Dopaminergic—cholinergic interactions in naloxone-induced circling in morphine-dependent rats with nigral lesions

European Journal of Pharmacology, 38 (1976) 39--54 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

39

DOPAMINERGIC--CHOLINERGIC INTERACTIONS IN NALOXONE-INDUCED CIRCLING IN MORPHINE-DEPENDENT RATS WITH NIGRAL LESIONS * EDGAR T. IWAMOTO, H.H. LOH and E. LEONG WAY

Langley Porter Neuropsychiatric Institute, and Department of Pharmacology, University of California, San Francisco, California 94143, U.S.A. Received 7 November 1975, revised MS received 4 March 1976, accepted 18 March 1976

E.T. IWAMOTO, H.H. LOH and E.L. WAY, Dopaminergic--cholinergic interactions in naloxone.induced circling in morphine-dependent rats with nigral lesions, European J. Pharmacol. 38 (1976) 39--54. 3--4 weeks after placement of a unilateral, electrolytic lesion of the substantia nigra zona compacta, rats were highly dependent on morphine by the s.c. morphine pellet implantation technique. Following challenge with a supramaximal naloxone dose of 20 mg/kg i.p., both continuous contralateral circling behavior and severe withdrawal signs in morphine-dependent, lesioned rats were elicited. After various drug pretreatments, the contralaeral circling behavior precipitated by naloxone was: (a) reversed to ipsilateral circling by i.p. apomorphine or damphetamine, (b) unaltered by i.p. haloperidol or intraneostriatal arecoline administered into the intact neostriatum, and (c) reversed to ipsilateral circling by the administration of atropine into the intact neostriatum. Atropine, apomorphine and amphetamine all interfered with the manifestation of naloxone-precipitated abstinence. These data suggest that a diminution of dopaminergic or an enhancement of cholinergic activities, or both, occur at the level of the neostriatum during naloxone-precipitated withdrawal in morphine-dependent rats. Naloxone-precipitated withdrawal Morphine-dependent rats

Electrolytic SNC lesions Circling behavior

1. Introduction

We have previously reported that contralateral circling behavior was precipitated by naloxone in morphine-dependent rats with unilateral lesions of the substantia nigra zona compacta, SNC (Iwamoto et al., 1976a). The similarity between naloxone-precipitated and dopamine receptor antagonist-induced contralateral circling behavior led to the hypothesis that during naloxone-precipitated withdrawal there may be a sudden, temporary diminution of dopaminergic activities in the nigro-neostriatal pathway. Additional evidence for a decreased dopaminergic transmission during naloxone-precipitated morphine withdrawal * Request reprints of paper No. 75-914 from the Department of Pharmacology.

ACh antagonists DA antagonists

ACh agonists DA agonists

is suggested by data showing that both precipitated abstinence and the interruption of the dopaminergic projections in the SNC result in an elevation of neostriatal dopamine concentration (Iwamoto et al., 1973; Iwamoto et al., 1976a). Other data suggest that cholinergic regulation may also participate in withdrawal. It has been suggested by numerous workers that there exists, probably within the neostriatum, a cholinergic system neurochemically linked to the dopaminergic nigro-neostriatal projection (Corrodi et al., 1972; Ladinsky et al., 1975; Guyenet et al., 1975; Trabucchi et al., 1975) and manipulations designed to elevate brain acetylcholine levels reduced naloxone-precipitated withdrawal jumping (Bhargava and Way, 1972; Iwamoto et al., 1973) whereas a lowering of acetylcholine enhanced the response (Bhargava and Way, 1975). In

40 view of these findings, it became of interest to investigate in depth the possible dopaminergic and cholinergic interactions during antagonistprecipitated morphine abstinence. Accordingly, the following study was designed to examine the effect of dopaminergic and cholinergic agonists and antagonists on naloxone-precipitared contralateral circling and withdrawal behavior in morphine-dependent rats with unilateral lesions of the substantia nigra zone compacta. 2. Materials and methods 2.1. Animals

Male Sprague--Dawley rats weighing between 157 and 300 g at the time of experimentation were used in the present investigations. Animals, housed two to a cage under uniform temperature, humidity and lighting conditions and acclimated for at least 3 days before use, were fed and watered ad libitum. 2.2. Stereotaxic techniques

Animals weighing 95--105 g were anesthetized with halothane--oxygen and unilateral electrolytic lesions and sham lesions were placed in the left SNC as previously described (Iwamoto et al., 1976a,b). The location and extent of the lesion was determined on every tenth animal (n = 31) using photographic methods of Thompson (1971). The resulting lesions centering on the dorso-lateral SNC were approximately 1.2--2 mm in diameter and included parts of the medial lemniscus, midbrain reticular formation and adjacent substantia nigra zona reticulata. Chemical destruction of dopaminergic cell bodies of the left SNC was accomplished after stereotaxic placement of 8 pg of 6-hydroxydopamine (6-OHDA, Regis Chemical Co., Chicago, Ill.) in 4 pl of artificial CSF (Palaic et al., 1967) using the procedures described previously in which an 80% depletion of forebrain dopamine was produced (Iwamoto et al., 1976b).

E.T. IWAMOTO ET AL. Bilateral, intraneostriatal drug administrations were performed with the aid of two stereotaxically m o u n t e d 10 pl Hamilton microsyringes using the techniques and rat brain coordinates (K~nig and Klippel, 1963) described previously. 2.3. Chemicals

All solutions of drugs were prepared on the day of use and doses refer to the free base; control animals received the appropriate vehicle of the corresponding drug. For intraneostriatal administration, the drugs used were arecoline hydrochloride (K and K Laboratories, Plainview, N.Y.), atropine sulfate (Sigma Chemical Co., St. Louis, Mo.) and lidocaine hydrochloride (Astra Pharmaceutical Products, Worcester, Ma.). All drugs in 3 pl artificial CSF were administered into the right neostriatum at a rate of 0.2 #l per 15 sec; the same volume of vehicle was concurrently administered into the left neostriatum. 24 h after the injections, every fifth animal was prepared for histological examination of the needle tracts using the photographic techniques of Thompson (1971). All injections were verified to have been in the b o d y of the nucleus caudatus--putamen and are in agreement with previous results (Iwamoto et al., 1976b). All other drugs were administered i.p. except for naloxone which was given s.c. The following drugs were dissolved in sterile saline: apomorphine hydrochloride (Merck, Sharp and Dohme, Philadelphia, Pa.), damphetamine hydrochloride (K and K Laboratories, Plainview, N.Y.), naloxone hydrochloride (generously supplied by Endo Laboratories, Garden City, N.Y.). Haloperidol (generously supplied by McNeil Laboratories, Forth Washington, Pa.) was dissolved in a gluc o s e - a c e t i c acid mixture described previously by And6n et al., 1970. 2.4. Behavioral observations

S t e r e o t y p y induced by the various drugs was evaluated by non-parametric, rank-scoring

MORPHINE WITHDRAWAL CIRCLING methods of Naylor and Olley (1972), cataleptic behavior by the procedures of Costall et al. (1972), and circling behavior according to Naylor and Olley (1972) and Iwamoto et al. (19765). 12--14 days after lesioning, all animals were screened for asymmetric body posturing and circling behavior after the administration of a challenge dose of d-amphetamine: After a 15 min acclimation period in opaque plastic observation cages (base area 40 × 25 cm, height 12.5 cm), 4 mg/kg d-amphetamine was administered and circling behavior evaluated using the criteria and ranking methods previously described. Rats that failed to attain a circling behavior score of 3 within 30 min were discarded.

