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Multifocal brain sites for apomorphine-induced circling and other stereotyped motor behaviour in the 6-hydroxydopamine-lesioned rat
N t ~ / m g , e / . , ~ o ' s , 34 (1982) 277-282 E h e v ~ Scientific Publishers Ireland Ltd.
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M U L ~ ~ S ~ FOR APOMORPmNE-INDUCED CIRCLING AND,: ~ , ~ ~ ' . n MOTORBEHAVIOUR IN THE 6-HYDRO.X ~ A M I N E - L F ~ ! O N E D RAT _
M.S. STA]RR and. M.. SUMMERHAYES Depw~r, ent of P&~nacology, The School of Pharmacy, 29/39 Brunswick Square, Lond~,n W C I N lAX
(u~.) (Received August 20th, 1982; Revised version received October 26th, 1982; Accepted November 8th, 1982)
Key words: stereotaxic injection - 6-hydroxydopamine - apomorphine - circling - stereotypy
Rats pre~reated with 6-hydroxydopamine (6-OHDA, 8~g in 4 ~) in one medial forebrain bundle exhibited an ~ { of stereotyped activities and pronounced circling towards the unlesioned side when al~omorphine was administered either subcutaneously (0.5 mg/kg), or by discrete stereotaxic injection (5 pg in 0.2 ~1) into the caudate nucleus, nucleus nccumbens, amygdala, lateral habenula, ventromedial thalamus, sub~Jmtta nigra zona reticulata, periaqueductal grey or superior colliculus (but not a variety of other areas) ipsilateral to the lesion. These rotational responses were absent in unlesioned animals and, where tetled, were attenuated by intraperitoneal haloperidol (0.5 mg/kg). It is suggested that multiple brain sites become sensitive to apomorphine followin$ dopamine depletion by 6-OHDA.
Lesk ning the dopamine (DA) neurones on one side of the brain produces an animal that moves in circles when administered a DA agonist [20]° Originally emplo3 ed to distinguish between DA agonists acting at pre- and post-synaptic central sites, more recently this behavioural model has gained wider application as a means for investigating the neural substrates of DA-mediated behaviours (e.g. refs. 4, 8 and 15). These have generally been regarded to be principally of striatal origin, such that secondary lesions may attenuate the contraversive circling elicited by systemic apomorphine in the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat by impainag striatal o~-~tflow.However, the observation that intranigral apomorphine will eficit locomotor asymmetry in 6-OHDA-denervated, but not in intact, rats suggests that ~his pretreatment generates new sites outside the striatum via which DA agonists may modify motor behaviour [11]. We should therefore not ignore the possiblecontribution to circfing of supersensitive DA receptors in other brain areas normslly receiving a spar.~er DAergic innervafion, such as the frontal cortex [19], h ~ h a l a m u s [9], amygdala [5], habenuia [14], pefiaqueductal grey [21], spinal 0304-3940/82/0000-0000/$ 02 75 © 1.982 Elsevier Scientific Publishers Ireland Ltd.
278
cord [2], etc., and perhaps even the ventromedial nucleus of the thal~unus [1]. To investigate this proposal we studied the behavioural consequences of injecting apomorphine stereotaxically ~nto various brain regions of control rats and rats pretreated unilaterally with 643HDA, O u r findinp ~point to the d e v e l o p m ~ t of multiple apomorphine sensitive sitesin the 6,OHDA d e n ~ rat, that are Other wise absent in the intact animal. We therefore suggest that the striatocentric view of apomorphine-evoked motor activity in this behavioural model should be revised. For this study male Wistar albino rats weighing 140-160 g were anaesthetized with 1.5% halothane in oxygen, secured in a Kopf stereotaxic frame and injected with 6-OHDA hydrobromide (8 ~g base in 4 ~ physiological saline) into the medial forebrain bundle CA 4.1, L 1.4 and V - 3 . 1 ) [10]. One week later the rats were screened for supersensitive DA receptors by noting the occurrence and frequency of contraversive circling co systemic apomorphine hydrochloride (0.5 mg/kg s.c. as base) when placed singly in a flat-bottomed test box, 0.7 m diameter and 0.3 m high. The responders received a second, stereotaxic injection of apomorphine (0.1-5 ~g in 0.2 ~1, or 25 ~g in 2 ~d saline) in the denervated hemisphere 7 days later under light halothane anaesthesia, into either the caudate nucleus (A 7 4, L 2.5 and V 0.5), nucleus accumbens CA 9.4, L 1.2 and V -1.0), amygdaia CA 4.6, L 3.8 and V -2.95), lateral habenula (A 3.3, L 0.5 and V 1.1), ventromedial thalamus (A 4.6, L. I. 15 and V - 1.2), substantia nigra zona reticulata (A 1.8, L 2.0 and V -2.2), superior colliculus (A 1.0, L 1.5 and V 0.8) or periaqueductal grey (A 1.0, L 0.6 and V 0.0). A separate group of previously untreated rats were similarly injected and served as controls. Circling was scored as turns/rain whilst stereotyped activity was noted but not quantified. All injection sites were verified histologically in 20 ~ n thick coronal brain sections stained with cresyl violet. In a separate experiment brains fcom 6-OHDA or vehicle-injected rats were dropped into solid CO2/acetone mixture and, using a razor blade, samples of rostral striatum Capprox. 