EFFECTS OF MORPHINE ON SPONTANEOUS AND EVOKED

ACTIVITY IN THE CORPUS STRIATUM

by

TAVYE CELESTE NAPIER, B.S.

A DISSERTATION

IN

PHARMACOLOGY

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Approved

Accepted

May, 1982 -<,J

ACKNOWLEDGEMENTS

I am most grateful to Dr. James H. Pirch for his ceaseless con­ cern and guidance during the research for this project and the prepa­ ration of this manuscript. I would like to express my appreciation to

Dr. Jean C. Strahlendorf and Dr. Howard K. Strahlendorf for their encouragement and excellent technical advice. Gratitude is also extended to Dr. Charles D. Barnes, Dr. David E. Potter and Dr. Thomas

E. Tenner, Jr. for their assistance and suggestions concerning this project.

I am deeply indebted to my parents, Dr. and Mrs. Gayle and Grace

Napier; my sisters. Ginger, Marsha and Genevieve; and my fiance,

David Gay, for their unending moral support during my educational studies. I dedicate this manuscript to my grandmother, Mrs. Ida Lou

Black, whose patience and endurance I sincerely respect.

Finally, I am grateful to Tarbox Parkinson's Disease Institute at

Texas Tech University Health Sciences Center for supporting this research.

11 ABSTRACT

This study consisted of three investigations. The first inves­ tigation was concerned with unit responses in globus pallidus and caudate nucleus to cumulative doses of morphine. A semichronie, phenobarbital anesthetized rat preparation was used. It was deter­ mined that systemieally-administered morphine caused a naloxone- antagonized depression of spontaneous neuronal activity in 79% of recorded pallidal cells (n=24) whereas only 27% of caudate cells

(n=26) were similarly affected. A Chi Square analysis comparing the number of cells depressed and not depressed in pallidum and caudate was significant (p < 0.005), clearly verifying area differences in

response to morphine.

Effects of systemieally administered morphine on cortically-

evoked unit activity in the caudate nucleus was the concern of the

second investigation. A semichronie rat preparation, similar to the

previous investigation, was used. Only 4 out of 36 caudate units

demonstrated specific (naloxone-antagonized) morphine-induced altera­

tion of evoked activity. These results indicate that eorticostriatal

pathways may not be involved in actions that systemic morphine has in

the caudate.

Effects of microiontophoretically-applied naloxone on striatally-

induced suppression of pallidal unit activity was assessed in the

third investigation. Twenty-three pallidal cells whose activity was

altered by caudate stimulation were recorded in chloral hydrate

anesthetized rats. With microiontophoretic application of naloxone

an increase in frequency of occurrence of action potentials during

ill caudate-induced suppression of pallidal activity was often observed.

The ability of naloxone to counteract caudate-induced suppression of pallidal activity indicates an enkephalinergie component in electro­ physiological events occurring in the globus pallidus following caudate stimulation.

These results demonstrate the ability of an exogenous opiate

(morphine) to alter unit activity in caudate nucleus and globus pallidus. Unit activity alterations produced by morphine, and alteration of stimulation-induced inhibition by naloxone, indicate an involvement of endogenous opiates (e.g., enkephalins) in neuronal events of the corpus striatum. Failure of morphine to alter corti- eally-evoked caudate activity suggests that enkephalin effects within the caudate are not mediated through an alteration of the

influence of cortical inputs on striatal neurons.

IV TABLE OF CONTENTS

ACKNOWLEDGEMENTS 11

ABSTRACT 111

LIST OF TABLES X

LIST OF FIGURES XI

I. INTRODUCTION

Review of Previous Research ....

The Anatomy and Physiology of the Corpus Striatum .... 2

The Neostriatum 3

Morphology of the Striatum 3

Eleetrophysiology of Striatal Intrinsic Neurons. . . 5

Neurotransmitters Found in Striatal Neurons 6

Acetylcholine 6

y-Aminobutyric Acid 7

Substance P 8

Enkephalin 8

Striatal Afferents 13

The Nigrostriatal Projection 14

The Thalamostriatal Projection 16

The Corticostriatal Projection 17

Other Striatal Afferent Projections 20

Striatal Efferents 21

The Striatonigral Projection 21

The Striatopallidal Projection 22

The Globus Pallidus 25

Morphology of the Globus Pallidus 25 Neurotransmitters Found in Pallidal Neurons 26

Acetylcholine 26

y-Aminobutyric Acid 26

Enkephalin 27

Pallidal Afferents 29

The Subthalamopallidal Projection 30

Pallidal Efferents 30

The Pallidosubthalamic Projection 31

The Pallidonigral Projection 32

Other Pallidal Efferent Projections 32

Motor Functions of the Corpus Striatum 33

Motor Effects of Abalations or Activation

of the Corpus Striatum 33

Motor Effects of Striatal and Pallidal Lesions ... 34

Motor Effects of Striatal and Pallidal Stimulation 35 The Relationship of Neuronal Activity to Movement. . 35 The Role of the Corpus Striatum in Pharmacol­ ogically-Induced Motor Dysfunctions 37

Neuroleptie-Induced Catalepsy 38

Narcotic-Induced Catalepsy 38

Purpose and Scope of This Investigation 40

Experiment I: Unit Responses in Globus Pallidus and Caudate Nucleus to Cumulative Doses of Morphine. . . 41

Experiment II: The Effects of Morphine on Cortieally- Evoked Unit Activity in the Caudate Nucleus 41

Experiment III: Naloxone Effects on Striatally-Induced Suppression of Pallidal Unit Activity 42

VI II. METHODS AND PROCEDURES 43

General Methods 43

Experimental Animals 43

Guide Cannulas and Electrodes 43

Implantation Procedures - Semichronie Preparations. ... 44

Experimental Procedures 45

Animal Preparations 45

Semichronie Preparations 45

Acute Preparations 48

Positioning the Microelectrodes 48

Recording, Stimulation and Mieroiontophoresis. ... 49

Experimental Drugs 50

Histology 50

Specific Experiments 52

Experiment I: Unit Responses in Globus Pallidus and Caudate Nucleus to Cumulative Doses of Morphine. . . 52 Experiment II: The Effects of Morphine on Cortically- Evoked Unit Activity in the Caudate Nucleus 53

Experiment III: Naloxone Effects on Striatally-Induced

Suppression of Pallidal Unit Activity 54

III. RESULTS 60

Experiment I: Unit Responses in Globus Pallidus and

Caudate Nucleus to Cumulative Doses of Morphine. . . 60

Globus Pallidus 60

Caudate Nucleus 67

Comparing Morphine Effects in the Globus Pallidus and Caudate Nucleus 76

Vll Experiment II: The Effects of Morphine on Cortically- Evoked Unit Activity in the Caudate Nucleus 77

Experiment III: Naloxone Effects on Striatally-Induced

Suppression of Pallidal Unit Activity 87

IV. DISCUSSION 100

General Discussion. 100

The Semichronie Preparation 100

The Influence of Anesthetics on Morphine Actions. . . 101

Phenobarbital 101

Chloral Hydrate 103

Multiple Dosing Schedule 104

Brain Concentrations of Systemically-Administered

Morphine in Rats 105

Acute Tolerance 106

Excitatory Effects of Morphine 106

Excitatory Effects Antagonized by Naloxone. . . . 107

Nonspecific Excitatory Effects 109

Naloxone and Opiate Receptor Specificity 110

Naloxone Antagonism of Opiates and Subsequent Increases in Unit Activity 113 Specific Experiments 116 Experiment I: Unit Responses in Globus Pallidus and Cau­ date Nucleus to Cumulative Doses of Morphine . . . 116

Globus Pallidus 116

Caudate Nucleus 118

Comparing Morphine Effects in the Globus Pallidus and Caudate Nucleus 121

Experiment II: The Effects of Morphine on Cortically- Evoked Unit Activity in the Caudate Nucleus. . . 123

viii Experiment III: Naloxone Effects on Striatally-Induced Suppression of Pallidal Unit Activity 125

Significance 129

LIST OF REFERENCES 132

IX LIST OF TABLES

Table Page

1. Specifications used for surgically implanting stimulating electrodes and/or microelectrode guide cannulas in the semichronie preparations 46

2. Response patterns of spontaneous pallidal activity to systemic morphine 66

3. Response patterns of spontaneous caudate unit activity to systemic morphine (Experiment I) 75

4. Response patterns of spontaneous caudate unit activity to morphine (Experiment II) ^0

5. Pattern of morphine-induced change in cortically-evoked activity of caudate nucleus units °2

6. The effects of morphine on respiratory rate 83 LIST OF FIGURES

Figure Page

1. Firing frequency of a pallidal neuron following a 100 mg/kg i.p. injection of sodium phenobarbital 47

2. A representative poststimulus time histogram showing caudate-induced suppression of unit activity from a cell located in the globus pallidus 57

3. Pallidal areas from which unit recordings were obtained . 61

4. Examples of typical locations of pallidal recording sites 62

5. Action potentials from single units located in the globus pallidus 63

6. Cumulative integrator recordings from spontaneously firing single units in the globus pallidus 65

7. A representative pallidal unit which demonstrated a morphine-induced depression 68

8. Cumulative dose-related effects of morphine on globus pallidus and caudate neurons which demonstrated depression antagonized by naloxone 69

9. Areas in the caudate nucleus from which unit recordings

were obtained in Experiment I 70

10. Examples of locations of striatal recording sites .... 71

11. Action potentials from single units located in the caudate nucleus 72 12. Cumulative integrator recordings of spontaneously active units in the caudate nucleus 74

13. Areas in the caudate nucleus from which recordings were obtained in Experiment II 78

14. Typical examples of the three types of caudate unit responses to cortical stimulation 79

15. The areas of the globus pallidus from which caudate- induced suppression of unit activity was observed .... 88

XI 16. Examples of pallidal activity following caudate nucleus stimulation 89

17. The effects of microiontophoretically-applied morphine on pallidal unit activity 91

18. The effects of morphine and morphine during naloxone on pallidal unit activity 94

19. Examples of caudate-induced inhibition of pallidal activity which was counteracted by microion­ tophoretically-applied naloxone 95

20. Naloxone-induced alterations of caudate-induced suppression of pallidal cells 98

21. The difference between 0 and E counts for pallidal units whose activity was inhibited by caudate stimulation ... 99

Xll I. INTRODUCTION

The history of man's use of opium dates back to the third and fourth centuries B.C. In recent years endogenously released peptides which demonstrate morphine-like actions have been isolated from nervous tissue. The discovery and subsequent characterization of these endogenous opiates have stimulated new interest in the actions of morphine-like compounds in the central nervous system.

The corpus striatum regulates various behavioral functions, predominantly those functions which are related to motor behaviors.

It is known that systemieally administered morphine, when given in doses higher than those required for analgesia, can cause certain motor abnormalities. An involvement of opiate systems in the corpus

striatum has been implicated in motor dysfunctions induced by high

doses of morphine. If the corpus striatum is involved in opiate-

induced motor dysfunctions then exogenously administered morphine

should be able to alter neuronal activity in this brain area. Mor­

phine-induced alterations of neuronal activity in the corpus striatum might indicate the types of action that endogenous opiates (e.g.,

enkephalins) have on these cells. Naloxone antagonism of neuronal

events elicited by stimulation of afferent projections might also

indicate the type of involvement of endogenous opiates in the activi­

ties of the corpus striatum. Based upon these postulates the present

study explores the effects of morphine and its antagonist, naloxone,

on neuronal events occurring in the corpus striatum. Review of Previous Research

The Anatomy and Physiology of the Corpus Striatum

In 1912 S.A.K. Wilson published a lengthy essay meticulously characterizing a motor dysfunction caused by a highly localized lesion in the brain. In this essay the terminology "extrapyramidal motor disease" was coined for the first time. Literally, the ex­ trapyramidal system denotes all of the brain's effector systems which do not involve the pyramidal tract (Nauta and Domesick, 1979). This terminology is extremely general however, and since "extrapyramidal" was not definitively characterized anatomically by its author, it is often used interchangably with another and, in fact, older anatomical designation, the basal ganglia. The basal ganglia constitutes a massive subcortical complex, which involves most of the non-cortical

gray matter of the telencephalon. Despite the longevity of the term,

disagreement remains concerning which specific nuclei should be

included in the basal ganglia. The present study is concerned with

an area of the basal ganglia considered to be involved with somatic motor function. This area is called the corpus striatum. The desig­ nation of corpus striatum distinguishes the neostriatum, or caudate-

putamen, and the paleostriatum, or globus pallidus, from the rest of

the basal ganglia (Carpenter, 1976a).

The importance of the corpus striatum in skeletomuscular func­

tion has been recognized for decades, as exemplified by the following

quote from Wilson's (1912) study:

In pure, uncomplicated, bilateral lesions ... of the corpus striatum, provided they are of sufficient size and of adequate duration, the clinical symptoms are bilateral involuntary movements, practically always of the tremor variety; weakness, spasticity or hypertonicity (sometimes spasmodic contractions) and eventually con tracture of the skeletal musculature; dysarthria or anarthria and dysphagia, and a degree of emotionalism; but without any sensory disturbance, without any true paralysis, and without any alteration in the cutaneous reflexes.

However, an adequate explanation for the precise nature of functional disorders manifested during pathology of the corpus striatum has not been presented to date. Similarly, the specific motor function in which the corpus striatum is involved also remains elusive.

The following sections will present a brief overview of the anatomy, biochemistry and physiology of the corpus striatum. Theories concerning the involvement of the corpus striatum in motor functions will be discussed. Elaboration of the areas of direct interest to this study will be presented in the discussion.

The Neostriatum

The neostriatum (or striatum) is by far the largest component of the basal ganglia. In primates the striatum is separated by the internal capsule which allows distinct designation of the caudate nucleus and the putamen. In rats this division does not occur and the striatum (or caudatoputamen) is a single anatomical entity.

Cytologically, the caudate and putamen have been considered to be identical, and like the rat striatum, the various cell types gen­ erally show a homogenous distribution (Graybiel and Ragsdale, 1979).

Morphology of the Striatum

Striatal cells are densely packed, exhibit no striations or clusters, and classically are considered to be of two anatomical groups: (1) small, achromatic neurons and (2) large multipolar neurons. Estimations concerning the ratio of these two major cell types vary considerably, ranging from 20:1 to 160:1, (E(ier, Vizkelety and Tombol, 1980); however, it is of general consensus that the small cells constitute the bulk of the striatal neuronal population.

Recent investigations utilizing the Golgi silver impregnation tech­ nique suggest further divisions based on the presence or absence of dendritic spines. The small cells appear to be mainly ovoid, ap­ proximately 10-18 y in diameter (Eder et al., 1980; Rafols and Fox,

1979) and usually have spiny dendrites and axons which arborize

locally (Carpenter, 1976b; Kemp and Powell, 1971a). E^er et al.

(1980) and Rafols and Fox (1979) have also reported aspiny small

cells and small cells which appeared to project extrastriatally. The

large neurons are most frequently aspiny (Eder et al., 1980), range

in size from 18-44 ]i in diameter, and are triangular or spindle-

shaped (Carpenter, 1976b; E^er et al., 1980). These cells have

thick, myelinated axons the termination of which is uncertain (Pasik

and Pasik, 1980) but is probably extrastriatal. Medium-sized neurons

with a diameter falling in the 20-25 ]i range have also been described

(E^er et al., 1980; Rafols and Fox 1979). Medium-sized cells possess

both spiny and aspiny dendrites. The spiny neurons are likely to be

efferent in nature (Pasik and Pasik, 1980). As many as nine subtypes

of striatal cells have been characterized, demonstrating the com­

plexity of striatal cyto-architecture. In a comparative study of

nerve cells of the rabbit, cat, monkey and human caudate nucleus. E^er et al. (1980) report that the number of the types and subtypes of caudate neurons increases as the phylogenetic series increases.

Eleetrophysiology of Striatal Intrinsic Neurons

Characteristics of intrinsic striatal cells have been described further by using electrophysiological approaches. Negative field potentials could be evoked from caudate cells by stimulating the nucleus at a distance of 1.5 mm from the recording site in cats with

either an intact or chronically isolated caudate nucleus (Marco,

Copack, Edelson and Oilman, 1973b) and 1 mm from the recording site

in rat striatal slices (Misgeld, 1979). The negative wave was com­ monly of 3-5 msec duration with an average latency of 10 msec. The

long latency to evoked response onset could be attributed to the slow

conduction velocity of the thin, unmyelinated axons of the small

interneurons. Unit recordings demonstrated evoked action potentials

of 1.5-2 msec duration frequently superimposed on the negative field

potential wave (Marco et al., 1973b). Spontaneously firing caudate

unit activity, however was both excited and inhibited by local stimula­

tion. In those units in which spontaneous activity was suppressed,

the inhibition usually lasted 150 to 200 msec (Marco et al., 1973b).

Studies using intracellular recordings have demonstrated excitatory

postsynaptic potentials (EPSPs) alone (Misgeld, 1979) and EPSPs

followed by inhibitory postsynaptic potentials (IPSPs) (Marco, Copack

and Edelson, 1973a; Lighthall, Park and Kitai, 1981) upon stimulation

of striatal interneurons. Also, activation of striatal cells has

been shown to result in recurrent inhibition (Park, Lighthall and

Kitai, 1980). These studies indicate both excitatory and inhibitory components of striatal interneuron activation.

Neurotransmitters Found in Striatal Neurons

Several candidates have been presented as neurotransmitters responsible for intrinsic communication in the caudate. The fol­ lowing is a brief summary of potential transmitters.

Acetylcholine. The striatum has been demonstrated to contain high levels of acetylcholine (Macintosh, 1941), choline acetyltransferase

(Hebb, 1957), and acetylcholinesterase (Burgen and Chipman, 1951). A marked decline in acetyltransferase activity in the striatum occurs following kainic acid treatment (Schwarcz, 1976). (Kainic acid is a neurotoxin whose action is supposedly limited to neuronal perikaya while sparing axons of passage and afferent nerve endings in the

injected area.) These studies suggest a significant cholinergic component within the intrastriatal neuroregulator system. The acety- choline-containing cells are presumably aspiny (Pasik and Pasik,

1980).

Iontophoresis of acetylcholine onto striatal neurons results in both excitation and inhibition (Bloom, Costa and Salmoiraghi, 1965;

McLennan and York, 1966). The predominant effect of acetylcholine, however, is excitation. Responses of caudate neurons to acetylcho- line, whether excitatory or depressant, were blocked by previous

iontophoretic application of atropine, and both types of response could be elicited by acetyl-3-methyl choline (McLennan and York,

1966). These results indicate that the cholinergic receptors in the striatum are in part muscarinic in nature. Field potentials evoked in striatal slices by intrastriatal stimulation demonstrate an increase in amplitude and a prolonged duration upon the addition of 10 ^ M physostigmine to the bathing medium (Misgeld, 1979). In contrast to the iontophoretic studies nicotinic antagonists blocked the evoked potential whereas atropine had no effect (Misgeld, 1979).

