CHARACTERIZATION OF THE SUBTYPES OF SIGMA RECEPTORS: ELECTROPHYSIOLOGICAL STUDIES IN THE RAT DORSAL HIPPOCAMPUS

Richard BERGERON

Student number: 9002559

Neurobiological Psychiat ry Unit and

Department of Neurology and Neurosurgery

McGill University

Montréal, Québec, Canada

1996

Thesis subrnitted for Ph.D. in Neuroscience

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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. The entire work, practical and theoretical, presented in this Ph.D. thesis was achieved at the Neurobiologicai Pcychiatry Unit of McGill University under the supervision of Drs.

Claude de Montigny and Guy Debonnel. 1 wish to thank both of my supervisors for providing me with such a stimulating milieu. Their exceptional personal competence in neuroscience and their contagious enthusiasm were greatly appreciated. They were very kind in accepting me as a graduate student.

1 wish ro thank Lise Martin for secretarial assistance, Claude Bouchard for his help and advice in preparing the slides and figures, and Doreen Wiggins for correcting my English. 1 would like ro thank al1 the graduate students and postdoctoral fellows, who have been working with me during these past yean, for the discriminating scientific environment they created around me. 1 would like to express my gratitude to Diane and Michel for their practical support in providing me with the use of their apartment for the duration of this thesis. In advance I would like tu thank the members of rny comrnittee for their rime evaluating my t hesis.

Finally, 1 would like to thank Dominique my wife, and Noérnie my daughter, who have been very understanding and suppoxtive dl through these years. For mosr weekends in the last five years 1 have been absent from home from six o'clock in the morning until late afternoon. This Ph.D. has been a project of our whole family. PREFACE

The review of the literature and the studies presented in this Ph.D. thesis concern the characterization of the subtypes of the sigma (O) receptors.

Eight experimental series are presented in this thesis. From these results eight original articles are published, are in press or have been submitted for publication. 1 have done al1 the expenmental work, analyzed the statistics, drawn the figures and wrinen the manuscripts for six articles (Chapters 2,3,6,7,8 and 9), and 1 completed ar least 50% of the experimental work reported in the remaining two articles (Chapters 4 and 5).

It is the policy of the Faculty of McGill University to allow the student to include as chapters original publications concerning the thesis research project. The Faculty regdations cm be summarised as foliows:

Candidates have the option, subject to the approvul of their Department, of

including, as pan of their thesis, copies of the text of a pape+) submitted /or

publication, or the clearly-duplicated text of published pape+), provded that these

copies are bound as an integrdl part of the thesis. Ifthis option is chosen, connecting

tsrts, provzding logical bridges between the different papers, are mandatory The

thesis must still confonn to aZ! othm requiremmts of the "Guidelines Conceming

Thesis Prtpration" and should be in a literav fom that is more than a mere

collection of manuscripts published or to be publisbed. 7he thesis must inchde, as

separate chapters or sections: (1) a table of contents,

(2) a gmeral abstract in Englzsh and hch,

(3) an introduction whicb clearly States the rationale and

o&ctives of the stztdy,

(4) a comprehmsive review of the background lirerature to the

suhect of the thesis, whm this review is appropriate,

(5) a jnai overall concl~iszonand/or summary.

Additional material (procedural and design data, as weIl as desc+tùns of equipment used) must be provided whme appropria te and in suficien t detail (e.g. in appendices) to aZZow a ciear and precise judgemmt to be made of the importance and originality of the researcb reported in the thesis.

In the case of manuscripts coautbored by the candidate and otbers, the candidate is required to make an expiicit statmmt in the theris of who contributed to sucb work and to what ewtmt; supaiisors must attest to the accuracy of such claims at the Ph.D. oral defense. Since the tmk of the eraminers is made more difimit in tbese cases, it is in the candidate's interest to make perfectiy cieur the responsibilities of the differmt authors of coauthored papers. III LIST OF PUBLICATIONS

Published Abstracts

1. BERGERON. R., DEBONNEL, G. and de MONTIGNY, C. Modulation of the response of rat hippocampal pyramidal neurons to NMDA by antidepressant sigma ligands: Evidence for different subtypes of sigma receptors. ~~111'~Collegium Internationale Neuropsychopharmacologicum, 18: 0-12-20, 1992.

2. BERGERON, R., DEBONNEL, G. and de MONTIGNY, C. Modification of NMDA response by antidepressants. Research Day, Dept. of Psychiatry, McGili University, 1992.

3. BERGERON. R., DEBONNEL, G. and de MONTIGNY, C. Modulation of NMDA response by sigma ligands. Research Day, Montreal Neurologicd Institute, McGili University, 1992.

4. BERGERON, R., DEBONNEL, G. and de MONTIGNY, C. Biphasic effects on NMDA response of rwo antidepressants with high affinity for sigma sites. Society Neuroscience Abstract 18: 16.9, 1992.

DEBONNEL, G., BERGERON. R. and de MONTIGNY, C. E1ectrophysiologica.I evidence for the existence of subtypes of sigma receptors in the rat dorsal hippocarnpus: Antagonistic effects of high doses of senraline, clorgyline and L-687,384. Society Neuroscience Abstract 18 :16.10, 1992.

BERGERON, R., DEBONNEL, G. and de MONTIGNY, C. Effects of chronic treatments with sigma ligands on the NMDA response. Research Day, Dept. of Psychiatry, McGill University, 1993.

BERGERON. R., DEBONNEL, G. and de MONTIGNY, C. Chronic treatrnents with sigma ligands acting as agonists. Research Day, Montreal Neurological Institme, McGill University, 1993.

BERGERON. R., DEBONNEL, G. and de MONTIGNY, C. Effects of chronic treatments with sigma ligands on the NMDA response. Canadian College of Neuropsychopharmacology, 16: M 33, 1993.

BERGERON. R., DEBONNEL, G. and de MONTIGNY, C. Biphasic effects of selective sigma ligands on the NMDA response. Society Neuroscience Abstract 19: 177.4, 1993.

10. DEBONNEL, G., BERGERON- and de MONTIGNY, C. Long-tem treatments with sigma ligands modify their effect on the NMDA response. Society Neuroscience Abstract 19: 177.5, 1993. DEBONNEL, G., BERGERON. R., GRONIER, B., MONNET, F.P. and de MONTIGNY, C. Effects of a pertussis toxin pretreatment on the modulation of the NMDA response by sigma ligands and NPY. American College of Neuropsychopharmacology, 32: 142, 1993.

BERGERON. R., DEBONNEL, G. and de MONTIGNY, C. Short and long term treatments with sigma ligands modify their effect on the NMDA response. American College of Neuropsychopharmacology, 32: 141, 1993. de MONTIGNY, C., BERGERON. R., MONNET, F.P. and DEBONNEL, G. The modulation of the NMDA response by sigma ligands and NPY in the dorsal hippocampus is mediated via different subtypes of sigma receptors. American College of Neuropsychopharmacology, 32: 141, 1993.

BERGERON. R., de MONTIGNY, C. and DEBONNEL, G. Progesterone suppresses the potentiation of NMDA response induced by sigma ligands. Society Neuroscience Abstract 20: 314.1 1, 1994.

DEBONNEL, G., GRONIER, B. BERGERON. R. and de MONTIGNY, C. Neuropeptides, neurosteroids and sigma ligands: Interactions in the modulation of the NMDA response of hippocampal neurons. American College of Neuropsychopharrnacology, 33: 141, 1994.

BERGERON. R., de MONTIGNY, C. and DEBONNEL, G. The potentiation of the NMDA response induced by sigma ligands is markedly reduced during pregnancy. Society Neuroscience Abstraa 21: 63 1.6, 1995.

DEBONNEL, G., BERGERON. R., GRONIER, B., LAVOIE, N., RETTORI, M.C. and GUARDIOLA, B. Modulation of NMDA neuronal response by sigmal and sigma? ligands. Society Neuroscience Abstract 21: 63 1.7, 1995.

DEBONNEL, G., BERGERON. R. and de MONTIGNY, C. DHEA and progesterone modulate the NMDA response via sigma receptors. International Symposium on DHEA transformation into androgens and est rogens in target tissues: Endocrinology, 1995.

BERGERON. R., de MONTIGNY, C. and DEBONNEL, G. Pregnancy prevents the potenriation of the NMDA response induced by sigma agonists. American College of Neuropsychopharmacology, 1995.

DEBONNEL, G., BERGERON. R. GROMER, B. and LAVOIE, N. Modulation of NMDA neuronal response by sigma, and sigma, ligands and neurosteroids: evidence for the existence of several types of sigma receptors. XXth Collegium Internationale Neuropsychopharmacologicum, 158 (546-2), 1996. v Published Articles

BERGERON. R., DEBONNEL, G. and de MONTIGNY, C. Modification of the N-methyl-D-aspartate response by antidepressant sigma receptor ligands. Eur. J. P harmacol., 240: 3 19-323, 1993.

MONNET, F., DEBONNEL, G., BERGERON. R., GRONIER, B. and de MONTIGNY, C. The effects of sigma ligands and of neuropeptide Y on N-Methyl-D- aspartate induced neuronal activation are differentialy affected by pertussin toxin. Br. J. Pharmacol., 112: 709-715, 1994.

BERGERON. R., de MONTIGNY, C. and DEBONNEL, G. Biphasic effeas of sigma ligands on the N-methyl-D-aspmate response. Naunyn-Schniedeberg's Arch. Phannacol., 351: 252-260, 1995.

BOUCHARD, A., MONNET, F.P., BERGERON, R., ROMAN, F., JUNIEN, J.L., de MONTIGNY, C., DEBONNEL, G. and QUIRION, R. In vivo modulation of sigma receptor sites by calcitonin gene-related peptide in the mouse and rat hippocampal formation: A radioligand binding and electrophysiological studies. Eur. J. Neurosci., 7: 1952-1962, 1995.

BERGERON. R., de MONTIGNY, C. and DEBONNEL, G. Potentiation of neuronal NMDA response induced by dehydroepiandrosterone and its suppression by progesterone an effect mediated via sigma receptors. J. Neurosci., 16: 1193-1202, 1996.

DEBONNEL, G., BERGERON. R., MONNET, F.P. and de MONTIGNY, C. Modification of the NMDA response by sigma ligands differentially affected by colchicine lesions. Neuroscience, 71: 977-987, 1996.

DEBONNEL, G. BERGERON. R. and de MONTIGNY, C. DHEA and progesterone modulate the neuronal response to NMDA in the CA, region of the dorsal hippocampus via sigma recepton. J. Endocrinol., 1996 (in press).

BERGERON. R., and DEBONNEL, G. Effects of low and high doses of sigma ligands: Further evidences suggesting different subtypes of sigma receptors. Psychopharmacology, 1996 (in press).

BERGERON. R., de MONTIGNY, C. and DEBONNEL, G. Short and long-term treatments with sigma ligands modiS their effects on the NMDA response-induced neuronal activation in the CA, region of the rat dorsal hippocampus. Brit. J. Pharmacol., 1996 (in press).

BERGERON. R., de MONTIGNY, C. and DEBONNEL, G. Pregnency markedly reduces brain receptor sigma funcrion. 1996, (submitted). LIST OF ABREVIATIONS

2-APHB 2-amino-pyrimidine-hydrobromide 2-APHB 2-amino-4-p hosp hono buty rate ~-APP 2-amino-3-phosphono propionic acid (+)3-PPP (+)3-[3-hydroxyphenyll-N-(1-propyl)piperidinehydrochloride ACh Acetylcholine ACPD Amino-1-3syclopentane dicarboxylate ACTH Adrenocorticotrophic hormone AdipG 1-(1-adamanty1)-3-(2-iodophenyl)guanidine AMPA a-Amino-3-hydroxy-5-methyl-isoxazole4propionicacid

AP-5 2-amino-5-p hosp- honovalerate Ar-7 2-amino-7-p hosp hono heptanoate MDQ 2-amino-3-p henol-dihydro quinazoline B, Maximum number of ligand-binding site complexes BD-737 (+)tis-N-methyl-N{2(3,4dichlorop henyl)ethyl]-2-(l-p yrrolidinyl) cyclohexy lamine [3HJendo-N-(8-met hyl-8-azabicyclo[3.2.1 .]oct-3-y1)-2,3-dihydro-3- ethyl-2-0x0-1H-benzimidazole-isarboxamide hydrochlonde BMY 14802 c~-(~fluoro~hen~l)-~-(5-fluoro-2-~~imidin~1)-~~~erazinebutanol BSA Bovine serurn albumin BW-234V Rimcazo le CA, region Supeior field of the Ammon's horn CA, region Inferior field of the Ammon's horn CAMP Cyclic adenosine monophosphate cDNA Complementar desoxyribonucleic acid cGMP Cyclic guanosine monophosphate (cis 2,3-PDA) cis-2, 3 piperidine dicarboxilic acid CCK Cholecysto kinine CNS Central nervous system CPP 3-[Zcarboxypiperazin-4-yl]propyl-1-phosphonate CPPP 3-(karboxyl pipendin4yl) propyl- 1-phosphonate CRF Corticot rophin releasing factor DA DAA D-a-aminoadipate DHEA Dehydroepiandrosterone DOPAC Dihydrop henylacetic acid yDGG y-D-glutamylglycine D,L-APS DL-2-amino-5-phosp honovahic acid DM (+ )-3-methoxy-N-met hylmo rp hinan DnBg 1Jdi-(2-norborny1)guanidine DTG Di(Ztoly1)guanidine DuP 734 1-(cydo~ro~~lmethyl)4(2-(4.fluoro~henyl)-2-oxoethy1)piperidine EAA Excitatory amino acid EKC Et hylketocyclazocine EPS Extrapyramidal symptorns EPSPs ~xcit&or~postsyna~tic potentids FH-5 10 5'8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol mono hydrochloride FPLC Fast protein liquid chromatography GABA yamino-butyric acid GAMS y-D-glutamyl amino methyl sulfonate GDEE L-glutamic diethylester acid GDP ~uanosinediphosphate GMP-PNP 5'-(B, y-imino)triphosp hate GPPCNH)p 5'-guanyl-yl-imidodip hosp hate G proteins Guanine nucleot ide-binding proteins GTP Guanosine triphosphate GTPys Guanosine 5'-O-(3-thiot rip hosp hate) HA-966 3-amino-1-hydroxy-p yrrolidin-Zone HPA axis Hypothalamic-pituitary-adrenal axis ['HIPPAP N-(3{'Wphenyl-n-propyl) 1-phenyl-2-amino propane hydrochloride HR-375 3-(4-(3(4-fluorobenzoyl) -propyl-piperazino- 1-yl-isoquinolino HVA Homovanillic acid HW 173 ICV Intracerebrovent ricular IPSPs Inhibitory postsynaptic potentials 1.p. Intraperitoneal 1.v. IACoc JO-1784 (+)~-cyclo~ro~~hnethy~-~-rnethyl- ~+di~henyl-kt hyl-but-3en-1- ylamine hydrochloride KA Kainate KC 9172 3,7diazabicyclo[3.3 Ilnonane-2,4,6,8-tet raon Kd Dissociation constant of the ligand-receptor complex LCGU Local cerebral glucose utilization L-AP3 D,L-2-amino-3-ë hosphono propionate L-AP4 L-2-amino-+phosphono butanoic acid L-CCG-1 2S,3S,4S a-carb~xyc~clopropylglycine LMMP Longitudinal muscle-myenteric plexus L-SOP O-phosp ho-L-serine LSD ~Ser~icacid diet hylarnide LTP Long-term potentiation L-687,384 [Lbenzylspiro[î ,î,3,+tetrahydro-1 ,4-piperidine]] MK-801 (+)3-trimethylsilyl-5-methyl-IO,11-dihydro-5H dibenzo[a,d]cycloheptene-5, leimine Messenger ribonucleic acid Molecular Weight Mego hm 2Jdehydroxy-6-nitro-7sulfomoyl-benzoquinoxaline Noradrenaline nA Nanoamper NANM N-dlyl-normetazocine nC Nanocoulomb NE Norepinephrine NE-100 N,N-dipropyl-2-[4-met hoxy-3-(2-p heny let hoxy)]-et hylamine - - monohydrochioride NEM N-et hylmaleimide WC- 16377 6-[6-(4-Hydroxypiperidinyl)hexyloxyl-3-met hlavone HC1 NTS Nucleus of the solitary tract NMDA N-mechy 1-D-as partate MPTP N-methyl4-p henyl-1,2,3,6-tetrahydr~p~ridine NPY Neuropeptide Y NT Neurotensin PB Piperonyl butoxide PCP P hencyclidine[î-(1-p henylcyclohexyl) piperidine PCPA Para-chlorop henylalanine NTS Nucleus of the solitary tract PCR Polymerase chain reaction PET Positron ernission tomography PI P hosphoinositide [lE]PIP AG [lLSl]-i-(piodopheny1)-3-(1-adamanty1)guanidine PKC Protein Kinase C PP Pancreatic polypeptide PON PPAP PRE-O84 hydrochlo ride PRL Prolactine QNB Quinuclidinyl benzilate QUIS Quisqualate SAR Stmcture-activity relationships S.C. Su b-cutaneous SDS Sodiumdodecyl sulphate SKF-10,047 N-dlylnormetazocine SL-82.07 15 (4-a-(4-ch10 rop heny l-4-(4-fluorop henyl)met hy1)-1-piperidine ethanol cis-3-(hexahydroazepui-1-yl) i-(3chloro-kyclo hexylp henyi) propene i ,hydrochloride TCP 1-[ 1-(2-t hieny1)cyclohexyupiperidine U-50,488H ((A-trans-3,4dichloro-N-methyl-N-12-(1-pyrro1idinyI)cyclo- hexyl]bebzeneacetamide met hane sulfonate) VTA Ventral tegmantai XJ 448 14-(2'-(4"-cyanopheny1)-2'-oxoethyl) 1- ABSTRACT

Previous studies in this laboratory have demonstrated that low doses of some selective o ligands such as DTG act as "agonists" by potentiating the neuronal response of rat CA, dorsal hippocampus pyramidal neurons to NMDA. Other o ligands such as act as

" antagonists" by reversing this potentiation. At doses in between 1-1000 &kg, i.v., several selective o ligands and antidepressant o ligands present a bell-shaped dose-response curve, which effect cannot be explained by a rapid desensitization. These o ligands that behave as "agonists" at low doses act as "antagonists" at the dose of 1OOO &kg, i.v.

To date, two subtypes of a receptors (a, and oj are acknowledged. However, data presented in this t hesis have shown that pertussis pret reatment abolished the potentiation induced by 50-1784 but not that induced by (+)pentazocine. Moreover, the injection of colchicine in the dentate gyrus abolished the potentiation induced by 50-1784, but not that of (+)pentazocine. These data suggest that the subtype of o, receptor on which JO-1784 is acting is related to a G-pro~einand is located presynaptically whereas the subtype of o, receptor on which (+)pentazocine is acting is not related to a G-protein and is located posisynaptically .

Because progesterone possesses high affinity at the o receptors, we have determined the effects of neuroactive steroids on NMDA-induced excitation. Low doses of DHEA potentiated the NMDA response selectively and d~sedependentl~.Progesterone had no effect by itself but reversed, at low doses, the potentiation of the NMDA response induced by DHEA as well as those induced by selective o ligands. The degree of the potentiation induced by 1 pg/kg, i.v. of DTG is significantly greater in ovariectorized rats than in control rats. Moreover, in late pregnancy, ten-fold higher dose of DTG is required to obtain a selective potentiation of the X

NMDA response. Conversely, the potentiation of the NMDA response induced by DTG is greater at day 5 post-partum than in control rats.

The data presented in this thesis suggest that: (1) more than two subtypes of o receptors exist in the mammdian brin; (2) neuroacxive steroids such as progesterone and DHEA modulate the NMDA response via o receptors. ABRÉGÉ

Des études faites dans ce laboratoire ont démontrées que de faibles doses de ligands

sélectifs pour les récepteurs a tel que le DTG agissent comme "agonistes" en la

réponse neuronale des neurones pyramidaux de l'hippocampe dorsal de la région CA,. D'autres

ligands pour les récepteurs o agissent comme "antagonistes" en renversant cette potentialisation.

A des doses variant de 1-1000 pg/kg, i.v., plusieurs ligands sélectifs présentent une courbe dose-

réponse ayant un aspect de courbe en cloche, lequel effet ne peut être expliqué par une

désensibilisation rapide. Ces ligands o qui agissent comme " agonistes" à faibles doses, agissent comme antagonistes à la dose de IOOO &kg, i.v.

A ce jour, deux sous-types de récepteurs o (a, et 03 sont reconnus. Cependant, des

données présentées dans cette thèse démontrent qu'un traitement à la pertussis abolit la potentialisation induite par le JO4784 mais pas celle induite par la (+)pentazocine. De plus,

l'injection de colchicine dans le gyrus dentelé abolit la potentialisation induite par le JO-1784

mais pas celle induite par la (+)pentazocine. Ces données suggèrent que le sous-type de

récepteur a, sur lequel agit le 50-1784 est relié à une protéine G et est situé au niveau présynaptique alors que le sous-type de récepteur o, sur lequel agit la (+)pentazocine n'est pas

relié à une protéine G et est situé au niveau postsynaptique.

Parce que la progestérone possède une haute affinité pour les récepteurs a, nous avons déterminé l'effet des neurostéroides sur la réponse neuronale induite par NMDA. De faibles doses de DHEA potentialisent la réponse au NMDA de façon sélective et dose-dépendente. La

progestérone n'a pas d'effet par elle-même mais elle renverse, à faibles doses, la potentialisation de la réponse au NMDA induite par le DHEA et par des ligands sélectifs pour les récepteurs

a. Le degré de la potentialisation induite par 1 pg/kg, i.v. de DTG est significativement plus grand chez les rats ovarieaornisées que chez les rats controles. De plus, en fin de grossesse, une dose dix fois plus élevée de DTG est nécessaire pour induire une potentialisation sélective de la réponse au NMDA. Par contre, la potemidisation de la réponse au NMDA induite par le

DTG est plus élevée au Jour 5 post-partum que chez les rats controles.

Les données présentées dans cette thèse suggèrent que (1) plus de deux sous-types de récepteurs cr existent dans le cerveau des mammifères; (2) neurostéroides comme la progestérone et le DHEA modulent la réponse neuronale au NMDA via les récepteurs a. TABLE OF CONTENTS

Review of literature ...... 1

1 . INTRODUCTION ...... 1 1.1 Histoncd perspectives ...... 1 1.2 Differentiation of sigma receptors ...... 2 1.2.1 with opiate receptors ...... 2 1.2.2 with PCP receptors ...... 3 1.3 Interaction with the glutamatergic system ...... 5

2. SUBTYPES OF SIGMA RECEPTORS ...... 6

3 . ANATOMY OF SIGMA RECEPTORS ...... 8 3.1 Ligands for sigma receptors ...... 8 3.2 Distribution in the centrai nervous system ...... 10 3.3 Distribution in the periphery ...... 11 3.4 Species differences ...... 13 3.5 Cellular locdization ...... 14

4 . PHYSIOLOGY OF SIGMA RECEPTORS ...... 15 4.1 Neural transmission ...... 15 4.2 Interaction with neuropeptides ...... 16 4.2.1 Neurotensine ...... 16 4.2.2 Neuropeptide Y ...... 17 4.2.3 Cholecystokinine ...... 18 4.3 Interaction with other drugs ...... 18 4.3.1 Anticonvulsant drugs ...... 18 4.3.2 Antidepressant drugs ...... 19 4.3.3 hntihistarninic drugs ...... 19 4.3.4 Antimuscarinic dmgs ...... 20 4.3.5 drugs ...... 21 4.3.6 Antitussive drugs ...... 22 4.3.7Cocaine ...... 23 5 . PHARMACOLOGY OF SIGMA RECEPTORS ...... 24 5.1 Biochemical properties of sigma receptors ...... 24 5.2 Behavioral propenies of sigma receptors ...... 24 5.3 Electrophysiological properties of sigma receptors ...... 25 5.4 Concept of agonisthntagonist at sigma receptors ...... 27 5.5 Up/down-regulation properties of sigma receprors ...... 28

6- CHARACTERISTICS OF SIGMA RECEPTORS ...... 30 6.1 Molecular biolog ...... 30 6.2 Second messenger ...... 32 6.3 Cytochrome P., ...... 34 6.4 Endogenous ligands for sigma receptor ...... 35

7 . CLINICAL IMPLICATIONS OF SIGMA RECEPTORS ...... 37 7.1 Ischernic ...... 37 7.2 Dementia ...... 38 7.3 Movement disorder ...... 40 7.4 Psychosis ...... 40 7.5 Cough ...... 42 7.6 Gastroenterology ...... 42

CONCLUSION ...... 43 CHAPTER II Modification of the Kmethy 1-D-aspartate response by ant idepressant sigma receptor ligands ...... 82

CHAPTER III Biphasic effects of sigma ligands on the neuronal response to N-methyl-D-aspartate ...... 9 1

CHAPTER N Differential effects of sigma ligands on the NMDA response in the CA1and CA3 regions of dorsal hippocampus: Effect of mossy fiber lesioning 112

CHAPTER V The effects of sigma ligands and of neuropeptide Y on N-methyl-D-asparrate- induced neuronal activation are differentially affected by pertussis toxin ...... 134

CHAPTER VI Effects of low and high doses of sigma ligands: Further evidence suggesting the existence of different subtypes of sigma receptors ... 153

CHAPTER VI1 Short-term and long-term treatments with sigma ligands modify the Mmethyl-D-aspartate response in the CA3 region of the rat dorsal hippocampus ...... 179

CHAPTERVIII Potentiation of neuronal NMDA response induced by dehydroepiandrosterone and its suppression by progesterone: Effects mediated via sigma receptors ...... 203

CHAPTER M Pregnancy markedly reduces the brain sigma receptor function ... 228

CHAPTER X GENERAL DISCUSSION ...... 239 CHAPTER 1 Review of literature

1.1 Historïcal perspectives The existence of o recepton was first postulated by Mmin and CO-workers20 years ago to explain the inherent psychotomimetic effects of (+)SU-10,047and related benzomorphans in the chronic spinal dog mode1 ". The fact chat the behavioral actions of (*)SKF-10,047were not blocked by opioid receptor antagonists such as naloxone and naltrexone 36J84 suggest that they were non-opioid in nature. Furthermore, the (+)benzomorphans were found to be more potent than the (-) benzomorphans with respect to these behavioral and physiological effects, thus indicating a reverse in the phannacology classically observed with the opioid receptor ligands "J499. The hypothesis that the o binding sites mediate the behavioral actions of psychotomimetic compounds, such as SKF-10,047, cyclazocine and pentazocine, has stimulated a great deaf of interest. The a receptors have a unique profile compared to any other receptors in the CNS for several reasons: (1) this binding site is widely and abundantly distributed outside the CNS including prnicularly high densities in spleen '", liver 326 and endocrine tissue such as adrenal, testes and ovaries 'IL; (2) a number of pharmacologically unrelated compounds bind with high affinity to the site, (several antipsychotic drugs 'j2, the monoamine oxidase inhibitor clorgyline the selective serotonin reuptake inhibitor sertraline "', the neuroprotective ifenprodil 19*, some steroids such as progesterone "', the antitussive de~rornethor~han" and neuropeptide Y "7; (3) some compounds inhibit binding with different affinity constants when tested against a variety of o specific radioligands; (4) the (+)stereoisomers of benzomorphans such as pentazocine, cyclazocine and SKF-10,047displace some tritiated o ligands with low Hill coefficients, suggesting that these cornpounds recognize heterogeneity within a population of a binding sites. 1.2 Differentiation of sigma receptors

1.2.1 With opiate receptors The first opiate antagonist in wide clinical use was nalorphine (N-dlyln~rmor~hine), derived from naturally occurring levorotatory morphine by replacing the N-methyl with an N-allyl substitute. For many years, naorphine was employed exclusively as an opiate antagonist to treat opiate overdose. Opiates have a wide range of pharmacological effects. To account for the different properties of some opiates, multiple opioid recepton were hypothesized. The four opiate receptor subtypes that have been proposed are p, 6, k, and a.

It has been proposed that p receptors mediate analgesia 5'35; that k receptors mediate analgesia and sedation 17"; that 6 receptors mediate satisfaction, reward and seizure and that o receptors mediate p~~chotomimeticeffects 198243.Certain benzomorphan opiates such as SKF-10,047 and cyclozacine, aside from analgesia, cause hallucinations, depersonalization, drunkenness and other psychotomimetic effects in humans 198. In monkeys, dogs and rodents, the psychotomimetic opiates cause behavioral and autonornic effects that are unlike those observed with administration of classical opiates such as morphine or the opioid peptides '6-'". These observations have suggested the existence of specific a-type "opioid" receptors that mediate such atypical effects "'. The existence in the rat CNS of a type of receptor that is biochemically and topographically distinct from the k, p and 6 opioid receptors has been characterized 365. Other opiates such as pentazocine, cyclozacine and bremazocine, which bind the p and 6 receptors, also bind the o receptors. Behavioral effects of SKF-10,047 and other agents, including mydriasis, tachycardia, tachypnea, and manic-like hyperact ivity , were characterized in dogs 2".

Su '58 examined the binding of racemic ['H]SKF-10,047 in the presence of the opiate etorphine ro elirninate binding to p-opiate receptors. He observed t hat the stereoselectivir y of SKF-10,047 at these sites was opposite that of conventional opiate receptors, as (+)SKF-10,047 was substantially more potent t han (-) SKF-10,047. Using [3H]ethylketocyclazoxine in the presence of naloxone of (+)['HISKF-IO,O47, Tarn 365 demonstrated a high-affinity a receptor in rat brain that had a regional distribution which was different from that any opioid receptors. Tarn and

Cook 367 noted that (+)['H]SKF-10,047 and [3H]haloperidol label a single site, apparently the same one observed by Su ''' with selectivity for (+)SKF-10,047. The u binding site labelled with C3H]DTG is clearly not related to conventional (p, 6, k) opioid recepton. This site is naloxone-insensitive and shows stereo~electivit~for dexrrorotatary isomers of benzomorphan drugs '? Therefore, since the publication of that study 3B, cr receptors are not referred to as u "opioid" receptors. Unlike the C( and 6 binding sites, which are sensitive to sodium ions "J9', the (+)[3HJSKF binding site was not affected by 200 rnM Na+ or K+ions. While Na' decreased agonist affinity for the p receptor, it improved the correlation between p agonist binding affinity and in vivo analgesic activity.

1.2.2 With PCP receptors In the CNS, glutamate acts as an excitatory neurotransmitter. On the basis of pharmacological characteristics, three families of glutamate-gated ion channels have been identified: NMDA, KA and AMPA receptors "'. AMPA and KA receptors mediate fast excitat ory postsy naptic potentials whereas NMD A receptors serve a more modulato ry funaion since they are recruited by depolarization 15'. The NMDA receptor has been extensively characterized with regard to the cornplex modulatory sites of pharmacological imporc 6U81J9g. In addition to the recognition site for glutamate, there is a second site to which glycine binds. Other modulatory sites include a binding site for polyamines and a binding site for zinc. The NMDA receptor channel is voltage dependent with magnesium occluding the channel at resting membrane potential. Finally, there is a binding site within the channel that becomes accessible, upon channel opening to MK-801 and related drugs, PCP and ketarnine which act as noncornpetit ive antagonists L)3. was first synthesized in 1957 as an anesthetic by the Parke Davis pharmaceutical Company 'O. PCP was thought initially to be an ided anesthetic drug because it produced ;uialgesia and anesthesia with minimal respiratory or cardiovascular effects 13? Unfortunately PCP also produces psychotomimeric effects that are often long lating Today, PCP (angel dust) is one of the most abused drugs due in pan to its ability to alter perception 5*94*"5J5'. Phencyclidine also induces violent behavior and psychosis that resembles schizophrenia '. It has even been suggested that the use of PCP would providc a better drug mode1 than amphetamine for the study of schizophrenia Evidence that PCP acts through its own receptor first came from Zukin and Zukin when a specific, relatively high affinity site for [-'H]PCPbinding was discovered '88-420. This site bas biochemical characteristics of a receptor since binding is saturable, reversible and selective -. NO known neurotransrniners or neuromodulators tested to date have high affinity for this site. Additionally, proteolytic enzymes and heat decrease binding, indicaring the proteinaceous nature of the site. The distribution of PCP binding throughout the brain is heterogeneous, 4 with highest density in the cortex and hippocampus and lowest in the brain stem and spinal cord 299. Data showing that some a ligands have both a high affinity for [3w-PCP-binding sites and induce PCP-like behavioral effects have led many to believe that the PCP and o receptor were identical and it has been referred to as the "PCP/sigmaWreceptor "? Studies examining the binding of SKF-10,047 to brain membranes have produad conflining results. In some smdies the drug specificity of (k) or (+)['HISKF-10,047 binding resembles thac of PCP receptor binding sites suggesting that SKF-10,047 and PCP may be labelling the same site '59258. However, other repom indicate that o binding sites labelled by (+)['H]3-PPP, (+)['H]SKF-10,047 and ['HJmloperidol have a dmg specificity quite distinct frOm that of PCp sites Wl~137.139215316$67J86$87 It is now clear that PCP binding sites are different from the (+)r3WSKF-10,047binding sites '". The reasons are: (1) ['H]PCP binding is decreased in the presence of sodium ions "O but (+)['H]SKF-10,047 binding is nor; (2) the two binding sites have different drug selectivity; (3) the PCP binding sites show low affinity and little stereoselectivity towards SKF-10,047, whereas these dmgs are highly stereoselective towards the (+)['H]SKF-10,047 binding sites and (4) the regional distribution of (+)['H]SKF-10,047 binding sites and PCP bindings sites in the rat central nervous system is different ".71JW15J65. The greatest density of PCP receptors is found in the cortex, hippocarnpus and dentate gyrus, with very few sites in the brainstem and hypothalamus 'lm. In contrast, there is a significant density of

215347358. With the synthesis of several new selective o ligands such as BD-737 BMU Dur 734 75*187*'88,FN-510 37lJ72, HR-375 LA L.687,384 11262, NE-100 49*28+a7 NPC-16377 57,344 SR 31747 46"2, XJ 448 '12, etc.. . it is easier to characterize the properties of cr receptors. Finally, PCP and a receptors display quite distinct developmental profiles. PCP recepton show a progressive increase in their B,, during postnatal development with the greatest rate of increase occ~rringafter P.O.N. 14. These findings are consistent with the results of autoradiographic studies of the ontogeny of NMDA receptors, as well as with other studies which reveal a marked increase i~.the rate of development of glutamatergic synapses between day 12 and 21 in the rat hippocampus ''O. During this same tirne pied it has also been shown that pyramidal neurons in the CA, area of the hippocampus become increasingly sensitive to the excitatory amino acid NMDA la, which provides physiologicd evidence for the maturation of NMDA receptor systems, and consequenrly of the NMDA receptor-linked ion channels containing the PCP binding sites. These findings dso correspond with the tirne course of the development of rat brain glutamatergic sysrems as determined by binding studies measuring total ['Hlglutamate in rat brain membranes 'O8. It should be noted that the results and conclusions reported above differ somewhat from those of another study Majewska et al. who concluded chat while both the affinity and the density of o receptors remain constant throughout the developmental period tested (postnatal day 1 to 1 year), the density of PCP binding sites increases from the time of birth, reaching the adult level by postnatal day 14.

1.3 Interaction with the glutamatergic system Severd lines of evidence suggest a functional modulation of the events mediated by the NMDA receptor cornplex by ligands with affinity for the u receptors. These include: (1) neuroprotective action of BMY 14802 68*'71, ifenprodil and SL-82.0715 45-125, dextromethorphan

%, dextrorphan 51 and (2j reversal of a ligand-mediated increases in plasma adrenocorticotrophic hormone (ACTH) and striatal DA turnover by the cornpetitive NMDA antagonist CPP 166*167; (3) modulation of NMDA-rnediated electrophysiological responses in CA, hippocampal pyramidal neurons by o ligands (4) antagonism of NMDA-induced seizures in mice by (+)pentazocine and (+)SU-10,047 Y6; (5) antagonism of ischemia-induced glutamate release " and (6) modulation of NMDA-dependent striatal DA release IM. Reversal of NMDAdependent, but not quisqualatedependent, cGMP responses by WY 47384 ' and

BMY 14802 3m dso support seleaive fincrional interaction between NMDA and a receptors. The presence of a receptors in the hypothalamus, pituitary and endocrine organs suggests a role for these receptors in modulating neuroendocrine funaion "bA1l. (+)Pentazocine and (+)SKF-10,047 stimulated ACTH release potently after both peripheral and central administration '". The HPA ais is rnodulated by o receptors independent of the opioid recepton, since ndoxone pretreatment did not block these effeas. More inrerestingly, the effects of both o ligands were prevented by pretreatments with CPP, implying a functional interaction between u receptors and NMD A receptors. In keeping with previous observations

16', 16', dl of the four ligands ((+)Sm-10,047, (+)pentazocine, (+)3-PPP and benzomorphans), tested increased release of ACTH Several selective o ligands @TG, 50-1784,(+)pentazocine, APDQ, AdipG, DnBG, JO- 1783) were tested in an in vivo elearophysiological paradigm. They act as "agonists" since the intravenous administration of low doses (pg/kg) of these a ligands, as well as their microiontophoretic applications potentiate the neuronal response to NMD A in the CA, region

of pyramidal neurons in the rat dorsal hippocampus t'JbsJ'l. Uthers a ligands (haloperidol, BMY-14802, (+)3-PPP) have no effect when they are injected intravenously but they act as "antagonists" since they suppress the potentiation of the NMDA response induced by a "agonists". , which has a binding profile similar to that of hdoperidol except for its low affinity for the o receptors 3g,does not reverse the potentiation of the NMDA response induced by o "agonists". The functiond modulation of the NMDA receptor cornplexdependent responses by a ligands is demonstrated by several observations *la. The neurochemical pathways and substrates involved in the funaional interaction between the NMD A receptor complex and o ligands are nor understood at this time.

2. SUBTYPES OF SIGMA RECEPTORS Two subtypes of a binding sites have been acknowledged based on their physical charaaeristics, tissue distribution and G protein coupling L98. Sigma, binding sites are charaaerized by high affinity for haloperidol, DTG and (+)benzomorphans such as (+)pentazocine and (+)SKI?-10,047. Sigma, bindings sites are characterized by high affinity for hdoperidol and DTG but low affinity for the (+)benzomorphans "j. Studies of the pharmacology and function of a, binding sites have been harnpered by the lack of a selective radioligand. The rank order to distinguish the two sites are as follows O,: (+)pentazocine = hdoperidol > DTG = (+)3-PPP > (+)SKF-10,047 > (-)pentazocine > PCP > (-)SKF-10,047;

0,: DTG = halopeI-idol > (+)3-PPP = (-)pentazocine > PCP > (+)pentazocine > (-)SIG- 10,047. Besides the differences in binding profile, the subtypes cm also be differentiated on the bais of the behaviord studies 392. Weber et al. '" using [JH]DTG and ['H](+)WPP concluded chat the drug selectivity profiles of the two binding site populations in guinea pig brain were virtually indistinguishable. A later report, using a more complex experimental design, delineated two binding sites in guinea pig brain whose affinities for E3wDTG differed only by 3-fold (and whose B,, values were similar), precluding their separation with only ['HJDTG saturation studies '?Subsequent studies have provided strong evidence for the existence of pharmacologically distinct a binding sites 14SJMJg0. Karbon et al. '" reporred that ['H](+)3-PPP binding sites in guinea pig brain membranes exhibited at least IO-fold higher affinity for (+)- than (-)benzomorphans, and that ['E-IJ(+)WPP but not ['WDTG binding sites exhibited stereoselectivity for benzomorphans in favor of the (+) enantiomen. The autoradiographic distribution of rHJ(+)pentazocine and [-'HJDTG in guinea pig brain was similar, but not identical, providing evidence for more than

one o receptor j9'. Caramiphen and dextromethorphan also were more potent inhibitors of ['W(+)3-PPP than [)H'JDTG binding, whereas haloperidol and BMY 14802 were each

equipotent at these sites. Itzhak and Khouri 16' and Beart et al. " have reported that in rat brain membranes u agents such as (+)SKF- 10,047 cm differentiate two [313J(+)3-PPPbinding sites on the bais of shallow displacement cunres as well as GTP sensitivity. Phenytoin, a widely used anticonvulsant dmg, increased the specific binding of ['H](+)3-PPP but not E3WDTG, although it markedly increased the potency of dextromethorphan to inhibit

['HJDTG binding 19'. These findings suggest that r3W3-PPP and ['WDTG label pharmacologically distinct but functionally coupled sites.

Using ['H](+)SKF-10,047, Itzhak and Alerhand 15' first reported that chronic haloperidol treatment produced a reducrion in a binding sites. Subsequemly, Karbon and

Naper i92 reported that repeated haloperidol administration reduced ['HJ(+)WPP but not ['HJDTG binding in membranes prepared from guinea pigs receiving halopend01 for 14 days. A selective reduction in ['H]3-PPP binding was reported by Itzhak and Stein (1991) in rats treated with haloperidol(4 mg/kg) once daily for 14 days. The distinction between the binding sites labelled with [)H]DTG and those labelled with (+)['H]3-PPP is also indicated by the following findings. (+)3-PPP binding in C57BL/6 mouse brain membranes displays the pharrnacological specificity and stereoselectivity of "O-binding", i.e. the (+) isomers of SKF- 10,047 and 3-PPP are significantly more potent inhibitors of (+)['H]3-PPP binding than the

(-) isorners la. However, C3H]DTG binding shows not only reduced stereospecificity towards 3-PPP isorners, but is also essentially insensitive to the prototypic "a ligand" (+)SKF-10,047. Further evidence suggests a distinction between the DTG and (+)SKF-10,047/(+)cyclazocine binding sites. It has been reported that DTG inhibits serotonin- and electrically-induced contractions of the guinea pig ileum, whereas (+)SKF-10,047 and (+)cyclazocine potentiate the contractions 42. These observations could not be explained as differences between "aagonists"

and "antagonists" nor by assurning an interaction with the PCP binding sites 42, but rather implies different mechanisms of action. The possible existence of " o receptor subtypes" is also suggested by the studies of Musacchio and CO-workers.They have reported that the antitussive dmg dextr~merhor~han(DM) labels multiple o/ha.loperidol-sensitive sites, whereas additional DM binding sites may be distinct from the (+)3-PPP binding sites. The authors have proposed a multiple dDM binding site receptor model. Since DM does not induce or block psychotomimetic effects, these findings imply that some subtypes of the o receptor may not be associated with psychotropic effeas. Previous work has demonstrated that o recepton are most highly concentrated in brainstem motor areas, with lower levels in the basal ganglia and cortex lJSW.The ratio of [3H](+)pentazocine binding to ['HIDTG binding varies according to brain regions. These marked differences in ratios between regions are consistent with recent findings demonstrating the presence of multiple types of a receptors. Computer-assisted, simultaneous analysis of self- and cross- displacement experiments demonstrated the existence of several binding sites in guinea pig brain for demromethorphan,

(+)3-PPP and DTG 206. Dextromethorphan binds with high affinity to two sites (RI Kd 50-83 and R, Kd 8-19 nM) and with low affinity to two additional sites (R, Kd24-36 nM). DTG binds with dmost identical high affinity to two different sites (RI % 22-24 and Kd 13-16 nhd). These results confirm that dextromethorphan, (+)3-PPP, and DTG bind to the cornmon DM,/q receptor (RJ and hdopendol displaces labelled Ligands from both high-affinity DTG sites (R, and R,). Thus, haloperidol sensitivity should not be used as the single criterion to identify a putative receptor.