2.5. Effect of drug treatments of naloxone. precipitated withdrawal in morphine-dependent rats The s.c. morphine pellet implantation technique was used to render the lesioned rats highly morphine dependent (Way et al., 1969). 1--2 weeks after pretesting for circling behavior with d-amphetamine, rats were implanted with one morphine pellet in the s.c. tissue of the lower back under halothane--oxygen anesthesia. 2 pellets were implanted 24 h later; all animals used in experiments were tested after 70--74 h of pellet implantation. Animals implanted with placebo pellets containing only the tablet filler served as the sham-implanted controls. On the day of experimentation, separate rats were administered i.p. saline, apomorphine or haloperidol. Other groups were anesthetized with halothane and administered artificial CSF, arecoline or atropine by intraneostriatal injection as previously described. 20 min after apomorphine or arecoline and 30 min after haloperidol or atropine, morphine abstinence was precipitated by a s.c. 20 mg/kg dose of naloxone and the behavioral signs of precipitated abstinence were assessed for 10 min and scored according to Wei and Loh (1972) using non-parametric, rank-scoring

41 methods. In all experiments, catalepsy, stereot y p y and circling behavior were assessed during the 20 or 30 min period~ before and during the 10 min after the injection of naloxone; standardized scoring sheets greatly simplified the scoring procedures.

2.6. Diffusion of intraneostriatally administered 3H-atropine 3 #l of artificial CSF, containing 27 nCi of 3H.(G).atropine (289 mCi/mmol, AmershamSearle, London, England) and 130 pg (free base) of atropine sulfate, were administered unilaterally into the caudate--putamen nucleus using the same coordinates described previously for intraneostriatal drug administration. At 5 and 30 min after completion of the injection, the animals were sacrificed by decapitation. Their brains were excised and blocked in a frontal plane at the level of the pineal within 1 min after removal and frozen onto microtome object discs at --5 ° C. Serial frozen sections were taken on an International-Harris cryostat 100 /am thick through at least 3.5 mm on either side of the injection tract. Each 100 pm thick section was hemisected into left (injected) and right sides, collected in scintillation vials, and dissolved in 1 ml of a tissue solubilizer ('NCS', Amersham-Searle, Arlington Heights, Ill.) overnight, neutralized with 100 #l glacial acetic acid, shaken with 10 ml of scintillator cocktail ('Aquasol', New England Nuclear, Boston, Ma.) and counted in a Beckman LS-150 counter 5 days later. The counting efficiency for tritium in these solubilized tissue sections was 35%. The locations of the caudate--putamen nucleus, the tip of the needle tract and the anterior commissure of each brain sample were recorded by comparing the anatomical features of the cut section to the photographs of frontal sections depicted in the Ktinig and Klippel atlas.

2. 7. Statistical analysis The two-tailed Student's t-test for unpaired data was used to evaluate differences between

42

E.T. I W A M O T O E T AL.

arithmetic means of data obtained from different animals. The Mann--Whitney U-test was used to evaluate the behavioral responses of catalepsy, stereotypy, circling and withdrawal behavior (which were assessed using ranking methods) by comparing the highest scores achieved by each animal between groups (Snedecor and Cochran, 1967).

0

3. Results

3.1. Effect o f dopaminergic agonists on naloxone-precipitated contralateral circling in morphine-dependent rats with unilateral S N C lesions Apomorphine reversed the direction of circling precipitated by naloxone and also diminished precipitated withdrawal signs in both the lesioned and sham-lesioned groups. Fig. 1 shows circling behavior before and after naloxone and precipitated withdrawal scores in both lesioned and sham-operated groups pretreated with saline and 3 doses of apomorphine. Apomorphine prior to naloxone produce ipsilateral circling behavior in lesioned, morphine-dependent animals which increased in intensity with higher doses but not in the sham-operated controls. Moreover, the contralateral circling induced by naloxone in lesioned, dependent animals was blocked by apomorphine. As can be seen in fig. 1, naloxone precipitated contralateral circling in lesioned, but n o t in sham-operated, salinetreated animals as previously observed (Iwam o t o et al., 1976a). However, after pretreatm e n t with apomorphine, the naloxone-elicited contralateral circling behavior gradually diminished in intensity with increasing dose a n d was reversed to ipsilateral circling with the highest apomorphine dose. It should be noted that the naloxone-precipitated withdrawal signs were significantly reduced by apomorphine pretreatment in both sham-operated and lesioned animals. However, apomorphine was found to be more effective in blocking withdrawal signs in sham-operated

N:6-8

[]

left snc lesion

[ ] sham

Fig. 1. Effect of i.p. apomorphine on circling behavior in sham-operated and left-SNC-lesioned morphinedependent rats before and after naloxone and on withdrawal b e h a v i o r a f t e r n a l o x o n e . W i t h d r a w a l was prec i p i t a t e d by 20 m g / k g n a l o x o n e a d m i n i s t e r e d 20 m i n a f t e r a p o m o r p h i n e or saline. T h e solid lines c o i n c i d e n t w i t h t h e p l a n e of t h e figure i n t e r c o n n e c t i n g circling a n d w i t h d r a w a l b e h a v i o r c o l u m n s d e m a r c a t e groups of animals p r e t r e a t e d w i t h d i f f e r e n t a p o m o r p h i n e doses. Saline (0) a n d doses of a p o m o r p h i n e are d e n o t ed b y n u m e r a l s ( m g / k g of free base) o b l i q u e to the figure plane. Circling b e h a v i o r c o n t r a l a t e r a l t o t h e lesion is i n d i c a t e d b y columy, s a b o v e t h e figure p l a n e ; ipsilateral circling b e h a v i o r c o l u m n s lie b e l o w t h e plane. All c o l u m n h e i g h t s r e p r e s e n t the m e a n of maxi m u m scores o f circling or w i t h d r a w a l b e h a v i o r achieved by each a n i m a l in each g r o u p o f at least 6 rats. D i a m o n d s o n t h e c o l u m n t o p s r e p r e s e n t signific a n t d i f f e r e n c e s f r o m t h e respective s a l i n e - t r e a t e d c o n t r o l groups: (}, p < 0 . 0 5 ; 0, p < 0.01, as determ i n e d b y t h e M a n n - - W h i t n e y U-test.