These results support the notion that acetylcholine is released upon activation of aspiny interneurons in the striatum. The released

acetylcholine causes an excitation of the postsynaptic cell. The

type of cholinergic receptor involved in this excitation, however,

remains uncertain.

y-Aminobutyric acid (GABA). The striatum contains moderately

high concentrations of GABA and its synthetic enzyme, glutamic acid

decarboxylase (GAD) (Fahn, 1976). Unilateral cortical or thalamic

lesions in rat brains failed to significantly alter mean striatal GAD

levels on the lesioned side (McGeer and McGeer, 1975). Interruption

of the nigral-striatal fibers also failed to reduce striatal GAD and

y-aminobutyric acid transaminase (another enzyme involved in the

synthesis of GABA) levels below the contralateral controls (Kataoka,

Bak, Hassler, Kim and Wagner, 1974). Extensive lesioning of globus

pallidus (the major efferent projection of the striatum), and hemi-

transections between the globus pallidus and striatum, did not sig­

nificantly depress GAD levels in the caudate (Hattori, McGeer, Fibi-

ger and McGeer, 1973; McGeer, McGeer, Wada and Jung, 1971). Auto­

radiography of [^H] GABA uptake indicates that 52% of the grains accumulated in striatal boutons and 28% were located in the cell soma

(Hattori et al., 1973) indicating an intranuclear origin for the neurotransmitter. GABA has been considered to be the transmitter responsible for the recurrent inhibition observed after local stimu­ lation of striatal neurons (Park et al., 1980).

These studies indicate a substantial striatal population of neurons which release GABA intrinsically. These cells may, however, have efferent projections with collaterals that remain in the cau­

date. The synaptic characteristics of these GABA-ergic cells are

different from those of the cholinergic interneurons as was demon­

strated by the inability of bicuculline and L-glutamic acid diethyl

ester to block intrastriatally-evoked caudate potentials which were

effectively blocked by cholinergic antagonists (Misgeld, 1979) .

Substance P. Substance P-containing fibers have cell bodies which originate in the anterior striatum and terminate in the zona

reticulata of the substantia nigra (Brownstein, Mroz, Tappaz and

Leeman, 1977; Gale, Hong and Guidotti, 1977). These fibers probably

have collaterals which terminate in the entopeduncular nucleus (for

review see Cuello, 1980).

Enkephalin. Of the many neurotransmitters which regulate

neuronal activity in the caudate nucleus, the present study pre­

dominantly deals with the enkephalinergie system. The enkephalins

are endogenously-produced opiate peptides whose actions are similar

to exogenous opiates. Since the recent discovery of endogenous opiates a flurry of research has been conducted concerning the loca- tion of the enkephalin peptide and its receptor in the caudate. A brief review of the results from these investigations is presented below.

In 1973 Pert and Snyder demonstrated the specific binding of various opiates and their antagonists to receptors in the mammalian brain. The discovery of opiate receptors not only demonstrated a site of action for exogeneously administered opiates but also indi­ cated that these receptors are present for the purpose of providing a

site of action for an endogenous opiate-like compound. In order to more fully understand the functional significance of both exogenous

and endogenous opiates, it became important to determine which areas

in the brain contain these receptors. It was shown by Pert and

Snyder (1973) and subsequent studies by these and other investigators

that the caudate possesses a high to moderately high opiate receptor

density (Kuhar, Pert and Snyder, 1973; Hiller, Pearson and Simon,

1973; Pert, Kuhar and Snyder, 1975; Pert, Kuhar and Snyder, 1976;

Atweh and Kuhar, 1977). Autoradiographic studies located "streaks"

and "dense clusters" of high density binding interspersed in a back­

ground of low density binding, throughout the striatum. The clusters

were spaced irregularly over the striatum whereas the streaks occurred

in the striatal edges, along the dorsal edge of the caudate next to

the corpus callosum (Pert et al., 1975; Atweh and Kuhar, 1977).

Results from studies performed in vitro on isolated organ pre­

parations (Hutchinson, Kosterlitz, Leslie, Waterfield and Terenius,

1975; Lord, Waterfield, Hughes and Kosterlitz, 1977) led to the

concept of multiple opiate receptors. Based on pharmacologic and behavioral profiles elicited by various morphine-like compounds in humans and spinally transected dogs, Martin and colleagues distin­ guished three opiate receptor effects which they designated as mu, kappa and sigma (Martin, Fades, Thompson, Huppler and Gilbert, 1976).

Each of these receptors possesses a specific ligand: morphine, keto- cyclazocine and a benzomorphan derivative (SKF 10047), respectively.

According to the displacement potency of various opiates in receptor binding studies, sigma opiate receptors seemed to populate the stria­

tum in a denser fashion than kappa and mu opiate receptors, with the

latter two types demonstrating approximately equal striatal concen­

tration (Delia Bella, Casacci and Sassi, 1978) .

Another pharmacologically distinct opiate binding site was

distinguished from the mu receptor by selective binding of separate

I-labeled enkephalin analogs (Chang, Cooper, Hazum and Cuatre-

casas, 1979). This receptor was identified as a delta opiate re­

ceptor (Lord, Waterfield, Hughes and Kosterlitz, 1976). The delta

receptor is more sensitive to the enkephalins than morphine, whereas

the reverse is true for the mu receptor (Goodman, Snyder, Kuhar and

Young, 1980). The ratio of the delta vs mu receptor population in

the striatum has been shown by autoradiographic methods to be unitary

(Goodman et al., 1980).

In 1975 Hughes published a work identifying a compound endo­

genous to the brain which had pharmacological characteristics similar

to morphine (Hughes, 1975). He determined that this morphine-like

substance was unevenly distributed in the central nervous system,

with substantial concentrations occurring in the striatum. This endogenous substance has been identified as a pentapeptide with an amino acid sequence of tyrosine-glycine-glycine-phenylalanine and either methionine or leucine (Hughes, Smith, Kosterlitz, Fothergill,

Morgan and Morris, 1975) and has been subsequently called met-enkepha-

lin and leu-enkephalin, respectively.

Since Hughes' initial observation concerning the presence of

enkephalins in the striatum, numerous investigations have been

conducted to further characterize striatal enkephalin content.

Studies utilizing radioimmunoassay techniques have shown that the

caudatoputamen has high quantities of the morphine-like pentapep-

tides (Miller, Chang, Cooper and Cuatrecasas, 1978; Yang, Hong, and

Costa, 1977; DiGiulio, Majane and Yang, 1979). Additional studies in

which more brain regions were investigated demonstrated that the

striatal enkephalin concentration was actually quite moderate when

compared to brain areas such as the hypothalamus and the globus

pallidus (Hong, Yang, Fratta and Costa, 1977b; Simantov, Kuhar, Uhl

and Snyder, 1977). Enkephalin-like immunoreactivity (ELI) has been

demonstrated by light microscopic immunocytochemistry techniques to

be localized in terminal varicosities (Elde, Hokfelt, Jahansson and

Terenius, 1976; Watson, Akil, Sullivan and Barchas, 1977). Perikarya

containing ELI have also been observed in the neostriatum of animals

which were pretreated with colchicine to arrest axonal transport of

the peptide (Sar, Stumpf, Miller, Chang and Cuatrecasas, 1978; Hokfelt,

Elde, Johansson, Terenius and Stein, 1977). Neuronal perikarya and

processes showing enkephalin-like immunoreactivity are unevenly distributed throughout the neostriatum with greatest density in the ventro- and caudo-lateral (Pickel, Sumal, Beckley, Miller and Reis,

1980) and dorsal (Simantov et al., 1977) portions of the nucleus.

The labeled perikarya measure 10-15 y in diameter, are heterogen- eously distributed, and constitute about 15-20% of the total neuronal population in the striatum (Pickel et al., 1980). Perikarya which show ELI are much less numerous than dendrites and dendritic spines showing ELI.

Due to the overlap between perikarya and terminals showing ELI in the striatum, the possibility of the enkephalin-containing cells serving interneuronal functions is certainly feasible. This view is supported by the presence of ELI in unmyelinated axons in striatal neuropil (Pickel et al., 1980). However, ELI was also detected in myelinated axons which were located near the pallidal border of the striatum (Pickel et al., 1980). Pickel and colleagues, therefore, proposed a theoretical model of the ELI containing neurons in which a single long axon emits multiple collateral branches within the striatum and then becomes myelinated near the pallidal border to terminate at a more distal site (Pickel et al., 1980). This hypo­ thesis will be pursued further in the section concerned with opiates and striatofugal projections.

By measuring the incorporation of [^H] tyrosine into enkephalin peptides in isolated striatal slices, it was determined that striatal enkephalins are locally produced (McKnight, Hughes and Kosterlitz,

1979). It has also been demonstrated that depolarization of striatal slices and synaptosomal preparations by high potassium concentrations in the bathing medium causes the release of enkephalins (Henderson,

Hughes and Kosterlitz, 1978; Richter, Wesche and Frederickson, 1979;

Osborne and Herz, 1980). This evoked release is calcium-dependent and is totally inhibited by high magnesium concentrations (Henderson et al., 1978; Richter et al., 1979; Osborn and Herz, 1980).

In summary, these studies demonstrate the presence of opiate receptors and the endogenous opiates, enkephalins, in the striatum.

These observations along with the ability of potassium to evoke re­ lease of enkephalins from in vitro striatal preparations, and the ability of striatal slices to incorporate tyrosine into enkephalin peptides, strongly support the contention that enkephalins may serve as striatal neuromediators.

Striatal Afferents

The caudate receives the principal afferent connections of the corpus striatum. The caudatoputamen acts, therefore, to integrate

the massive input communications and reduce them down to efferents which project only to the globus pallidus or the substantia nigra.

The striatopetal fibers arise primarily from the substantia nigra, the intralaminar thalamic nuclei and the cerebral cortex

(Carpenter, 1976a). The nigral, thalamic and cortical inputs have been characterized electrophysiologically, and suprising similarities

in the evoked caudate response have been revealed. Intracellular recordings have been obtained from caudate neurons upon activation of each of these input areas in cats (Buchwald, Price, Vernon and Hull,

1973) and in rats (VanderMaelen and Kitai, 1980). The intracellular 14 response pattern, regardless of which input was activated, consisted of a fast-rising EPSP with a latency of about 12 msec followed by a slow IPSP of approximately 150-250 msec duration. This pattern was occasionally followed by a slow-rising EPSP. These studies indicate that striatal cells receive excitatory influences from each of the major inputs and, due to the onset latency of the fast EPSPs, these inputs seem to be monosynaptic in nature. The slow IPSP's can be explained as being generated via adjacent caudate cells (Buchwald,

Price, Vernon and Hull, 1973; Hull, Bernard; Price and Buchwald,

1973).

The Nigrostriatal Projection. Lesions of the striatum produce retrograde cell changes and cell loss in the pars compacta of the substantia nigra (Bedard, Larochelle, Parent and Porier, 1969). This finding was substantiated by biochemical and flourescent histochemi- cal techniques which provided convincing evidence that the large cells of the pars compacta give rise to nigrostriatal projections

(Anden, Carlsson, Dahlstrom, Fuxe, Hillarp and Larsson, 1964; Dahl- strom and Fuxe, 1964).

Stimulation of the substantia nigra produces an increased release of dopamine in the ipsilateral caudate (Portig and Vogt,

1969; Hedreen and Chalmers, 1972). Chemical (6-hydroxydopamine) and electrolytic lesions of the substantia nigra produce a degeneration of the nigrostriatal pathway with a concomitant loss of dopamine and dopaminergic terminals in the striatum (Hedreen and Chalmers, 1972).

Microiontophoretic studies in which dopamine has been applied directly 1555555 to caudate cells have demonstrated both excitation and inhibition of the caudate units (Spencer and Havlicek, 1974; Bevan, Bradshaw and

Szabadi, 1975). The exact influence of dopamine on striatal neurons is an area of considerable debate, which is beyond the scope of the present investigation. Discrepancies of striatal responses to micro- iontophoretically applied dopamine may be due to the type of anaes­ thetic used in the experimental preparation (Spencer and Havicek,

1974), to the population of striatal units sampled, and a host of other experimental variables. Therefore, both possible responses to dopamine will be considered in relation to the present study.

As previously mentioned, intracellular responses of caudate neurons to stimulation of the substantia nigra are generally char­ acterized by an EPSP-IPSP sequence (Hull et al., 1973; VanderMaelen and Kitai, 1980). Intracellular recordings combined with intracel­

lular dye injections show that monosynaptic EPSPs produced by nigral

stimulation occur in medium-sized intrinsic neurons (Kitai, Sugimori, and Kocsis, 1976b). These striatal cells demonstrate converging

evoked responses from thalamic nuclei and the cerebral cortex (Hull et al., 1973).

Microiontophoretic application of horseradish peroxidase onto striatal neurons in the rat and tracing of the afferents to the area of origin demonstrated that the nigrostriatal projection appears to be organized along an oblique longitudinal neostriatal axis (Veening,

Cornelissen and Lieven, 1980). Thus, the substantia nigra conveys its inputs in a highly organized topographical fashion on the striatum, The Thalamostriatal Projection. Retrograde cell degeneration can be observed in the intralaminar nuclei of the thalamus after ipsilateral lesions of the striatum in monkeys (Powell and Cowan,

1956; Mehler, 1966). Fibers can be traced from discrete lesions in the centromedial nucleus, through other thalamic areas, to widespread terminations in the putamen and the body of the caudate (Mehler,

1966). Retrograde studies of microiontophoretically applied horse­ radish peroxidase onto striatal cells in rats demonstrate that the anterior portion of the caudatoputamen appears to receive projections from additional thalamic nuclei such as the mediodorsal, ventro­ medial, rhomboid and gelatinosus nuclei, and that these inputs are

topographically organized (Veening et al., 1980). Electron micro­

scopic evidence suggests that most thalamostriate fibers terminate upon the spines of spiny striatal neurons (Kemp, 1968).

The placement of electrolytic lesions in the parafascicular

(Simke and Saelens, 1977) and centromedian-parafascicular complex

(Kim, 1978) of the thalamus results in a significant reduction of

choline acetyltransferase in rat caudatoputamen. These studies

implicate acetylcholine as a neurotransmitter in the thalamostriate

projection.

Electrophysiological studies generally demonstrated EPSP-IPSP

sequences in caudate cells after stimulation at the lateral posterior

and the centromedian nuclei of the thalamus (Buchwald et al., 1973;

VanderMaelen and Kitai, 1980), although pure EPSP's were frequently

evoked by centromedian-parafascicular stimulation (Buchwald et al.,

1973). The EPSP has been attributed to a monosynaptically mediated 17 excitatory input from the thalamus, whereas the IPSP probably re­ flects inhibitory inputs from neighboring caudate interneurons that were also activated upon thalamic stimulation (Buchwald et al.,

1973). Collectively these studies indicate that stimulation of the centromedian-parafascicular complex of the thalamus activates thalamo­ striate fibers which release acetylcholine to cause a depolarization of striatal cells.

The Corticostriatal Projection. Studies in which various corti­ cal regions were lesioned indicate that virtually all regions of the neocortex contribute fibers to the striatum and that all parts of the striatum receive fibers from the cortex (Webster, 1961; Carman,

Cowan and Powell, 1963; Webster, 1965; Kemp and Powell, 1970). These corticostriate fibers impinge on the striatum in a topographical fashion, in both its mediolateral and anteroposterior dimensions

(Webster, 1961; Carman et al., 1963; Webster 1965; Veening et al.,

1980). These projections terminate on medium size spiny neurons

(Kitai, Kocsis, Preston and Sugimori, 1976a). A great deal of con­ vergence occurs, with inputs from the cortex, substantia nigra and thalamus frequently making synaptic contacts with the same striatal neuron (Hull et al., 1973; VanderMaelen and Kitai, 1980). It was observed with intracellular recordings of evoked striatal activity, that EPSPs evoked from thalamic or nigral stimulation were inhibited by cortical IPSPs, whereas cortical EPSPs were left intact or even enhanced when superimposed on an IPSP of a conditioning stimulation to the other striatal input areas (Hull et al., 1973; VanderMaelen 18 and Kitai, 1980). Hull et al. (1973) and VanderMaelen and Kitai

(1980) concluded that the ability of the cortical stimulus to remain intact during spatially combined stimuli from other input areas indicates a "prepotency" of cortical influence on evoked potentials in the caudate nucleus. A portion of the present work is concerned with the corticostriatal pathway.

Extracellular unit activity of striatal cells after cortical stimulation has been assessed by Rocha-Miranda (1965) in cats and

Schultz and Ungerstedt (1978) and Spencer (1976) in rats. Following a supramaximal stimulation, caudate cells tended to respond initially with a single spike, although several spikes per cortical stimulus were recorded in both studies. Additionally, response to stimulus

ratios of less that 1:1 were reported by Schultz and Ungerstedt

(1978) in rats. Latencies for the evoked caudate response varied

from 3-14 msec for rats and 8-48 msec in cats, depending on the

cortical site being stimulated. Schultz and Ungerstedt (1978) reported

that these evoked responses were followed by a period of depressed

impulse activity lasting several hundred milliseconds with a subse­

quent rebound excitation.

Fry and Zieglgansberger (1979) studied effects of various drugs

microiontophoresed on caudate cells which responded to cortical

stimulation in rats. These authors enhanced unit activity by con­

tinuous microelectrophoretic application of 8-27 nA of L-glutamate.

Under these experimental conditions, cortical stimulation typically

evoked a response of 2-6 msec latency and 8-30 msec duration followed

by a 160-250 msec inhibitory phase. The inhibition was then followed by a late response that merged into the unit's spontaneous activity.

Similar descriptions of spike responses of caudate neurons to cere­ bral cortical stimuli have been mentioned in several reports (Laursen,

1961; Liles, 1973; Liles, 1974; Spencer, 1976; Katayama, 1978).

These response patterns presumably reflect the postsynaptic poten­ tials (EPSP-IPSP sequence) recorded intracellularly upon activation of the corticostriatal pathway.

Godukhin, Zharikova and Novoselov (1980) demonstrated that L- glutamic acid was released from rat neostriatum following stimulation of the frontal cortex. Glutamate has been proposed as a possible neuroregulator in the central nervous system (Barchas, Akil, Elliott,

Holman and Watson, 1978) and is present in high concentrations in the striatum (10-12 ymole/g; Johnson, 1972), a concentration second only to that found in the cortical ectosylvian gyrus (12.4 ymole/g).

Additional studies supporting the view that glutamate is the neuro­ transmitter in the corticostriatal pathway include the following: mieroiontophoresis of the glutamic acid diethyl ester (an antagonist of glutamic acid) inhibits the response of striatal neurons to corti­ cal stimulation (Spencer, 1976); destruction of the corticostriatal pathway decreases the uptake of glutamic acid by ipsilateral neo­ striatal slices (Divac, Fonnum and Storm-Mathisen, 1977; McGeer,

McGeer, Scherer and Singh, 1977); and destruction of the frontal cortex causes a specific decrease in striatal glutamic acid concen­ trations (Kim, Hassler, Hang and Paik, 1977).