3. ANATOMY OF SIGMA RECEPTORS

3.1 Ligands for sigma receptors The use of haloperidol as an antipsychotic drug has increased drarnatically in North America over the pas1 10 years. Although the mechanism of action and antipsychotic efficacy of hdopendol is generally attributed to the blockade of the D, receptors 339, its high affinity for o receptors and the known psychotomimetic and motor effeas of a agonists have Ied to the suggestion that blockade of the o recepton may also account for some of the therapeutic actions and side effects 352367. Haloperidol's receptor binding profile is cornplex. For example, it binds to D, receprors (KD = 2.7 nM 9 to (T receptors (KD = 0.95 nM and to or, adrenergic receptors (KI,= 22 nM Although hdoperidol lacks specificity for any single receptor system, it is one of the most potent o ligands yet reported and has been used as a radioligand for mapping cr receptor populations in vivo. PET study of halopend01 binding represents the direct measurement of the total u~take,regional distribution and kinetics of this antipsychotic drug in normal and schiz~~hrenichurnan brain '?The major findings of that study were the following: first, haloperidol has a high uptake and retention in normal brain; second, the distribution of haloperidol is more extensive than the regional distribution of dopamine D2 receptors, with the highest concentration in basal pglia, cerebellum, and thalamus; and third, the pattern of haloperidol distribution presents a rapid washout from brain. The regional distribution of binding sites labelled by ['H]haloperidol, in the presence of excess spiroperidol, was compared to the regional distribution of receptors labelled by L3HJSCH 23390 and [3H] ". ['HJSCH 23390 and ['Hlsulpiride labelled distinct nuclei such as the olfactory tubercle, caudate, globus pallidus, substantia nigra, and inferior and superior colliculi. In contrast, the distribution of binding sites labelled by ['HJhal~~eridol,in the presence of excess spiroperidol, were much more extensive. Some areas containing the highest density of sites labelled by ['H]haloperidol were the external plexiform layer of the olfactory bulb, the cerebral cortex, the paraventricular nuclei, the interpeduncular nucleus and the superior colliculus. The distribution of nondopaminergic binding sites labelled by haioperidol was clearly quite different from that labelled by ligands. The distribution of (+)['Hl-3-PPP binding sites does not correlate with the receptor distribution of any recognized neurotransrnitter or neuropeptide. However, there is a notable similarity between the *listributionof (-)['H]-3-PPP sites and high affinity binding sites for the benzomorphan (+)SKF-10,047 138. Largent et al. 216 have shown rhat the localization of o receptors overlaps with those of the sites labelled by ['H]3-PPP and dopaminergic receptors.

Gundlach et al. lJ8, by producing lesions with quinolinic acid, have shown that ['Hl)-PPP cleariy labels the pyramidd cells of the hippocampus together with the granular cells of the dentate gyrus. After the administration of 6-hydro~~doparnineinto the striatum to destroy the doparninergic terminals these sites are affected, which suggests that their localization on the dopaminergic fibers is likely. Receptor binding and autoradiographic studies have demonstrated that the distribution of o receptors is unique U4-'50-'65. Further, through the use of the selective a receptor ligand (+)3-PPP and neurotoxins selenive for monoamine neurons, Gundlach et al.

13' demonstrated that a recepton are associated with dopamine neurons of the substantia nigra pars compacta but not with noradrenergic neurons of the locus coeruleus or serotonergic neurons of the raphe nuclei. 3.2 Distribution in the central nervous system The first anatornic evidence for discrete localization of rr binding sites in the CNS was obtained by Gundlach and coworkers 13'. Other groups have carried out autoradiographic studies with V~~OUSradioligands and have visualized o binding sites in the grey matter of many brain regions. McLean and Weber carried out autoradiographic surveys of guinea pig and rat brain with a synthetic a-selective derivative of guanidine, ['H]DTG and E3H]haloperidol ? Both radioligands were found to have broad binding distributions wirhin the forebrain and hindbrain but both also displayed enhanced binding in a subset of regions identified as parts of the limbic and motor sysmm. Loyer levels of a binding sites were observed over nonlimbic nuclei, wit h the 10 west levels of binding demonstrated over extrapyramidal brain regions. The anatomical pattern for the a receptor labelled with [3H]DTG was shown to be similar to the distribution of sites labelled by (+)['H]3-PPP '16. Recent autoradiographic studies in the guinea pig brain dernonstrate further that the distribution pattern for (+)['HJ3-PPP agrees well with the localization of the potent and seleccive a ligand (+)[3H]pentazocine 88. Results of binding studies in the rodents and humans brain suggest the presence of o receptors in the hippocarnpus, the prefrontal correx, the substantia nigra, the red nucleus, the cerebellum, the dorsal horns of the spinal cord, the pituitary and the dorsal tegmental nuclei of the hindbrain 69,138,174,256,iûI

Previous studies in animals have shown that the cerebellum has a high density of cr recepto rs 138J58J65. The cerebellum appears to be an ideal region to study ['H]haloperidol binding to o receptors as it has been shown to have a low density of dopamine binding sites in several species, including humans ". The human cerebellar binding site labelled with 1 nM ['flhaloperidol in the presence of 50 nM spiperone shows stereoselectively and a marked affinity for dnigs such as (+)Sm-10,047, DTG and (+)-pentazocine. Sigma receptors are highly concentrated in the gray matter region and have a low density in the white rnatter areas. It seems that a receptors are found in many areas of the brain populated by large neuronal ce11 bodies (e.g. Purkinje ce11 layer of the cerebellum, pyramidal ce11 layer of the hippocampus, cranial nerve nuclei and red nucleus). Extremely high densiries of a receptors are observed in motor nuclei of cranial nerves such as the trigeminal motor nucleus, as well as the hypoglossal, facial, and red nuclei. Throughout the brin and spinal cord o receptors occur almost exclusively in neuronal ce11 bodies '. 3.3 Distribution in the periphery

Studies have revealed that the o binding sites are not unique for brain "l but are found at high concentrations in membrane fractions of spleen, liver, adrenal cortex and on lymphocytes 326-+11. Subcellular fraction shows that the a binding sites are located in plasma membranes and microsornes 1M2103+.'553U. A dense distribution of o binding sites was observed in the mucosa and in the submucosal plexus, particularly at the level of the fundus and duodenum "'. Muscular layers were only marginally labelled. No phencyclidine binding site could be demonstrated in any area. This selective distribution suggest a funaional role of o receptors mainly in the control of endocrine or exocrine secretions, or both. ['H]haloperidol binding sites have been identified on human peripheral blood leucocytes with kinetic and pharmacologie characteristics comparable to a receptors in rat brin '12. These

0 receptors are present in unfractionated human peripheral blood leucocytes in comparable density to that of rat cerebellum, a brain region which contains the highest density of o receptors in the CNS. Sigma receptors were dso identified in lymphocytes using the specific ligand ['NDTG ", whose binding characteristics were comparable to those of cr receptors in rat brain. ['HIDTG dso labelled a higher number of sites in B cells than in T cells and a good correlation was found between the lymphocytic binding of DTG 'l'. Membrane preparations of rat hearts displayed specific binding activity for the ['HJDTG binding site 95. The discovery of dense concentrations of o binding sites in organs outside the nervous system 172~17'~6J61.4L1 raised the question of whether these penpheral sites are the same entity as those found in the brain. The results of a study previously mernioned 17' indicate chat 'penpheral' ['WDTG binding sites respond to chronic haloperidol in a manner similar ro ['WDTG binding sites in the brain. This increases the probability that these sites are biologically active receptors and that central and peripheral L3H]DTG binding sites are similar entities.

A high concentration of o recepton was seen in the pineal gland 17'. This result is of interest as both o receptors and the pineal gland have recently been shown to play a role not only in the nervous system but also in the immune and endocrine systems. Some corticosteroids and sex hormones have been shown to bind to o receptors 361. These sites are found in high concentrations in the hypothalamic-pituitary area *6, in peripheral endocrine glands *11 and also on lymphocytes "'. The occurrence of these receptors in the pineal provides further evidence ro support a possible role for these sites in endocrine control. In addition to its psychotomimetic effects in the CNS, SKF-10,047 has been reported to alter neuroendocrine function. Specificdly, this compound has been reported to stimulate hypothalamic-pituitary-adrenocorcical secretion L4293J94 and suppress LH 294 and PRL secretion in rats. ['HJSKF-lO,O47-binding sites have been demonstrated in the anterior pituitary

365 and SKF-10,047 also binds to cultured pituitary cells and alters LH release in vitro "'. The distribution of o receptors in the rat has also been examined. In the adrenal glands, o receptors were localized primady in the adrenal cortex, with significantly lower concentrations in the medulla. Studies in hypophysectomized rats demonstrated that cr recepton in the adrenal cortex were present on cells that were not dependent on maintenance by trophic pituitary hormones, since the densities of a binding sites were not decreased after hypophyseaomy. Thus, o binding sites in the adrenal gland do not appear to be localized to glucocorticoid-producing cells. While o receptor agonists, including SKF-10,047, have been reported to stimulate corticosterone secretion in mice and rats 26 these effects appear to be primarily mediated via pituitary ACTH regulation, rather than through direct actions at the adrenal gland, since they could be blocked by dexamethasone administration 26. The role of o binding sites in the adrenal gland in modularing other adrenal functions remains to be elucidated. There was a high density of a-binding sites in the testis. Sigma binding sites were uniformly distributed in the seminiferous tubules and were notably absent in the interstitial areas and the contents of the tubule lumens. Similar to the adrenal cortex,

3.5 Cellular localization The distribution of o recepton in subcellular fractions of rat brain homogenates was extensively characterized ". In synaptosomal fractions, enriched in choline acetyltransferase activity, o receptors were present in lower concentrations than in whole brain homogenates. It is concluded that rat brain o receptors are not concentrated at synaptic regions of plasma membrane. However, the possibility that o receptors are localized to speciaiized areas of nonsynaptic plasma membrane cannot be excluded. Another study zis demonstrated that liver o receptors are enrïched in the microsomai fraction of the tissue homogenate and their distribution correlates with that of the endoplasrnic reticulum marker. No similarity was found between the distribution of a receptors and that of the nuclear and mitochondrial markers. The distribution of o receptors also differed from that of the enzymic markers for the bile canalicular plasma membrane. It is important that there was no similarity between the distribution of a receptors and that of the enzymic marker for the basolateral plasma membrane that may be present in the microsomal fraction. These findings suggest that cr receptors are not located on vesicles formed from the membrane, but ~robablelocus is the endoplasmic reticulum. On the other hand, the possibility that o receptors may be located on Golgi vesicles, those also present in the microsomaI fraction, cannot be ruled out based on the presented data.

4. PHYSIOLOGY OF SIGMA RECEPTORS The most common a ligands have multiple funnional effects. Pan of the uncertainty over the multiplicity of o receptor actions may be attributed to the large number of structurally diverse compounds (e-g., , guanidines, butyrophenones and phenylpiperidines) that display moderate to high affinities but poor specificity for the o receptors.

4.1 Neural transmission Although the exact physiological functions of the o receptors are still unclear, recent obsenrations have suggested significant roles of these receptors in the modulation of neural transmission in the CNS and peripheral organs. Some investigators have used several different types of functional assays in an attempt to elucidate a physiological role for the o receptors, yet there is no conclusive evidence that any of the observed effects are mediated by o receptor activation 53. Thus, O ligands have been shown in vitro to produce inhibition '' or potemiation

*1285 of neurogenic smooth muscle contraction, negative modulation of brain phosphoinositide metabolism 'a4' and NMDA enhancement of cerebellar cyclic GMP formation 'O3. In vivo application of o agents has also been reported to cause behavioral '", biochemicd lb6 and electrophysiological M1lO1lnlJM changes in rodents. Many o ligands inhibit the contractile response of the rat tail artery to norepinephrine with different potencies The u recepton have been variously described as modulating phosphatidylinositol turnover "", altering neuronal electrical activity "*'14, inhibiting ion transport in the gut "' and suppressing nicotine-stimulated catrcholamine release from chromaffin cells Il7. Reported physiological effects of o compounds include regulation of neurotransmitter release in peripheral neurons

", rubidium efflux from rat brain synaptosomes 'O7, control of neuronal excitability "9 and changes in deoxyglucose uptake in rat brain ". Although the physiological actions of a wide range of o ligands have been studied, the lack of absolute selectivity of the a dnigs has led to difficulcies in defining the consequences of a receptor activation.

4.2 Interactions with neuropeptides

4.2.1 Neurotensin A substantial body of preclinical and clinical evidence implicates neurotensin (NT) in the pathogenesis of schizophrenia and/or the mechanism of action of antipsychotic dmgs. The blockade of o receptors produces a pattern of alterations in regional NT concentrations similar to that observed following treatments with clinically efficacious butyrophenone and antipsychotic dmgs. The regulation appears to be mediated via

NT concentrations were assessed "*? To ascertain that the NT alterations produced by BMY- 14802 were not the result of nonspecific activity on D, receptors, the effeas of the selective D, antagonist sulpiride were evaluated. Neither acute nor chronic treatment with the seleaive D, antagonist sulpiride produced the pattern of NT alterations observed afrer treatment with BMY-14802. The selective o ligand SR 31742A has also been found to increase the level of neurotensin in the nucleus accumbens '13.

4.2.2 Neuropeptide Y Sigma receptors are distributed in the same brain areas as neuropeptide Y (NPY) in rats and guinea pigs '.'". It has been reported that NPY and PWhave high affinity for the a receptors "'. Moreover, despite the fact that NPY and PW are generally asssumed to have very similar physiological properties and to interact with cornmon receptors, they differ in their affinity for ['WSKF-10,047 and for ['HITCP binding sites 3L8. Thus NPY appears to be a relatively specific ligand for the a binding site whereas PYY has an equal affinity for the o and for the PCP binding sites. Another group using an in vitro receptor-binding assay "found that NPY and PW at up to 500 nM did not bind to o or PCP receptors under different binding conditions including temperatures, membrane preparations, presence of a protease inhibitor and sources of the peptides. Despite the controversy in the results from the in vitro receptor-binding assays, the evidence for the interaction of NPY and o receptors in vivo is quite convincing '67-U03'.For example, in isolated mouse intact jejunum, NPY and JO-1784 reduced ion transport "O? This effect was antagonized by haloperidol, but not by spiperone, thus suggesting a reaaion mediated via o receptors. Moreover, NPY and 50-1784 suppressed both psychological stress- induced and corticotropin-releasing factor-induced colonic motor activation in rats The effects were believed to be mediated through a receptors and the effeas were shown to be G protein-linked '? An in vivo receptor displacement assay was used to examine the interaction of NPY and o receptors. Neuropeptide Y was found to displace close to 40% of the specific finding of ['H](+)SKF-10,047 to a receptors in the mouse hippocampus ". Using an in vivo electrophysiological model, NPY and the fragment NPY,,,, potentiated the NMDA response in the CA, region of the rat donal hippocarnpus. In contrast, PWand the fragment NPY,,,, 18 had no effect by themselves but antagonized the effect of NPY. Al1 othen NPY fragments and NPY-related peptides tested, which have activity at Y,, Y,, or Y, receptors, were without effect on the NMDA response.

4.2.3 Cholecystokinin Sigma ligands have been shown to enhance selectively the colonic motor response to feeding in dogs. This effect is blocked by devazepide, a CCK, antagonist, suggesting the

involvement of CCK in this response lm. Similarly in rats, the stimulation of duodend alkaline secretion by o ligands is blocked by devazepide 291. Moreover, devazepide also blocks the effects of JO-1784 and NPY lY, supporting the hypothesis that the effects of o ligand or NPY depend upon the central release of CCK and/or the activation of supraspinal CCK neurons. This is in agreement with previous findings showing that devazepide administered centrally suppress the enhancement of colonic motor response to feeding in dogs induced by JO-1784 ''O, a response associated with the CNS release of CCK '". Moreover, the potentiation of the NMDA response induced by a "agonists" is abolished by selective CCKh antagonists but nor by selenive CCKs antagonist. CCK-8S, applied with a low current, insufficient to induce by itself an increase of the firing activity, markedly potentiated the neuronal response to NMDA without affeaing significantly that of quisqualate. These results suggest the existence of a hnctional interaction between CCK and a receptors '".

4.3 Interactions with other drugs

4.3.1 Anticonvulsant drugs Phenytoin was first reported to stimulate the binding of ['H-Jdextrornethorphan to membranes from guinea pig brain in 1983 13'. At that time it was known that dextrornethorphan binds to ['HJSKF-10,047-labelled 0 receptors but the involvement of o receptors in phenytoin-stimulated [3~dextromethorphanbinding was not certain. It appears that the stimulatory effect of phenytoin on o ligand binding is specific for only certain o ligands. The finding that ['WDTG binding is not stimulated by phenytoin is in agreement with a recent report by Karbon lg3 indicating that phenytoin does not stimulate either

['HJDTG or ['HJhaloperidol binding 19'. Of al1 the

4.3.2 Antidepressant dmgs Repeated but not acute administration of caused a reducrion in the B, of ['HJDTG u binding sites in the striatum, hippocarnpus and cerebral cortex without alteration of the dissociation constants (Kd) Chronic fluoxetine trearment also causes a sirnilar diminution of the B, of ['HJDTG binding. However, repeated administration of desipramine failed to affect ['H-JDTG binding in these brain areas. Opipramol (OP) is a tricyclic anridepressant compound that was characterized as a potent a ligand with minimal affinity for dopamine and PCP receptors U9301.Opipramol, a clinically effective antidepressant whose structure resembles the major tricyclic antidepressants, is inactive as an inhibitor of NE and 5-HT uptake Unlike most tricyclic antidepressants, the therapeutic actions of OP do not involve biogenic amino uptake. The major finding of a study was that ['HIOP labels two binding sites in rat brain membranes with similar high affinity lM. One site involved o receptors.

Another study lMextended preliminary findings indicating that clorgyline and other MAO inhibitors display high affinity for the (+)3-PPP/a-binding sites in C57BL/6 mouse brain 16'. Among the various MAO inhibitors tested, it seems that dmgs that are considered MAO-A, such as clorgyline and harrnaiine, display relatively selective high affinity for the a binding sites. However, the affinities of some of the dmgs tested for the a-binding sites do not imperatively correlate wit h t heir potencies as MAO-A inhibit ors in enzyme assays. In vitro, semaline displays surprisingly high affinity for u binding sites labelled wich (+)[3H]3-PPP '". Since sertraiine lacks substantial affinity for other CNS binding sites, except for the serotonin transporter, it may be a paitimlarly useful compound for the funher study of a recepton. Furthermore, the combination of antidepressant activity and antagonism of cr receptors may warrant clinical investigation of sertraline in the treatment of psychiatric disturbances where affective disorder and psychosis coexist, such as delusional depression and schizophrenia with prominent negative symptoms.

4.3.3 Antihistamine dmgs Many antihistamines bear a strong structural resemblance to the tricyclic antidepressants and neuroleptic agents. Several antihistarnines have undesirable central nenrous system side effects 12'. The acute dystonias elicited by the antihistamines have an identical clinical presentation to neuroleptic-induced reactions. In addition, prolonged use of antihistamines such as chlorpheniramine, brompheniramine and phenindramine, has led to the development of tardive dyskinesia '? Neuroleptic-induced tardive dyskinesias and dystonias have been generally thought to result from excessive dopaminergic activity in nigrostriatai pathways. However, there has been evidence presented which suggests a role for the a receptors in the motor effeas of antipsychotic dmgs '". Since the antihistamines bear both a structural and clinical resemblance to those neuroleptia which have demonstrated affinity for the o receptor 367, the possibility that antihistamines with a receptor affinity could dso induce O-like behaviors " in the rat has been inves~igated.The results of that study demonstrated that many Hlantihistarnines can interact with the a receptors in addition to their known affinities at histarninic and muscarinic sites. It is possible that some of the central side effects that are common to both classes of drugs,(e.g. acute dystonia and tardive dyskinesia) that are seen with prolonged use of high doses of these agents may be due to these similar affinities.

4.3.4 Antirnuscarinic drugs A link between a and muscarinic binding sites has been proposed following the observation that a compounds inhibit carbachol stimulated phosphoinositide (Pl) '' turnover and carbachol-induced contractions of guinea pig ileum "'. Agents such as caramiphen, levetimide and dexetimide bind with high affinity to both the muscarinic M, receptor and the

O recognition site l". The g-uinea pig ileum longitudinal muscle/myenteric plexus (LMMP) preparation contains a receptors IS6 and its use as an in vitro bioassay for the cr receptors has been described. It has been reported that some a receptor ligands have affinity for the muscarinic receptors in radioligand binding assays and may interaa direaly with junaional muscarinic receptors in the LMMP "'. Sigma receptor ligands were found to inhibit LMMP contractions to electrical stimulation, and the potency of numerous compounds for these effects correlated highly with their binding affinit~to the o receptors ". This inhibition of stimulated contractions was hypothesized, but not shown, to be via inhibition of stimulated ACh release from cholinergie nerve terminais. DTG and its congeners (SKF-10,047,(+)pentazocine and haloperidol) were found to inhibit acerylcholine-induced depolarization in the guinea pig myenteric neurons lf3 and carbachol-induced contraction of guinea pig ileum "" whereas JO1784 potentiated ['HJACh release from rat hippocarnpd slices lg2. These results suggest that there is a funaional role of the a receptors on cholinergic nerve terminas, or within the myenteric plexus, and that these recepton can inhibit stimulated ACh release through an unknown mechanism. It has also been reporred chat o receptor ligands inhibit the stimulated release of ['HIACh from cholinergic nerve terminas in the guinea pig ileum LMMP ". The inhibitory effea of the prototypic a ligand ['H]SKF-10,047 and DTG on ['HJACh release was shown to be concentrationdependent on 5-HT for the electrical stimulation. Six additional selective, high affinity a receptor ligands were also found to inhibit electrically stimulated ['HJACh release. The nature of this mechanism at a molecular or cellular Ievel has not been demonstrated. The effect was not mediated via neuronal muscarinic receptors because the ligands tested in this study included those that do not bind to muscarinic receptors '". The potent antimuscarinic benzetimide and its resolved stereoisomers dexetimide and levetirnide were tested for their affinities at o receptors labelled by ['13j(+)pentazocine or ['WDTG '"". Levetimide was a potent and stereoselective inhibitor of [3HJ(+)pentazocine binding, with a ki of 2.2 nM,while dexetimide was nine-fold less potent (ki = 19 nM). These observations support the hypothesis of an indirect interaction between o and muscarinic receptors 87J55.

4.3.5 Antipsychotic dmgs Some data suggest that o bindiq sites modulate DA activity: (1) using autoradiographic methods, o binding sites have been detecred in DA cell body regions of the rat '" and car brain; (2) in electrophysiological and biochemicd studies, compounds that have high affinity for a binding sites have been shown to either decrease or increase DA activity 110*166*219356.The exact mechanism for the interaction between a receptors and dopaminergic systems has not been clearly elucidated despite research demonstrating an interaction between a and dopaminergic systems in anatornical 129*21S, behaviord 373374, neurochemical 'bbJ75 and elect ro p h ysiological studies '8JYJS6Jm. Gundlach et al. "' demonstrated that o receptors are associated with dopamine neurons of the substantia nigra pars compacta. The results of several studies imply a potential interaction between the o receptors and the midbrain dopamine neurotransmitter system iio*'iiJ49*2g6J~Jm~419.Support for this hypothesis also cornes from studies that demonstrate that munerous neuroleptics possess high affinity for a receptors. A number of antipsychotic drugs representing several chernical classes were found to bind to the (+)-['HJSKF-10,047 binding site with high to moderate affinity. These drugs include a butyrophenone, a benzimidazolinone, a tetrahydroindolone, and all the phenothiazine tested. The rank order of binding potency is halopend01 > > fluphenazine > > > 2 a thioridazide 2 chlorpromazine 2 . However, there are other antipsychotic dmgs such as spiperone, thiothixene, , and that had much Iower or no affinity for the (+)-[3H]SK.F-I0,047 binding site 367. Using animal behavioral paradigms predictive of antipsychotic efficacy, a number of candidate antipsychotic drugs have been identified which may possibly lack the comrnon side- effects associated with neuroieptic therapy. Three of these promising agents, rimcazole (BW

234U) (BMY 13859) '73m and have undergone clinical trials with positive therapeutic results in schizophrenic patients. Rimcazole and remoxipride, two drugs which display clinical antipsychotic actions i3~2iJ6*93~97~ios~202*TU~U7~276*Z96,are quite selective for o receptors. Remoxipride, whose affinity for o receptors has not been previously reponed, is particularly potent and discrirninating for o receptors. Given the psychotomimetic nature of opioid dmgs active at o receptors and rhe notable a receptor affinity found in common arnong these potential antipsychotic agents, the blockade of o receptors represents a novel, promising strategy for the development of new antipsychotic drugs.

4.3.6 Antitussive drugs DM is a synthetic dextrorotary analogue of codeine. While it lacks the analgesic, the respiratorydepressant, and the addinive propeaies of opiate analgesic drugs '5, numerous animal and clinical tests have shown that DM is an effective, centrally acting anritussive, in some cases e-al in potency to codeine. Although the mechanism by which DM exem its antitussive effects is unknown, the fact that DM is structurally related to codeine, the most widely used opiate cough suppressant, may suggest that DM acts at the same central sites as codeine to elevate the cough threshold. Since DM and codeine possess opposite stereoconfigurations, it could be implied that receptors responsible for cough suppression lack the strict stere~s~ecificityassociated with other pharmacological actions of opiates and therefore represent a distinct subclass of opiate binding sites 74. Furthermore, it has been reporred that pentazocine exhibits a potent antitussive effect lS6. The o receptor is found in al1 23 areas throughout the brain, including the nucleus of the solitary tract (NTS) z6. The NTS is the site of the first central synapse for primary afferent fibers which originate from airway receptors and play an important role in the reylarion of respiration. Moreover, the NTS is also adjacent to the site that controls the basic centra mechanism of the cough reflex "'. Thus, it is possible that O-receptors might be involved in the control of the cough reflex. One study demonstrated that (+)SKI?-10,047 and DTG, have marked cough depressant effect in rats lg4. The antitussive effects of these dmgs were antagonized by pretreatment with haloperidol. Furthermore, the antitussive potencies of o-ligands were similar to that of dextromethorphan. Therefore, these results raise the possibility that haloperidol-sensitive O- receptors are involved in the regulation of cough. This possibility is supported by the finding that the antitussive effect of (*)pentazocine and dextromethorphan was significantly reduced by pretreatment with haloperidol. Klein et al., 'O8 reporred that prototypic cr-ligands such as haloperidol, (+)pentazocine and (+)SKF-10,047 displace ['H]dextromethorphan from the high- affinity site with a rank order of potency that is typical of the o receptor. Furthemore, there is a high correlation betweer, the potency of compounds to displace ['H]3- PPP and ['HJdextromethorphan Based on these results, Musacchio et al. contended chat the a-receptor and the high-affinity dextromethorphan binding site are identical. Thus, these data also support the contention that the o receptors are involved in the regulation of the cough reflex.

4.3.7 Cocaine

Cocaine is a p~werfull~addicting dmg of abuse 19. However, cocaine use and abuse, especially when ingested in the form of crack, have increasingly been associated with toxic consequences in humans 'OJo7, and in animal models "*116*'n.Cocaine produces its toxic effects and a variety of physiological and behavioral effens through its interactions with several distinct CNS receptors. It is well established thar cocaine inhibits neuronal reuptake of dopamine, norepinephrine and serotonin and that the transporters for these neurotransrnitters appear to be labelled by ['HJcocaine 16935j-'". In addition, receptor binding studies have shown that (-)cocaine interacts with both cr and muscarinic cholinergic receptors in che brain "2m. Additional findings of interest are that cocaine does not appear to inhibit ligand binding to GABA, NMDA,phencyclidine or BZ receptors arnong others, receptors related to neuronal systems typically associated wirh seizures, as well as to the efficacy of various anticonvulsant compounds 'O8. Cocaine and some related psychostimulants interact with o receptors in binding assays. Their K, values are compatible with blood concentrations reported to produce psychosis in human volunteers. It is proposed that the psychotomimetic effects of some psychostimulants

may derive, at least in part, from their interaction with o receptors "93". As previously mentioned, it has been demonstrated that persistent supersensitivity of u recepton occurs after repeated administration of methamphetamine '". Furthermore, BMY 14802 prevents the

development of methamphetamine-induced sensitization 'J78. Cocaine is classified as a potent, indirect sympathomimeric with substantial abuse potential. This agent is characterized as a monoamine uptake blocker and its effects on CNS systems have been extensively studied. The possibility that cocaine influences other CNS neurotransrnitter systems, such as neuropeptide pathways, has received little attention. For example, stimulation of the release of DA from the nigral-striatal neuronal axis causes significant increases in the concentration of several striatal and nigral neuropeptides ".

5. PHARMACOLOGY OF SIGMA RECEPTORS

5.1 Biochernicaf properties of sigma receptors Numerous drugs from different pharmacological classes bind to o receptors (for review see These compounds interact with o receptors probably because they al1 possess a common pharmacophore required for the interaction. The basic structural requirements for a a ligand are a hydrophobic area, an unprotonated nitrogen and an intermediate side chain with modifications in each area resulting in different activity 136'179241. Numerous s~srematicSAR studies of o receptor activity have been conduned 50~56~~-84~98~118~12B122~1U~I43~1U~2O1229~261~32.1$2~$373~8$~1.J07~.M8~.115FrOM the results of these stu&es severaf models of a receptors have been proposed, but at the present tirne there is no mode1 which appears to be a unifying hypothesis.

5.2 Behavioral properties of sigma receptors Following the biochemical demonstration of the existence of a receptors 105315~24U5U65~3673~,t here were attempts to demonstrate distinct behaviors elicited by a receptors in animals using ligands possessing high affinity for these receptors. Although a number of behavioral studies have been conducted, the precise involvement of a receptors in each of those behavion needs to be fully established. There are many possible reasons for potential difficult ies in hilly establishing u receptor-mediated behavior in animals. The most O ften used o drug in the earlier behavioral studies was (+)SKF-10,047 which dso interacts with PCP receptors. In squirrel monkeys and rats trained to discriminate PCP from saline (+)SKF-lO,O47 resembles PCP, but (-)SKF-IO,O47 fails to generalize to the PCP stimulus 36J41. Similarly, in rhesus monkeys trained to self-administer cocaine, (+), but not (-), isomers of SKF-10,047 and cyclazocine, as well as PCP, are self-administered 349.

A senes of recent reports 37'3g0 on the behavioral effects caused by more selective cr ligands deserves attention. The behavioral supersensitivity to chronic administration of metharnphetamine has been understood to be a possible mode1 for human psychosis lm.BMY 14802 was able to prevent the development of the behavioral supersensitivity induced by methamphetamine. Interestingly, animals that are supersensitive to the methiunphetamine were also behaviorally sensitive to a a receptor ligand (+)fPPP '". It was further demonstrated that animals behaviorally sensitized to cocaine were also sensitized to the behavioral effects of (+)3- PPP '". The sensitized effects of (+)3-PPP could be blocked by 0 antagonist like BMY 14802 Thus, it appears that chronic treatments with either metharnphetamine or cocaine cnggers the receptor-mediated behaviors. This means that the neuronai basis for the development of supersensitivity to both metharnphetamine and cocaine may share a cornmon pathway. Certain o ligands also have been shown to antagonize the acute locomotor stimulating effect induced by cocaine 39.

5.3 ElectrophysiologicaI properties of sigma receptors (+)SKF-10,047 has been shown to rnarkedl~stimulate the aaivity of ventral tegmental

(VTA) A,, DA neurons in the midbrain of the rat "O. (+)Pentazocine is more selective for cr us PCP sites than (+)SKF-10,047 '=Jg9. High doses of (+)pentazocine (8 to 16 mg/kg) had weak stimulatory effects on less than haif of the nigrostriatal DA neurons tested '19. These effects were of lower potency and efficacy than those observed previously with the enantiomers of SKF-10,047 and cyclazocine on nigral DA neurons "O. One study found however, that (+)pentazocine (2 to 30 mg/kg, i.v.) dosedependenrly inhibited the firing rate of A, DA neurons '%. The effects of (+)pentazocine on the entire group of 11 mesoaccumbai neurons were greater than those recently reported for ventral tegmentd area DA neurons 'Il. Because not al1 DA neurons respond to (+)pentazocine, it is possible that this discrepancy is the result of cell-sarnpling differences between the two studies. DTG and JO 1784 are o ligands with very low affinity for the PCP receptor The lack of effea of these compounds on the electrophysiological activity of nigrostriatal and mesoaccumbd DA neurons is consistent with the notion that alteration of DA ce11 firing rate by acute administration of PCP/u ligands is a non-a phenomenon related to the action at the

PCP receptor lm. (+)3-PPP and BMY 14802 bind to a receptors with low nanomoIar affinity and produce marked inhibitory and excitatory effects respectively , on the firing rate of DA neurons. However, these compounds are known to have pharmacological properties unrelated to o receptors. (+)3-PPP for example, was originally identified as a preferential DA autoreceptors agonist '". The electrophysiological and biochernical effects of (+)3-PPP on nigral DA neurons are typical of such an agonist. (+)J-PPP-induced inhibition of nigral DA neurons was prevented and reversed by the DA antagonist (+) but not by the a ligand (- )butaclamol '". The same study has dso been shown in the same study that two other DA antagonists, spiperone and (-)eticlopride, also reversed (+)3-PPP-induced inhibition. Thus, although (+)3-PPP has much lower affinity for DA receptors in cornparison to u recepton '16, the inhibitory effects of (+)3-PPP on DA neuronal firing rate are due specifically to the DA agonist properties of the drug. It has been reported that BMY 14802 increases the firing rate of A, and A,, DA neurons "9-'"3MJW. BMY 14802 is a 5-HT,, recepror agonist of modest potency OC,, 150-200 nM) and binds with lower potency to a, receptors and other neurotransrnitter receptors 382. It is concluded that the marked effects of certain o ligands on DA ce11 electrophysiology are likely due to their non-cr propexties. Extracellular recordings have shown that action potential firing rate was depressed by

(+)3-PPP in rat Purkinje cells 39b, but increased by (+)Sm-10,047 in rat A,, neurons ". Intracellular recording from rat hippocampal CA, neurons showed that (+)3-PPP had no effect on membrane potential and input resistance, but potentiated the depolarization and generation of action potentials induced by NMDA U9. In contrast, in the rat locus coeruleus (k)SKF-

10,047 inhibited the depolarization evoked by NMDA 214. In the hybrid cells (mouse neuroblastoma-Chinese golden hamster brain) of the NCB, line both (+)3-PPP and (+)SKF- 10,047 induced a depoiarization, but the mechanism underiying this was not determined U. Several selective high affinity o ligands such as DTG were tested in an in vivo electrophysiological mode1 using extracellular recording from pyramidal neurons of the dorsal hippocarnpus of anesthetized rats. The effects of these o ligands on the neuronal firing activity induced by rnicroiontophoretic applications of NMDA were compared to those of QUIS and KA-induced activations. Low doses of DTG (lpg/kg, i.v.) as well as its rnicroiontophoretic applications enhanced selectively the neuronal response to NMDA z6. A similar potentiation of the NMDA response is also obtained with other selective a ligands such as 10-1784, JO-1783 (+)pentazocine, APDQ, Adip G and DnBG). This potentiation was suppressed by the administration of o ligands such as haloperidol, BMY-14802 and (+)3-PPP ". Spiperone, which has a similar binding profile to that of haloperidol, except for its low affinity for o receptors, did not reverse the potentiation of the NMDA response induced by low doses of o iignads such as DTG.

5.4 The concept of agonidantagonia at sigma receptors It should be kept in mind that the concept of "agonist/antagonist" at a receptors has not been completely established. Recent behavioural, electrophysiological receptor binding 20J15 and peripheral bioassay data 385 have provided evidence that haloperidol has the properries of an antagonist on a receptors, whiie rhere have been reports that hdopendol can act as an agonist on o receptors 3~9.1. Extracellular recording techniques were used to study the effeas of the selective o receptor agonist (+)3-PPP and selective cr receptor antagonist BMY 14802 on DA neurons of the substantia nigra. Intravenous administration of (+)3-PPP produced a dosedependent inhibition of DA neuron firing rate. Total inhibition of DA neurons produced by (+)3-PPP could be completely reversed by administration of BMY 14802. Also, pretreatment wirh BMY 14802 shifted the (+)3-PPP dose response curve to the right. These data demonstrate a relationship of the o receptor with the DA system, even though the mecanism underlying this phenornenon is not elucidated. A major obstacle for behavioral research with o ligands appears to be the lack of well defined agonists and antagonists at o receptors. DTG, for exarnple, has been called both an agonist and antagonist at o receptors '56. There have been, however, few systematic behaviorai studies with cr ligands that have attempted to define a agonists and antagonists. Using a discrirninative stimulus procedure, Steinfels et al. "j have shown that the interoceptive cue produced by (+)SKF-10,047 in rats was mimicked by (+)3-PPP, PCP and (-)NANM and anragonized fully by haloperidol and partially by . Balster 16, however, reported thar halopend01 did not antagonize the discriminative stimulus effects of (+)SKF-10,047, correlated well with relative affinities for the PCP receptors. Holtzman lY, also using a discrimination procedure, found that the interoceptive me produced by DTG was mimicked by PCP and o ligands and by selected opioids, yet the effects of DTG were neither mimicked nor antagonized by halopendol, (-) butaclamol and naltrexone.

5.5 The up- and down-regulation properties of sigma receptors It is well documented that chronic treatments with halopend01 decrease the number of

a receptors in the mouse, rat and human brains 1"*17'3'7JJb.M9. These findings have generally been interpreted in terms of down-regulation of cr receptors. Of the therapeutically used antipsychotic agents, halopendol is the only one which has been found to reduce the number

of o recognition sites in the brain. Riva and Cresse 'O9 reported that the antipsychotic drugs clozapine and which, in contrast to haloperidol have quite low &inities for the o

binding sites, did not change the number of 0 binding sites after repeated treatmenr. Repeated t reatment wit h chlorpromazine, fluphenazine or remoxipride, antipsychotics

with moderate affinity for o receptors, did not affect o receptors in the brain IW1. Reynolds et al. '& reported that patients treated with chlorpromazine or fluphenazine had, in contrast to those treated with haloperidol, no signifiant decrease in a receptors detennined with DTG binding in cerebellar correx membranes from post-mortem human brains. Both these antipsychotic benzamide derivarives " with 14 times higher affinity for the o binding sites than for the D, receptors in the rat brain '18, were found in that study not to alter the B,, or KD of the

receptor and the a binding sites "6? Thus, that study "' shows chat at similar D, receptor occupancy, remoxipride, in contrast to haloperidol, did not alter the a binding sites. Furchermore, remoxipride did not antagonize the action of haloperidol, an effect which could be expected if haloperidol and remoxipride compete for the sarne binding sites in vivo. Since the compounds were administered via osmotic pumps giving brain concentrations producing similar behavioral changes, pharmacokinetic differences between the two compounds were excluded. It should also be stressed that remoxipride appears to bind more potently to a, than to q recognition sites, since it is more active in inhibiting the binding of ['H](+)SKF-10,047 than that of L3H]DTG 'Il. Chronic treatment with (+)pentazocine, a putative cr agonist, produced no change in o receptors in the rat brain However, similar treatment was reported to increase the Kd value of the labelled ligand to a receptors without affecting the density an effect which was not reproduced by a more recent study ". In accordance with a possible regulation of the o recognition sites are the findings of Beart et al. that repeated treatment of rats with the cr ligands rimcazole and DTG produced increased B,, and decreased KD values of the binding of [3H](+)3-PPP to cortical homogenates which is the typical response to chronic exposure to an antagonist. A significant up-replation of ['HI(+)pentazocine binding sites was observed in cerebellum, substantia nigra and frontal cortex of animals exposed to methamphetamine (130 to 145Oh of control Ba.No apparent change in the affinity of ['H](+)pentazocine for the cr recepton was observed between the two groups in any of the brain regions examined. A study has demonstrated that a similar schedule of methamphetamine-treatment produced a marked sensitization to the effects of (+)fPPP as determined by several behavioral paradigms in rats '". A more recent study supports the notion that met hamp hetarnine-induced sensitization is associated with the up-regulation of a receptors. Two of the brain regions that sustained cr receptor up-regulation, the substantia nigra and frontal conex, correspond to the nigrosrriatal and mesolimbic dopaminergic systems respectively. These findings suggest that supersensitivity of a recepton in critical brain regions may be associated with the development of psychomotor sensitization ro methamphetamine, and thus a receptors may have an important role in methamphetamine-induced psychosis. The finding that methamphetamine and haloperidol, that have diametric effects on dopamine neurotransmission regulate the o receptors in "opposite directions" 16' supports the involvernent of dopaminergic transmission in cr receptor funaion. The response of the peripheral o receptors indicates that "peripheral" ['WDTG binding sites respond to chronic haloperidol in a manner similar to ['WDTG binding sites in the brain '". This increases the probability that these sites are biologically active receptors and that central and peripheral [3H]DTG binding sites are sirnilar entities, rather than fundamentally different in a manner resembling the central and peripheral benzodiazepine receptors. It is well documented that behavioral sensitization, or reverse tolerance after repeated methamphetamine administration, is an animal mode1 of vulnerability to acute exacerbation in methamphetamine psychosis and schizophrenia 8JwJ'4. In view of the significant affinity of some atypicai neuroleptics for a recepton and the modulating effects of o receptor-related compounds on dopamine neurons "J", a receptors might be involved iil psychosis and methamp hetamine-induced behavioral sensit izat ion. Another study '*, has shown that repeated administration of halopend01 to rats induces a transient down-regulation of o binding sites labelled with (+)[-'H]3-PPP but not ['HJDTG. In addition, ( + ) 3-PPP binding sites express a marked plasticity towards the regdatory effects of guanine nucleotides. Three major findings were presented in that study: (1) o receptor dow n-regulation following repeated exposure to halo pend01 is transient and the receptor functionaily recovers 4 weeks after the treatment is ceased; (2) the down-regulation of a receptor is accompanied by reduced responsiveness to guanine nucleotides; (3) the differential effects of halopend01 and guanine nucleotides of (+)['H]3-PPP and ['WDTG binding sites hnher support rhe hypothesis of existence of a receptor subtypes. Chronic treatment with SKF-10,047 decreased proenkephalin mRNA levels in the caudate putamen and the nucleus accumbens by 20 to 22% relative to controls. Chronic treatment with pentazocine also decreased proenkephalin mRNA levels by 20 to 25% in the same brain regions. The results demonstrate that a-receptor activity cm exert tonic effects on proenkephalin mRNA expression in the striatum and rhe nucleus accumbens of the rat 9.

