly or classically- conditioned stimuli. We have found similar results in rats using the presently- described multimodal stimulus (see above; Moore et al., 1993a), as well as with classically-conditioned auditory signals presented in an operant box

(unpublished observations).

It is possible that BZR ligands, acting in the basal forebrain, selectively modulate stimulation of cortical ACh release by environmental changes. It appears unlikely that the BZR ligand infusions significantly affected basal efflux 59

since in preliminary studies, these specific drug treatments had no effect on basal cortical efflux 15-30 min following infusion into the basal forebrain (see

Chapter IV, Section B, above), and in the present study, the drugs had no effect on basal efflux 5 to 10 min post-infusion (see Figure 8, bottom panel, left).

Similarly, Pirch et al.(1991) found that infusion of GABA into the basal forebrain blocked ACh-mediated augmentation of evoked firing in cortical cells, while not affecting the basal firing rate. The activity-selective efficacy of BZR ligands may be a function of their effects on GABA transmission, since the increased chloride conductance at the activated GABAa/BZ receptor stabilizes the membrane potential of the neuron (Nicoll et al., 1990) by counteracting depolarizing currents. Moreover, the GABAa current increases proportionally with increases in depolarizing currents, thus leading to a "use-dependent" inhibition of cell activity (Kaila, 1994). CHAPTER V

INTERACTIONS OF DOPAMINE RECEPTOR ANTAGONISTS WITH

THE GABAa/BZR PARTIAL INVERSE AGONIST IN THE

MODULATION OF CORTICAL ACETYLCHOLINE EFFLUX

A. Background and rationale

Release of ACh to the cortex and release of DA in the nACC are stimulated under similar environmental/behavioral conditions (Fibiger & Day, palatable mean; Hernandez & Hoebel, 1988; Young, Joseph & Gray, 1992;

McCullough & Salamone, 1992a; McCullough et al., 1993). A primary mechanism for the increase in cortical ACh release during release of DA into the nACC may be decreased GABAergic inhibition of the basal forebrain cholinergic cells as a result of activation of DA receptors in the nACC. This mechanism has been postulated on the basis of the disinhibitory effects of DA agonists applied into the nACC on the cell activity in the basal forebrain

60 (i.e. ventral pallidum and substantia innominata; Yang & Mogenson, 1989;

Mogenson et al., 1991). Moreover, this is supported by multiple studies demonstrating increases in cortical ACh efflux as a result of systemic administration of DA indirect and direct agonists that are blocked by DA receptor antagonists (Day & Fibiger, 1992, 1993; Acquas, et al., 1994).

Increases in DA transmission in the nACC and ACh transmission in the neocortex have been postulated to underlie important psychological processes, such as stimulus-reward association formation (i.e. Everitt & Robbins, 1992;

Cador et al., 1991; Taylor & Robbins, 1984) and selective attention to conditioned stimuli (i.e. Ashe & Weinberger, 1991; Muir et al., 1992a,b). It is therefore important to examine how these systems interact when they are both activated. FG 7142 is a BZR partial inverse agonist known to stimulate both

ACh (see Chapter III) and nACC DA efflux (McCullough & Salamone, 1992b).

This drug, thus, allows testing of the hypothesis that under such conditions in which nACC DA release and cortical ACh release are stimulated, the stimulation of cortical ACh release is dependent on the activation of dopamine receptors in nACC. In the following studies, this hypothesis was tested by testing the ability of dopamine receptor antagonists to block FG 7142-stimulated cortical ACh efflux. 62

Few studies have examined the role of different DA receptor sub-types in the regulation of the basal forebrain cholinergic cells, and when D l- versus

D2-selective DA receptor ligands were tested, agonists were used (i.e. Yang &

Mogenson, 1989). Thus, conclusions about the role of endogenous dopamine at different receptor sub-types in the effects have not been examined. In order to examine this issue with the present experiments, selective DA receptor antagonists were used. At present, the pharmacology of DA antagonists allows selectivity for two receptor sub-type families, the Dl-like (Dl and D5) and the

D2-like (D2, D3, D4) receptors (see Jackson & Westlind-Danielsson, 1994;

Leysen et al., 1993). For the present study, the Dl-like receptor antagonist,

SCH 23390 (Alburges et al., 1992) and the neuroleptic haloperidol were tested.

The anti-psychotic properties of haloperidol are generally attributed to its antagonistic properties at D2-like receptors (Seeman, 1981; but see

Leysen et al., 1993; Lidow & Goldman-Rakic, 1994). Therefore, to determine the extent to which the effects of haloperidol were mediated by D2 receptor blockade, the effects of the selective D2-Iike antagonist, clebopride

(Leysen et al., 1993), on FG 7142-stimulated cortical ACh efflux were also assessed. 63

B. Experimental design and methods

B.l. Effects of systemic administration of DA antagonists on FG 7142-

stimulated cortical ACh efflux

Four-month-old Fisher 344/Brown Norway FI rats (NIA colony at

Charles Rivers or Harlan) that had been habituated to the testing chamber were implanted with guide cannulae (0.65 mm o.d.) into the left frontoparietal cortex according to the procedure described in Chapter II. Following the 3-day recovery period (see Chapter II), animals were assigned to one of three groups tested on four sessions with FG 7142 and varying doses of one of the following

DA receptor antagonists: the Dl-selective antagonist SCH 23390, the neuroleptic halporidol or the D2-selective antagonist clebopride. The drug combinations tested and doses of drugs used are summarized in Tables 1 and 2.

The dose of FG 7142 was chosen on the basis that it produces a reliable increase in cortical ACh efflux (see Chapter III) and is comparable to a dose that increases DA efflux in the nACC (McCullough et al., 1992a). Doses of

DA antagonists were based on doses comparable to those shown to interact with effects of systemic DA agonists on cortical ACh efflux (Day & Fibiger, 1992, 64

1993). Drug combinations were counterbalanced across test sessions; test

sessions were separated by a “wash-out” day (see Chapter II).

The test session followed the general procedure outlined in Chapter II.

Animals were perfused with the standard aCSF at 2.0 /xl/min with 15-min collection periods. After 4 baseline dialysates were collected, animals were injected with the DA antagonist or its vehicle and dialysates were collected for an additional 15 (for SCH 23390) or 30 (for clebopride or haloperidol) min, prior to injection of FG 7142 or its vehicle (See Table II). Dialysates were collected for 45 min following injection of FG 7142 (or vehicle, on DA- antagonist-alone tests).

ACh was quantified in the dialysates according to the methods in

Chapter II. Efflux values were expressed as a percent change from the median baseline. Percent change values were then subjected to statistical analysis. The effect of each DA antagonist on FG 7142-stimulated cortical ACh efflux was analyzed separately by subjecting the 4 different DA antagonist/FG 7142 combinations to a DOSE x COLLECTION PERIOD repeated measures

ANOVA. The analyses were constructed to test whether the DA antagonist blocked the expected increase in cortical ACh efflux following FG 7142 administration. Based on the experiments in Chapter III, peak FG 7142-induced 65

increases were expected within the first 2 collection periods following

administration. Given this expected time course for FG 7142, and to minimize variance due to different pharmacokinetics of the DA antagonists, the levels of

COLLECTION PERIOD were limited to the last baseline and the first two collections following FG 7142, the window in which the greatest differences in efflux over time were expected as a function of dose of DA antagonist. Main effects and interactions were further analyzed with the appropriate paired t-tests.

B. 1. Effect of DA antagonists co-perfased in nACC or cortical probes on

FG 7142-stimulated cortical ACh efflux

In well-habituated 4-mo-old Fisher 344/Brown Norway FI rats (NIA colony at Harlan), microdialysis guide cannulae (0.65 mm, see Chapter II) were placed into the left medial prefrontal cortex and the ipsilateral nACC using the general surgical procedure outlined in Chapter II modified to employ two stereotaxic arms as follows. The medial prefrontal cannula was implanted by rotating the stereotaxic arm in the saggital plane 10° anterior from vertical, then lowering the cannula 0.7 mm from a point on the dural surface 3.0 mm anterior to Bregma and 0.6 mm lateral to the midline (Paxinos & Watson, 1985). The nACC cannula was placed by rotating the stereotaxic arm in the saggital plane 66

10° posterior away vertical, then lowering the cannula 6.5 mm from a point on the dural surface 0.5 mm posterior to Bregma and 0.9 mm lateral to the midline. The cannulae and a small hook for attachment of microdialysis lines were secured to the skull with skull screws and dental acrylic. Post-surgery procedures were performed as outlined in Chapter II. Cannula placements in anterier cingulate cortical areas 1 and/or 3 and in the nACC were verified by examination of Nissl- and AChE-stained sections.

Following recovery, animals were tested on four sessions with systemic injection of FG 7142 (8.0 mg/kg, i.p.) and local perfusion of either the Dl- selective antagonist SCH 23390 or the neuroleptic haloperidol. Table 3 outlines the procedure used to test the effects of intra-accumbens or intracortical DA antagonist perfusion on FG 7142-stimulated efflux. As shown, two doses of the

DA antagonist were tested in the nACC, with only the higher dose tested in the cortex. Doses of DA antagonists chosen are comparable to those shown to significantly modulate ACh efflux in the neostriatum (Johnson & Bruno, 1995).