Anatomically, the corticostriatal pathway appears to be mono- 20 synaptic in nature. This was demonstrated by the presence of horse­ radish peroxidase-containing cell bodies in the neocortex following

intrastriatal horseradish peroxidase injections (Hedreen, 1977) and

after iontophoretic application of the tracer substance in the striatum

(Veening et al., 1980).

It would seem, therefore, that the corticostriatal pathway

involves the axons of neurons which are located in the neocortex and

which release glutamate on striatal cells upon activation. Glutamate

acts on the striatal neuron to produce the fast EPSP component of

cortically-evoked postsynaptic events. It is speculated that the

slower IPSP is due to inhibitory interneurons, located within the

striatum, which are activated by cortical stimulation and which

impinge on the recorded caudate cell to cause hyperpolarization

(Buchwald et al., 1973). The slower onset of the IPSP is due to the

longer time course for polysynaptic events as compared to the direct

monosynaptic nature of the fast EPSP.

Other Striatal Afferent Projections. As research on the striatum

progresses, novel striatopetal connections have been characterized.

One example is the serotonergic projection from the dorsal raphe

nucleus (Lorens and Guldberg, 1974). Serotonin (Bogdanski, Weissbach

and Udenfriend, 1957; Ternaux, Hery, Bourgoin, Adrien, Glowinski and

Hamon, 1977) and its receptors (Schwarcz, Bennett and Coyle, 1977)

are located in the striatum. Conflicting evidence suggests that

serotonin is either inhibitory (Herz and Zieglgansberger, 1968), or

excitatory (VanderMaelen, Bonduki and Kitai, 1979) in the striatum.

Other such examples could be mentioned; however, such a detailed 21 anatomical, physiological and pharmacological characterization of striatal connections is outside the objectives of this essay.

Striatal Efferents

The neostriatum has two major efferent connections, one to the substantia nigra and the other to the globus pallidus. The present study is more concerned with the striatopallidal projection, but since both of these systems emanate from the caudatoputamen and each indirectly influences the functions of the other, a brief description will be presented for both pathways.

The Striatonigral Projection. Fibers from the head of the caudate nucleus terminate in the rostral substantia nigra (Voneida, 1960) in a mediolateral topographical fashion (Szabo, 1962). The majority of the striatonigral fibers terminate in the region of the substantia nigra designated as the pars reticulata; however, some also make connections in the pars compacta area (Nauta and Mehler, 1966).

Numerous investigations have demonstrated that stimulation of the ipsilateral caudate results in a reduction of nigral neuronal activity (Feltz, 1971; Yoshida and Precht, 1971; McNair, Sutin and

Tsubakawa, 1972; Grossman, Walker and Woodruff, 1973). Lesion studies indicate that GABA is a likely candidate as the inhibitory transmitter released in the nigra upon ipsilateral caudate activation

(Fonnum, Grofova, Rinvik, Storm-Mathisen and Walberg, 1974). Picro- toxin and bicuculline reduce the inhibition of neuronal activity in the substantia nigra during caudate stimulation (Precht and Yoshida, 22

1971; Grossman et al., 1973; Dray, Gonye and Oakley, 1976), thus supporting the concept of GABA involvement in striatonigral fibers.

Substance P has been located in the pars reticulata of the substantia nigra (Cuello and Kanazawa, 1978). Disruption of stria­ tonigral fibers results in a loss of substance P immunoreactivity in the substantia nigra (Hong, Yang, Racagni and Costa, 1977) which

suggests an involvement of this peptide in striatofugal projections

to the substania nigra.

The Striatopallidal Projection. The globus pallidus receives

the majority of striatal efferents and serves as the principal output

for the basal ganglia (Graybiel and Ragsdale, 1979; Nauta and Dome-

sick, 1979). Striatal efferent fibers radially encroach on the

pallidum "like the spokes of a wheel" (Papez, 1941) and, as evidenced

by fiber degeneration studies, demonstrate a topographical organi­

zation with the striatum projecting to both segments of the pallidum

(Szabo, 1962; Cowan and Powell, 1966). These fibers entwine around

the dendrites of the large pallidal neurons covering them with boutons

en -passage (Fox and Rafols, 1976; Kemp and Powell, 1971b). It is

thought that this longitudinal, axodendritic pattern demonstrates a

controlling influence on the pallidal cells by their afferent con­

nections (Fox and Rafols, 1976; Kemp and Powell, 1971b).

Electrophysiological studies in the globus pallidus of awake

monkeys have demonstrated either an excitation or an inhibition-

excitation sequence following striatal stimulation (Ohye, Guyader,

and Feger, 1976). In unanesthetized, paralyzed cats 27% of the

observed pallidal cells exhibited a cessation of firing for 60-230 23 msec following stimulation of the head of the caudate, 16% demon­ strated an excitatory response (3-5 msec latency) followed by inhibi­ tion, and 15% were facilitated (Noda, Manohar and Adey, 1968). Investi­ gations utilizing intracellular techniques have shown that pallidal cells respond to stimulation of the caudate head with pure EPSPs, pure IPSPs and EPSP-IPSP sequences (Malliani and Purpura, 1967;

Levine, Hull and Buchwald, 1974a). Involvement of intranuclear collaterals in recorded potentials is probably not as significant in the globus pallidus as they are in the caudate nucleus (Levine et al., 1974a).

The exact nature of the transmitters released from the striato­ pallidal fibers remains rather elusive. Among the various candidates that have been recently proposed are acetylcholine, GABA, enkephalins and substance P.

Studies identifying cells containing acetylcholinesterase pro­ vide evidence that the large striatal neurons are the source of a cholinergic striatopallidal projection (Olivier, Parent, Sinard and

Poirier, 1970; Poirier, Parent, Marchand and Butcher, 1977). Elec­ trolytic lesions of the caudate nucleus cause a decrease or disap­ pearance of cholinesterase in the ipsilateral globus pallidus (Oli­ vier et al., 1970).

Substantial losses of pallidal glutamic acid decarboxylase have been reported after striatal hemitransections (Fonnum, Gottesfeld and

Grofova, 1978a; Nagy, Carter and Fibiger 1978; Staines, Nagy, Vincent and Fibiger, 1980) and following striatal ablation by electrolytic lesions (Nagy et al., 1978) or suction (Kim, 1978). These observat- 24 ions lend support to a possible GABA-ergic striatopallidal projection.

Of particular interest to the present study is the potential involvement of opiates in the striatopallidal projection. Destruc­ tion of fiber connections between the globus pallidus and striatum produces a dramatic decrease in pallidal enkephalin concentrations

(Cuello and Paxinos, 1978; Staines et al., 1980), with a concomitant increase in striatal levels (Staines et al., 1980). Intrastriatal kainic acid injections also reduce enkephalin content in the globus pallidus (Uhl, Kuhar, Goodman and Snyder, 1979). Pallidal enkephalin release is increased after striatal stimulation, whereas no accelera­

tion of release can be detected when the septal or preoptic regions

are activated (Bayon, Shoemaker, Lugo, Azad, Ling, Drucker-Colin and

Bloom, 1981). The idea of an enkephalinergie striatopallidal pro­

jection is also supported by a study showing retrograde transport of

an immunofluorescence dye from the globus pallidus to the caudate by

enkephalin containing neurons (Brann and Emson, 1980).

Both the striatum and globus pallidus contain substance P. A

decrease in pallidal levels of substance P which coincided with an

increase in levels of the peptide in the striatum has also been

reported following isolation of the globus pallidus (Staines et al.,

1980).

Collectively, these studies indicate that the neurons involved

in the striatopallidal pathway may contain a variety of possible

neuroregulators which are capable of influencing pallidal cells by

excitation and/or inhibition. Further characterization of the

striatal inputs to the globus pallidus is essential before it will be 25 possible to understand the interaction between these two structures.

The Globus Pallidus

The globus pallidus lies medial to the striatum and, like the striatum, has different conformations in primates and non-primates.

In all species, however, the pallidum is divided into two main parts.

In primates these parts are separated by a thin fiber plate (the internal medullary lamina) and are designated as the internal and external pallidal segments. In rodents and other non-primates the two main segments of the globus pallidus are less compact and are more clearly distinguished as separate structures than in primates.

In these species the recognized equivalent of the internal segment is called the entopeduncular nucleus, and the external segment is called the external pallidum or simply the globus pallidus. Since the globus pallidus is a primary area of interest for the present study, the majority of the following discussion will be concerned with this nucleus.

Morphology of the Globus Pallidus

Neurons located in the globus pallidus are generally large fusiform or multipolar cells of about 12 x 18 y to 15 x 40 y in diameter (Pasik, Pasik and DiFiglia, 1979) with exceptionally long, smooth dendrites (Fox, Andrade, LuQui and Rafols, 1974). These large neurons are generally long-axoned and are candidates for the pallidal output projections (Levine et al., 1974a). Golgi studies also reveal a lesser population of small pallidal cells (about 12 y in diameter) which have axons that arborize locally (Pasik, Pasik and DiFiglia, 26

1979). It is due to this scarcity of interneurons that electro­ physiological activity recorded in the globus pallidus most likely reflects direct afferent (i.e., striatal) influence (Levine et al.,

1974a).

Neurotransmitters Found in Pallidal Neurons

Acetylcholine. Cell bodies containing acetylcholinesterase have

been located in the ventromedial portions of the globus pallidus

(Jacobowitz and Palkovits, 1974). These cells are generally medium-

sized (20-25 y) triangular or fusiform neurons (Parent, Gravel and

Oliver, 1979). Most of the globus pallidus only moderately demon­

strates nerve processes which stain for acetylcholinesterase (Jaco­

bowitz and Palkovits, 1974; Parent et al., 1977; Parent et al.,

1979). An exception to this generality occurs in the ventromedial

extreme of the central pallidus where a slightly higher concentration

of staining for acetylcholinesterase can be observed (Jacobowitz and

Palkovits, 1974).

y- Aminobutyric Acid (GABA). High pallidal concentrations of

glutamate decarboxylase (Lowe, Robins and Eyerman, 1958; Albers and

Brady, 1959; Miiller and Langemann, 1962; Walaas and Fonnum, 1979) and

GABA (Fahn and Cote, 1968) have been reported in several mammalian

species. The glutamate decarboxylase levels in the globus pallidus

were observed to be at least twice the concentrations observed in the

striatum (Lowe et al., 1958; Miiller and Langemann, 1962). Pallidal

GABA may originate from more than one source. GABA may be released

onto pallidal cells via terminal endings of afferent striatal fibers 27

(Fonnon, Gottesfeld and Grofova, 1978a; Nagy et al., 1978) and pos­ sibly via fibers projecting from the nucleus accumbens (Walaas and

Fonnum, 1979). Autoradiographic studies have indicated, however, that afferents are probably not the major source for pallidal GABA

(Hattori, McGeer, Fibiger and McGeer, 1973). These studies demon­ strated that less than 30% of [^H] GABA grains could be detected in boutons but 42% were located in pallidal cell soma, thus suggesting an intranuclear origin for the neurotransmitter. Pallidal GABA neurons are also likely origins for efferent fibers such as pal­ lidonigral (Hattori, Fibiger and McGeer, 1975) and pallidosubthalamic

(Fonnum, Grofova and Rinvik, 1978b) projections.

Enkephalin. In both radioimmunoassay and immunocytochemical studies the globus pallidus contains a higher concentration of enkephalins than any other brain region examined (Miller, Chang,

Cooper and Cuatrecasas, 1978; Watson et al., 1977; Simantov et al.,

1977; Sar et al., 1978). However, in contrast to the striatum, perikarya containing enkephalin-immunoreactive staining were not demonstrated in the pallidus in colchicine pretreated rats (Sar et al., 1978) or in non-pretreated rats (Pickel et al., 1980). The greatest concentration of enkephalins seems to be located within fibers and terminals in the globus pallidus.

In contradiction to thes^ conclusions, other investigators report that pallidal enkephalin concentrations decrease significantly following kainate lesions of the nucleus, indicating an intrinsic source for the peptides (Correa, Innis, Hester, Childers and Snyder, 28

1979). The lack of a decline in pallidal enkephalin content fol­

lowing large electrolytic lesions of the anterior caudate nucleus

refuted the contention of a striatopallidal enkephalinergie pathway

while substantiating the idea that interneurons serve as the sig­

nificant source of enkephalin in this nucleus (Correa et al., 1979).

The possibility of a pallidostriatal enkephalinergie pathway has also

been proposed (Hong, Yang and Costa, 1977a).

Both methionine- and leucine-enkephalin are released from

perfused slices of the globus pallidus in a Ca -dependent manner

(Bayon, Rossier, Mauss, Bloom, Iversen, Ling and Guillemin, 1978;

Iversen, Iversen, Bloom, Vargo and Guillemin, 1978). Using a push-

pull cannula method Bayon and colleagues recently characterized the

release of pallidal enkephalins in unanesthetized-freely moving rats

and eats (Bayon et al., 1981). As in the in vitro studies, the in

vivo enkephalin release was K -activated and Ca -dependent.

Receptor binding and autoradiographic studies have demonstrated

that the globus pallidus also contains stereospecific receptors for

opiates; however, these are found only in moderately dense concen­

trations (Kuhar et al., 1973; Hiller et al., 1973; Pert et al., 1976;

Atweh and Kuhar, 1977). This finding seems inconsistent with the

large number of enkephalinergie terminals presumed to be located in

the pallidum. To date, no satisfactory explanation has been pre­

sented for this enigma.

An interesting phenomenon concerning a head turning response

elicited by electrically stimulating the striatum was observed after

unilateral pallidal injections of several opiates (Slater and Longman, 29

1980). Morphine, leucine enkephalin and D-ala^, D-leu^-enkephalin had no effect on the head turn, but ethylketocyclazocine caused a naloxone-sensitive decrease of the motor response. These results indicate that the globus pallidus contains a significant population of kappa-type opiate receptors. This study does not necessarily preclude the possibility of additional opiate receptors which are sensitive to the other morphine-like compounds. For example, cells located in the globus pallidus have recently been shown to respond to microiontophoretic applications of morphine with a depression of both spontaneous activity and glutamate evoked activity (Huffman and

Felpel, 1981). Even though the currents necessary to elicit this depression by morphine were rather high, a naloxone antagonism of the morphine effect was demonstrated suggesting a specific opiate re­ ceptor action for the mu receptor agonist.

Pallidal Afferents

Classically, the afferent projections to the globus pallidus have been considered to arise from two structures, the neostriatum and the subthalamic nucleus (Carpenter, 1976b). Recent studies, however, indicate that additional structures may also contribute to pallidal afferent fibers. For example, Lindvall and Bjorklund (1979) contend that the dopaminergic nigrostriatal pathway sends collaterals to the globus pallidus. The major afferent influence on pallidal neurons is via the striatopallidal projection, as previously dis­ cussed. The following section will deal briefly with subthalamic

inputs to the globus pallidus. 30

The Subthalamopallidal Projection. Autoradiographic studies utilizing isotope transport techniques have demonstrated a fiber system arising from the subthalamic nucleus and projecting to all parts of the globus pallidus (Nauta and Cole, 1974) . Investigations utilizing horseradish peroxidase indicate that subthalamopallidal fibers predominantly originate from the medial two-thirds of the subthalamic nucleus and terminate in the globus pallidus in a topo­ graphical fashion (Carpenter, Batton, Carleton and Keller, 1981).

These authors also propose that cells in specific subthalamic loca­ tions project to either the entopeduncular nucleus or the globus pallidus in the monkey but not to both pallidal segments. Recently it has been shown that the predominant response of pallidal neurons to stimulation of the subthalamic nucleus is inhibition, even though excitation was also observed (Perkins and Stone, 1980).

Pallidal Efferents

Efferent projections of the globus pallidus form three major bundles: (1) the ansa lenticularis, (2) the lenticular fasciculus, and (3) the pallidosubthalamic projection (Carpenter, 1976b). The lateral pallidus gives rise to the subthalamic projection whereas the ansa lenticularis and the lenticular fasciculus originate from the medial segment (or entopeduncular nucleus). Entopeduncular pro­ jections terminate in various thalamic nuclei and constitute the major efferent projections of the corpus striatum (Nauta and Mehler,

1966). The majority of these fibers project to the anterior ven­ trolateral (Kuo and Carpenter, 1973) and central medial thalamic nuclei (Jones, 1981) which subsequently project to cortical area 6, 31 and the frontal and motor cortex, respectively (Jones, 1981). These pathways serve to integrate a variety of motor functions and to 'fine tune' movements dictated in a grosser fashion by other brain areas.

The medial pallidal segment also gives rise to several minor projections relating the pallidus to other nuclei which modulate somatic motor function (entopeduncular-nigral fibers terminating in the caudal pars compacta [Grofova, 1975] and releasing GABA [Hattori et al., 1973] have been proposed). Minor entopedunular projections may also serve to integrate the corpus striatum with the limbic system (e.g., a pallidohabenular connection has been reported [Nauta,

1974; Parent and Boucher, 1978]). Since the present study deals predominantly with the globus pallidus proper, i.e., the lateral or external segment, only the outputs from this nucleus will be dis­ cussed in detail.

The Pallidosubthalamic Projection. The topographically or­

ganized pallidosubthalamic fibers terminate in the rostromedial and

centramedial regions of the subthalamic nucleus (Carter and Fibiger,

1978; Nauta, 1979; Carpenter et al., 1981). Pallidal stimulation

generally causes an inraiediate depression of subthalamic neuronal

activity lasting 10-20 msec (Rouzaire-Dubois, Hammond, Humon and

Feger, 1980). This inhibition can be either blocked or decreased by

microiontophoretically applied bicuculline or picrotoxin (Rouzaire-

Dubois et al., 1980), implicating GABA as the transmitter in the

pallidosubthalamic pathway. These results are in agreement with

biochemical studies where a decrease in subthalamic glutamate de­

carboxylase was observed following lesions in the globus pallidus 32

(Fonnum et al., 1978b).

The Pallidonigral Projection. Classically, it was believed that the cells of origin for nigralpedal fibers were predominantly located in the neostriatum. These neurons were thought to provide collaterals to pallidal cells while coursing through the nucleus on the way to the substantia nigra. In recent years, however, investigators using various techniques have provided evidence for a pallidal origin for at least a portion of the nigral afferent projection (McGeer et al.,

1971;Hattori et al., 1973; Grovofa, 1975; Hattori et al., 1975;

Carter and Fibiger, 1978; Nauta, 1979). This pallidal output seems to terminate in both the pars compacta and pars reticulata of the substantia nigra (Carter and Fibiger, 1978; Nauta, 1979).

The transmitter believed to be released from pallidonigral fibers is GABA (McGeer et al., 1971; Hattori et al., 1973). Pallidal activation presumably releases this transmitter onto the dopamine containing neurons of the substantia nigra (in the pars compacta)

(Hattori et al., 1975) to cause a slowing of neuronal activity in the nigra.

Other Pallidal Efferent Projections. Recent studies utilizing

both autoradiographic and histochemical techniques have shown the

globus pallidus to have efferent fibers which terminate in areas of

the brain previously thought to be independent of direct pallidal

influences. These areas include the entopeduncular nucleus (Carter

and Fibiger, 1978), the nucleus reticularis thalami (Carter and

Fibiger, 1978; Nauta, 1979), the caudate nucleus and putamen, and 33 even the neocortex (Nauta, 1979). These tentative projections greatly

increase the complexity of the interrelationships involving the

corpus striatum; however, even if substantiated these fibers are

certainly minor when compared to the pallidosubthalamic projection.