6. CHARACTERISTICS OF SIGMA RECEPTORS

6.1 Molecular biology The structure-affinity relationships of o binding sires support their distinction from conventional opioid receptor subtypes. The absolute configuration at the chiral carbon atom bound to the nitrogen for the preferred (+)-benzomorphans is opposite to that for classical opioid compounds, such as morphine and ndoxone, which lack affiniry for o binding sites '58. A study has described stmcnire-affinity relationships for various groups of compounds at o binding sites These binding sires were labelled wirh (+)-I3HJ3-PPP.Several srnicrural requirements are evident. Firstly, the prirnary pharmacophore at o binding sites seems to be the phenylpiperidine moiety(3- or ~~henylpiperidine)which is present in most compounds that are potent at o binding sites. Secondly, affinity is markedly influenced by the N-alkyl substituent with more lipophilic substitutions affording greater affinity for o receptor binding sites. Thirdly, compounds of many different srnicrural classes demonstrate substantial affinity for o binding sites, suggesting that certain intramolecular distances (e.g., N to aromatic ring) 31 are not subject to rigid constraints. Calculated imrarnolecular distances for several compounds support this conclusion. Two polypeptides have been identified (molecular masses of 22 and 27kDa) in the endoplasmic reticulum of liver, kidney, lung and adrenal gland that bind phenyldkylamine

Ca2+antagonists with nanomolar affinity L9692b133"37. There is evidence that the photoiabelling of the 27-kDa polypeptide is blocked by nanomolar concentrations of o ligands [order of potency: haloperidol > pentazocine > DTG > dextromethorphan > (+)SKF-1O,O47] '". The apparent affinities of these and other drugs closely correspond to those for ['H]DTG binding sites. A radiolabelled azido-derivative of DTG recently was used to isolate a 29-kDa polypeptide that may represent the intact a receptor complex lY7. Recent studies have associated the o receptor with monoamine oxidase type A '" or the cytochrome F4&d (dibrisoquine hydroxylase) on the basis of SKF 525a and substrate binding

320. These findings also indicate that the o binding sites are distinct from the enzyme binding sites. However, a possible relationship between the o binding sites and MAO is apparent from the subcellular distribution of the receptor and enzyme binding sites. The low molecular weight of the a receptor in comparison with monoamine oxidase type A or the cytochrome P-JId (borh > 45 kDa) argues against the identity of the o receptor with either of these enzymes '". A high proportion of cr recognition sites were found in the microsomal fraction of the guinea-pig brain, a location which indicates that many of these sites rnay be intracellular. This confirms the work of McCann and CO-workers 53.m, and extends it to the four other radioligands widely used to label the a recognition sire. Whilst it is conceivable that these sites represent internalized or recently synthesized receptors, the possibility that the o receptor is involved principdly with some intracellular function must be considered. The presence of high concentrations of a recognition sites in liver ''O and in other peripheral tissues 3'5J26~'" and the paucity of evidence implicating the o receptor in synaptic transmission support this view. Despite numerous studies of a receptors a number of basic questions about their nature remain to be answered. Although the results of subcellular fraction studies indicate rhat a receptors are not concentrated in synaptosomes, mitochondria or nuclei the precise subcellular location of o receptors remains to be determined. Ultimately any mode1 of u receptor structure of subtype composition will have to be substantiated by biochemical evidence. Kavanaugh et al. 19' have demonstrated labelling of an M, = 29,000 polypeptide in guinea-pig brin membranes using a radiolabelled azido derivative of DTG. This protein presumably represents the proposed halopendol site or subunit of the o receptors. Very recently, the a, binding site has been clonedl". It appeared to be a single 3O-KDa protein from guinea pig liver. (+)['Wpentazocine was used as specific probes. This binding protein retained its high affinity for haioperidol, DTG and (+)pentazocine. This protein seemed to be unique since no other known mammalian proteins presented similar amino acid sequence. Moreover, this protein shared homology with fungal proteins involved in sterol synthesis, in agreement with the ability of some steroicls, such as progesterone, to interact with o, binding sites.

6.2 Second messengers In binding studies, one cannot differentiate agonists from antagonists unless some second messenger system can be shown to influence ligand binding. There have been some suggestions of an association of o receptors with second messenger systems, including the phosphoinositide cycle and cyclic GMP. Some reports indicate a regulation of a receptor binding by guanine nucleotides 20*"J57*161. Such regulation presumably reflects a role for G proteins in mediating o effects. If a receptors act through G proteins, one rnight anticipate influences on cyclic AMP or phosphatidylinositol second messenger systems. One study reported the identification of two affinity states of the a receptors and the formation of a high affinity u-ligand-receptor and a high affinity a-ligand-receptor-G protein complex 'j7.The identity of the G proteins coupled to the o receptors is not clear, and furcher studies are required to characterize these regulatory binding proteins. However, the findings that GTP inhibits the binding of (+)E3HI3-PPP'OJ6', and r3HJDTG s9 significantly reduces the regulatory effect of Gpp(NH)p, may suggest the G proteins that are coupled to the o receptor have the characteristics of Gi/G, regulatory proteins 'O. Studies with either radioligand indicated that haloperidol, chlorpromazine, DTG, rimcazole, (+)SIG-10,047, (-)SKF-I0,047, (+)3-PPP, (i)pentazocine, (+)cyclazocine, PCP and dextromethorphan, in the absence of Gpp(NH)p when o, receptors were in a high affinity state, al1 ligands exhibited nanomolar K, values and stereoselectivity consistent with rhose seen at al receptors. (+)Isomers were more potent than (-)isomers. However, in the presence of Gppop,which rendered o, receptors in a low-affinity state, at least one study " has shown that stereoisomers of SKF-10,047 lost their stereoselectivity (ie., (+)SKF-10,047 was almost equipotent to (-)SKF-IO,O47). In addition, the Ki values for both isomers were in the 33 micromolar range ". These results, obtained from the low affinity state of a, receptors, are strikingly similar to those found at "low affinity o receptors" reported in NCB-20 cell lines "' (Le., micromolar K,s and a lack of stereoselectivity). Could it be that the "low affinity cr receptors" demonstrated in NCBZO cells are the GTPcoupled low-&nity o receprors with characteristics similar to those reported in the NCB-20 cells in human and guinea pig brain ". No data are available yet regarding whether u, receptors are linked to GTP-binding regdatory proteins. Nevertheless, several physiological responses were shown to be linked to GTP- binding regulatory proteins. The rnechanism of cation effects on ligand binding to o receptor subtypes and the potential interaction of cations wirh G proteins have not been studied in depth. Such information may indicate whether o receptor subtypes are independemly coupled to either G proteins, ion channels, or both. Others have demonstrated similar regulation of cerebellar cyclic GMP in intact animals, when cr ligands reverse the increase of cyclic GMP produced by administration of harmaline of d-serine 'O3. Dmgs parricularly effective include the putative antipsychotic o drug BMY 14802, as well as opiprarnol, a and ifenprodil, a dmg originally developed as adrenergic antagonist 'O2. Ifenprodil and opipramol like haloperidol, have very high affinity for o receptors 1m?194.tZ0. The effect of guanine nucleotides and ions of ((+)['H]3-PPP), ((+)['KJSKF-10,047) and

(['HJPCP-)-OH) specific binding to rat brin membranes was examined 16'. These three compounds are proposed as protorypical ligands for the labelling of the

6.3 Cytochrome P,,,

The high density of o receptors in rnicrosomal fractions L53, coupled with the observation that proadifen, a prototypic cytochrome P,, blocker "', potently inhibits ['WSKF- 10,047 binding to o recepton in rat brain and liver 'Io led to the hypothesis that a a receptor subtype may be a form of cytochrome P,, 17. A wide variety of compounds induce the synthesis of cytochrome P,, forms and related proteins several-fold in the rnitochondria and endoplasrnic reticulum of the liver and other peripheral tissues *. A characteristic feature of P,, enzymes is their broad and overlapping drug specificities and their heterogenous localization throughout the brain lg9. The binding properties, affinities and drug specificities of a "specific" o receptor ligand were identicai in membranes from rat brain and liver microsomes, suggesting that the o receptor is, in fact, a P,, isozyme 320. Based on the findings that most compounds binding with high affinity to the o receptor are tertiary of secondary amines, McCann discussed the possibility that these sites are a form of microsomal flavinsontaining monooxygenase, or alternatively, cytochrome P,tS3. As long ago as 1983, Craviso and Musacchio questioned whether the a binding site might not be a metabolic enzyme rather than a receptor. This was based on its selective subcellular localization to rnicrosomes rather than synaptic plasma membranes 52. A number of hypotheses spring to mind. First, it is possible that proadifen is just one more drug which has a properties unrelated to its therapeutic and bestcharacterized pharmacological mechanisrn of action. Another possible explanarion is that the NMDA receptor, the cr receptor and cytochrome P,, (the site of action of proadifen) contain very sirnilar arnino acid sequences. However, an in vivo electrophysiological study has shown that the potentiation of the NMDA response induced by cr "agonists" such as DTG is not reversed by proadifen nor by piperonyl butoxide. Unlike o "agonists", neither proadifen nor piperonyl butoxide affected the neuronal response to NMDA in the CA, of dorsal hippocarnpus pyramidal neurons '". Using cytochrome P,,, a study has confirm that u binding site is not identical to cytochrome P,, ".

6.4 Endogenous ligands for sigma receptors Evidence that the cr binding site may be a site of action for an endogenous transmitter was suggested by the demonstration of a component in brain extracts that cm displace

[3H]DTG binding to o receptors 67-'M. The structure of the putative "a factor" is not yet known, but the physiological relevance of the putative a factor was supponed by work showing that depolarization of live brain slices caused the calciumdependent reduction in radioligand binding to the haloperidol-sensitive a receptor 2g3. Using porcine brains, which have already been used to isolate and purify an endogenous ligand for the PCP receptor, a factor has been isolated that inhibits the binding of ['HJ-(+)SKF-10,047 and not the binding of ['Hl- PCP. This factor appears to be a peptide or protein because incubation of the active fraction with pronase, a nonspecific peptidase, elirninated the ability of the porcine fractions to inhibit the binding of [3H]-(+)SKF-10,04ï. A study demonstrated the possibility of the existence of endogenous ligands for the o receptors in the brain j6). Fractions from peak 1 and peak II in that study likely contain such ligands. Those fractions were more potent at competing for a receptor binding for p and k opioid receptor binding. Furthermore, rhey were al1 almost inactive in the phencyclidine receptor binding assay. It was suggested that the term "sigmaphins" be used to denote endogenous ligands which interact specifically with the a opioid receptors. Peaks 1 and II obtained in that study therefore may contain sigmaphins. Although the chemical nature of sigmaphins was still unknown, there are points that can be made. Inasmuch as there are at least two different peaks which cm interact preferentially with the o receptors, it is likely that sigmaphins may be heterogenous. Alrhough the possibility that one is the breakdown product of the other is difficult to rule out definitively. In an attempt to isolate and examine endogenous brain substances that interact with o recepton, an active substance has been tentatively identified '" which had several unusual characteristics: first, it was small, with a molecular mass of about 480 Da; second, it contained no nicrogen, and third, it potentiated electrically induced contractions in a guinea pig vas deferens preparation. Steroids also have some of these propexties. Among more than 40 steroids tested, only a few were able to inhibit binding of the a receptor ligand Pm(+)-SKF-10,047 to guinea pig brain and spleen membrane preparations L55J61-"6. These were progesterone, testosterone, de~x~corticosterone,11&hydroxyprogesterone, pregnenolone sulp hate and corticosterone. The Hill coefficients derived from the binding curves were al1 close to one, indicating absence of cooperativity. Furthermore, progesterone reduced the affinity of C3H](+)-

SKF-10,047 for CF receptors without affecting the totd number of o binding sites 3531*416. Although steroids bind to the o receptor, this receptor is not the traditionally recognized cytosolic steroid receptor '". First, a receptors must be membrane bound, because brain and spleen membrane preparations were used in the ligand binding assays. Second, several steroids that are good ligands for cytosolic steroid receptors, including promegestone, oestradiol, RU27987 and RU486 were almost inactive in the a receptor binding assay. Third, the Kd values of these steroids are in the range of 0.1 to 10 nM at cytosolic steroid receptors '" but are much higher (200nM to 3pM) at cr receptors. Recently, it has been reported that neurosteroids interact with o receptors ? It was also proposed that neuropeptide Y (NPY) and peptide YY (PYY)could act as endogenous ligands for a binding sites, as both NPY and PWcornpeted with high affinity (nanomolar) for ['H](+)SKF-10,047 binding sites in rat brain membrane homogenates "'. It thus appean that NPY, PW and a selective Y, agonist can interact in a concentration- dependent manner with in vivo ['H](+)SKF-10,047 labelling in the mouse hippocarnpal formation. This effect demonstrates select ivity as a Y2agonist, unrelated peptides and adrenalin failed to alter in vivo a labelling. Others '" were unable to obtain clear evidence for the existence of in vitro receptor binding interactions between a variety of a- and NPY-related molecules. Various peptidase inhibitors are generdly used in in vitro binding assays to insure the integriry of NPY peptides used either as radioligands andior cornpetitors. Could it be that an endogenously generated NPY metabolite is able to act as a o ligand in vivo? While clear evidence is currently lacking to supporr this hypothesis, Contreras et al. ' proposed the existence of endogenous a ligands of peptidergic nature. It is thus of interest that only certain NPY-related peptides were able to modulate the in vivo labelling of ['HI(+)SKF-10,047 in the mouse hippocampus ". Evidence has been provided that depolarization of brain slices releases an endogenous ligand that competes with the binding of the o radioligands ['H'JDTG and

(+)L3H)3-PPP 60J83. These results support the hyporhesis that a form of the o binding site may act as a receptor for both a drugs and an endogenous neurotransmitter. The mossy fiber tracts of the hilus contain large arnounts of ionic zinc, which has been demonstrated to be released by synaptic stimulation or tissue depolarization '"'". The ability of ionic zinc to displace o ligands from their binding sites in rat brain was examined and compared its effects with those of other divalent cations. There are two principal conclusions from the data presented in that study. First, these results suggest that the cation Zn" has a 50- fold greater affinity for a, than a, receptors, and second, they imply that ionic zinc may act as an endogenous ligand at the receptors. This was the first dernonstration of selective effects of a divdent cation on radioligand binding to the a receptor subtypes and provides evidence that endogenous Zn2' may be responsible for previously observed decreases in the binding of ['H]DTG to hippocampd slices after focal elearical stimulation of the hilar region of the slice in vitro 61. High frequency stimulation of seleaed pathways in the hippocampus caused the Ca2+dependent reduction of ['HJDTG and ['H](+)3-PPP binding to the haloperidol-sensitive o binding site 60. Theoretically, a reduction in radioligand binding could reflect either a competition between ['HIDTG and an endogenous o ligand or a depolarization-induced change in the conformation of the o binding site that alters its affinity for ['H-JDTG. The results observed show that the reduction in ['HJDTG binding was Ca" dependent, produced by depolarization under a variety of conditions (KI, veratridine) ? The observation that both perforant path and mossy fiber stimulation gave rise to significant displacement of C3H]DTG lead to the speculation that the endogenous

7. CLNCAL IMPLICATIONS OF SIGMA RECEPTORS

7.1 Ischernic It has been proposed that the neurotoxicity of glutamate, or related excitotoxic compounds, may play a key role in the pathophysiology of cerebral ischaemia, at least partidly via overstirnulation of the NMDA subtype of excitatory receptor ". Since NMDA antagonism is one prornising pharmacologie approach to preventing neuronal necrosis associated with cerebral ischaemia Il7, compounds possessing NMDA anragonist activity might prove beneficid in stroke or other clinicd situations causing brin ischaemia. Recent findings indicate that the neuroprotective and polyamine sire of the NMDA receptor complex antagonists ifenprodil and eliprodil have high affinity for the a recepton labelled by [3H]3-PPP 50th in vitro 19432 and in vivo studies ". These compounds, as well as other o ligands such as BMY 14802, haioperidol, opipramol and caramiphen, prolong survival time in a hypoxic environment in the mouse 45*a*297-Vm and have been taken as evidence to support an involvement of a receptors in neuroprotection The fact that o ligands modulate electrophysiological and biochemicd NMDA receptor-mediated responses "'JOWM and reduce glutamate release in rat hippocampal slices subjected to ischemia-like conditions is consistent with a funaional antagonism by a ligands of NMDA receptor mediated transmission. Taken together, these data raise the possibility that o ligands may have therapeutic potentid as neuroprotecrive agents in brain ischemia 203*U'.

7.2 Dementia A diverse range of recepton ha been shown to be altered in the hippocarnpus in

Alzheimer's disease (AD) '5~176but the status of o binding sites in this disease has just been investigated recently. Lesion studies in the rat suggest that o binding sites in the hippocampus occur on large pyramidal neurons The distribution of o binding sites in the animal L56 and human brain 17' indicates that these binding sites are frequently associated with the ce11 bodies of large cells. The cells in the hippocampus are particularly vulnerable to Alzheimer-type pathology 15. While cognitive enhancers may eventudly prove useful in AD, the major clinicd problem in hospitalized patients is behavioral control. The antipsychotic dnig hdoperidol, which has a very high affinity for o binding sites ", is one of the most cornmonly prescribed substances for the hospitalised AD patient. The developrnent of new antipsychotic agents with fewer side effects will thus be of immediate practical use for managing AD. Some of these compounds may have high affinity for a binding sites and little affinity for DA receptors (e.g. BMY-14802) It was thus of interest to consider the status of a binding sites in AD.

Seven post-morrem brains with a diaposis of AD were used in a study 17' (4 rndes, 3 females; mean age 74 6 years; mean postmortem delay 14 5 h). The diagnosis was based on the clinical history and an examination of brain tissue. Al1 cases showed numerous plaques and neurofibrillary tangles in the absence of other neurological disease. Care was taken to ensure that none of the cases used in this study had been given dmgs which may function as a ligands in the 6 weeks preceding death. The densities of E3H'JDTGbinding to a binding sites in the CA, stratum pyramidal region in 7 hippocampi affected by Alzheimer's disease were compared with densiries in 7 normal hippocampi 17'. There was an average reduction of 26% 39 in VHJDTG binding in this area which was correlated with an average 2g0/0 pyramidal celi loss in the sarne region. The central cholinergic systems ~laya pivotal role in rnemory functions as -. - -- demonstrated by the marked amnesia induced by administration of muscarinic or nicotinic receptor antagonists such as scopolamine. Several clinicopatological observations support these studies, in particular the profound deficits in corrical cholinergic activity observed in patients from Alzheimer's disease and in pathological aging. NMDA receptors also play a cmcial role in the neurophysiological processes underlying learning and memory in the hippocarnpus. These receptors are implicated in the induction of long-term potentiation in hippocampal CA, pyramidal neurons, a form of synaptic plasticity that may be involved in the encoding of memory. Blockade of NMDA receptors by competitive antagonists or noncornpetitive ones, acting either through the glycine modulatory site or through the PCP binding site, suppresses the induction of long-term potentiation, without affecting baseline synaptic transmission. Recent studies have provided convincing evidence that o, receptor ligands exert a modulation of learning and memory processes, through interactions with both the cholinergic and glutamatergic systems W"? Scopolamine, a cholinergic receptor blocker that impairs memory, has been shown by several investigators to produce a transient memory disorder in humans which mimics the cognitive impairment associated with aging or dementia 3992G. In a study 96 the effect of JO- 1784, a seleaive o ligand wac investigated in the rat scopolamine mode1 of memory impairment in comparison to the o ligands. That study demonstrated that pre-administration of 50-1784 (0.25 to 16 mg/kg i.p.) to rats reversed, in a dose dependent manner, the amnesic effect produced by scopolamine in the passive avoidance test in the rat. (+)3-PPP at a dose range from 0.25 to 4.0 mg/kg also antagonized the memory deficit following scopolamine administration. In contrast, DTG (0.25 to 8 mgkg) ~roduceda bel1 shaped dose-res~onsecurve with maximum effect between 1 and 4 mg/kg. Piracetam, a reference drug with no affinity for the o receptor, administered in the same test conditions always antagonized the action of scopolamine in agreement with previous results "'. Conversely, administration of rimcazole and (+)Sm-10,047resulted in no signifiant effect on the retention deficits. From these results it was proposed that o receptors play a role in the memory processes through their NMDA enhancing effect in hippocampal region and that JO-1784 might be a putative candidate for the treatment of cognitive dysfunctions. 7.3 Movement disorder

Walker et al. 394 have shown that the injection of DTG into the red nucleus of the rat causes a marked dystonia. Sirnilar effects were found when halopend01 or (+)SKF-I0,047 were injected into this region, whereas PCP and clozapine had no effect. Further studies by these investigaton showed that microinjections of DTG into the substantia nigra produced vigorous contralateral turning behaviour at very low doses (0.2% 8.6 nmol). Such findings suggest that a receptors represent biologically functiond recepton that are active in the control of movement 18*1"*146-'91. Furthermore, since haloperidol and many other antipsychotic drugs exhibir an affinity for o receptors that is equivalent to their affinity for dopamine receptors, it seems possible that these receptors are involved in the motor side effects of antipsychotic drugs. Further support to this hypothesis is provided by the obsenration that a genetically dystonic rat strain also has an increased density of haloperidol-sensitive o receptors rhan their non-dystonic controls; the o receptors in these dystonic rats also had a higher affinity for the ligand 'j.

7.4 Psychosis Initial studies in the 1980's indicated that the dysphoric effects elicited in humans, or the excitatory effects elicited in rats, by SKF-10,047 or cyclazocine are similar to those induced

by the psychotomimetic dmg PCP 15L1537198. Although the regional distribution of o receptors has been characterized in guinea pig 138*116256,rat lYa6 and human 174*a3 brain, the mechanism and circuitry through which o ligands produce bi01o~ica.leffects are poorly undersrood. One way to address this issue is by using the 2-deoxy-D-[l-'*C]glucosemethod (["CD, which provides a means for evaluating changes in regional metabolic activity, thereby enabling the identification of neural substrates of behavior distinct from the sites of initial receptor interaction L89*413.Della Puppa and London 9'*92 used the ["CIDG method to quantify the cerebral metabolic effects of a single dose (30 mg/kg) to three putative o ligands, BMY 14802, BW 234U (rimcazole) and (+)pentazocine. This study reported increases in local cerebral glucose utilization (LCGU) for BMY 14802 and (+)pentazocine in various brain regions and decreases in LCGU for BW 234U. A role of o recepton in the LCGU effects described in that study seemed probable because the most profound effects on cerebral metabolism were observed in motor regions that are rich in o receptors 13836. The marked changes in LCGU within brainstem motor circuits are also consistent with behavioral data that identify a role for motor control in these regions. For example, in the rat microinjection of u ligands in the substantia nigra induces contralateral

circling lUJg4, while microinjection in the red nucleus did not show significant changes in LCGU after treatment with DTG. However, the red nucleus projects to rnany cerebellar and related nuclei that were significantly altered by this ligand. These include the deep cerebellar nuclei (the cerebellar output neurons), the olivary nuclei (the source of the cerebellar climbing fibers) and the vestibular neuclei (a terminal field of the o-rich cerebellar Purkinje cells). The neuronal circuitry suggest that DTG-induced changes in the afferent supply to these hindbrain structures resulted in the observed increases in regional metabolic activity. The marked increases in LCGU in the CNS are suggestive of a-mediated increases in synaptic activity in projections terminating on nigrostriatal dopamine neurons. This in turn

would be expected to alter the activity of dopamine neurons. Indeed, a role of O recepton in nigrostriatal neurotransmission has been suggested by data from microdialysis experirnents w hich have demonst rated a dopamine-releasing effect of o ligands adrninistered parenterally . DTG (1 mg/kg) produced a 40% increase in extracellular dopamine @A) levels in the caudate

395. Previous data on dopamine metabolites dso suggest a dopamine-releasing action of a ligands

29*'66*'". The possibility that increased LCGU in the CNS reflects O-induced activation of nigrostriatal dopamine neurons has also been suggested in behavioral experirnents. Circling behaviour induced by low doses of DTG, following unilateral microinjection in the substantia nigralD, was dependent upon an intact nigrostriatal dopamine system. Since changes in glucose utilization mainly reflect metabolic activity in synaptic terminas, one might hypothesize that the changes in the substantia nigra reflect increased excitatory or inhibitory synaptic activity of nigral afferents. This would lead to a change in firing of dopaminergic neurons which in turn might be expected to lead to significant changes in striatd LCGU. In spite of these limitations, the findings of that study indicate that low doses of parenterally administered DTG significantly alter cerebral metabolism in discrete regions of rat brain. These alterations occurred extensively in motor and limbic structures that are rich in a receptors. Alrhough the available data are compelling, the metabolic effects of multiple doses of a variety of selective a ligands must be examined in order to determine whether the effects reported here are indeed a-mediated. The aboved rnentioned studies with the fact that many neuroleptics bind with high affinities at the a receptors suggest the involvement of these receptors in psychosis. 7.5 Cough Dextromethorphan, a comrnonly available non-opioid antitussive drug, has a high affinity for the o binding sites 205JW. Furthermore, it has been reported that penrazocine exhibits a potent antitussive effect Ia6. The o receptors are found in al1 areas throughout the brain, including the nucleus of the solitary tract (NT) 256. The NTS is the site of the first central synapse for primary afferent fibers which originate from airway recepton and play an important role in the regulation of respiration. The NTS is also adjacent to the site that controls the basic central mechanism of the cough reflex 'IL. Thus, it is possible chat a receptors might be involved in the control of the cough reflex. However, no detailed investigations of the possible role of o receptors in the regulation of the cough reflex have yet been reported.(+)SKF-10,047 and DTG also have a marked cough depressant effect in rats 18*.

Klein et al. 'Oa reported rhat o ligands such as haloperidol, (+)pentazocine and (+)SKF-10,047, displace [3131dexrromethorphan frorn the high-affinity site "*La1*205~*" with a rank order of potency chat is typical of the a receptor. Furthermore, there is a high correlation between the potency of compounds to displace ['H]3-PPP m6 and ['HJdextromethorphan Therefore, these results raised the possibility that o receptors are involved in the regulation of cough 3,185 .

7.6 Gastroenterology DTG and (+)Sm-10,047 potently and dose-dependenrly stimulate duodenal aikaline secretion in the rat duodenal mucosa 291 and this stimulation appears mainly related to the o- binding properties of these compounds. The stimulant activity of DTG on bicarbonate duodenal secretion is independem of p-opioid, prostaglandin, dopamine, az adrenergic and muscarinic receptors. (+)Sm-10,047, DTG and JO4784 induce a selective potentiation of postprandial colonic motility without affecting the fasted motor profile '". Haloperidol blocks

(+)SKF-10,047-, DTG- and JO-1784induced srimulation of colonic motility 133Jn. These results reinfome the hypothesis of selective effects on colonic motility through a receptors The failure of these compounds to influence colonic motility when adrninisrered LC.V. is in agreement with a penpheral site of anion, a faa also supported by the presence of ['H](+)SKF- 10,047 binding sites in the myenteric plexus and a high density of a binding sites in the colonic mucosa and smooth muscle 316. CONCLUSION

Studies have suggested that o receptors may represent the site of action for a number of important drugs. The research on the o receptors has opened up new areas of receptor investigation and also new strategies for developing antipsychotic drugs. In addition, o receptors have been noted in guinea pig splenoqes which are sensitive to haloperidol and progesterone, suggesting a link between endocrine, nervous and immune systems. Additional therapeutic targets for o ligands include epilepsy and brain ischernia. Although the p harmacologicai roles of o receptors are not totally understood at present, evidences indicate that o receptors may have several biologicd functions. REFERENCES

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128 Gray, N. M., Contreras, P. C., Allen, S. E. and Taylor, D. P., Hl antihistamines interact with central sigma receptors, L$ Sci, 47 (1990) 175-180. 129 Graybiel, A. M., Besson, M. J. and Weber, E., Neuroleptic-sensitive binding sites in the nigrostriatal system. Evidence for differential distribution of sigma sites in the subtantia nigra, pars compaaa of the car, /.Neurosci., 9 (1989) 324-338.

130 Greiffenstein, F. E., DeVault, M., Yoshitake, J. and Gajewski, J. E., A study of 1-arylcyclohexylamine for anesthesia, Anesth.Anai., 37 (1958) 283-294.

131 Gronier, B. and Debonnel, G., Electrophysioiogical evidence for the implication of cholecystokinin in the modulation of rhe N-methyl D-aspartate response by sigma ligands in the rat CA, dond hippocampus, Na~np-SchmiedebwgsArch. of Phammoi., 353 (1996) 382-390.

132 Gudelsky, G. A., Nash, J. F., Berry, S. A. and Meltzer, H. Y., Basic biology of clozapine: Electrophysiological and neuroendocriniological studies, Psycbopharmatofogy, 99 (1989) 5 13-517.

133 Gue, M., Gleizes-Escala, C., Del Rio-Lacheze, C., Junien, J. -L. and Bueno, L., Revend of CRF- and dopamine-induced stimulation of colonic motility by CCK and igmesine (JO 1784) in the nt, Br.J Phannacoi., 111 (1994) 930-934.

134 Gue, M., Junien, J. L., Del Rio, C. and Bueno, L., Neuropeptide Y and sigma ligand 00 1784) suppress stress-induced colonic motor disturbances in rats through sigma and cholecystokinin receptors, j. Phannacoi. Exp. Ther., 26 1 (1992) 850-855.

135 Gue, M., Yoneda, M., Monnikes, H., Junien, J. L. and Taché, Y., Central neuropeptide Y and u ligand 00 1784) reverse conicotropin-releasing factor-induced inhibition of gastric acid secretion in rats, Br.JPhamacoi., 107 (1992) 642.

136 Gund, T. M., Shukla, K. and Su, T. -P., Molecular modeling of sigma receptor ligands: a mode1 of binding based on conformational and eIectrostaric considerations, J.Marh.Chem., 8 (1991) 309.

137 Gundlach, A. L., Largent, B. L. and Snyder, S. H., Phencyclidine and o opiate receptors in brain: biochemical and autoradiographic differentiation, Eur./.Phamacol., 113 (1985) 465-466.

138 Gundlach, A. L., Largenr, B. L. and Snyder, S. H., Autondiogaphic localization of sigma receptor binding sites in guinea pig and rat central nervous system with (+)['w3-(3-Hydroxyphenyl)-N-(1-propyl)piperi&ne, /.Newosci, 6 (1986) 1757-1770.

139 Gundlach, A. L., Largent, B. L. and Snyder, S. H., Phen~~clidine(PCP) recepton: a~toradio~raphic Iocdization in bnin with the selective ligand, [3H-JKP, Brain Res., 386 (1986) 266-279. 140 Hamon, B. and Heinemann, U., Developmentd changes in neuronal sensitivity to excitatory amino acids in area CA1 of the rat hippocarnpus, Dw.Brain Re-., 38 (1988) 286-290.

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142 Hara, H., Tanaka, K., Handa, Y-and Sukamoto, T., Sigma receptor-mediated effects of a new antiulcer agent, KB-5492, on experimentaI gasrric mucosal lesions and gastric alkaline secretion in rats, /. Pharmacol. Lcp. nm., 269 (1994) 799-805.

143 He, X. S., Bowen, W. D., Lee, K. S., Williams, W., Weinberger, D. R. and de Costa, B. R., Synthesis and binding characteristics of ~otentialSPECT imaging agents for sigm2.-1 and sigma-2 binding sites, /.Med.Chem., 36 (1993) 566-571.

144 He, X. S., Raymon, L. P., Mattson, M. V., Eldefnwi, M. E. and de Costa, B. R., Synthesis and bioiogical evaluation of 1-[I-(2-benzo[b]thienyl)cyclohexyl]pip~dinhomologues at dopamine-uptake and phencyclidine- and sigma-binding sites, /.Med.Chm., 36 (1993) 1188-1193.

145 Hellewell, S. B. and Bowen, W. D., A sigma-like binding site in rat pheochromocytoma (PCl2) cells: decreased affinity for (+)benzomorphans and lower molecular weight suggest a different sigma receptor form from that of guinea pig brain, Brain Res., 527 (1990) 244-253.

146 Higashi, H.,Inanaga, K., Nishi, S. and Uchimua, N., Enhancement of dopamine actions on rat nucleus accum bens neurons in vitro after methamphetamine pre-treatment, j. Pbysioi., 408 (1989) 587-603.

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151 Hollmann, M. and Heinemann, S., Cloned glutamate receptors, Ann.Rev.Neurosci, 17 (1994) 31-108.

152 Holtzman, S. G., Phencyclidine-Iike discriminative effects of opioids in the rat, J.Phannacoi.E~.7%e~., 214 (1980) 614-619.

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154 Holtzman, S. G., Opioid- and phencyclidine-like discriminarive effects of ditolylguanidine, a selective sigma ligand, J Phamacoi.Exp, Ther., 248 (1989) 1054-1062.

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156 Itzhak, Y., Pharmacological specificity of some psychotornimetic and antipsychotic agents for the sigma and PCP binding sites, Life Sci, 42 (1988) 745-752.

157 Itzhak, Y., Multiple affinity binding States of the sigma receptor: effect of GTP-binding protein-modifying agents, Mol.Phammcol., 36 (1989) 5 12-5 17.

158 Itzhak, Y. and Alerhand, S., Differential regdation of sigma and PCP receptors after chronic administration of haioperidol and phencyclidine in rnice, FASEBJ., 3 (1989) 1868-1872.

159 Itzhak, Y., Hiller, J. M. and Simon, E. J., Characterization of specific binding sites for (+)[3HJN-aIlylnormetazocine in rat brain membranes, Mol.Pbannacol., 27 (1985) 46-52.

160 Itzhak, Y. and Kassim, C. O., Clorgyline displays high affinit~for sigma binding sites in C57BL/6 mouse brain, Ecrr.jPhamzacol., 176 (1990) 107-108.

161 Itzhak, Y. and Khouri, M., Regdation of the binding of sigma and phencyclidine (KP) receptor ligands in rat brain membranes by guanine nucleotides and ions, NeurosciLert, 85 (1988) 147-152. 162 Itzhak, Y., Mash, D., Zhang, S. H. and Stein, I., Characterization of N-rnethyI4phenyl-1,2,3,&tetrahy&opyridine (MPTP) binding sites in C57BL/6 mouse brain: mutual effects of monoamine oxidase inhibitors and sigma ligands on MPTP and sigma binding sites, Mol.Pbamcol., 39 (1991) 385-393.

163 Itzhak, Y. and Stein, E., Regdation of sigma receptors and responsiveness CO guanine nucleotides following repeated exposure of rats to haloperidol: further evidence for multiple sigma binding sites, Brain Res., 566 (1991) 166-172.

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420 Zukin, S. R. and Zukin, R. S., Specific UHIphencyclidine binding in rat central nervous system, Proc.Nad.Acad.ScUSA, 76 (1979) 5372-5376. 82 CHAPTER II Modification of the N-met h y 1-D-as parcare respo nse by antidepressant sigma recepto r ligands

It had been shown previously in Our laboratory that low doses of certain selective o ligands, such as DTG, potentiate the neuronal response to NMDA in the CA, region of the rat dorsal hippocampus and that this potentiation is suppressed by other u ligands such as haloperidol. Trollus and Skolnick reported in 1990 that cornpetitive and non-competitive NMDA antagonists were presenting antidepressant properties in a behavioral paradigm suggesting that the glutamatergic system might be involved in the neurobiology of depression. It has also been reported chat the selective serotonin reuptake inhibitor (SSRI) sertraline and the monoamine oxidase inhibitor (MAOI) clorgyline have high affinity at the cr receptors. Surprisingly, other antidepressant drugs belonging to the same pharmacological family, such as paroxetine (an SSRI) and tranlycypromine (an MAOI), have no affinity at the u receptors.

The purpose of my first study was to assess the effects of antidepressant o ligands on the NMDA response. In the present micle 1 compared the effects of the two groups of antidepressant dmgs on the NMDA response. The first group has high affinity at the o receptors and included the SSRI sertraline and the MAOI clorgyline. The second group of antidepressants, the SSRI paroxetine and the MAO1 tranlycypromine, has no affinity at the a receptors. MODIFICATION OF THE N-METHTL-D-ASPARTATERESPONSE BY ANTIDEPRESSANT SIGMA LIGANDS Richard Bergeron, Guy Debonnel and Claude de Montigny

European Journal of Pharmacology, 240: 319-323, 1993.

SUMMARY Sertraline, a selective serotonin rcuptake inhibitor, and clorgyline, a monoamine oxidase

inhibitor, both having high affinity for sigma (O) receptors, were assessed in an elearophysiological model. In keeping with previous data obtained with other o ligands, low doses of sertraline and of clorgyline ~otentiatedselectively with a bell-shaped dose-response curve the effect of N-rnethyl-D-aspartate (NMDA) on pyramidal neurons in the CA, region of the rat dorsal hippocampus. This potentiation was reversed by the o ligands haloperidol and BMY-14802.The selenive serotonin reuptake inhibiror paroxetine and the monoamine oxidase inhibitor tranylcypromine, borh devoid of affinity for a receptors, had no effects on the NMDA response. These data suggest that the effects of sertraline and clorgyline on the NMDA response are due to their affinity for the o receptors.

Key words: Sigma, NMDA, Antidepressant, Selective serotonin reuptake inhibitor (SSRI) Monoarnine oxidase inhibitor wOI)

INTRODUCTION The hypothesis that serotonin plays a role in mediating the therapeutic effect of antidepressant treatments has been well documented in the last two decades (for review see Blier, De Montigny and Chaput, 1990). However, there is evidence that other neuronal systems, such as the noradrenergic, the dopaminergic and the glutamatergic systems, might also be involved. The glutarnatergic excitatory neurotransmission is mediated via the activation of three main receptor subtypes, labelled according to their most effective agonist: quisqualate, kainate and N-met hyl-D-asparrate (NMD A) (Foster and Fagg, 1984). Recently, it has been shown that a cornpetitive and a noncornpetitive NMDA antagonist mimicked the effects of clinicdly effective antidepressant dmgs (Trullas and Skolnick, 1990). These findings raised the possibility that the glutamatergic system might be involved in the pathophysiology of affective disorder, or might be a potential target for antidepressant treatments. In Our laboratory, it has been shown, using an electrophysiological paradigrn of extracellular unitary recording of dorsal hippocarnpd pyramidal neurons of the CA, region, that o ligands modulate selectively the neuronal response to NMDA (Monnet et ai. 1990; Monnet, Debonnel and De Montigny, 1992). Some anridepressant dmgs such as serrraline, a select ive serotonin reuptake inhibit or, and clorgyline, a monoamine oxidase inhibit or, have a high affinity for o receptors (Schmidt et al. 1989; Itzhak and Kassim, 1990), whereas the selective serotonin reuptake inhibitor paroxetine and the monoamine oxidase inhibitor tranylcypromine have a very low affinity for these receptors (Schmidt et al. 1989; Itzhak and Kassim, 1990). The present studies were undertaken to investigate the putative modulation of NMDA- induced activation of hippocampal pyramidal neurons by searaline and clorgyline. These experiments were carried out in the CA, region of the dorsal hippocampus, a region with high densities of o and NMDA receptors, using an in vivo electrophysiological paradigm whereby the neuronal responsiveness to microiontophoretic applications of NMD A, quisqualate and acetylcholine can be quantified by extracellular unitary recordings.

MATERIALS AND METHODS Adult male Sprague-Dawley rats weighing 200 to 250 g were kept on a 12:12 h lighddark cycle. Animas were anesthetized with urethane (1.25 g/kg, i.p.), mounted in a stereotaxic apparatus and body temperature was maintained at 37OC throughout the experiment. Microiontophoretic applications and extracellular unitary recordings were performed with five-barrelled glas micropipettes. For rnicroi~nto~horesis,the side barrels were filled with NMDA (10 mM in 200 mM NaCl, pH: 8), quisqualate (1.5 rnM in 400 rnM NaCl, pH: 8) and acetylcholine (20 rnM in 200 mM NaCl, pH: 5). Extracellular recordings were obtained at a depth of 3.5 to 4.5 mm from the cortical surface in an area defined stereotaxically as 4.2 0.2 mm anterior to lambda and 4.2 I 0.2 mm lateral to midline (Paxinos and Watson, 1986). Pyramidal neurons were identified by their long duration (0.8 - 1.5 ms) and large amplitude (0.1 - 0.5 mV) action potentials, and by the presence of "cornplex spike" discharges. Alternate 50 s microiontophoretic applications of NMDA, quisqualate, and acetylcholine were carried out with ejecting currents adjusted to obtain a physiological firing activity. These currents were maintained constant for the reminder of the experiment. The effects of the microiontophoretic applications of NMDA, quisqudate and acetylcholine, expressed as the nurnber of spikes generated/nanoCoulomb (nC; 1 nC being the charge generated by 1 nA applied for 1 s), were calculated by an one-line cornputer averaging the effects of three successive applications of each of the excitatory substances. Only one dose of one drug was tested in one rat. In a first series of experiments, the seleaive serotonin reuptake inhibitors sertraline and paroxetine, and the monoamine oxidase inhibi~orsclorgyline and tranylcypromine were injected via a catheter installed in a lateral tail vein, at doses ranging from 1 pg to 1 mg/kg. The effect of the drug studied was assessed by determining the ratio (NJNJ of the number of spikes generatedhc by NMDA, quisqualate and acetylcholine before (NJ and afrer (NJ its intravenous injection. In a second series of experiments, sertraline (10 mM in 200 mM NaCI, pH: 6) and clorgyline (10 mM in 200 rnM NaCI, pH: 5) were applied by rnicroiontophoresis. The currents used were between + 10 to + 12 nA. The effeas of sertraline and clorgyline were measured by calculating the number of spikes generatedhl of NMDA and quisqualate before, during, and after their microiontophoretic application. The degree of statisticd significance was calculated using the paired Student's t-test.

RESULTS At al1 doses tested, neither sertraline nor clorgyline had any effea on the spontaneous firing aaivity of CA, dorsal hippocampus pyramidal neurons nor on the activations of CA, pyramidal neurons induced by acetylcholine or quisqualate. As illustrated in figure 1, both sertrdine and clorgyline potentiated selectively the excitatory effect of NMDA, NOpg/kg i.v. of searaline or clorgyline inducing a four-fold increase of the response to NMDA. With both drugs, this effea was dosedependent, the maximal potentiation being obtained with 200- 250 pg/kg, i.v. (figure 2). At doses higher than 500 pg/kg i.v., sertraline and clorgyline had no potentiating effect on the NMDA response. Thus, in between 1 pg to 1 mg/kg i.v., sertraline and clorgyline potentiated with a bell-shaped curve the NMDA response (figure 2). The dose- response cunres were obtained with the software Tablecurve (version 3 .O, Jandel Scientific, 1991), the best fit equation for sertraiine being: Y = (a + cx + ex3 / (1 + bx + dd (r = 0.93); and that for clorgyline: Y = a + exp2 (-0.5 (1 n (x/c)/d) (r = 0.89). The potentiating effect of low doses of serrraline and clorgyline were reversed by hdopendol (IO pg/kg, i.v; n = 5), a high affinit~u ligand (Zukin and Zukin, 1981) (figure 1). The effects of low doses of sertraline and clorgyline were also suppressed by the cr ligand BMY- 14802 [a-(4-8uorophenyl)~(~-horo-2-pynmidinyl)--piperinebutanol] (100 ,ug/kg i.v.; n = 5) (Taylor and Dekleva, 1988). The pnor administration of haloperidol (10 kg, i.v.; n = 6) and BMY-14802 (100 pg/kg, i.v.; n = 5) totally prevented the effects of the low doses of semaine and clorgyline. As a further verification chat the potentiating effects of low doses of sefiridine and cforgyline were mediated via a receptors, we derermined the effect of spiperone, another butyrophenone, which has the same radioligand binding profile as haloperidol, except for its low affinity for o receptors (Taylor and Dekleva, 1988). Spiperone (10 &kg, i.v.) failed to reverse the potentiating effects of senraline (100 pg/kg, i.v.; n = 5) and of clorgyline

(100 pg/kg, i.v.; n = 5). The same dose of spiperone also failed to prevent the potentiating effect of both drugs (n = 6 for each dnig). Paroxetine and tranylcypromine, bot h having very low affinity for o receptors, had no effect on NMDA-induced activation at doses ranging from 1 pg to 1 mg/kg, i.v. (figure 2). In a second series of experiments, to determine if the effects of sertraline and clorgyline were exened locally, these two anridepressant dmgs were applied microiontophoretically. Small currenrs of 10-12 nA did not modify the spontaneous firing activity of CA, pyramidal neurons, but increased the NMDA-induced activation by two-fold (spikes generatedhl of NMDA pnor to sertraline: 1.86 t 0.18; during sertraline: 3.98 * 0.32, n = 10, P < 0.001 - prior to clorgyline: 1.56 0.09; during clorg~line3.21 0.13, n= 8, P < 0.001). Neither drug modified the neuronal response to quisqualate. The NMDA potentiating effects of sertraline and clorgyline applied by micr~ionto~horesiswere prevented by haloperidol (10 &kg, i.v.) administered prior to the rnicroiontophoreric applications of serrraline (spikes generated /nC of NMDA prior sertraline: 1.09 O. 11; during sertraline: 1.14 * 0.08, n= 8, P > 0.9) and clorgyline (spikes generated /nC of NMDA prior to clorgyline: 1.72 0.16; during clorgyline: 1.66 0.14, n=6, P > 0.9).