than in lesioned rats. Coincident with the ipsilateral circling induced by apomorphine in morphine-dependent animals was the appearance of stereotyped behavior which also increased in intensity with increasing dose. However, there were qualitative differences in stereotypy between the sham and lesioned groups of dependent animals. Table 1 shows the mean scores of apomorphine-induced stereotypy recorded before and after naloxone from the same dependent animals used in fig. 1. Administration of 3 mg/kg apomorphine consistently elicited stereotypy scores of 3 or 4 in the sham-oper-

MORPHINE WITHDRAWAL CIRCLING

43

TABLE 1 A p o m o r p h i n e s t e r e o t y p y before and after n a l o x o n e in m o r p h i n e - d e p e n d e n t rats with unilateral SNC lesions * Drug dose (mg/kg)

Saline 1.5 3.0 6.0

Group (n)

S t e r e o t y p e d behavior, group m e a n score Prenaloxone

Post naloxone

L (8)

0

1

s (8)

0

1

L(6) S (6) L (6) S (6) L (6) S (6)

0 2.67 1.33 3.83 1.83 3.83

** # ** # **,-~

1 2.5 * * , t 1 3.67 * * , t 2.83 # 3.67 **

* Data represent the c o m p i l e d results 4 separate experiments o f s t e r e o t y p y scores achieved by the same animals used in fig. 1. Rats weighing 2 2 7 - 294 g were s.c. i m p l a n t e d with 3 m o r p h i n e pellets 3--4 weeks after p l a c e m e n t o f an electrolytic lesion (L) in the left SNC or a sham-operation (S). On the third day of pellet implantation, saline or a p o m o r p h i n e was administered i.p. 20 min before n a l o x o n e , 20 m g / k g s.c. S t e r e o t y p y scores depicted above represent an average of the m a x i m u m scores achieved by each group within t h e 20 rain pre-naloxone and 10 rain post-naloxone periods. ** Significantly different f r o m saline control, p < 0.01, as d e t e r m i n e d by the Mann--Whitney U-test. "~ Sham-operated group significantly different f r o m lesioned group receiving the same a p o m o r p h i n e dose, p < 0.01. # Significantly different f r o m saline control, p < 0.05.

ated group {mean score, 3.83). On the other hand, a lower degree of stereotypy was observed in the SNC-lesioned animals (mean score, 1.33). Thus, unilaterally lesioned rats appeared less responsive to apomorphine and this observation, in part, is consistent with previous work showing a reduction of apomorphine stereotypy after chronic, bilateral electrolytic SNC lesions (Costall and Naylor, 1973). It was also apparent during the above observations that the stereotyped behavior induced by apomorphine may have been inter-

fering with the expression of withdrawal behavior precipitated by naloxone. Apomorphine markedly reduced the precipitated withdrawal signs in sham-operated, dependent rats at all 3 doses used and diminished withdrawal intensity in the lesioned group at the 6 mg/kg dose. As indicated in table 1, these groups of animals were exhibiting a high degree of stereotypy which lasted for as long as 30 min after naloxone. Increased intensities of stereotyped behavior with increasing doses of apomorphine in both sham and lesioned animals appeared to be associated with a diminished appearance of naloxone-precipitated withdrawal signs. Contralateral circling precipitated by naloxone in morphine-dependent, SNC-lesioned rats was also reversed to ipsilateral circling by d-amphetamine. Table 2 shows circling and stereotyped behaviors before and after naloxone and withdrawal after naloxone in morphine-dependent, unilaterally SNC-lesioned rats. In contrast to controls, which did not respond to a saline injection, amphetamine induced ipsilateral circling (mean score, 2.62) and a moderate degree of stereotypy (mean score, 2.87). 30 min after naloxone, both ipsilateral circling (mean score, 1.3) and stereot y p e d behavior (continuous sniffing and exploring) in the amphetamine group were reduced (stereotypy mean score, 2.0) and the intensity of precipitated withdrawal behavior was mild (mean score, 1.25). In comparison, the controls after saline pretreatment exhibited naloxone-precipitated contralateral circling and withdrawal scores of 3. Also, the amphetamine plus naloxone-treated animals were extremely irritable to touch and, in contrast to the apomorphine pretreatment, all animals died within 6 h after amphetamine due to hyperthermia. In a separate experiment, 3 placebo and 3 morphine pellet-implanted rats were administered 4 mg/kg d-amphetamine i.p. 30 min before 20 mg/kg s.c. naloxone. Placebo-implanted rats exhibited elevated rectal temperatures {0.4, 0.7 and 1.0°C) above a baseline of 38.2 + 0.4°C (S.D.) 15 min after naloxone. Rectal temperatures

44

E.T. IWAMOTO ET AL.

TABLE 2 d-Amphetamine-induced circling and stereotypy before and after naloxone-precipitated withdrawal in morphinedependent rats with unilateral SNC lesions * Treatment

Saline (n = 4 ) d-Amphetamine 4 mg/kg i.p. (n 8)

Score before naloxone

Score after naloxone

Circling

Stereotypy

Circling

0

0

C : 3

1

3

I : 1.62 **

2.87 **

I : 1.3 **

2 ***

1.25 ***

Stereotypy

Withdrawal

=

* Data represent the results of a single experiment. Animals weighing 250--283 g at the time of experimentation were implanted with morphine pellets four weeks after placement of an electrolytic lesion in the left SNC. On the third day of implantation, d-amphetamine or saline was administered 30 min before precipitation of withdrawal with 20 mg/kg s.c. naloxone. All behavioral scores are an average of the maximum scores achieved within the 20 min and 10 min observation periods before and after naloxone, respectively (I: ipsilateral, C: contralateral to the lesion). ** Significantly different from saline control, p < 0.01, as determined by the Mann--Whitney U-test. *** Significantly different from saline control, p < 0.05.

of morphine-dependent a n i m a l s 15 m i n a f t e r n a l o x o n e w e r e s i g n i f i c a n t l y e l e v a t e d 1.5, 1 . 9 a n d 2 . 3 ° C a b o v e a c o n t r o l b a s e l i n e o f 3 8 . 5 -+ 0.5°C. In similarity with the results previously o b s e r v e d , all m o r p h i n e - d e p e n d e n t rats given amphetamine and naloxone died within 6 h after naloxone.