Motor Functions of the Corpus Striatum

Since Wilson's initial description of the motor deficits caused

by lesions in the extrapyramidal system, numerous researchers have

been concerned with characterizing those specific functions which are

under the influence of the basal ganglia. The basal ganglia, and

therefore the corpus striatum, is intimately related to the motor

cortex. This relationship occurs directly via afferents in the

corticostriatal pathway and indirectly via entopeduncular-thalamic-

cortical connections. These anatomical interconnections strongly

implicate the corpus striatum in somatic motor function.

Various approaches have been utilized in attempts to elucidate

the functional role of the corpus striatum in motor behavior. Each

of these approaches has contributed to the understanding of motor

dysfunction and the contributions of the extrapyramidal system in

normal motor activity.

Behavioral Effects of Ablation or Activation of the Corpus Striatum

Generally, small discrete lesions of either the globus pallidus

or the striatum are without obvious long-term motor effects, even

when bilateral. However, when the lesions are sufficiently larger

(but still localized to the area of interest) various dysfunctions

have been reported. 34

Motor Effects of Striatal or Pallidal Lesions

The effects of striatal lesions on motor abilities are generally nondramatic and the reported deficits vary considerably among re­ searchers. This variation is likely to be due to factors such as anatomical differences among species, diffuseness of the structure

(especially in rodents), and the difficulties in avoiding damage to adjacent structures such as the internal capsule and corpus callosum.

One of the earliest studies concerning the effects of striatal injury was done by Mettler and Mettler (1942). These authors reported that bilateral lesions of the caudate nucleus in cats resulted in motor deficiencies such as hypertonia and tremors. Tremors and gross eye movement defects were reported following bilateral caudate lesions in monkeys (Denny-Brown and Yanagisawa, 1976). The eye movement defects were attributed to the loss of an "activating 'set' or 'pump primer' for a certain act" following caudate ablation.

The long term motor deficits caused by large bilateral lesions

in the globus pallidus are minor, and the reported dysfunctions cover a variety of behaviors. In monkeys, bilateral pallidal lesions cause a decrease or fragmentation of normal display behavior (MacLean,

1978), hyperextended and weakened limbs (Ransom and Berry, 1941) and tremors (Kennard, 1944). Hyperactivity during a 12 hour dark cycle was reported in rats with bilateral pallidal lesions, with hypoactivity observed during the 12 hour light cycle (Norton, 1976).

Kainic acid injections in the globus pallidus of monkeys trained to perform various "reaching" tasks caused long term flexor drift and difficulties in executing movements in the contralateral arm (DeLong 35 and Coyle, 1979). In studies where unilateral reversible cooling of the pallidum was conducted in trained monkeys, a disruption of motor performance was observed with a flexion deviation of the contralateral limb similar to that observed with kainic acid injections (Hore,

1977). Electromyograph observations taken during pallidal cooling revealed co-contraction of limb antagonist muscles.

Motor Effects of Striatal or Pallidal Stimulation

Electrical stimulation of the caudate nucleus, where the currents are localized to the nucleus, evokes contraversive turning of the head and body (Forman and Ward, 1957; Laursen, 1962). It has been reported in both cats (Hassler and Dieckmann, 1968) and monkeys

(Delgado, Delgado-Garcia, Amerigo and Grau, 1975) that a contra­ lateral turning of the head can be elicited with unilateral globus pallidus activation.

The Relationship of Neuronal Activity to Movement

With the advancement of electrophysiological techniques the capability of recording neuronal activity in awake behaving animals has become increasingly available. These techniques provide a valuable means by which the functional role of the corpus striatum in motor behavior can be assessed more clearly. Studies monitoring unit activity during movement have confirmed classical views concerning the involvement of these nuclei in. movement and have provided a means by which the specific relation of unit activity to movement as well as the somatotopic organization of these cells can be determined.

Striatal neuronal activity in monkeys significantly increases 36 during performance tasks involving various limb movements (Buser,

Ponderoux and Mereaux, 1974). In a study in which monkeys were trained to perform flexion-extension movements in a visuomotor task, approximately 50% of the observed caudate cells responded during some component of the motor task (Anderson, Aldridge and Murphy, 1976).

In a subsequent study these authors reported that many caudate units

in the monkey demonstrate an altered activity with the appearance of

the visual display in the context of the visuomotor task (Aldridge,

Anderson and Murphy, 1978). In a delayed task paradigm about 26/84

caudate units in monkeys showed activity which correlated with the

delay period (Soltysik, Hull, Buchwald and Fekete, 1975). These

studies indicate that the caudate is indeed involved with motor

behavior and perhaps also acts to provide a neuronal 'bias' for the

initiation of movement, with the movement itself being more directly

controlled by other brain areas.

Neuronal activity in the cat globus pallidus has been studied

during performance of a self-paced elbow-flexion task (Neafsey, Hull

and Buchwald, 1978). Thirty to 40% of the movement-related units

demonstrated modulation of discharge which preceded the self-ini­

tiated flexion movement by more than 500 msec. In monkeys performing

self-paced alternating limb movements many of the recorded pallidal

units exhibited modulation of unit activity relating to limb movements

(DeLong, 1971). Most of the movement-related cells fired in associa­

tion with contralateral limb movement. To determine whether pallidal

cells became activated before the onset of movement monkeys were

trained to perform a visuomotor reaction-time task (DeLong, 1972). 37

An alteration in the activity of many pallidal cells was shown to occur before any detectable change in electromyographic activity of limb muscles. These studies, like those of the striatum, seem to indicate pallidal involvement in the initiation of specific movements by an animal.

Neurons in the globus pallidus demonstrate an alteration of activity relating to extremely discrete movements of the arm or leg, or during chewing and licking (Georgopoulos and DeLong, 1978; DeLong and Georgopoulos, 1979). These neurons are organized somatotopically with cells relating to arm movements generally being located ventral

to those relating to the leg and dorsal to neurons relating to chewing

and licking movements (DeLong and Georgopoulos, 1979).

The Role of the Corpus Striatum in Pharmacologically- Induced Motor Dysfunctions

The concentrations of various neuroregulators in the corpus

striatum are known to be altered in many neurological dysfunctions.

An understanding of the types of transmitters present and their

mechanism of action is essential for understanding the role of the

corpus striatum in normal and abnormal motor behaviors.

Many drugs whose predominant site of action is believed to be

localized within the transmitter system of the corpus striatum cause

profound alterations in the motor capabilities of an animal. An

example of a motor dysfunction produced by exposure to various pharma­

cological agents is catalepsy. Catalepsy is characterized by a loss

of voluntary motion with a plastic rigidity such that an animal can

be placed in bizarre postures, and it will retain the position for an 38 indefinite period of time.

Neuroleptie-Induced Catalepsy

The ability of a drug to induce a cataleptic state in an animal is commonly used as a laboratory indicator of neuroleptic activity.

However, this action is not specific for neuroleptic drugs (Costall and Naylor, 1973) as will be discussed in future sections. Neuro­ leptic catalepsy can be markedly reduced or eliminated by lesions in the corpus striatum (Fog, Randrup and Pakkenberg, 1970; Costall and

Olley, 1971a&b; Koffer, Berney and Hornykiewiez, 1978). Unilateral injections of haloperidol (a dopamine antagonist) into either the caudatoputamen or the globus pallidus cause an ipsilateral circling behavior in rats, with the globus pallidus being the most sensitive

(Costall, Naylor and Olley, 1972). A dose-dependent catalepsy can be observed after bilateral injection of haloperidol in both nuclei of the corpus striatum, with the pallidum again being most sensitive to the drug (Costall et al., 1972). These studies indicate an involve­ ment of the monoaminergic systems of the corpus striatum in neuro­ leptic- induced catalepsy.

Narcotic-Induced Catalepsy

Morphine, in doses greater tlian 5 mg/kg i.p. (expressed as the base), produced a cataleptic state in rats, the intensity of which increased in a dose-dependent fashion with increasing doses of mor­ phine (Costall and Naylor, 1973). The duration of the cataleptic effect was also dose-dependent. Intracisternal injections of 3- endorphin produced a catatonic-like rigidity in rats which was na­ loxone reversible (Bloom, 1973). 39

In 1978 Wilcox and Levitt presented observations which indicated a striatal involvement in opiate-induced rigidity. They demonstrated

that microinjection of naloxone into the head of the caudate nucleus

reversed the cataleptic effects of 80 mg/kg i.p. morphine in rats.

Havemann and colleagues conducted a series of experiments which also

indicated a striatal site for opiate-induced catalepsy (Havemann,

Winkler and Kuschinsky, 1980b; Havemann, Winkler, Gene and Kusehin-

sky, 1980a). These authors demonstrated that intrastriatal injec-

0 ^ tions of morphine, D-ala -met -enkephalinamide and levorphanol in­

duced continuous electromyographical activity in the ipsilateral and

contralateral gastrocnemius-soleus muscle in rats. These effects

were reversed by systemic administration of naloxone (1-2 mg/kg

i.p.) and could not be mimicked by intrastriatal administration of

dextrorphan (an inactive stereoisomer). Therefore, the opiate effect

was stereospecific and mediated via opiate receptors located within

the caudate nucleus. A similar electromyographical activation was

also induced by systemic administration of morphine (15 mg/kg i.p.).

This activation could be reversed by either systemic or intrastriatal

administration of naloxone. Bilateral kainic acid lesions of the

caudate also abolished the electromyographical activation produced by

systemic morphine. These studies present convincing evidence for a

role of the striatum in the catatonic effects of morphine; however,

other authors concede that the predominant cataleptic actions of

morphine are mediated through structures outside the extrapyramidal

system (Pert, 1978; Koffer et al., 1978; Browne, Derrington and

Segal, 1979; Dunstan, Broekkamp and Lloyd, 1980). 40

Purpose and Scope of This Investigation

The corpus striatum serves as the functional center for the extrapyramidal system. The massive afferent projections to the corpus striatum predominantly impinge upon neurons located in the caudate nucleus. The caudate reduces these divergent inputs into two major efferent projections, the striatonigral and the striatopallidal fibers, with the latter receiving the bulk of the caudate projections.

The destinations of the pallidal outputs, like the origins for the striatal afferents, are very diverse anatomically. The corpus striatum, therefore, serves as a complex integrator, "biasing" input messages from, for example, the cortex, substantia nigra and thalamus before relaying these messages on to other regions such as the substantia nigra and subthalamic nucleus.

Numerous neuroregulators are involved in the functions of the corpus striatum. Recently, enkephalins and opiate receptors have also been demonstrated within nuclei of the corpus striatum. The present study was designed to evaluate pharmacologically the actions of an exogenously administered opiate, morphine and/or its antago­ nist, naloxone, on the unit activity of cells located within the corpus striatum. More specifically, the study addressed the fol­

lowing questions: (1) What are the effects of systemieally adminis­

tered morphine on unit activity in the globus pallidus and caudate nucleus? (2) Are there area differences in morphine-induced effects between these two nuclei? (3) Does morphine act on caudate activity

by influencing excitatory inputs, such as cortically-evoked activity?

(4) Is there an enkephalinergie component in the striatopallidal 41 projection? This study consisted of three separate investigations.

The objectives of each investigation will be discussed in further detail in the following sections.

Experiment I: Unit Responses in Globus Pallidus and Caudate Nucleus to Cumulative Doses of Morphine

Opiate actions in the caudate nucleus have been studied by

numerous investigators using a variety of techniques. At the time

this study was conducted opiate effects on pallidal activity were not

characterized. The objectives of this study were two fold: 1) to

examine the effects of incremental doses of systemieally-administered

morphine on spontaneously active single units in the rat globus

pallidus; and 2) to compare the effects of morphine on pallidal units

with effects on units in the caudate nucleus.

Experiment II: The Effects of Morphine on Cortieally-Evoked Unit Activity in the Caudate Nucleus

This study was designed to evaluate the effects of systemieally-

administered morphine on unit activity in the caudate nucleus which

was evoked by stimulating the frontal and motor cortex in rats.

Evoked activity in other brain regions has been shown to be sensitive

to the depressant effects of morphine (for example, the reticular

formation, Hosford and Haigler, 1980). Striatal release of glutamic

acid (the putative neurotransmitter of the eorticostriatal pathway)

is thought to be be modulated presynaptieally by dopamine (Mitchell

and Doggett, 1980). It has been proposed that enkephalins regulate

the release of various transmitters by a presynaptic mechanism (for

example: Biggio, Casu, Corda, DiBello and Gressa, 1978). If an 42 enkephalinergie regulation is present at corticostriatal terminals, then systemieally-administered morphine should be able to alter cortically-evoked activity in the striatum.

Experiment III: Naloxone Effects on Striatally- Induced Suppression of Pallidal Unit Activity

The possibility of an enkephalinergie striatopallidal projection was examined in this study. Caudate stimulation frequently produces inhibition of pallidal activity. If this inhibition is due to the release of enkephalin, then naloxone should reduce the inhibition.

This investigation was designed to test the effects of iontopho­ retically-applied naloxone on caudate-induced suppression of pallidal unit activity. II. METHODS AND PROCEDURES

General Methods

Experimental Animals

Male and female rats of the Sprague-Dawley strain (King Labora­

tories) weighing from 230-475 grams were used in these studies. For

the first two experiments the rats were housed individually following

implant surgery. They were housed in pairs or groups of three for

the last experiment. All animals were maintained under a 12-hour

light-dark cycle. Food (Purina rat chow) and water were provided ad

libitum.

Guide Cannulas and Electrodes

Microelectrode guide cannulas used in the first two investiga­

tions were constructed from 22 and 16 gauge stainless steel needles.

The cannula length was approximately 13-15 mm. Styli were construc­

ted from 30 and 19 gauge needles for insertion into the 22 and 16

gauge cannulas, respectively, to prevent oceulsion.

Insulated tungsten microelectrodes (Frederick Haer & Co.), with

a shaft diameter of .005 inches, were most frequently used in the

first two studies. The electrodes generally used for pallidal re­

cordings had a 25y exposed tip with a factory stated impedance of approximately 4 megohms. Recordings from caudate units were gen­

erally performed with tungsten electrodes having a 5y exposed tip with an impedance of about 12 megohms. These higher impedance elec­ trodes facilitated isolation of the small striatal cells. Stainless steel microelectrodes with characteristics similar to those described

43 44 for the tungsten electrodes were occasionally used, primarily in initial studies, due to the advantages of recording site verification

(to be discussed in subsequent sections).

Seven-barrel micropipettes (R & D Optical Systems, Inc.) were used for mieroiontophoresis. Micropipettes were loaded with strands of fiberglass before pulling in order to facilitate drug filling by capillary action (Tasaki, Tsukahara, Ito, Wayner and Yu, 1968). The tip of the micropipette was broken back to a diameter of approxi­ mately 7y under microscopic control. The central recording barrel was filled with a 4 M NaCl solution saturated with fast green dye.

Drug solutions used in filling the remaining barrels are described in later sections. Impedance of the recording barrels, measured across physiologic saline at 1 KHz by an impedance cheek module (Frederick

Haer & Co.), was determined to be 4.6 ± .7 (S.D.) megohms.

Bipolar stimulating electrodes were constructed from two 200y insulated stainless steel wires. Each wire was soldered to a female connector pin and the pair was twisted together. The electrode ends were bluntly cut. The wire tip separation varied up to 1 mm.

Implantation Procedures - Semichronie Preparations

For implantation of microelectrode guide cannulas and stimu­ lating electrodes, the animals were anesthetized with sodium pento­ barbital, intraperitoneally, in a dose of 50 mg/kg for the male rats or 36 mg/kg for the females. Supplements of anesthesia were provided utilizing ether as needed after atropine pretreatment (5 mg/kg, administered subeutaneously) to control secretions.

The animal's head was placed into a stereotaxic instrument 45

(David Kopf Instruments) and a midline scalp incision was made.

Bregma was considered as zero and all stereotaxically-located co­ ordinates were determined from this zero point. The coordinates for placement of the cannulas and stimulating electrodes are presented in

Table 1. Guide cannulas, stimulating electrodes and stainless steel anchoring screws were fixed in place with Caulk Grip cement and embedded in dental acrylic. Earbar holders (PE tubing) were also permanently embedded into the implant. The rats were allowed at least one week post-surgery recovery time before experimentation was initiated.

Experimental Procedures

Animal Preparations

Semichronie Preparations

Sodium phenobarbital was administered intraperitoneally in a dose of 140 mg/kg at the onset of the experiment. The choice of phenobarbital as the anesthetic for the recording sessions was based on several observations noted during preliminary experiments. For example, since phenobarbital has a long duration of action supple­ mentations were rarely necessary. If supplemental anesthetic was needed, a dose of 10-15% body weight (i.v.) was used. However, no anesthetic was administered during the recording periods. Also, a stable baseline firing rate could be maintained for hours after the initial injection (see Figure 1).

Approximately one hour after phenobarbital administration the animal's head was placed into a stereotaxic instrument with the earbars inserted into the implant earbar holders. This procedure 46

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into the auditory canal. A padded nose clamp was also used. These

steps were necessary because of the light level of anesthesia. The

tail was anesthetized locally by subcutaneous injections of 2% lido-

caine hydrochloride, and a tail vein cannula was inserted for the

administration of the experimental drugs.

Acute Preparations

For microiontophoretic studies the rats were initially anes­

thetized with 400 mg/kg of chloral hydrate intraperitoneally. A tail

vein cannula was inserted, and the animals were subsequently main­

tained at a surgical level of anesthesia by intravenous infusion of

chloral hydrate at approximately 190-210 mg/kg/hr. The trachea was

intubated in order to maintain airway patency. The animal's head was

then placed into a stereotaxic instrument, with the interaural line

set 5 mm below the upper incisor bar. A midline scalp incision was

made and a 3 mm burr hole was drilled through the skull 0.8 mm

anterior and 3 mm lateral to bregma, according to Pellegrino, Pel-

legrino and Cushman (1979). This exposed the dural surface dorsal to

the head of the caudate nucleus and the globus pallidus.

Positioning the Microelectrodes

Microelectrodes were clamped to an electrode holder which was

mounted in a calibrated electrode carrier. Metal electrodes were

visually centered over the guide cannulas and zeroed to the cannula

top in the semichronie preparations. The cannula length was measured

before implantation and therefore the location of the brain surface 49 could be determined. In the acute studies the glass pipettes were zeroed at bregma and subsequently located stereotaxically over the burr hole dorsal to the globus pallidus. The dorsal brain surface was determined by the diminution or disappearance of recorded elec­ trical noise.

Electrodes were lowered by the carrier to a point approximately

1 mm above the area of interest. Further electrode advancement was conducted by means of a Trent-Wells hydraulic mierodrive, which allowed advancement of the microelectrode in increments of 1 y.