DISCUSSION The present results show that, at doses in between 1-500 ~g/kgix, sertraline and clorgyline rnodified neither the spontaneous firing rate of CA, pyramidal neurons nor their responses to microiontophorecic applications quisqualate or acetylcholine, but increased their response to NMDA (figures 1, 2). In contrast, at doses in between 0.5-1 mg/kg i.v., sertraline and clorgyline did not potentiate effect of NMDA (figure 2). Since these dmgs also exert monoaminergic effects, ir was deemed essential to verify wherher this potentiation of NMDA was attributable to their affinity for o recepton. The fact that paroxetine and tranylcypromine which have monoaminergic profiles sirnilar to those of sertraline and clorgyline respectively, except that they are devoid of affinity for o receptors, did not affect the NMDA-induced activation (figure î), clearly shows that the potentiation of the NMDA response by clorgyline and sertraline is not related to their effects on monoaminergic systems. Two hirther arguments strongly suggest that the potentiating effects obtained with these antidepressant drugs are mediated via o receptors: 1) the potentiation of the NMDA response by low doses of sertraline and clorgyline (figure 2) is similar to chat observed previously with low doses of several o ligands, such as DTG [1,3di-O-tolylguanidine],50-1784 [(+)N- cyclopropylmet hyl-N-methyl-i ,4-dip henyl-1st hyl-b~t-3-en-l-~larninehydrochloride], and (+)pentazocine (Monnet, Debonnel and De Montigny, 1992); 2) low doses of the two high- affinity a ligands haloperidol and BMY-14802 prevented and reversed the potentiating effects of searaline and clorgyline on the NMDA response, as they do for other a ligands (Monnet, Debonnel and De Montigny, 1992). Moreover, spiperone, a butyrophenone having a binding profile similar to that of halopend01 except for its low affinity for o sites, did not reverse the effects of sercraline or clorgyline. The bell-shaped curves obtained with sertraline and clorgyline (figure 2) cannor be explained with any degree of certainty at present. The lack of potentiating effects of high doses of sertraline and clorgyline on the NMDA response rnight suggest that the low and high doses of these drugs exert their effects via distinct receptors. It is now accepted that at least two subtypes of o receptors exisr in the mammdian central nervous system (Quirion et ai. 1992). In keeping with this notion, previous data obtained in Our laboratory have shown that, in the CA3 region of the donal hippocampus, low doses of the o ligand DTG potentiate the NMDA response in intact rats; however, after the destruction of the mossy fibre, DTG reduces the NMDA response, indicating that DTG acts on, at least, two subtypes of receptors: a presynaptic one potentiating, and a postsynaptic one reducing, the NMDA response (Debonnel, Monnet and De Montigny, 1992). It may be hypothesized that a similar phenomenon could account for the present data. At low doses, clorgyline and serrraline might activate a first subtype of a recepton for which they would have high affinity, and which potentiate the effect of NMDA; at higher doses, these drugs rnight activate another subtype of a receptors for which they would have a lower affinity, and which would counteract their potentiating effects. An alternative explanation might be a rapid desensitization of the a receptor involved in the potentiation of the NMDA response. Even though this possibility cannot be mled out, it does not appear ro be likely since the potentiating effects obtained with the intravenous administration of CT ligands have been found to last for as long as 30 min (Monnet, Debonnel and De Montigny, 1992). Thus, the precise mechanism whereby clorgyline and sertraline produce these bell-shaped dose-response curves remains to be elucidated.

REFERENCES

Blier, P., C. de Montigny and Y. Chaput, 1990, A roie for the serotonin system in the mechanism of action of antidepressant treatmenrs: Preclinical evidence, J. Clin. Psychiarry 51 (Suppl.4), 14.

Debonnel, G., F.P. Monnet and C. de Montigny, 1992, Differentid effects of high affinity sigma ligands and of neuropeptide Y on the NMDA response within rat hippocampus subfields, Clin. Neuropharmacol. 15 (Suppl.), 11B.

Foster, A.C. and Fagg, G.E., 1984, Acidic amino acid binding sites in mammalian neuronal: Their characteristics and relationship to synaptic receptors, Brain Res. Rev. 7: 103.

Itzhak, Y. and C.O. Kassim, 1990, Clorgyline displays high affinity for sigma-binding sites in C57BY6 mouse brain, Eur. J. Pharmacol. 176, 107.

Monnet, F.P., G. Debonnel, J.L. Junien and C. de Montigny, 1990, N-Methyl- D-aspartate-induced neuronal activation is selectively modulated by sigma receptors, Eur. J. Pharmacol. 179, 441.

Monnet, F.P., G. Debonnel and C. de Montigny, 1992, In vivo eIectrophysiologicai evidence for a selective moduIation of N-mechyl-D-aspartate-inducedneuronal activation in rat CA, donal hippocampus by sigma ligands, J. Pharmacol. Exp. Ther. 261,123.

Paxinos, G. and C. Watson, 1986, The rat brrin in stereota.xic coordinates (Academic Press, San Diego, CA).

Quirion, R., W.D. Bowen, Y. Itzhak, J.L. Junien, J.M. Musacchio, R.B. Rothman, T.P. Su, W. Tam and D.P. Taylor, 1992, A proposai for the classification of sigma binding sites, Trends Pharmacol. Sci. 13, 85.

Schmidt, A., L. Lebel, B.K. Koe, T. Seeger and J. Heym, 1989, Sertraline potently displaces I3H](+)3-PPPbinding to sigma-sites in rat brain, Eur. J. Pharmacol. 165, 335.

Taylor, D.P. and J. Dekleva, 1988, BMY 14802: A potential antipsychotic agent that selectively binds to sigma receptors, in: Sigma and Phencyclidine like Compounds as Molecdar Probes in Biology, eds. E.F. Domino and J.M. Kamenka (NPP Books, Ann Arbor) p. 345.

Trullas, R. and P. Skolnick, 1990, Functional antagonists at the NMDA receptor complex exhibit antidepressant actions, Eur. J. Pharmacol. 185, 1.

Zukin, R.S. and S.R. Zukin, 1981, Demonstration of ['H]cyclazocine binding to multiple opiate receptor site, Mol. Phaacol. 20, 246. ACh NMDA QUIS 12 -1 1 -6 mu -0

CLORGYLINE HALOPERIDOL 100 pglkg, i.v. 10 pglkg, Î.v.

ACh NMDA QUlS 8 -1 0 - 3 rn 0 = m 0 =

2 min ' t t SERTRALINE HALOPERIDOL 100 pglkg, i-v. 10 pg/kg. i.v.

Figure 1 Integrated firing rate histograms of CA, dorsal hippocampus pyramidal neurons showing the effects of microiontophoretic applications of acetylcholine, quisqualate and NMDA, before and after the intravenous adminisrration of a low dose of clorgyline (A) and of sertraline (B) and the suppression of clorgyline- and sertraline-induced potentiations of the NMDA response by a low dose of haloperidol. Each neuronal response to each excitatory agent represents the cornputer-generated mean of the effects of three successive applications. The two circIes represent an interval of two to five minutes. Time base applies to both traces. O Clorgyline Tranylcypromine

Dose (pglkg, Lv.)

Sertraline A Paroxetine

1 10 1 00 1O00

Dose (pglkg, i.v.)

Figure 2 Dose-response curves of the effects of the intravenous administration of clorgyline and tranykypromine (A), and of sertdine and paroxetine (B) on the neuronal activation of CA3dorsal hippocampus pyramidal neurons induced by microiontophoretic applications of NMDA. The effects of these drugs were assessed by determining the ratio (N2/NI)of the number of spikes generatedlnc of NMDA before (NI) and after (Nd the injection of the hg.One dot represents the effect of one dose of the dnig administered to one nt while recording from one neuron. 91 CHAPTER III Biphasic effect of sigma ligands on the neuronal response to N-methyl-D- aspart ate

The previous study had demonstrated that two o ligands sertraline and clorgyline presented bell-shaped dose-response curves of their potentiation of the NMDA response. These results obtained with sertraline and clorgyline bnng new information about the potentiation of the NMDA response induced by o ligands. Indeed, serrraline and clorgyline porentiate selecrively and markedly the NMDA response. However, the potentiation of the NMDA response reaches a plateau around 200 pg/kg i.v. At higher doses the potentiation progressively decreases and disappears at doses higher the 500 pg/kg i.v. These results raise the important question of whether selective a ligands also presenr this type of biphasic effect. Review of the lirerature suggests that in other paradigms selective cr ligands may also produce this biphasic effect. For exarnple, it has been reported in a behavioral paradigm that BD-737 induced a biphasic effect on punished and unpunished responses in rats.

The goal of my second study was to determine whether higher doses of selective o ligands, aiready tested in our laboratory, may also present the biphasic effect on the NMDA response obtained with the antidepressant cr ligands sertraline and clorgyline. BIPHASIC EFFECTS OF SIGMA LIGANDS ON THE NEURONAL RESPONSE TO MMETHYL-D-ASPARTATE Richard Bergeron, Claude de Montigny and Guy Debonnel

Naunyn-Schmiedeberg's Archives of P harmaco! ogy, 3 5 1: 252-260, 1995.

Previous studies from our laboratory have demonstrated rhat low doses of selective sigma (O) ligands potentiate the neuronal response to N-rnethyl-D-aspartate (NMDA) in the CA, region of the rat dorsal hippocarnpus. Serrraiine and clorgyline, two antidepressant dmgs with a high affinity for a receptors, also potentiate, at low doses, the NMDA response; however, when adrninistered at higher doses, the degree of potentiation induced by these two o ligands progressively decreases (Bergeron et al., 1993). In the present experiments, the selenive o ligands DTG, (+)pentazocine, BD-737, JO-1784 and L-687,384 were studied to determine if they would also generate bell-shaped dose-response curves. These cr ligands were administered intravenously at doses ranging from 1 pg/kg to 1 mg/kg or applied by microiontophoresis. They potentiated selectively, with bell-shaped dose-response curves, the NMDA-induced activation of pyramidal neurons in the CA, region of the rat dorsal hippocampus. The potentiation of the NMDA response following the intravenous administration of a low dose of a o ligand persisted for at lest 45 min, at which point in tirne a second injection of the same dose induced the sarne degree of potentiation. Moreover, a sustained potentiation was obtained during prolonged microiontophoretic applications of a o ligand. These two latter series of observations suggest that the lack of effect of the high doses of o ligands is not related ro a rapid desensirisation of a receptors. This biphasic effect of o ligands rnight be due to the concomitant action of these ligands on distinct subtypes of a

recept O rs.

Key words: Sigma receptors, NMDA receptors, electrophysiology, hippocampus, DTG, (+)pentazocine, BD-737, JO-1784, L-687,384, halopend01

INTRODUCTION The functional role of sigma (0) receptors has been the focus of considerable attention in recent years (for review see: Walker et al. 1990; Su 1993; Debonnel 1993). The existence of cr recepton was postulated in 1976 to account for the actions of SKF-10,047 and related benzomorphans (Martin et al. 1976). It has been suggested that o receptors might be involved in the pathophysiology of psychosis (Fudenberg et al. 1984; Snyder and Largent 1989) since some potent neuroleptic agents, such as halopend01 (Su 1982; Tarn and Cook 1984) as well as atypical anripsychotic agents, such as remoxipride (Magnusson and Fowler 1989) and rimcazole (Ferris et al. 1986), exhibit high affinity for these receptors (Snyder and Largent 1989). At low doses, several selective high affinity o ligands such as 1,3-di(2-toly1)guanidine (DTG) (Weber et al. l986), (+)N-cyclopropylmet hyl-N-met hyl- 1+dip henyl- 1-ethy l-but-3sn-l- ylamine hydrochloride 00-1784) (Roman et al. 1990), and (+)pentazocine (Steinfels et al. 1988), selectively and dosedependently potentiate the neuronal response produced by the rnicroiontophoretic application of NMDA in the CA3 region of the rat dorsal hippocarnpus (Monnet et al. 1990b; Monnet et al. 1992b). This effect is suppressed by other o ligands such as haloperidol, a-(+fluorophenyl)+(5-fluoro-2-pyrimidinyl)-I- butanol @MY-14802) (Taylor and Dekleva 1987) and 3[3-hydroxyp henyll-N-(1-p ropyl) piperidine [(+)3-PPP] (Largent et al. 1984). Previous data obtained in Our laboratory have also shown that the selective serotonin reuptake inhibitor (SSRI) sertraline and the monoamine oxidase inhibitor (MAOI) clorgyline, two antidepressant drugs with high affinity for o receptors (Schmidt et al. 1989; Itzhak and Kassim IWO), also potentiate selectively, at low doses, the NMDA response (Bergeron et al. 1993). Paroxetine (another SSRI) and tranylcypromine (another MAOI) have a sirnilar binding profile to sertraline and clorgyline respectively, except that they are devoid of aflinity for o receptors (Schmidt et al. 1989; Itzhak and Kassim 1990). Paroxetine and tranylcyprornine do not affect the NMDA-induced activation when adrninistered in the same range of doses as sertraline and clorgyline, suggesring that the potentiating effects of semaline and clorgyline on the NMDA response are mediated by a receptors (Bergeron et al. 1993). When used at higher doses, the magnitude of the potentiation induced by these two o antidepressants, after reaching a maximai effect at doses around 200 &kg i.v., progressively decreases to findly disappear at doses higher than 500 pg/kg i.v. (Bergeron et al. 1993). The present experiments were undertaken to determine if other o ligands such as DTG, (+)pentazocine, [(+)-cis-N-methyl-N-[2-(3,Cdichlorophenyl) ethyll-2-(1-pyrrolidinyl cydohexylarnine] (BD-737) (Contreras et 4. 199I), 50-1784 and 1-benzylspiro[l,2,3,4- tetrahydronaphthalene- I,+piperidine] (L-687,3 84) (Middlerniss et al. 199 1; Barnes et al. Wî), already tested at low doses in Our model, could dso present the same type of biphasic effects on the NMDA response when administered at higher doses. These experiments were carried out in vivo in the CA, region of the rat dorsal hippocarnpus, where the responsiveness of pyramidal neurons to rnicroiontophoretic applications of NMDA and QUIS were quantified by extracellular unitary recording.

MATERIALS AND METHODS Preparation of animals Male Sprague-Dawley rats weighing 200-250 g were housed three to four per cage, two or three days before the experiments, with free access to food and water. They were rnaintained at constant temperature (2S°C) under a 12h-Eh lightdark cycle. For electrophysiological expenments, the rats were anesthetized with urerhane (1.25 g/kg, i.p.) and mounted in a stereotaxic apparatus. Body temperature was maintained at 37OC throughout the experiment .

Preparation of dmgs The following substances were used. 50-1784, a generous gift from J.L. Junien (Institut de Recherche Jouveinal, Fresnes, France); BD-737, a generous gift from W.D. Bowen (Laboratory of Medicinal Chernistry, NIDDK, MH, USA); L-687,384, a generous gifi from L.L. Iversen (Merck-Sharp and Dome, Tyler Park, UK); (+)pentazocine ('BI) Natick, MA, USA); N-Methyl-D-Aspartate (NMDA) (Sigma, St-Louis, MO, USA); quisqualate (QUIS) (Tocris Neuramin, Buckburst Hill, Essex, UK); Acetylcholine (ACh) (Sigma, St-Louis, MO, USA); DTG (Aldrich Chemicals, Milwaukee, WI, USA); haloperidol (McNeil Laboratories, Sroufiille, Ontario, Canada).

Prcparation of m icropipettes Microiontophoretic applications and extracellular unitary recording were performed with five-barrelled glass rnicropipettes prepared in a conventional manner (Haigler and Aghajanian, 1974). Two of side barrels used for microiontophoresis, were filled with NMDA (10 mM in 200 rnM NaCl, pH: 8), (1.5 mM in 400 mM NaCl, pH: 8) for QUIS. The concentration for QUIS was lower than that of NMDA since pyramidal neurons are much more sensitive to QUIS. A third side barrel was filled with either ACh (20 mM in 200 mM NaCl), pH: 5) or with the a ligands DTG (1 mM in 200 mM NaCl, pH: 8) or JO-1784 (1 mM in 200 mM NaCl, pH: 8). The remaining side barrel, filled with 2 mM NaCl, was used for automatic curent bdancing. The central barrel, used for extracellular unitary recording of the firing activity of CA3 dorsal hippocampus pyramidal neurons, was filled with a 2 mM NaCl solution.

Recordings from dorsal hippocampus pyramidal newons The rat hippocampus, which contains high densities of o and NMDA receptors (Cotman and Monaghan 1988), is a region of choice for studying the interaction between these receptors. Extracellular recordings were obtained in the CA3 region at a depth of 3.5 to 4.5 mm below the cortical surface in an area defined stereotaxically as 4.2 * 0.2 mm anterior to lambda and 4.2 0.2 mm lateral to midline (Paxinos and Watson, 1986). The action potential wave form was monitored on an oscilloscope. For a given neuron, alternate rnicroiontophoretic applications of the excitatory substances NMDA, QUIS were carried out, adjusting the ejecting currents to obtain physiologicd firing frequency between 7 and 15 Hz (Ranck, 1975). These currents were thereafter maintained constant for the remainder of the recording. The currents used for ejecting NMDA ranged from -8 to -15 nA, from -2 to -6 nA for QUIS and from 7 to 12 nA for Ach. The duration of the rnicroiontophoretic ejections of NMDA, QUIS and ACh was kept constant at 50 sec. The intensity of the ejecting current and the duration of the microiontophoretic applications as well as the number of action potentials generated were stored on an on-line computer.

Expm-mental series In the first series of experiments, al1 drugs were prepared in physiological saline and administered via a lateral rail vein. Only one dose of each drug was administered to one rat while recording from one neuron. Severai doses of each drug were tested to generate dose- response curves. In these series, DTG, (+)pentazocine, BD-737,JO-1784 and L-687,384 were tested at doses ranging from 1 pg to 1 mg/kg. In the second senes of experiments, DTG (1 mM in 200 rnM NaCl, pH: 8) and JO-1784 (1 mM in 200 mM NaCi, pH: 8) were applied by microiontophoresis with different ejecting currents (between 5 nA and 100 nA). These cr ligands were chosen because of their high affinity for the o receptors and because they have no or a negligible affinity for dopaminergic, serotoninergic or PCP receptors (Weber et al. 1986; Steineis et al. 1988; Romarn et al., 1990; Contreras et al. 1991; Middlemiss et al., 1991). Caladations The computer calculated the effecx of each 50 sec rnicroiontophoretic application of an excitatory substance, as the total number of spikes generatedhc (1 nC being the charge generated by 1 nA applied for 1 sec.). Each value was calculated by the compurer as the mean of the effect of three consecutive applications. The effects of the intravenous administration of o ligands were assessed by determining the ratio (Nz/NJ of the number of spikes generatedhl of each the three excitatory substances ACh, QUIS and NMDA before (NI)and after (Nd the injection of the o ligand. The dose-response curves of the effeas of intravenous administration of o ligands were obtained by fitting experimentd data to general logistic equations obtained with the software Tablecurve (version 3.0, Jandel Scientific, 1991). The effects of the rnicroiontophoretic applications of DTG and JO-1784 were also assessed by calculating the ratio (N2/NI),NZ being the number of spikes generated per nC of QUIS and NMDA du&g the rnicroiontophoretic applications of DTG or JO-1784.

RESULTS Intravmous series The effect of the intravenous administration of (+)pentazocine was assessed in a first series of experiments. At low doses, (+)pentazocine potentiated selectively and dose- dependently the NMDA response. The maximal potentiation was obtained at the dose of 50 pg/kg i.v. (Fig. IA). At higher doses, the degree of potentiation progressively decreased and, at the dose of 500 ~rg/kg i.v., no potentiation could be observed (Fig. IB). In between 1- 1000 &kg i.v., a bell-shaped dose-response curve of the potentiation of the NMDA response was obtained (Fig. 2A). A similar curve was obtained with 10-1784 (Fig. 2B), with BD-737 (Fig. 2C) and L- 687,384 (Fig. 2D) ar. doses ranging between 1 pg and 1 mg/kg. With 50-1784, the maximal potentiation was obtained at the dose of 4 pg/kg i.v. and disappeared from the dose of 500 &kg i.v. (Fig. 2B). Following the intravenous administration of BD-737 (Fig. 2C), the maximal degree of the potentiation has been obtained at the dose of 100 pg/kg, and, at the dose of 1 mg/kg i.v., the potentiation disappeared. L-687,384 behaved in Our mode1 as the most potent agent tested thus far since a potentiation of the NMDA response could be observed with a dose as low as 0.1 ~rg/kgi.v. (Fig. 2D). Moreover, the maximal degree of potentiation was obtained at the dose of 1 pg/kg i.v. At that dose, the ratio N2/Ni was above 3 (Fig. 2D). At doses higher than 1 &kg, the potentiation of the NMDA response progressively decreased and disappeared at doses higher than 50 pg/kg (Fig. 2D). Findly, as previously observed (Monnet et al. 1990b), at the dose of 1 pg/kg i-v., DTG induced a +fold potentiation of the NMDA response, which was reversed by 10 pg/kg i.v. of haloperidol (Fig. 3A). At doses higher than 3 pg/kg i.v., the effect of DTG could nor be quantified because, following the intravenous injection of DTG, the microiontophoretic applications of NMDA induced an epileptiform activity. It is noteworthy that DTG is to date the only cr ligand ever rested in our mode1 giving rise to NMDA-induced epileptic aaivity. This phenornenon was observed at doses in between 3 and 40 pg/kg ix, but when DTG was administered at doses higher than 40 &kg i.v., the neurons could be recorded again. At doses of DTG between 40 and 100 pg/kg, ix, a small potentiation of NMDA-induced response was observed, whereas, at doses higher than 100 &kg i.v., DTG had no effect on the NMDA response. At the dose of 1 mg/kg i.v., DTG did not modify the NMDA response and the subsequent injection of 10 pg/kg i.v. of haloperidol did not have any effect (Fig. 3B). Thus, notwithstanding the gap in between 3 and 40 pg/kg, a bell-shaped dose-response curve was also obtained with DTG at doses in between 1-1000 pg/kg i.v. (Fig. 3C). The dose response curves of (+)pentazocine, BD-737, 50-1784 and L-687,384 were 2 fitting the equation: y = a + b(-0.5(Ln'dC)'4)and the r values were: 0.94 for 50-1781, 0.89 for (+)pentazocine, 0.93 for BD-737, and 0.93 for L-687,384. It is noteworthy that despite the fact that no data could be obtained with doses in between 3 and 40 &kg i.v., the best fit equation representing the dose-response curve for DTG was the same than that found for (+)pentazocine, BD-737,504784 and L-687,384, with a r value of 0.99. To determine if the lack of effects of high doses of the a ligands could be due to a rapid desensitization of the cr receptors, the duration of the effect of the intravenous administration of 1 &kg of DTG was determined. The maximal degree of potentiation was obtained wirhin

10 min and lasted for at lem 60 min with a ratio NJN, = 3.5. During the following 30 min, the potentiation gradudly disappeared (Fig. 4A). The injection of a second dose of 1 pg/kg of DTG at that tirne produced a potentiation of the NMDA response of a similar magnitude than that produced by the first injection. Similar experiments were also carried out with 4 &kg, i.v. of 50-1784. In that case, the maximal degree of potentiation was obtained within 15 min and was present for 90 min with a ratio NJN, = 3.0 and then progressively vanished (Fig. 4B). Two h after the first injection, the neuronal response to NMDA was back to baseline. At that time, a second injection of 4 pg/kg, i.v. of JO-1784 induced the sarne degree of potentiation of the NMDA response as the first one. In none of these series, were the effeas of QUIS and ACh modified (Fig. 1,3,4).

Micro ion topho reric smkî Alternate microiontophoretic applications of NMDA and QUIS were carried out. After the obtention of stable responses for three consecutive cycles of NMDA and of QUIS, DTG or 50-1784 were applied by microiontophoresis with a current of 5 nA for 5 min. The current was then increased gradually to 10, 25, 50, 75 and 100 nA. For each current, the microiontophoretic application of each cr ligand was done for a period of 5 min. DTG applied with a current of 5 nA did not modify significantly the NMDA response. However, with a current of IO nA, a two-fold potentiation was noted and the degree of potentiation of the NMDA response gradually increased to a maximal response with a current of 50 nA (NJN,

= 2.5) (Fig. 5A). At higher currents, the magnitude of the potentiation decreased and finally no effect was produced by a current of 100 nA (Fig. SA). At that tirne, the application of DTG was stopped and restarted with a current of 50 nA. A potentiation similar to that produced previously by the sarne current of DTG was obtained (Fig. 6A). Ir is noteworthy that DTG when applied by microiontophoresis did not induce epileptoid activity. The same paradigm was used with JO-1784. With a current as low as 5 nA a selective potentiation of the NMDA response was observed (Fig. 5B). Upon increasing gradudly the current to 100 nA, a response similar to that previously observed with DTG was obtained.

The maximal potentiation was obtained at 25 nA &/NI = 3). At higher currents, the potentiation decreased, and, at 100 nA, chere was no potentiation of the neuronal response to NMDA (Fig. 5B). When 50-1784 was applied again with a currenr of 25 nA, the neuron displayed again the same degree of potentiation of its response to NMDA (Fig. 6B). In a third series of experiments, we assessed the possibility of maintaining a long-lasting potentiation of the NMDA response using microiontophoretic applications with low currents of a ligands. After three consecutive cycles of NMDA and QUIS, DTG was applied with a current of 25 nA and a very stable potentiation of the NMDA response could be obtained for more than two hours (Fig. 7A). A similar long-lasting potentiation of the NMDA response was also obtained with 50-1784 using the current of 25 nA (Fig. 7B). In none of these series, was the effect of QUIS modified (Figures 5,7). DISCUSSION The present data suggest that the o ligands DTG, (+)pentazocine, BD-737,501784 and L-687,384 modify the neuronal response to NMDA of CA3 pyramidal neurons of the rat dorsal hippocampus in a similar way. Administered intravenously or applied with low microiontop horetic currents, t hese ligands potent iated selectively and d~sedependentl~the neuronal response to NMDA (Fig. 2,3,5) in keeping with previous observations in this and other laboratories (Monnet et al. 1990b; Monnet et al. 1992b; Martin et al. 1992; Walker and Humer 1994). However, high doses of the same u ligands did not modiS the NMDA response. At doses ranging from 1 &kg to 1 mgkg, and rnicroiontophoretic currents from 10 nA to 100 nA, the a ligands modified selectively the NMDA response with bell-shaped dose-response curves. We have also previously reported chat the two antidepressant drugs with high affinity for a receptors sertraline and ciorgyline, at doses ranging from 1 pg to 1 mg/kg i.v., potentiated the neuronal response to NMDA with a bell-shaped dose-response curve (Bergeron et d. 1993). Similar biphasic effects of a ligands have been observed by other investigaton in different models. For exarnple, low concentrations of DTG suppress in vitro murine splenocyte natural killer activity, while high concentrations of DTG enhance their killer activity (Carr et al. 1992). In behavioral models, drugs with high &nity and s~ecificityfor o recepton such as BD-737 (Steinfels et ai. 1988) and (+)3-PPP (Largent et al. 1984) generate bell-shaped dose- response curves on punished and unpunished responses in rats (McMillan et al. 1991). DTG has also been found to produce a bell-shaped dose-response curve in experimentally-induced amnesia using a passive avoidance task in the rat (Earley et al. 1991). The monitoring of cerebellar cGMP levels has been used to determine in vivo the modulation of the NMDA receptor (Wood, 1991). Using this paradigm, it has been shown that several a ligands modulate NMDA receptor function, particularly (+)pentazocine and rimcazole, which were found to modulate the levels of cGMP in a biphasic manner (Rao et al. 1991). It has also been shown that BMY-14802 presents a biphasic effect on levels of dopamine in the mesolimbic and in the nucleus accumbens (Taylor et al. 1989). On NMDA-evoked release of [3wnoradrenalinefrom preloaded rat hippocarnpal slices, a bell-shaped profile of the concentration-response curves with JO-1784 and (+)3-PPP has been obtained (Monnet et al. 1992a). The effect of neuropeptide Y which appears to activate a subtype of o receptors (Roman et al. 1989; Monnet et al. 1990a; Bouchard et al. 1993) in the same mode1 also presents a bell-shaped profile (Roman et 4. 1991). It is known that the o receptors are not present only in the central nervous system 100 but also in the peripheral system with a particularly high density in the digestive tract Vam 1983; Roman et al. 1988; Su et 4. 1988). The intravenous administration of DTG has been found to produce a biphasic effect on postprandial colonic motility in fasted and fed dogs Uunien et d. 1990). Finally, very recently, Nabeshima's group reported data virtually superimposable to ours (Maurice et al. 1994a; Maurice et ai. 1994b). In a behavioral paradigm measuring the degree of amnesia induced by the NMDA antagonist MK-801, they showed that doses of DTG or (+)pentazocine between 1 and IOOO &kg had no behavioral effect in the naive rats, but d~sede~endentlyrevened the effect of MK-801. In this range of doses, the same type of bell- shaped dose-response curves was obtained with maximal effects at the dose of 100 rg/kg of DTG and of (+)pentazocine. The lack of effect of DTG and (+)pentazocine in this mode1 in the naive rats and their biphasic effects following a reduction of the NMDA response by MK- 801 constitutes a strong argument in keeping with our electrophysiological observations. This suggests that o receptors modulate the NMDA response and that, under basal conditions, no effect of the o ligands can be detected and that such an effect is present only when NMDA receptors are involved. Three explanations can be envisaged to account for the biphasic effects observed in the present experiments. First, a rapid desensitization of the o receptors involved in the potentiation of the NMD A-induced activation is not likely since the potentiat ing effeas obtained with the intravenous administration of low doses of DTG or JO-1784 lasted as long as 60 and 90 min respectively. A second intravenous administration of the same dose of the same o ligand at these times induced similar potentiations of the NMDA response. Furthermore, microiomophoretic applications of DTG or 50-1784 induced a potentiation of the NMDA response which lasted as long as rwo hours. Second, another explanation could be that several a ligands also have affinity for the PCP binding site (Largent et al. 1986). For instance, at the high dose of 5 mg/kg i.v., DTG reduces the NMDA response presumably due to the low affinity of DTG for the PCP binding site since this inhibition was not reversed by haloperidol (Monnet et al. 1992b). However, the phenornenon observed in the present experiments cannot be due to the occupation of PCP binding sites since it was observed with BD-737 and L-687,384 which do not have any affinity for PCP binding sites. Third and finally, the most likely explanation for the biphasic effects of o ligands on the NMDA response might be related to the existence of different subtypes of o receptors. During the last years, evidence has accumulated for the existence of multiple specific o binding sites in the central nervous system, (for reviews see: (Itzhak and Stein 1990; Thomas et ai. 1990; Itzhak et al. 1991; Iyengar et al. 1991; Walker et al. 1992). At present, there is a consensus that at least two subtypes of a receptors exist in the rnarnmalian brain (Quirion et al. 1992). It is possible that the o ligands studied in the present experirnents act, at low doses, on a subtype of o receptor for which they have high affinity, resulting in a potentiation of the NMDA response; at higher doses, they rnight act on another o receptors for which they would have a lower affinity, and which they would cancel out the potentiation of the NMDA response. The epileptoid activity produced by microiontophoretic applications of NMDA follnwing the systemic administration of DTG is in keeping with previous observations from our laboratoxy (Monnet et al. 1990a) but remains intriguing. DTG is the only o ligand we tested thus far, which produces such a phenomenon. L-687,384, BD-737, JO-1784 and (+)pentazocine are known to be selective for ai receptor whereas DTG binds to both al and 02 receptors. Thus it is possible that the epileptoid phenomenon is induced by the activation

of oz receptors or by the coactivation of O, and 0, receptors. Further studies using selective q ligands should clarify this issue. Even when applied with high currents, the rnicroiontophoretic applications of DTG never induced any epileptoid activity. This could be due to the fact that such an epileptoid activity appean only when a large number of neurons are exposed to DTG since epileptoid activity results from the synchronous firing of a population of neurons.

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(+) PENTAZOC INE HALOPERIDOL 10 pgkg. i.v. 10 pgkg, i.v.

ACh NMDA 11 I

I I 2 min (+) PENTAZOCINE HALOPERIDOL

Figure 1 Integrated firing are histograms of CA3 dorsal hippocampus pyramidal neurons showing the effecu of microiontophoretic applications of ACh, QUIS and NMDA before and aher the intravenous administration of a low dose of (+)pentau>cine (A) and of a high dose of (+)pentazocine (B) followed by the intravenous administration of a low dose of haloperidol. In this and subsequent figures: bars indicate the duration of applications for which currents are given in RA; each neuronal response to each excitatory agent represenrs the computed-generated mean of the effem of three successive applications; the two circles represent an interval of 5 min. Dose (pglkg. i.v.) of (+)PENTAZOCINE

Dose (pg/kg. i.v.) of BD-737 Oose (pglkg. i.v.1 of L-687.384

Figure 2 Dose-response curves of the effecü of inravenous administrations of (+)pentazocine (A), JO-1784 (B), BD-737 (C)and L-687,384 (D) on the neuronal activation of CA, dors$ hippocampus pyramidal neurons induced by microiontophoretic applications of NMDA. The effecü of o ligands were assessed by determining the ratio (N-J of the nurnber of spikes genented per nanocoulomb by NMDA before (NI) and after (Nd the injection of one dose of each of the drugs. Each dot represenls the effect of one dose of the o ligand administered to one rat while recording from one neuron. The equation used to generate these curves is given in the text. A ACh NMDA QUIS 7 -12 -2 0 In-0

I DTG HALOPERIDOL 1 pg/kg, i-v. 10 ygjkg. i.v.

ACh NMDA QUIS 11 .8 - 3 O -10 1-0

DTG HALOPERtDOL 1000 pglkg. I.V. 10 pglkg, i.v.

Dose (pg/kg, i.v.) of DTG

Figure 3 Integrated firing rate histograms showing the response of CA, dorsal hippocampus pyramidal neuron to microiontophoretic applications of ACh, QUIS and NMDA before and after the i.v. injection of a low dose (A) or a high dose (B) of DTG followed by the i.v. injection of a low dose of 10 pg/kg i.v. of haloperidol. C. Dose-response curve of the effect of intravenous administration DTG on the neuronal activation of CA3 dorsal hippocampus pyramidal neurons by microiontophoretic applications of NMDA. See legend ro figure 2. QUlS NMDA -3 -1 0 œ r? ~13 - u -

DTG DTG 1 pg/kg, i.v. 1 p@g. i.v.

QUlS NMDA -3 -1 O - C œ

I 1 2 min

Figure 4 Integrated firing rate histogams of CA3 dorsal hippocampus pyramidal neurons showing the effecu of microiontophoreric applications of QUIS and NMDA before and aher the intravenous administration of a low dose of o ligand. In (A), a fint injection of 1 pg/kg of DTG was administered. // indicate a 60 min period. A second injection of i pg/kg of DTG at that tirne induced a effect similar to that of the fim one. In (B), the injection of 4 pg/kg of 10-1784 was administered. // indicate a 90 min period. A second injection of 4 pg/kg of 50-1784 produced a similar effect as the first one. DTG

NMDA QUIS -10 -3

JO-1 784

NMDA QUIS -10 -3 O n, oua u 0- 0-

- 2 min

Figure 5 Integrated firing rate histograms of CA, dorsal hippocampus pyramidal neurons showing the effects of microiontophoretic applicaiions of NMDA and QUIS before, during and after the microiontophoretic application of DTG (A) or JO-1784 (B) at different currents (5 - 100 nA). In both traces the microiontophoretic applications of DTG or JO-1784 were stopped after which DTG and JO-1784 were applied again. SPIKES GENERATEDInC 01 NMDA rn SPIKES GENERATEDInC ol NMDA D DTG --25 QUIS NMDA -3 -1 0 n I 101n

NMDA -1 O O

. r 2 min

Figure 7 Integrared firing rate histognms of neurons of the CA3 region showing the effect of microiontophoretic applications of QUIS and NMDA before, during and after long-laring microiontophoretic applications of DTG (25 nA) (A) and JO-1784(25 nA) (B). // indicares an interruption of 10 min. 112 CHAPTER IV Differential effects of sigma ligands on the NMDA response in the CA, and CA, regions of dorsal hippocampus: Effect of mossy fiber lesioning

The potentiation of the NMDA response previously reponed was obtained in the CA, region of the rat dorsal hippocampus. This potentiation is obsenred with several a ligands. These results raised another question. Do the potentiation obtain in the CA, area may also be observed in other brin regions?

The initiai aim of this third study was to assess whether the potentiation of the NMDA response induced by a agonists already identified in the CA, region of the donal hippocampus may also be obtained in the CA, region. A secondary goal was to determine the involvement of the mossy fiber syrtem in this phenornenon. Indeed, colchicine has been shown to be neurotoxic for the granular cells of the hippocampus dentate gyrus. The mossy fiber lesioning should provide us with new information about the localization (pre- or post-synaptic) of the a receptors in the CA, region of the dorsal hippocampus. If the potentiation of the NMDA response induced by a agonists is obtained by an activation of the a receptors, located pre- synaptically on the mossy fibers, this potentiation should be abolished by pretreatment with colchicine and should not be found in the CA, region of the hippocampus. DIFFERENTIAL EFFECTS OF SIGMA LIGANDS ON THE NMDA RESPONSE IN THE CAI AND CA3 REGIONS OF THE DORSAL HIPPOCAMPUS: EFFECT OF MOSSY FIBER LESIONING Guy Debonnel, Richard Bergeron, François P. Monnet and Claude de Montigny

Neuroscience, 7 1: 977-987, 1996.

SUMMARY In the CA, region of rat dorsal hippocampus, several sigma ligands, such as DTG, (+)pentazocine and 50-1784, administered intravenously at low doses, potentiate selectively the pyramidal neuron firing activity induced by microiontophoretic applications of N-methyl- D-aspartate, without affecting those induced by quisqualate, kainate or acetylcholine. A similar potenriation of the NMDA response has ais0 been found with rnicroiontophoretic applications of neuropeptide Y, an effect exerted via sigma receptors. The present experiments were carried out to determine the effects of these sigma ligands and of neuropeptide Y, in the CA, and CA, regions following a prior unilateral destruction by a local injection of colchicine of the mossy fiber system which is a major afferent to CA, pyramidal neurons. In the CA, region, DTG, 50-1784 and neuropeptide Y did not potentiare the activation induced by microiontophoretic applications of NMDA. However, (+)pentazocine potentiated the NMDA response, similady to its effect in the CA, region on the intact side. In the CA, region, on the intact side, (+)pentazocine, DTG, JO-1784 and neuropeptide Y induced a selective potentiation of NMDA-induced activation, in keeping with previous reports. On the lesioned side, the effect of (+)pentazocine on the NMDA response was still present, but those of DTG, JO-1784 and neuropeptide Y were abolished. These resulrs suggest that (+)pentazocine on the one hand, and DTG, 50-1784 and neuropeptide Y on the other hand are not acting on the sarne subtype of sigma receptors. Since (+)pentazocine, 50-1784 and neuropeptide Y have been suggested to am on the sigma, subtype of receptors, these data suggest the existence of two subtypes of sigma, receptors. They also suggest that the subtype on which DTG, JO-1784 and neuropeptide Y acts, is located on the mossy fiber terminals in the CA, region and is absent in the CA, region.

Key words: DTG, 50-1784, (+)pentazocine, neuropeptide Y, ele~tro~hysiology INTRODUCTION

Previous data from our laboratory have shown that several sigma (0) ligands, when applied by microiontophoresis at low currents or admnistered intravenously at low doses, potentiate selectively the excitatory effect of microiontop horetic applications of N-methyl-D- aspartate (NMDA) ont0 pyramidal neurons of the CA, region of the rat dorsal hippocampus in vivo 13. This potentiation by o ligands of the NMDA response appeared to be selective as the activations induced by quisqualate (QUIS) or kainate were not affected '*'. A similar potentiation of the NMDA response is also produced by microiontophoretic applications of neuropeptide Y (NPY), an effect mediated via an activation of o receptors and not of the YI,

Yz or Y3 subtypes of NPY receptors lJ. In the CA, region, the potentiation of the NMDA response by the a ligands 1,3di(2-toly1)guanidine @TG) ', (+)Ncyclopropylmethyl-N-methyl-

1,kiiphenyl-bethyl-but-3-en-I-ylarninehydrochloride 00-1784) and NPY 6J is mediated via a subtype of o receptor linked to a Gi/, protein, whereas the potentiation induced by the cr ligand (+)pentazocine ' is mediated via another subtype of o receptor not linked to a Gv, protein 9. The first series of experiments was carried out to assess the effects of these o ligands and of NPY in the CA, region of the dorsal hippocampus, to determine if these two subtypes of o receptors were also present in this region. The second series was carried out in the CA, region which receives a massive input from the dentate gynis mossy fiber system of granular cells of the dentate gyms 'O. Experiments were carried out in naive rats and following a lesion the of the dentate gyrus granule cells by a local injection of colchicine, which has been shown to be neurotoxic for certain populations of neurons in the brain, in particular for the granular cells of the hippocampal dentate gyrus 'l.

MATERIAL AND METHODS Preparation of dmgs The following substances were used: 50-1784, a generous gift from J.L. Junien (Institut de Recherche Jouveinal, Fresnes, France); MY,a generous gift from A. Fournier, (I.N.R.S. Santé, Pointe-Claire, Qc, Canada); Colchicine (Fisher, Ottawa, ONT, Canada); (+)pentazocine (RBI, Natick, MA, USA.); NMDA (Sigma, St-Louis, MO, USA); QUIS (T.ocris Neurarnin, Buckburst Hill, Essex, UK); ACh (Sigma); DTG (Aldrich Chemicals, Milwaukee, WI, USA); haloperidol (McNeil Laboratories, Stouffville, ONT, Canada). Anirnals Male Sprague-Dawley rats weighing 175-200 g were obtained from Charles Rivers (Saint- Constant, Qc). They were maintained at constant temperature (2S°C) under a 12h-12h light- dark cycle, with free access to food and water. Al1 efforts were made to minimize animal suffering and reduce the number of animals used. Animd care was according to protocols and guidelines approved by McGill University and the Canadian Council for Animd Care.

Colchicine lesions Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic apparatus. A burr hole was drilled at 4 mm anterior to lambda and 2 mm lateral from midline. The tip of a 5 pl Hamilton synnge was lowered in the dentate gynis, 3.1 mm under the cortical surface. Colchicine (3 pg in 3 pl of distilled water) was slowly injected over a 3 min period in the right dentate gyms. The needle was left in place for 1 min after the end of the injection to prevent a backflow of the solution through the needle track. During the 72 h following the surgery, animas were adrninisrered, twice daily, a solution of glucose 5% and NaCl 0.9 O/O (1 ml, i.p.). Electrophysiological experiments were carried out 10 to 15 days after the colchicine injection.

Elect ropbysio~ogicalexpm'men ts Rats were anesthetized with urethane (1.25 g/kg, i.~.),and mounted in a stereotaxic apparatus. Body temperature was mainrained at 37°C throughout the experiment. Microiontophoretic applications and extracellular unitary recordings were performed with five barrelled glass micropipettes, preloaded a-ith fibreglas strands in order to promote capillary filling, as previously described 12. The central barrel, used for extracellular unitary recordings of the activity of the CA, or CA, hippocampal pyramidal neurons, was filled with a 2 mM NaCl solution saturated with fast green FCF. One side barrel, filled with 2 rnM NaCl, was used for current balancing. The ~therside barrels, used for microiontophoresis, were filled with three of the following solutions: NMDA (10 mM in 200 rnM NaCl, pH: 8); QUIS (1,s rnM in 400 mM NaCl, pH: 8); ACh (10 m in 200 mM NaCl pH: 8); NPY (OJ rnM in 150 NaCl and 0,1% BSA, pH: 8); DTG (1 mM in 200 mM NaCl, pH: 8); 50-1784 (1 mM in 200 mM NaCI, pH: 8); (+)pentazocine (0,2 rnM in 200 mM NaCl, pH: 8).

