A role for the subthalamic nucleus in 5-HT2C-induced oral dyskinesia

Pergamon 0306-4522(95)00548-X

Neuroscience Vol. 72, No. 1, pp. 117-128, 1996 Elsevier Science Ltd

Copyright © 1996 IBRO Printed in Great Britain. All rights reserved

0306-4522/96 $15.00 + 0.00

A ROLE FOR THE SUBTHALAMIC NUCLEUS IN 5-HT2c-INDUCED ORAL DYSKINESIA

K. E B E R L E - W A N G , * I. L U C K I * t and M.-F. CHESSELET*:~

Departments of *Pharmacology and ]'Psychiatry, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.

Abstract--The 5-hydroxytryptamine2c serotonin receptor is broadly distributed in brain, however its functional role is unknown. Peripheral administration of drugs acting at the 5-hydroxytryptamine2c receptor induces abnormal oral dyskinesias, hyperkinetic motor disorders that often result from dysfunction of the basal ganglia. The subthalamic nucleus, a brain region anatomically and functionally related to the basal ganglia, has been implicated in oral dyskinesia. The subthalamic nucleus contains messenger RNA encoding 5-hydroxytryptamine~c receptors, suggesting its potential role in 5-hydroxytrypt- aminezc-mediated oral dyskinesia. Both systemic administration and local unilateral infusion of the 5-hydroxytryptamine2c/l B agonist, 1-(m-chlorophenyl)piperazine into the subthalamic nucleus increased orofacial movements. Oral movements following subthalamic infusion of 1-(m-chlorophenyl)piperazine were blocked by systemic administration of the 5-hydroxytryptamine2c/2 A antagonists mianserin, ketanserin and mesulergine but were not altered by systemic pretreatment with either the 5-hydroxytryptamine~A/2 A and dopamine antagonist spiperone or the 5-hydroxytryptaminelA/l B antagonist pindolol. Co-infusion of mesulergine with l-(m-chlorophenyl)piperazine into the subthalamic nucleus blocked 1-(m-chlorophenyl)piperazine-stimulated oral movements. Oral bouts following systemically administered 1-(m-chlorophenyl)piperazine were markedly reduced following bilateral subthalamic infusion of either mesulergine or the selective 5-hydroxytryptamine2c antagonist SDZ SER 082. The findings indicate that stimulating 5-hydroxytryptamine2c receptors in the subthalamic nucleus elicits orofacial dyskinesia in the rat.

These data are novel in providing a behavioral model for central 5-hydroxytryptamine2c receptor stimulation attributed to a specific anatomical location, and suggest that antagonists at the 5-hydroxy- tryptamine2c receptor could be useful in treating hyperkinetic motor disorders.

Key words: l-(m-chlorophenyl)piperazine, serotonin, 5-hydroxytryptamine, SDZ SER 082, basal ganglia, hyperkinesia, orofacial dyskinesia.

Orofacial dyskinesias are a severe side effect of pro- longed treatment with antipsychotic drugs and are also present in a number of hyperkinetic disorders of the basal ganglia in humans. Increased orofacial movements have been elicited in rats by peripheral administrat ion of dopaminergic antagonists, ~2'5° agonists, 42 and more recently, agonists of the 5-hy- droxytryptamine2c (5-HT2c) receptorY This latter finding is of particular interest because it suggests that endogenous serotonergic (5-hydroxytryptamine, 5-HT) pathways may participate in the generation of orofacial dyskinesia and that antagonists at the 5- HT2c receptor may be of potential therapeutic value in treating orofacial dyskinesia. Furthermore, it

:~To whom correspondence should be addressed. §The International Nomenclature Committee of the

Serotonin Club has reclassified the 5-HT~c receptor as a 5-HT2c receptor and renamed the classical 5-HT 2 receptor as a 5-HT2A receptor.

Abbreviations: CA3, CA3 hippocampus; CP, cerebral peduncle; 5-HT, 5-hydroxytryptamine; LH, lateral hypothalamus; m-CPP, 1-(m-chlorophenyl)piperazine; STh, subthalamic nucleus; ZID, dorsal zona incerta; ZIV, ventral zona incerta.

suggests a potential function for 5-HT2c receptors in motor control.

Al though the 5-HTzc receptors (formerly the 5- HT~c receptor§), were originally thought to be expressed exclusively in the choroid plexus, 39 more recent studies have revealed a more widespread expression of the receptor and its m R N A in brain. 1°'2°'3° The functions subserved by these brain 5-HT2c receptors, however, remain unknown. In particular, it is unclear which brain regions contain- ing 5-HT2c receptors mediate the effects of agonists on orofacial movements.

Orofacial dyskinesias often result from alteration in the function of the basal ganglia, a group of subcortical brain regions involved in the control of movement. 9'5° In the basal ganglia, the m R N A encoding 5-HT2c receptors is expressed in the ventrolateral striatum, the caudal substantia nigra pars reticulata, the entopeduncular nucleus, and the subthalamic nucleus (STh). 1°'2°'3° This latter region is of particular interest because recent work in our laboratory has demonstrated that stimulating dopaminergic receptors within the STh by local administration of apomorphine elicits orofacial dyskinesias with similar