R~

°

3 o

0.$

"

3.2. Effect o f haloperidol on naloxone-precipitated contralateral circling Haloperidol, which invariably produces c o n t r a l a t e r a l c i r c l i n g a n d c a t a l e p s y in d e p e n dent and non-dependent animals, did not modify naloxone-induced circling or precipit a t e d w i t h d r a w a l in S N C - l e s i o n e d , m o r p h i n e dependent animals. Fig. 2 shows circling behavior before and after naloxone and the w i t h d r a w a l s c o r e p r e c i p i t a t e d b y n a l o x o n e in lesioned and sham-operated animals 30 min after saline, 0.5, 1 or 2 mg/kg haloperidol pret r e a t m e n t . A d m i n i s t r a t i o n o f h a l o p e r i d o l in morphine-dependent rats with SNC lesions produced contralateral body asymmetry and contralateral circling movements of low intens i t y ( m e a n s c o r e , 1.6, a f t e r 2 m g / k g ) . H o w ever, the contralateral circling, precipitated by n a l o x o n e in t h e l e s i o n e d b u t n o t t h e s h a m -

[]

left snc

lesion

[ ] sham

Fig. 2. Effect of i.p. haloperidol on circling behavior in sham-operated and left-SNC-lesioned morphinedependent rats before and after naloxone and on withdrawal behavior after naloxone. Withdrawal was precipitated by 20 mg/kg naloxone 30 min after haloperidol or saline administration. The solid lines coincident with the plane of the figure interconnecting circling and withdrawal behavior columns demarcate groups of animals pretreated with different haloperidol dose which are denoted by numerals (mg/kg, 0 = saline) oblique to the figure plane. Circling behavior contralateral to the lesion is indicated by columns above the figure plane. All column heights represent the mean of maximum scores of circling or withdrawal behavior achieved by each animal in each group of at least 7 rats. Diamonds represent significant differences from saline control as in fig. 1. N = 7-8.

MORPHINE WITHDRAWAL CIRCLING

45

operated group, was n o t modified by increasing doses of haloperidol with respect to either intensity or direction. Similarly, the withdrawal behavior precipitated by naloxone remained unmodified after pretreatment. As previously mentioned, haloperidol produced dose-dependent cataleptic behavior. Table 3 summarizes haloperidol-induced cata-

TABLE 3 Haloperidol catalepsy before and after naloxone in morphine-dependent rats with unilateral SNC lesions * Drug dose (mg/kg)

0 0.5

1.0 2.0

Group (n)

Cataleptic behavior, group mean score Prenaloxone

Postnaloxone

L(7) S (7) L (7)

0 0 0.71 **

0 0 0

S (7)

0 . 8 6 ***

0

L (8) S (8) L (7) S (7)

1.00 1.75 1.43 2.29

0.50 0.88 0:43 1.57

*** *** *** ***

t **'t t ***'t'#

* Data represent the compiled results from 4 separate experiments of catalepsy scores achieved by the same animals used in fig. 2. Rats weighing 229--290 g were implanted with 3 morphine pellets 3--4 weeks after placement of an electrolytic lesion (L) in the SNC or a sham operation (S). On the third day of implantation, acetic acid--glucose vehicle or haloperidol were administered i.p. 30 min before 20 mg]kg s.c. naloxone. Catalepsy scores before naloxone injection represent the maximum scores achieved within the 30 min prenaloxone period. Catalepsy scores after naloxone represent the maximum scores achieved 1 rain after the re-onset of catalepsy. ** Significantly different from vehicle control, p < 0.05. *** Significantly different from vehicle control, p < 0.01. t Naloxone precipitated withdrawal behavior (refer to fig. 2) which was followed by cataleptic.behavior appearing 1--10 min after naloxone administration. # Sham-operated group significantly different from the lesioned receiving the same haloperidol dose, p < 0.05.

lepsy scores observed before and after naloxone in the same animals used in fig. 2. Increasing doses of haloperidol increased the intensity of catalepsy observed in both sham and lesioned groups of rats. In fact, some of the animals receiving the 1 and 2 mg/kg doses of haloperidol were still cataleptic at the time of naloxone administration, and as a consequence, the intensity of catalepsy in these group may have been underestimated. Somewhat surprising was the elicitation of precipitated withdrawal by a naloxone challenge 30 min after haloperidol injection since at this time most of the animals appeared sedated and 3 rats were still cataleptic. After naloxone administration, the cataleptic rats exhibited l o c o m o t o r activity and withdrawal behavior for 1--10 min before returning to a milder cataleptic state (table 3). It is conceivable that the severe catalepsy observed in the sham-operated group after the 2 mg/kg dose of haloperidol before the naloxone injection may have interfered with the expression of certain precipitated withdrawal signs, such as escape attempts. It should be noted, however, that the predominant withdrawal signs that appeared after naloxone in both the sham-operated and lesioned groups of animals receiving the 2 mg/kg haloperidol pretreatment were wet shakes and teeth chattering in addition to ptosis, abnormal posturing, ear blanching and vocalization to touch. Additionally, escape attempts were manifested by both groups of rats receiving 0.5 and 1 mg/kg haloperidol. Thus, haloperidol pretreatment, at the doses administered in morphine-dependent shamoperated or lesioned rats, did not modify appreciably either the withdrawal signs after naloxone or the precipitated contralateral circling. 3.3. Effect o f naloxone on apomorphine- and haloperidol-induced circling behavior in nondependent rats with unilateral S N C lesions Naloxone administration in placebo pelletimplanted animals with unilateral SNC lesions did not alter circling behavior induced by apo-

46

E.T. IWAMOTO ET AL.

TABLE 4 Effect of naloxone on amorphine-induced stereotypy, haloperidol-induced catalepsy and on drug-induced circling behavior in non-dependent rats with unilateral, electrolytic SNC lesions ** Group (n)_

Pre-naloxone Post-naloxone

S (4) L(4) S (4) L(4)

Score after apomorphine ***

Score after haloperidol -~

Stereotypy

Circling

Catalepsy

Circling

4.00 3.25 4.00 3.25

0 I : 2.75 0 I : 3.00

2.50 0.25 2.75 0.25

0 C:1# 0 C :1#

* Animals were implanted with 3 placebo pellets, 2 pellets 24 h after the first, for 3 days. At the time of experimentation, 3 weeks after placement of a sham (S) or left-SNC lesion, animals weighed 207--290 g. ** 3 mg/kg i.p. apomorphine or 1 mg/kg i.p. haloperidol were administered 20 or 30 min, respectively, before a 20 mg/kg s.c. challenge or naloxone. Apomorphine stereotypy and circling behavior scores are an average of the maximum scores achieved within the 20 and 10 min observation periods before and after naloxone, respectively. Haloperidol catalepsy and circling behavior scores are an average of the maximum scores achieved within the 30 and 10 min observation periods before and after naloxone. *** Stereotypy onset: 3--5 min; circling onset: 2--3 min. I: ipsilateral to the lesion. J- Catalepsy onset: 11--20 min, circling onset: 15--20 min. C: contralateral to the lesion. # Provoked movement by pinching the tail. All sham vs. lesioned animal observations significantly different, p < 0.05, except apomorphine stereotypy.