The neuronal firing activity was monitored as previously described 13. In brief, action potentials were detected and square pulses were fed to a computer which generated integrated firing rate histograrns. Pyramidal neurons from the CA, or CA, region of the hippocampus were identified according to the criteria of Kandel and Spencer '*: long durarion (0.8-1.5 ms), large amplitude (0.5-2 mV) action potentials, and presence of characteristic complex spike discharges alternating with simple spike activity.

Culculations Alternate microiontophoretic applications of 50 s of each excitatory substance (NMDA, QUIS and ACh) separated by 50 s retention penods were carried out continuously during the whole duration of the recording. The duration of the microiontophoretic applications and the intensity of the currents used were also stored in the computer. The effeas of their applications on pyramidal neuron firing activity were expressed as the number of spikes generated/nanocoulomb (1 nC being the charge generated by 1 nA applied for 1 s). After a neuron was isolated, it was recorded for a period of at least 20 min before any experimentd dmg was administered. The recording was stored on the computer without interruption for the whole duration of the experiment. Five to six applications of each excitatory substances were carried out before the a compounds or NPY were injected or applied microiontophoretically. The effects of the drugs studied occurred within 10 min after their intravenous injections or the beginning of the rnicroiontophoretic application. The data were calculated when the maximal effen of the dmg was observed (within the first 20 min). The computer calculated the effect of each microi~nto~horeticapplication of an excitatory substance, as the total number of spikes generared/nC (1 nC being the charge generated by 1 nA applied for 1 sec). Each value was calculated by the computer as the mean of the effects of three consecutive applications. The effects of the intravenous administrations of a ligands were assessed by comparing the number of spikes generated/nC of each the three excitatory substances ACh, QUIS and NMDA before and after their injection. The effects of the microiontophoretic applications of the a ligands and NPY were also assessed by comparing the the number of spikes generated/nC of ACh, QUIS and NMDA before and during the microiontophoretic applications of the a ligands or NPY.

Statistical anabis Results are expressed as rneans * S.E.M. The means were compared using the paired Student's t-test. Probability values smaller than 0.05 were considered as significant. Each series of experirnents was carried out in 8 to 12 rats. Histologicaf prqûarat ions At the end of each experiment, a Fast Green deposit was left at the last recording site by passing a -25 pA mrrent for 20 min through the recording barrel. The animal was sacrificed by decapitation and the brain was quickly removed, frozen in methyl-butane at -3j°C, and kept at -70°C. Subsequently, 40 pm-thick cryostat sections were prepared and stained with a Timm's staining for heavy metal which specificdly marks the zinc containing mossy fiber terminals "3and with cresyl violet to evaluate the extent of dentate gyrus lesion produced by the colchicine injection and to verify the Iocalization of the Fast Green deposit.

Al1 recordings were obtained from the stratum pyramidale of the CA, or CA, areas of the dorsal hippocarnpus, as confirmed by histological verification of the Fast Green FCF deposit left at the end of each experiment. On the lesioned side, the local colchicine injection in the dentate gyms produced a massive destruction of the mossy fiber terminals, as evidenced by a near complete disappearance of these terminals on the Timm's stained histological preparations (data not shown). This lesion produced some shrinking of the Hamon's horn so that CA, pyramidal neurons were located more medially on the lesioned side. Microiontophoretic applications of NMDA, QUIS and ACh increased the firing activity of al1 CA, or CA, pyramidal neurons tested. None of the a ligands tested had any effect on the spontaneous firing activity of pyramidal neurons in the CA, or CA, region. Microiontophoretic applications of NMDA, QUIS and ACh increased the firing activity of al1 CA, or CA, pyramidal neurons tested. None of the a ligands tested had any effect on the spontaneous firing activity of pyramidal neurons in the CA, or CA, region.

CA,region Microiontophoretic applications of DTG (20 nA) as well as of 50-1784 (20 nA) and NPY (-20 nA) had no potentiating effect on the response of pyramidal neurons to NMDA (Figs 1A and 2). In contrast, the microiontophoretic applications of (+)pentazocine (20 nA) produced a selective two-fold potentiation of the NMDA response (Figs IB and 2). Sirnilarly, when administered intravenously, neither DTG (1 pg/kg) nor JO-1784 (5 Irg/kg) had any effect on NMDA-induced activation (Fig. 3), whereas a low dose (5-10 pg/kg, i.v.) of (+)pentazocine potentiated the NMDA response by two-fold (Fig. 3), as has been observed in previous studies in the CA, region '. This effect of (+)pentazocine, whether applied by microiontophoresis or administered intravenously, was reversed by the injection of a low dose (10-20 ~g/kg,i.v.) of halopend01 (Fig. 3 and Table 1). None of the a ligands nor NPY modified significantly the neuronal response ro QUIS.

CA, region On the intact side, in keeping with previous data from Our or other laboratories, the microiontophoretic applications of the o ligands DTG, (+)pentazocine (Fig. 4A), JO-1784 as well as of NPY (Fig. 5A), induced a selective potentiation of the pyramidal neuron response to NMDA without affening those to QUIS. Sirnilarly, when administered intravenously, DTG (1 ~g/kg),(+)pentazocine (IO pg/kg) and 50-1784 (5 &kg) potentiated the NMDA response without affecting those to QUIS and ACh (Tables t and 3). On the lesioned side, rnicroiontophoretic applications of NMDA, QUIS or ACh produced an activation of al1 ~~ramidalneurons tested similar to that observed on the inract side (Table 3). As illustrated for NPY in figure 5, on the lesioned side, microiontophoretic applications of the a ligands DTG (10 and 20 nA), 50-1784 (10 and 20 nA) and NPY (-20 nA) had no effect on NMDA-induced activation of CA, pyramidal neurons (Fig. 6A,B,D), whereas the responsiveness to NMDA was still potentiated by (+)pentazocine (20 nA) (Fig. 6C). The systemic administration of (+)pentazocine (IO pg/kg, i.v.) also increased by two-fold the excitatory effect of NMDA (Table 3). Whether applied by rnicroiontophoresis or administered intravenously, the effects of (+)pentazocine on the lesioned side were not statistically different from those observed on the intact side. As was the case in the CA, region, following its intravenous administration or its microiontophoretic application, the potentiating effect of (+)pentazocine was reversed by the administration of a low dose of haloperidol(10 ~g/kgi.v.; Table 3). The intravenous administration of JO-1784 (5 ~g/k~,i.v.) did not modify the NMDA response (Table 2). However, the administration of DTG (1 pg/kg, i.v.) induced a significant reduction of NMDA-induced activation; this effect of DTG was not reversed by the subsequent injection of haloperidol (IO ~g/kg,i.v.; Table 2).

DISCUSSION

As previously reported 11, the local injection of colchicine in the dentate gyrus induced a selective lesion of the mossy fiber system. Previous reports have shown that colchicine is not selective for the mossy fibers system and have reported an important loss of cholinergie innervation in the dorsal hippocampus following local injections of colchicine in the septum or following i.c.v. administration 17? These two studies have shown t hat the cholinergie system is not altered by a local injection in the dentate gyrus. This is in keeping with the present observation that the neuronal response to microiontophoretic applications of ACh was unchanged on the lesioned side. In the CA, region, on the intact side, the intravenous administration and the microiontophoretic application of the o ligands DTG, (+)pentazocine, JO-1784, as well as the microiontophoretic application of NPY, induced a selective potentiation of the pyramidal neuron response to NMDA, without modifying those to QUIS or Ach. This is in keeping with previous reports from our and other laboratories In the CA, region, which is not innervated by mossy fibers, the effect of (+)pentazocine was similar to that obtained in the CA, region on the intact side. However, there was no potentiation of the NMDA response by DTG, 50-1784 and NPY. In the CA, region, on the lesioned side, the ~otentiatingeffect of (+)pentazocine was unchanged but the potentiation of the NMDA response by DTG, JO-1784 and NPY was abolished. The existence of different subtypes of a receptors is now widely accepted and the distinction between ai and oz subtypes has been clearly established (+)Pentazocine and JO- 1784 are thought to be selective for the alsubtype whereas DTG is presumed to act on both a1 and oj subtypes ? The suppression by a colchicine pretreatment of the effects induced by DTG, 50-1784 and NPY but not of that induced by (+)pentazocine constitutes a clear indication that (+)pentazocine is acting on a subtype of a receptor different from that activated by 50-1784, and consequently supports the existence of at least two subtypes of a, receptors. This hypothesis is fully consistent with previous data from Our laboratory, showing that: 1) the effects of 50-1784 (+)pentazocine is mediated via a recepton since they are reversed by haloperidol and by the selective a, antagonist NE-100 (Debonnel et aL, submitted) but not by spiperone '; 2) a penussis toxin pretreatment, which inactivates GV, coupled recepton, prevents the potentiation of the NMDA response induced by DTG, 50-1784 and NPY but does not affect thar induced by (+)pentazocine 9. These data suggest that, in the CA, region of the dorsal hippocampus, one subtype of al recepror is linked to a G;,, protein, whereas that activated by (+)pentazocine is not. These data are also in keeping with that previous results suggesting that (+)pent;wcine binds to a subset of a, recepton or a a receptor differem from the classical a, and q receptors "34. However, it cannot be completely ruled out rhat a small part of the potentiating effea of (+)pentazocine is mediated via the same subtype of o, receptor activated by JO4784 and NPY, given the slight (non significant) decrease of the potentiating effect of (+)pentazocine on the lesioned side and the recent report of an heterogeneity of the binding of [WJ(+)pentazocine The disappearance of the potentiating effects of DTG, JO-1784 and NPY following a colchicine lesion suggests that the effects of these o ligands, in the CA, region, are mediated via a subtype of o receptor located pre~yna~ticallyon mossy fiber terminals. These data are in keeping with previous autoradiographic studies on o receptors which showed that a colchicine lesion of the dentate gyrus did not change the binding parameters of ['H]3-PPP, a a ligand with a preferential affinity for a, receptors, and a reduction of r3Hj3-PPP binding densities by a quinolinic acid injection in the CA, region, which destroys pyramidal neurons. Interestingly, the local injection of kainic acid which produces the sarne effect did not change

[,H]DTG binding 26*L7. On the other hand, autoradiographic studies have also shown high densities of NMDA receptors distributed throughout the hippocarnpal formation. In the CA, region, most of them are not located presynaptically on the mossy fiber tenninals since a colchicine lesion of the dentate gyrus produced not significant reduction of r3WTCP binding Thus, the potentiation of the postsynaptic NMDA response, induced by the activation of o receptors located pre~ynapticall~is presumably mediated via the release frorn the mossy fiber terminas of an unidentified substance which would in turn induce a potentiation of the postsynaptic NMDA receptors. Chavkin et al., have reporced that the electrical stimulation of the mossy fiber system can displace of E3H]DTG binding ". These authors concluded that this electrical stimulation of the mossy fiber system induced the release of an endogenous o2 ligand for receptors labelled with ['HJTITG. Taken together, these data would suggest that the release of a cr ligand acting postsynaptically on oz receptors is modulated via p resynaptic a, receptors. The persistence of t he effect of (+)pentazocine following colchicine pretreatment clearly indicates that the 0, subtype of receptor mediating the potentiating effect of t his a ligand is not located on the mossy fiber terminals. The most plausible possibility is that this subtype of o receptor is located posrsynaptically on the pyramidal neurons and represents the binding site of ['H]3-PPP. However, it cannot be excluded that these cr receptors are located on interneurons or on other terminals. At present, there is no evidence suggesting that this subtype of o receptor is located on the NMDA receptor-complex nor is there any evidence for an allosteric modulation of NMDA receptors by a ligands. DTG has a low affinity for the phencyclidine binding site ' whose ligands are non- competitive NMDA antagonists "jl. The suppressant effect on the NMDA response exerced by DTG in the colchicine lesioned side cannot be ascribed to the affinity of DTG for the phencyclidine site' given the low dose (1 &kg i.v.) administered. This inhibitory effect is in keeping with our previous results obtained following a pertussis toxin pretreatment where DTG also induced a reduction of the NMDA response, which was not reversed by haloperidol '. This effect of low doses of DTG has been aiso reported in other areas such as the rat prepiriform cortex 32. This effect could either be related to a subtype of a receptor not sensitive to haloperidol, such as the R4 subtype proposed by Zhou and Musacchio " or to an effea of DTG on a non-a receptor. In the CA, region, the lack of effect of DTG, JO 1784 and NPY on NMDA-induced response is in agreement with previous reports of Colmers et and Connick et ai.," who have shown that, in this region, NPY and DTG do not porentiate the NMDA response. The fact that (+)pentazocine potentiates the NMDA response in this region constitutes another argument suggesting that JO4784 and NPY act on a subtype of o receptor different from that on which (+)pentazocine is acting. In conclusion, the present data ~rovidenew functional evidence for the existence of distinct subtypes of a receptors and for their differential localization. They suggest that two subtypes of al receptors can affect pyramidal neuron response to NMDA, one on which JO-1784, DTG and NPY are acting, located presynaptically on terminas of the mossy fiber system and not present in the CA, region, and another, on which (+)pentazocine is acting, presumably located postsynaptically in both CA1 and CA, regions of the dorsal hippocampus.

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35. Connick J.H., Addae J.E., Nicholson C.D. and Stone T.W. (1992) The sigma ligand 1,3-di-o-tolylguanidine depresses amino acid-induced excitation non-selectively in rat brain. Ew.J Pharmacol. 2 14, 169-173. DTG QUlS NMDA 20

HALOPERIDOL 10 pglkg, i.v.

(+)PENTAZOCINE QUlS NMDA 20

1 2 min HALOPERIDOL 10 pglkg, i.v.

Figure 1 Integrated firing rate histograms of CA, dorsal hippocampus pyramidal neurons showing the effect of microiontophoretic applications of NMDA and QUIS before and during the rnicroiontophoretic application of DTG (A) or (+)pentazocine (B). In this and the subsequent continuous integrated firing histograms, ban indicate the duration of applications for which currenü are given in nA. Open circles (00) represents an interruption of the illustration of the continuous recording. Time base applies to both traces. NMDA OUlS NMDA OUlS

OS 1

Cunents (nA) of (+)pentazoane Cunents (nA) of JO-1784

NMDA NMDA i OS 1 *

OMO

Currents (nA) of DTG Currents (nA) of NPY

NMDA NMDA + sigma ligand NMDA + sigma ligand + haloperidol (10 pg/kg. 8.v.)

Figure 2 Responsiveness, expressed as the number of spikes generated/nanocoulomb (mean + S.E.M.)of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA and QUIS before (open columns) and during (hatched columns) the microiontophoretic applications of (+)pentazocine (A), DTG (B), JO-1784 (C) or NPY (D) and following the intravenous administration of haloperidol (black columns). * p < 0.05 uing the paired Student's r test. ACh NMDA QUIS 10 -15 -2 œ 0 0 0

t+)PENTAZOCINE HALOPERIDOL

NMDA QUIS -19 - 5 na= 010I 01 II

I I JO-1784 (+)PENTAZO.CINE HALOPERIDOL 5 pglkg, i.v. 5 pg/kg. I.V. 20 pglkg, i.v.

Figure 3 Integrated firing rate histograms of CA, dorsal hippocampus pyramidal neurons showing the effects of microiontophoretic applications of ACh, QUIS and NMDA before and after the intravenous administration of (+)pentazocine followed by haloperidol (A); and the effects of microiontophoreric applications of QUIS and NMDA before and after the injection of JO-1784, followed by (+)pentazocine and haioperidol @) (see legend co figure 1). (+)pentazocine (20 nA) ACh QUIS NMOA 0 8 4 -16 5o=anoaaoazin~o

ACh QUlS NMDA 8 -4 -16 (Zourzao5oanu;i

- 1 min

Figure 4 Continuous integrated firing rate histogram of a CA, dorsal hippocarnpus pyramidal neuron illustrating the effect of microioncophoretic applications of NMDA, QUIS and ACh before, during and after a microiontophoretic application of (+)pentazocine on the intact side. Time base applies to the three traces. ACh QUIS NMDA 9 -3 -12 IczaOIanU -mu I[LZIULZDO

- 1 min

Figure 5 Continuous integrated firing rate histograms of CA, dorsal hippocarnpus pyramidal neuron, illustrating the effect of microiontophoretic applications of NMDA, QUIS and ACh before, during and after microiontophoretic applications of (+)pentazocine on the colchicine-lesioned side. Time base applies to the three traces. NPY NMDA -20 QUlS

NPY NMDA -20 QUlS

-- 2 min

Figure 6 Integrated firing rate histograrns showing the response of CA, dorsal hippocarnpus pyramidal neurons to microiontophoretic applications of NMDA and QUIS before, during and after microiontophoretic applications of NPY on the intact side (A) and on the lesioned side (B) (see legend to Fig.1). CONTROL COLCHICINE B COMROL COLCHICINE

O 10 20 O 10 20 O 10 20 O 10 20 Currents (nA) of DTG Currents (nA) of JO-1784

COMROL COLCHICINE D CONTROL COLCHICINE *

O 20 20 O 20 20 O 20 Currents (nA) of (+)pentazocine Currents (nA) of NPY

NMDA NMDA + sigma ligand NMDA + sigma ligand + haloperidol (IO pgkg. Lv.)

Figure 7 Responsiveness of CA3 dorsal hippocampus pyramidal neurons to microiontophoretic applications of NMDA before and during microiontophoretic applications of DTG (A), JO-1784 (B), (+)pentuocine (C) and NPY (D) on the intact side and on the lesioned side. * p < 0.05 using the paired Student's t test. Table 1 Responsiveness, expressed as the number of spikes generared/nC (mean * S.E.M.), of CA, dorsal hippocarnpus neurons to microiontophoretic applications of NMDA, QUIS and ACh before and aker the intravenous administration of a low dose of DTG and 10-1784.

DTG (1 pg/kg, i.v.; n = 10)

NMDA

NMDA + sigma ligand

NMDA + sigma ligand

QUS + sigma ligand

QUIS + sigma ligand + haloperidol

ACh

ACh + sigma ligand

ACh + sigma ligand

* p < 0.05 using the paired Student's t test. Table 2 Responsiveness of CA3 dorsal hippocampus pyramidal neurons to microiontop horetic applications of NMD A before and after the intravenous administration of a low dose of DTG and 10-1784 in intact animals and in colchicine-treated rats.

DTG JO-1784 (1 pg/kg, i.v.; n= 10) (1 pg/kg, i.v.; n= 10) Colchicine II Control 1 Colchicine I NMDA

NMDA + sigma ligand

NMDA + sigma ligand + haloperidol

+ p < 0.05 using the paired Student's t test. Table 3 Responsiveness of CA3 dorsal hippocampus pyramidal neurons to rnicroiontophoretic applications of NMDA, QUIS and ACh before and after intravenous administration of a low dose of (+)pentazocine in the control and colchicine-treated rats.

Control Colchicine

NMDA

NMDA + sigma ligand

NMDA + sigma ligand + haloperidol

+ sigma ligand I

-- - ACh + sigma ligand

II ACh + sigma ligand II + haloperidol * p < 0.05 using the paired Student's t test. CWTER V The effects of sigma ligands and of neuropeptide Y on N-methyl-D-aspmate- induced neuronal activation are differenrially affected by pertussis toxin

In 1989 Itzhak reponed that at least a subpopulation of o binding sites were coupled to G proteins. In 1992 a general consensus was reached by experts in the field of o receptors research on the distinction of at least two subtypes of o receptors labelled o, and o,. In this classification, a ligands such as (+)SKF 10,047, (+)pentazocine and 50-1784 are selective for the u, receptors, whereas others a ligands such as DTG and halopend01 have equivalent affinity for both subtypes of receptors. Both o, and subtypes are present in the brain but they differ in their &inities for a drugs, tissue distribution and GTP sensitivities.

The purpose of this founh study was to determine the role of Gy, proteins in the potentiation of the NMDA response obtained with certain a ligands and NPY. This study may provide functional evidence that o ligands, acting on distinct a receptors, rnay also be differentiated by their sensitivity to pertussis toxin. THE EFFECTS OF SIGMA LIGANDS AND OF NEUROPEPTIDE Y ON MMETHn-D-ASPARTATE-INDUCEDNEURONAL ACTIVATION OF CA3 DORSAL HIPPOCAMPUS NEURONES ARE D1FFER.E-Y AFFECTED BY PERTUSSIS TOXIN François P. Monnet, Guy Debonnel, Richard Bergeron, Benjamin Gronier and Claude de Montigny

British Journal of Pharmacology, 112: 709-715, 1994.

SUMMARY The in vivo effects of the high affinity sigma ligands I,3di(2-tolyl)guanidine @TG), (+ )N-cyclopropylmethyl-N-methyl-1,4diphenyl, -1sthyl-but-kn-1-ylamine hydrochloride 00-1784), (+)pentazocine and haloperidol, as well as of those of neuropeptide Y (NPY), on N-methyl-D-aspartate (hMDA)- and quisqualate (QUIS)-induced neuronal activations of CA, pyramidal neurones were assessed, using exrracellular unitary recording, in control rats and in rats pretreated with a local injection of pertussis toxin (PTX), to evaluate the possible involvement of Gy, proteins in mediating the potentiation of the neuronal response to NMDA by the activation of sigma receptors in the dorsal hippocampus. Microiontophoretic applications as well as intravenous injections of (+)penrazocine potentiated selectively the NMDA response in control rats as well as in PTX-pretreated. In contrast, the PTX pretreatrnent abolished the potentiation of the NMDA response by DTG, JO-1784 and NPY. Moreover, microiontophoretic applications of DTG induced a reduction of NMDA-induced neuronal activation. Neither in control nor in PTX-treated rats, did the sigma ligands and NPY have any effect on QUIS-induced neuronal response. In PTX-treated rats, the potentiation of the NMDA response induced by (+)pentazocine was suppressed by haloperidol, whereas the reduction of the NMDA response by DTG was not affected by haloperidol. This study provides the first in vivo functional evidence that sigma ligands and NPY modulate the NMDA response by acting on distinct receprors, differentiated by their PTX sensitivity .

Key words: NMDA - Gy, protein - Pertussis toxin - Sigma receptors - Haloperidol - DTG - JO-1784 - (+)Pentazocine - NPY - BMY-14802 136 INTRODUCTION The recent synthesis of selective and high affinity sigma ligands has led to the identification of some of the p harmacological characteristics of sigma receptors. We have shown that several sigma ligands seleaively and rnarkedly potentiate N-methyl-D- aspartatc(NMDA)-induced neuronal activation of rat CA, donai hippocarnpus pyramidal neurones using an in vzvo eIectrophysiological paradigm (Monnet et al., 1990; 1992b). This potentiation of the NMDA response is suppressed by other sigma ligands such as haloperidol, (BMY 14802) and (+)N-n-propyl-3-(3-hydroxyphenyl) piperidine [(+)WPP] which by themselves, at low doses, do not affect the NMDA response. Therefore, the former are tentatively denoted agonists and the later antagonists in the present manuscript. These results suggest the existence of a funaional interaction berween sigma and NMDA receptors. Further support to this notion has been provided by data obtained in both A9 and A10 regions (Iyengar et al., 199Oa) and in the hypothalamic-pituitq-adrenal axis (Iyengar et al., 1990b; Iyengar et al., 1991). Neuropeptide Y (MV),which has been reported to bind to sigma receptors (Roman et aL, 1989, 1993; Bouchard et al., 1993), behaves like sigma agonists in our in vzvo electrophysiological mode1 (Monnet et al., 1992d; 1992c) as well as in various in vivo and in vitro paradigms (Roman et al., 1989; Roman et al., 1991 b; Riviere et ai., 1990), suggesting that it might aa on sigma receptors. Guanosine triphosphate (GTP)-binding regulatory G proteins (guanine nucleotide- binding proteins) might be involved in the biologieal effects of several high affinity sigma ligands (Chattarji et al., 1989; Itzhak, 1989; Itzhak & Khouri, 1988). Indeed, in rat brain membrane preparations, GTP and Gpp(NH)p reduce the high affinity component of the binding of the sigma ligands (+)N-allylnormetazocine [(+)SKF-10,0471, [(+)3-PPP] and pentazocine, this phenomenon being attributed to the conversion of sigma receptors from a high to a low affinity state (Beart et al., 1989; Itzhak & Khouri, 1988). As Gpp(NH)p reduces the slower dissociative component of sigma binding, it has been proposed that, in their high affinity state, at least a subpopulation of sigma receptors are coupled to G proteins (Itzhak, 1989). In addition, a pret reatment with pertussis toxin (PTX), which inactivates Gi/, proteins by ADP-ribosylation, reduces the high affinity binding component and prevents the effects of Gpp(NH)p on ['W(+)3-PPP binding (Itzhak, 1989), suggesting the coupling of sigma receptors to G, proteins (Gilman, 1987; Fredholm & Lindgren, 1988; Heming & Allgaier, 1988).

Sigma receptors have been separated, to date, into two classes, denoted a, and 0, (Quirion et al., 1992). The sigma ligands haloperidol, 1,3di(2-toly1)guanidine (DTG) and (+)3 PPP, do not discriminate between o, and oz sites, whereas the (+)benzomorphans (+)SIG-10,047 and (+)pentazocine as well as JO-1784, bind preferentially to a, sites with a nanomolar affinity (Quirion et al., 1992). The god of the present study was thus to assess, in vivo, whether the effect of sigma ligands on the NMDA response involved a Gd, type of prorein. To this end, using in vivo extracellular unitary recording of CA, dorsai hippocampus pyramidal neurones we compared, in control rats and in rats pretreated with PTX, the capacity of high affinity sigma ligands @TG, JO-1784, (+)pentazocine) and of NPY, to potentiate NMDA-induced neuronal activation in the CA, dorsal hippocampus.

Pertussk toxin pretreatment Male Sprague-Dawley rats (175-200 g) were obtained from Charles Rivers (Saint- Constant, Québec, Canada) and housed four per cage. They were kept on a 12:12 h lighddark cycle with free access to water and Purina chow. Following anesthesia with chlord hydrate (400 mg/kg, i.p.), the rats were mounted in a stereotaxic apparatus. The pretreatment with PTX (1 pg in t pl of physiological saline, Sigma Chernical Co., St-Louis, MO, USA) consisred in lowering unilaterally the tip of a 5-~1Hamilton syringe into the dorsal hippocampus at A: 4.5, L: 4 and D: 4, according to the atlas of Paxinos & Watson (1986). Pertussis toxin was slowly injected over a penod of 5 min. Control rats received an equal volume of physiological saline solution. In vivo electrophysiological experiments were carried out 3 to 11 days later. This interval was chosen because it was observed, in the sarne in vivo electrophysiological experiments, that the efficacy of rnicroiontophoretic applications of serotonin in suppressing the firing activity of CA3 hippocampal pyramidal neurones, an effect which is G;/, protein- dependent (Andrade et al., 1986), was drastically reduced during this tirne ~eriod(Blier et al., 1992).

Recordings from CA, dorsal hippocampus pyramidal neurones Recordings were obtained as previously descnbed (Monnet et al., 1992d). In brief, rats were anesthetized with urerhane (1.25 g/kg, i.p.), and mounted in a stereotaxic apparatus. Body temperature was maintained at 370 C chroughout the experiment. Five-barrelled glas micropipettes were used for extracellular unitary recordings of the activity of CA, dorsai hippocampus pyramidal neurones. One side barrel, filled with 2 mM NaCl, was used for 138 automatic current balancing. The other side barrels, used for microiontophoresis, were filled with NMDA (10 mM in 200 mM NaCl, pH: 8), QUIS (1.5 mM in 400 mM NaCl, pH: 8), acetylcholine (ACh) (1.5 mM in 200 mM NaCl, pH: 8) and one of the following solutions: DTG (1 rnM in 200 rnM NaCl, pH: 4), JO4784 (1 rnM in 200 rnM NaCl, pH: 4), (+)pentazocine (0.5 rnM in 200 mM NaCl, pH: 4), NPY (0.1 mM in 150 mM NaCl and bovine semm albumin 0.l0/o, pH: 4), or haloperidol (0.2 mM in 50 mM NaCl, pH: 3.5). The micropipette was lowered into the CA, region of the dorsal hippocampus (L: 4.2 mm and A: 4.2 mm, at a depth of 3.5 to 4.5 mm from the cortical surface; Paxinos & Watson, 1986). Action potentials, rnonitored on an oscilloscope, triggered square pulses fed into a computer. The duration of the microiontophoretic applications and the intensities of the currents used were also stored in the computer, permitring the calculation of the total number of spikes generated/nC. For a given neuron, the currents of NMDA and QUIS were adjusted to obtain a firing frequency in between 7 and 15 Hz and were thereafter maintained constant for the rerninder of the experiment. Al1 applications of NMDA and QUIS were of 50 s, while those of the sigma ligands and NPY were of 15 to 20 min.

In a first series of experiments, since NPY does not cross the blood brain barrier and could only be applied by microiontophoresis, the sigma ligands DTG, JO-1784, (+)pentazocine and haloperidol were also applied by microiontophoresis in control and PTX-treated animals. The effeas of the rnicroiontophoreric applications of the sigma ligands were assessed by determining the number of spikes generatedhc of NMDA and QUIS before and dunng the microiontop horetic application of the substance studied. In a second series, DTG, (+)pentazocine and haloperidol were administered intravenously. The effeas of these sigma ligands were assessed by determining the number of spikes generatedinc of NMDA and QUIS before and after the injection. Only one neuron was tested in each rat which received only one dose of DTG or (+)pentazocine.

Dmgs The following substances were used: NMDA and PTX (Sigma Chernical, St. Louis, MO, U.S.A.), QUIS (Tocris Neuramin, Buckhurst Hill, Essex, U.K.),DTG (Aldrich, Milwaukee, WI), hdoperidol (McNeil Laboratories, Stoufhrille, Ont, Canda). JO-1784 [(+) N- cyclopropylmethyl-N-rneth~l-i,4-dip henyl-I-erhyl-but-3-en-i-ylamine,hydrochloride] was kindly provided by Dr. J.L. Junien (Institut de Recherche Jouveinal, Fresnes, France), (+) pentazocine by Dr. B.C. deCosta (N.I.H., Bethesda, MD, U.S.A.) and NPY was a generous gift from Dr. A. Fournier (Institut National de la Recherche Scientifique-Santé, Pointe-Claire, Qc, Canada).

Statzsticai analyszs Al1 results are expressed as the mean t SEM of the number of spikes generatedhc of NMDA or QUIS, n being the number of neurones tested. Statisrical significance was assessed using the paired Student's t-test with the Dunnett's correction for multiple cornparisons. Probability dues smaller than 0.05 were considered as significant.

RESULTS Microiontophoretic applications of NMDA and QUIS produced consistent activation of al1 CA, pyramidal neurones recorded. Pertussis toxin, which inactivates Gi/, proteins by ADP-ribosylation, was used to document the ~ossibleinvolvement of these proteins in the modulation of NMDA-induced neuronal activation in the CA, region of the rat donal hippocarnpus by high affinicy sigma ligands and NPY. The in vivo PTX prerreatment affected neither the spontaneous firing activity nor NMDA- or QUIS-indiiced neuronal activity. None of the sigma ligands tested nor NPY had any effect on the sponraneous activity of pyramidal neurones in the CA, region, consistent with previous observations (Brooks et ai., 1987; Lodge et al., 1988; Monnet et ai., 1992b; 1992c; 1992d).

Effects of rn icroion tophoretzc applications of JO-2784, NP Y, D TG and (+)pztazocine. In a first series of experiments, the high affinity sigma ligands JO-1784 (Roman et ai., 1990b), (+)pentazocine (Su, 1982), DTG (Weber et ai., 1986), as well as NPY (Roman et a., 1989) were applied rnicroiontophoretically for successive penods of IO to 20 min, with 5, 10 and 20 nA ejecting currents, in control and PTX-treated rats. In naive rats, 50-1784 (from 10 and 20 nA) produced a selective and currentdependent enhancement of the NMDA response, 20 nA induciq a three-fold increase of NMDA-induced firing activity (figures 1A, 2A). In PTX-treated rats, JO-1784, even at a currem of 20 nA, failed to induce any significant potentiation of the NMDA response (figures lB, 2A). As in cornroi rats, 50-1784 did not modify either the neuronal response ro QUIS. Similarly, when applied rnicroiontophoretically with a current of 10 nA,NPY produced in control rats a two-fold enhancement of the NMDA response (figure 2B), but had no effect on the QUIS response. In PTX-treated rats, the potentiating effect of NPY on the NMDA response was abolished (figure 2B). In control animals, microiontophoretic applications of DTG produced a greater than two-fold increase in the neuronal activation induced by rnicroiontophoretic applications of NMDA (figure 2C). Afcer a pretreatment with PTX however, DTG induced a slight but significant reduction of the NMDA-induced firing activity (figure ZC). The QUIS response was not modified by the rnicroiontophoretic application of DTG in both control and PTX- pretreated rats (Figure 2C). The microiontophoretic application of (+)pentazocine produced, in naive rats, a 150°h increase of NMDA-induced neuronal activation (figure 2D) without affecting the response to QUIS. In PTX-treated rats, (+)pentazocine, contrarily to the ocher sigma ligands, still produced a potentiation of the NMDA response (figure 2D) without modifying that of QUIS. Similarly to what was obtained in previous studies, in control animals, the microiontophoretic application, as well the int ravenous administration of a low dose (10 ~rg/kg)of haloperidol concurrently with the application of DTG, (+)pentazocine,10-1784 or NPY, suppressed the potentiation of the NMDA response induced by these sigma ligands (data not shown). In PTX-treated rats, the potentiation induced by the microiontophoretic application of (+)pentazocine was also su~pressedby haloperidol (IO &kg, i.v. figure 2D). However, the DTG-induced attenuation of the NMDA response obtained in PTX-t reated rats was not reversed by haloperidol (figure ZC).

Effects of the intrmenotrs administrations of DTG and (+&ntazocine. In a second series of experiments, the two sigma ligands DTG and (+)pentazocine, which were found to modulate the NMDA response when applied by rnicroiontophoresis in PTX-treated rats, were administered intravenously. As illustrated in figures 3A and 4A, DTG, at a dose of 1 pg/kg, and (+)pentazocine, at a dose of 5 pg/kg, selectively enhanced by three- fold the firing activity of CA, pyramidal neurones to microiontophoretic applications of NMDA in control rats (figure 5), consistent with previous reporcs (Monnet et ai., 1990; 1992b). When injected in PTX-treated rats, DTG (1 pg/kg, i.v.) neither enhanced nor reduced the NMDA-induced neuronal activation (figures 3B, 5B). However, in PTX-treated rats, (+)pentazocine (5 pg/kg, i.v.) produced a marked and selective potentiation of the NMDA 141 response, sirnilar to that obtained in the control animas (figures 4, 5A). The subsequent intravenous administration of a low dose of halopend01 (IO pg/kg) completely abolished the (+)pentazocine-induced potentiation of the NMDA-induced firing activity of CA, pyramidal neurones in PTX-treated rats (figures 4B,5A). Neither the intravenous administration of DTG nor (+)pentazocine did modify the neuronal activation induced by microionotophoretic applications of QUIS or ACh in control and PTX-treated rats (Figures 3-5).

DISCUSSION The present results obtained in vivo, in the CA3 region of the rat dorsal hippocampus, show that the inactivation of Gi/, proteins by PTX abolished the potentiating effects of DTG, 50-1784 and NPY on the NMDA response, but not that of (+)pentazocine. In addition, in PTX-treated rats, haloperidol reversed the potentiating effect of (+)pentazocine on the NMDA res ponse. The unaltered effects of NMDA and QUIS on the firing activity of CA, pyramidal neurones, following G;,, protein inactivation by a PTX pretreatment, are consistent with previous observations in rat striatal neurones (Sladeczek et al., 1985), cerebellar granule cells (Nicoletti et aL, 1986), hippocampal slices (Beaudry et al., 1986) and forebrain synaptosomes (Recaens et al., 1987) suggesting that the excitatory effects of these aminoacids are not mediated by G proteins. The observation that microiontophoretic applications of DTG, (+)pentazocine, JO-1784 and NPY, as well as the intravenous administration of low doses of DTG and (+)pentazocine, potentiated NMDA-induced neurond activation of CA, dorsal hippocampus in control animds is consistent with previous in vivo studies (Monnet et al., 1990; 1992b; Martin et al., 1992) and with the data obtained by Iyengar et al. (1990b; 1991), who demonstrated that several sigma ligands also potentiated NMDAdependent adrenocorticotropic hormone and prolactin release in pituitary cells as well as dopamine turnover in the striatum and olfactory tubercles. The lack of effect of the PTX-treatment on the (+) pentazocine-induced potentiation of the NMDA response (figures 2, 4 and 5) suggests that this sigma ligand activates a subtype of sigma receptor not coupled to Gu, proteins. HaIoperidol is known to bind with high affinity to doparninergic, a,-adrenergic, serotoninergic, muscarinic and sigma binding sites (Bus et al., 1977; Peroutka et al., 1977; Su, 1982). However, the only binding sites that haloperidol, DTG, JO-1784 and (+)pentazocine share are the sigma sites (Su, 1982; Weber et al., 1986; Roman et al., 1990b). Therefore, the suppression by haloperidol of the potentiating effect of (+)pemazocine on the NMDA response in the present series of experiments constitutes a conducive evidence that (+)pentazocine potentiates the NMDA response by activating sigma receptors. These results are in apparent discrepancy with the reporc of Itzhak (1989) that the binding of racernic pentazocine to sigma sites labelled with ['H]3-PPP was altered by GTP and Gpp(NH)p suggesring that these sites were coupled to G proteins. Furthemore, (+)pentazocine is considered to bind selectively to al receptors which have been suggested to be associated with a Gu, protein (Quirion et al, 1992). However, DeHaven-Hudkins et al. (1992) have reported that the binding of [3HJ(+)pentazocine was insensitive to GTP and Gppop,suggesting that ['HJ(+)pentazocine binds to sigma receptors not coupled to Guo proteins. The apparently discrepant data of these two binding studies could be explained by the different forms of pentazocine and/or species used (guinea pigs vs rats). The present results, in keeping with the observations of De Haven-Hudkins et al. (1992), suggest that the potentiating effect of (+)~entazocineon the NMDA response is mediated by a subtype of a, receptor not coupled with a G, protein and sensitive to haloperidol. The high &nity sigma ligand 50-1784, as well as WY, have previously been shown to enhance in vivo as well as in vitro the NMDA response (Riviere et aL, 1990; Roman et a!., 1991b; Monnet et al., 1992a; 1992b; 1992d), the effect of NPY being mediated via a non-Y,, non-Y,, non-Y, receptor, probably corresponding to a subtype of sigma receptor (Monnet et al., 1992c; 1992d). The similariry of some of the effects of 50-1784 and hPY has been reported in other models (Riviere et aL, 1990; 1993; Roman et al.; 1991a; 1991b; Gué et al., 1992a; 1992b; Pascaud et ai., 1993). Thus, the prevention by a PTX pretreatment of the potentiating effect of 50-1784 and NPY (figures 1 and 2) is fully consistent with previous observations suggesting that they exerr their effeas on the NMDA response via a subtype of sigma receptor coupled to Gi/, proteins Ounien et al., 1991; Monnet et ai., 1992a). Following the inactivation of Gi/, proteins by PTX, the intravenous administration of DTG did not modiG the neuronal response to NMDA and QUIS, whereas the rnicroiontophoretic application of DTG ~roduceda slight but significant reduction of the excitatory effect of NMDA, but not of QUIS, (figures 2, 3 and 5). Presently, we do nor have a definite explanation for the differential effects of DTG when applied by microiontophoresis and intravenously. The suppression of the potentiating effect of DTG following PTX is consistent with the results obtained with 50-1784 and NPY. However, in addition, PTX pretreatment unveiled a second component (Gi/, protein-insensitive) of the effect of DTG on the NMDA response, consisting in reduction of the NMDA response. Some reports have descnbed an inhibitory effea of high doses of sigma ligands on NMDA-induced effects, aibeit to a weaker degree than phencyclidine (PCP)-like dmgs (Anis et aL, 1983; Lodge & Anis, 1984; Mdouf et aL, 1988). This inhibitory effect of sigma ligands has been ascnbed, however, to their low affinity for the PCP binding site (Lodge et al, 1988; Mdouf et al., 1988; Church & Lodge, 1990; Monnet et al., 1992b). Two groups have reported an inhibitory effect of DTG at low doses. First, Roth et al. (1993) have observed in the rat prepiriform cortex that concentrations of DTG as low as 1 nM produced an anticonvulsant effect via a reduction of the NMDA response (Roth et af., 1993). Second, Connick et al. (1992a) reported a reduction of the NMDA response by low doses of DTG in the CA, region of the rat dorsal hippocampus. One intriguing finding here is that the suppressant effect of DTG was not reversed by haIopend01 (figure 2c). In this and in previous experirnents hdoperidol consistently suppressed the potentiating effects of sigma ligands. The reversal of the effects of (+)pentazocine, DTG and 50-1784 by haloperidol has also been observed in the peripheraI nervous system (Massamiri & Duckles, 1990), in behaviourd models (Tarn et al., 1988; Steinfels et al., 1989) as well as in the central nervous system both in vivo (Tarn et al., 1988; Monnet et al., 1990; 1992b) and in vitro (Roman et al., 1989; Connor et al., 1992). Hence, since DTG and haloperidol both bind to both o, and a2 receptors (Quirion et al., 1992), it is possible that the suppressanr effect of DTG on the NMDA response in PTX-treated rats rnight not be ascribed to sigma receptors. However, some repons have provided evidence for the existence of more than two subtypes of sigma receptors. Zhou and Musacchio (1991) have suggested the existence of at lem four subtypes of sigma receptors, one of them, R4,having a moderately low affinity for DTG and very low affinity for haloperidol. Connick et al. 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MONNET, F.P., BLIER, P., DEBONNEL, G. & DE MONTIGhY, C. (1992a). Modulation by sigma ligands of N-methyl-D-aspmate- induced [WJnoradrenaline release in the nt hippocampus: Gprotein dependency. Naunyn-Schmredeberg 's A rch. Pharmacol., 346, 32-39.

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ZHOU, G.Z. & MUSACCHIO, J.M. (1991). Cornputer-assisted modelling of multiple dextromethorphan and sigma binding sites in guinea pig brain. Ew. /. Phamacol.: Mol. Phamacol., 206, 261-269. NMDA

QUIS NMDA -2 -17

2 min

Figure 1 Integrated firing rate histograms of a CA, donai hippocampus pyramidal neuron showing the effecu of microiontophoretic appiications of NMDA and QUIS before and during the microiontophoretic application of JO-1784 in a conrrol rat (A) and in a rat pretreated with PTX (8). Bars indicate the duration of applications for which currents are given in nA and docs correspond to 10-15 min interruption, of the trace, in this and subsequent figures. Time base applies to both traces. CONTROL PERTUSSlS TOXlN CONTROL PERTUSSIS TOXlN

atamo

Currents (nA) of JO-1784 Currents (nA) of NPY

CONTROL PERTUSSIS TOXIN D CONTROL PERTUSSIS TOXlN *

IO nt" HAL 10 pglkg.1.v Currents (nA) of DTG Currents (nA) of (+) PENTAZOCINE

Figure 2 Responsiveness expressed as the number of spikes generated per nanoCoulomb (nC; mean t S.E.M.) of CA, dorsal hippocampus neurons to microiontophoretic app1ica:ions of NMDA before (open column), during (hatched columns) microiontophoretic applications of JO-1784 (A), NPY (B),DTG (C) and (+)pentnzocine (D), and following the microiontophoretic application or the intravenous administration (black column) of haioperidoi in control and PTX-treated rats. The number at the bottom of the fint column of each histognm in this and in subsequent figures indicates the nurnber of neurons tested. In al1 series of experiments, the same neurons were recorded from during the complete sequence. Al1 applications of NMDA and QUIS were of 50 S. * P c 0.01. ACh NMDA QU l S 12 -1 1 - 6 n n ru

DTG HALOPERlDOL 1 pglkg, i.v. 10 pg/kg. i.v.