For each session, the aCSF used throughout the entire session was mixed prior to probe insertion. After baseline collection, the DA antagonist was mixed with the aCSF and a syringe, tubing and alternate swivel channel were filled with the

DA antagonist-containing aCSF. The inlet to the probe was then quickly 67 transferred to the inlet of the DA antagonist channel. The dead volume of the inlet was calibrated so that the antagonist would begin perfusing the brain area

12 min after the inlet was transferred. Further references to the onset of DA antagonist perfusion correct for this dead volume.

Cortical dialysates were analyzed for ACh according to the methods outlined in Chapter II. A limited number of dialysates from the nACC were also analyzed for GABA content, according to the GABA analysis procedure described in Chapter II. Because the data are preliminary, analyses of the data were averaged over collection periods considered to directly test 1) whether the local perfusion of the antagonist had a primary effect on cortical ACh efflux and

2) whether co-perfusion of the antagonist interacted with the peak effect of

FG 7142. Efflux was expressed as percent change from median baseline, and summary statistics were compiled for efflux changes 1) in the last baseline collection period, 2) in the collection period reflecting the first 15-17 min of

DA antagonist co-perfusion, and, 3) in the collection period 15-30 min following FG 7142 injection (the time of the expected peak increase in efflux). 68

C. Results

C .l. Effects of systemic administration of DA antagonists

on FG 7142-stimulated efflux

Cortical microdialysis probes were confirmed to lie within the frontal parietal cortex, between 1.4 and 2.2 mm anterior to Bregma (Paxinos &

Watson, 1985). Sections from these animals (not shown) appeared nearly identical to the sections shown in Plates I and II.

The median baseline efflux averaged over subjects in all three test groups was 0.085 ± .015 pmol/min. Baseline efflux did not differ across DA antagonist groups. As has been observed in previous experiments (see Chapter

III), there was a trend for a decrease in baseline efflux across sessions

(for SCH 23390 and haloperidol groups, F[3,30] = 2.34, p = .089).

Figure 9 shows the effect of SCH 23390 and FG 7142 on cortical ACh efflux. Administered alone, FG 7142 had the expected stimulatory effect on cortical ACh efflux. SCH 23390 administered alone, had no effect on efflux, but was able to attenuate FG 7142 stimulated efflux, though both doses appear equally effective. The DOSE x COLLECTION PERIOD ANOVA revealed main effects of DOSE (F[3,15] = 7.27, p < .01) and COLLECTION PERIOD 69

(F[2,10] = 28.06, p < .001), with a trend for an interaction between the two factors (F[6,30] = 2.0, p = .097).

The incomplete nature of the attenuation by SCH 23390 was reflected by the main effect of COLLECTION PERIOD and the lack of a fully significant interaction. To confirm that the SCH 23390-mediated attenuation contributed to the trend for interaction (rather than the interaction being due to variance from the FG 7142-alone and SCH 23390-alone conditions), planned simple comparisons were performed to reveal the basis for both main effects and the trend for the interaction. Analyses of each drug condition (refer to Table 2) over time indicated that all drug conditions except the SCH 23390- alone condition were associated with an increase in efflux (F[2,10]'s > 7.0, p's < .05) in the post-FG 7142 collection periods, relative to the last baseline

(all paired t[5]'s > 3.0, p's < .05). To see if the DOSE effect or the trend for the DOSE x COLLECTION PERIOD interaction was due, in part, to the attenuation of the FG 7142-induced increase in efflux, the mean increase in efflux during the first 30 min after FG 7142 injection (averaged over two collection periods) was compared across the 4 dose conditions. As expected, the increase in efflux in the FG 7142-alone condition was significantly greater than the increase in efflux following vehicle in the SCH 23390-only condition 70

(t[5] = 10.0, p < .001). However, FG 7142-stimulated efflux was also

significantly reduced following pre-administration with the higher dose of

SCH 23390 (t[5] = 2.53, p < .05), and showed a trend for attenuation with preadministration of the lower dose (t[5] = 1.55, p = .09). Though somewhat indirect, these statistics show the basis for a reliable but incomplete antagonism of FG 7142 by the Dl antagonist.

Figure 10 shows the ability of haloperidol to block FG 7142-stimulated cortical ACh efflux. Haloperidol appeared to fully block the increase in cortical

ACh efflux induced by FG 7142, being most reliable at the higher dose of

0.9 mg/kg. This effect was revealed in the DOSE x COLLECTION PERIOD

ANOVA which showed a main effect of COLLECTION PERIOD

(F[2,10] = 8.53, p < .01), as well as a significant DOSE x COLLECTION

PERIOD interaction (F[6,30] = 2.73, p < .05). Within-dose-condition comparisons of collection periods revealed that only in the FG 7142-alone condition was post-FG 7142 efflux significantly elevated over baseline (for

FG 7142-alone, F[2,10] = 10.5, p < .05; other F[2,10]'s < 3.0, p > .2).

The FG 7142-induced increase in efflux was significantly greater than the change in efflux following vehicle in the haloperidol-alone condition

(t[5] = 2.44, p < .05). Moreover, post-FG 7142 efflux following either dose 71 of haloperidol was not different than efflux in the haloperidol-alone condition

(t[5]'s < 1.5, p's > .25). Direct comparisons of the increase in post-FG 7142 efflux revealed that the effect of FG 7142 was significantly reduced following pre-administration of either 0.15 mg/kg (t[5] = 3.11, p < .05) or 0.9 mg/kg

(t[5] = 2.2, p < .05) of haloperidol.

As an indication of whether blockade of the FG 7142-induced increase in cortical ACh efflux is mediated by D2 receptors, data from 3 animals tested with the D2-selective antagonist clebopride are presented in Figure 11. It is clear from the identical time courses of FG 7142-stimulated efflux that the D2 antagonist had absolutely no ability to block the effect of FG 7142, nor did it have any primary effect on cortical ACh efflux.

A summary of the effects of systemically-administered SCH 23390, haloperidol and clebopride is shown in Figure 12.

C.2. Effect of intra-accumbens or intra-cortical perfusion of DA antagonists on

FG 7142-stimulated efflux

Example positions of the dialysis probes are shown in schematic diagrams of saggital sections in Figure 13. All data presented are from animals in which the placement of the probe tips were confirmed to lie within the medial 72 prefrontal cortex (areas Cgl, Cg3 or IL) and the nACC according to the coordinates of Paxinos and Watson (1985). However, in 1 animal in each of the SCH 23390 and haloperidol groups, the nACC apparent perfusion area of the probe extended into the rostral ventral pallidum.

Efflux values reflect changes from median baseline efflux, which averaged 0.16 (± 0.08) pmol/min over all subjects. The effects of SCH 23390 perfused into the nACC (n=3) are shown in Figure 14. The left-most panel shows that efflux was similar to the median baseline efflux during the last baseline collection prior to perfusion with either regular aCSF or aCSF with

SCH 23390. The middle panel shows the effect of perfusion of SCH 23390 into the accumbens on cortical ACh efflux. The lower dose (10 /xM) of the Dl antagonist perfused into the nACC appeared to increase cortical ACh efflux, but this effect was more variable at the higher dose (middle panel). On sessions in which regular aCSF was perfused in the nACC, efflux appeared slighly elevated following the switch to a different syringe (middle panel), then appeared markedly increased above baseline following in the 15-30 min collection period following administration of FG 7142. During co-perfusion of either dose of

SCH 23390 into the nACC, following FG 7142, ACh efflux was not increased further from the level attained following addition of SCH 23390 to the aCSF, 73 and did not appear different from FG 7142-elevated efflux in the control condition (i.e. regular aCSF co-perfusion).

Figure 15 shows the effects of intra-accumbal perfusion of haloperidol on basal and FG 7142-stimulated cortical ACh efflux (n=3). As with

SCH 23390, the baselines on each session were similar to each other and to the median baseline (left-most panel). While haloperidol appeared to have only a minor inhibitory effect on basal cortical ACh efflux at the higher dose (middle panel), it dose-dependently blocked the ability of FG 7142 to increase cortical

ACh efflux (right-most panel).

Figure 16 shows the effects of SCH 23390 (n=2) or haloperidol (n=2) perfused locally into the cortex on basal and FG 7142-stimulated cortical ACh efflux. The top panel shows that relative to baseline (left-most bar),

SCH 23390 produced a local increase in cortical ACh efflux (middle bar), with

FG 7142 further increasing efflux (right-most bar). By contrast, haloperidol did not appear to affect basal efflux, nor did it appear to block the FG 7142-induced increase in cortical ACh efflux.