m o r p h i n e or h a l o p e r i d o l . Table 4 s u m m a r i z e s s t e r e o t y p y and circling b e h a v i o r b e f o r e and a f t e r n a l o x o n e in b o t h sham and lesioned animals given a p o m o r p h i n e . A high degree o f s t e r e o t y p y was observed after a p o m o r p h i n e in b o t h groups o f rats. A l t h o u g h a lesser degree o f s t e r e o t y p y was observed in the lesioned groups o f animals, the difference was n o t statistically d i f f e r e n t f r o m c o n t r o l . Ipsilateral circling b e h a v i o r was elicited a f t e r a p o m o r p h i n e o n l y in rats with unilateral SNC lesions as previously o b s e r v e d (fig. 1). A f t e r challenge with n a l o x o n e , n e i t h e r the a p o m o r p h i n e - i n d u c e d s t e r e o t y p y n o r the ipsilateral circling behavior was altered. As i n d i c a t e d in table 4, 30 min after 1 mg/ kg h a l o p e r i d o l in animals with unilateral SNC lesions, the c o n t r a l a t e r a l circling score elicited u p o n tail-pinching was n o t significantly altered b y the a d m i n i s t r a t i o n o f n a l o x o n e . Likewise, h a l o p e r i d o l - i n d u c e d c a t a l e p s y and circling behavior were n o t altered by a naloxone challenge. A l t h o u g h the degree o f halop e r i d o l - i n d u c e d catalepsy was significantly

less in the lesioned g r o u p o f rats, n a l o x o n e h a d n o e f f e c t on the i n t e n s i t y o f c a t a l e p s y in either s h a m or lesioned animals. This is in c o n t r a s t to the results p r e s e n t e d previously with m o r p h i n e - d e p e n d e n t rats (fig. 2 and table 3) in w h i c h h a l o p e r i d o l - i n d u c e d catalepsy was d i s r u p t e d by n a l o x o n e .

3.4. Effect o f apomorphine on naloxone-precipitated withdrawal in morphine-dependent rats with 6-OHDA-induced unilateral SNC lesions A p o m o r p h i n e p r e t r e a t m e n t , w h i c h prod u c e d a high degree o f s t e r e o t y p y , was f o u n d t o diminish n a l o x o n e - p r e c i p i t a t e d w i t h d r a w a l signs in m o r p h i n e - d e p e n d e n t rats with unilateral, 6 - O H D A - i n d u c e d SNC lesions. Table 5 s u m m a r i z e s s t e r e o t y p y and circling behavior b e f o r e and after n a l o x o n e challenge and withdrawal b e h a v i o r after n a l o x o n e in m o r p h i n e d e p e n d e n t animals p r e t r e a t e d with saline or 6 m g / k g a p o m o r p h i n e . As previously observed and in c o n t r a s t t o animals with unilateral elec-

MORPHINE WITHDRAWAL CIRCLING

47

TABLE 5 Effect of apomorphine on naloxone-precipitated circling and withdrawal behavior in morphine-dependent rats with 6-OHDA lesions of the SNC * Group

Saline, 6-OHDAlesioned Apomorphine, sham-operated Apomorphine, 6-OHDA-lesioned

Pre-naloxone score

Post-naloxone score

Stereotypy

Circling

Stereotypy

Circling

Withdrawal

0

0

1.00

C : 2.50

3

3 **

0

3.50 **

0 **

0 **

3 **

C : 3 **

3.25 **

C : 3.25

0 **

* Data represent the results of a single experiment using 4 rats in each group. Animals, weighing 157--300 g at the time of experimentation, were implanted with 3 morphine pellets 3 weeks after placement of 6-OHDA or artificial CSF into the left SNC. On the third day of pellet implantation, saline or 6 mg/kg i.p. apomorphine was administered. Behavioral scores depicted above are an average of the maximum scores achieved within the 20 and 10 rain observation periods before and after naloxone, respectively. C: contralateral to the lesioned side. ** Significantly different from saline control, p < 0.05.

t r o l y t i c SNC lesions, a p o m o r p h i n e administration p r o d u c e d c o n t r a l a t e r a l circling ( I w a m o t o et al., 1 9 7 6 b ) . B o t h a p o m o r p h i n e - i n d u c e d c o n t r a l a t e r a l circling and s t e r e o t y p e d behaviors were n o t altered a f t e r n a l o x o n e ; h o w e v e r , in a g r e e m e n t with previous d a t a (fig. 1), apom o r p h i n e p r e t r e a t m e n t r e m a r k a b l y diminished the n a l o x o n e - p r e c i p i t a t e d withdrawal signs. Thus, these data s h o w t h a t d o p a m i n e r gic s t i m u l a t i o n b y a p o m o r p h i n e ameliorates rather than exacerbates naloxone-precipitated a b s t i n e n c e in 6 - O H D A SNC-lesioned, morp h i n e - d e p e n d e n t rats.

3.5. Effect o f unilateral, intraneostriatal arecoline administration on naloxone-precipitated contralateral circling in SNC-lesioned, morphine-dependent rats Arecoline, which was a d m i n i s t e r e d unilaterally i n t o the n e o s t r i a t u m , p r o d u c e d circling behavior t o w a r d s the injected side and did n o t m o d i f y the n a l o x o n e - p r e c i p i t a t e d withdrawal signs. Fig. 3 shows circling behavior a f t e r nal o x o n e in b o t h s h a m - o p e r a t e d and left-SNClesioned rats 20 min a f t e r 0, 32.5, 65 or 130 pg arecoline a d m i n i s t e r e d i n t o the right, in-

t a c t n e o s t r i a t u m . In animals r e n d e r e d depend e n t b y m o r p h i n e pellet i m p l a n t a t i o n , arecoline p r o d u c e d circling behavior of mild intensity t o w a r d s the side o f drug administration in b o t h s h a m - o p e r a t e d and left-lesioned rats. T h e c o n t r o l group injected with artificial CSF bilaterally did n o t e x h i b i t circling behavior. N a l o x o n e p r e c i p i t a t e d circling b e h a v i o r towards the c o n t r a l a t e r a l side in the left-SNClesioned, artificial CSF-injected groups as previously observed (figs. 1 and 2). Increasing doses o f arecoline, which itself p r o d u c e d circling m o v e m e n t s c o n t r a l a t e r a l to the SNC lesion, did n o t m o d i f y the d i r e c t i o n or the int e n s i t y o f the contralateral circling t h a t was p r e c i p i t a t e d b y n a l o x o n e . Additionally, intraneostriatal arecoline also failed to m o d i f y nal o x o n e - p r e c i p i t a t e d withdrawal signs in all groups o f animals.