ACh NMDA 8 -14 ami-

- 1 min DTG HALOPERIDOL 1 pglkg, i.v. 10 pglkg, i.v.

Figure 3 Integrated firing rate histogram showing the response of a CA, dorsal hippocampus pyramidal neuron to microiontophoretic applications of ACh, NMDA and QUIS before and following the intravenous administration of DTG and following the intravenous injection of haloperidol, in a control (A) and a PTX-treated nt @)- NMDA -25 nm

(+) PENTAZOCINE 5 pglkg, i-v.

NMOA -20 O=

t t 1 min (+) PENTAZOCINE HALOPERIDOL 5 pg/kg, i.v. 10 pglkg. i-v.

Figure 4 Integrated fixing rate histognm showing the response of a CA, donai hippocvnpus pyramidal neuron to microiontophoretic applications of NMDA and QUIS before and following the intravenous administration of (+)pentazocine and following the intravenous injection of haloperidol, in a control (A) and a PTX-treated rat (B). CONTROL PERTUSSIS TOXIN

- - Figure 5 Responsiveness expressed as the number of spikes genented per nanocoulomb (mean I S.E.M.) of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (open columm) and after (hatched columns) the intravenous administration of (+)pentazocine (A) and DTG (B) ((dark coloumn). Since (+)pentazocine still induced a potentiation of NMDA response in PTX-treated rats, a subsequent injection of haloperidol was administered. 153 CHAPTER VI Effects of low and high doses of sigma ligands: Further evidence suggesting the existence of different subrypes of sigma receptors

By this stage of my Ph.D. research the concept of "biphasic effea" of o ligands was well documented. A low dose of some selective ligands potentiate the NMDA response, and the degree of potentiation progressively decreases with higher doses until it disappean at doses higher than 500 ,ug/kg i.v.

The first aim of my fifth study was to determine the effect of high doses of a ligands that behave as agonists at low doses. Our hypothesis was that if the biphasic effect of a ligands could be explained through the sequentid activation of different subtypes of a receptors, then high doses of specific a ligands would present antagonistic effects. A second goal was to determine if the subsequent administration of two o agonists would produce an additive effect. Our hypothesis was that if the potentiation induced by one cr agonist is obrained via the activation of a specific subrype of a receptor, and if another o agonist activates the sarne subtype of cr receptor, then the subsequent administration of these two o agonists would not produce an additive effect . 154 EFFECTS OF LOW AND HIGH DOSES OF SELECTIVE SIGMA LIGANDS: FURTHER EVIDENCES SUGGESTING THE EXISTENCE OF DIFFERENT SUBIYPES OF SIGMA RECEPTORS

Richard Bergeron and Guy Debonnel

Psychopharmacologyy in press.

SUMMARY Several high affinity sigma (0) ligands, such as DTG, JO-1784, (+)-pentazocine, BD-737 and L-687,384, administered at low doses act as agonists by potentiating N-methyl-D-aspartate (NMDA)-induced activation of pyramidal neurons in the CA3 region of the rat dorsal hippocampus. This potentiation is dosedependent at doses between 1 and 1000 &kg, i.v. but bell-shaped dose-response curves are obtained. Other o ligands Iike haloperidol, BMY-14802, (+)3-PPP and NE-100 administered at low doses aa as a antagonists since they do not modify the NMDA response but suppress the potentiation of the NMDA response induced by o agonists. Because high doses of the o agonists do not potentiate the NMDA response, the present experiments were undertaken to assess if at high doses, these o ligands could also act as a amagonists and suppress the potentiation induced by low doses of a agonists. High doses of DTG, 50-1784, BD-737, and L-687,384 administered acutely, had an effect sirnilar to that of low doses of haloperidol, by suppressing and preventing the potentiation induced by low doses of DTG, JO-1784, BD-737, L-687,384 and (+)-pentazocine. High doses of (+)-pentazocine suppressed the effect of a low dose of (+)-pentazocine but did not affect the potentiation induced by a low dose of the other o agonists. The potentiation induced by a low dose of a a, agonist was not furcher increased by the subsequenr administration of another low dose of a o, agonist. Al1 together, these results strongly suggest that more than two subrypes of o receptors exist in the CNS.

Key Words: N-Merhyl-D-Asparcare (NMDA), DTG, pentazocine, 50-1784, L-687,384, electrophysiology, hippocampus INTRODUCTION The existence of sigma (a) receptors was originally proposed by Martin and coworkers to account for the psychotomimetic actions of N-allyl-normetazocine (Martin et al. 1976). We have previously proposed that one of the functions of the a receptors might be to modulate the NMDA response. We have already shown (Monnet et al. 1990; Monnet et al. 1992; Bergeron et al. 1993; Bergeron et al. 1995), using in vivo extracellular unitary recordings, a marked potentiation of NMDA-induced activation of CA3 dorsal hippocampus pyramidal neurons by low doses of severai 0 ligands. These ligands included DTG [di@-tolyl)guanidine] (Weber et al. l986), JO-1784 [(+)N-cyclopropylmethyl-N-methyl-i,+diphenyl-1-ethyl-but-3-en- 1-ylamine hydrochloride] (Roman et al. 1990), (+)pentazocine (Steinfels et al. 1988), BD-737 [( + )lis-N-meth y l-N{2-(3,4dichlorop hen y 1) et hyll2-(i-p y rrolidiny l cyclohexy lamine] (Contrem et al. 199 1b) and L-687,384 [I benzylspiro(l,2,3,4-tetrahydronaphthalene-1+piperidine)] (Middlemiss et al. 1991). This potentiation is selective since no signifiant effects are observed on the neuronal responses induced by quisqualate (QUIS), acetylcholine (ACh) and kainate (Monnet et al. 1990; Monnet et al. 1992; Bergeron et al. 1993; Bergeron et al. 1995). Administered at doses in between 0.1 to 500 pg/kg, i.v., these a ligands potentiate, with beil-shaped dose-response curves, the exciratory effeas of microiontophoretic applications of NMDA (Bergeron et al. 1993; Bergeron et al. 1995). When administered at doses higher than 200 pg/kg, i.v., the degree of the potentiation induced by these o ligands progressively decreases to finally disappear at doses higher than 500 pg/kg, i.v. (Bergeron et ai. 1995). The potentiation of the NMDA response induced by the low doses of these rr ligands is reversed by low doses of haloperidol, BMY-14802 [a-(4-fluorophenyl)-4-(5-fluoro-2- pyrirnidiny1)-1-piperazine butanol], (+ )-3-PPP [3 (3-hydroxyp heny1)-N-(1-propyl)piperidine] and of NE-100 (N,N-dipropyl-2-(4-met hoxy-3(-pheny1ethoxy)p heny1)-ethylarnine rnon~h~drochloride).These al1 share a high affinity for a receptors (Taylor and Dekleva, 1987; Taylor et al. 1993; Okuyama et al. 1993) which suggests that this potentiation is mediated via the a receptors (Monnet et al. 1990; Monnet et al. 1992; Bergeron et al. 1993; Bergeron et al. 1995). Since haloperidol, BMY-14802 and (+)3-PPP have no effect by themselves on the NMDA response but suppress the effeas of the o agonists rhey were considered as putative antagonists, whereas the a ligands inducing a potentiation of the NMDA response at low doses were considered as putative agonists (for review, see Debonnel and De Montigny, 1996). It is now accepted chat a receptors exist in at lest two subtypes denoted a, and 0, (Quinon et al. 1992). The fact that a potentiation of the NMDA response was obtained with none selective ol/q ligands (DTG) as well as with selective o, ligands (JO-1784, (+)pentazocine, L-687,384 and BD-737) suggests that this effect is probably mediated via the activation of o, receprors. Since high doses of the o agonists DTG (1000 pg/kg, i.v.), JO-1784 (1000 pg/kg, i.v.), (+)pentazocine (1000 pg/kg, i.v.), BD-737 (1000 pg/kg, i.v.) and L-687,384 (1000 ~g/kg,i.v.) do not produce any potentiation on the NMDA response (Bergeron et al. 1995), the present senes of experiments were undertaken to assess if, when administered ar these high doses, these o ligands may have antagonistic properties. A second series of experiments were underraken to assess the additive effects of two low doses of two different o agonists on the NMDA response.

MATERIAL AND METHODS Prepuration of dmgs The following dmgs were used: NMDA and ACh (Sigma Chemical Co., St-Louis, MO, USA); QUIS (Tocris Neuramin, Buckburst Hill, Essex, UK); DTG (Aldrich Chemicals, Milwaukee, WI, USA); JO-1784, a generous gift from Dr. J.L. Junien (Institut de Recherche Jouveinal, Fresnes, France); (+)-pentazocine (Research Biochemical International, Natick, MA, USA); BD-737, a generous gift from Dr. W.D. Bowen (Laboratory of Medicinal Chernistry, NIDDK, NIH, USA); L-687,384, a gift from Dr. L. Iverson (Merck-Sharpe and Dome, UK); and haloperidol (McNeil Laboratories, Stoufhille, Ontario, Canada). Ail dmgs were dissolved in saline.

Preparatzon of micropipettes Microiontop horetic applications and extracellular unitary recordings were performed with five-barrelled glass micropipettes for most of the experiments and with seven-barrelled glass micropipettes when needed. They were ~ulledin a conventional manner (Haigler and Aghajanian, 1974) and their tips broken back to 8 to 12 Pm under microscopie control. Three or five side barrels were used for microiontophoresis and filled with NMDA (10 mM in 200 mM NaCI, pH: 8), QUIS (1.5 mM in 400 rnM NaCl, pH: 8) and ACh (20 rnM in 200 mM NaCl, pH: 5) or filled wirh o ligands DTG, 50-1784, BD-737, L-687,384 (10 mM in 200 mM NaCl, pH: 8) or (+)pentazocine (2 mM in 150 mM NaCl, pH: 6). The impedance of these three or five barrels was typically between 30 and 50 Ma. The fourth side barrel, containing a 2 mM NaCl solution with an impedance between 20 and 40 Mn, was used for automatic current balancing. The central barrel of the glas micropipette, filled with 2 rnM NaCl solution saturated with Fast Green FCF, was used for extracellular unitary recording of the firing activity of CA, dorsal hippocampus pyrarnidai neurons. The impedance of the central barrel was typically between 2 and 5 Mn.

Prtparation of unimals and recordings from CA, dorsal hippocampus pyramidal neurons Adult male Sprague-Dawley rats, weighing 200 to 250 g, were housed three to four per cage three or four days pior to the experiments. They were kept on a UA2 h lighddark cycle. For the electrop hysiological experiments, animals were anestherized wit h urethane (1.25 g/kg i.p.) and mounted in a stereotaxic apparatus. Body temperature was maintained at 37°C throughour the experiment and a catheter was installed in a laterai tail vein. Al1 dmgs were prepared in physiological saline prior to their administration. A burr hole arasdded in the skull at 4 mm anterior to lambda and 4 mm lateral to midline. After careful removal of the dura mater, the micropipette was lowered into the CA3 region of the dorsal hippocampus (L +4.2 mm and A, +4.2 mm us. lambda: H, -3.5 to -4.5 mm) from the cortical surface. This region was chosen because of the high density of o (Graybiel et ai. 1989) and NMDA receptors (Foster and Fagg, 1984). Pyramidal neurons were identified according to their long duration (0.8-1.5 msec) and large amplitude (0.5-2 mV) action potenrials, and by the presence of characteristic complex spike discharges alternating with simple spike activity (Kandel and Spencer, 1961). Neuronal firing activity was rnonitored on an oscilloscope afier signal magnification by a high input- impedance amplifier. Action potentials were detected by a differential amplitude discriminator generating square pulses fed in a computer from which integated firing rate histograms were generated and displa~edon a paper chan recorder. The duration of the microiontophoretic application and the intensities of the currents used were also stored in the computer. The duration of the micr~ionto~horeticejections of the three excitatory substances NMDA, QUIS and ACh was kept constant at 50 sec. When a neuron was isolated it was kept for a period of at lean 30 min to establish a stable baseline. About 15 to 20 applications of each excitatory substances were carried out before the o compounds were administered. The recording was without interruption. The effeas of the cr ligands ocmrred within 5-10 min following their administration with the maximal effect being obtained within 20-30 min. The effect of the application of each of the substances studied on pyramidal neuron firing activity was expressed as the number of spikes generated per nanocoulomb (1 nC being the charge generated by 1 nA applied for 1 sec). At the end of each experiment, a -27 pA currem, through the central barrel, was applied for 20 minutes to permit a Fast Green deposit for subsequent histological verification of the last recording site.

Experirnental series The following series of expriments were performed:

1- Effect of a high dose of DTG (1000 pg/kg, i.v.) on the effect s of low dos JO-1784, (+)~entazocine,L-687,384 and BD-737; 2- Effect of high a dose of 50-1784 (1000 &kg, i.v.) on the effeas of low doses of DTG, (+)pentazocine, L-687,384 and BD-737; 3- Effect of a high dose of L-687,384 (1000 pg/kg, i.v.) on the effects of low doses of DTG, 50-1784, (+)pentazocine, and BD-737; 4- Effect of high dose of BD-737 (1000 pg/kg, i.v.) on the effeas of low doses of DTG, 50-1784, (+)pentazocine, and L-687,384; 5- Effect of a high dose of (+)pentazocine (1000 pg/kg, i.v.) on the effects of low doses of DTG, 50-1784, BD-737 and L-687,384; 6- Additiveeffectsoflowdosesoftwooagonists.

Calculations Each value was calculated by the computer as the mean of the effect of three consecutive applications of the same excitatory substance. The effects of the a ligands were assessed by determining the number of spikes generated per nC of NMDA, QUIS and ACh before and after their intravenous administration, or before and during the microiontophoretic applications when their m&md effect was observed.

Statisticai analysis Al1 results are expressed as means + S.E.M. of the number of spikes generated per nC of NMDA, QUIS or ACh, n being the number of neurons tested. Statistical significance was assessed using the Student's t-test with the Dunnett's correction for multiple comparisons. Probability values smdler than .OS were considered as significant. Each series of experiments was carried out in 5 to 10 rats. RESULTS Efects of a high dose of DTG As previously reported and as illustrated in figure LA, the intravenous administration of DTG (1000 pg/kg) did not modify the neuronal response to microiontophoretic applications of NMDA. The potential antagonistic effect with this dose of DTG was assessed by comparing the degree of the potentiation of the NMDA response induced by low doses of five different selective o ligands (DTG: 1 pg/kg, i.v.; JO-1784: 4 pg/kg, i.v.; (+)pentazocine: 10 pg/kg, i.v.; BD-737: 10 pg/kg, i.v. or L-687,384: 1 pg/kg, i.v.) before and after the intravenous administration of the high dose of DTG. When administered at a dose of 1000 /~g/kg,i.v.,

DTG completely suppressed (n = 5) the potentiating effects of dl selective a agonists tested. Moreover, when this dose of DTG was adrninistered pnor to the injection of the low dose of the cr agonists, it completely prevented the potentiating effect of these compounds (Fig. 1 B, C, D, E). The intravenous administration of the same high dose of DTG (1000 pg/kg), also reversed the potentiation of the NMDA response induced by microioncophoretic applications of DTG, JO-1784 or (+)pentazocine with a current of 20 nA (Table 1).

Effects of a high dose of /O-1 784 and BD-737 The effects of a high dose of JO-1784 and BD-737 (1000 &kg, ix), which did not modify by themselves the NMDA response, were dso assessed prior to or following the intravenous administration of a low dose of DTG, 50-1784, (+)pentazocine, BD-737 or L-687,384. Sirnilar to the results obtained with the high dose of DTG, high doses of JO-1784 and BD-737 prevented and reversed the potentiating effects of the low doses of al1 the a agonists tested (Fig. 2). The intravenous administration of 1000 pg/kg of JO-1784 and BD-737 also reversed the potentiation of the NMDA response induced by microiontophoretic applications of DTG, 10-1784 and (+)pentazocine with a current of 20 nA (Table 2).

Effets5 of a high dose of L-687,384 We have previously reported that L-687,384 appears to be the most potent o ligand tested thus far in our laboratory, since a potentiation of the NMDA response could be obtained with doses as low as 0.1 &kg (Bergeron et al. 1995). The maximal potentiation was observed at 1 pg/kg and no potentiation was obtained at doses higher than 50 pg/kg. Thus, for this ligand, a dose of 100 pg/kg, i.v. was considered as a high dose. At that dose, L-687,384 prevented and revened the potentiating effects of DTG but had no effect on the potentiations of the NMDA response induced by 50-1784, (+)pentazocine and BD-737 (Fig. 3). The effect of IOOO pg/kg, i.v. of L-687,384 was also assessed. At that dose, L-687,384 revened the potentiating effects of low doses of JO-1784, (+)pentazocine and BD-737 (Fig. 4).

Effects of a hzgb dose of (+)pentazocine Finally, the effects of a high dose of (+)~entazocine(1000 Cig/kg, i.v.) were assessed. At that dose, (+)~entazocinedid not modify NMDA-induced activation, neither did it prevent nor reverse the ~otentiationinduced by low doses of DTG, JO-1784 and L-687,384 (Fig. 5). However, this high dose of (+)pentazocine revened the potentiation induced by a prior administration of a low dose of (+)pentazocine. While the high dose of (+)pentazocine did not affect the potentiation of the NMDA response induced by microiontophoretic applications of DTG (20 nA) or 50-1784 (20 nA), it did reverse the potentiation of the NMDA response induced by microiontophoretic applications of (+)pentazocine 20 nA (Table 3).

Additive effeects of Zow doses of a ligands Three series of experiments were undertaken to assess the effeas of two subsequent injections of a low dose of two a agonists. In the first series of experiments, we assessed the effects of the subsequent intravenous administration of a low dose of 50-1784 (1 pg/kg, i.v.) followed by that of a low dose of (+)- pentazocine (2.5 pg/kg, i.v.). Neither JO-1784 nor (+)pentazocine, when administered alone, rnodified the NMD A response. However, t heir sequential administration induced a significant potentiation of the NMDA response. The injection of JO-1784 (2 pg/kg, i.v.) induced a significant potentiation of the NMDA response. The subsequent administration of (+)- pentazocine (5 pg/kg, i.v.) induced a three-fold increase of the NMDA response (Fig. 6) similar to that obtained from an single dose of 50-1784 (4 pg/kg, i.v.) or by a single dose of (+)- pentazocine (10 pg/kg, i.v.; figs. 3,4,7). In the second series, an injection of 1 pg/kg, i.v. of L-687,384, which represents the dose inducing the maximal potentiation of the NMDA response, was administered following that of 1 kg, i.v. of DTG. Following the second injection, an epileptoid activity was obtained upon the microiontophoretic application of NMDA. This epileptoid activity prevented the calculations of the number of spikes generated. The administration of 1 pg/kg, i.v. of DTG following that of 1 &kg, i.v. of L-687,384 produced the same epileptoid phenomenon. This epileproid activity upon applications of NMDA was also observed when a low dose of (+)pentazocine (5 pg/kg, i.v.) or 50-1784 (4 pg/kg, i-v.) was administered following that of 1 pg/kg, i.v. of DTG. In the third series, an injection of 4 pg/kg, i.v. of JO-1784 (Fig. 7A) or 5 pg/kg, i.v. of (+)pentazocine (Fig. 7B) did not modib the potentiation of the NMDA response induced by a prior administration of a low dose of 1 pg/kg, i.v. of L-687,384. Moreover, 4 pg/kg, i.v. of JO-1784 induced a two-fold potentiation of the NMDA response which was not modified by the subsequent injection of 5 pg/kg, i.v. of (+)pentazocine (Fig. 7C). In each experimental series, the intravenous administration of IO pg/kg of hdoperidol reversed the potentiation of the NMDA response obtained with the intravenous administration of the o agonist.

Effects of iow and hzgb mmts of o ligands To rule-out the possibility of a desensitization of the a receptors by the high doses of the o ligands, a last series of experiments was underraken. Using a seven-barrelled glas micropipene, JO-1784 was applied microiontophoretically with a current of 20 nA which induced a potentiation of the NMDA response. The potentiation was maintained for 15 minutes. DTG was than applied with a current of 75 nA and we observed an attenuation of the potentiation of the NMDA response. After another 15 minutes, the microiontophoretic application of both o ligands were discontinued for 5 minutes. Following that period, JO-1784 was applied again with a current of 20 nA and induced the same level of potentiation of the NMDA response (Fig. 8) as previously observed.

DISCUSSION The intravenous administration of low doses (1-10 pg/kg) of the o ligands DTG, JO- 1784, ( + )pentazocine, BD-737 and L-687,384 induced a selective potentiation of the NMD A response of CA, hippocampal pyramidal neurons in keeping with previous results (Monnet et al. 1990; Monnet et al. 1992; Bergeron et al. 1993; Walker and Hunter, 1994; Bergeron et ai. 1995). Higher doses of the same ligands (500-1000 pg/kg, i.v.) were ineffective in potentiating the NMDA response as already reported (Bergeron et al. 1995). It has been shown that this lack of effect from high doses of these o ligands was not due to a rapid desensitization of the o recepton but rather appeared to be related to the simultaneous activation of several subtypes of o receptors (Bergeron et al. 1995). There is accumulating evidence for cr receptor heterogeneity (for reviews see: Walker et d. 1990; Su, 1993). Two forms of o receptors are actudly acknowledged (Quirion et al. 1992) and both a, and a, subtypes of receptors are present in the brain. They differ by their affinities for a drugs, tissue distribution and GTP sensitivities (Quirion et al. 1992). A senes of experiments camed out in Our laboratory suggest that o, ligands act on at least two different o receptors. We have previously shown that a pretrearment with a penussis toxin suppresses the potentiation of the NMDA response induced by 50-1784 but not the potentiation induced by (+)pentazocine (Monnet et al. 1994). Though 50-1784 and (+)pentazocine have been classified as selective o, ligands (Quirion et al. 1992), these results suggest that JO-1784 is acting on a o reîeptor coupled to a Gd. protein and (+)pentazocine on another u receptor not coupled to a Gv, protein. Moreover, a unilaterd destruction of the mossy fiber system, by a local injection of colchicine, abolishes the potentiating effect of the NMDA response induced by low doses of DTG and JO-1784 but has no effect on the potentiation induced by low doses of (+)pentazocine (Debonnel et al., 1996). In the CA, region of the dorsd hippocampus JO- 1784 does not modify the NMDA response, whereas (+)pentau>cine induces a potentiating effect similar to that observed in the CA, area (Debonnel et al., 1996). Findly, following a long-tem treatment with a low dose of JO-1784, a supersensitivity of o receptors is observed which is not presenr following a longterm rreatment with a low dose of (+)~entazocine (Bergeron et al. 1996). These different series of data constitute convincing evidences that o, receptors should be considered as a population of at least two types of u receptors. They ais0 suggest that, in the CA, region of the dorsal hippocampus, the potentiation induced by DTG and 50-1784 is obtained via a a receptor located pre~ynapticall~on the mossy fiber terminais, whereas the potentiation induced by (+)pentazocine is obtained via a o receptor presurnably located in the postsynaptic region. In the present series of experiments the five cr agonists, adrninistered at high doses, acted as antagonists in different ways. DTG is a selective ligand for a receptors (Weber et al. 1986), with a similar affinity for both ol and oz subtypes of receptors, whereas 50-1784 and BD-737

have been found to be selective for 0, (Quirion et al. 1992). These three o ligands @TG, JO- 1784 and (+)pentazocine), administered at high doses, suppressed the potentiating effect of low doses of u agonists, whereas (+)pentazocine, which is also classified as a a, ligand, did not modify the potentiation effect of the o agonists when administered at high doses. L-687,384 has high affinity and is selective for o receptors (Middlerniss et al. 1991; Mclanon et al. 1994). We have previously reported that this o ligand acts as an extremely potent agonist since a potentiation of the NMDA response is obtained with doses as low as 0.1 pg/kg, i.v. (Bergeron et al. 1995). The maximal potentiation of the NMDA response was obtained at the dose of 1 pg/kg, i.v., and no effect was observed on the NMDA response at doses higher than 50 adkg, i.v. In the present experimental series the dose of 100 pg/kg, i.v. suppressed the potentiation of the NMDA response induced by a low dose of DTG but not those induced by a low dose of 50-1784, BD-737 or of (+)pentazocine (Fig. 3). However, the dose of 1000 pg/kg, i.v. of L-687,384 prevented and reversed the porentiation of al1 four a agonists. This observation could suggests that L-687,384 at the dose of 100 pg/kg, i.v. acts on a subtype of a receptor distinct from that on which it acts at the high dose of IOOO &kg. The observation that at low doses DTG, JO-1784, L-687,384 and BD-737 induce a potentiation of the NMDA response whereas when administered at high doses, the sarne a ligands suppress and prevent this potentiation may appear surprising. Several interpretations could explain the results obtained in the present study. It could be postulated that high doses of a ligands induce a desensitization of a receptors. This would explain the lack of effect of the high doses of a ligands on the NMDA response and the reversa1 of the potentiation induced by the low doses. This hypothesis appear however unlikely since we have previously shown that, sirnilarly to the intravenous administration, the microiontop horetic application of a high current, a o agonist does not induce any potentiation of the NMDA response, but that the subsequent application of a low current will still induce the same level of potention as observed in the control situtation (Bergeron et al. 1995). Moreover, the data obtained in the present series show that a high current of DTG prevents the potentiating effect of a low current of 50-1784, which is still able to induce a potentiation of the NMDA response of the cessation of the application of DTG. It is also possible that high doses of o ligands induce a suppressant effect on the NMDA response by acting on a subtype of o receptor for which they would have a lower affinity. The lack of potentiation of the NMDA response with high doses could then be the result of the addition of the potentiating effect obtained with low doses and the suppressant effects obtained with high doses. For example, since some o receptors are located presynaptically, it is possible that the activation of these receptors by low doses of o agonists may induce, directly or indirectly, the release of an endogenous substance which would act as a modulator on the NMDA receptor complex and potentiate the NMDA response. A high dose of the sarne

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DTG (+)Pentatocine 1000 pgkg, i.v. 10 pmg, i.v.

- wO 5 W 5 0 0.5 LuVI 2E "'O

4 (+)~entazocine.10 pgkg. i-v. CL-687.384. 1 pgkg. i.v. t DTG, 1000 pgkg, i.v. t DTG, 1000 pg/kg, i.v.

Figure 1 (A) Integrated firing rate histogram of CA, dond hippocampus pyramidal neurons showing the effects of microiontophoretic applications of ACh, QUIS and NMDA before and after the intravenous administration of a high dose of DTG and aher the intravenous administration of a low dose of (+)pentazocine. In this and subsequent figures, bars indicate the duration of applications for which the currents are given in nA. Each neuronal response to each excitatory agent represenü the computed-generated mean of the effects of three successive applications. Open circles (00) represent an interruption of the illustration of the continous recording in this and the following integated firing rate histognms. Responsiveness, expressed as the number of spikes generated/nC (mean SEM) of CA, dorsal hippocarnpus neurons to microiontophoretic applications of NMDA before (open columns) and after (grey columns) the intravenous administration of a low dose of a agonists 00- 1784 (B), (+)pentazocine (C)]and after the subsequent intravenous administration of high dose of DTG (dark columm). In the last group of bar histogams, the high dose of DTG wu injected before (grey colurnns) the intravenous administration of a low dose of a agonists [(+)pentazocine (D) and L-687,384(E)]. The number at the bottorn of the first column indicates the nurnber of neurons tested. The number beneath ACh, QUIS and NMDA represents the intensity of the cunent used for the microiontophoretic applications of these excitatory substances. * p < 0.01, using the Student's r test. A ACh NMDA QUIS 9 -1 1 -5 Ii010

DTG JO-1 784 1 pglkg. i.v. Io00 pglkg, i.v.

L DTG, 1 pglkg. i-v. (+)Pentazocine. 10 pg/kg, i.v.

Figure 2 (A) Integrated firing rate hisrogram of CA, dorsal hippocampus pyramidal neurons showing the effects of rnicroiontophoretic applications of Ach. QUIS and NMDA before and after the intravenous administration of a low dose of DTG and after the intravenous administration of a high dose of JO-1784. Responsiveness, expressed as the number of spikes generated/nC (mean SEM)of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (open columns) and afier (grey columns) the intravenous administration of a low dose of IT agonists DTG (B), (+)pentazocine (C)] and after the subsequent intravenous administration of high dose of JO-1784(dark columns). In the last group of bar hisrograms, the high dose of BD- 737 (D) was injected before (grey columns) the intravenous administration of a low dose of o agonists PTG (D) and L-687,384(E)]. + p < 0.01, using the Student's t test in B and C. A ACh NMDA QUIS 7 -12 -2 In-9 O O

a Vi

0 0 0 O 00 + 4 4 2 min

- f f 4 Haloperidol. 10 pgkg. i-v.

1 DTG, 1 pgkg, i.v.

Figure 3 (A) Integrated firing rate histogram of CA, dorsal hippocampus pyramidal neurons showing the effects of microiontophoretic applications of ACh, QUIS and NMDA before and after the intravenous administration of a Iow dose of JO-1784 and after the intravenous administration of a 100 pg/kg, i.v. of L-687,384. Since that dose of L-687,384 was ineffective in reversing the potentiation induced by a low dose of 50-1784, a low dose of haloperidol was subsequently injected. Responsiveness, expressed as the number of spikes generared/nC (mean SEM) of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (open columns) and after (grey columns) the intravenous administration of a low dose of 50-1784 (B) and (+)pentazocine (C). Since the dose of 100 pg/kg, i.v. of L-687,384 was ineffective in reversing the potentiation induced by low doses of JO-1784 (B) and (+)pentazocine (C) (dark columns), a low dose of haloperidol was injected (black columns). Responsiveness, expressed as the number of spikes generared/nC (mean SEM) of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (open columns) and after (grey columns) the intravenous administration of a low dose of DTG (D) and before and aher the intravenous administration 100 &kg, i.v. of L-687,384 (E). * p < 0.01, using the Student's t test in B, C and D. Figure 4 Responsiveness, expressed as the number of spikes generatedlnc (mean SEM), of CA, donal hippocampus neurons to microiontophoretic applications of NMDA before (open columns) and after ~he intravenous administration of low dose of DTG (A), 50-1784 (B), (+)pentazocine (C) or BD-737 (D) (dark columns) and after the intravenous adminisrmion of a high dose of L-687,384 (black columns). * p < 0.01, uring the Student's t test in A, B, C and D. O Control ÜI] Controi W DTG i pmg. i.v. (+)Pentazocine 1M)O pgikg. i-v. (+)Psntazocine 1000 pgikg. i-v. 50-1784 5 pgll

Haloperidol 10 pg/kg. i.v. Haloperidol 1O pgkg. i.v.

CI Controi (+)Pentazocine 1O pg/kg. i.v.

(+)Pentazocine 1000 pghg. i.v-

Figure 5 (A) Responsiveness, expressed as the number of spikes generated/nC (mean SEM),of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (open columns) and after the intravenous administration of low dose of DTG (grey columns) followed by the intravenous administration of high dose of (+)pentazocine (dark colurnns) and followed by the intravenous administration of a lov dose of haloperidol (black columns). (B) Responsiveness, expressed as the number of spikes generated/nC (mean SEM), of CA, dorsal hippocampus neurons to microionrophoretic applications of NMDA before (open columns) and after the intravenous administration of high dose of (+)pentazocine (grey columns) followed by the intravenous administration of a low dose of JO-1784 (dark columns) and followed by the intravenous administration of a low dose of hdopendol (black columns). (C) Responsiveness, expressed as the nurnber of spikes genented/nC (mean SEM),of CA, donal hippocampus neurons to microionrophoretic applications of NMDA before (open columns) and after the intravenous administration of low dose of (+)penrazocine (grey columns) followed by the intravenous administration of a high dose of (+)pentazocine (dark colurnns). * p < 0.01, using the Student's r test in A B and C. Control Control

JO-1 784. 1 pgkg i.v. JO-1784, 2 pgkg i.v. (+)Pentazocine, 5 pg/kg i-v.

Figure 6 Responsiveness, expressed as the number of spikes generated/nC (mean SEM), of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (open colwnns) and after (grey and dark columns) the intravenous administration of different low doses of two a agonists. * p < 0.01, using the Student's t test in A and B. SPIKES GENERATED/nC OF NMDA

SPIKES GENEAATEDlnC OF NMDA - - O wl 2

SPIKES GENERATEDlnC OF NMDA DTG (75 nA)

JO- 1 784 (20 nA)

- 1 min

Figure 8 Integrated firing rate histogram of CA, dorsal hippocarnpus pyramidal neurons showing the effects of microiontophoretic applications of ACh, QUIS and NMDA before and after the microiontophorecic applications of low current of JO-1784 and after the rnicroiontophoretic applications of high current of DTG. Microiontophoretic applications of both a ligands were stopped for 5 minutes. After that period, 10-1784 was applied again. Table 1 Effects of the intravenous administration of a high dose (1 mg/kg) of DTG on the potentiation of the NMDA response induced by microiontophoretic applications of low currents of o agonists.

I CONTROL I DTG (1 mg/kg, i-v.) Spikts gcncrated/nC of NMDA man 5 Spi& genented/nC of NMDA mcan i S.E.M. S.E.M. 1 NMDA 0.41 + 0.09 0.39 1 0.07 NMDA + DTG (20 nA) 0.98 + 0.19* I 0.33 & 0.09 NMDA 1

NMDA + Pentazocine (20

* P < 0.01 using the Student t test Table 2 Effens of the intravenous administration of high doses (1 mg/kg) of JO-1784or BD-737 on the potentiation of ~heNMDA response induced by microiontophoretic applications of low currents of o agonists.

CONTROL JO-1784 BD-737 Spikes generared/nC (1 mg/kg, i-v.) (1 mg/kg, i.v.1 of NMDA mmî Spikes genenrdnl Sp~ktsgenetlrcd/nC S.E.M. of NMDA mean * of NMDA mtan IS.E.M. S.E.M.

-- - NMDA 0.46 + 0.08 NMDA + DTG (20 nA) 0.92 * 0.14' NMDA 0.42 & 0.07 NMDA + 10-1784 (20 nA) 0.87 + 0.09')

NMDA 0.39 I0.06 NMDA + (+)pentazocine (20 0.84 + 0.07* nA)

* P c 0.01 using the Student t test Table 3 Effects of the intravenous administration of a high dose (1 mgkg) of (+)pentazocine on the potentiation of the NMDA response induced by rnicroioncophoretic applications of low currents of o agonists.

CONTROL ( + )PENTAZOCINE Spikes generated/nC (1 mg/kg, i.v.) of NMDA mean I S.E.M. Spikes generated/nC of NMDA mean i S.E.M.

II NMDA + DTG (20 nA) NMDA II NMDA + 50-1784 (20 nA) NMDA NMDA + (+)pentazocine (20

* P < 0.01 using Srudent t test 179

CHAPTER VI1 Short-term and long-term treatments with sigma ligands modify the N- methyl-D-aspartate response in the CA, region of the rat dorsal hippocampus

The second phase of my Ph.D. (Chapter 7-8-9) was focussed on the potential clinical implications of a receptors. It is common knowledge that antidepressant and anripsychotic drugs may require a penod of two to three weeks before the appearance of clinical effects. Binding studies have indicated rhat long-term treatments with high-affinity o ligands modtfy the B, andlor KD values of the o binding sites. Moreover, it has been demonstrated, by several invesrigators, that long-term treatment with haloperidol produces a down-regulation of the o receptors in rnice, rats and humans.

The firsr aim the following study was to assess the effects of long-term treatment with four different o ligands. Haloperidol is a very potent, but not seleaive, ligand at the o receptors. In Our paradigrn this compound behaves as an antagonist at the o receptors at both low and high doses (Chapters 2, 3, 4, 5). High doses of DTG and 50-1784, which also act as antagonists (Chapter 6), will be assessed. Finally, low and high doses of (+)pentazocine, which appears to have a distinct pharmacological profile (Chapters 3 and 4) will be evaluated. The second aim of this study was to assess the effects of shorc-term treatrnents and the effea of "on board" treatmenr with a ligands. Our working hypothesis was that long-term treatment with o antagonists would desensitize the o receptors, while long-term treatment with o agonists would supersensitize the cr receptors. It is noteworthy that this is the first funaional study to address this issue. 180 SHORT-TERM AND LONG-TERM TREATMENTS WITH SIGMA LIGANDS MODIFY THE N-METHYL-D-ASPARTATE RESPONSE IN THE C4, REGION OF THE RAT DORSAL HIPPOCAMPUS Richard Bergeron, Claude de Montigny and Guy Debonnel

British Journal of Phannacology, in press.

SUMMARY Long-term treatment with the sigma (o) ligand halopend01 decreases the density of o receptors in mammalian CNS. We have shown that o ligands, such as DTG, potentiate dose- dependently, with bell-shaped dose-response curves, the neuronal response of pyramidal neurons to N-methyl-D-aspartate (NMDA) in the CA3 region of the rat dorsal hippocampus. Sigma ligands producing such a potentiation were denoted "agonists". This potentiation is suppressed by low doses of other a ligands denoted "antagonists". High doses of DTG and JO- 1784 do not modify the NMDA response but act as "antagonists" by suppressing the potentiation induced by a "agonists". Following a 21day treatment with haioperidol as well as wirh high doses of DTG or JO-1784, after a 48-h washout, the acute administration of o "agonists" failed to induce any ~otentiationof the NMDA response. Following a 21day treatment with a low dose of DTG or JO-1784, afier a 48-h washout, the neuronal response to microiontophoretic applications of NMDA was markedly increased. A Xday treatment with low or high doses of (+)pentazocine, after a 48-h washout, did not produce any change. Following a twoday treatment with a high dose of haioperidol, DTG, 50-1784 and (+)pentazocine, after a 24-h washout, the potentiation of the NMDA response induced by the acute administration of the a "agonists" was unchanged. With the minipumps on board, with DTG and JO-1784, a dose dependent enhancernent of the NMDA response but no effect was obtained in the groups of rats treated at the same doses with haloperidol or (+)pentazocine. The present data suggest that a long-term with o "antagonists" induce a desensitisation of the a receptors, whereas a long-term treatment with a "agonists" induce a supersensitivity of the a receptors.

Key words: Sigma receptors, haloperidol, DTG, JO-1784, pentazocine, hippocampus, elect rophysioiogy, long-term treatment INTRODUCTION Many neuroleptics, several antidepressants and some neuroactive steroids as well as psychotomimet ic drugs, such as pentazocine and cocaine, exhibit high to moderate affinity for sigma (0) receptors, suggesting that these receptors might be involved in the control of behavioral and emorional States (for review see: Walker et al., 1990; Su, 1991; Debonnel, 1993). Moreover, some recently developed neuroleptic dmgs, having a low affinity for dopamine receptors and high to moderate affinities for a recepton, were reported to exert a therapeutic effect in schizophrenia (Snyder & Largent, 1989; Munetz et al., 1989; Ashwood et al., 1992). Post-mortern studies have shown a decrease of the densities of a binding sites in severai regions of the brain of schizo~hrenicpatients (Weissman et al., 1988). Moreover, a down-regdation of a binding sites has been observed following long-term halopend01 administration in animals (Itzhak & Alerhand, 1989; Matsumoto et al., 1990; Reynolds et al., 1991). Binding studies using selective o radioligands have provided evidence for the existence of at lest two subtypes of a receptors (for review see: Hellewell & Bowen, 1990; Quirion et aL, 1992). The most commonly used o ligands, including DTG [di@-toly1)guanidinJ(Weber et aL, 1986) and haloperidol (Tam & Cook, 1984) do not discriminate between a, and q. In contrast, (+)pentazocine (Steinfels et al., 1988) and 50-1784 [(+)N-cyclopropylmethyl-N-methyl-1,4-diphenyl-l-ethyl-but-3-en-i- ylamine hydrochloride] (Roman et aL, 1990) are more selective for the u, subtype (Bowen et al., 1989; Hellewell & Bowen, 1990; Rothman et al., 1991). We have previously shown chat the acute administration of low doses of selective a ligands, such as DTG, 50-1784, (+)pentazocine, BD-737 (Contreras et al., 1991) and L-687,384 [l-benzylspiro[1,2,3+tetrahydronap hthdene-1 ,+piperidine (Middlemiss et al., 199 1; Barnes et al., 1992), potentiate selectively the neuronal response of CA, dorsal hippocampus pyramidal neurons to microiontophoretic application of NMDA Monnet et aL, 1990; Monnet et aL, 1992; Bergeron et al., 1993; Bergeron et aL, 1995). These ligands were denoted o "agonists". This potentiation can be suppressed by other o ligands such as haloperidol, BMY-14802 [a-(+ fluorop henyl)4(5-fluo ro-2-pyrimidinyl)-1-piper ne butanol] (Taylor & Dekleva, 1987) and (+)3-PPP (Largent et al., 1984) which were thus denoted cr "antagonists". The degree of the potentiation obtained with a "agonists" is dosedependent and presents a bell-shaped dose- response curve (Bergeron et al., 1995). For most of the o "agonists", the maximal potentiation is observed at doses in between IO& 200 pg/kg i.v. At higher doses, the degree of the potentiation progressively decreases to finally vanish at doses higher rhan 500 pg/kg i.v. At the dose of 1000 &kg i.v., u ligands such as DTG and 50-1784 do not modify the NMDA response but t hen act as "antagonists" by preventing and suppressing the potentiarion induced by u agonists (Debonnel et al., 1992; Bergeron et al., 1995). It is noteworthy that these selective cr ligands have negligible &nity for any of the binding sites of the NMDA receptor cornplex (Walker et al., 1990). The purpose of the present study was to determine the effects of acute administration of low doses of the ligands DTG, JO-1784, (+)~entazocineand L-687,384 on NMDA-induced activation, following long- and short-term treatrnents with low or high doses of several a ligands. These experiments were carried out in the CA, region of the rat dorsal hippocarnpus, a region with high densities of o and NMDA receptors (Cotman & Monaghan, 1988), using an in vivo electrophysiological paradigm whereby the neuronal responsiveness to microiontophoretic applications of NMDA, quisqualate (QUIS) and acetylcholine (ACh) can be quantified by extracellular unitary recording.

MATERIAL AND METHODS Long and sholr-tom treatments Male Sprague-Dawley rats (125-150 g) were anesthetized with halothane for the subcutaneous implantation of osmotic minipumps. For the long-term treatments, saine or the following o ligands were delivered at the doses of 200 &kg/day or 2 mg/kg/day for haloperidol, 50-1784 and (+)pentazocine and of 100 pg/kg/day or 1 mg/kg/day for DTG. In a first study, rats were treated for 21 days followed by a washout period of rwo days before the electrophysiological experiments. In a second study, animas received one of the following o ligands: haloperidol, 50-1784 or (+)pentazocine at the dose of 2 mg/kg/day or DTG at the dose of 1 mg/kg/day for two days, followed by a washout period of one day before the experirnents. In a third study, animas were treated for two days and the electrophysiological experiments were carried out while the rninipumps were sri11 on board. For this last series, DTG, 50-1784 and haloperidol were administered to the rats, at the doses of 1, 10, 100 or iOOO pg/kg/day.