Recording, Stimulation and Mieroiontophoresis

Only those action potentials which could be adequately isolated from background activity were used in this study. Single neuron spikes were displayed on a Tektronix 5111 storage oscilloscope after amplification via a Grass P15 AC microelectrode preamplifier and a secondary amplifier. Individual spikes were discriminated with a

Frederick Haer amplitude analyzer, and the window output was fed into a Coulbourn R22-10 printing counter, as well as a cumulative in­ tegrator. A Frederick Haer pulsar stimulator was used to deliver monophasic square wave pulses to a stimulus isolator (W-P Instru­ ments, Inc.). The stimulus isolator, set to deliver a constant cur­ rent, was connected to the stimulating electrodes via a channel selector which allowed individual selection of each bipolar elec­ trode. The evoked activity was displayed on an oscilloscope with the aid of a raster/stepper (W-P Instruments, Inc.). The raster/stepper takes the action potential and converts the signal to a TTL pulse.

The pulse is subsequently visualized as a dot on a storage oscil- 50 loscope with each successive oscilloscope sweep being displayed below

the preceding sweep. Trigger pulses, action potentials and window

pulses were recorded on magnetic tape (Vetter instrumentation re­

corder) for later reference and analysis. A six channel Dagan

current generator was used for drug ejection from the glass pipettes

in the mieroiontophoresis studies. Where appropriate, experimental

data were analyzed by means of a Nieolet MED-80 Data Acquisition and

Analysis System.

For all statistical analysis performed in these experiments a

significance level of p < 0.05 was selected a priori. However, the

probability level obtained from the calculated statistical coeffi­

cient will be presented for each analysis.

Experimental Drugs

Drugs used in this study include the following: atropine sul­

fate. United States Biochemical Corporation; chloral hydrate, Sigma;

ether, J.T. Baker Chemical Co.; glutamic acid (monosodium salt),

Sigma; morphine sulfate, Mallinckrodt; naloxone hydrochloride, Endo

Laboratories; sodium pentobarbital, Sigma; sodium phenobarbital,

Mallinckrodt; and 2% lidocaine hydrochloride (Xyloeaine®), Astra.

All drug doses were calculated as the salt, unless otherwise stated.

The following stains were used: cresyl violet acetate, Eastman; fast

green, Fisher Scientific Company; and neutral red, Fisher Scientific

Company.

Histology

For histological verification of the recording sites in the 51 semichronie studies, a lesion was generally made by applying a cur­ rent of approximately 20 yA to the metal microelectrode (electrode positive) for 45 sec to 1 min 45 sec. If stainless steel electrodes were used, the rats were perfused with a potassium ferrocyanide/10% formalin solution via the ascending aorta. Potassium ferroeyanide reacts with iron ions, which are deposited in the tissue during current passage, to form a blue "spot". The brains were removed, preserved in formalin, frozen, sliced in 50-75y sections, mounted on glass slides and stained with cresyl violet. In those experiments where observable lesions did not result from current application, the position of the recording site was determined by the electrode track and the depth was estimated from the vertical stereotaxic coordinate recorded during the experiment.

In the microiontophoretic studies the stereotaxic position of all neurons was recorded. At the end of each experiment, the verti­ cal location of the micropipette tip was recorded (usually being the same as the last recorded unit) and a negative current of approxi­ mately 40 yA was passed for 10-25 minutes through a pipette barrel containing fast green solution. This procedure deposited the fast green in the vicinity of the pipette tip, and produced a "spot" up to

200y in diameter. The brains were removed and prepared for staining as previously described. The brain slices were stained with either cresyl violet or neutral red. The location of each recording site was determined by using a graticule in the eyepiece of the microscope to measure the distance from the fast green reference point to the recorded position of each unit. 52

Specific Experiments

Experiment I: Unit Responses in Globus Pallidus and Caudate Nucleus to Cumulative Doses of Morphine

Both male and female rats weighing from 240-475 grams were used in this study. Bilateral microelectrode guide cannulas were perma­ nently implanted on the dural surface dorsal to the caudate nucleus or globus pallidus as described above. One week after surgery the rats were lightly anesthetized with sodium phenobarbital and prepared for the recording session. A metal microelectrode was lowered through the guide cannula and a spontaneously active caudate or pallidal cell was located. After a 10- to 15-minute control period of stable activity, morphine sulfate dissolved in physiological saline was injected intravenously in doses of 1, 4, 5, 10 and 10 mg/kg to pro­ vide cumulative doses of 1, 5, 10, 20 and 30 mg/kg. Morphine was administered at 10-minute intervals and the neuronal firing rate was determined for each 60-second interval. Naloxone hydrochloride, 1 mg/kg, dissolved in saline was administered intravenously at the end of each experiment. This dose of naloxone has been shown to antago­ nize the effects of systemieally administered morphine on neuronal activity in rats (Dafny, Brown, Burks and Rigor, 1979). Naloxone antagonism, defined as the return of firing rate at least to control level, was used to verify that morphine's action was on opiate re­ ceptors. Control experiments were identical to the drug experiments except that saline was substituted (in equal volumes) for morphine.

Statistical significance of morphine's effect on single unit activity was determined by analysis of variance comparing treatment effects on the last five one-minute samples obtained during the 53 interval between doses. A morphine effect was considered significant if the cell response met two criteria: (1) at least a 15% change from control and (2) a significant F test (p < 0.05).

Experiment II: The Effects of Morphine on Cortieally-Evoked Unit Activity in the Caudate Nucleus

Male rats weighing 310-380 grams at the time of surgery were used. The animals were permanently implanted with a 16 gauge guide cannula on the dural surface dorsal to the caudate nucleus. Three bipolar stimulating electrodes were embedded in the ipsilateral frontal and motor cortex. After allowing a week post-surgery re­ covery time, the animals were lightly anesthetized with sodium pheno­ barbital and placed into the stereotaxic instrument. A tungsten microelectrode was lowered through the guide cannula and a spon­

taneously active caudate neuron was located. Monophasic square wave pulses (0.25-5 mA; 0.3 msec; 1 Hz) were applied to each stimulating

electrode for cortical activation. The optimal cortical site and

threshold current necessary for driving the unit were determined as

those which produced a consistent stimulus to evoked response ratio without interference of stimulus artifact in the evoked activity.

Before administering drug, a 5-minute control recording of spon­

taneous activity and a control sample of evoked activity (64 stimuli) were obtained. Morphine sulfate, in cumulative doses of 1, 5, 10, 20 and 30 mg/kg, was injected at 10-minute intervals via a tail vein cannula. Naloxone hydrochloride (1 mg/kg i.v.) was given at the end of the experiment. Respiratory rate was recorded (as inspirations/60 sec) 5 minutes after each morphine administration and approximately 2 54 minutes after naloxone. Cortical stimuli (64 stimuli/sample) were

applied beginning 8.5 minutes after each respective dose of morphine

and 1 minute after naloxone administration. Spontaneous activity was

monitored throughout the experiment except during the recording of

evoked activity. A morphine effect on spontaneous rate was con­

sidered significant if the cell response met two criteria: (1) at

least a 15% change from control and (2) a significant F test (p <

0.05). In order to establish criteria for evaluation of drug effect

on evoked activity, a control series (n=10 cells) was performed in

which six repeated samples of 64 stimuli were obtained for each cell.

The total number of evoked spikes for each sample (of 64 stimuli) was

expressed as percent of the evoked activity obtained in the first

sample. Variability in the evoked responses of the 10 cells for each

repetition was determined by calculating the standard deviations.

The standard deviations for the repetitions were large, ranging from

18 to 34%. The 34% value was chosen as the criterion for considering

the drug-induced change in evoked activity as a real effect. The

drug effect on spontaneous or evoked activity was considered "spe­

cific" for opiate receptors if it could be antagonized (i.e., re­

turned to control level) by naloxone.

Experiment III: Naloxone Effects on Striatally-Induced Suppression of Pallidal Activity

Male rats weighing 230-300 grams were anesthetized with 400

mg/kg i.p. chloral hydrate and prepared for the recording sessions as

described above. Two bipolar stimulating electrodes, spaced 0.5 to 1

mm apart, were lowered 1.5 mm into the head of the caudate nucleus. 55

Caudate stimulation was achieved with 0.3 msec monophasic pulses applied at a frequency of 1 Hz to each separate stimulation electrode.

The optimal stimulating electrode was subsequently selected. The

current range used varied from 0.2-4.75 mA. Optimal caudate stimu­

lation current was determined as that which produced a generally

consistent depression of pallidal unit activity.

Drug solutions used for mieroiontophoresis included morphine

sulfate (0.05 M; pH 4.3 ± 0.07 [S.D.]), naloxone hydrochloride (.05

M; pH 4.2 ± 0.1) and glutamic acid (monosodium salt; 0.2 M; pH 6.9 ±

0.03). The impedance of the drug barrels was 12.8 ± 3 megohms. An

additional barrel was filled with a 4 M NaCl solution (saturated with

fast green; 10.7 ± 2 megohms) and was used for automatic balancing of

the current at the tip of the pipette.

The glass pipette was stereotaxically positioned over the globus

pallidus and lowered through the brain tissue to about 1 mm above the

dorsal aspect of the globus pallidus. At this time the iontophoretic

currents for each drug barrel were set and checked by the microion­

tophoretic current generator. The automatic ejection sequencer was

then turned on, which applied current to each barrel serially. The

electrode was advanced by means of a hydraulic mierodrive with the

sequencer in operation until the top of the globus pallidus was

reached. This process prevented drug ions from migrating away from

the electrode tip and theoretically decreased the "warm-up" period

often seen with microiontophoretic application of drugs.

The paradigm generally used in the recording sessions was as

follows: (1) A spontaneously active or glutamate activated (9-25 nA) 56 pallidal cell was located. (2) Determination of caudate-induced inhibition was made. If the cell was not affected by caudate stimu­ lation, it was not subjected to further study. (3) Morphine was microiontophoretically applied (15-120 nA) to the cell. If the unit firing rate was altered by morphine then the next step was conducted; if the rate was not changed, then the cell was eliminated from the

study. (4) A control sample (64-128 stimuli) of caudate-induced

depression of pallidal activity was obtained. (5) Naloxone was microiontophoretically applied for 5-15 minutes and effect on the

morphine-induced depression of activity was subsequently determined.

If naloxone failed to antagonize the effects of morphine, then the

cell was eliminated from the study. (6) The naloxone current which

antagonized microiontophoretically applied morphine was then used to

determine the effect of naloxone on caudate-induced depression of

pallidal activity. Naloxone currents used ranged from 3-30 nA, with

an approximate mode of 5 nA. A sample of the pallidal unit responses

to 64-128 caudate stimulations was obtained during naloxone appli­

cation.

A poststimulus time histogram was generated for each sample of

64 or 128 stimuli, using bin widths of 5, 10 or 15 msec. In order to

establish a treatment effect on the caudate-induced depression of

pallidal activity (i.e., antagonism by naloxone), the following

analysis was conducted (refer to Figure 2): The mean counts per bin

was determined for a period of the control interstimulus interval

occuring 760-850 msec after caudate stimulation. The caudate-induced

depression of pallidal activity was then defined as terminating at 57 58

^

K LTL — mean al W

ru U u H L J inhibitory period 90 m sec

Nal bl Con cts=E Con bl 59 the bin which contained the same number of counts as the control mean counts per bin. Since naloxone-induced changes in interstimulus baseline firing rates might give a false indication of treatment effects, an "expected value" (E) was calculated. E was defined as the number of action potentials (or counts) which would be expected to fall into the inhibitory period if naloxone had no effect on caudate-induced suppression. The number of counts occurring during the 760-850 msec post-stimulus period in the naloxone sample was divided by those counts observed during the same period in the con­

trol sample. This value was multiplied by the number of counts which

occurred during the inhibitory period in the control sample to obtain

E. E was then compared via a paired-t test to the number of counts

actually observed during the inhibitory period in the treatment

sample. Ill. RESULTS

Experiment I: Unit Responses in Globus Pallidus and Caudate Nucleus to Cumulative Doses of Morphine

Globus Pallidus

Twenty-two rats were implanted with guide cannulas overlying the globus pallidus. Twenty-nine cells, verified histologically to be located in the globus pallidus, were successfully recorded. Five of these units served as saline controls and 24 units constituted the treatment population. The pallidal areas from which unit recordings were obtained are presented in Figure 3. A typical recording site within the globus pallidus is shown in Figure 4. Representative action potentials of recorded pallidal cells are presented in Figure

5; A, B and C. The action potentials were biphasic with both nega­

tive and positive initial deflections. The control (i.e., pretreat­ ment) firing rates varied from 0.7-13.5 spikes/sec, with a mean rate

of 5.5 spikes/second.

The patterns of unit activity of pallidal cells fell into three major catagories (Figure 5; D, E and F): continuous regular firing,

irregular firing, and some short bursting activity. The units with a

continuous firing pattern demonstrated both slow and fast activity.

Often the activity of one continuously firing cell was superimposed

upon the activity of another cell with a smaller action potential

amplitude (Figure .5; E). The majority of the small-amplitude units

had a much greater rate of activity than those with large amplitude

action potentials, but the firing patterns of the two cells were

usually similar.

60 61

0.8

02

Figure 3.. Pallidal areas from which unit recordings were obtained.

Drawings of the rat brain transverse sections were taken from

Pellegrino et al., 1979. The numbers to the right of each brain

section denote the anterior-posterior position of that section

(mm), with bregma as zero. CA, anterior commissure; CC, cor­

pus callosum; CI, internal capsule; CO, optic chism; CPU, cau­

date nucleus-putamen; FX, formix; GP, globus pallidus; OT,

optic tract; V, ventricle. Calibration is 1 ram. 62

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In the control experiments the unit activity remained very stable during the periods of saline administration (Figure 6; A).

None of the saline controls met the criteria for a treatment effect

(i.e., at least a 15% change from the untreated rate and a signi­ ficant F test, p<0.05). At the end of the control experiments, naloxone (1 mg/kg, i.v.) was administered to determine its actions on pallidal neuronal activity. In 3 out of 5 units naloxone failed to alter firing frequency. Naloxone caused a decrease in neuronal

activity (greater than 50% from the untreated rate) in one cell, and

in another cell an increase (also greater than 50%) in rate was

observed.

The general types of pallidal neuronal activity response to

systemic morphine are presented in Table 2. Seventy-five percent of

the pallidal cells responded to morphine with a depression of firing

rate which was counteracted by naloxone; 21% were not altered by the

doses of morphine used in this study (1, 5, 10, 20 and 30 mg/kg,

i.v.). Only 1 cell showed a naloxone-antagonized excitation (30%

above pretreatment levels) following morphine treatment.

To determine if the control rate of a pallidal cell was related

to the maximum unit activity response elicited by morphine, a Spear­

man's rank correlation test for nonparametric associations was per­

formed. The association proved to be nonsignificant at the 95% level

of confidence (r = .406, 21 pairs). Also, no correlation was ob- s

served between the wave form (initial positive vs negative deflec­

tion) and control rate or between wave form and response pattern to

morphine. 65

B

SALINE I mg/kg I mg/ng 5 I mg/kg H—10 min- }*—Omin-

D

I mq/kg

Figure 6. Cumulative integrator recordings from spontaneously firing

single units in the globus pallidus. Treatments (either saline or

morphine and naloxone) were administered at ten minute intervals.

Morphine and naloxone doses are in mg/kg, i.v. Saline injections

(i.v.) were in volumes equal to the respective morphine injections,

(A) A representative saline control. (B), (C) and (D) Examples of

pallidal single units which demonstrated a significant depression

of firing rate upon administration of cumulative doses of mor­

phine. 66

Table 2. Response patterns of spontaneous pallidal unit activity to

systemic morphine.

Response Pattern % Total Cells*

Depression Naloxone Antagonized 75%

Stimulation Naloxone Antagonized 4%

Depression Not Antagonized

Stimulation Not Antagonized

No Change 21%

* Total n = 24. 67

Oscilloscope traces from a typical pallidal cell which responded to morphine with a naloxone-antagonized decrease in unit activity are shown in Figure 7. Cumulative integrator recordings showing activity of additional pallidal units which demonstrated a naloxone-antagonized morphine-induced depression are found in Figure 6; B, C and D. These representative units exemplify the inverse relationship often observed when comparing pallidal spontaneous activity with increasing doses of systemieally administered morphine. A dose-response curve using data from those cells whose activity demonstrated a naloxone-antagonized morphine depression (n = 18) is presented in Figure 8. This curve, which had a significant linear regression (F = 39.6, p<0.01), indi­ cates that the magnitude of pallidal activity suppression was related to the dose of morphine administered.

Caudate Nucleus

Eighteen rats were implanted with bilateral cannulas overlying the caudate nucleus. Thirty-three caudate units were recorded; 7

served as saline controls and 26 units were observed during morphine

treatment. The striatal areas from which unit recordings were ob­

tained are shown in Figure 9. A typical recording site within the

caudate nucleus is shown in Figure 10. Representative action po­

tentials are presented in Figure 11 (A, B, C and D).

The majority of the caudate neurons demonstrated very slow

firing rates (20 out of 33 units had a frequency of less than 1 Hz)

with an area mean of 2.0 spikes/second. Bursting activity was often

encountered. The trains usually contained 2 to 4 spikes (see Figure 68

Control

Morphine 1 mg/kg iv 5 mg/kg 10 mg/kg

20mg/kg 30 mg/kg

Naloxone 1 mg/kg iv

Figure 7. A representative pallidal unit which demonstrated a mor­

phine-induced depression. Each trace is a single five-second

sweep. The morphine traces were taken 5 minutes after the ad­

ministration of each respective dose. The naloxone trace was

taken 1 minute after administration of the drug. 69

100 H CAUDATE GLOBUS PALLIDUS o-J Q: o o li. o 50-

oUJ Q^ 25 GL

T ~i r- 5 10 20 30 MORPHINE SULFATE (mg/kg)

Figure 8. Cumulative dose-related effects of morphine on globus pal­

lidus and caudate neurons which demonstrated depression antago­

nized by naloxone. For globus pallidus, n=18, linear regression

p < 0.01. For caudate, n=7; linear regression p < 0.025. 70

3.6 2.8 •'''. CPU \\ "•

3.2 2.6

3.0 24

Figure 9. Areas in the caudate nucleus from which unit recordings

were obtained in Experiment I. Brain illustrations from Pelleg­

rino et al. , 1979. The numbers at the right of each section de­

note the anterior-posterior position (mm), with bregma as zero.

CA, anterior commissure; CC, corpus callosum; CPU, caudate

nucleus-putamen; V, ventricle. Calibration is 1 mm. 71

cd o o

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«•»—«i»**-"«Bi«*

IL 5m»

iL «^L 5 mwc

IL so mMC SOntMC 5 msec

Figure 11. Action potentials from single units located in the caudate

nucleus. (A), (B), (C) and (D) illustrate the action potential

wave duration and form of striatal neurons. Oscilloscope traces

(E) and (F) are single sweeps, and represent the major firing

patterns observed in the caudate nucleus. Trace (G) illustrates

the decreasing amplitude of the action potential often observed

with multiple-spiked bursts in the caudate. Upward deflection

is positive. 73

11; E and F) but, contrary to bursting activity usually observed in the globus pallidus, trains containing 4 and more spikes were also recorded. In these trains the spike amplitude generally demonstrated a progressive decrease with each successive action potential (see

Figure 11; G).