GABA efflux in the nACC was quantified in the regular aCSF and high antagonist dose conditions for 3 animals in the SCH 23390 group and 2 animals in the haloperidol group. Average median baseline GABA efflux over all 74 subjects from both drug conditions was 0.32 ± .05 pmol/min. The summary of effects of co-perfusion of the antagonists into the nACC on basal and post-FG 7142 GABA efflux in the nACC are shown in Table 4. The average change in efflux (from median baseline) in the collection periods during 30 min of antagonist perfusion was used to assess effects of the co-perfusion on "basal

GABA efflux". The average change in efflux during the first 30 min following

FG 7142 injection was used to represent "post FG 7142 efflux" . These values are shown in Table 4. The process of switching syringes and inlet lines appeared to have no consistent effect on basal GABA efflux. Similarly, co perfusion of SCH 23390 (100.0 ptM) had little effect on basal GABA efflux, while co-perfusion of haloperidol (100.0 jtM) appeared to produce a moderate decrease in GABA efflux in the nACC. In the control condition, systemic injection of FG 7142 produced an increase in GABA efflux in the nACC. This post-FG 7142 increase in GABA efflux was also observed with co-perfusion of

SCH 23390 (100.0 ^M). During co-perfusion of haloperidol, however, efflux did not appear to exceed basal levels. However the increase from the haloperidol-suppressed basal levels represents an increase of similar magnitude to that observed in the other conditions. 75

D. Discussion

These experiments showed that the stimulation of cortical ACh efflux by the BZR partial inverse agonist FG 7142 can be attenuated by systemic administration of either a dopamine Dl-like selective antagonist or the neuroleptic haloperidol (see Figure 12 and Table 5). The greater effectiveness of haloperidol indicates that its actions may be more direct than those of the Dl- like receptor antagonist in interacting with FG 7142-stimulation of cortical ACh efflux. In addition, systemic administration of the D2-selective antagonist did not antagonize FG 7142, indicating that the effect of haloperidol involves a mechanism other than systemic blockade of dopamine D2 receptors.

Intracranial perfusion of these antagonists via microdialysis probes placed in the nACC and mPFC revealed that the local effects of SCH 23390 in the nACC and cortex may not interact with FG 7142, but, rather, may produce increases in basal cortical ACh efflux. By contrast, intra-accumbens perfusion of haloperidol replicated its systemic effects in that it dose-dependently blocked the stimulation of cortical ACh efflux by the BZR partial inverse agonist. Local cortical perfusion of haloperidol did not block FG 7142-stimulated efflux.

While it cannot be ruled out that the relatively high systemic dose of 76

SCH 23390 used in the present study affected receptor populations other than

DA D l receptors (i.e. 5-HT receptors; Alburges et al., 1992), the attenuation of

FG 7142-stimulated cortical ACh efflux by the Dl-selective antagonist is consistent with multiple studies demonstrating that the stimulation of neo- and allocortical ACh release by systemic DAergic agonists is mediated by dopamine receptor activation (i.e. Day & Fibiger, 1992, 1993; Acquas et al., 1994;

Imperato et al., 1993; Casamenti et al., 1986). Unlike the presently observed effects on FG 7142-stimulated efflux, Day and Fibiger (1992) found that amphetamine-stimulated increases in cortical ACh efflux could be antagonized with either Dl or D2 receptor antagonist. It is not clear why D2-receptor blockade did not attenuate FG 7142-stimulated cortical ACh efflux. It is possible that FG 7142, through its effects on a sub-population of GABAa receptors, selectively "primes" Dl receptors or reduces the sensitivity of D2 receptors, although the basis for these actions can only be speculated. It is also possible that the very high concentrations of DA produced by amphetamine

(Cooper et al., 1990) are necessary to reveal or elicit the effects of D2-receptor blockade. Along these lines, the direct D1/D2 agonist, apomorphine, is less effective in stimulating cortical ACh efflux, and its effects are rather selectively blocked by D l receptor blockade (Day & Fibiger, 1993). 77

The interactions of Dl receptor blockade with apomorphine and

FG 7142 are consistent with the finding that stimulation of allocortical ACh release by systemic administration of either a Dl-selective or a D2-selective agonist can also be blocked with a Dl-selective antagonist (Imperato et al.,

1993). Allocortical and neocortical ACh inputs are both likely to be modulated by GABA/DA interactions involving parallel dopaminergic pathways from the ventral tegmental area to the lateral septum and nACC, respectively

(Onteniente, et al., 1987). Thus, unlike the opposing actions that Dl-like and

D2-like receptors have on striatal ACh intemeurons, Dl and D2 receptor activation appears to synergistically increase cortical ACh release

(Imperato, et al., 1993), and as long as Dl receptors are activated, D2 receptor activation is not necessary. This conclusion is further supported by the finding that Dl and D2 agonists applied into the nACC act synergistically to disinhibit ventral pallidal cells, and that Dl receptor activation is necessary for the effects of the D2 receptor agonist (Yang & Mogenson, 1989).

Based the effects of D l agonists in the accumbens on ventral pallidal cells presumed to include the cholinergic projection cells (see above), the nACC appears to be a likely site for the stimulation of cortical ACh release via

Dl receptor activation. The present findings do not support this, however. In 78

fact, if anything Dl-receptor activation in the nACC under basal and, possibly,

FG 7142-stimulated conditions in the nACC appears to inhibit cortical ACh efflux. This is evidenced by the disinhibitory effects of SCH 23390 perfused into the nACC. The basis for this is not clear, but these effects are not inconsistent with evidence that activation of Dl receptors located on

GABAergic projection neurons in the nACC can suppress inhibition (i.e. suppress IPSPs), depending on the size of the synaptic potential (Meredith et al., 1993). Interestingly, unlike its lack of action in the nACC on FG 7142- stimulated cortical ACh efflux, SCH 23390 appeared in some animals (data not shown) to block FG 7142-induced orofacial tremor (see Chapter III; Koene et al., 1993), a behavior that is mediated primarily by Dl activation in the nACC

(Koene et al., 1993; but see also Parry et al., 1994).

An alternative site at which Dl receptor activation could mediate

FG 7142-stimulated ACh release is in the vicinity of the basal forebrain cholinergic soma. However, although a moderate density of Dl receptors is found in the VP and SI (Huang et al., 1992), DA receptor stimulation in these basal forebrain areas has not been demonstrated to have effects on the cortical cholinergic afferents (see Casamenti et al., 1986). Other possible sites for D l-

GABAa interactions include the ventral tegmental area and medial substantia 79 nigra, and the frontal cortex. These sites project to the basal forebrain (see

Chapter I) and possess D l receptors (Huang et al., 1992). Moreover, both are known sites of significant DA/GABA interactions (i.e. Cameron & Williams,

1993; Scheel-Kruger & Willner, 1991; Santiago et al., 1993). The possibility of the frontal cortex as a major site for dopamine Dl receptor-mediated disinhibition of cortical ACh release is not supported by the presently observed local effects of SCH 23390 on basal and FG 7142-stimulated ACh efflux. On the other hand, complex GABA-GABA and DA-GABA interactions have been demonstrated in the substantia nigra and ventral tegmental area (i.e. Cameron &

Williams, 1993; Grace & Bunney, 1979; Grace & Bunney, 1985) that could possibly mediate the facilitatory effects of D l receptor blockade on the disinhibition of cortical cholinergic afferents. However, the mechanism can only be speculated at this point.

Based on the high selectivity of clebopride for the dopamine D2-like receptor sub-type (Leysen et al., 1993), the lack of interaction of clebopride with FG 7142 is likely to reflect that DA D2 receptor activation is not necessary for stimulation of cortical ACh efflux by a BZR inverse agonist. This leads to the rather surprising conclusion that haloperidol blocks this stimulated ACh efflux either through DA Dl receptors, or through a non-dopaminergic 80 mechanism. The first possibility is not supported by the present finding that an antagonist with a greater selectivity for Dl-like receptors produced a less complete blockade of of FG 7142-stimulated cortical ACh efflux than did haloperidol. However, subtle, yet synergistic, interactions between Dl and D2 receptors that were not revealed with the selective blockade of one sub-type (i.e. with clebopride or SCH 23390) might be revealed by a less selective dopamine receptor antagonist such as haloperidol (Leysen et al., 1993). Examination of this putative D1-D2 synergism will require use of combinations of selective antagonists. Alternatively, haloperidol suppressed stimulated ACh efflux by acting at sigma receptors, a class of receptors to which haloperidol binds more potently than to dopamine receptors (Walker et al., 1990; Leysen et al., 1993).

Sigma receptors appear to have significant interactions with amino acid transmitters (Walker et al., 1990). Moreover, haloperidol reduces the release of glutamate into the striatum via sigma receptors (Ellis & Davies, 1994), an effect that would likely extend to the nACC, given the higher density of sigma receptors (Wallace & Booze, 1995). This presumed reduction in glutamatergic stimulation of nACC GABAergic neurons would be expected to lead to a decrease in GABA in the nACC, an effect previously observed in vitro

(Belleroche & Gardiner, 1983), and apparent at the higher dose of haloperidol 81

in the present study (see Table 4). A haloperidol-mediated reduction in

glutamate release could have diverse effects on the circuitry of the nACC, given the multiple sites of glutamatergic inputs (Meredith et al., 1993). That the net outcome would be a reduction in the activity of basal forebrain cholinergic neurons is supported by the presently-observed effects of intra-accumbal haloperidol on FG 7142-stimulated cortical ACh efflux (see Figure 15 and

Table 5), and by the fact that cortical ACh efflux stimulated by sigma receptor agonists is reduced by haloperidol (Matsuno et al., 1993).

Taken together, these results, a portion of which is still preliminary, indicate that attenuation of FG 7142-stimulated cortical ACh efflux mediated by the blockade of dopamine Dl receptors is mediated neither in the nACC nor the cortex. The antagonistic actions of haloperidol, do, at least in part, take place in the nACC; however, they do not solely reflect D2 receptor blockade.