117

118 K. Eberle-Wang et al.

cha rac t e r i s t i c s to t hose o b s e r v e d af te r per iphera ' , ad - m i n i s t r a t i o n o f 5-HT2c agon i s t s . 37'47 T h e S T h pro jec t s

to, a n d in f luences the ac t iv i ty o f the m a j o r o u t p u t nucle i o f the basa l gangl ia : the g l o b u s pa l l idus (exter-

na l pa l l i dum) , the e n t o p e d u n c u l a r n u c l e u s ( in te rna l

p a l l i d u m ) a n d the s u b s t a n t i a n i g r a p a r s re t icu- lata. 1324"36"43"49 In b o t h h u m a n s a n d p r i m a t e s , un i la t -

eral l e s ions o f the S T h resu l t in h e m i b a l l i s m w h i c h is

c h a r a c t e r i z e d by i n v o l u n t a r y , hype rk i ne t i c m o v e - m e n t s o f the c o n t r a l a t e r a l l imbs . 32'51 T h e i m p o r t a n c e

o f the S T h in the c o n t r o l o f m o t o r b e h a v i o r is f u r t he r

i l lu s t r a t ed by the reversa l o f p a r k i n s o n i a n s y m p t o m s

by les ions o f the S T h in m o d e l s o f P a r k i n s o n ' s d i sease

in p r i m a t e s Y

W e have e x a m i n e d the h y p o t h e s i s tha t the S T h p lays

a role in oral m o v e m e n t s i nduced by 5-HTzc agonis ts .

C o m b i n a t i o n s o f se ro tonerg ic d rugs were admin i s t e r ed sys temica l ly a n d / o r directly into the S T h via ch ron ic

indwel l ing cannu l a e , a n d orofac ia l m o v e m e n t s were

recorded in consc ious , u n r e s t r a i n e d adu l t rats.

EXPERIMENTAL PROCEDURES

Surgery

All procedures were performed on male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, U.S.A., 250 275 g) in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local animal care committee. Prior to surgery, rats were maintained on a 12:12 hour l igh t , l a rk schedule with food pellets and water ad libitum. Rats were anesthetized with equithesin (prepared as per instruction of Janssen-Salbutry laboratories, Kansas City, MO) and implanted with cannulae for unilateral or bilateral microinfusion of drug into the subthalamic nucleus ( A P = + 4 .9mm; M L - + 2 . 5 m m relative to interaural zero and DV - 7.4 below the surface of the cortex). 3s Unilateral cannulat ion was performed using the left STh. For bilateral cannulation, two guide cannulae were positioned using a copper rectangular plate (5 x 15 mm, with two holes centered 5 mm apart) which was implanted within the headblock during surgery. Details of the surgical procedure, infusion cannulae and injection equipment have been described previously? 7 Briefly, the guide shaft was made of a 22-gauge cannula and the injector was made of a piece of fused silica ( o . d . - 1 5 0 / ~ m , i.d. - 75 #m) threaded through a 28-gauge internal cannula such that a 1 mm tip of fused silica extended from the end of the cannula shaft. Behavioral testing began one week following surgery.

Behavioral testhlg

On the day of behavioral testing, each rat was adapted to a quiet room in a clear plastic circular cage (12" diameter x 18" high) for 1 h prior to drug (or vehicle) infusion into the STh. Systemic or local pretreatment with various serotonergic antagonists (mianserin, ketanserin, mesulergine, SDZ SER 082, spiperone, or pindolol) or their respective vehicles was administered during this 60-min period. Following the 60-min adaptat ion and immediately prior to the onset of behavioral observations, the serotonergic agonist, l- (m-chlorophenyl)piperazine (m-CPP) or its vehicle was administered either systemically (i.p.) or locally into the STh. The effect of local or systemic administration of m-CPP to stimulate oral movements was confirmed by an observer blind to the treatment conditions. In the case of microinfusion into the STh, the awake rat was gently hand-held and the drug was infused directly into the STh via an injector placed within the surgically-implanted guide cannula. A total volume 0.1 t~l was infused over 54 s. The

injector was left in place for an additional 60 seconds to permit diffusion of the drug away from the injector tip. In the case of bilateral cannulation, the drug infusions were done serially starting with the left STh. Previous observations following infusion of Pontamine Blue (P. Brun and M.-F. Chesselet, unpublished observations), or of the neurotoxin kainic acid, 8'37 revealed that this volume and method of infusion results in delivery of drug to an area confined within the STh.

Following local administration of drugs into the STh, the rats were returned to the testing cage and observed continu- ously for 60 min for bouts of orofacial movements. An oral bout was defined as any combination of continuous orofacial movements including vacuous chewing, jaw tremor, and tongue darting, distinct from ingestive or grooming behaviors. The frequency of oral bouts was quantitated over the 60 min observation period. The duration of each bout was not simultaneously measured. Additional behaviors which were quanti tated during the 60 min testing period included: grooming, turning, sniffing, rearing, wet dog headshakes, cage licking, and yawning. In the present experiments, as in previous s tudies) 7 orofacial movements were recorded in unrestrained animals. This allowed for the potential dctcc- tion of any competing behavior such as stereotypy or sedation which could influence the oral dyskinesia. 27 The serotonin syndrome, a behavioral model of central 5-HT~- type receptor stimulation, 48 did not occur during any of the experiments. In all studies, at least two drug-free days intervened between test days using m-CPP, with a total of one week between experiments using the various antagonists. During the course of behavioral testing (one to eight weeks post op.), vehicle- and m-CPP-induced responses were re-evaluated regularly to check for possible sensitiz- ation or loss of responses.