None of the saline controls (n = 7) met the criteria for a treatment effect (i.e., at least a 15% change from the untreated rate and a significant F test, p<0.05). An illustration of caudate unit activity in a control experiment is shown in Figure 12; A. Naloxone, administered at the end of the control experiment, was ineffective in altering the baseline activity of the cells.

The general types of caudate unit response to systemic morphine are presented in Table 3. Only 27% of the caudate cells demonstrated a morphine-induced depression of activity which could be antagonized by naloxone. Eleven percent of the recorded units showed an activa­ tion after morphine that was antagonized by naloxone. Some changes in spontaneous activity met the criteria for a treatment effect after morphine, but these alterations were not antagonized by 1 mg/kg i.v. naloxone. In 42% of the cells, morphine did not significantly alter the rates. No correlation was observed between the response to morphine and the wave form (initial positive vs negative deflection) of the caudate units.

A cumulative integrator recording of one of the three cells whose activity was increased by morphine, with the increase being antagonized by naloxone, is presented in Figure 12; B. The dose- response relationship for this population of cells did not have a 74

II- 10- 9 -

e -

UJ

« 5 - u a 4- 3- 2- Ik ill iiliii iilM SALINE I mg/hg |<-IOr

C 7 •

Figure 12. Cumulative integrator recordings of spontaneously active

units in the caudate nucleus. Treatments were administered intra­

venously at ten-minute intervals. Saline injections were in

volumes equal to the respective morphine injections. (A) A repre­

sentative saline control. (B) An example of a caudate neuron which

demonstrated a significant morphine-induced increase in activity

that was antagonized by naloxone. (C) A caudate unit whose

activity was significantly depressed upon administration of cumu­

lative doses of morphine, with the depression being counteracted

by naloxone. 75

Table 3. Response patterns of spontaneous caudate unit activity to

systemic morphine (Experiment I).

Response Pattern % Total Cells*

Depression Naloxone Antagonized 27%

Stimulation Naloxone Antagonized 11%

Depression Not Antagonized 12%

Stimulation Not Antagonized 8%

No Change ^2%

* Total n = 26 76 significant linear regression. A representative recording for the caudate cells which demonstrated a (naloxone-antagonized) morphine- induced depression of activity is shown in Figure 12; C. The dose- response curve for this population of caudate cells (n = 7) had a significant linear regression (F = 17.9, p<0.025), indicative of a dose-related depression of firing rate (see Figure 8).

Comparing Morphine Effects in the Globus Pallidus

and Caudate Nucleus

In comparing the population of cells sampled from the globus

pallidus and caudate nucleus, the globus pallidus contained more

neurons which responded to morphine with a depression of activity. A

Chi Square analysis comparing the number of cells depressed and not

depressed in the pallidum and caudate was significant (X = 10.36,

p<0.005), clearly verifying area differences in response to systemic

morphine. However, if the population of cells which showed a na­

loxone-antagonized morphine depression are compared in the two areas,

the dose-response relationships are very similar (Figure 8). The

cumulative dose of morphine required to produce a 50% depression from

control level was 20 and 23 mg/kg (as determined from linear re­

gressions of the curves in Figure 8) for the globus pallidus and

caudate nucleus, respectively. The similarity of these doses sug­

gests that for those cells which were depressed by morphine, the

potency of morphine was the same in both areas of the corpus stria­

tum. 77

Experiment II: The Effects of Morphine on Cortieally- Evoked Unit Activity in the Caudate Nucleus

Eighteen rats were implanted with cortical electrodes and a cannula over the ipsilateral caudate nucleus. Cortically-evoked unit activity was recorded in 46 caudate cells. Ten of these cells were used for control experiments, and treatment effects on evoked activity were determined for 36 units. Treatment effects on spontaneous

activity were also monitored in 33 units. Striatal areas from which

recordings were obtained are shown in Figure 13.

Cortical stimulation typically evoked a caudate unit response of

2-20 msec latency with a mode of 5 msec. The latency varied con­

siderably, depending upon the cortical site being stimulated and the

location of the recorded caudate unit. The characteristics of the

evoked response varied from a 1:1 stimulus-response ratio, to a

loosely associated stimulus-response pattern demonstrating both

greater and lesser than a 1:1 ratio (see Figure 14; I and II). The

cells which demonstrated a 1:1 stimulus-response ratio were those

with very slow rates of spontaneous activity (generally less than 1

Hz). In those cells with sufficiently high rates of activity the

evoked response was often followed by an inhibitory phase, usually

lasting approximately 100-250 msec before returning to the control

spontaneous rate (see Figure 14; III B and C).

The general types of spontaneous activity response to systemic

morphine are presented in Table 4. Only one fourth of the cells 78

3.4

2.4

3.2

.8

30

L

Figure 13. Areas in the caudate nucleus from which recordings were ob­

tained in Experiment II. Brain illustrations and anterior-posterior

locations of each section (mm; numbers at the right) according to

Pellegrino et al., 1979. CA, anterior commissure; CC, corpus cal­

losum; CI, internal capsule; CO, optic chiasm; CPU, caudate

nucleus-putamen; LS, lateral septal nucleus; GP, globus pallidus;

V, ventricle. Calibration is 1 mm. 79

B

10 msec

50 msec

Figure 14. Typical examples of the three types of caudate unit re­

sponses to cortical stimulation (I, II, HI). The arrow indicates

the stimulus artifact. Each oscilliscope trace represents 10

superimposed sweeps (lA, IIA, IIIA and B). Each raster stepper

trace represents 64 sweeps (IB, IIB, IIIC). The oscilliscope

traces were obtained during the same recording period as the re­

spective raster traces for each cell. Stimulation parameters:

0.3 msec; 1 Hz; I, 1.5 mA; II, 1.3 mA; III, 1.0 mA. 80

Table 4. Response patterns of spontaneous caudate unit activity to

morphine (Experiment II).

Response Pattern % Total Cells*

Depression 24% Naloxone Antagonized

Stimulation 6% Naloxone Antagonized

Depression 12% Not Antagonized

Stimulation 9% Not Antagonized

No Change 48%

* Total n = 33. 81

recorded from the caudate nucleus demonstrated a dose-dependent morphine depression which was antagonized by naloxone. The majority

of the units were unaffected by morphine. These results are very

similar to those obtained in the previous study concerning effect of

morphine on spontaneous caudate activity. In 56% of the cells,

morphine was ineffective in altering cortically-evoked activity (see

Table 5). Only 3 out of 36 caudate units demonstrated a specific

(naloxone-antagonized) morphine-induced depression of evoked activity,

and 1 out of 36 was specifically stimulated by morphine. One third

of the units demonstrated changes in the evoked activity which met

the criteria for a treatment effect (i.e., at least a 34% change from

control evoked activity; for explanation see METHODS) but which were

not antagonized by the dose of naloxone used in this experiment.

This rather large number of nonspecific responses (i.e., those not

reversed by naloxone) are difficult to interpret physiologically.

Possible explanations will be considered in the DISCUSSION.

The respiratory rate of the experimental animals was determined

for a 60-second period before administering injections, 5 minutes

after each morphine dose and, when possible, about 2 minutes after

naloxone administration. These results are shown in Table 6. Mor­

phine depressed the respiration rate in virtually every instance

tested. This indicates that morphine was pharmacologically active

even in those experiments where no change in striatal spontaneous

and/or evoked activity was observed.

This study indicates that the caudate nucleus contains only a

limited population of units which are influenced by systemieally 82

Table 5. Pattern of morphine-induced change in cortically-evoked

activity of caudate nucleus units.

Response Pattern % Total Cells*

Morphine Depressed Naloxone Antagonized 8%

Morphine Stimulated Naloxone Antagonized 3%

Morphine Depressed Not Antagonized 11%

Morphine Stimulated Not Antagonized 22%

No Change 56%

* Total n = 36. 83

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Experiment III: Naloxone Effects on Striatally-Induced Suppression of Pallidal Unit Activity

Twenty-three pallidal cells whose activity was altered by cau­ date stimulation were recorded from 12 rats. The areas of the globus pallidus from which unit recordings were obtained are presented in

Figure 15.

In approximately 50% of the encountered pallidal cells, caudate stimulation produced an inhibition of activity which lasted 20 to 300 msec. In some neurons this inhibition consisted of a marked arrest of activity, whereas in other cells, the firing was simply attenuated following activation of the caudate (see Figure 16). An initial excitation, usually consisting of one or two spikes, was sometimes observed before the onset of the inhibitory period. Occasionally the inhibitory period was followed by a potentiation of activity which plateaued at a lower rate before the onset of the next stimulus artifact.

In 20 cells, the effect of microiontophoretically applied mor­ phine on unit activity (either spontaneous or glutamate-activated) was assessed. In those units where morphine influenced nondriven pallidal activity, the only response observed was that of a decrease

in firing frequency. The currents used to apply morphine onto pal­

lidal cells and the effects of this drug on firing rates of these 88

CC

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X)| 1.2 ^1

02

CPU

08 CI GP

T CC -02 :i' CPU \\ iCI

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Figure 15. The areas of the globus pallidus from which caudate-induced

suppression of unit activity was observed. Brain sections and an­

terior-posterior locations (in mm; indicated by the numbers of each

section) are from Pellegrino et al., 1979. CA, anterior commissure,

CC, corpus callosum; CI, internal capsule; CO, optic chiasm; CPU,

caudate nucleus-putamen; FX, fornix; LS, lateral septal nucleus; GP,

globus pallidus; OT, optic tract, V, ventricle. Calibration is 1 mm, 89

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In order to establish a current level for naloxone when as­

sessing the antagonist's ability to counteract caudate-induced sup­

pression of pallidal activity, the ability of naloxone to block the

depressant actions of morphine was first determined. Naloxone was

applied to a cell and the firing rate was recorded. Morphine was

subsequently applied at currents which had previously depressed the

cell's activity. If this current of morphine was no longer able to

produce the previously observed depression, then it was determined

that a naloxone antagonism had been demonstrated. These results are

shown in Figure 18. This same naloxone current was subsequently

applied during the sampling period of caudate-induced suppression of

pallidal activity. In those cells (n = 3) where this paradigm was

not followed, the current of naloxone used was that which could be

maximally applied without the demonstration of current effects (e.g.,

changes in action potential amplitude or dramatic changes in activity).

With microiontophoretic application of naloxone, an increase in the

frequency of occurrence of action potentials during caudate-induced

suppression of pallidal activity was often observed. Examples of

naloxone counteracting caudate-induced inhibition are shown in Figure

19; I and II.

In some pallidal cells the baseline firing rate was altered

during naloxone application. To compensate for any influence the

baseline shift might have on spike occurrence during the inhibitory 91 92

MORPHINE CURRENT (nA) 93 94

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CZE :H: 97 period, an "expected" value (E) was calculated (see METHODS). In

Figure 20 the E number of counts (i.e., action potentials) is com­

pared to the number of counts observed during the inhibitory period

with naloxone. For most units, the observed counts were larger than

the expected counts. This is more clearly illustrated in Figure 21,

which depicts the differences between 0 and E. The majority of the

pallidal cells (12 units) responded to naloxone with a substantial

increase in the number of spike occurrences during the inhibitory

period. These cells, therefore, demonstrated a large 0-E difference.

In 4 units naloxone caused a decrease in the frequency of spike

occurrence while there were minimal changes in 7 units. A paired-t

test was performed in which the expected value was compared with the

observed value for each pallidal cell. The test proved significant

at the p<0.05 level ( t = 2.83, 23 pairs). This study therefore

indicates that microiotophoretically applied naloxone significantly

counteracts the suppression of pallidal activity produced by caudate

stimulation. 98

225-1

200-

I 175 UJ O. ^ 150 P CD =F I25H

CO 100-

8 75-

50-

25-

0-"

Figure 20. ' Naloxone-induced alterations'of caudate-induced suppression

of pallidal cells. Each pair of bars represents one pallidal cell

(n=23). The open bars illustrate the number of counts (spikes)

which would be expected to occur during the inhibitory period if

naloxone had no effect (i.e., the calculated E value, described in

METHODS, Figure 2). The closed bars illustrate the number of counts

occurring in the same time period of inhibition during naloxone ap­

plication (i.e., the observed value, "0"). 99 I60n

140-

120-

100- in 80- oO UJ 60-1 oI

40-

20-

0- jrrTTf

-20-

-40-'

Figure 21. The difference between 0 and E counts for pallidal units

whose activity was inhibited by caudate stimulation. E=expected

number of counts; O=observed number of counts (for further explana­

tion see METHODS, Figure 2). Each bar represents one pallidal

cell. IV. DISCUSSION

General Discussion

The Semichronie Preparation

The semichronie preparation described in the present report provides an experimental approach which appears to reduce some of the problems accompanying acute procedures. Some of the observed ad­ vantages include the following: (1) The semichronie preparation

eliminated painful stimuli to the animal during the experimental

sessions and allowed for a light anesthetic level. (2) The light

anesthetic level decreased the synergistic action between the anes­

thetic and morphine on respiratory depression and thereby allowed the

administration of larger morphine doses. This allowed an assessment

of unit activity of the corpus striatum at those dose levels which

cause muscle rigidity in rats. (3) By using an anesthetic with a

long duration of action, a stable baseline firing rate could be

maintained for hours after the inital injection. This allowed

determination of a dose-response relationship over a wide range of

dosages with the assurance that the alteration of unit activity

occurring during the treatment period was not related to fluctuating

baseline activity. (4) The i.v. cannula provided a means of adminis­

tering drugs without disturbing the animal. This is a problem en­

countered in chronic studies, where the animals are handled and

injected for each dose of drug used. This added stress could po­

tentially alter the neuronal activity under investigation. (5) The

problems involved in locating spontaneously firing cells in the corpus striatum have been noted by several investigators (Schultz and

100 101

Ungerstedt, 1978; Frederickson and Norris, 1976; Nicoll, Siggins,

Ling, Bloom and Guillemin, 1977; Huffman and Felpel, 1981). With the lighter anesthesia, spontaneously active single units could be lo­ cated in both the globus pallidus and caudate nucleus. By sampling only spontaneous activity, the homogeneity of the sample population should increase; however, this is also a type of a sampling bias

(Skirboll and Bunney, 1979).

There are also distinct drawbacks using this semichronie pro­ cedure and the cumulative dose-response technique: (1) The effect of anesthetic introduces a variable of undetermined influence on the action of morphine in the corpus striatum. (2) Tachyphalaxis may develop when using cumulative doses such as those administered in this study, and this could influence the ability of morphine to alter unit activity. These problems will be discussed in subsequent sections.

The Influence of Anesthetics on Morphine Actions

When a preparation necessitates the use of an anesthetic, the possibility that the anesthetic will affect the results must be considered. The anesthetics used in this work were phenobarbital for

the semichronie studies and chloral hydrate for the acute experi­ ments.

Phenobarbital

The primary pathway of morphine metabolism in most mammalian

species, including rats, is glucuronidation by the liver (Fujimoto and Way, 1957). It has been recently demonstrated that phenobarbital

(90 mg/kg, i.p.) administered daily for 4 days slightly enhances the 102 production of morphine glucuronide in rats (Liu and Wang, 1980).

Since liver enzyme induction may influence the actions of morphine on unit activity, at least two weeks were allowed to lapse between recording sessions in the semichronie studies.

Several investigations have dealt with the central interactions of barbiturates and opiates. Multiple injections of naloxone (10 mg/kg) were shown to protect against the lethality of phenobarbital in mice (Ho and Ho, 1979). In rats, acutely administered naloxone (1 mg/kg) delayed the development and decreased the duration of the loss of righting reflex caused by 35 mg/kg pentobarbital (Furst, Foldes

and Knoll, 1977). These results suggest that the barbiturates and

opiates may have common underlying mechanisms of action.

Other investigators oppose the contention of similar barbiturate-

opiate mechanisms. Bhargava (1979) reported that neither naloxone

nor naltrexone (1, 5 and 10 mg/kg) administered 5 minutes prior to a

challenge dose of pentobarbital (75 mg/kg) was able to modify the

duration of pentobarbital-induced anesthesia in mice. Morphine (1,

2.5 and 5 mg/kg) was also ineffective in modifying the onset or

duration of anesthesia. Acute pretreatment with phenobarbitone (5-20

mg/kg) did not alter the antinociceptive effect of morphine (Wong,

Roberts and Wai, 1980). Lawrence and Livingston (1981) detected no

effect of acutely administered naloxone on the loss of righting

reflex (naloxone doses of 10, 20 and 50 mg/kg) or analgesia (naloxone

dose up to 20 rag/kg) produced by pentobarbitone.

Even if there is minimal barbiturate-opiate interaction, other

possible influences of the barbiturate anesthetic on experimental 103 results are possible. For example, an anesthetic may selectively silence neuronal activity of a particular subset of cells while leaving another relatively intact. This obviously creates a sampling bias which in turn may bias the results. No preparation provides a totally unbiased sample of cells within an area (e.g., there is a greater probability of recording from a large cell than from a smal­ ler one); therefore, the main consideration of these experiments was to devise a preparation appropriate for the questions being asked.

In studies using semichronie preparations, the objective was to examine alterations of unit activity using dose-response techniques.

This necessitated a light level of anesthesia (to reduce S3mergistic effects between the anesthetic and morphine on respiratory depres­ sion) and a stable baseline of recorded activity. Phenobarbital anesthesia in semichronie animals provided such a preparation.

Chloral hydrate

Because of the size of the multibarreled glass pipette an elec­ trode guide cannula embedded within an implant was not suitable for microiontophoretic experiments. Chloral hydrate had been used successfully in rats as the anesthetic for microiontophoretic appli­ cation of opiates in our laboratory and for similar studies in other laboratories (Haigler, 1976; Nicoll et al., 1977). Chloral hydrate was therefore chosen as the anesthetic for the acute preparations.

As with phenobarbital, the use of chloral hydrate should be considered as an uncontrolled variable which may alter the results.

For example, neuronal sampling bias may be a consequence of chloral 104 hydrate anesthesia. Interactions between chloral hydrate and opiates is also a potential problem. It has been suggested that chloral hydrate has additive effects with microiontophoretically-applied morphine in some brain areas of rats (Haigler, 1976) but reduces the effects of morphine in other areas (Haigler, 1978). In a study characterizing effects of systemieally-administered morphine on caudate unit activity in chloral hydrate anesthetized rats, the doses of morphine necessary to produce a significant alteration of activity were approximately one-tenth those reported in the present study, but the number of cells which were affected by morphine was also less

(Peterson, Napier, Pirch; unpublished results). These results could be explained as responses to morphine by different neuron population subsets which were selectively recorded under the different anes­ thetic conditions. These observations may also indicate a morphine- chloral hydrate interaction. In the present study, however, most recorded pallidal units were suppressed by microiontophoretically- applied morphine. These results were very similar to microionto­ phoretic studies of Huffman and Felpel (1980) with alpha chloralose- urethane anesthetized rats and to the previously presented results concerning pallidal unit responses to systemic morphine in pheno­ barbital anesthetized rats.