3.6. Effect of unilateral, intraneostriatal atropine administration on naloxone-precipitated contralateral circling in SNC-lesioned, morphine-dependent rats A t r o p i n e , a d m i n i s t e r e d unilaterally into the right, i n t a c t n e o s t r i a t u m o f rats with left-SNC

48

E.T. I W A M O T O E T AL.

R 3

N=6-10

[]

left snc

lesion

[ ] sham

Fig. 3. E f f e c t of u n i l a t e r a l , i n t r a n e o s t r i a t a l arecoline a d m i n i s t r a t i o n i n t o t h e r i g h t n e o s t r i a t u m o f shamo p e r a t e d a n d left-SNC-lesioned m o r p h i n e - d e p e n d e n t rats o n circling b e h a v i o r b e f o r e a n d a f t e r n a t o x o n e precipitated withdrawal and on withdrawal behavior a f t e r n a l o x o n e . W i t h d r a w a l was p r e c i p i t a t e d b y 20 m g / k g n a l o x o n e 20 rain a f t e r a r e c o l i n e a d m i n i s t r a tion. T h e solid lines c o i n c i d e n t w i t h t h e p l a n e of t h e figure i n t e r c o n n e c t i n g circling a n d w i t h d r a w a l behavior c o l u m n s d e m a r c a t e groups o f a n i m a l s p r e t r e a t e d w i t h d i f f e r e n t a r e c o l i n e doses. Doses are d e n o t e d b y n u m e r a l s (pg o f free base, 0 = CSF) o b l i q u e t o t h e figure plane. Artificial C S F was a d m i n i s t e r e d conc u r r e n t l y i n t o t h e left n e o s t r i a t u m . Circling b e h a v i o r c o n t r a l a t e r a l t o t h e lesion is i n d i c a t e d b y c o l u m n s a b o v e t h e figure plane. All c o l u m n h e i g h t s r e p r e s e n t t h e m e a n o f m a x i m u m scores of circling or withdrawal b e h a v i o r a c h i e v e d b y each a n i m a l for each g r o u p of at least 6 rats. D i a m o n d s o n t h e c o l u m n t o p r e p r e s e n t significant d i f f e r e n c e f r o m t h e respective C S F - t r e a t e d c o n t r o l g r o u p s : 0, P < 0 . 0 5 ; 0, P < 0.01, as d e t e r m i n e d b y t h e M a n n - - W h i t n e y U-test.

lesions, produced circling behavior towards the opposite side of injection and markedly diminished naloxone-precipitated withdrawal signs. Fig. 4 shows circling behavior before and after naloxone and withdrawal behavior after naloxone in both sham-operated and left-SNC-lesioned animals 30 min after 0, 32.5, 65 or 130 pg atropine administered into the right intact neostriatum. In dependent animals, increasing doses of atropine produced dose-dependent circling behavior towards the left side in both sham-operated and left-SNC lesioned animals. As observed previously, naloxone precipitated contralateral circling to-

ward the intact neostriatum in the lesioned group of rats. In contrast to arecoline, unilateral atropine administration into the right neostriatum, which itself produced circling to the left, not only diminished naloxone-preciptared contralateral circling but also reversed the direction of circling towards the side ipsilateral to the lesion at the 65 and 130 pg atropine dosages. Furthermore, atropine reduced naloxone-precipitated withdrawal signs at the 65 pg dose and virtually abolished them at the 130 pg atropine dose in both lesioned and sham-operated groups of dependent rats. Indeed, of the total of 14 rats given 130 pg atropine, the behavior observed after naloxone

C,l~O~.~0

~

r~,~

@e°'~"

o

N=6-9

[ ] left snc lesion

[ ] sham

Fig. 4. E f f e c t of u n i l a t e r a l i n t r a n e o s t r i a t a l a t r o p i n e a d m i n i s t r a t i o n i n t o t h e r i g h t n e o s t r i a t u m of s h a m o p e r a t e d a n d left-SNC-lesioned m o r p h i n e - d e p e n d e n t rats o n circling b e h a v i o r b e f o r e a n d a f t e r n a l o x o n e precipitated withdrawal and on withdrawal behavior a f t e r n a l o x o n e . W i t h d r a w a l was p r e c i p i t a t e d b y 20 m g / k g n a l o x o n e 30 m i n a f t e r a t r o p i n e a d m i n i s t r a tion. T h e solid lines c o i n c i d e n t w i t h t h e p l a n e o f t h e figure i n t e r c o n n e c t i n g circling a n d w i t h d r a w a l behavior c o l u m n s d e m a r c a t e g r o u p s o f a n i m a l s p r e t r e a t e d w i t h d i f f e r e n t a t r o p i n e doses w h i c h are d e n o t e d b y n u m e r a l s (/lg o f free base, 0 = C S F ) o b l i q u e t o t h e figure plane. Artificial C S F was a d m i n i s t e r e d c o n c u r r e n t l y i n t o t h e left n e o s t r i a t u m . Circling b e h a v i o r c o n t r a l a t e r a l t o t h e lesion is i n d i c a t e d b y c o l u m n s a b o v e t h e figure p l a n e ; ipsilateral circling b e h a v i o r c o l u m n s lie b e l o w t h e plane. All c o l u m n h e i g h t s repr e s e n t t h e m e a n o f m a x i m u m scores a c h i e v e d b y each a n i m a l for e a c h g r o u p o f at least 6 rats. Diam o n d s r e p r e s e n t significant d i f f e r e n c e s f r o m C S F c o n t r o l as in fig. 3.

MORPHINE WITHDRAWALCIRCLING was n o t that of precipitated withdrawal but a continuation of pre-naloxone behavior after atropine, namely, continuous circling behavior to the left. However, 1 rat out of 9 in the lesioned group given 130 ttg atropine did exhibit teeth chattering for a score of 2.

49

Io

10-

5 MIN

5-

3.7. Effect o f naloxone on intraneostriatal arecoline- and atropine-induced circling in non-dependent rats with left-SNC lesions In animals implanted with 3 placebo pellets for 72 h, naloxone failed to alter circling behavior elicited after intraneostriatally administered arecoline or atropine. Circling behavior rank scores were identical before and after 20 mg/kg naloxone in animals administered arecoline or atropine intraneostriatally. Atropine, 130 pg into the intact neostriatum, produced continuous circling behavior towards the side of the lesion (onset of circling after injections, 5--15 min; group mean score, 2.75; n = 4) for about 2 h; the maximum scores in circling intensity achieved before naloxone were identical to those recorded after the naloxone challenge. Likewise, the circling behavior induced by 130 pg arecoline towards the intact, injected neostriatum (onset of circling after injections, 5--10 min; group mean score, 1.75; n = 4), which continued for up to 1.5 h after injection, remained unchanged after naloxone. Thus, in n o n - d e p e n d e n t animals, "intraneostriatal arecoline- and atropine-induced circling are unaffected by naloxone.