Verification o f cannula placement

At the completion of the behavioral studies for each surgical group, all rats were anesthetized and killed by decapitation. Brains were removed, rapidly frozen on pow- dered dry ice, and stored at - 2 0 ° C . Twenty micrometer thick sections were sectioned on a cryostat through the STh. Tissue sections from each brain were fixed for 30 min in 4% paraformaldehyde and stained with Cresyl Violet for verification of cannula placement within the STh. Only data from rats in which accurate cannula placement was verified were retained for analysis. The criterion for inclusion was clear evidence that the tip of the cannula tract was located within the STh. Successful placement of the cannula was seen in > 90% of all of the surgeries performed. A representative tissue section depicting cannula placement in the STh is shown in Fig. 1A. The high power photomicrograph shown in Fig. 1B shows that minimal damage occurs in the STh even in rats which received multiple drug injections.

Materials

1-(rn-Chlorophenyl)piperazine HC1 (m-CPP, FW = 196.7) was prepared in dH20 and the final pH was adjusted to 6.6 with NaOH. m-CPP was stored at - 2 0 C in aliquots and thawed for use on each experimental day. Mesulergine HCI (FW - 398.9), mianserin HCI (FW = 337.3) and SDZ SER 082 (FW free base = 228) were dissolved in dH20. ( + / )Pindolol ( F W - 248.3) was dissolved in 0.2% (v/v) glacial acetic acid. Ketanserin HC1 ( F W = 4 3 1 . 5 ) and spiperone (FW = 395.5) were wetted with Tween-80 and suspended in dH20. Drug dosages were calculated with respect to the free base weight of each compound, m-CPP was purchased from Aldrich Chemical Co. (Milwaukee, WI). Mianserin and mesulergine were purchased from Re- search Biochemicals Inc. (Natick, MA). Spiperone, ketanserin, and pindolol were purchased from Sigma Chemical Co. (St Louis, MO). SDZ SER 08234 was generously donated by The Preclinical Research Division of Sandoz Pharma LTD, Basel, Switzerland.

Subthalamic 5-HT2c receptors elicit oral dyskinesia 119

?

%

. . s

2 . " ~ " a ' , .

Fig. 1. (A) Low power photomicrograph of a cannula tract showing tip placement within the subthalamic nucleus• The three arrowheads highlight the vertical pathway of the cannula tract terminating in the subthalamic nucleus (arrow). (B) High power photomicrograph showing the tip of an injection cannula in a rat which received multiple drug injections. Note that the damage due to the injector is confined to a minimal area within the nucleus (gliosis at arrow). STh, subthalamic nucleus; cp, cerebral peduncle. Scale

bar A = 500/~m; scale bar B = 200 #m.


1 O0

~= 80

m ~- 60 ~'~ O o ~ 40

i-- 2o

G Veh 1.0 2.0 4.0 8.0

m-CPP dose (mg/kg)

m-CPP B (mg/kg, ip)

30-

~o 20-

~ 10"

I--

0 10 20 30 40 50 60

Time Post-Injection (min)

Fig. 2. (A) Systemic administration of m-CPP (0, 1.0, 2.0, 4.0 and 8.0mg/kg, i.p.) stimulated oral bouts in a dose- related manner. The data represent the mean + S.E.M. from n = 8 rats. The doses of m-CPP (1.0, 2.0, 4.0, 8.0 mg/kg) were administered randomly over four test days with a minimum of two intervening, drug-free days--such that at the end of the four test days all rats had received each of the four doses of m-CPP. All eight rats were then tested for baseline responses following injection of vehicle (0 mg/kg) one week following the fourth drug-test day. *P < 0.05 when compared to the Veh group with ANOVA and Dunnett's test. (B) Time course of oral dyskinesia following systemic administration of m-CPP. These time-course data are from the same experiment described in A. The number of oral bouts was grouped by 10-min observation periods for each drug treatment. At the 1.0 mg/kg probe dose, 82% of the total oral bouts occurred between 10 and 50 min of

the 60-min observation period.

Statistical analysis For each study, the total number of oral bouts was

calculated for the 60 min observation period and was analysed with the Statview 512+ Interactive Statistics and

Graphics Package (Version 1.0, Abacus Concepts). Analysis of Variance (ANOVA) was followed by: Dunnett's test for comparison of treatment means to a control. Duncan's multiple range test was used for comparisons between treatment groups. The threshold for significance was P < 0.05.

RESULTS

Systemic administration of 1-(m-chlorophenyl) piperazine produced oral dyskinesia

Confi rming previous reports, 47 systemic injection of the 5-HTzc/m receptor agonist m - C P P (1.0-8.0 mg/kg, i.p.) s t imulated oral bouts in a dose- related m a n n e r (Fig. 2A). The est imated EI950, 1.0 mg/kg (51.5 + 6.2 oral bouts /60 rain), was used as a probe dose in fur ther studies. Each oral bou t was a con t inuous episode of m o u t h movements which might include any combina t ion of non-directed chewing, jaw tremors, tongue dar t ing and large ampl i tude chews (gaping), characterist ically lasting 2 10 s. These orofacial movements were nondirected as they were not or iented towards any object or purpose, such as g rooming or ingestion.

Analysis of the time course of the oral bouts elicited by peripheral adminis t ra t ion of m - C P P at 1 .0mg/kg revealed tha t increased oral bouts were reliably observed within 10min of drug adminis- t rat ion, and 82% of the total oral bouts occurred between 10 and 50 min following inject ion (Fig. 2B). The elicitation of abnorma l orofacial movements by systemically adminis tered m - C P P had no effect on ongoing exploratory or g rooming behaviors.