Lastly, the preliminary analysis of GABA efflux in the nACC indicates that under some conditions (i.e. following administration of a BZR inverse agonist), GABA efflux in the nACC and cortical ACh efflux are elevated in parallel. This implies either that GABA efflux in the nACC does not reflect output of the GABA efferents to the basal forebrain, or that GABA efflux and cortical ACh efflux in the basal forebrain are not necessarily reciprocally modulated by BZR ligands. Given recent evidence that inhibitory synaptic responses of striatal cells are mediately almost exclusively by GABAergic interneurons and not collaterals of the GABAergic projection neurons (Jaeger &

Wilson, 1994), the former is likely to be the case. CHAPTER VII

GENERAL DISCUSSION

The data presented establish the following: 1) in vivo microdialysis can be employed in a repeated testing paradigm to examine changes in neurotransmitter efflux, 2) benzodiazepine receptor ligands that negatively modulate GABAergic transmission at the GABAa receptor increases cortical

ACh release, 3) depending on its efficacy at the GABAa/BZ receptor, systemic administration of a BZR inverse agonist may selectively enhance stimulated (i.e. during behavioral activation) cortical ACh release , 4) BZR ligands that positively modulate GABA selectively suppress stimulated ACh release, 5) the basal forebrain is most likely the site of action for the bidirectional modulation of stimulated cortical ACh efflux by BZR ligands, 6) stimulation of cortical

ACh efflux by BZR inverse agonists is partially dependent on dopamine Dl receptor stimulation. The involvement of D2 receptors may be more complex.

Systemic administration of a D2 antagonist indicates that D2 receptors are not

83 necessary, but opposing interactions between structures receiving mesolimbic and mesocortical inputs from the ventral tegmental area may not allow determination of the site at which D2 stimulation is necessary for the increase in

ACh efflux by BZR ligand. 7) The neuroleptic haloperidol can block the ability of a GABAa/BZR negative modulator to increase cortical ACh release. The fact that these results are inconsistent with the systemic effect of a D2 antagonist indicates that the actions of the neuroleptic on cortical ACh efflux are not mediated solely via D2 receptors. The non-D2 profile of haloperidol is also reflected by its local effects on GABA efflux in the nACC. These conclusions are discussed below.

A. In vivo microdialvsis; general limitations and

application in repeated-testing, within-subiects experimental designs

Dialysis probes are designed to act as perfusion and ultrafiltration vessels allowing both the delivery of drugs and the monitoring of extracellular neurotransmitter levels. The structure of the probe determines whether a constant flow rate and pressure gradient are maintained between the inlet and outlet cannulae. These conditions, in turn, maintain a constantly low level of 85

analyte in the perfusion fluid. In the present studies, the use of a very low dead

volume concentric probe with a perfusion rate ranging from 1.3 to 2.0 /il/min

were most likely near optimal for maintenance of near-zero levels of analyte in

the dialysis membrane loop (see Johnson & Justice, 1983; Wages et al., 1986).

Besides probe design, major factors affecting the validity of the dialysis

procedure for measuring neurotransmitter release include the perfusion fluid and

impact of the dialysis probe on the surrounding tissue. In order to maintain the buffering capacity of the extracellular fluid (ECF) and maintain physiological

ionic gradients across cell membranes in the brain microenvironment, the ionic composition of the perfusion medium should be identical to that of ECF

(Benvenista & Huttemeier, 1990). Of particular importance are potassium ion levels and the ratio between calcium and magnesium ([Ca2+] and [Mg2+]) ion concentrations (Osborne et al., 1991; Timmerman & Westerink, 1991), since increased levels of the former depolarizes nerve terminals and even small changes in the ratio between the divalent ions affects calcium-dependent synaptic neurotransmitter release (Kandel, et al., 1990; for review, see Bernath,

1991). Further, addition of glucose to the perfusion medium, as was done in the present studies, is required in order to prevent progressive depletion of the nerve terminals of their primary energy source (see Hansen, 1985). Furthermore, ACh and GABA recovered by the present methods were demonstrated to be sensitive to blockade of voltage-gated sodium channels and, thus, derived from transmitter released from axonal depolarization (see

Benvenista & Huttemeier, 1990). Finally, in the case of both accumbens

GABA and cortical ACh in control rats, it was generally-observed that the slope of the efflux pattern, as a function of hours of perfusion, was near zero. Thus, there is no indication that the perfusion media used in dialysis of ACh and

GABA in the present experiments altered the interstitial fluid as to significantly disrupt neuronal release of these transmitters.

Two further considerations specific for recovery of ACh are, however, effects of 1) choline depletion and 2) tonic perfusion of an acetylcholinesterase

(AChE) inhibitor on the release of ACh from surrounding nerve terminals.

Neuronal synthesis of ACh requires the high-affinity uptake of choline (Cooper et al., 1991). Extracellular levels of choline greatly exceed the concentration required to saturate the uptake sites, and interstitial choline is constantly replenished by diffusion across blood vessels (Brunello et al., 1982; Jope,

1982). Thus, ACh release is rarely affected by changes in interstitial choline levels (i.e. Brunello et al., 1982).

In in vivo dialysis of cortical ACh, it is typically necessary to add an 87

AChE inhibitor, such as neostigmine or physostigmine, to the aCSF, since

levels of ACh recovered without an AChE inhibitor are near the limit of

detection of most ACh analytical methods (5-10 nM; Messamore et al., 1993).

Tonic AChE inhibition potentially leads to accumulation of non-hydrolyzed

ACh in the synapse which would, in turn, tonically modulate release via

activation of muscarinic and nicotinic autoreceptors (Raiteri et al., 1984).

Supraphysiological levels of ACh primarily inhibit ACh release via stimulation

of M2 receptors on cholinergic terminals, as deduced from the similar actions of

oxotremorine (Szerb & Somogyi, 1973). While there is evidence that AChE

inhibition changes the ability of muscarinic antagonists and agonists to increase

and decrease, respectively, ACh release (de Boer et al., 1990; Messamore et al., 1993), there is also evidence that tonic autoreceptor stimulation does not

interact with modulation under other conditions. For example, across multiple experiments using a 100-fold range of neostigmine concentrations (0.5 to

5.0 nM), I have observed similar magnitudes in increased ACh levels during behavioral activation. The ability of BZR ligands to bidirectionally modulate stimulated cortical ACh efflux has also been replicated at different levels of

AChE inhibition (Moore et al., 1992b,c; 1993a,b; unpublished observations).

Furthermore, even with relatively high levels of esterase inhibitors in the aCSF 88

(i.e. 75 - 100 fiM physostigmine), neo- and allocortical ACh efflux shows

expected variation during diurnal cycles (Kametani & Kawamura, 1991; Mizuno et al., 1994) and is increased with manipulations that stimulate activity in the basal forebrain (Bertorelli et al., 1991; Kurosawa et al. 1989; Rasmusson et al.,

1992). Morever, serotonin-induced decreases in ACh efflux can also be demonstrated under conditions of presumed suppressed release by AChE inhibition (Rada et al., 1993). Thus, it is likely that use of an AChE inhibitor tonically suppresses ACh release via activation of the muscarinic autoreceptor.

However, under these conditions, behaviorally- and pharmacologically-relevant changes in ACh release can be accurately monitored.

The final methodological issue raised by the present experiments deals with the effects of repeated insertion and perfusion of the dialysis probe. It is possible that this procedure could lead to tissue alterations that significantly affect the accuracy of our measurement of changes in neuronally-derived ACh and GABA. The multiple findings discussed in Chapter III indicate, however, that cortical ACh is TTX-sensitive and equally sensitive to modulation by BZR ligands and other manipulations for up to four sessions in which the probe is inserted and perfused for up to 7 hours. Other investigators have also effectively employed repeated insertion and perfusion of a concentric probe. 89

Keller et al (1992) found that this method, despite being associated with a loss of responsivity to cocaine, revealed sensitization of striatal DA release in cocaine-treated versus saline-treated, multiply-dialyzed rats. Their experiment also dissociated correlations between basal and stimulated efflux of DA and its metabolites as a function of cocaine or repeated dialysis sessions.

While it appears that microdialysis designs employing reinsertion of concentric probes are valid for measuring changes in cortical and striatal ACh release. It should be noted that this conclusion cannot be extended to designs that employ other, larger probe designs (see Westerink et al., 1988), nor to studies employing continuous or intermittent perfusion of a permanently- implanted membrane. Permanent membrane preparations result in a substantial glial accumulation around the membrane (see Hamburger, 1983; Shuaib et al.,

1990), resulting in debilitating losses of recovery of both basal and dynamic efflux (Hamburger et al., 1983; Robinson & Camp, 1991; Westerink et al.,

1987). By contrast, reinsertion of the membrane results in moderate, poorly- organized gliosis in the perfusion area that appears to have less impact on recovery (see Georgieva et al., 1993; Moore et al., 1992c). Along these lines

Fumero et al. (1994) recently found that repeated microdialysis sessions with removable concentric probes was a viable method for examining relative 90 changes in dopamine efflux during sexual behavior as a function of experience.

By contrast, permanently-implanted dialysis membranes were found not be acceptable in this paradigm, due to loss of patency and neurological impairments over chronic implantation (see Osborne, 1995).