I

,~lll

i

JILL

i

2

1

0

~ca, 1

,

3.8. Effect o f intraneostriatal lidocaine on circling and withdrawal behaviors in morphinedependent, SNC-lesioned rats Intraneostriatal lidocaine, which was administered to test the possible influence of a local anesthetic action, did not alter the contralateral circling nor the withdrawal syndrome precipitated by naloxone in left-SNClesioned rats implanted with morphine pellets for 3 days. 50 tzg lidocaine injected into the intact, right neostriatum produced mild circling to the left side (group mean scores, 1.0

10-

0

30

MIN

5-

I 2

,,,ll

lilt,,,

I

I

~C,AA

I

I

0

I

2

Fig. 5. The diffusion of 3H-atropine in the rat forebrain (left, injected side) 5 and 30 min after intra-

neostriatal injection (n = 5 at each time point). The point of reference for the frontal sections was CA, commissura anterior. The rostral--caudal spread of label is indicated in ram.

and 1.0) in both sham and lesioned groups of animals (n = 4 each). Continuous circling to the right was still precipitated by naloxone 30 min after lidocaine in the lesioned group as previously observed; however, both groups of animals exhibited severe withdrawal behavior (mean withdrawal sign scores, 3.0 and 3.0). Thus, it is unlikely that the reversal of contralateral to ipsilateral circling and the attenuation of appearance of naloxone-precipitated withdrawal signs observed after intraneostriatal atropine (fig. 4) in SNC-lesioned rats were caused by a local anesthetic action.

3.9. Diffusion o f 3H-atropine after unilateral intraneostriatal injection Intraneostriatally administered 3H-atropine appeared to diffuse in a normal distribution pattern but remained mostly localized in the injected neostriatum even 30 min after injec-

50 tion. The actual recovery of labelled material 5 and 30 min after administration was 90 and 87%, respectively, of the amount initially injected. As shown in fig. 5, the label had spread approximately 2.9 mm after 5 min; 30 min after injection, 97% of the recovered label was found within 3.4 mm. The right, uninjected side contained radioactivity which was indistinguishable from blank values. With reference to the anterior commissure (Anterior 6800 #m to 7200/am), the center of the neostriatal injections was located at a b o u t Anterior 8200 pm, the approximate center of the body of the caudate--putamen nucleus (6400 pm to 9800 /am). Since the label was minimally transported into the bloodstream and the bulk of the radioactivity was recovered within the confines of the caudate--putamen nucleus and anterior globus pallidus of the injected side, it may be concluded that at 30 min after injection, the intraneostriatally administered atropine had its primary effect in the brain area containing the neostriatum and paleostriatum. 4. Discussion The data presented in this communication indicate that naloxone-precipitated withdrawal in morphine-dependent rats may be accompanied by a diminution of dopaminergic activities or enhancement of cholinergic activities, or both, in the nigro-neostriatal pathway. Using unilaterally SNC-lesioned rats as the model, we found that the direction of circling (contralateral) observed after a naloxone challenge in morphine-dependent rats was the same as that observed in lesioned, non-dependent animals after haloperidol administration (see also Andgn et al., 1966). This finding supports the hypothesis that a blockade of dopaminergic activity occurs in the nigro-neostriatal projection during naloxone-precipitated withdrawal (Iwamoto et al., 1976a). A decrease of dopaminergic transmission conceivably could lead to a diminished release with subsequent accumulation of dopamine in neostriatal terminals, which in fact, was ob-

E.T. IWAMOTO ET AL. served in morphine-dependent mice and rats during naloxone-precipitated withdrawal (Iwamoto et al., 1973; Iwamoto et al., 1976a). Moreover, two dopamine agonists, apomorphine and d-amphetamine, reversed the direction of contralateral circling precipitated by naloxone in rats with unilateral electrolytic SNC lesions and markedly reduced the appearance of naloxone-precipitated withdrawal signs. In contrast, both withdrawal behavior and naloxone-precipitated circling were unaffected by dopamine receptor blockade with haloperidol. Involvement of cholinergic neostriatalpaleostriatal activities is also suggested by these studies. Unilateral administration of arecoline into the intact neostriatum did n o t modify either the contralateral circling or the appearance of withdrawal signs precipitated by naloxone in morphine-dependent rats with unilateral SNC lesions. In contrast, both the circling and withdrawal behaviors were markedly attenuated by intraneostriatally administered atropine whereas the local anesthetic, lidocaine, was without effect. These results suggest that increased cholinergic activity might occur at the level of the neo-paleostriatum during naloxone-precipitated morphine withdrawal. Recent evidence support an hypothesis that the dopaminergic neurons of the nigro-neostriatal pathway may inhibit cholinergic interneurons in the neostriatum (McGeer et al., 1974; Groves et al., 1975). Dopamine (DA) receptor stimulation should then diminish acetylcholine (ACh) utilization (Ladinsky et al., 1975; G u y e n e t et al., 1975; Trabucchi et al., 1975). Conversely, dopamine receptor antagonism would be expected to enhance cholinergic activity (Stadler et al., 1973). Our present results are in agreement with the notion of an ACh-DA interaction at the level of the neostriatum and extend previous findings to include the possibility that the precipitated morphine withdrawal state may involve such an interrelationship (Way et al., 1975). That is, a diminution of DA activity during precipitated with-

MORPHINE WITHDRAWAL CIRCLING drawal may result in an enhancement of cholinergic mechanisms as evidenced by recent reports of increased ACh utilization and/or decreased total brain ACh content of morphine-dependent mice and rats during abrupt withdrawal (Domino and Wilson, 1973) and during naloxone-precipitated withdrawal (Bhargava and Way, 1975; Domino and Wilson, 1975). Since intraneostriatal atropine diminished the appearance of precipitated withdrawal signs (fig. 4), these data indicate that increased neostriatal cholinergic activity accompanies precipitated withdrawal. The results on the effects of peripherally administered anticholinergic drugs are less clear since both an exacerbation (Bhargava and Way, 1972; Brase et al., 1974) and an amelioration (Jhamandas and Dickinson, 1973) of the narcotic abstinence syndrome in mice have been reported. An explanation must await further experiments in view of other findings which indicate that the varying effects on neostriatal dopamine turnover observed after anticholinergic administration may depend on the site of administration (Bartholini and Pletscher, 1971; Javoy et al., 1974). Cholinergic neurons may exist in the SNC (Butcher et al., 1975) as well as in the neostriatum (Corrodi et al., 1972). These findings, therefore, may provide an explanation for opposing effects of intraneostriatally and peripherally administered anti-. cholinergic agents on precipitated withdrawal. Also, it cannot be stated at this time whether the attenuation of withdrawal by apomorphine and amphetamine is due to enhanced neostriatal DA activity or to DA receptor stimulation of neostriatal or SNC cholinergic interneurons resulting in a decrease of ACh release at these sites. Additionally, a participatory role for 3,-hydroxybutyric acid should also be considered (McGeer et al., 1974). In similarity with out findings, Herz et al. (1974) observed that a large dose of apomophine, 5 mg/kg, interfered with levallorphaninduced withdrawal jumping behavior in morpine-dependent rats. Our results differ from theirs at the lower doses (1.5--3.0 mg/kg) of