Bilateral infusion of the 5-hydroxytryptamine2c antagonists, mesulergine or SDZ SER 082 into the subthalamic nucleus reduced oral movements following systemicall.v -administered 1 -(m-chloro - phenyl)piperazine

To determine whether s t imulat ion of 5-HTzc receptors in the STh were involved in the orofacial dyskinesia induced by peripheral adminis t ra t ion of m-CPP, the 5-HT2c antagonis ts (Table 1) mesulergine and SDZ SER 082 were adminis tered into bo th subtha lamic nuclei immediately before a systemic injection of m - C P P (Table 2). Local izat ion of bo th left and right cannula placements with respect to the STh is depicted for each animal in Fig. 3. With the

Table 1. Summary of serotonergic drugs used in this study

5-HT2c Other receptors

Agonist m-CPP + 5-HTIB

Antagonists Mesulergine + 5-HT2A Mianserin + 5-HT2A Ketanserin + 5-HT2A ~-adrenergic SDZ SER 082 + Spiperone - 5-HTIA/2A, DA Pindolol 5-HTIA/I B fl-adrenergic

Based on Refs 16, 23, 31, 34.

Subthalamic 5-HT2c receptors elicit oral dyskinesia

Table 2. Summary of the effect of drugs on 1-(rn-chlorophenyl)piperazine-induced oral dyskinesia

121

Systemic antagonist Local STh antagonist

Systemic m-CPP

Subthalamic m-CPP l Mesulergine + Mianserin + Ketanserin + Spiperone - Pindolol --

Mesulergine + t SDZ SER 082 + ]"

Mesulergine + *

+ Reduces m-CPP-induced oral dyskinesia. -Does not reduce rn-CPP-induced oral dyskinesia. *Infused unilaterally into the left STh. tlnfused bilaterally into the STh.

technique used, damage caused by the microinjector was minimal in the STh (Fig. 1). Bilateral, subthalamic infusion of mesulergine (0.5 and 1.0/~g/each STh) reduced oral movements following systemic injection of m-CPP (1.0 mg/kg) in a dose-dependent manner (Table 3). Four weeks following the completion of the study with mesulergine, rats received a bilateral infusion of 0.1 #1 dH20, and return to the baseline level of responding was verified in all animals (mean + S.E.M. = 7.0 + 1.9, n = 7). In these animals, bilateral infusion of SDZ SER 082 (0.35 and 0 .50#g/each STh) following return to the baseline, also significantly reduced oral movements following systemic injection of m-CPP (1.0mg/kg, i.p.;

Table 3). Subthalamic administration of the higher doses of each of these 5-HT2c antagonists, mesulergine (1.0/lg) and SDZ SER 082 (0.50/~g), was sufficient to block 64 and 50%, respectively, of the response to peripherally administered m-CPP.

Unilateral administration of 1-(m-chlorophenyl) piperazine in the subthalamic nucleus produces oral dyskinesia

To confirm that stimulation of 5-HT2c receptors in the STh elicit orofacial dyskinesia, 5-HT2c agonists were locally infused within this region. These experiments were performed with unilateral infusions within the left STh, because previous studies had

Fig. 3. Diagram of tip placements after bilateral cannulation of the subthalamic nucleus. The location of injector tip placements for each of seven rats are depicted in schematic coronal sections. Each of the seven different symbol pairs on the diagram correspond to placements within the right and left subthalamic nucleus for each of the seven individual animals. Numbers on the upper right of the sections indicate mm from bregma, based on the atlas of Paxinos and Watson. 38 cp, Cerebral peduncle; CA3, the CA3 of the

hippocampus; STh, subthalamic nucleus.


Table 3. Bilateral administration of 5-hydroxytryptamine2c antagonists, mesulergine or SDZ SER 082 into the subthalamic nucleus significantly reduced oral bouts following systemic administration

of 1-(m-chlorophenyl)piperazine

Total number of oral bouts/60 min i.p. STh Mean _+ S.E.M. n

Vehicle Vehicle 3.6 + 1.2 7 m-CPP Vehicle 56.0 + 7.0* 7 m-CPP Mesulergine (0.5) 28.7 _+ 2.6** 7 m-CPP Mesulergine ( 1.0) 20.3 _+ 4.4** 6

Vehicle Vehicle 7.0 +_ 1.9 6 Vehicle SDZ SER 082 (0.35) 7.8 -+- 3.7 6 m-CPP Vehicle 53.7 _+ 13.3" 6 m-CPP SDZ SER 082 (0.35) 28.5 -+- 6.9** 6 m-CPP SDZ SER 082 (0.5) 26.5 -+- 5.5** 6

Rats with bilateral cannulae were infused with mesulergine (0.5, 1.0 #g) or vehicle immediately preceding a systemic injection of m-CPP (1.0 mg/kg, i.p.). One week following this experiment, the same group of bilaterally cannulated rats was tested with the 5-HTzc antagonist SDZ SER 082 (0.35, 0.5/~g) using a similar experimental design. All rats were tested with the same drug treatment combination on each of the test days in the order listed above, with at least one week between successive administrations of each antagonist. *P < 0.05 when compared to the Veh-Veh group with ANOVA and Dunnett's test. **P < 0.05, compared to Veh-m-CPP, with ANOVA and Duncan's multiple range test.