Repeated-dialysis methodological designs may not be useful for all pharmacological manipulations with a neurotransmitter system. For example,

Robinson & Camp (1991), who conducted a comprehensive study of the effects of repeated and continuous dialysis on striatal dopamine efflux, found that although the abilities of cocaine and TTX to increase and decrease DA efflux, respectively, were unaltered, the stimulatory effect of amphetamine was significantly reduced by continuous or repeated dialysis. This paradox illustrates that repeated-microdialysis designs may need to be validated as a function of the independent variable.

Taking into account the factors that minimize the effects of repeated dialysis on surrounding tissue, there is strong evidence that changes in neurotransmitter release can be accurately measured with dialysis using a multiple-session experimental design. Added to effective use of this method to study neurochemical correlates of sensitization (Keller et al., 1992) and sexual experience (Fumero et al., 1994), is our most recent use of a repeated-dialysis, 91 within-subjects to study cortical ACh efflux in animals performing in operant tasks (see below). Thus, with appropriate control groups or counterbalancing of experimental conditions, such designs may be powerful for examining multiple or cumulative pharmacological or behavioral determinants of neurotransmitter release.

B. Modulation of cortical ACh release bv BZR ligands: dependence on the

activity level of basal forebrain cholinergic cells

The results in Chapter IV support that BZR ligands bidirectionally modulate cortical ACh release via their actions in the basal forebrain.

Importantly, the pharmacological actions of BZR ligands in the basal forebrain were significantly more effective in modulating the stimulation of cortical ACh efflux, as opposed to changing efflux from resting levels. This apparent selectivity was also observed with systemically-administered BZR ligands.

These findings are summarized in Figure 17. The BZR full agonist, CDP, at a systemic dose half of which was shown to have no effect on resting efflux

(10 mg/kg; Moore et al., 1992c), blocked the stimulation of efflux by the 92 darkness/cereal stimulus. The agonist had equally selective effects on stimulated efflux when administered into the basal forebrain. On the other hand effects of systemically-administered BZR inverse agonists appeared to depend on the intrinsic activity of the agonist at the GABAa/BZR, in that only the weak inverse agonist ZK 93 426 was selective in significanly increasing stimulated ACh efflux. However, the full inverse agonist (P-CCM) infused directly into the basal forebrain enhanced stimulated efflux, like the systemic effects of the weaker BZR inverse agonist, had little effect on resting efflux.

Moreover, a higher dose (9.0 /xg/hemisphere) of the BZR full inverse agonist in the basal forebrain was no more effective, but was equally selective, in enhancing stimulated release (data not shown). Thus, the increased efficacy of

BZR ligands in modulating activated cortical ACh release may be a rather specific characteristic of their actions on their target neurons in the basal forebrain.

Several studies have indicated that GABAergic modulation of cortical

ACh release is not tonic, but may correlate with the level of activation of SI cells. For example, contrary to what would be predicted from the GABAa agonist effects (see above), Blaker (1985), found that administration of the

GABAa antagonist, bicuculline, into the SI did not increase cortical ACh levels, 93 indicating that GABAergic modulation of cortical ACh transmission was not tonic. However the regulatory mechanism for this possibly phasic interaction has been largely unexamined. Moore et al. (1993a) recently found that chlordiazepoxide (CDP), a benzodiazepine positive modulator of GABAergic transmission, blocked the increase in cortical ACh release produced by presentation of an appetitive stimulus; however, CDP had no effect on cortical

ACh efflux in resting animals (Moore et al., 1992c). Moreover, the action of

CDP on cortical ACh efflux was not related to locomotor activity, but appeared to depend on the exposure of the subjects to a behaviorally-significant stimulus

(Moore et al., 1993a). Thus, it is likely that GABA normally modulates cortical ACh efflux only when the cholinergic cells of the SI are activated by a psychologically-significant stimulus.

Electrophysiological support of this hypothesis has been provided by

Pirch et al. (1986, 1991) who showed that classically-conditioned bidirectional changes in cortical neuronal activity were dependent on cortical ACh, and were blocked by administration of GABA into the SI/NBM. In contrast, spontaneous cortical activity was not appreciably affected by intra-SI GABA administration

(Pirch et al., 1991).

BZR ligands may be especially effective pharmacological tools to study 94

the activity-dependent effects of changes in GABA transmission for several

reasons. First, the actions of BZR ligands on the GABAa receptor complex

result in a change in the frequency of the opening of the anion channel gated by

the receptor, without changing the permeability or other conductance properties

of the channel (Haefely, 1989; Kaila, 1994). They may have this effect, in

part, by altering the affinity of the receptor for GABA (Haefely, 1989). In this

way, their effects can be thought of as being analogous to changes in synaptic

levels of GABA or post-synaptic responsivity to GABA. Thus, their actions

can be interpreted in terms of the role of endogenous GABA and GABAa

receptors. Agonists of the GABA site, and even other allosteric modulators,

such as pentobarbital, impose GABA-independent changes in the conductance properties of the receptor channel (Kaila, 1994). This is consistent with several examples in which the behavioral (Dudchenko & Sarter, 1992), neurochemical

(Tanganelli et al., 1985) and electrophysiological effects (Ross et al., 1982;

Waszczak et al., 1980) of BZR agonists do not resemble those of direct agonists. Moreover, when compared, the BZR agonists are more predictive of the effects of GABA (see Tanganelli et al., 1985). 95

C. Functional implications of the modulation of simulated cortical ACh

release bv BZR ligands

Based on behavioral data, it has been postulated that attentional performance depends on the activation of cortical cholinergic afferents as evidenced by the selective impairment of attentional performance by disruption of cortical cholinergic transmission with muscarinic blockers or basal forebrain lesions (Sarter & Bruno, 1994). There is also evidence that performance which depends on activated cortical ACh transmission (i.e. visual attention, see above) is also sensitive to BZR agonists (i.e. McGaughy & Sarter, 1995; McGaughy, et al., 1994). In contrast, cognitive performance involving "habit-like" response rules does not appear to depend on cortical ACh transmission (see Sarter,

1990), and is resistant to the effects of BZR ligands (i.e. Muir et al., 1992b,

Moore et al., 1992a, Dudchenko & Sarter, 1992). This evidence supports the idea that certain components of attentional processing require the co-activation of cholinergic and excitatory sensory inputs to the cortex (see above), and that

BZR ligands, acting at GABAa receptors in the SI/NBM, affect attentional performance via their blockade or enhancement of the activation of cortical cholinergic afferents. This is consistent with the action of GABA, which is to 96

stabilize the neuron at its resting potential (Nicoll et al., 1990), resulting in the greatest effect when the target neuron receives simultaneous excitatory input.

The behavioral correlates of basal forebrain cell activity and the electrophysiological actions of ACh in the cortex, also support the idea that cortical cholinergic transmission mediates processing of biologically-significant stimuli. During performance of cognitive tasks, corticipetal basal forebrain cells respond more robustly during presentation of conditioned stimuli, relative to during retrieval of reward (i.e. unconditioned stimuli) or during inter-trial periods (i.e. Wilson, 1991; Richardson & DeLong, 1990). Application of ACh into the cortex or stimulation of the SI/NBM reliably and selectively augments the responsivity of cortical projection cells to excitatory (i.e. depolarizing) inputs (i.e. Metherate & Ashe, 1993; Tremblay et al, 1990a,b; McCormick,

1990). ACh applied to the cortex also shifts receptive fields such that the cells are more responsive to conditioned stimuli (i.e. Ashe & Weinberger, 1991;

Hars et al., 1993). The most direct evidence that GABAergic transmission in the SI/NBM affects cortical neuronal correlates of attentional processing is provided by Pirch et al. (1991) who found that infusion of GABA into the NBM blocked the increase in firing rate of cortical cells induced by a conditioned stimulus. This electrophysiological evidence, coupled with the results from the 97 well-designed lesion studies cited above, indicates strongly that cholinergic input to the cortex mediates attention.

These and other findings support the rationale for developing BZR inverse agonists as therapeutic agents in attenuating age-related attentional impairments (Sarter et al., 1990). The possibility that BZR inverse agonists act in the basal forebrain to selectively enhance ACh release from activated cholinergic projection cells has important implications for this therapeutic strategy. Specifically, it may be possible to develop drugs which selectively enhance cortical ACh release during the activation of the cholinergic inputs by behaviorally-relevant stimuli (see Sarter & Bruno, 1994).

Given these implications, it is important to identify the stimulus properties that modulate cortical ACh release, and to determine whether activation of cortical ACh release is modifiable by BZR ligands in a way that affects stimulus processing. Towards this aim, preliminary steps have been taken to inter-correlate attentional demands, behavioral performance and cortical ACh release. In a recent collaboration among several investigators in the Psychobiology Area of the Psychology Department at The Ohio State

University, it was observed that changes in cortical ACh efflux can be reliably measured in rats performing an operant task designed to measure visual 98

attentional abilities (Sarter et al., 1995). The experimental design allowed

changes in efflux to be temporally correlated with performance during periods

of high and low attentional demands. Preliminary results indicated that ACh is

released into the medial prefrontal cortex in response to the transfer of the

animal to the operant box and at the onset of the task. Introduction of a visual

distractor was used to increase the level of attention required to maintain

performance. Regardless of performance, ACh was higher during this period of

increased attentional demands, relative to the subsequent task period. Periods

of increased ACh release corresponded to periods of increased population spike

activity in the prefrontal cortex. No firm conclusions can be drawn from these

results. However, they are consistent with the idea that the basal forebrain

cholinergic system is activated in response to stimuli as temporally non-specific

as a change in context. This is consistent with the concept that ACh acts in the cortex to "release" cortical cells from their current receptive fields (or, in the case of association cortices, from their current restraints on activity) and, thus, allows biologically significant stimuli to reshape the responsivity pattern of the cortical cell (i.e. Ashe & Weinberger, 1991; Metherate & Weinberger, 1989).