51 apomorphine in that we still observed a decrease in withdrawal intensity (fig. 1) whereas a profound enhancement of withdrawal jumping behavior was observed by Herz at 2.5 mg/kg apomorphine. However, these workers did observe a diminution of other levallorphan-precipitated withdrawal signs after ~heir d-amphetamine or apomorphine pretreatment which is in agreement with our results. We can offer no reason for the discrepancy except to note that our animals were implanted with a lower dose of morphine and for a shorter period of time (1125 mg/kg morphine over 72 h'versus 2250 mg/kg morphine over 240 h). Additionally, our sham-operated, morphine-dependent animals were observed to be quite stereotypic and almost all animals exhibited continuous sniffing and licking, biting or gnawing behavior before and after naloxone even at the lowest dose of apomorphine pretreatment, 1.5 mg/kg, and these daSa are similar to those previously reported in naive rats (Iwamoto et al., 1976b). It is conceivable, therefore, that drug-induced stereotyped behavior 'occludes' naloxone-precipitated signs (Herz et al., 1974), and our results support this interpretation (table 1). However, if increased dopaminergic transmission and dopamine release actually do occur during precipitated withdrawal, one might expect apomorphine and d-amphetamine to enhance the intensity of withdrawal signs. Since a reduction in withdrawal sign frequency was observed in our animals after these dopamine-mimetic agents, the results are more compatible with the interpretation that diminished dopaminergic transmission occurs during precipitated withdrawal. If a diminution of dopaminergic transmission occurs during naloxone-precipitated withdrawal, one might expect that blockade of dopamine receptors with neuroleptic drugs should produce behavior similar to withdrawal. We have observed that certain withdrawal signs in the rat do bear, at least, ~superficial resemblance to some neuroleptic-induced behavioral signs such as ptosis, abnormal (hunched) posturing, vocalization to touch and blanch-

52

ing of the ears. These same signs generally occurred after the first 4--5 min of naloxoneprecipitated withdrawal in morphine-dependent rats. However, such a simplification of the precipitated morphine-withdrawal syndrome does not seem complete since increased exploratory activity, wet shakes and escape attempts are observed during the first 4 - 5 min after naloxone (see also Wei et al., 1973; Blasig et al., 1973; Domino and Wilson, 1975). A rather peculiar finding we have observed in morphine-dependent rats was the momentary disruption of haloperidol-induced cataleptic behavior shortly after a naloxone challenge (see present results, table 3) during which animals exhibited withdrawal behavior lasting from 1 to 10 min. Since a reversal of cataleptic behavior was observed, this finding suggested that dopaminergic transmission may be enhanced. However, we believe that this is n o t the case since the 2 mg/kg dose of haloperidol used was more than adequate to block completely the stereotypy induced by 3 mg/ kg apomorphine or 3.2 mg/kg d-amphetamine, doses of which produce profound stereotyped behavior alone (Iwamoto et al., 1976b). It is presently u n k n o w n h o w haloperidol catalepsy can be quickly reversed during naloxone-precipitated withdrawal. However, neuroleptic-induced catalepsy and alterations of DA metabolism appear to require an intact nigro-neostriatal pathway (Costall and Olley, 1971; Nyb~ck and Sedvall, 1971). Lesions of the nigro-neostriatal pathway at the level of the globus pallidus or SNC greatly diminish haloperidol-elicited catalepsy in rats (Costall and Olley, 1971). Thus, one possible explanation for the disruption of haloperidolelicited catalepsy during naloxone-precipirated withdrawal may be a presynaptic blockade of the nigro-neostriatal projection. Additionally, recent data suggest that haloperidol, narcotic agonists and antagonists may interact with the same tissue binding site of rat brain homogenates (Clay and Brougham, 1975). However, this observation still does not explain the failure of narcotic antagonists to re-

E.T. I W A M O T O E T AL.

verse neuroleptic-induced catalepsy in naive animals (Kuschinsky and Hornykiewicz, 1972). It is thus apparent that the initial events of precipitated withdrawal, during which haloperidol catalepsy was disrupted by the naloxone challenge, differ both in character and in time sequence from withdrawal behavior appeaxing later. The sequence of neurochemical events during precipitated withdrawal cannot be determined as this time. However, our finding that naloxone disrupts haloperidol catalepsy only in morphine-dependent rats may provide a basis for further investigations of narcotic withdrawal (compare tables 3 and 4). In addition to a participation of both dopamine and acetylcholine, possible involvement of serotonin and noradrenaline in the circling behavior model has been suggested (Cools and Janssen, 1974; Costall and Naylor, 1974; Pycock et al., 1975). Discussion of our present data has been limited to ACh--DA interactions in naloxone-precipitated circling behavior during morphine withdrawal in rats; the roles of serotonin and noradrenaline in this behavior must await additional experiments. In summary, using the circling behavior model to differentiate dopamine agonistic and antagonistic effects, it was observed that the administration of apomorphine and d-amphetamine systemically and atropine intraneostriatally in morphine-dependent rats with unilateral, electrolytic SNC lesions either reversed the direction or decreased the intensity of the naloxone-precipitated contralateral circling behavior while concomitantly causing a diminution of the intensity of the precipitated withdrawal syndrome. It is proposed that naloxone-precipitated withdrawal may be accompanied by either a diminution of dopaminergic activities or an enhancement of cholinergic activities, or both, at level of the neostriatum.

Acknowledgements This w o r k was s u p p o r t e d in p a r t b y research grants f r o m t h e N a t i o n a l I n s t i t u t e o f Drug A b u s e D A 0 0 0 3 7

MORPHINE WITHDRAWAL CIRCLING and DA 00564. Portions of this paper were presented at the 58th Annual Meeting of the Federation of American Societies for Experimental Biology (FASEB), Atlantic City, N.J., April 7--12, 1974 and at the Western Pharmacology Society Meetings, Honolulu, Hawaii, January 1975.

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