shown tha t uni lateral drug infusions in the STh were sufficient to elicit robus t orofacial dyskinesia. 37 Local infusion of 5-HT (1.0/~g, 5-HT creatine sulfate) into one STh st imulated oral bouts (56.0 + 13.8, n = 7) in awake rats. However, the use of 5-HT in fur ther studies was limited by its relatively poor solubility, and lack of receptor selectivity. To test the hypothesis tha t s t imula t ion of 5-HT2c receptors within the STh is sufficient to induce an increase in orofacial movements , m - C P P was adminis tered directly into the STh on one side of the brain. Uni la tera l infusion of m - C P P (0 .5 -4 .0#g) into the left STh st imulated nondi rec ted orofacial movements in a dose-related manner , with the m a x i m u m n u m b e r of oral bouts occurr ing with 1.0/*g of m - C P P (Fig. 4A). A sub- maximal dose of 0.75/~g was chosen from the dose- response curve as a p robe dose for fur ther behaviora l studies. The oral bouts observed following local sub tha lamic infusion of m - C P P resembled those following systemic inject ion of this c o m p o u n d in the previous experiment, except for the notable absence of large ampl i tude chewing or gaping behavior . The stability of baseline responses was reproducible within and across exper imental groups. The baseline frequency of oral bouts was not affected by either the surgical or the infusion procedures because compar- able levels of orofacial movements were observed in intact rats (11.4 _+ 1.9, n = 8), and in cannula ted rats after gentle res t ra int with no infusion (14.8 _+ 3.3, n = 8).

Analysis of the t ime course of m-CPP-s t imula ted oral dyskinesia taken from the same experiment revealed tha t m - C P P (0.5, 1, 2 and 4 # g ) increased oral bouts within the first 10 min following subthalamic infusion, with ~ 70% of the total n u m b e r of oral movements occurr ing between 20 and 40 min following infusion (Fig. 4B). No changes in the frequencies of turning, sniffing, grooming, or rearing were ob-

served dur ing the testing period as a funct ion of dose of m-CPP. The ana tomica l selectivity of m-CPP ' s behaviora l effects at the level of the STh are fur ther suppor ted by observat ions tha t deliberately infusing m - C P P (0.75/~g) into other bra in regions proximal to but outside the STh, the dorsal zona incerta (ZID, n = 4) and the lateral hypotha lamic area (LH, n = 1: Fig. 5), failed to elicit nondirected oral movements above baseline levels: vehicle 11 .4+ 5.1 vs m - C P P 4.6 _+ 2.1 oral bouts /60 min (n = 5).

Peripheral administration of the 5-hydroxyto,pta- mine2c/2 A antagonists mianserin, ketanserin and mesulergine, blocked oral movements following inJusion of 1-(m-chlorophenyl)piperazine into the subthalamie nucleus

To confirm a role of 5-HTzc receptors in the effect of local adminis t ra t ion of m - C P P into the STh, drugs sharing antagonis t effects at this receptor were administered peripheral ly pr ior to local infusion of m - C P P into the STh. Systemic pre t rea tment with mianser in (1.0 mg/kg, s.c.), a nonselective antagonis t of 5-HT2A and 5-HT2c receptors (Table 131) completely blocked oral dyskinesia following unilateral infusion of m - C P P (0 .75pg) into the left STh (Table 4). In the same group of five animals, pretreatment with ketanser in (5.0 mg/kg, s.c.) similarly pre- vented oral bouts following subtha lamic infusion of m - C P P (0.75/*g). This relatively high dose of ketanser in is sufficient to block bo th 5-HT2A and 5-HT_,c receptors. 16.23

Fur the r suppor t ing the involvement of the 5-HTzc receptor in this effect, systemic adminis t ra t ion of mesulergine (0.25 and 0.5 mg/kg, s.c) reduced in a dose-related m a n n e r oral bouts induced by unilateral infusion of m - C P P (0.75 #g; Table 4). Mesulergine has an approximate ly three-fold higher affinity for 5-HTzc than for 5-HT m receptors ~6 and is considered


a relatively selective an tagonis t of 5-HT2c receptors (Table 131). The higher dose of mesulergine (0.5 mg/kg) completely blocked m-CPP-s t imula ted orofacial movements (Table 4: mesulergine [0.5]/m- CPP), bu t did not by itself alter baseline levels of orofacial movemen t s (Table 4: mesulergine [0.5]/vehicle). A l though bo th doses of mesulergine produced marked reduct ions in the incidence of m-CPP-s t imula ted orofacial movements , the reduct ion of abno rma l orofacial movements was not due to sedat ion because nei ther dose of mesulergine changed the incidence of normal exploratory and grooming behaviors . The higher dose of mesulergine (0.5 mg/kg), however, did produce some in termi t ten t headdown sniffing and cage-licking in four of the seven rats which did not appear to interfere with the elici tat ion of the oral bouts.