Future experiments will continue to temporally correlate cortical ACh efflux with cognitive performance. The ultimate goal will be to determine the 99

behavioral and pharmacological conditions in which pharmacological

modulation of cortical ACh release affects attentional processes.

D. Actions of dopamine receptors and neuroleptics in the modulation of

stimulated cortical ACh efflux

The findings reported in Chapter V indicate that attenuation of FG 7142- stimulated cortical ACh efflux mediated by the blockade of dopamine D1 receptors is mediated neither in the nACC nor the cortex. Moreover, while the antagonistic actions of haloperidol are mediated in the nACC, they are likely to involve subtle interactions between dopamine receptors, or actions at non- dopaminergic receptors. At present, these results do not allow strong conclusions to be drawn about the modulation of GABAergic afferents to the basal forebrain by dopamine in the nACC. Given the highly reciprocal interconnections between the ventral tegmental area, nACC, basal forebrain, limbic cortices, and amygdala, it is clear that GAB A/D A interactions affecting cortical cholinergic afferents may occur at a number of sites. While various components of this circuit could potentially mediate dual regulation of cortical

ACh release by dopamine and GABAa receptors (see Chapter V, Discussion) the present results do not provide the straightforward "neurochemical confirmation1' of an interaction between the nACC and basal forebrain. Future studies will continue to compare the effects of selective dopamine receptor antagonists within this circuit on stimulated cortical ACh efflux. Given the related behavioral or psychological correlates of stimulated mesoaccumbal DA and cortical ACh efflux (see above), it will be important to study the effects of

DA antagonists on behaviorally-activated cortical ACh release, as well as ACh release disinhibited by GABAa/BZR inverse agonists. Table 1: Drugs and Doses Used in Chapter V

The drugs tested Experiments 1 and 2 of Chapter V are listed in the left most column. Their primary pharmacological actions are given in the first row of the middle column, other possible actions indented in parentheses. The doses used for systemic admnistration are listed in the right-most column. FG 7142 was administered systemically in both experiments. The doses used for intracranial perfusions of the DA antagonists are given in Table 3.

PrttS Primary pharmacological action Systemic doses (secondary actions)

FG 7142 BZR partial inverse agonist 8.0 mg/kg

SCH 23390 DA D1 antagonist 0.1, 0.3 mg/kg (5HT[ agonist) haloperidol DA D2 antagonist 0.15, 0.9 mg/kg (sigma receptor antagonist) (DA D1 antagonist) clebopride DA D2 antagonist 10.0 mg/kg

101 Table-2._ Drue Treatment Combinations for Chapter V. Experiment 1

Treatments given on each of the four test sessions in Chapter V, Experiment 1. Each DA antagonist was tested in a separate group of animals. Following baseline collections, either SCH 23390, haloperidol, or clebopride or the antagonist’s vehicle was administered. After 1 or 2 subsequent collection periods (see text), FG 7142 was injected. Dialysates were then collected for 3 more collection periods. Treatments were counterbalanced across session.

Vehicle for DA antagonist (saline) + FG 7142

DA antagonist (low dose) + FG 7142

DA antagonist (high dose) + FG 7142

DA antagonist (high dose) + Vehicle for FG 7142 (Cremephor EL)

102 Iabte.3. Drue Treatment Combinations for Chapter V. Experiment 2

Treatment given on each of the four test sessions in Chapter V, Experiment 2. Each DA antagonist was tested in a separate group of animals. Following baseline collections, either SCH 23390 or haloperidol was added to the aCSF. After the DA antagonist had been co-perfused for 30 min, FG 7142 was administered (8.0 mg/kg, i.p.). The DA antagonist continued to be perfused until the end of the experiment. Treatments were counterbalanced across session, except for the intra-cortical antagonist session, which was always the fourth session.

Systemic FG 7142 preceeded by 30 min of perfusion of

- standard aCSF in PFC and nACC

- aCSF in PFC, DA antagonist in nACC (10.0 /xM)

- aCSF in PFC, DA antagonist in nACC (100.0 (M )

- DA antagonist in PFC (100.0 pM) + std. aCSF in nACC

103 Table 4. Changes in GABA efflux in the nucleus accumbens during local co-perfusion of DA antagonists.

Shown are the average changes in efflux during perfusion of the regular aCSF or CSF with 100.0 /zM of either the D1 antagonist SCH 23390 or the neuroleptic haloperidol. Effects on ’'basal" efflux were assessed by averaging the change in efflux (relative to median baseline efflux) during co-perfusion of the antagonist, prior to the administration of FG 7142. Effects on "post-FG 7142" efflux were assessed by averaging the changes in efflux during the first 30 min following systemic injection of FG 7142 (8.0 mg/kg).

Co-perfusion Change in Change in condition basal efflux post-FG 7142 efflux (percent change from median baseline efflux mean(S.E.M.)

regular aCSF +66 (67) + 423 (119)

SCH 23390 (100.0 /zM) - 22 (23) + 171 (90)

haloperidol (100.0 /iM) - 61 (24) + 72 (77)

104 Table 5. Summary of the effects of dopaminereceptor antagonists on frontal cortical ACh release and accumbal GABA release

Shown are the directions of effects of the Dl-selective antagonist, SCH 23390, the neuroleptic haloperidol, and the D2-selective antagonist, clebopride, on prefrontal cortical ACh efflux and accumbal GABA efflux. Effects on "basal" efflux are represented by changes in efflux 15 min after admnistration of the antagonist. Effects on "post-FG 7142" efflux are represented by changes 15-30 min after systemic administration of FG 7142.

105 TableS

Systemic Nucleus Accumbens Prerontal Cortex

basal post-FG 7142 basal post-FG 7142 basal post FG 7142

Dl-selective antagonist SCH 23390

Cortical ACh efflux no change t no change \ ^(additive)

Accumbens GABA efflux no change no change

Haloperidol

Cortical ACh efflux no change no change no change

Accumbens GABA efflux

D2-selective antagonist Clebopride H O C T i Cortical ACh efflux no change no change Figure 1. Schematic time course of a microdilavsis session

Schematically illustrated is the order of events in a standard microdialysis session. In each session, the probe was inserted and perfused with an artificial CSF. After a 3-hour washout/equilibration period (see text), baseline samples were collected every 15 min for at least one hour. Then, one or several manipulations was made in the next 1 to 3 collection periods, including insertion of intracranial drug infusion needles, onset of intracranial drug infusion, systemic administration of drug, or presentation of the behaviorally-activating stimulus (darkness/fruit cereal). One manipulation per collection period was made, within the first minute of that period, and dialysate was collected for up to 13 min thereafter. Dialysates were then collected for 45 - 120 min after the last manipulation.

107 I

EXPERIMENTAL DESIGN

VEH VEH VEH DRUG

cannula surgery DRUG VEH VEH DRUG

habituation r S e s s 1 S ess 2 S e ss 3 S e ss 4 4 histology day 0 day 4 day 6 day 8 day 10

Fiewe-I 108 Figure 2. Experimental design for assessing repeated microdialvsis testing

Schematically illustrated is the experimental design used to assess the effect of four microdialysis sessions within an animal on basal and FG 7142- stimulated cortical ACh efflux. Animals were implanted with guide cannulae as described in Chapter 2, then were allowed 3 days to recover and be re- habituated to the testing chamber. Two groups of animals were then dialyzed for 4 sessions, each separated by a day. One group was administered vehicle (Cremephor EL, see text) on Sessions 1, 2 and 3, then administered FG 7142 on Session 4. The second group was administered FG 7142 on Session 1 and Session 4 and vehicle on Sessions 2 and 3. Effects of repeated dialysis testing on stimulated cortical ACh efflux were determined by comparing efflux following injection of FG 7142 on Session 4 in Group 1 with that on Session 1 in Group 2.

109 TEST SESSION

reed'«s stimulus in serted probe inserted baseline \ drug 11 hr discard period

Figure 2 Figure-3. Effect of FG 7142 on cortical ACh efflux.

Increases (mean ± S.E.M.) in cortical ACh efflux as a function of time following injection (i.p.) of FG 7142 or its vehicle (CEL). ACh efflux is expressed as percent change from the median baseline efflux (i.e. pre-drug; 0.15 ± 0.04 pmol/min). There was a significant increase in the duration of the increase as a funciton of dose.

I l l ACh EFFLUX (% change from b aselin e ) 200 0 0 3 0 0 4 0 0 5 0 0 1 0 0 6 SN 15 BSLN IE mi ATR INJECTION AFTER ) in (m TIME 30 560 45 Figure 3 Figure Vehicle G74 ( kg) g /k g m 6 (1 FG7142 FG 7142 ( 8 m g /k g ) ) g /k g m 8 ) g ( /k g m 4 ( 7142 FG 7142 FG 75

0 0 120 105 90 112 Figure 4. Effect of FG 7142 on orofacial tremor and locomotor activity.