Recording from CA3dorsal hippocampus pyramidal neurons Rats were anesthetized with urethane (1.25 g/kg i-p.) and mounted in a stereotaxic apparatus. Body temperature was maintained at 37' C throughout the experiments. Five- barrelled glas micro pipettes, p reloaded wit h fibreglass strands in order t O promote capillary filling, were pulled in a conventional manner (Haigler & Aghajanian, 1974) and their tips broken back to 8 to 12 pm under rnicroscopic control. The central barrel, used for extracellular unitary recordings of the activity of CA, dorsal hippocarnpus pyramidal neurons, was filled with a 2 mM NaCl solution as well. The impedance of the central barrel was typically between 2 and 5 Mn. One side barrel, filled with 2 rnM NaCl, was used for current balancing. The other side barrels, used for microiontophoresis, were filled with NMDA (IO mM in 200 mM NaCl, pH: 8), QUIS (1.5 mM in 400 mM NaCl, pH: 8) and ACh (29 rnM in 200 rnM NaCl, pH:4). After removal of the dura mater, the micropipette was lowered into the CA, region of the dorsal hippocampus (lateral: 4.2 mm and anterior: 4.2 mm at a depth of 3.5 to 4.5 mm from the corrical surface (Paxinos & Watson, 1986). Action potentials were deteaed by a differential amplitude discriminator generating square pulses which were fed to a computer and to a counter from which integrated finng rate histograms were generated and displayed on a Gould paper chart recorder (mode1 RS 3200). Pyramidai neurons were identified according to their long duration (0.8-1.5 msec) and large amplitude (0.5-2 mV) action potentids, and by the presence of characteristic "cornplex spike" discharges alternating wit h simple spike activity (Kandel & Spencer, 1961). The duration of the rnicroiontophoretic applications and the intensity of the current used were also stored in the computer. The effect of the microiontophoretic applications of either NMDA, QUIS or ACh on pyramidal neuron firing activity was expressed as the number of spikes generated per nC (1 nC being the charge generated by 1 nA applied for 1 sec). The duration of rnicroiontophoretic ejeaions of these excitatory substances was kept constant at 50 sec. The currents used for ejeaing NMDA ranged from -8 to -20 nA, from -2 to -6 nA for QUIS and 5 to 10 nA for Ach. For a given neuron, the current was adjusred to obtain a firing frequency in between 7 and 15 Hz and was thereafter maintained constant for the rerninder of the experiment. The effects of long and short-term treatments were rneasured by cornparing the neuronal response to NMDA, QUIS and ACh in rats treated with o ligands or saline. The effects of these treatments were also assessed by comparing the degree of potentiation of the NMDA response following the acute intravenous administration of a Iow dose of a

The computer calculated the effect of each 50 s microiontophoretic application of an excitatory substance as the total number of spikes generatedhl. Each value was calculated by the computer as the mean of the effect of three consecutive applications of the same excitatory 184 substance. The effects of the intravenous administration of a ligands were assessed by deterrnining the ratio (NJNJ of the number of spikes generatedhl of each for the three excitatory substances NMDA, QUIS or ACh before (NJ and after (NJ the injection of the a ligand.

Stutzstzcal analyses Al1 results are expressed as the mean * S.E.M. of the number of spikes generated/nC of NMDA, QUIS and Ach. Statistical significance was assessed using the Student's t test with the Dunnett's correction for multiple comparisons. Probability values smaller than -05 were considered as significant. Covariance analyses were used to compare the degree of the potentiation of the NMDA response in the group of rat treated with different doses of DTG and 50-1784 while keeping the minipumps on board.

RESULTS The short- and long-term treatrnents with various doses of the o ligands studied did not change the spontaneous firing rate of CA, pyramidal neurons nor their response to QUIS and ACh nor did they modify the baseline neuronal response to NMDA except for the series experiments carried out with the minipumps on board.

Effects of 22-&ytreatmmts with differmt doses of/O-2784followed by a 48 h washout. Several series of long-term treatments with 50-1784 (50, 200, 500, 1000, 2000 pg/kg/day) were performed. Following 2Iday treatments and after a 48-h washout, no significant differences were found between the degrees of potentiation of the NMDA response induced by microiontophoretic applications of JO-1784 in the control animals and the rats treated with 50 and 500 ~g/kg/day(fig. 1). However, in the group of rats treated with the dose of 200 pg/kg/day, a sipificant enhancement of the potentiation of the NMDA response was observed (fig. 1). In the group of rats treated at the dose of IOOO and 2000 ~g/kg/day, no potentiation of the NMDA response was found following the administration of 50-1784 (fig. 1). This type of bell-shaped dose-response curve is very similar to those already found following acute treatments with a "agonists" (Bergeron et al., 1995). Therefore, in the following series of experiments, the dose inducing the maximal degree of potentiation and a high dose were chosen. Effects of 2I-&y treatments with high doses of DTG,jO-1784 and (+)pmtazocine followed by a 48 h wushout. Following Xday treatments with 1000 pg/kg/day of DTG or 2000 pg/kg/day of JO-1784, after a 48-h washout, neither the intravenous administration of low doses of DTG, JO-1784 or (+)pentazocine nor the rnicroiontophoretic application of these o ligands induced any potentiation of the NMDA response (fig. 2a, b). However, following a 48 h washout, the ïlday treatment with the high dose of ZOO0 Irg/kg/day of (+)pentazocine, the degree of potentiation of the NMDA response induced by low doses of DTG, 50-1784 and (+)pentazocine was similar in treated and in control rats (fig. Zc).

Effects of 2I-day treatments with low doses of DE,10-1784 and (+)pentazon'ne followed by a 48 h washout. Following a Xday treatment with DTG at the dose of 100 pg/kg/day, after a 48-h washout, the effects of the acute administration of DTG was significantly increased. In control rats, the potentiation of the NMDA response induced by microiontophoretic application of DTG (20 nA), was of 2-fold (fig. 3a), whereas, in rats treated with DTG, rnicroiontophoretic application of this ligand with the same current induced a ~foldincrease of the NMDA response (fig. 3a). In these DTG-treated rats, the intravenous administration of DTG (1 pg/kg) induced an epileptoid activity upon NMDA application (data not shown), a phenornenon observed only with doses higher than 3 pg/kg, i.v. in naive rats (Monnet et al., 1992; Bergeron et al, 1995). The acute administration of JO-1784 (4 pg/kg, i.v. or 20 nA) (fig. 3c) and chat of (+)pentazocine (10 @g/kg, i.v. or 20 nA) (fig. 3c, d) induced a potentiation of the NMDA response similar to that produced in naive rats. Following a long-term treatment with 50-1784 at the dose of 200 pg/kg/day for 21 days, afrer a 48-h washout, the intravenous administration of 1 pg/kg of 50-1784, (a dose which did not induce any modification of the neuronal response to NMDA in the control rats), ~roduceda Zfold increase of the NMDA response (fig. 4a). However, the subsequent administration of 4 pg/kg, i.v. of 50-1784 did not induce any further enhancement of the NMDA response (fig. 4b). In contrast, the injection of 10 pg/kg, i.v. of (+)pentazocine induced a potentiation of the NMDA response similar to that obtained in control rats (fig. 4c). Moreover, as was the case for rats treated with a low dose of DTG, in JO-1784-treated rats, the acute administration of DTG (1 &kg, i.v.) induced epileptoid activity upon microiontophoretic application of NMDA (data not shown). Following a long-term treatment with the dose of 200 Ccg/kg/day of (+)pentazocine, after a 48 h washout, no significant difference was found between the degrees of potentiation of the NMDA response induced by microiontophoretic applications or by intravenous adminisirations of low doses of DTG, (+)pentazocine or 50-1784 in the treated and control rats.

Effects of 21-&y treatmmts with a ~OWor a high dose of halopmWSdolfollowed by a 48 h washout. In a first series of experiments, rats were treated for 21 days with haloperidol, (200 or 2000 pg/kg/day s.c.), or with saline, delivered subcutaneously by osmotic minipumps. After a 48-h washout (removal of the minipump), the effects of the intravenous administration and of the microiontophoretic applications of the three seleaive and high affinity u ligands DTG (1 pg/kg, i.v. or 20 nA), JO-1784, (4 pg/kg i.v. or 20 nA) or (+)pentazocine (10 &kg, i-v. or 20 nA) were assessed. As illustrated for DTG in figure 5, no potentiation of the NMDA response was induced by the intravenous administration of a low dose of DTG (fig. 5a) or by microiontophoretic applications of DTG (fig. Sb) in the rats treated with haloperidol for 21 days at the dose of 200 &kg/day and at the dose of 2000 pg/kg/day. Similarly, the intravenous administration of a low dose of 50-1784 or (+ )pentazocine or the microiontophoretic application of 10-1784 and (+)pentazocine failed to potentiate the NMDA response in the rats treated for 21 days with 200 or 2000 pg/kg/day of halopendol (data not shown). To rule out the possibility that the absence of potentiation of the NMDA response was due to the residual halopend01 despite the 48 h washout, a group of rats was treated with the same doses of haloperidol (200 or 2000 &kg/day) for 21 days but the electrophysiological experiments were carried out following a 7-day washout. In this series, DTG injected inrravenously (1 pg/kg, i.v.; fig. 6a) or applied by rnicroiontophoresis (20 nA) and (+)pentazocine (IO pg/kg, i.v.; fig. 6b) or applied by microionrophoresis (20 nA), failed to induce any potentiation of the NMDA response.

Eflects of 2-day treatmmts wzth DTG, JO-1784, (+)pentazocine and halopmdol followed by a 24 h washotrt. In this series of experiments, rats were treated for 48 h with DTG, JO-1784, (+)pentazocine and haloperidol at the high dose of 2000 &kg/day followed by a 24-h washout. In rats treated with 2000 gg/kg/day of haloperidol, the intravenous administration of DTG (1 pg/kg, i.v.) and JO-1784 (4 gg/kg, i.v.) or the microiontophoretic application of DTG or 50-1784 with a currenr. of 20 nA, induced a potentiation of the NMDA response similar to that obtained in the control rats (fig. 7a). Sirnilarly, the 48-h treatments with DTG (2000 pg/kg/day, s.c.), with JO-1784 (2000 pg/kg/day, s.c.) or with (+)pentazocine (2000 pg/kg/day, s,c,) did not modify the degree of the potentiation of the NMDA response induced by the intravenous administration of low doses or by rnicroiontophoretic applications of DTG or 50-1784 (figure Tb, c).

Effects of 2-day treatmmts with DTG, 10-1784, (+)pentazocine and halopmdol with minipumps on board.

In a lasr series of experiments, doses of 1, IO, 100 and IOOO ~~/k~/day,S.C. of each of the four o ligands studied [DTG, 50-1784, (+)pentazocine and hdoperidol] were administered for 48 h. Electrophysiological experiments were carried out while the minipumps were still on board. In rats treated with DTG or 50-1784, the neuronal response to microiontophoretic application of NMDA was increased. As shown in figure 8, the degree of activation of CA, pyramidal neurons induced by rnicroiontophoretic application of NMDA, using the rame ejecting current, was related to the dose of the o ligands administered during the 48 h. The degree of activation by NMDA in the group of rats treated with DTG (10 ~g/kg/day)could not be quantified because the rnicroiontophoretic application of NMD A induced an epileptoid activity. This epileptoid activity was not affected by diazepam (5 mg/kg, i.v.), but was completely reversed by a very low dose of hdoperidol (IO pg/kg, i.v.). In rats treated with (+) pentazocine, the neuronal response to rnicroiontophoretic applications of NMDA was not increased (data not shown). In rats treated for 48 h with low doses of halopend01 (1 and 10 pg/kg/day), the neuronal response to microiontophoretic applications of NMD A was sirnilar to t hat of control rats. In rats treated with higher doses of hdoperidol (100 and IOOO pg/kg/day), the rnicroiontophoretic applications of DTG or (+)pentazocine failed to induce a potentiation of the NMDA-induced activation. DISCUSSION Twenty-one day treatments with various doses of the o "agonist" 10-1784 modified the effects of the acute administration of the sarne o ligand. The dose response curve of this effea presented a bell-shaped aspect similar to that observed following an acute treatment. Twenty-one day treatments, followed by a 48-h washout, with a low or a high dose of the high affinit~o ligand haloperidol, as well as with high doses of DTG or 50-1784, prevented the potentiation of the NMDA response by an acute administration of low doses of o "agonists". In our electrop hysiological paradigm, we have previously shown that halopendol is acting as a o "antagonist" since it does not have any effect on NMDA-induced activation by itself, but prevents and reverses the potentiation of the NMDA response induced by the acute administration of low doses of o "agonists". The possibility that the lack of potentiation by the acute administration of low doses of o "agonis&' in rats treated with a low or a high dose of haloperidol, could have been due to residual halopend01 following the 48-h washout is ruled out by the obsenration of the same phenornenon following a sevenday washout. A more plausible explanation could be that long-term treatment with haloperidol induces a down-regulation of the

be easily verified when seleaive 0, ligands become available. Long-term treatments with either a low or a high dose of (+)pentazocine did not modify the effens of the acute administration of a low dose of

treatment with a low or a high dose of (+)pentazocine does nor alter the sensitivity of 0, receptors. When the minipumps were removed 24 h before the electrophysiological experiments, none of the two-day treatments with al1 four of the o ligands tested (haloperidol, DTG, 50-1784 and (+)pentazocine) did modify the potentiation of the NMDA response induced by the acute administration of the o "agonists". These results suggest that the modification at the u ligands induced by the o ligands cannot be achieved over a short period. In contrast, in the ex~erimentdseries carried out with the minipumps on board, DTG and 50-1784 (administered for 48-h at each of the following four doses: 1, 10, 100 or 1000 pg/kg/day, s.c.) enhanced the NMDA response in a dose-dependent manner, whereas in the group of rats treated with halopend01 with the same four doses, no potentiation of the NMDA response has been observed. Moreover, 48-h treatments with 1 and 10 ~g/kg/day,S.C. of halopendol, did not prevent the potentiation induced by rnicroiontophoretic applications of DTG and 50-1784 with a current of 20 nA, whereas in the group of rats treated with 100 and IOOO &kg/day, S.C. of haloperidol, no potentiation of the NMDA response has been observed upon the microiontophoretic applications of the same two ligands with the same current. This suggests that when the experiments were performed with the minipumps on board delivering haloperidol at doses of 100 and 1000 &kg/day, s.c., the arnount of haloperidol in the brain was sufficient to occupy the a receptors and consequently prevent the action of the a "agonists" DTG and JO-1784. In rats treated with (+)pentazocine (at any of the four doses administered), the microionrophoretic applications of NMDA induced the same degree of neuronal activation. Over the last few years, we have observed that (+)pentazocine presents a pharmacological profile distinct from that of other o ligands: 1) a pertussis toxin pretreatment abolishes the potentiation induced by 50-1784, L-687,384 and NPY, but not that induced by (+)pentazocine (Monnet et al., 1994); 2) following a unilateral lesion of the mossy fiber system by a local injection of colchicine, the potentiating effect of JO-1784 is abolished a-hereas the effect of (+)pentazocine on the NMDA response is still present (Debonne1 et al, 1996); 3) high doses of the o, ligands 50-1784, BD-737 and L-687,384 (which are acting as "agonists" at low doses) prevent and suppress the potentiation of the NMDA response induced by low doses of the o "agonists" whereas high doses of (+)pentazocine fail to produce such an effect. These observations, in keeping with the present results, suggest that (+)pentazocine, a selective O, receptor (Quirion et al., 1992), act on distinct subtype of o, receptors. However, the present electrophysiological data do not allow to conclude to ascertain that the metabolism of the drugs used may contribute to the changes observed following long-term treatments.

CONCLUSION In conclusion, the results of the present study suggest rhat long-terrn treatment with o "agonists" induces a supersensitivity of cr recepton whereas long-term treatment with a "antagonists" induces a desensitization of cr receptors. REFERENCES

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KIZU, A., YOSHIDA, Y. & MNAGISHI, T. (1991). Rat cortical sigma receptors differentially regulated by pentazocine and haloperidol. j. Neural Transm., 83, 149-153.

LARGENT, B.L., GUNDLACH, A.L. & SNYDER, S.H. (1984). Psychotomimetic opiate recepton labelled and visualized with (+) ['Hl-3-(3-hydroxypheny1)-N-(1-propyi)piperidine. Proc. Nad. Ach. Sci. USA, 81, 4983-4987.

MATSUMOTO, R.R., BOWEN, W.D. & WALKER, J.M. (1990). Down-regulation of sigma receprors by chronic haloperidol. Prog. Clin. Biol. Ru.,328, 125-128.

MIDDLEMISS, D.N., BILLINGTON, D., CHAMBERS, M., HUTSON, PH., KNIGHT, A., RUSSELL, M., THORN, L., et d (1991). L-687,384 is a potent, selective ligand at the centrai sigma recognition site. Br. j. Pharmacol., 102-153.

MONNET, F.P., DEBONNEL, G., BERGERON, R., GRONIER, B. & DE MONTIGNY, C. (1991). The effects of sigma ligands and of neuropeptide Y on iV-rnethyl-D-aspanate-inducedneuronal activation of CA, donal hippocampus neurones are differentially affected by perrussin toxin. Br. j. Phamacol., 112, 709-713.

MONNET, F.P., DEBONNEL, G. & DE MON?IGNY, C. (1992). In vivo electrophysiological evidence for a selective modulation of N-methyl-D-aspartate-inducedneuronal activation in rat CA, dorsal hippocampus by sigma ligands. j. Pharmacoi. Exp. Ther., 261, 123-130.

MONNET, F.P., DEBONNEL, G., JUNIEN,J.L. & DE MONTIGNY, C. (1990). N-methyl-D-aspanate-induced neuronal activation is selectively modulated by sigma receptors. Ezrr. /. Phamacol., 179, 441145.

MUNETZ, M.R., SCHULZ, S.C., BELLIN, M. & HARTY,1. (1989). Rimcazole (BW234U) in the maintenance teatment of outpatients with schizophrenia. Dmg Dm.Res., 16, 79-83.

PAXINOS, G. & WATSON, C. (1986). The rat brain in stereotaxic coordinates. Academic Press., 2nd Ed., Orlando, Florida.

QUIRION, R., BOWEN, W.D., ITZHAK, Y., JUNEN, J.L., MUSACCHIO, J.M., ROTHMAN, R.B., SU, T.P.,et al (1992). A proposa1 for the classification of sigma binding sites. Trends Phannacol. Sci, 13, 85-86. REYNOLDS, G.P., BROWN, J.E. & MIDDLEMISS, D.N. (1991). [3~tolyIguanidinebinding to human brain a sites is diminished after haloperidol treatment. Eur. /. Phamcol., 194, 235-236.

RIVA, M.A. & CREESE, 1. (1990). Effects of chronic administration of dopamine receptor antagonists on Dl and D2 dopamine recepton and sigma hdoperidol-binding sites in rat brain. Mol. Nerrrophamuicol., 1-17.

ROMAN, F.J., PASCAUD, X., MARTIN, B., VAUCHE, D. & JUNIEN, J.L. (1990). 10-1784, a potent and selective ligand for rat and mouse brain sigma sites. j. Pham. Pbamcol., 42, 43940.

ROTHMAN, R.B., REID, A., MAHBOUBI, A., KIM, CH., DE COSTA, B.R., JACOBSON, A.E. & RICE, K.C. (1991). Labelling by [3w1,3-di(2-toly1)gwnidine of rwo high affinity binding sites in guinea pig brain: evidence for allo~teri~regdation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands. Mol. Phamzacol., 39, 222-232.

SNYDER, S.H. & LAKGENT, B.L. (1989). Receptor mechanisms in antipsychotic drug action: focus on sigma receptors. j. Neuroprychiatry Clin. Neurosci, 1, 7-15.

STEINFELS, G.F., ALBERICI, G.P., TAM, S.W. & COOK, L. (1988). Biochemical, behavioral and electrophysiologic actions of the selective sigma receptor ligand (+)pentazocine. Neuroprychophnnacology, 1, 321-327.

SU, T.P. (1991). Sigma recepton. Putative links between nervous, endocrine and immune systems. Eur. j. Biochem., 200, 633-642.

TAM, S.W. & COOK, L. (1984). Sigma opiates and cenain antipsychotic drugs mutually inhibit (+)[3H]SKF-10,047 and [3H]haloperidol binding in guinea pig bain membranes. Pmc. Nad. Acad. ScL USA, 81, 5618-5621.

TAYLOR, D.P. & DEKLEVA, J. (1987). Potentid antipsychotic BMY 14802 selectively binds to sigma sites. Dmg Dar. Ru., 1 1, 65-70.

WALKER, J.M., BOWEN, W.D., WALKER, F.O., MATSUMOTO, R.R., DE COSTA, B. & RICE, K.C. (1990). Sigma receptors: bioIogy and function. Phamtacol. Rev., 42, 355-402.

WEBER, E., SONDERS, M., QUARUM, M., MCLEAN, S., POU, S. & KEANA, J.F. (1986). 1,3-Di(2-15-3H]rolyl)guanidine: a selective ligand that labels sigma-type receptors for psychotomimetic opiates and antipsychotic dnigs. Proc. Nad. Ac&. Sci USA, 83, 8784-8788.

WEISSMAN, A.D., SU, T.P., HEDREEN, J-C. & LONDON, E.D. (1988). Sigma receptors in post-mortem human brain5.j. Phannacol. Fxp. Th.,247, 29-33.

WEISSMAN, A.D. & DE SOUZA, E.B. (1991). Chronic treatment of rats with the specific o ligand D-pentazocine fails ro modulate dopamine Dz and a binding in brain. Eux j. Pharmacol., 195, 163-165. JO-1784 (21-day treatment)

Figure 1 Responsiveness expressed as the nurnber of spikes geneated per nanocoulomb (nC:mean S.E.M.) of CA, dorsal hippocampus pyramidal neurons to micmiontophoreric applications of NMDA in control and treated rats for 21 days with the dose of 50, ZOO, 500, 1000 and 2000 pg/kg/day of 50-1784. * p < 0.05, using the Student's t test. Control DTG * (1 000 pglkglday x 21 days, SC.) r 1.0,

O 10 O 10 Current (nA) of DTG

Control DTG (1 000 pgkgiday x 21 days, s-c.)

O 20 O 20 Current (nA) of JO-1 784

Control (+) pentazocine (2 000 pg/kg/day x 21 days, s.c.)

O aW W Wz

U1W NMDA

NMDA + sigma ligand O 20 O 20 Current (nA) of (+)Pentazocine

Figure 2 Responsiveness expressed as the number of spikes generated per nanocoulomb (mean S.E.M.) of CA, donai hippocampus pyramidal neurons to microiontophoretic applications of NMDA before and during microiontophoretic applications of DTG (A), JO-1784 (B) and (+)pentazocine (C) at current of 10 nA in control and rats treated with high dose of DTG (A and B) or in control and rats treated with high dose of (+)pentarocine (C) for 21 days following a washout penod of two days. ' p < 0.05, using the Student's t test. Control DTG Control DTG (100 pgkglday x 21 days, s.c.) (1 00 pglkgiday x 21 days, s-c.) 8

O 10 O 10 O 20 0 20 Current (nA) of DTG Current (nA) of JO-1 784

Contml OTG Control DTG (100 pgBcg/day x 21 days. SC.) (100 pgkglday x 21 days. s.c.)

O 20 0 20 Current (nA) of (+)pentazocine

NMDA + sigma ligands

Figure 3 Responsiveness expressed as the number of spikes generated per nanocoulomb (mean S.E.M.) of CA, dorsal hippocampus pyramidal neurons ro microiontophoreric applications of NMDA before and during the microiontophoretic applications of DTG (A). JO-1784 (B) or (+)pentazocine (C) or before and afrer the intravenous administration of (+)pentazocine (D). * p c 0.05, using the Student's t test. Control JO-1 784 (200 pg/kg/day* x 21 days, s.c.)

Control JO- 1784 (200 pgkglday x 21 days, s.c.)

Control JO-1 784 (200 pg/kg/day x 21 days, s.c.)

% 0.8 0.8 O ctW 0.6 0.6

W Wz 0.4 0.4 0 NMDA Ws a 0.2 0.2 NMDA + sigma ligand

O O NMDA + sigma ligand + haloperidol (10 pg/kg, i.v.)

Figure 4 Responsiveness exprssed as the nurnber of spikes generated per nanocoulomb (mean S.E.M.) of CA, dorsai hippocampu pyramidal neurons to microiontophoretic applications of NMDA before, during and after the intravenous administration of a low dose of 50-1784 (A and B) and (+)pentazocine (C) in control and rats treated with a low dose of JO-1784 for 21 days following a two-day washout. The potentiation obtained following the intravenous administration of a cr ligand is reversed by the intravenous adminstration of 10 tcg/kg of haloperidol. * p < 0.05, using the Student's t test. ACh NMDA QUIS 8 -11 -4 eZ¶iI'==OiL--l

4 4 DTG HALOPERIDOL 1 pgkg, i-v. 10 pg/kg, i-v.

Control Haloperidol Haloperidol (200 pg/kg/day x 21 days, SC.) (2 000 pg/ke/day x 21 days. s.c.)

Current (nA) of DTG Current (nA) of DTG Current (nA) of DTG

Figure 5 A) Integnted firing rate histogram of CA, dorsal hippocampus pyramidal neurons showing the effects of microiontophoretic applications of ACh, QUIS and NMDA before and aher the intravenous administration of a low dose of DTG in rats treated with 200 &kg/day for 21 days of haloperidoi following a washout period of two days. Ban indicate the duation of applications for which the currents are given in nA. Each neuronal response ro each excitatory agent represents the computed-generated mean of the effects of three successive applications. The open circles (00) represent an interruption of the illustration of the continuous recording in this and the following integrated firing rate histognms. B) Responsiveness expressed as the number of spikes generated per nanocoulomb (mean S.E.M.) of CA, dorsal hippocampus pyramidal neurons to microiontophoretic applications of NMDA before and during microiontophoretic applications of DTG at current of 10 nA in control and treated rats with low or high dose of halopetid01 for 21 days. + p c 0.05, using the Student's t test. ACh NMDA QUIS 9 -1 1 -5 omr1u

Haloperidol (2 000 pglkglday x 21 days. s.c.)

Figure 6 A) Integrated firing rate histogram of CA, dorsal hippocampus pyramidal neurons showing the effects of microiontophoretic applications of ACh, QUIS and NMDA before and after the intravenous administration of two consecutive low doses of DTG in rats treated with 200 Ccg/kg/day for 21 days of hdopendol following a washout period of seven days. B) Responsiveness expressed as the number of spikes generated per nanocoulomb (mean S.E.M.) of CA, dorsal hippocampus pyramidal oeurons to microiontophoretic applications of NMDA before and after intravenous administration of (+)pentazocine in rats treated with low or high dose of haloperidol for 21 days, following a seven day washout. + p < 0.05, using the Student's t test. SPIKES GENERATEDlnC SPIKES GENERATEDlnC SPIKES GENERATEDlnC A JO-1784 (x 2 dayç, with minipurnpç on board)

DTG (x 2 days, with minipumps on board)

CONTROL 1 pgkg

Figure 8 Responsiveness expressed as the number of spikes generated per nanocoulomb (nC:mean S.E.M.) of CA, dorsal hippocampus pyramidal neurons to microiontophoretic applications of NMDA in control and treated rats for two days with the dose of 1, 10, 100 and 1000 pg/kg/day by osmotic minipumps with 50-1784 (A) or DTG (B). The effect of microiontophoretic applications of NMDA in the group of rats treated with 10 pg/kg/day of DTG codd not be determined as they induced epileptoid activity. Experiments were carried with the minipumps on board. + p < 0.05, using the Student's t test. CHAPTERVIII Potentiation of neuronal NMDA response induced by dehydroepiandrosterone and its suppression by progesterone: Effects mediated via sigma recepton

The search for endogenous o receptor ligands has been the interest of research of several groups in the lasr decade. Some sreroids, pmicularly progesterone, have been reported to have high affinity at the o receptors. Steroid binding at the o receptors suggests that these may mediate some aspects of steroid-induced mental disturbance. To date, no funaional studies on the effect of progesterone and other neuroactive steroids at the o receptors have been undertaken.

The purpose of this study was to determine the effect of neuroactive steroids on the NMDA response. Our hypothesis was that if progesterone behaves as an agonist by potentiating the NMDA response, then this potentiation could be reversed by a selective antagonist such as NE-100. Altematively, if progesterone behaves as an antagonisr then it should suppress the potentiation induced by selective a ligands such as DTG. In Our paradigm neuroactive steroids thar have no affinity at the o receptors, such as pregnenolone and pregnenolone-sulfate, should have neither an agonist nor antagonist effect. 204 POTENTIATION OF NEURONAL NMiDA RESPONSE INDUCED BY DEHYDROEPIANDROSTERONE AND ITS SUPPRESSION BY PROGESTERONE: EFFECTS MEDIATED VIA SIGMA RECEPTORS Richard Bergeron, Claude de Montigny and Guy Debonnel

The Journal of Neuroscience, 16(3): 1193-1202, 1996

We have previously shown that low doses of selective sigma (a) receptor ligands potentiate the excitatory response of pyramidal neurons to NMDA in the CA3 region of the dorsal hippocampus in the rat. Since progesterone competitively displaces the binding of the o ligand [3H](+)SKF-10,047,the present studies were undertaken to determine in vivo the effect of neuroactive steroids on the NMDA-induced excitation of rat CA, pyramidai neurons. Low doses of DHEA potentiated selectively and dosedependently the NMDA response. The effect of DHEA was reversed by the selective cr antagonist NE-100 and by haloperidol, but not by spiperone. Progesterone had no effect by itself but revened, at low doses, the potentiation of the NMDA response induced by DHEA, as well as those induced by non-steroidd o ligands. Neither pregnenolone nor pregnenolone sulfate had any effea on the NMDA response, nor did they antagonize the potentiation of the NMDA response induced by DHEA and by non-steroidal o ligands. A pertussis toxin pretreatment, which inactivates Gd,-proteins, abolished the potentiating effects of DHEA. Ovariectomy enhanced the potentiation of the NMDA response by the non-steroidal a ligand DTG. There was a reciprocal occlusion of the effects of DHEA and DTG: DTG did not further potentiate the NMDA response following DHEA, and DHEA did not do so following DTG. These results suggest chat some neuroactive steroids modulate the NMDA response via a receptors.

Key words: Neurosteroids, Hippocampus, Electrophysiology, Ovariectomy, Haloperidol, DTG

INTRODUCTION

Sigma (O) receptors are present in high density in the central nervous system (Walker et 4.1990) and in peripheral organs with a particularly high density in the ovaries (Wolfe et d.1989). A wide variety of psychotropic drugs such as antipsychotics and antidepressants have high affinity for o receptors (Su, 1982; Snyder and Largent, 1989; Schmidt et d.1989; Itzhak and Kassim, 1990; Ferris et al. 1991). Several sreroids, in particular progesterone, also bind with high affinity to a receptors (Su et d.1988). Previous studies have demonstrated that low doses of selective a ligands, such as DTG [di@-tolyl)guanidine](Weberetal. l986), L-687,384[î-ben~lspiro[l,2,3,4-tetr&ydro-aphthalene- 1,4-piperidine]] (Middlemiss et al. 19%; Barnes et al. 1992),50-1784 [(+)N-cyclopropylmethyl-N- methyl-î,4-diphenyl-i-ethyl-but-3-en-I-ylaminehydrochloride] (Roman et al. 1990) and (+)pentazocine (Steinfels et d.1988) potentiare selectively the response of rat CA, dorsal hippocarnpus pyramidal neurons to microiontophoretic applications of N-rnethyl-D-aspartate (NMDA) (Monnet et al. 1990; Monnet et d. 1992; Martin et al. 1992; Walker and Hunter, 1994). This effect is reversed by other a ligands such as hdoperidol, (+)3-PPP [(3-hydroxyphenyll-N- (1-p ropy 1) piperidine] and BMY- 14802 [a-(4-fluorophenyl)+(5-fluoro-2-pyrimidinyl)- 1-piperazine butanol] (Tm and Cook, 1984; Largent et d.1984; Taylor and Dekleva, 1987), but not by spiperone which has a binding profile similar to that of halopend01 except for its low affinity for a receptors (Monnet et 4.1990; Monnet et d.1992). We have also recently tested the novel and very selective o ligand NE- 100 ~dipropyl-2-(4-methoxy-3-(2-p henylet ho)pheny1)- ethylamine monohydrochloride] (Okuyama et al. 1993). It proved extremely potent in blocking or revening the potentiation of the NMDA response induced by o ligands such as DTG (Debonnel et al, 1995). Therefore, a ligands acting like DTG have been tentatively denoted o

"agonias " and o ligands acting like halo pend01 "antagonistsfl (Monnet et al. 1990). These data and several other reports from other laboratories using biochernical, neuroendocrinological and behavioral models suggest the existence of a functional interaction between a and NMDA receptors even though the exact mechanism is not fuily understood (for review see: Debonnel, 19%). There are several types of o receptors; those classified as o, and a, have been best characterized (Quirion et al. 1992). The most commonly used a ligands, including haloperidol and DTG, do not discriminate between a, and o, receptors (Quirion et al. 1992), whereas drugs such as (+) pentazocine, 50-1784, L-687,384, (+)SKI?-10047 [N-dlyl-normetazocine] and NE-100 are selective for a, receptors (Quirion et al. 1992; Chaki et al. 1994). However, recent data obtainedn in our laboratory have shown that a pertussis toxin (PTX) pretreatment abolishes the potentiation induced by JO-1784, but not that induced by (+ ) pentazocine (Monnet et d.1994), suggesting that these two o ligands act on different o receptor subtypes. Moreover, the injection of colchicine in the dentate gyrus, which destroys the mossy fiber system (a major afference to CA, pyramidal neurons), abolishes the potentiation of the NMDA response induced by DTG and 50-1784 but not that of (+)pentazocine, suggesting that the subtype of o receptors on which DTG and 50-1784 are acting is located presynaptically, on the mossy fiber terminal whereas the subtype of a receptors on which (+)pentazocine is acting is located postsynaptically. Several reports have shown that progesterone acts as a cornpetitive inhibitor of [WJ(+)SKF-10,047 or [WJhaloperidol (Su et al. 1988; McCann et al. 1994; Yarnada et al. 1994; Rarnarnoorthy et al. 1995) at o receptors and have prompted us to assess the potential agunistic or antagoninic properties of progesterone, dehydroepiandrosterone (D HEA) and or her neuroactive steroids (Paul and Purdy, 1992) in an in vivo electrophysiological paradigm.

MATERIALS AND METHODS The experiments were carried out in vivo in the CA, region of the rat dorsal hippocampus where the responsiveness of pyramidal neurons to microiontophoretic applications of NMDA, quisqualate (QUIS) and acetylcholine (ACh) was assessed using extracellular unitary recordings.

Prtparation of animals Male Sprague-Dawley rats weighing 200-250 g were obtained from Charles River, (St- Constant, Québec, Canada). Females of the same weight were obtained at days one or three of the menstrual cycle or two weeks following ovariectomy (OVX). Rats were housed three to four per cage with free access to food and water. They were maintained at constant temperature (25OC) under a 12:12h 1ight:dark cycle.

Pertassis pretreatmmt The pretreatment with PTX (1 pg in 2 pl of physiological saline; Sigma Chemical CO, St-Louis, MO, U.S.A.) consisred in loweriq unilaterdly the tip of a 5 pl Hamilton syringe into the right dorsal hippocampus of chloral hydrate-anesthetized rats at A: 4.5, L: 4 and D:4 according to the atlas of Paxinos and Watson (1986). Pertussis toxin was slowly injected over a period of 5 min. Control rats were injected an equal volume of physiological saiine under the same conditions. In vivo electrophysiological experiments were carried out 5-7 days later. 207 hgs The following substances were used: DTG (Aldrich, Milwaukee, WI, USA); 50-1784, a generous gift from J.L. Junien (Institut de Recherche Jouveinal, Fresnes, France); L-687,384, a generous gift from L.L. Iversen (Merck-Sharp and Dhome, Tyler Park, UK); NMDA and acetylcholine (ACh) (Sigma, Milwaukee, Wi, USA); quisqualate (QUIS) (Tocris Neuramin, Buckburst Hiil, Essex, UK); halopend01 (McNeil Laboratories, Stouffville, Ontario, Canada); spiperone,(+)pentazocine, progesterone, pregnenolone, pregnenolone-sulfate and DHEA @BI, Natick, MA, USA); NE-100, a generous gifi from S. Okuyama (Taisho P harmaceutical, Ohmiya, Japan) .

Preparation of micropipettes Microiontop ho retic applications and extracellular unitary reco rdings were performed with five-barrelled glas micropipettes prepared in a conventional manner (Haigler and Aghajanian, 1974). Three of the side barrels used for microiontophoresis were filled with NMDA (10 mM in 200 rnM NaCl, pH: 8), QUIS (1.5 mM in 400 rnM NaCl, pH: 8) and ACh (20 mM in 200 mM NaCl, pH: 4). The remaining side barrel, used for automatic current balancing, and the central barrel, used for extracellular unitary recording, were filled with a 2 mM NaCl saturated with Fast Green FCF (Aldrich, Milwaukee, WI, USA).

Recordings from CA, dorsal hippocampus pyramidal neurons For the electrop hysiological experiments, rats were anest hetized wit h uret hane (1.25 g/kg, i.p.) and mounted in a stereotaxic apparatus. Body temperature was maintained at 37°C throughout the experiment. After removal of the dura mater, the micropipette was lowered into the CA, region of the dorsal hippocampus (L: +4.2 mm and A +4.2 mm from lambda and H -3.5 to -4.5 mm from the cortical surface. Pyramidal neurons were identified by to their long duration (0.8-1.5 ms) and large amplitude (0.5-2 mV) action potentials, and by the presence of characreristic complex spike discharges aiternating with simple spike activity (Kandel and Spencer, 1961). Neuronal firing activity was monitored on oscilloscope after signal magnificarion by a high input-impedance amplifier. Action potentials were detected by a differential amplitude discriminator generating square pulses which were stored in an on-line computer and forwarded to a paper chart recorder generating inregrated firing rate histograms. Alternate microiontophoretic applications of 50 s of each excitatory substance (NMDA, QUIS and ACh) separated by 50 s retention periods were carried out continuously dunng the whole duration of the recordkg. The duration of the microiontophoretic applications and the intensity of the currents used were also nored in the computer. The effects of their applications on pyramidal neuron firing activity were expressed as the number of spikes generated/nanocoulomb (1 nC being the charge generated by 1 nA applied for 1 s). After a neuron was isolated, it was recorded for a period of at least 20 min before any drug was injected. The recording was stored on computer without interruption for the whole duration of the experiment. Five to six applications of each excitatory substances (NMDA, QUIS, ACh) were carried out before the studied drugs were injected or applied microiontophoretically . The effects of dmgs studied appeared within a period of 10 min after their intravenous injection. The calculations were carried out when the maximal effect of the drug was achieved (within the first 20 min). At the end of the experiment, a -27 FA current was passed for 20 min. through the central barre1 to deposit Fast Green FCF for subsequent histological verification of recording sites.

Experimmtal series Neuroactive steroids were prepared in a solution of 40% polyethylene glycol. The

Stat ist ical analyses Al1 results are expressed as means SEM of the number of spikes generatedhc of NMDA or QUIS or Ach. Statistical significance was assessed using the Student's t test with the Dunnett's correction for multiple comparisons. Covariance analysis were used to compare the degree of the potentiation of the NMDA response induced by DTG (1 pg/kg, i.v.) in male and in non-OVX and OVX female rats. Statistical differences smaller than 0.05 were considered as significant.

RESULTS Al1 recordings were obtained from the stratum pyramidale of the dorsal hippocampus CA3 region, as confirmed by histological verification of the Fast Green FCF deposit left at the end of each experiment. The intravenous injection of the vehicle (40% polyethylene glycol), used for preparing the solutions of neuroaaive steroids, affected neither the spontaneous firing activity of dorsal hippocampus CA, pyramidal neurons nor their response to NMDA, QUIS and Ach.

Effects of DHEA The intravenous administration of 100 pg/kg of DHEA induced a two-fold increase of the response of dorsal hippocampus CA, pyramidal neurons to microiontophoretic applications of NMDA without affecting their response to QUIS (Fig. LA). The effect of DHEA was dose- dependent and a maximal potentiation was obtained at a dose of 500 pg/kg, i.v. (Fig. IB). No further potentiation was obtained by increasing the dose of DHEA up to 2 mg/kg, i.v. (Fig. 1). This enhancing effect of DHEA lasted for more than 40 min. The potentiation of the NMDA response by DHEA was reversed by hdopendol(20 pg/kg, i.v.; Fig. lC), but not by spiperone (20 g/kg, i.v.; Fig. ID) and not by the intravenous administration of saline (Fig. 1E). Moreover, NE-100, a novel selective o antagonist (Okuyama et d.1993), at the dose of 25 &kg, i.v., also suppressed the potentiation of the NMDA response induced by DHEA (Fig. 14.

Effects of progesterone At doses ranging from 1 to 2000 pg/kg, i.v., progesterone affected neither the neuronal response induced by microiontophoretic applications of NMDA (Fig. 2A,B) nor by that of QUIS or Ach. However, progesterone, at the dose of 20 pg/kg, i.v., prevented and reversed the potentiation of the NMDA response by DHEA (250 &kg, i.v.; Fig. 2C). To investigate the possibility that this effea of progesterone could be mediated via o receptors, low doses of the selective cr ligands DTG (1 pg/kg, i.v.; Fig. 3), L-687,384 (1 pg/kg, i.v.), 50-1784 (5 pg/kg, i.v.) and (+)pentazocine (IO pg/kg, i.v.) were administered to naive rats as illustrated for DTG in figure 3. Progesterone (20 pg/kg, i.v.; Fig. 4A,B,C,D) revened the potentiation induced by the non-steroidal CF ligands. Halopend01 (20 pg/kg, i.v. Fig. 4E), but not spiperone (20 pg/kg, ix.; Fig. 4k), also reversed the effect of DTG, as previously reported (Monnet et d.1990; Bergeron et al. 1993).

Effects of pregnenolone and pregnmolone sulfate The effects of pregnenolone and pregnenolone sulfate were assessed in the same paradigm. At doses ranging from 1 to 2000 pg/kg, i.v., rhese two neuroactive steroids did not modify NMDA-, QUIS- or ACh-induced neuronal responses. The efficacy of these two neuroactive steroids in reversing the potentiation of the NMDA response induced by a prior administration of DHEA (100 &kg, i.v.), as well as by other non-steroidal a agonists, was also tested. Pregnenolone and pregnenolone sulfate neither reversed nor prevented the potentiation of the NMDA response induced by DHEA (Fig. 5A,B). Similarly, neither pregnenolone nor pregnenolone sulfate suppressed the potentiation of the NMD A response induced by the non-steroidal cr agonists DTG (1 pg/kg, i.v.; Fig. 5C,D), L-687,384 (1 pg/kg, i.v.), 50-1784 (5 pg/kg, i.v.), and (+)pentazocine (IO pg/kg, i.v.) (data not shown for the latter three compounds). Efect of coadministration of DHEA and DTG To put to the test the notion that DHEA and non-steroidal a ligands rnight both activate o receptors, we have examined the possibility of a reciprocal occlusion of the effeas of DHEA and DTG. The microiontophoretic applications of DTG (20 nA) produced, as previously observed (Monnet et a1.1990; Bergeron et a1.1995), a two-fold increase of the neuronal response to NMDA. The injection of 200 pg/kg i.v. of DHEA failed to furcher increase the NMDA response. Conversely, the intravenous administration 200 pg/kg of DHEA also induced a three-fold increase of the neuronal response to NMDA and the subsequent rnicroiontophoretic applications of DTG (20 nA) failed to further increase the NMDA response (Fig. 6).