Multiple Dosing Schedules

In preliminary experiments discussed by Dafny, Brown, Burks and

Rigor (1979b), it was noted that single doses of morphine were not sufficient to demonstrate differences in response to the opiate among various brain regions. These authors employed five incremental doses 105 of morphine in order to fully characterize the actions of morphine on multiunit activity. Cumulative doses of systemic morphine were also used in the present study. Two questions can be raised concerning this type of dosing schedule: 1) do the second and subsequent treat­ ments reflect the actual dose (5, 10, 20 or 30 mg/kg morphine) and 2) does acute tolerance develop to the latter treatments? Ideally, each dosage level of morphine should be individually tested in separate animals. This standard is difficult to meet in studies using elec­ trophysiological events as indicators of treatment effects. There­ fore, the effects of cumulative doses of morphine were observed for each recorded cell. The aforementioned questions are the concern of the following sections.

Brain Concentrations of Systemically- Administered Morphine in Rats

Dahlstrom and Paalzow (1975) demonstrated that maximum striatal concentration of morphine was obtained in 15-20 minutes after intra­ venous injection (2.5 mg/kg) in unanesthetized rats. These authors also showed that 50% of the peak morphine concentration was still present in the striatum 50 minutes postinjection. In preliminary

experiments where spontaneous pallidal activity was monitored after a

single intravenous injection of morphine, a peak depression was observed 10-15 minutes after morphine administration. These obser­ vations indicate that systemic morphine is able to affect neuronal activity within the 10 minute interdose-interval used in the present

study and that most of the injected morphine is present in the corpus

striatum for the duration of the experiment. 106

Acute Tolerance

In the present study, when morphine was effective in decreasing unit activity, the magnitude of the decrease was generally dose- related (see Figure 8). Dose-related effects of systemic morphine on

striatal multiunit activity following incremental dosing has also

been reported by other investigators (Dafny, Brown, Burks, and Rigor,

1979a; Dafny et al., 1979b). Striatal neurons in the rat have been

reported to develop tachyphylaxis to the specific (naloxone-antago­

nized) depressant effects of microiontophoretically-applied enkepha­

lins on spontaneous and glutamate-evoked activity (Fry, Zieglgansberger

and Herz, 1980). The loss of responsiveness to enkephalins occurred

within a few minutes after repeated or continuous application of the

peptides. Dafny and Rigor (1980) observed similar effects of single

doses of 10 mg/kg i.p. morphine on striatal cells as those obtained

from incremental cumulative studies at 10 mg/kg i.p. in rats. These

results indicate that development of tachyphylaxis with systemieally

administered morphine may be minimal. Perhaps acute tolerance to

morphine is more likely to develop in striatal cells when opiate

receptors are exposed to high concentrations of the drug, such as

those produced with microiontophoretic application. These morphine

concentrations would not be achieved at receptors located in the

caudate with systemic administration of the drug. It is interesting,

however, that no decrease in responsiveness to microiontophoretic

morphine on pallidal cells was observed in the present study.

Excitatory Effects of Morphine

In the present study, the caudate nucleus contained a population 107 of units which demonstrated an increase in spontaneous and/or corti­ cally-evoked activity following systemieally-administered morphine.

Even though a suppression of activity is the predominant response reported in the literature, several investigators have observed excitatory effects subsequent to opiate administration. Both naloxone- antagonized and naloxone-insensitive excitations have been reported, and both were observed in the present study. The morphine-induced changes in neural activity which are antagonized by naloxone should be distinguished from those changes which are not reversed by naloxone

(Klemm, 1981). Therefore, a brief review of previously reported excitatory effects of morphine and possible explanations for both types of excitations will be presented.

Excitatory Effects Antagonized by Naloxone

In awake rats, excitatory and inhibitory multiunit responses to

systemieally-administered morphine were reported for several brain

areas, including the caudate nucleus (Dafny et al., 1979b). Naloxone

antagonized both responses. In a study using decerebrate rats, low

doses of morphine (5 mg/kg) generally excited neurons in the midbrain

reticular formation, whereas high doses (15 mg/kg) generally de­

pressed activity (Mallari and Klemm, 1978). Both of these effects

were blocked by naloxone. Microiontophoretically-applied endorphins

and enkephalins were shown to excite hippocampal cells, with the ex­

citation being blocked by naloxone (Nicoll et al., 1977). Systemic

administration of morphine (15 mg/kg) to rats slightly accelerated

amygdala neuron firing rates in a specific manner (Klemm and Mallari,

1978). Microiontophretically-applied morphine was shown to excite 108 the majority of the recorded Renshaw cells in cats, with naloxone antagonizing this excitation (Duggan, Davies and Hall, 1976). Ex­ citation was the predominant effect observed after opiates were microiontophoretically-applied onto neurons in the nucleus reticu­ laris paragigantocellularis in the rat (Satoh, Akaike and Takagi,

1979). This effect was antagonized by naloxone. In cat spinal and rat cortical neurons, a naloxone-antagonized morphine-induced ac­ celeration of activity was observed (Davis and Dray, 1978). It was speculated that the excitatory effects resulted from activation of a different population of opiate receptors than those which mediated

specific morphine-induced depression of activity.

One possible explanation for morphine-induced excitation is that

opiates depress activity of an inhibitory interneuron which was

tonically suppressing the recorded cell. This phenomenon is called

disinhibition. Disinhibition has been presented as an explanation

for the excitatory effects of morphine in the hippocampus (Ziegans-

berger, French, Siggins, and Bloom, 1979). The proposed interneurons

may be basket cells which use GABA as their neurotransmitter. If one

is recording activity from large pyramidal cells and iontophoreti­

cally applying opiates, the morphology of the hippocampus is such

that diffusion of the drug from the pipette tip could activate opiate

receptors located on the basket cells and inhibit their activity.

This would in turn allow the pyramidal cell to fire more frequently

(Zieglgansberger et al., 1979). In a series of experiments performed

on hippocampal slices by Hoffer and colleagues this theory was sub­

stantiated (Dunwiddie, Mueller, Palmer, Stewart and Hoffer, 1980; 109

Lee, Dunwiddie and Hoffer, 1980). The ability of naloxone to antago­ nize the depressant effect of enkephalins on basket cells and the subsequent pyramidal cell excitation was demonstrated.

Another explanation for opiate stimulation of neuronal activity was presented by Duggan and colleagues (1976). These authors sug­ gested that opiate-induced excitation was mediated by cholinergic receptors. Morphine excited those cells which contained nicotinic receptors for acetylcholine. Naloxone and nalorphine reduced the action of morphine and acetylcholine on these cells but did not antagonize the effects of excitatory amino acids. These results might be particularly significant in interpreting the observations in

the present study since the striatum has been shown to contain nico­

tinic cholinergic receptors (see INTRODUCTION). Nicoll and col­

leagues (Nicoll, Alger and Jahr, 1980) have presented additional

specific opiate actions which could result in enhancement of neuronal

activity. These actions include blockade of dendrodendritic and/or

presynaptic inhibition.

Nonspecific Excitatory Effects

Both excitation and inhibition of cortical activity were demon­

strated in a microiontophoretic study in rats (Satoh, Zieglgansberger,

Fries and Herz, 1974). In those cells where morphine facilitated

unit activity, naloxone application did not reverse the increase.

Excitation and inhibition were also observed in rat brain stem neur­

ons during microiontophoretic application of various opiates (Bradley

and Bramwell, 1977; Hosford and Haigler, 1980). The excitation was

unaffected or potentiated with naloxone application. 110

The possibility that morphine-induced excitation results from

the opiate acting at a different receptor population than that re­

sponsible for naloxone-antagonized depression of activity has been

suggested by Bradley and Bramwell (1977). These authors demonstrated

that naloxone was able to antagonize the inhibitory phase, but not

the excitatory phase of biphasic neuronal responses to morphine.

Even though excitatory effects of morphine may not be mediated by a

receptor which has an affinity for naloxone, these effects may be

physiologically significant (Jacquet, Klee, Rice, lijima and Minami-

kawa, 1977; Haigler and Spring, 1978; Hosford and Haigler, 1980;

Bell, Sharpe and Berry, 1980). For instance, morphine injected into

the periaqueductal gray and mesencephalic reticular formation of rats

produced a behavioral hyperactivity that was not antagonized by

naloxone (Jacquet et al., 1977; Haigler and Spring, 1978). Even

though no direct observation was made by these authors concerning

unit activity changes during the period of behavioral hyperactivity,

these studies demonstrate a functional manifestation of morphine-

induced excitation.

Because of the possible functional significance of each of the

morphine-induced effects on unit activity in the corpus striatum, all

response patterns observed in the present study were reported. To

fully assess the reasons why these various response patterns oc­

curred, further experimentation concerning the events subsequent to morphine-receptor interactions are necessary.

Naloxone and Opiate Receptor Specificity

Naloxone was initially introduced as a narcotic antagonist which Ill lost its specificity for opiate receptors only at doses at least a hundred fold higher than those necessary to block narcotic actions

(Blumberg and Dayton, 1971). Naloxone, like morpine, demonstrates a high affinity for mu opiate receptors, and a lower affinity for delta

(or enkephalin) receptors (Lord et al., 1977; Wuster, Schulz and

Herz, 1979). It is generally conceded that naloxone antagonism is

essential for demonstration of opiate receptor specificity for ef­

fects observed following exposure to opiate agonists. Several in­

vestigators have recently challenged the idea of naloxone specifi­

city, as reviewed by Sawynok, Pinsky and LaBella (1979). These

authors reported that naloxone blocked the antinociceptive actions of

several non-narcotic agents (e.g., nitrous oxide, halothane, acetyl­

choline, haloperidol, somatostatin and reserpine). However, Sawynok

and colleagues (1979) suggested that these seemingly nonspecific

actions of naloxone may result from antagonism of enkephalins re­

leased by the non-narcotic agents.

The possibility that naloxone acts as a GABA-antagonist has also

been presented (Furst et al., 1977; Dingledine, Iversen and Breuker,

1978; for review see Sawynok et al., 1979). Naloxone delayed the

development and decreased the duration of the loss of righting reflex

caused by pentobarbital (Fiirst et al., 1977). Dingledine et al.

(1978) provided three lines of evidence which support the possibility

that naloxone can act as a GABA antagonist: (1) High doses of na­

loxone (100-200 mg/kg, i.p.) caused convulsions in rats. (2) Na-

loxone, morphine, levorphanol and dextrorphan displaced [ H] GABA

from GABA receptor sites in homogenates of human cerebellum, rat 112 cerebellum and rat forebrain with a low potency. (3) Iontophoretic naloxone antagonized GABA-induced depression of activity in the rat olfactory tubercle nucleus accumbens region. In the present study a naloxone dose of 1 mg/kg, i.v., was sufficient to block the depres­ sant effect of morphine in the majority of pallidal cells. This same dose, administered intraperitoneally to awake rats, generally antago­ nized morphine actions on striatal multiunit activity (Dafny et al.,

1979b) . It is not surprising that the doses of naloxone used by

Dingledine and colleagues (1978) produced nonspecific effects since they were greater than one hundred times the concentration shown to be necessary for narcotic antagonism. In our laboratory, we were not able to demonstrate naloxone antagonism (using iontophoretic currents

from 3-30 nA) of the suppresion of pallidal activity produced by

iontophoretically applied GABA (in currents as low as 1 nA) . Law­

rence and Livingston (1981) observed no effects of naloxone (in doses up to 20 mg/kg, i.p.) on the analgesic activity of pentobarbitone (30 mg/kg, i.p.) in rats. The present study supports Lawrence and Living­

ston's observations since in the control experiments of the semi-

chronic preparation, the rats remained anesthetized and respiratory

rates remained at pretreatment levels following naloxone administra­

tion. If the systemic doses of naloxone used in the present study

were sufficiently low as to maintain naloxone specificity, then

perhaps the failure to antagonize morphine-induced effects are due to

morphine initiating a sequence of events which, once activated,

cannot be antagonized by naloxone acting at the opiate receptor. 113

Naloxone Antagonism of Opiates and Subsequent Increases

In Unit Activity

The dose of systemieally administered naloxone used in the first two experiments was 1 mg/kg i.v. In those cells where naloxone counteracted the effects of morphine, 61% of pallidal units and 70% of caudate units demonstrated an increase in activity greater than

15% above control levels. A naloxone-induced hyperactivity has been previously reported for striatal neurons following iontophoretic application of opiates (Fry, Zieglgansberger and Herz, 1980). The ability of naloxone to augment firing rate above control levels following morphine treatment suggests that naloxone may have blocked opiate receptors which were occupied by endogenously released en­ kephalins as well as those occupied by morphine. Low doses of na­

loxone, such as the dose used in the present study, have been shown

to counteract physiological phenomena considered to be a consequence

of enkephalin and/or endorphin release. For example, electrocon-

svulsive shock supposedly causes the release of endorphins (Belenky

and Holaday, 1979). Naloxone, 1 mg/kg i.p., blocked the cardiovas­

cular changes and respiratory depression produced by electroconvul­

sive shock in rats (Belenky and Holaday, 1979). Naloxone, in doses

of 0.1-1.0 mg/kg has been reported to enhance responsitivity to noxious

stimuli (Pomeranz and Chiu, 1976; Frederickson, Burgis and Edwards,

1977; Jacob and Ramabadran, 1977). Low doses of naloxone have also

been shown to antagonize stimulation-induced analgesia in rats (1 mg/kg; Akil, Mayer, and Liebeskind, 1976). In Experiment II morphine-

induced respiratory depression was monitored. Naloxone antagonism of

this depression was often dramatic with rate and/or depth of inspira- 114 tion frequently surpassing control levels (see Table 6). These studies support the contention that the low doses of naloxone used in the present investigation may be sufficient to block actions of endogenously released opiates. If a tonically active enkephalin system modulates activity in the corpus striatum, then the "over­ shooting" observed after naloxone may be due to antagonism of en­ dogenously released opiates.

If the enkephalinergie system of the corpus striatum is toni­ cally active, and if naloxone, in doses of 1 mg/kg, i.v., is suf­ ficient to antagonize endogenous opiates, then an increase in unit activity should be observed when naloxone is administered after

saline as well as when it is given after morphine. However, only 1

out of 5 saline controls in the globus pallidus and none of the 7

caudate controls showed an increase in unit activity following na­

loxone. Similar results were reported by Klemm and Mallari (1978)

where systemic naloxone was ineffective in altering striatal activity

after saline in rats. It was also observed in the present study that

the animal's respiratory rate generally did not change when naloxone

was administered after saline.

To account for the "overshooting" actions of naloxone in treat­

ment conditions, it could be speculated that dual effects (i.e.,

excitation and inhibition) were produced by morphine, and naloxone

was more efficacious in antagonizing the depressant effect. The

excitation produced by naloxone following morphine treatment would

reflect both the disinhibitory actions of naloxone and the excitatory

effects of morphine. The nonspecific (i.e., not antagonized by na- 115 loxone), excitatory actions of morphine would not be present in the saline controls (i.e., where saline was substituted for morphine); consequently, naloxone would not be likely to precipitate increases in neuronal activity. Such morphine-naloxone interactions have been postulated to explain the production of withdrawal symptoms by na­ loxone following a single injection of morphine (Jaquet et al.,

1979; Stevens and Klemm, 1979).

It has recently been shown that analgesic doses of morphine may alter membrane mechanisms, thereby reducing postsynaptic efficacy of

GABA (Moises, Yeh and Woodward, 1981; Werz, Baum, Young and Mac- donald, 1981). This decreased GABA efficacy was expressed as an increase in unit activity subsequent to morphine administration. The excitation was not antagonized by naloxone, and levorphanol and dextrorphan were equipotent in blocking the GABA responses. These results indicated that the ability of morphine to reduce GABA re­ sponses probably was not mediated by opiate receptors, since these effects were not antagonized by naloxone nor were they stereospe- cific. Similar mechanisms may be involved in the corpus striatum, which would result in the observed excitatory effects of naloxone following morphine treatments.

Even though non-drug levels of activity were frequently exceeded by activity recorded during postmorphine naloxone treatment, the underlying mechanisms remain unclear. Each of the aforementioned proposals shed light on the enigma; however, none explains all of the naloxone-induced response patterns observed in this study. Adequate explanations may not be possible until the receptor (and/or membrane) 116 actions of morphine and naloxone are more clearly elucidated.

Specific Experiments

Experiment I: Unit Respones in Globus Pallidus and Caudate Nucleus to Cumulative Doses of Morphine

Globus Pallidus

The patterns of pallidal unit activity observed in this study

(see Figure 5; D, E and F) have also been observed in awake monkeys

(Filion, 1979) and in paralyzed, awake cats (Noda et al., 1968).

Noda and colleagues (1968) described large amplitude pallidal units with a slow activity rate being superimposed upon the activity of another pallidal cell with a smaller spike amplitude and a faster firing rate. This phenomenon was also observed in the present study in the globus pallidus.

Spontaneous neuronal activity in the globus pallidus demon­ strated a dose-related depression subsequent to intravenously-adminis­ tered morphine. The depression was shown to be due to morphine activating opiate receptors since the depression was antagonized by naloxone. The location of opiate receptors responsible for morphine- induced suppression may be within the globus pallidus (Pert et al.,

1976), in the caudate nucleus (Pert and Snyder, 1973) which directly influences pallidal activity (Fox and Rafols, 1976) or in other areas of the brain which indirectly influence pallidal activity. The depression of activity observed following microiontophoretic applica­ tions of morphine in the pallidus (see RESULTS, Experiment III) indicates that at least a portion of the depression seen after systemic morphine could be attributed to intrapallidal opiate re- 117 ceptors. Recent studies by Huffman and colleagues have also demon­ strated naloxone-antagonized suppression of pallidal activity with microiontophoretically-applied morphine and methionine-enkephalin

(Huffman and Felpel, 1981; Frey and Huffman, 1981). These authors reported that 65% of the glutamate-activated cells and 79% of the spontaneously active units were depressed by morphine. None of the pallidal neurons were reported to be stimulated by morphine. These results are similar to the present observations following either systemieally-administered or microiontophoretically-applied morphine.

It therefore appears that exogenously-administered morphine can act on opiate receptors located within the globus pallidus to cause a depression of pallidal unit activity.