Top panel. Effect of FG 7142 (4.0, 8.0, & 16.0 mg/kg, i.p.) on the probability for a bout of orofacial tremor in each 15-min collection period. Orofacial tremor was recorded in 2 2-min observation periods within each 15 min collection period. Probability represents percentage of animals displaying a bout. Bottom panel. Effect of FG 7142 on locomotor activity. Activity was rated with the scale described in Chapter n. Following the first collection period following injection, activity scores reflected mostly forepaw and head movements.

113 114

4.0 m g/kg 3 8 .0 m g /k g o 03 0.9 1 6 .0 m g /k g O' o 0.8 2 UJ 0.7 t—0£ 0.6 0.5 gO cc 0.4 o u. 0.5 o £ 0.2 0.1 0.0 a:o q . 0 1530 45 60 75 105 12090

TIME (m in ) FOLLOWING INJECTION

4.0

3.5 ce o 3.0 2.5 £u 2.0 < cc o 5 2 1.0 o a 0.5

0.0

0 1530 4560 75 90 105 120

TIME (m in) FOLLOWING INJECTION

FigureJ Figure 5. Effect of repeated dialysis testing on basal and FG 7142- stimulated cortical ACh efflux

Top panel. Baseline cortical ACh efflux (mean ± S.E.M.) on Session 1 and Session 4. Efflux was corrected for in vitro recovery and expressed as a rate for each collection period; the median of the four pre-injection baseline collections was then averaged over subjects. Repeated dialysis was reliably associated with a small to moderate decline in basal ACh efflux. Bottom panel. Increases (mean ± S.E.M.) in cortical ACh efflux (percent change from median baseline) as a function of time after injection (i.p.) of FG 7142 (8.0 mg/kg) during perfusion of standard aCSF (closed symbols) or with co- perftision of TTX (1.0 /iM). The Session 1 group (circles) had never been previously dialyzed; the Session 4 group had been dialyzed on 3 previous sessions, each separated by a rest day. Repeated dialysis testing did not affect the ability of cortical ACh efflux to be stimulated by FG 7142, nor did it affect the ability of basal and stimulated efflux to be suppressed by TTX.

115 ACh EFFLUX (7. change from baseline) ACh EFFLUX (pm ol/m in 0 6 - 0 3 - 0 9 - . 5 0.0 0.10 15 .1 0 0.20 0.25 150 180 210 40 2 120 90 SN 5 0 0 5 0 0 120 105 90 75 60 5 4 30 15 BSLN IE mi) FE F 12 INJECTION 7142 FG AFTER in) (m TIME ESO 1 SESSION Figure 5 Figure s 4 —TTX 4 ess S TTX — 1 ess S s 4 4 ess S s 1 ess S ESO 4 SESSION 116 Figure 6. Stimulation of cortical ACh efflux bv behavioral activation and systemic administration of benzodiazepine receptor inverse agonists

A summary of results from several experiments testing the effects of behavioral activation and BZR inverse agonists on cortical ACh efflux. The maximal effects of systemically-administered vehicle or the BZR weak or partial inverse agonists, ZK 93 426 (5.0 mg/kg) or FG 7142 (8.0 mg/kg), respectively, are depicted with analogous responses to the "darkness/fruit cereal" stimulus following vehicle or ZK 93 426. The "darkness/cereal" stimulus presentation reliably produces approximately a 100% increase in cortical ACh efflux. Note that the weak inverse agonist is minimally effective on non-stimulated efflux, but, at the same dose, significantly enhances behaviorally-activated efflux. FG 7142, on the other hand, stimulates basal efflux (i.e. efflux in resting, non trained animals).

117 ACh EFFLUX (X change from baseline) 0 5 3

9 m m m m

811 Figure 7. Cortical dialysis probe and basal forebrain drug cannulae placements

Placements of the microdialysis probes in the cortex (right panel) and the corresponding ipsilateral drug infusion cannula in the basal forebrain (left panel). Each set of numbers represents placements for an individual subject. The combined length of the bar and symbols in the cortex represent the 2 mm membrane of the dialysis probe (0.5 mm o.d.) which was inserted through a guide cannula (not shown) at a 50° angle. Numeric symbols in the basal forebrain are placed at the site of the tip of the ipsilateral drug infusion needle (vertical; 30 ga).

119 120

Bregma 2.20 mm Bregma —1.80 mm

Bregma 2.70 mm

Bregma -1 ,4 0 mm

Bregma 3.20 mm

Bregma — 1 JO mm

Bregma 4.20 mm

Bregma 0.20 mm

Figurp2 Figure 8. Effects of BZR ligands infused into the basal forebrain on basal aud stimulated cortical ACh efflux

Top panel. Changes (mean ± S.E.M., relative to median baseline efflux) in cortical acetylcholine (ACh) efflux over consecutive collection periods. Following baseline collections, drug infusion needles were inserted into the basal forebrain guide cannulae and dialysate was collected for 15 min. During the middle 5 min of the collection period subsequent to needle insertion, an osmotically- ,pH-controlled vehicle (see text), p-CCM (3 /xg/hemisphere) or CDP (40 /xg/hemisphere) was infused into the basal forebrain. In the collection period that followed the drug infusion period, the darkness/cereal stimulus was presented. Following stimulus presentation, dialysates were collected for 45 additional min. Bottom panel Summary of changes in cortical ACh efflux relative to baseline during infusion of BZR ligands into the basal forebrain (left) and following the behaviorally-activating stimulus (right). The drug infusion period consisted of the 15 min collection prior to the presentation of darkness/fruit cereal. Neither CDP nor P-CCM had a significant effect on cortical ACh efflux during and 10 min after drug infusion. "Darkness Onset" efflux was measured during a 15-min collection following the darkness onset/cereal presentation. The stimulus-induced increase in cortical ACh efflux (vehicle - solid bar) was significantly enhanced by P-CCM and fully blocked by CDP.

121 .o M c a o a>

ACh EFFLUX (7. changet from baseline) ACh EFFLUX (7. change> from 140 100 160 120 20 60 0 8 40 0 160 40 0 8 - - ■ - - DRUGINFUSION bsln Vehicle CDP 0-CCM (3.0 ug/hemi) (3.0 0-CCM inserted needles Figure 8 Figure COLLECTION INTERVAL (40.0 ug/hemi) (40.0 infused drug Bt-C 3. ug/ i) m e /h g u .0 (3 Beta-CCM ■ Vehicle • CDP * Time (min)darkness/'Fruitloop'after DARKNESSONSET 153 30-45 5-30 1 5 t - 0 40. ug/ i) m e /h g u .0 0 (4 i 122 Figure 9. Effects of systemic SCH 23390 on FG 7142-stimulated cortical ACh efflux

Illustrated is cortical ACh efflux (mean ± S.E.M., expressed as percent change from median baseline) during the final baseline collection, and following administration of the Dl-selective antagonist SCH 23390 and the BZR partial inverse agonist FG 7142 (8.0 mg/kg). SCH 23390 (0.1, 0.3 mg/kg) or its vehicle was administered (solid arrowhead) 15 min prior to FG 7142 (or its vehicle). The D1 antagonist had no effects on resting cortical ACh efflux (squares); however pre-administration of SCH 23390 (triangles) attenuated, but did not fully block, stimulation of cortical ACh efflux by FG 7142.

123 ACh EFFLUX (X change from baseline) 200 250 0 0 1 150 300 50 IE ( n FOLLOWING FG7142 INJECTION OF TIME in) (m C 39 (. g/kg) m (0.3 SCH23390 G74 (. g/kg) m (8.0 FG7142 FG 7142 + SCH 23390 (0.3 m g/kg) g/kg) m (0.3 SCH + 23390 FG 7142 g/kg) m <0.1 SCH 23390 + FG7142 15 -1 SCH 23390 inject SCH 23390 Figure 9 Figure 0 ______

530 15 124 Figure 10. Effects of systemic haloperidol on FG 7142-stimulated cortical ACh efflux

Illustrated is cortical ACh efflux (mean ± S.E.M., expressed as percent change from median baseline) during the final baseline collection, and following administration of the neuroleptic and dopamine receptor antagonist haloperidol and the BZR partial inverse agonist FG 7142 (8.0 mg/kg). Haloperidol (0.15, 0.9 mg/kg) or its vehicle was administered (solid arrowhead) 30 min prior to FG 7142 (or its vehicle). Haloperidol dose-dependently antagonized stimulation of cortical ACh efflux by FG 7142.

125 ACh EFFLUX (% change from b aselin e) 200 0 5 2 0 0 1 0 5 1 50 TIME (m in) FOLLOWING INJECTION OF FG 71 42 71 FG OF INJECTION FOLLOWING in) TIME (m haloperidol AOEIO (.0mq/ ) q /k q m ) (0.90 g /k g HALOPERIDOLm (0.90 HAL + ) g 7142 FG /k g m 5 (0.1 HAL + 7142 FG G7 2 80mg/ ) g /k g m (8.0 FG7142 15 -1 iwe 10 Eigwre 0

530 15 126 Figure 11. Effects of systemic clebopride on FG 7,142-stimulated cortical ACh efflux

Illustrated is cortical ACh efflux (mean ± S.E.M., expressed as percent change from median baseline) during the final baseline collection, and following administration of the D2-selective antagonist clebopride and the BZR partial inverse agonist FG 7142 (8.0 mg/kg). Clebopride (10.0 mg/kg) or its vehicle was administered (solid arrowhead) 30 min prior to FG 7142 (or its vehicle). Unlike haloperidol, this high dose of the D2-selective antagonist had no effects on resting cortical ACh efflux (squares), nor did it affect stimulation of cortical ACh efflux by FG 7142.