Peripheral administration of the 5-hydroxytrypt- aminelA/2A dopamine antagon&t, spiperone, or of the 5-hydro~wtryptaminelA/~ B ~-adrenergic antagonist, pindolol did not reduce oral movements following unilateral infusion of 1-(m-chlorophenyl)piperazine into the subthalamic nucleus

Systemic pre t rea tment with a relatively high dose of spiperone (1.0 mg/kg, s.c.), a prototypical an tagon- ist of 5-HT~A,2A and D A 2 (dopamine2) but not 5-HT2c receptor subtypes (Table 13~) produced a small, non- significant enhancemen t of the frequency of oral bouts when given alone (Table 5: spiperone/vehicle). This increase in oral movements following spiperone t rea tment (1.0 mg/kg, s.c.) occurred in spite of the fact tha t this dose of the an tagonis t also produced characterist ic sedation, and therefore a notable

A

60-

4O

m

o I--

Veh 0.5 1.0

m-CPP Dose (pg)

2.0 4,0

15

10 O

~'~ O o

"6 I - 5 -

10 30 40

Time Post-Infusion (min)

m-CPP (l~g/STh)

s; 8;

Fig. 4. (A) Infusion of m-CPP into the STh stimulates oral dyskinesia in awake rats. Microinfusion of the serotonergic agonist, m-CPP (0.5, 1.0, 2.0 and 4.0 ~g; total volume 0.1/~1) or its vehicle (dH20) into the STh stimulated abnormal orofacial movements in a dose-dependent manner. Data (n = 6 rats) are expressed as mean number of total oral bouts per 60 min + S.E.M. Asterisks (*) indicate value differed significantly from the Veh group, according to Dunnett's test (P < 0.05). (B) Time course of oral dyskinesia following infusion of m-CPP into the STh. These time-course data are from the same experiment described in A. The number of oral bouts were grouped by 10-min observation periods for each drug treatment. While there was an enhancement of oral bouts within the initial 10 min, the majority of m-CPP-stimulated oral bouts ( ~ 70%) occurred between 20 and 40 min post-infusion, with a trend

towards baseline levels by 60 min.

NSC 7ZI E


f

Bregma -4.16 mm

Fig. 5. Schematic diagram ofcannula placements for central injection sites deliberately outside of the suhthalamic nucleus. Probe tips labeled by the five symbols on the diagram terminated in the dorsal zona incerta, the ventral zona incerta and the lateral hypothalamus. The schematic diagram, based on the atlas of Paxinos and Watson, 38 is a hemicoronal section at 4.16 from Bregma. cp, Cerebral peduncle: CA3, the CA3 of the hippocampus; STh, subthalamic nucleus; LH, lateral hypothalamus; ZID, dorsal zona

incerta; ZIV, ventral zona incerta.

reduct ion in turning, sniffing, g rooming and rearing behaviors. However, this t rea tment did not reduce the large increase in orofacial movements following local infusion of m - C P P (0.75 #g) into the left STh (Table 5: spiperone/m-CPP).

As with spiperone, systemic pre t rea tment pindolol alone, an an tagonis t of 5-HTIA,jB and / t-adrenergic receptors but not 5-HT2c receptors (Table 1), also produced a small increase in the frequency of oral bouts (Table 5: pindolol/vehicle). However, at a dose previously shown to be sufficient to block centrally mediated effects, " 'm~ systemically adminis tered pindolol ( l . 0 m g / k g , i.p.) did not a t tenuate oral bouts following local, unilateral infusion of m-CPP (0.75/ lg) into the STh (Table 5: p indolol /m-CPP) .

Co-in[usion ~] mesulergine with 1-(m-chlorophenyl)piperazine into the subthalamic nucleus reduced oral dyskinesia

The site of action of mesulergine in blocking orofacial dyskinesia induced by local adminis t ra t ion of m - C P P was confirmed by co-adminis ter ing mesulergine with m - C P P directly into the left STh. Co- adminis t ra t ion of mesulergine (0.5 #g) with m - C P P (0.75 pg, total combined volume of 0.1 ~1) directly into the STh blocked m-CPP-s t imula ted oral movements. The following drug solutions were infused into the STh in the order described: (i) vehicle (dH:O), (ii) mesulergine, (iii) m-CPP, (iv) mesulergine + m-CPP. Da ta are expressed as mean n u m b e r of total oral bouts per 6 0 m i n _ + S . E . M . ( n - 7 rats): vehicle 8.3 +_ 2.0; m - C P P 29.7 + 2.2*; m - C P P + mesulergine 8.7 + 2.4**. This dose of mesulergine (1.0 ~Lg) alone did not affect spontaneous oral movements (11.7 + 2 . 6 ) or appear to impair normal ongoing behaviors in these animals. *P < 0.05, differed significantly f rom the vehicle control group; **P < 0.05, differed significantly from m - C P P group.

Table 4. Systemic administration of mianserin, ketanserin or mesulergine, blocks oral dyskinesia following infusion of l-(m-chlorophenyl)piperazine into the subthalamic nucleus

Total number of oral bouts/60 min STh Mean _+ S.E.M. n

S.C. Vehicle Vehicle 7.5 _+ 2.3 8 Vehicle m-CPP 38.2 _+ 4.6* 5 Mianserin m-CPP 5.0 _+ 1.6"* 5 Ketanserin m-CPP 9.6 _+ 2.7** 5

i .p . Vehicle Vehicle 15.7 + 4.7 7 Mesulergine (0.5) Vehicle 16.7 _+ 3.1 7 Vehicle m-CPP 51.4 + 10.7" 7 Mesulergine (0.25) m-CPP 32.4 ± 2.2** 7 Mesulergine (0.5) m-CPP 16.6 + 4.7** 7

Thirty minutes prior to infusion of either m-CPP (0.75pg) or its vehicle (dH20) into the subthalamic nucleus, rats were injected subcutaneously with either mianserin (l.0mg/kg), ketanserin (5.0 mg/kg), or vehicle. In a separate group of animals, mesulergine (0.25 or 0.5 mg/kg, s.c.I or vehicle was injected 30 min prior to infusing m-CPP (0.75 #g) or vehicle into the subthalamic nucleus. All rats were tested with the same drug treatment combination on each of the test days in the order listed above, with at least one week between successive administration of each antagonist. *P < 0.05 when compared to the Veh-Veh group with ANOVA and Dunnett's test. **P <0.05, compared to Veh-m-CPP, with ANOVA and Duncan's multiple range test.