The functional significance of these receptors may relate to some of the previously mentioned motor activities which have been attributed to the globus pallidus. For example, following stimula­ tion of the caudate nucleus a contralateral head turn is observed in rats which is dependent in part upon the integrity of the striato­ pallidal pathway (Slater and Longman, 1980). Intrapallidal injec­ tions of ethylketocyclazocine (a kappa opiate receptor agonist) caused a pronounced, naloxone-sensitive slowing of the motor re­ sponse. Intrapallidal injections of morphine or leucine-enkephalin had no effect on the response. This indicates that pallidal kappa opiate receptors mediate head turning motor responses. Behavioral responses other than head tuning might be sensitive to changes in pallidal activity induced by morphine. Moroni and colleagues have shown that subcutaneously-administered morphine or 3-endorphin in- 118 jected intraventricularly increases the turnover rate of GABA in the

globus pallidus (Moroni, Cheney, Peralta and Costa, 1978). These

authors contend that the increase GABA turnover elicited by the

opiate agonists is associated with catalepsy since muscimol caused a

dose-related catalepsy when injected intrapallidally in rats. Even

though Moroni's study did not demonstrate that the increase in GABA

turnover was due to an activation of opiate receptors located within

the pallidus, these results do allow for such a possibility. It has

been shown that morphine is able to bind to receptors in the globus

pallidus (Kuhar et al., 1973) and it is generally believed that mor­

phine is a potent mu receptor agonist (Martin et al., 1976). There­

fore, perhaps the depression of pallidal unit activity by morphine is

due to the opiate acting at a mu receptor to subsequently cause an

increase in GABA turnover. The released GABA could then result in a

decrease in firing rates of the recorded postsynaptic cell. Assuming

that the premise of this conjecture is true the catalepsy reported by

Moroni et al. (1978) may indeed involve an activation of mu receptors

which would result in a suppression of pallidal activity.

Caudate Nucleus

The firing patterns of spontaneous caudate activity recorded in

this experiment are described in RESULTS (see Figure 11; E, F and G) .

Similar firing patterns for striatal neurons have been reported in

pentobarbital anesthetized rats (Lee, Wong and Chan, 1976) and in

paralyzed, awake cats (Levine, Hull, Buchwald and Villablanca, 1974b)

Numerous investigations have been conducted concerning the ef­

fects of opiates on neuronal activity in the striatum. In one study, 119 microiontophoretically-applied enkephalin depressed the activity of 6

out of 14 caudate cells and morphine depressed 3 out of 3 caudate

cells (Frederickson and Norris, 1976). It is unclear how many of the

cells were tested for naloxone sensitivity. Naloxone antagonism was

demonstrated in 2 of the cells whose activity was depressed by en­

kephalin. In another study, microiontophoretically-applied met-

enkephalin inhibited activity in 83% of the tested striatal cells, 3-

endorphin depressed activity in 86%, and normorphine caused inhi­

bition of 73% of the cells (Nicoll et al., 1977). Naloxone antago­

nized the opiate actions in the majority of cells tested. Using

pentobarbital anesthetized rats, Lee et al. (1976) reported that all

of the recorded caudate cells were inhibited by intracarotid in­

jections of morphine in doses up to 5.5 mg/kg. Similar morphine-

induced suppression of all recorded caudate cells was also reported

in subsequent studies by these authors (Lee, Wong and Chan, 1977;

Finnerty and Chan, 1981). This suppressive effect was antagonized by

naloxone. The median change from control rate after morphine (15

mg/kg, i.p.) reported for 15 striatal cells in paralyzed, unanes­

thetized rats was a 37.5% decrease in activity (Klemm and Mallari,

1978). After morphine, naloxone antagonism resulted in a 4.4% in­

crease in activity above control levels. In a study on 22 multiunit

recordings in awake rats, Dafny et al. (1979a) showed that 9 multi-

units demonstrated a naloxone-antagonized, morphine-induced depres­

sion of activity. Four recordings showed an increase in activity

which was antagonized by naloxone. Three recordings were nonspe-

cifically depressed and 6 were not changed after 10 mg/kg morphine 120 i.p. In additional studies using incremental doses of morphine

(ranging from .5 to 30 mg/kg, i.p.) in awake rats, Dafny et al.

(1979b) observed four response patterns of striatal multiunit activity to morphine. These patterns included both a decrease and an increase in activity. Naloxone (1 mg/kg i.p.) antagonized most of these responses. In a more recent study, Dafny and Rigor (1980) reported three types of multiunit response patterns to incremental doses of morphine: (1) a dose-related decrease (11 recordings); (2) a de­ crease in activity with low doses of morphine, with higher doses causing an increase in activity (6 recordings); and (3) increased activity with low morphine doses followed by an activity decrease

during higher morphine doses (9 recordings). In 6 recordings, mor­

phine was ineffective in altering multiunit activity. Naloxone

generally antagonized the morphine-induced effects.

The present study, in conjunction with the aforementioned in­

vestigations, demonstrates that opiates influence striatal neuronal

events. With the exception of the work by Lee and collaborators (Lee

et al., 1976; Lee et al., 1977; Finnerty and Chan, 1981), a variety

of neuronal response patterns to morphine has been reported. This

variety was also observed in the present investigation. In an

attempt to explain the different response patterns, Dafney et al.

(1979b) proposed that each pattern may be a result of morphine acting

on different cell types within different neuronal organizations. The

involvement of different opiate receptor populations and actions on

nonspecific receptors may also explain the gamut of observed response

patterns to morphine. The incongruity among study results may be due 121 to the influence of anesthetic and/or the animal preparation used.

Both of these influences have been shown to modify neuronal responses to morphine (Linseman, 1980). Differences in criteria set for a treatment effect (in many studies no such standard was even stated) could also account for the variety of response patterns reported.

Comparing Morphine Effects in the Globus Pallidus and Caudate Nucleus

With respect to the number of cells which demonstrated a signi­

ficant effect subsequent to morphine administration, a difference in

response patterns was observed between the globus pallidus and cau­

date nucleus. The potency of systemic morphine (i.e., the dose which

caused a 50% depression in rate from control level) was, however,

very similar for the two nuclei. This similarity in potency may

reflect similar affinities for the opiate receptor in the two nuclei.

On the other hand, the potency similarity may be due to an alteration

of neuronal activity of a common afferent input to the pallidus and

caudate. A morphine alteration of afferents could be reflected as a

change in the recorded electrophysiological events of these nuclei.

The dopaminergic fibers of the substantia nigra project to the

caudate nucleus with collateral connections in the globus pallidus

(Lindvall and Bjorklund, 1979). Systemic morphine increased activity

of substantia nigra, pars compacta neurons, while causing simul­

taneously recorded striatal cells to decrease firing rates (Lee, Wong

and Chan, 1977). The inhibitory effect of morphine on caudate neu­

ronal activity was prevented by pretreatment with pimozide (a dopa­

mine antagonist). Microinjection of morphine directly into the 122 substantia nigra, zona compacta induced a naloxone-reversible sup­ pression of striatal unit activity (Finnerty and Chan, 1981). These results indicate that the effects of systemieally administered mor­ phine on caudate spontaneous activity may be mediated, at least in part, by an activation of the dopamine-containing nigrostriatal pathway. Since collaterals from the nigrostriatal fibers terminate in the globus pallidus, a similar mechanism may also be involved in morphine-induced depression of activity observed in pallidal cells.

Another "indirect" mechanism by which morphine could similarly influence the activity of pallidal and caudate cells involves direct opiate modulation of dopamine terminals. The dopaminergic nerve endings in the striatum possess a large population of opiate re­ ceptors (Pollard, Llorens-Cortes and Schwartz, 1977). When the receptors were activated by mu receptor agonists a dose-dependent increase in dihydroxyphenylacetic acid (DOPAC) and homovanillic acid

(HVA) was observed in the rat striatum (Wood, Stotland, Richard and

Rackham, 1980; for review see Iwamoto and Way, 1979). These in­ creases in dopamine metabolites were prevented by naloxone pretreat­ ment. The increase in dopamine turnover induced by opiates is pre­ sumably mediated by opiate receptors located on dopaminergic nerve terminals, since intrastriatal opiate administration elicited similar increases in striatal dopamine metabolites (Biggio, Casu, Corda,

DiBello, and Gressa, 1978). If the dopamine terminals of the nigro­ striatal fibers in the caudate nucleus are modulated by enkephalins, then perhaps a similar modulation occurs at the dopamine terminals of the nigrostriatal fiber collaterals in the globus pallidus. If one 123 contends that dopamine can serve as an inhibitory transmitter in the corpus striatum then the morphine-induced increase in dopamine re­ lease would cause a suppression of neuronal activity. However, if dopamine has excitatory functions then increases in dopamine release by morphine would potentiate spontaneous activity. The excitatory

theory of dopamine function might explain the occurrence of morphine-

induced increases of activity in the striatum.

The differences in the number of cells which were significantly

depressed by morphine in the two nuclei might be a reflection of the

amount of interneuronal modification of dopaminergic (and/or other

neurotransmitter) actions. The caudate has a preponderance of inter­

neurons. Influences by interneurons on the rate effects of morphine

might explain the variety of morphine-induced response patterns

observed in the caudate. Likewise, the consistency of pallidal

responses to morphine may reflect the scarcity of interneurons lo­

cated in the globus pallidus.

Experiment II: The Effects of Morphine on Cortieally-Evoked Unit Activity in the Caudate Nucleus

The types of striatal response patterns to cortical stimulation

observed in this study (see Figure 15; I, II and III) agree with

reported studies on cortically-evoked caudate unit activity in

chloral hydrate (Schultz and Ungerstedt, 1978) and penthrane (Spen­

cer, 1976) anesthetized rats. The latencies for the evoked response

and the subsequent inhibition correspond to the EPSP-IPSP sequence

recorded from rat striatal neurons following cortical stimulation

(VanderMaelen and Kitai, 1980). 124

Spontaneous and cortically-evoked caudate unit activity were monitored following incremental systemic doses of morphine. Actions of morphine on spontaneous rates were similar to the previous study in that about 25% of the cells demonstrated a decrease in activity after morphine which was antagonized by naloxone.

Cortically-evoked activity showed a specific morphine-induced

depression in only 8% of the cells. This indicates that the action

of morphine on spontaneous activity does not correlate with its

effect on evoked activity. Since some of the cells whose cortically-

evoked activity was unaltered by morphine did show a decrease in

spontaneous activity after the drug, one might conclude that the

influences of driven cortical inputs were able to supersede the

ability of morphine to attentuate spontaneous activity. Such a

phenomenon would occur if the termination of cortical inputs on

striatal cells occur more proximal to the soma than those inputs or

receptors which are sensitive to morphine. Differential effects of

opiates on spontaneous and evoked activity have also been demon­

strated in the reticular formation in rats (Hosford and Haigler,

1980).

Schultz and Ungerstedt (1978) presented a study characterizing

the cortically-evoked responses of caudate neurons. The authors

recommended this method of recording activity in neuropharmacological

investigations of the caudate nucleus since the spontaneous rate of

caudate neurons is so low. However, the data obtained in the present

study indicate a necessity for caution when extrapolating pharma­

cological results from evoked activity to possible treatment effects 125 on spontaneous activity.

Fry and ZieglgKnsberger (1979) studied the effects of microion­ tophoretically-applied methionine-enkephalin on responses of striatal neurons evoked by cortical stimulation. Iontophoretically-applied enkephalin was reported to inhibit spontaneous activity in 14 out of

20 striatal cells at currents which caused no or only slight at­ tenuation of cortically-evoked activity. These observations con­ trasted with the results obtained during GABA application, where both activities were depressed.

The work by Fry and Zieglgansberger (1979) in conjunction with the present study indicates that opiates generally do not influence striatal activities produced by cortical stimulation. The failure of microiontophoretically-applied opiates to alter cortically-evoked caudate activity indicates that opiate receptors located within the caudate are not involved in modulation of cortical inputs. The failure of systemieally administered morphine to alter evoked activity indicates that the effect of morphine on either the cortex or the caudate, is not reflected as a change in the cortically-evoked activity in the caudate. Therefore, actions of systemieally administered morphine on caudate activity are probably not mediated through the ability of cortical inputs to influence striatal neurons.

Experiment III: Naloxone Effects on Striatally-Induced Suppression of Pallidal Unit Activity

The response patterns of pallidal cells to caudate stimulation observed in this study (see Figure 18) correspond to the patterns of intracellularly recorded postsynaptic potentials reported for monkeys 126

(Ohye et al., 1976) and for cats (Malliani and Purpura, 1967; Levine et al., 1974a). Levine et al. Cl974a) reported that 50% of the recorded pallidal cells responded to caudate stimulation. Fifty-nine percent of the cells responded with an EPSP-IPSP sequence, and 34% demonstrated only IPSPs. These authors also noted variability in evoked potential durations (4-100 msec for EPSPs, 20-600 msec for

IPSPs). The characteristics of the extracellularly-recorded inhi­ bition of pallidal activity observed in this study were very similar to those intracellularly-recorded potentials.

The effect of microiontophoretically-applied naloxone on the inhibition of pallidal activity following caudate stimulation was assessed in this study. Generally, naloxone increased the frequency of spike occurrence during the inhibitory period at currents which were demonstrated to block depressant effects of iontophoretically- applied morphine. These data lend electrophysiological and pharmaco­ logical credence to the immunofluorescence studies of Brann and Emson

(1980) which indicated the presence of enkephalinergie striatopal­ lidal fibers.

Naloxone was generally not able to totally eliminate the caudate- induced inhibition. This could be due to the activation of striato­ pallidal fibers which contain inhibitory transmitters other than enkephalin (e.g., GABAergic fibers). The pallidal inhibition would then be produced by more than one transmitter, and blockade of the enkephalinergie component would not be sufficient to totally elimi­ nate the suppression.

In four pallidal cells, the caudate-induced inhibition was 127 enhanced during naloxone application. Various experimental parameters were noted in an attempt to characterize and subsequently explain these responses to naloxone. Three of the cells were in the ventral anterior pallidum, while one cell was more medially located. The action potential amplitudes were similar to the rest of the sampled cells, and the currents used for caudate activation ranged from 1-4 mA. The duration of the caudate-induced inhibition varied among the four cells but fell within the range of that observed in the re­ mainder of the recorded pallidal cells. Two cells demonstrated regular firing patterns while the other two cells had bursting activity. There was a range of sensitivity to iontophoretic morphine among these cells, but the currents of naloxone necessary to block morphine-induced rate depression were generally higher (10, 10, 20 and 30 nA) than those required in other pallidal cells in the present studies.

Even though none of the above characteristics would provide a basis for grouping these four units together, it is possible that these cells are the small, sparsely located pallidal interneurons.

Because of the small size and sparsity of occurrence, the cells would be infrequently encountered by an electrode. Pallidal interneurons may not receive the same input influences as the medium sized neurons

(for review see Pasik, Pasik and DiFiglia, 1980) and therefore may not respond to naloxone in a similar fashion.

Another possible explanation for the observed responses to naloxone involves the position of the microelectrode in reference to

synaptic contacts of various inputs on the recorded pallidal cell. 128

It is most probable that recordings will be obtained from the largest segment of the most frequently occurring neuronal element. In the globus pallidus this would be the soma of the medium-sized efferent neurons. The cell bodies of these cells probably receive common input influences (Pasik et al., 1980). Microiontophoretically- applied naloxone presumably acts on opiate receptors located on the recorded cell, and/or on opiate receptors which may be located on presynaptic terminals which are within the "diffusion distance" of naloxone from the electrode tip. This situation would lead to a type

of pallidal activity response to naloxone, where the magnitude of the

response would depend on the number of opiate receptors blocked by

the antagonist. In some instances another portion of a pallidal cell may be recorded. For example, the dendritic trunk of the medium-

sized neurons would not be as frequently encountered as the soma.

The dendritic trunk (and not the soma) receives GABAergic fibers from

the caudate (Pasik et al., 1980). The response to naloxone observed

in this situation might be different from those observed when re­

cording from the soma because of the different input influences of

the cell in the vicinity of the electrode tip. Therefore, a continuum

of responses would be observed during naloxone application. The

direction and magnitude of the responses would be dictated by the

probabilities involved with electrode placement, by the location of

each input influence on the recorded cell, and by the diffusion

distance and number of applied molecules of iontophoresed naloxone.

This study demonstrates that, in the majority of pallidal cells

whose activity was suppressed following caudate stimulation, microion- 129 tophoretically-applied naloxone was able to attenuate the suppres­ sion. If naloxone antagonism of an electrophysiological event demon­ strates an enkephalinergie involvement in that event, then it can be postulated that caudate stimulation causes the release of enkephalins onto efferent pallidal cells which in turn suppresses the activity of these cells.

Significance

The present series of experiments is submitted as a contribution toward elucidating opiate actions in the corpus striatum. Charac­ terization of opiate involvement in the corpus striatum is an es­ sential link in understanding the functional role of enkephalins in the central nervous system. Considerable amounts of data have been collected concerning the effects of opiates on neuronal activity in the striatum; however, at the time the present research was conducted opiate actions on pallidal neuronal events had not been characterized.

Experiment I describes the alterations in pallidal activity following systemic administration of a range of morphine doses. The results obtained in the globus pallidus were compared to the effects of systemic morphine in a closely related, more characterized area, the caudate nucleus. No such comparison has been reported in the litera­ ture. It was observed that the responses to morphine of neurons located in the globus pallidus were distinctly different from those in the caudate nucleus. Area differences in response to morphine indicate that the pallidus and caudate might have separate functions in the behaviors manifested after systemic administration of mor­ phine. 130

The author is not aware of previous studies dealing with the influence of systemieally administered morphine on cortically-evoked unit activity in the caudate. The results of this study indicate that cortically-evoked activity does not respond to morphine in the same fashion as does spontaneous caudate activity. These results also suggest that the corticostriatal pathway may not be involved in the actions of systemic morphine in the caudate nucleus.

One might speculate that differences in responses of spontaneous activity to morphine reflect the extent of excitatory inputs to nuclei of the corpus striatum. For example, since the cortex sends a preponderance of excitatory inputs to the caudate, and since corti­ cally-evoked activity is generally uneffected by morphine, then perhaps one of the reasons why morphine-induced suppression is a predominant response in the globus pallidus but not the striatum is a lack of "direct" cortical control on pallidal cells.

There is considerable debate as to the presence of an enkephalin­ ergie component in the striatopallidal pathway. Support for enkep­ halinergie fibers projecting from the caudate to the pallidus is based on biochemical and anatomical evidence. Experiment III investi­ gated, with extracellular recording and microiontophoretic tech­ niques, the possibility that enkephalin is a transmitter in this pathway. Naloxone was shown to increase the frequency of spike occurrence during the caudate-induced inhibition of pallidal activity.

This supports the previously mentioned evidence for a striatopallidal enkephalin projection.

Endogenous opiates located in the basal ganglia have been pro- 131 posed to be involved in the etiology of several motor dysfunctions.

One such dysfunction is Parkinson's disease. Naloxone administration has been shown to provide significant clinical improvement of Parkin­ sonian symptoms (Vardi, Flechter, Regev, Borenstein and Shapira,

1979). A decrease in naloxone binding has been demonstrated in post­ mortem striatal tissue obtained from patients suffering from Parkin­

son's disease (Reisine, Rossor, Spokes, Iversen and Yamamura, 1979).

The studies cited above provided clinical evidence for a role of

the endogenous opiates in basal ganglia function and in the under­

lying symptomatology of Parkinson's disease. This work has provided

additional and new electrophysiological information regarding the

actions of the opiates in the corpus striatum. The nature of the

correlation between electrophysiological events, enkephalinergie

function and the symptomatology of Parkinson's disease and/or other

dysfunctions attributed to the corpus striatum remains to be eluci­

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