Efect of a pertussis toxin pretreatmmt on the potmtîution induced by DHEA Pertussis toxin, which inactivates Guoproteins by ADP-ribosylation, was used to assess the possible involvement of these proteins in the modulation of NMDA-induced neuronal activation in the CA, region of the rat dorsal hippocampus by DHEA. The in vivo PTX pretreatment affected neither the spontaneous firing activity of CA, pyramidal neurons nor their responsiveness to NMDA, QUIS nor ACh, in keeping with previous data (Monnet et al. 1994). DHEA, at the dose of 250 &kg i.v. (a dose producing a greater than two-fold increase of the NMDA response in control rats) failed to produce any potentiation of the NMDA response in PTX-pretreated rats (Fig. 7 A,B). We have previously reported rhat a PTX pretreatment abolishes the potentiation of the NMDA response by 50-1784 but not that induced by (+)pentazocine, this effect of (+)pentazocine being still revened by a low dose of hdopendol (Monnet et d.1994). In the present series, the potentiating effect of (+)pentazocine (IO Irg/kg, i.v.) was still present in PTX-treated rats; this effect of (+)pentazocine was readily reversed by progesterone (20 pg/kg, i.v.; Fig. 7 C,D).

Effect of ovariectorny on the potentiation induced by DTG In the last series of experiments, we measured the magnitude of the potentiation induced by DTG (1 pg/kg, i.v.) in male rats and in female rats at day one and three of menstrual cycle, as well as two weeks following OVX. As shown in figure 8, no signifiant difference was found between the degrees of potentiation induced by DTG in male rats and non-OVX female rats at either day one or day three of menstrud cycle. In these three groups, the potentiation of the NMDA response induced by DTG was completely suppressed by progesterone (20 pg/kg, i.v.) and by halopend01 (20 adkg, i.v.; Fig. 8). However, the degree of the potentiation produced by DTG in OVX rats was significantly greater than those observed in the three other groups (Fig. 8). Moreover, in OVX rats, one dose of 20 pg/kg, i.v. of progesterone reversed only partially DTG-induced potentiation. Such a dose of progesterone completely reversed the effect of DTG in male and non-OVX females rats (Fig 8). In OVX rats, a second dose of 20 &kg, i.v. of progesterone or a subsequent injection of 20 pg/kg, i.v. of halopend01 were required to obtain a complete suppression of the potentiation of the NMDA response induced by DTG (Fig. 8D).

DISCUSSION The present ïesults indicate that DHEA, at very low doses, potentiates selectively, in a dosedependent manner, the neuronal response to microiontophoretic applications of NMDA onto pyramidal neurons in the CA, region of the rat dorsal hippocarnpus (Fig. 1). This potentiation is reversed by NE-100, by halopendol but not by spiperone nor by saline (Fig. 1). Progesterone, at doses ranging from 1 pg to 2 mg/kg i.v., does not modify by itself the NMDA response, but suppresses at the very low dose of 20 ag/kg, i.v. the potentiation of the neuronal response to NMDA induced by DHEA as well as by several non-steroidal a ligands (Figs. 24). Pregnenolone and pregnenolone-sulfate neither modify the NMDA response nor prevent or suppress the potentiation of the NMDA response induced by DHEA and by non- steroidal a agonists (Fig. 5). Several interactions between the NMDA receptor complex and some neuroactive steroids have been documented (Wu et al. 1991; Irwin et al. 1992; Maione et al. 1992; Bowlby, 1993). In particular, pregnenolone sulfate augments NMDA receptor-mediated elevations in intacellular Ca2+in cultured rat hippocampal neurons, presumably via a porenriation of the glutamatergic activation of the NMDA receptor (Irwin et al. 1992). In this preparation, NMDA receptor activation produces a greater inward curent of Ca2+ in the presence of pregnenolone sulfate; this effea has been attributed to a direct action of the steroid on the NMDA receptor complex (Bowlby, 1993). Pregnenolone sulfate also enhances NMDA-gated currents in spinal cord neurons and significantly increases the proconvulsant activity of NMDA (Maione et al.1992). The mechanisms whereby neurosteroids affect glutamatergic transmission are not completely elucidated. A growing body of evidence suggests that many neuroactive steroids can rapidly alter the excitabilit~of neurons via a modulation of GABA, receptors acting as agonists (e.g. pregnenolone sulfate and oestrogen) or as antagonists (e.g. progesterone) (Majewska et al. 1986; Majewska and Schwartz, 1987; Majewska et al. 1990; Purdy et al. 1991; Lambert et al. 1987; Morrow et al. 1990; Mienville and Vicini, 1989; Wu et al. 1990). The interactions obsenred in the present study are unlikely to be related to a GABA, receptor modulation since pregnenolone and pregnenolone sulfate were inactive in Our mode1 (Fig. 5)) whereas these two neuroactive steroids are among the most active in paradigms involving the GABA, receptors (for review see: Baulieu, 1991). Other mechanisrns have aiso been suggested such as the existence of steroid recognition sites on the NMDA receptor complex itself (Irwin et al. 1992). However, such a mechanism could not account for the effect of progesterone in Our paradigm since it did not modify the NMDA response. Nonetheless, the possibility of a downstream action of progesterone at the level of effector mechanisms triggered by o receptor activation cannot be ruled out at present. Several observations suggest that the selective modulation of the NMDA response by the neuroactive steroids reported here is mediated via cr receptors. First , the potentiation induced by DHEA is suppressed by haloperidol but not by spiperone (Fig. IC). Indeed, spiperone binds with high affinity to dopaminergic, al-adrenergic, and serotonergic receptors as does hdopendol (Bun et al. 1977; Clark et al. 1985), but it has a low affinity for a binding sites (Su, 1982; Tarn and Cook, 1984; Weber et al. 1986; Steinfels et al. 1989). Second, a low dose (25 pg/kg, i.v.) of the selective o antagonist NE-100 also suppresses the potentiation of the NMDA response induced by DHEA (Fig. If), as well as the potentiation induced by non- steroidal o ligands (Debonnel et al, 1995). Third, low doses of progesterone prevent and suppress not only the potenriating effect of DHEA, but also those induced by the selective non-steroidal cr ligands DTG, 50-1784, L-387,684 and (+)pentazocine (Figs. 2-4). Fourth, pregnenolone and pregnenolone sulfate, which have low affinity for o receptors (Su et al. 1988), neither prevent nor suppress the potentiation of the NMDA response by DHEA and by the non-steroidal a agonists even when adrninistered at doses up to 2 mg/kg, i.v. Moreover, Our hypothesis is supported by a recent report showing that neuroactive steroids, via u receptors, modulate the ['H]norepinephrine release evoked by NMDA in the rat hippocampus (Monnet et al. 1995). Following the intravenous administration of DHEA (200 pg/kg), microiontophoretic application of DTG (20 nA) was ineffeaive in further enhancing the NMDA response Pig. 6). Moreover, when DTG was applied microiontophoretically (20 nA), the intravenous injection of DHEA was ineffective in further enhancing the NMDA response (Fig. 6). The occurrence of these bilateral occlusion phenornena provides further evidence that the potentiation of the NMDA response by DHEA is mediated by a receptors. We have previously reported t hat several non-steroidal

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[71 control Control DHEA (250 pg/kg. Lv.) DHEA (250 pog.i-v.)

Haloperidol (20 vglkg. I.v.) Spiperone (20 pog. 1.v.) F

Control 1DHEA (250 pghg. i-v.)

Saline

Figure 1 A) Integnted firing rate histogram of a CA, dorsal hippocampus pyramidal neuron illustrating the effects of microiontophoretic applications of NMDA and QUIS before and after the injection of DHEA, and after the subsequent administration of haloperidol. In this and the subsequent continuous integrated firing histograms, bus indicate the duration of applications for which currenis are given in nA. Open circles (00) represent an interruption of the illustration of the continuous recording in this and the following integrated firing rate histograms. Figure 1 (continued)

B) Dose-response curve of the effect of the intravenous administration of DHEA on the neuronal activation of CA, dorsal hippocampus pyramidal neurons induced by microiontophoretic applications of NMDA. Each dot represents the effea of one dose of the dnig administered to one rat whiie recording from one neuron in this and subsequent dose-response cwes. The effect was assessed by determining the ratio (N2/NI) of the number of spikes genented/nC of NMDA before (Nt) and after (Nz) the injection of the drug.

C) Responsiveness, expressed as the nurnber of spikes gener;ited/nC (mean * SEM) of CA, donal hippocampus neurons to rnicroiontophoretic applications of NMDA before (open colurnns) and after (grey columns) the administration of DHEA and after the subsequent administration of haloperidol (dark columns). The number at the bottom of the first columns indicates the number of neurons tested (one neuron per rat, in this and subsequent bar chart histograms). * p < 0.05, using the paired Student's t test.

D) Responsiveness, expressed as the number of spikes generated/nC (mean f SEM) of CA, dorsal hippocampus neurons CO microiontophoretic applications of NMDA before (open columns) and after (grey columns) the administration of DHEA and after the subsequent administration of spiperone (dark coiumns). The number at the bottom of the first columns indicates the number of neurons tested (one neuron per rat, in this and subsequent bar chan hisrognms). * p < 0.05, using the paired Student's t test.

E) Responsiveness, expressed as the number of spikes generated/nC (mean SEM) of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (open columns) and after (grey columns) the administration of DHEA and ;ifter the subsequent adrninistration of saline (dark columns). * p < 0.05, using the paired Student's t test.

F) Responsiveness, expressed as the number of spikes generated/nC (mean + SEM) of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (open coiurnns) and after (grey columns) the administration of DHEA and after the subsequent administration of the selective CT antagonist NE- 130 (dark columns). * p < 0.05, using the paired Student's t test. A ACh QUIS NMDA 9 -5 -11

4 4 2 min

Dose (pg/kg. i.v.) of progesterone

Figure 2 A) Integrated firing rate histogram of a CA, dorsal hippocampu pyramidal neuron illustrating the effect of microiontophoretic applications of NMDA, QUIS and ACh before and after the injection of progesterone and after the subsequent injection of hdoperidol (see legend to Figure 1). B) Dose-response curve of the effect of the injection of progesterone on the neuronal activation of CA, dorsal hippocampus pyramidal neurons induced by microiontophoretic applications of NMDA. C) Responsiveness, expressed as the number of spikes generated/nC (mean SEM), of CA, dorsal hippocampus pyramidal neurons to microiontophoretic applications of NMDA before (open columns) and after (grey columns) the administration of DHEA or progesterone and after the subsequent administration of either steroids (dark columns). + p < 0.05, using the paired Student's t test. ACh QUlS NMDA 7 -2 -12

Progesterone ACh QUIS NMDA 20 pgikg. i.v. 7 -2 -12

mr~CLZ]~~~U[L~~~(ZDO

- 1 min

Figure 3 Continuous integrated firing rate histogram of a CA, dorsai hippocampus pyramidal neuron illustrating the effect of microiontophoretic applications of NMDA, QUIS and ACh before and afrer the administration of DTG and after the administration of progesterone. Time base applies to the three traces. O controi

Progesterone (20 vgtkg, i.v.1 Progesterone (20 p@g. i-v.)

El Convoi L-687384 (1 pgkg. i-v.)

Progesterone (20 pglkg, i.v.)

Contrai

DTG (1 peg,i-v.)

Halopefidol (20 pgkg, i.v.1 Spiperone (20 vgikg. i.v.1

-- -- Figure 4 Responsiveness, expressed as the number of spikes generated/nC (mean 2 SEM), of CA, dorsal hippocampus pyramidal neurons to microiontophoretic applications of NMDA before (open colurnns) and after (grey colurnns) the intravenom administration of DTG (A), JO-1784 (B), (+)pentazocine (C),L-687,384 (D) and after the subsequent intravenous administration of progestemne (A-D), halopend01 (E) or spiperone (F) (dark colurnns). * p < 0.05, using the paired Student's t test. Conb'oi

gjl DHEA (250 p@g. i.v.1

Pregnenolone sulfate (250 pgkg. i.v.1

ContmI convoi Pregnenolone (250 pgkg. i.v.1 DTG (1 pg/kg. i.v.)

Dl G (1 ciqkg. i-v.) Pregnenolone sulfate (250 pg/kg. i.v.)

Figure 5 Responsiveness, expressed as the number of spikes genented/nC (mem ISEM) of CA, dorsal hippocarnpus pyramidal neurons to microiontophoretic applications of NMDA before (open columns) and aher (grey columns) the administrarion of DHEA (A), pregnenolone (B and C) or DTG (D) and aher the subsequent administration of pregnenolone sulfate (A and D), DHEA (B) or DTG (C) (dark columns). * p < 0.05, using the paired Student's t test.

[7 Connoi (rnak) C! Control (female. day 1 ) 5 DTG (1 pgkg .i.v.) Progesterone (20 pgkg. i.v.1 Progesterone (20 pgBrg. 1.v.)

Control (fernale, day 3) 0 14 days following OVX DTG (1 @cg,i.v.)

Progesterone (20 pykg. i.v.) Progesterone (20 cig'kg. i.v.1

Halopendol (20 ugkg. i.v.1

Figure 8 Responziveneçs, expressed as the number of spikes generatednc (mean f SEM), of CA, donal hippocampus pyramidal neurow to microiontophoretic applications of NMDA before (open columns) and after (grey columns) the administration of DTG and after the subsequent administration of progesterone (dark colurnns) and hdoperidol (black column). * p < 0.05, using the paired Student's t tesr. + < p 0.0001, comparing the effect of DTG to those in male and non-OVX female rats using covariance analysis. CHAPTER M Pregnancy markedly reduces the brain sigma receptor function

The finding that progesterone behaves as a potent antagonist and DHEA as an agonist at rhe a receptors raised several imporrant cruciai questions that may have clinicd implications in the treatment of some neuroendocrinologicai disorders such as post-partum depression and premenstrud dysp horic disorder.

The goal of this 1st study was to determine if the potentiating effects of cr ligands may be modified during pregnancy and post-partum period. Since the level of progesterone increases significantly in late pregnancy, we anticipated a reduction of the potentiating effect of DHEA and other selective a agonists, such as DTG and (+)pentazocine. In addition, there is a rapid drop in the levels of progesterone following delivery. The effect of DHEA and DTG will also be tested in puerperium. Our hypothesis was that 5, 10 and 15 days postpartum cr agonists should poduce their enhancing effect sirnilar to the effects observed in control rats. 229 PREGNANCY MARi;LEDLY REDUCES BRAIN SIGMA RECEPTOR FUNCTION Richard Bergeron, Claude de Montigny and Guy Debonnel

Report subrnitted to Scierce

SUMMARY A large percentage of women, with or without psychiatric history, experience emotional disturbances during post-partum period, whereas others suffering from psychiatric disorders improve during pregnancy. It has been generally assumed that the hormonal changes occurring dunng these periods may underlie these phenomena. We have previously reported that selective sigma (o) ligands, such as DTG and (+)pentazocine as well as the neuroactive steroid DHEA, at very low doses, act as "agonists" since they potentiate the excitatory response of pyramidal neurons to NMDA in the CA, region of the rat dorsal hippocampus via the activation of a receptors. Other a ligands such as hdoperidol and the selective o ligand NE-IO0 act as "antagonists" since they reverse the potentiations induced by DTG and DHEA. Moreover, low doses of progesterone suppress the potentiating effects of DTG (+)pentazocine and DHEA. Prompted by these latter findings, we investigated the effect of a "agonists" in female rats in late pregnancy (at which time progesterone levels are very high) and in the posr- partum period (at which cime there is an abrupt fa11 of the levels of progesterone). At Day 18 of pregnancy, ten-fold higher doses of DTG, (+)pentazocine and DHEA were required to obtain a selective potentiation of the NMDA response compared to non-pregnant female rats. Conversely, the potemiation of the NMDA response induced by the a "agonist" DTG was greater at 5 days post-partum than in control fernales. Ten and 15 days after delivery, the function of a receptors had normalized. The present data suggest t hat endogenous progesterone might act as a potent "antagonist" at the o receptors and changes in the function of o receptors occurring during this period may be implicated in emotional disturbances found in the post- partum period.

Sreroid hormones derive from cholesterol and emerged early in phylogenesis as growth regulators. They diversified later to sex steroids, gluco- and minerdocorticoids. Steroid hormones ensure maintenance of the organism's homeostasis, adaptability to the environment and reproductive capabilities (1). The high lipophilicity of steroids ensures their penetration of biologicai membranes, enabling access to al1 cells and organs, including the central nervous system (CNS). Steroid hormones which interact wit h the CNS (denoted neuroactive steroids) (1) produce a diversity of both rapid and delayed neuroendocrine and behavioral effects. Some of the effects of the neuroactive steroids in the CNS occur within minutes to hours and are mediated via the interaction with intracellular receptors (2). In 1976, Martin and CO-workerspostulated the existence of a receptors to account for the psychotomimetic effeas (hallucinations, depersondisation and excitement) of certain benzomorphans such as N-allyl-normetazocine (SKF 10,047), the prototypical o "agonisr" (3). Much of the interest in a receptors resulted from the early hypothesis that they may be involved in the pathophysiology of psychosis (4). The finding that progesterone can displace competitively, at nM concentrations, the binding of [3~(+)SKF-10,047to a receptors raised the possibility that progesterone may act as an endogenous ligand of the a receptors (5). Previous studies in our laboratory have shown that very low doses (pg/kg) of selective a ligands, such as DTG [di(2-toly1)guanidinel and (+)pentazocine, potentiate selectively the neuronal response of rat CA, dorsal hippocarnpus pyramidal neurons to microiontophoretic applications of N-methyl-D-aspartate (NMDA) (6). This effect is reversed by other a ligands such as the seleaive o "antagonist" NE-100 and haloperidol but not by spiperone which has a binding profile sirnilar to that of haloperidol except for its low affinity for cr receptors. We have recently reported that progesterone has no effect on the NMDA response when administered intravenously (1-1000 pg/kg, i.v.) but suppresses, at low doses (20 pg/kg, i.v.), the potentiation induced by selective a "agonists" such as DTG. We have also reported that the neuroactive sreroid dehydr~e~iandosterone(DHEA) potentiates selectively, in a dose- dependent manner, the neuronal response to NMDA. This potentiation is reversed by low doses of progesterone, NE-100 and haloperidol. Furthermore, in female rats, two weeks following ovariectomy, the degree of the potentiation of the NMDA response induced by DTG is significantly higher than in control female rats, suggesting that a receptors might be tonically inhibited by endogenous progesterone (7). Pregnancy in humans tnggers alterations of mood. Typicdly, during the last two trimesten of pregnancjr, many women report feelings of elation and tranquillity. Ar this point in time, they present a lower risk of developing emotional disturbances (8). In contrast, during the puerperal period, there is a higher incidence of psychiatric disorders (8). During pregnancy, 23 1 the levels of progesterone as well as those of other steroids are the highest ever in a woman's life (9). The plasma Ievels of progesterone in pregnant rats are increased by six-foid in late pregnancy but fdl very rapidly after deiivex-y (IO). The present experiments were undenaken to determine if these variations in the concentrations of progesterone could modify the funcrional properties of o receptors in modulating the neuronal NMDA response using an electrophysiological paradigm in the CA, region of the rat dorsal hippocampus (11). In dl experïments, cr ligands did not modify the spontaneous firing rate of CA, pyramidal neurons nor their responses to quisqualate (QUIS) and acetylcholine (ACh) as previously reporced (6).Moreover, the baseline responses to NMDA, QUIS and ACh were not altered in pregnancy and in post-parcum period (12). In control female rats, the dose of 1 pg/kg, i.v. of DTG induced a three fold increase of the NMDA response (figure lA), whereas the effect of the sarne dose of DTG was completely ineffective at Day 18 of pregnancy (figure IB). At that time, the administration of a dose of 10 &kg, i.v. of DTG induced only a two fold increase of the NMDA response (figures IB-2A). A dose of (+)pentazocine ten fold higher than in non-pregnant rats was also required to produce a potentiation of the NMDA response in pregnant rats (figure 2B). These potentiarions of the NMDA response obtained in pregnant rats with the dose of 10 pg/kg, i.v. of DTG or 100 pg/kg, i.v. of (+)pentazocine were suppressed by halopend01 (20 pg/kg, i.v.; figure 1A) and by progesterone (20 &kg, i.v.) but not by spiperone (20 pg/kg, i.v.; figure IA), a butyrophenone like haloperidol but devoid of affinity for o receptors. Moreover, in control rats, a dose of DTG higher than 3 ~g/kg,i.v. induce an epileptoid activity upon the microiontophoreric applications of NMDA (13), whereas this phenornenon was not observed in pregnant rats even with the dose of 10 pg/kg,

1.v. DHEA, at doses as low as 100 pg/kg i.v., induces a selecrive potentiation of the neuronal response to NMDA in control female rats (7). This effect of DHEA is dosedependent and a maximal effect is obtained at a dose of 500 pg/kg, i.v. This effect of DHEA is prevented and reversed by low doses of NE-100 (25 &kg, i.v.), progesterone (10 pg/kg, i.v.) and halopendol (10 @g/kg, i.v.). In pregnant rats, the dose of 100 pg/kg, i.v. of DHEA was totdly ineffective in inducing any potentiation of the NMDA response. Much higher doses (1000 ~g/kg,i.v.) induced a small but significant potentiation of the NMDA response, which was suppressed by the subsequent administration of NE-100 (figure ZC). The effects of DTG (1 &kg, v.) (+)pentazocine (10 pg/kg, ix.) and DHEA (100 pg/kg, i.v.) were also assessed at Day 5 in port-partum penod. At that time, the degree of the potentiation of the NMDA response induced by DTG (1 &kg, i.v.), by (+)pentazocine (10 pg/kg, i.v.) and by DHEA (100 pg/kg, ix) was significantly higher than in control female rats. At Days 10 and 15 in post-partum period, the degree of potentiation of the NMDA response was normalized (figure 3). The present data show that, in late pregnancy (at which time progesterone is at its highest level), the potentiation of the NMDA response induced by low doses of selective o "agonists" and of DHEA is markedly reduced. Much higher doses of these o "agonists" are required to produce a potentiation of the NMDA response. This potentiation of the NMDA response induced with higher doses is also reversed by the o "antagonists" NE-100, halopend01 and by progesterone, but not by the non-sigma butyrophenone spiperone. Conversely, early dunng the post-partum period, the degree of the potentiation of the NMDA response induced by low doses of DTG, (+)pentazocine and DHEA is significantly increased. The effects of DTG, (+)pentazocine and DHEA are normalized at Days 10 and 15 in the post-partum. In the last decade, the search for an endogenous ligand at the a receptors has been an area of intense research. Su and CO-workers(5) have identified several steroids, particularly progesterone, able to displace at very low concentrations [3w(+)-SKF-10,047 and ['Hlhaloperidol binding to a receptors. They have hypothesized that progesterone Mght be an endogenous ligand of cr receptors. Schwarz and CO-workers(14) challenged this hypothesis and have argued that the concentration of the endogenous progesterone are insufficient to occupy the a receptors in the brain, even in late pregnancy. However Our data suggest the opposite for several reasons. First, we have previously demonstrated that, in control rats, very low doses (pg/kg) of exogenous progesterone and of DHEA modulate the NMDA response via an effect at the a receptors even though it has been demonstrated rhat, at much higher doses or concentrations, neuroactive steroids exert their pharmacological effeas in the brain by various systerns such as GABA, or NMDA receptor complex (15). Second, we have previously shown that two weeks following ovariectomy, at which time the level of progesterone is reduced, the enhancing effect of DTG is significantly higher than in the control rats. The present results indicate that, in late pregnancy, ten times higher doses of DTG, (+)pentazocine and DHEA are required to induce a selective potentiation of the NMDA response whereas early in the post-partum period, the potentiation of the NMDA response induced by low doses of o "agonists" is even greater than in control female rats. These data provide novel argument that the modifications of progesterone levels during pregnancy, post- pmum penod or after ovariectomy are sufficient to modify the sensitivity of o receptors and chus that progesterone may act as an endogenous ligand for o receptors. Indeed, the abrupt fall in progesterone concentrations during post-panum period induces an enhanced responsiveness of the a receptors since doses of 1 &kg, i.v. of DTG, 10 pg/kg i.v. of (+)pentazocine and 100 pg/kg, i.v. of DHEA produced a greater potentiation of the NMDA response as compared to control female rats. It is noteworthy that the effect of 1 &kg, i.v. of DTG at Day 5 in the post-paxtum penod was greater than that observed with the dose of 10 &kg, i.v. during Late pregnancy, suggesting a greater than IO-fold increase of the sensitivity of the a receptors early in port-partum period. The fact that the potentiation of the NMDA response was back to its normal degree at Days 10 and 15, indicates that this enhanced responsiveness of a receptors induced by the abrupt fdl of progesterone levels after delivery is only transient. Pregnancy is a low-risk period for severe psychiatric disorders, even though depressions of lesser severity may be observed (16). It is generally agreed that a transient lability affects about 50% of women at around the fifth day post-partum and about 1045% of women suffer frorn depression after childbirth (17). Post-partum psychosis, which occurs in about 0.2O/0 of women, usually &ses in the first two post-patum weeks (18). A recurrent hypothesis that has been put forward to explain these phenornena is the important hormonal changes, particularly of progesterone, that occur in the few days following childbixth. In fact, progesterone has been advocated to be effective in the treatrnent of postpartum depression (19). Our data suggest that the reduction of a receptor function in pregnancy is due to the increase of progesterone levels. However, the implication of other neuroactive steroids cannot be completely nile out. The present data provide the first funaional evidence for the putative involvement of o receptors in the psychological changes t hat frequently emerged du ring pregnancy and post- partum period. The finding that progesterone behaves as a potent "antagonisr" at the o receptors strengths the suggestion that this hormone may act as an endogenous ligand to those receptors. REFERENCES

1. S. M. Paul, R. H. Purdy, FASEBJ. 6, 2311 (1992).

2. M. D. Majewska, Biochem. Phamcol. 36, 3781 (1987).

3. W. R. Martin, C. G. Eades, J. A. Thornpson, R. E. Huppler, P. E. Gilbert,/. Pharmacol. Exp. Ther. 197, 517 (1976).

4. S. H. Snyder, B. L. Largent,/. Neuropsychktry Clin. Neurosci 1, 7 (1989).

5. T. P. Su, E. D. London, J. H. Jaffe, Science 240, 219 (1988); D. J. McCann, A. D. Weissman, T. P. Su, Synapse 14, 182 (1994).

6. F. P. Monnet, G. Debonnel, J. L. Junien, C. de Montigny, Ew. j. Phamcol. 179, 441 (1990); F. P. Monner, G. Debonnel, C. de Montigny, j. Pharmacol. Exp. ihr. 261, 123 (1992); F. P. Monnet, G. Debonnel, P. Blier, C. de Montigny, Naunyn-Schmiedebergs Archives of Phamcology 346, 32 (1992); F. P. Monnet, G. Debonnel, R. Bergeron, B. Gronier, C. de Montigny, B:i& j. Pharmacol. 112, 709 (1994).

7. R. Bergeron, C. de Montigny, G. Debonne1,j. Nerrroscience 16(3), 1193 (1996); G. Debonnel, R. Bergeron, C de Montigny, /. of Endocrinoloay (in press).

8. M. W. O Hara, J. A. Schlechte, D. A. et al Lewis, Arch. Cm. Prychiatry 48, 801 (1991).

9. M. L. Casey, P. C. MacDonald, E. R. Simpson, in Williams's Textbook of Endocrinology, J. D. Wilson and D. W. Foster, Eds. (Saunders, Philadelphia, 1985), p. 422.

10. W. K. Morishige, G. J. Pepe, 1. Rotchild, Endocrinology 92, 1527 (1973).

11. These experiments were carned out in vivo, in the CA, region of the rat dorsal hippocampus where the responsiveness of pyramidal neurons to microiontophoretic applications of NMDA, QUIS and ACh was assessed using extracellular unitary recordings. For the electrophysiologicai experiments, rats were anesthetized with urethane (1.25 g/kg, i-p.) and mounted in a stereotaxic apparatus. Microiontophoretic applications and extracellular unitary recordings were performed with five-barrelled glass micropipettes prepared in a conventional manner. H. J. Haigler, G. K. Aghajanian, j. Phannacol. Exp. 73er. 168, 688 (1974). Pyramidal neurons were identified according to Kandel and Spencer, j. Neurophysiol 24, 243 (1961). The effects of alternate microiontophoretic applications of 50 s of each excitatory substance (NMDA, QUIS and ACh) separated by 50 s retention periods were expressed as the number of spikes generated/nanocoulomb (1 nC being the charge generated by 1 nA applied for 1 s). Al1 results are expressed as rneans, SEM of the number of spikes generatecünc of NMDA or QUIS or Ach. Statistical significance was assessed using the Student's t test with the Dunnett's correction for multiple comparisons. Covariance analyses were used to compare the degree of the potentiation of the NMDA response induced by DTG (1 crg/kg, i.v.) or by DHEA (100 pg/kg, i.v.) in control, pregnant and female rats in the post-partun period. Statistical differences smaller than 0.01 were considered as significant.

12. Neuroactive steroids (prepared in a solution of 40% polyethylene glycol) and a ligands were administered via a lateral tail vein. Only one dose of each dmg was administered to one rat while recording from one neuron.

13. R. Bergeron, C. de Montigny, G. Debonnel, Naunyn-Schmiedebergs Archives of Pbmmacology 351, 252 (1995). R. Bergeron, G. Debonnel, C. de Montigny, Eur. J Phannacol. 240, 319 (1993).

14. S. Schwarz, P. Pohl, G. Z. Zhou, Science 246, 1635 (1989).

15. B. S. McEwen, Trends Neurosci 12, 141 (1991). A. L. Morrow, R. H. Pace, R. H. Purdy, S. M. Paul, Mol. Phamcol. 37, 263 (1990). M. R. Bowlby, Mol. Pharmacol. 43, 813 (1993).

16. M. J. Gitlin, R. O. Pasnau, Am. j. Psychiarry 146, 1413 (1989). M. W. O Hara, AdGen. P~ychiatry43, 569 (1986). 17. R. Kendell, J Psychosorn Res. 29, 3 (1985).

18. A. Maggi, J. Perez, Life Sci 37, 893 (1983).

19. K. Dalton, International journal of Prenatul and Periwral Studies. 323 (1989).

20. This work was supported by the Medical Research Council of Canada and by the Fonds de la Recherche en Santé du Québec (F.R.S.Q.). G.D. is in receipt of a Scholarship from the F.R.S.Q.and R.B. is in receipt of a Feilowship from the F.R.S.Q. ACh 7 1Tipi\ -2 O0-

DTG HALOPERIDOL 1 pglkg, i.v. 20 pglkg, i-v.

ACh 7

4 4 - DTG DTG SPIPERONE HALOPERIDOL 1 min 1 pglkg, i.v. 10 pglkg. i.v. 20 pglkg. i.v. 20 pglkg. i.v.

Figure 1 Kntegrated firing nte histogram of a CA, dorsal hippocampus pyramidal neuron showing the effects of microiontophoretic applications of NMDA, QUIS and ACh before and dter the intravenous administration of DTG and after the intravenous administration of hdopendol in the control female rats (A) and after the intravenous administrations of DTG, folIowed by spiperone and halopend01 (B). Bars indicate the duration of applications for which currents are given in nA and open circles (00) indicate a 5-min interruptions of the recording. Ocseiine CI Baseline a DTG i p~g.LV. in controî (+)pentazocine 10pglkg. I.V. in contml DTG 1 pgkg. I.V. in pregnant rats (+)pentazocine10 pg'kg. i.v. in pregnant rats

DTG 10 rigkg. i.v in pregnant rats (+)peniazocine100 pgùg. I.V. in pregnant rats

DTG + progesterone 20 pg/kg. I.V. in pregnant rats (+)pentazoctne + haiopendol20pg~rg. 1.v. in pregnant rats

DHEA 100 pgkg. I.V. in pregnant rab

Figure 2 Responsiveness, expressed as the nwnber of spikes generated/nC (mean i SEM) of CA, dorsal hippocampus neurons to microiontophoretic applications of NMDA before (open columns) and aher (grey columns) the intravenous administration of 1 ~g/kgof DTG (A), (+)pentazocine (B) or DHEA (C) in control female rats, or in pregnant rats and after the intravenous administration of 20 ~g/k~of progesterone (A), of 20 ~g/kgof haloperidol (B) or 50 ~g/kgof NE-IOO.The number at the bottom of the first column indicates the number of neurons tested (one neuron per rat, in this and subsequent bar chart histograms). * p < 0.01, using the Student's t test. Baseline

DTG 1 pg/kg, i.v. in control

DTG 1 pgfkg, i.v. in puerpurium (5 days)

DTG 1 pgfkg, i.v. in puerpurium (1 0 days)

DTG 1 pgfkg. i.v. in puerpurium (1 5 days)

Figure 3 Responsiveness, expressed as the nurnber of spikes generated/nC (mean * SEM) of CA, dorsal hippocampus neurons to microiontophoretic applicatiom of NMDA before (open columns) and after (grey columns) the intravenous administration of 1 pg/kg of DHEA in control fernale rats, or after the intravenous administration of 1 pg/kg of DTG in the post-panum period at days 5, 10 and 15. CHAPTER X General discussion

The results presented throughout this Ph-D. thesis provide new information for the comprehension of the pharmacology of the a receptors. Two major conclusions cm be drawn from the data included in this document. First, these results support the notion that more than two subtypes of a receptors exist in rat brain. Second, these results also suggest that progesterone behaves as an endogenous ligand at the a receptors. The first study indicates that, at low doses, antidepressant o ligands potentiate the

NMD A response. The dose-response curves (1-1000 pg/kg, i-v.) O btained wit h antidepressant a ligands have a bell-shaped aspect whereas antidepressanc drugs, with no affinity at the a receptors, have no effect on the NMDA response in the sarne range of doses. The second study suggests that this biphasic effect is also found with severd selective a agonists and cannot be explained by a rapid desensitization of the a receptors but rather by the sequential activation of different subtypes of o receptors. The third and the fourth studies provide functional evidences that more than two subtypes of 0,receptors exist in the CNS. The fifth study clearly indicates that low doses of some selecrive o ligands aa as agonists whereas high doses of the same ligands act as antagonists. In the same theme of subtypes of o receptors, long-terrn .. . treatments with a agonists induce a supersensitivity of o receptors whereas long-term treatments with a antagonists induce a desensitization of o receptors. The last two studies on neuroactive steroids show thar progesterone is affecting the neurotransmission, which effect is clearly related to the a receptors. The in vivo data show that, at low doses, severaf selective

Several groups have reported similar biphasic effects with

symptoms 5.?5"6?19-55. AS the activation of the dopaminergic nigro-striatal pathways results in a presynaptic inhibition of srnatal glutamate release "", antipsychotics may act by enhancing glutamatergic activity ". In addition, decreasing the activation of NMDA receptors located on

striatal dopaminergic nerve terminas results in an inhibition of dopamine release 3', suggesting that enhancing NMDA-mediated neurotransmission should exert antipsychotic effects. Recently it has been reported that an agonist at the glycine site, added to neuroleptics, improves the

negative symptoms of schizophrenia lb. Al1 together, these observations would suggest rhat psychotic processes might occur during insufficient activation of NMDA receptors (PCP is a

powerful psychotomimetic agent) M. Several neurological and neuropsychiatric diseases may result from an excessive of NMDA receptors 17.19.2~6~3.+j.51. For example, amte neuronal ce11 loss in response to cerebral trauma, hypoglycaernia or hypoxia may result from the failure of an effective

regulation of NMDA receptor activity 'j. It has been reporred that BMY 14802 and ifenprodil

present some neuroprotective effects 9. The recent finding rhat ifenprodil, an NMDA antagonisr with anti-ischemic properties, potently interacts with a binding sites suggests that

a agents rnight influence excitatory amino acid neurotransmission 7*87'820? In addition, dexrromechorphan has displayed neuroprorective activity in both cultured neurons and in a

rat hypoxia mode1 '6. In keeping with the effea of o antagonists, it is possible that with the administration of these dmgs following cerebral trauma, hypoglycaemia or hypoxia, neuronal darnage could be reduced. Furthermore, the cerebellar degeneration causing sorne symptorns of Alzheimer's, Parkinson's, Huntington's diseases and primary lateral sclerosis, might be due to NMDA receptor dysregulation '. Additional therapeutic targets for a compounds include epilepsy. Dextromethorphan is able to inhibit NMDA-induced convulsions in mice 15. It is known that long-term treatments with haloperidol decrease the density of a receptors in the CNS. Since haloperidol behaves in our paradigm as a very potent antagonisr, we decided to test the effea of short- and long-term treatments of this ligand. Since high doses of DTG and JO-1784 dso behave as antagonists whereas low doses of these two ligands behave as agonists, a series of experiments were carried out to determine if short- and long-term treatments with selective o ligands modify the effects of acure administration of a agonists on the NMDA response. The results of that study suggest a supersensitivity of cr receptors after long-term treatments with o agonists and a desensitization of the o receptors after long-term treatrnents with a antagonists. Sigma receptor ligands have been reporred to have potency in animal models suggestive of antipsychotic, antidrug abuse, cognitive enhancing, neuroprotective and anxiolytic activities. Clinical studies confirmed a role for o ligands in the treatment of schizophrenia. However, many of the a ligands used are not very selective and the contribution by other receptors for biological activity is possible. Even with the mosr selective o compounds that have been developed to dace (such as NE-IOO), action on some yet unidentified receptors cannot be ruled out. The proof for therapeutic effects of cr ligands requires future clinical trials of highly potent and select ive o recepto r ligands. The denoted a, and receptor subtypes differ from each other with respect to: (1) dmg selectivity and stereospecificity; (2) modulation by guanine nucleotides; (3) association with signal transduction mechanisms; (4) regulation by certain anticonvulsants (e.g. phenytoin); involvement in psychotropic dmg action and motor behaviour. It is conceivable that the brain anatomical distribution, the subcellular localization and the rnolecular weight of the receptor subtypes are different. However, the data presented above strongly suggest the existence of several subtypes of a receptors. Cloning of the binding proteins and additional functional studies should enable a better understanding of the biological significance of each receptor subtype. Sigma receptor ligands have little or no effect on second messenger systems. They modify the function of agonists at other neurotransmitter receptors. The effects of o ligands on second messenger systems described to date have only been modulatory. This is the case with carbachol- and norepinephrine- stimulated phosphoinositide turnover, glutamate- stimulated cGMP formation, and K+- and nicotine-stimulated CA" influx. The putative G- protein-sigma receptor interactions could be due to some type of indirect coupling of other receptors. This modulatory theme extends to several cellular and physioiogical assessrnena of o receptor hnction, such as inhibition of serotonin-induced ileal contraction ', potentiation and inhibition of NMDA-stimulated noradrenaline release " and inhibition of nicotine- stimulated catecholarnine release +'. Given the wide distribution of o receptors in both central and peripheral tissues, this indicates potential for important tissue-specific regulatory roles for receptors. Even though this field was not the purpose of the present thesis, this remains a very important issue. In order for the o receptors to be fully accepted, it is imperative to identify an endogenous ligand. There have been several attempts to find this substance. There are evidence of the existence of several endogenous substances which can inhibit the binding of a-seleaive radioligands ro their binding site. However, none of these has been conclusively shown to be the ligand. Several group have reporred that progesterone acts as a competitive inhibitor of

['H](+)SKF-10,047 or ['Hjhaloperidol 38*'8*58-". SU and CO-workerswere the first to propose than progesEerone might be the endogenous ligand at the a receptors. The argument against this hypothesis is that cerebro-spind fluid levels of this hormone are too low to have a signifiant impact on neuronal responsiveness ". However, the data reported in the 1st two studies of this t hesis bring convincing arguments that progesterone acts as a potent modulator at o receptors. The inuavenous administration of low dose of progesterone (20 pg/kg) prevents and reverses the potentiation induced by several cr agonists whereas pregnenolone and pregnenolone-sulfate, which have very low affinity for o receptors, neither prevent nor reverse the potentiation of the NMDA response induced by several selective a agonists. Moreover, the degrees of the potentiation of the NMDA response induced by DTG (1 &kg, i.v.) is significantly greater in OVX rats. The more convincing argument remains the fact that the potentiation of the NMDA response induced by DTG (1 /~g/kg,i.v.) in pregnant rats is markedly reduced in late pregnancy, at which tirne there is a high level of progesterone. Conversely, the potentiation induced by DTG (1 pg/kg, i-v.) is greater five days post-partum than in control female rats. Ten and 15 days aker delivery, the funaion of o receptors is back to normal. Al1 together, these data suggest that progesterone behaves as a potent suppressor at cr receptors. These findings strength the suggestion that progesterone may act as endogenous ligand at those receptors, Sigma receptors are unique among CNS receptors because they bind a diverse array of structural classes of compounds including certain non-nitrogens compounds such as the steroids. This suggests a great deal of structural and steric tolerance. The minimum features required for high affinity binding at a receptors appear to be at least one basic nitrogen atom separated from an aromatic ring by at lest two methylene groups and also substituted with a lipophilic bulky substituent. One of the most prornising remaining avenues for o research is the functional Iink between cr and NMDA receptors. The different studies included in this thesis show that o ligands modulate the NMDA response. The importance of an appropriate control of the putative dysregulation of NMDA receptors in several acute and chronic disorders is sufficient to carry out furcher evahations on the relationship between o and NMDA receptors. For example, the clarification of the different locations of o and NMDA binding sites and the identification of the sequence of events which underlies this o/NMDA modulation are some aspects of particular relevance. It sholild be of great interest to determine if o ligands also rnodulate the expression of nuclear factors following NMDA receptor induction, i.e. modulate NMDA-induced cfos production ". Furthermore, it would be highly interesting to determine if LTP induction, which is triggered by the activation of NMDA receptors, might be sensitive to o ligands. This possibility could open new perspectives in the understanding and treatment of cognitive funct ions. The present results shed new light on the pharmacology of o receptors. The modulatory effect on the NMDA response of some o ligands tested in this thesis supports the notion of the heterogeneity of the o receptors. The data presented in this thesis bring numerous evidences that more than two subtypes of a receptors exist in the CNS. Amongst the unanswered questions regarding the nature of o receptors, the most urgent one is probably the identification of the endogenous ligands. The results in the lasr two chapters of this thesis scrongly suggest that progesterone is certainly one of the candidates. REFERENCES

1 Allen, R. M. and Young, S. J., Phencyclidine-induced psychoses, Am./.Psycbiatl, 135 (1978) 1081-1084.

2 Bean, P. M., O'Shea, R. D. and Manallack, D. T., Regdation of sigma recepton: high- and low-affinity agonist States, GTP shifts, and up-regdation by rimcazole and 1,3-Di(2-tolyl)guanidine, j.Neurochw., 53 (1989) 7ï9-788.

3 Bonhaus, D. W., Loury, D. N., Jakeman, L. B., To, Z., DeSouza, A., Eglen, R. M.and Wong, E. H., [3H]BIMU-1, a 5-hydroxyrryptamine3 receptor ligand in NGlO8 cells, selectively Iabels sigma-2 binding sites in guinea pig hippocampus, /. Pbannacol. Exp. 7%'1her., 267 (1993) 96 1-970.

4 Campbell, B. G., Scherz, M. W., Keana, J. F. and Weber, E., Sigma recepton regulate contractions of the guinea pig ileum longitudinal rnuscle/myenteric plexus prepantion elicited by both electrical stimulation and exogenous serotonin, /. Neurosci., 9 (1989) 3380-3391.

5 Carlsson, M. and Carlsson, A., Interactions between glutamatergic and monoaminergicsystems within the basal ganglia: implications for schizophrenia and Parkinson diseue, Trends rVeurosci., 13 (1990) 272-276.

6 Carr, D. J., Mayo, S., Woolley, T. W. and DeCosta, B. R., Immunoregulatory propenies of (+)-pentazocine and sigma ligands, Imrnunology, 7ï (1992) 527-531.

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