Subthalamic 5-HT2c receptors elicit oral dyskinesia

Table 5. Systemic administration of spiperone or pindolol did not block abnormal oral-facial movements following 1-(m-chlorophenyl)piperazine infusion into the subthalamic nucleus

125

Total number of oral bouts/60 min STh Mean _+ S.E.M. n

S,C, Vehicle Vehicle 13.0 _+ 2.6 9 Spiperone Vehicle 26.6 + 8.1 9 Vehicle m-CPP 49.5 + 9.3* 9 Spiperone m-CPP 60.2 + 7.6* 9

i .p . Vehicle Vehicle 9.8 __+ 3.8 6 Pindolol Vehicle 24.8 _ 5.9* 5 Vehicle m-CPP 41.8 ___ 7.7* 6 Pindolol m-CPP 46.0 _ 4.0* 6

Rats were pretreated systemically with spiperone (1.0 mg/kg, s.c.), an antagonist of 5-HTIA/2 A and DA 2 receptors or vehicle, 30min prior to infusing m-CPP (0.75 #g) or vehicle into the subthalamic nucleus. One week following this experiment, in the same group of animals, pindolol (l.0 mg/kg, i.p.) or vehicle was injected 30 min prior to infusing m-CPP (0.75 #g) or vehicle into the STh. All rats were tested with the same drug treatment combination on each of the test days in the order listed above, with at least one week between successive administration of each antagonist. *P < 0.05 when compared to the Veh-Veh group with ANOVA and Dunnett's test.

D I S C U S S I O N

The data indicate that serotonergic stimulation of the STh increased orofacial movements in awake rats by a 5-HT2c receptor-mediated mechanism, and that serotonergic receptors in the STh may, at least in part, mediate the effects of systemic infusion of the 5-HT2c/m agonist, m-CPP on orofacial movements.

Pharmacological specifici O'

The inverted U-shaped, dose-response relationship observed in this experiment resembles that reported previously for induction of purposeless chewing behavior following systemic m-CPP by Stewart et al. 47 Ascribing a specific function to activation of 5-HT2c receptors is hindered by the fact that there are no well-characterized drugs which act selectively at this receptor subtype 3~ (Table 1). m-CPP is an important pharmacological tool for characterizing 5-HT2c- mediated responses 6'28 because, in vitro, it has a higher affinity for 5-HT2c receptors than for 5-HT~A or 5-HTm receptorsfl ~ In the present studies, a 5-HTzc- mediated behavioral effect is suggested by our findings that the m-CPP-stimulated oral dyskinesias were blocked by mianserin, mesulergine, and ketanserin, drugs sharing 5-HT2c receptor antagonist activity, but not by spiperone or pindolol, which lack 5-HT2c receptor antagonist properties (Table 1; 16'22'28'31

Table 2). Furthermore, the selective 5-HT2c receptor antagonist SDZ SER 082 blocked m-CPP-induced oral dyskinesia.

Interpreting the lack of effect of pindolol and spiperone is complicated by the fact that each of these drugs by itself increased bouts of orofacial movements after peripheral injections, although the effect of spiperone was not significant. These antagonists could be altering orofacial dyskinesia by effects at

nonserotonergic receptors, such as spiperone's blockade of D2 receptors. 42 Although the dose of spiperone (1.0 mg/kg) was capable of producing marked sedation in these animals and the dose of pindolol (1.0 mg/kg) has been shown previously to be sufficient to significantly attenuate centrally mediated behavioral effects, u'15'19 the effectiveness of m-CPP ap- peared to be identical in the presence or absence of these antagonists. These observations suggest that the effects of m-CPP were not mediated by 5-HTIA/2 A or 5-HT1A/m receptors, which are blocked by spiperone and pindolol, respectively (Table 1).

Subthalamic nucleus and orofacial dyskinesia

The type of movements elicited by local and peripheral administration of m-CPP were remarkably similar, and differed from the type of orofacial dyskinesia induced by local administration of dopaminergic drugs in the ventrolateral striatum which were characterized by stereotyped, stimulus directed oral behaviors. TM In this study, the anatomical selectivity of the local drug injections was achieved by using very small volumes of injection (0.1/ll) delivered through a specially designed microinjector. 37 When delivered in this fashion, the potent neurotoxin kainic acid produces neuronal loss within most of the STh, with very little extension in the zona incerta, 8'37 indicating that the small volume of injection does not diffuse significantly outside the STh. Administration of m-CPP to adjoining regions did not produce orofacial dyskinesia, supporting the hypothesis that stimulation of 5-HT2c receptors within the STh is responsible for the effects of local m-CPP administration.