127 ACh EFFLUX (% change from b aselin e) 0 5 2 0 0 3 200 50 1 0 0 1 50 CLE80 inject TIME (m in) FOLLOWING INJECTION OF FG 71 42 71 FG OF INJECTION FOLLOWING in) TIME(m 30 -3 LBPIE 10. kg) g /k g m .0 0 (1 CLEBOPRIDE G74 ( 0 kg) g /k g m .0 (8 FG 7142 FG 7142 + CLEBO (1 0 .0 m g /k g ) ) g /k g m .0 0 (1 CLEBO + FG7142 15 -1 Figure Li Figure 0 15 30

45 128 Figure 12. Summary of effects of systemic dopamine receptor antagonists on FG 7142-stimulated cortical ACh efflux

Depicted is the maximal increase in cortical ACh efflux following administration of FG 7142 (8.0 mg/kg, i.p.) on sessions in which vehicle or dopamine receptor antagonists were pre-administered. The D1-selective antagonist SCH 23390 dose-dependently attenuated the stimulation of ACh efflux produced by FG 7142. Haloperidol also dose-dependently attenuated the effect FG 7142, blocking it fully at the highest dose. By contrast, a high dose of the D2-selective antagonist clebopride had no effect on FG 7142-stimulated cortical ACh efflux.

129 ACh EFFLUX (% change from baseline) 0 0 1 200 150 0 5 2 50 0 c £7777) n n F G 71 42 (8.0 mg/kg) FG (8.0 71 42 lbpie (1mg/kg) 0.0 Clebopride+ mg/kg) (0.30 SCH + 23390 + Haloperidol (0.90 mg/kg) Haloperidol (0.90 + Haloperidol (0.1mg/kg) + 5 mg/kg) (0.10 SCH + 23390 Figure 12 Figure

i 130 Figure 13. Schematic diagrams of placements of dialysis probes in the cortex and nucleus accumbens for measurement of ACh efflux in the medial pretrontal cortex and GABA efflux in the nucleus accumbens (adapted from Paxinos & Watson, 1985)

131 132 Figure 14. Effect of intra-accumbens perfusion of the Dl-selective antagonist on FG 7142-stimulated cortical ACh efflux

Shown is cortical ACh efflux (mean ± S.E.M.) at baseline, during perfusion of SCH 23390 through a dialysis probe placed in the nucleus accumbens, and following systemic injection of FG 7142. The dopamine antagonist was added to the aCSF of the accumbens probe and perfused contiuously from 30 min prior to FG 7142 admnistration until the end of the experiment. Shown are efflux changes during the last 30 min of baseline collection (left-most bar group), during the first 15 min of antagonist perfusion (middle bar group), and during 15-30 min following FG 7142 injection (right most bar group). Intra-accumbens SCH 23390 at the lower dose appeared to directly stimulate cortical ACh efflux, but this effect was more variable with the higher dose. Post-FG 7412 ACh efflux during intra-accumbens SCH 23390 perfusion was not different from control levels.

133 600 aCSF SCH 23390 1 0.0 uM 550 mmm SCH 23390 1 00.0 uM 500 450 400 350 300 250 200

150 mm W«v,V, 1 0 0

50 T 0 xiES&Si - 5 0 baseline SCH 23390 post-FG 7142 (i.p.) 134 infusion Figure 14 Figure 15. Effect of intra-accumbens perfusion of haloperidol on FG 7142- stimulated cortical ACh efflux

Shown is cortical ACh efflux (mean ± S.E.M.) at baseline, during perfusion of haloperidol through a dialysis probe placed in the nucleus accumbens, and following systemic injection of FG 7142. The dopamine antagonist was added to the aCSF of the accumbens probe and perfused contiuously from 30 min prior to FG 7142 admnistration until the end of the experiment. Shown are efflux changes during the last 30 min of baseline collection (left-most bar group), during the first 15 min of antagonist perfusion (middle bar group), and during 15-30 min following FG 7142 injection (right most bar group). Intra-accumbens haloperidol had no effect on basal efflux. However, it dose-dependently blocked stimulation of cortical ACh efflux by the BZR partial inverse agonist.

135 550 vehicle 500 IZ/y/d haloperidol 1 0.0 uM 450 haloperidol 1 00.0 uM 400 350 300 250 200 150 100 50

0 1 1 - 5 0 mm 136 baseline haloperidol post-FG 71 42 (i.p.) infusion Figure 15 Figure 16. Effects of intra-cortical perfusion of dopamine receptor antagonists on FG-7142-stimuiated. cortical ACh.efflux

Shown is cortical ACh efflux (mean ± S.E.M.) at baseline, during co perfusion of SCH 23390 (top panel) or haloperidol via the cortical dialysis probe, and following systemic injection of FG 7142. The dopamine antagonist was added to the aCSF of the cortical probe and perfused contiuously from 30 min prior to FG 7142 admnistration until the end of the experiment. Shown are efflux changes during the last 30 min of baseline collection (left-most bar group), during the first 15 min of antagonist perfusion (middle bar group), and during 15-30 min following FG 7142 injection (right-most bar group). In contrast to its effects when administered systemically or into the nucleus accumbens, intracortical SCH 23390 (100.0 jaM; upper panel) appeared to increase basal cortical ACh efflux . Administration of FG 7142 produced an increase that was apparently additive to that produced by the intracortical D l- antagonist. Intracortical haliperidol (100.0 j*M) had no effect on basal efflux. FG 7142-stimulated efflux was of similar magnitude (right-most bar) but apparently shorter duration (not shown) with intracortical haloperidol; thus the right-most bar shows efflux during the first 15 min after FG 7142 injection.

137 ACh efflux (7. change from boseline) ACh efflux (7. change from baseline) 200 - 0 5 2 100 - 0 5 1 - 0 0 3 - 0 5 3 - 0 0 4 - 0 5 4 - 0 0 5 0 5 5 200 100 0 5 2 0 5 1 0 0 3 0 0 4 0 0 5 0 5 3 0 5 4 0 5 5 - 0 5 0 - - elne hal i post 7142 (. .) (i.p 2 4 1 7 G F t- s o p l o rid e p lo a h e lin se o b . Figure C 23390 9 3 3 2 SCH 16 infusion nf i n sio fu in -FG 7 ip.) (i.p 2 4 71 G F t- s o p 138 Figure 17. Summary of effects of BZR ligands on non-stimulated and stimulated cortical ACh efflux

This figure summarizes the ability of BZR ligands to modulate cortical ACh efflux under resting and behaviorally-activated conditions. The left two groups of bars represent the effects of systemically-administered BZR ligands on efflux in resting animals (left-most panel) an in animals activated by the "darkness/cereal" stimulus presentation. The BZR weak inverse agonist, ZK 93 426 produced a modest increase in basal ACh, while the BZR full agonist CDP had no effect in this condition. However, BZR ligands effectively modulated activated efflux evidenced by a significant enhancement by ZK 93 426 and a complete blockade by CDP of the stimulus-evoked increase. The two right- hand bar groups show the analogous effects of the BZR inverse agonist P-CCM and the full agonist infused into the basal forebrain. Similar to systemically- administered ligands, the action of BZR ligands in the basal forebrain had minimal effects on cortical ACh efflux in resting animals, but significantly modulated stimulated efflux. This indicates that the basal forebrain is a suffcient site of action for the effects of BZR ligands on stimulated cortical ACh efflux.

139 ACh EFFLUX (% change from baseline) 250 200 225 175 150 0 0 1 125 50 25 75 0 - ■ _ >6<^BCM(. he ; i.e.) i; em /h g u (3.0 B-CCM n ^ < 6 t> /A 7 7 V Vehicle i H YTM C INTRACRANIAL SYSTEMIC . i NON-ACTIVATED Vehicle D 50mgk; i.p.) g/kg; m (5.0 CDP 426 ZK93 CDP 4. ghmi i.c.) i; ug/hem (40.0 50mgk; i.p.) g/kg; m (5.0 10mlk; i.p.) l/kg; m (1.0 05L/e ; i.e.) i; /hem (0.5uL Figure 17 Figure YT MC INTRACRANIAL SYSTEMIC

ACTIVATED 140 141

Plate I. Nissl-stained section of cortical probe placement

Shown is a Nissl-stained coronal section (level: 1.7 mm anterior to bregma; Paxinos et al., 1985; magnification 20x) taken through the center of the tract formed by the guide cannula and dialysis probe. 142

Plate II. Acetylcholinesterase-stained section showing cortical probe tract

Photomicrograph of a coronal section (level: 1.7 mm anterior to bregma; Paxinos et al., 1985; magnification lOx) showing extent of probe tract and density of acetylcholinesterase-positive fibers following four microdialysis sessions. The section shows a relatively large vacuous tract caused by the chronic guide cannula with additional, less severe, tissue damage extending beyond this tract associated with the probe membrane tip. Tissue within 1 to 2 mm of the membrane tract, presumably the area of perfusion, contained a normal density of AChE-positive fibers. BIBLIOGRAPHY

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