Further support for a role of the STh in mediating 5-HT2c agonist-induced orofacial dyskinesia comes from evidence that blockade of 5-HT2c receptors in


this region by local administration of mesulergine or SDZ SER 082 partially antagonizes the effects of peripherally administered rn-CPP. Like mianserin and ketanserin, mesulergine acts on other 5-HT receptors in addition to the 5-HTzc. The higher affinity of mesulergine for 5-HT2c sites over 5-HTzA sites, ~6"31 however, has made it one of the few well- characterized drugs for the pharmacological identifi- cation of both 5-HT2c-mediated behaviors 2~'z8 and binding sites. 3° In contrast, SDZ SER 082 has been less extensively studied, but appears to be specific for 5-HTzc receptors. 34

Previous studies have suggested that the effect of m-CPP on oral movements is mediated by stimulation of central 5-HT2c receptors, 47 but the exact sites of action of the drug remained unknown. Be- cause of the small injection volumes used in this study to insure anatomical specificity, mesulergine may not have reached all receptors within the STh, explaining the incomplete blockade of peripheral m-CPP effects. Alternatively, stimulation of 5-HTzc receptors in other brain regions could play a contributing role in oral behavior. 5-HT2c receptors are present in the ventrolateral striatum, the internal pallidum and the substantia nigra pars reticulata, and these regions could participate in the generation of orofacial dyskinesia. J°'3°

Serotonin #z the subthalamic nucleus

The presence of 5-HT has been detected in the rat STh in biochemical 35'44 and immunohistochemical studies? TM In the cat and primate, injection of horse- radish peroxidase into the STh revealed retrograde labeling of the dorsal raphe, 41 however, further trac- ing studies are necessary to determine whether this region is also the origin of serotonergic projections to the STh in rats. Although the involvement of 5-HT as a neurotransmitter in basal ganglia function is well-documented, 45 the actions of 5-HT within the STh are unclear.

The presence of both mRNA and binding sites for 5-HT2c receptors in the STh suggests that these receptors are expressed by intrinsic subthalamic neurons. The major target areas of these neurons are the pallidum and substantia nigra pars reticulata, the main output structures of the basal ganglia. 36 The STh plays a critical role in the control of movements by way of its glutamatergic projections to these regions. 9 Lesions of the STh in primates induce hemiballistic movements of the contralateral limbs. 32'5~ In rats, unilateral lesions of the STh with kainic acid increase the duration of locomotor activity and sniffing, and the frequency of oral dyski- netic movements characterized by transient mouth opening and closing (gaping) during the first few days following the lesion. 4 Thus hyperkinetic motor disorders appear to result from a decreased activity of efferent subthalamic neurons. The effects of stimulating 5-HT2c receptors in the STh on the electrophysio-

logical activity of these neurons, however, are not known.

Potential monoaminergic 5-hydroxytryptamine / dopamine &teractions within the subthalamic nucleus

A previous report from this laboratory has demonstrated that local administration of apomorphine within the STh increased orofacial movements through stimulation of D t receptors. 3v The movements elicited by subthalamic administration of apomorphine were very similar to those produced by serotonergic stimulation of the same region. Behav- ioral studies by Gong et al. ~7 have demonstrated that D l receptor-stimulated oral activity in 6-hydroxy- dopamine-lesioned rats was mediated via the activity of 5-HT2c receptors. In primates, 5-HT reuptake blockers produced oral hyperkinesia resembling hu- man tardive dyskinesia which was intensified by amphetamine and blocked by haloperidol. 26 Clinical observations also suggest that altering both serotonergic and dopaminergic neurotransmission may be required to reduce the affective and motor symp- toms of certain hyperkinetic disorders. 29 The observation that stimulation of either dopamine or 5-HT2c receptors by local drug administration within the STh elicit similar orofacial dyskinesia raises the possibility that some of these dopaminergic/serotonergic inter- actions could take place within this brain region.

In contrast to mRNA for the 5-HT2c receptor, D~ receptor mRNA is not detected by in situ hybridization histochemistry in subthalamic neurons. ~4 Fur- thermore, kainic acid lesions of the STh, which destroy neuronal cell bodies, do not reduce the number of dopamine DI binding sites in this region, suggesting that D 1 receptors are located presynapti- cally on afferent terminals. 37 Therefore, orofacial dyskinesia produced by serotonergic and dopaminergic drugs are likely to result from direct or indirect alterations in the activity of subthalamic neurons, respectively.

CONCLUSION

In conclusion, the data provide evidence for a behavioral response mediated by 5-HT2c receptors in a discrete brain region. This provides a unique para- digm to examine the regulation of these receptors in relation to a functional response in vivo. Furthermore, the data suggest that serotonergic mechanisms within the STh are necessary and sufficient to elicit abnormal orofacial movements and thus may play a role in the pathogeny of hyperkinetic movements disorders. The STh has been implicated in neuroleptic-induced tardive dyskinesia, 2'j8 and in the pathogeny of chorea in Huntington's disease. ~'9 Major increases in 5-HT and its metabolite 5-HIAA have been measured in the basal ganglia of patients with Huntington's disease, suggesting abnormal serotonergic transmission. 4° The


data presented here raise the possibility that increased st imulation of 5-HT2c receptors within the STh may play a role in the symptomatology observed in these patients, and suggest that antagonists at the 5-HT2c receptor could be useful in the t reatment of hyperkinetic motor disorders.

Acknowledgements~his work was supported by PHS grants MH-48125 and MH-44894, and a fellowship from the Tourette Syndrome Association (KEW). We thank Dr Daniel Hoyer, Sandoz Pharma Ltd, for the gift of SDZ SER 082, Drs A. Frazer, S. Mayaani, and P. Molinoff for stimulating discussion of the data and Mr James Eberle and Mr Arpesh Mehta for their excellent technical assistance.

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(Accepted 14 November 1995)

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A role for the subthalamic nucleus in 5-HT2C-induced oral dyskinesia