20 rag) were dissected from a frozen coronal section and assayed for DA [17]. Microinjection of up to 5 ~g apomorphh~-, (in 0.2 ~d saline) into the caudate nucleus (CN) of control rats failed to modify their motor behaviour. A 5-fold higher dose (25 ~g in 2 ~l saline) elicited a pronounced contralateral posture of the head and trunk immediat(.ly ur)on recovery from the anaesthetic (approximately 5 rain), ,'hich then graduated into weak contraversive circling, reaching a peak at 15-20 rain (7.2 ± 2.8 turns/rain, n = 7) and disappearing after 30--40 rain. Administration of the smaller dose (5 ~g) of apomorphine into the nucleus accumbens (NA), amygdala (~MY), ventromedial thalamus (VM), substantia nigra zona reticulata (SNR)~ superior c6Iliculus (SC), periaqueductal grey (PAG) or lateral habe~ula (LH), or of ~..2 v~ saline into any of the~e regions in control rats, neither affec:ed the animal's posture or locomotion, nor induced stereotyped movements. The nigher dose (25 #g) delivered into the NA p?oduced low intensity stereotypies (sniffing and head bobbing) over the period 20-35 rain post-injection, but not actual circling. At 14 days post-lesion with 6-OHDA the DA concentration in denervated striata
279 averaged 0.SZ~ =l: 0.04 ~ g / g wet wt. (n = 10), c o m p a r e d to 6.03 ± 0.22 ~ g / g wet wt. in the:contralateral hemisphere ( P < 0 . 0 0 1 , n = 10) a n d 6.39 =1:0.27 ~ g / g wet wt. in vehicle-injectcdcontrols ( P < 0.001,, n = 10), The assay pcocedure employed here was not sufficiently sensitive t o detect claanges in D A levels in extrastriatal tissues. Seven d a d prior t o t h e biochemical d e t e l m i n a t i o n the 6 - O H D A - t r e a t e d g r o u p responded to 0.5 m g / k g a p o m o r p h i n e s.c. with a mean turning score of 15.6 =l= 2.9 t u r n s / m i n , recorded at 20 rain after injection. Other 6-OHDA-injected rats were accordingly screened with s.c. a p o m o r p h i n e and selected for f u ~ h e r behavioural testing if they circled with a similar intensity (i.e. within the range 10-26 t u r n s / m i n ) . Stereotaxic injectio~ o f 5 ~g a p o m o r p h i n e into the CN ipsilateral to a 14 day-old 6 - O H D A lesion evoke.t a p r o n o u n c e d contralateral posture and circling that lasted 6 0 - 8 0 rain (Table I). "l~ne f, equency of rotation was maximal over the period 2 0 - 4 0 min and was clearly dc se-related as similar, though less intense circling was recorded after injections o f 0.5 ~g (5.7 =t: 1.9 turns/rain, n = 5) and 2 ~,g a p o m o r p h i n e (18.1 ± 3.3 t u r n ~ / m i n , n = 5) directly into a supersensitive CN. Some rats which responded only weakly to systemic a p o m o r p h i n e often exhibited robust circling following intracaudate administration of the c o m p o u n d . Stereotyped sniffing, g r o o m i n g and teeth chattering were also observed intermittently.
TABLE I BEHAVIOURAL F:.ESPONSESTO APOMORPHINE INJECTED STEREOTAXICALLY INTO DIFFERENT BRAIN AREAS OF RATS PRETREATED UNILATERALLY WITH 6-OHDA Rats were prepared by injecting 6-OHDA (8/~g in 4 tA) into one medial forebrain bundle and screened for contraversive c rcling to s.c apomorphine (0.5 mg/kg) one week later, l~'.espondersthen r"o:ived 5 st apomorphine (in 0.2 ~,1).aereotaxically into one of 8 brain areas on the same side and the animal's bchaviour noted, Results show average circling scores (turns/min at 20 min post-injection) and range of response (in parenthesis), and the nature of any other stereotyped motor behaviour. All circling reported here was in the contralateral direction. Brain area
n
Circling (turns/min)
Posture
Stereotypy SniffT'~g,glooming, biting Sniffing, biting Sniffing, biting, licking Sniffing, biting, grooming Occasional suiffing and b:,t.ing Occasional sniffing Occasional sniffing ann biting Uccasional sniffing and biting
Candat¢ nucleus
44 27.9 (12-52)
Very tight
Naclcus accumhens Amygdala
9 9
13.6(5-21) 16.0(9-24)
Very tight Very tight
Ventromedial thalamu~ Substaa:2a nigra zona reticulata Superior colliculus Periaqueducml grey
8
21.0 (9-44)
Tight
$
14.5 (4-18)
Tight
8 7
20.4 (I 5-=,2) 23.2 (6-~,6)
Tight Very tight
Lateral habenula
8
18.8(12-30)
Very tight
280 As can be seen from Table I, tightly postured contraversive rotations were also elicited by 5 jt~ apomorl~hine applied discretely to the NA, AMY, VM, SNR, SC, PAG and LH on the side bearing the 6-OHDA lesion. In these experiments the circling was less well sustained and usually subsided after 2 0 - 4 0 min, though at: this lime stroking the animal's fur was often sufficient to reinstate short bursts of intense circling (up to 28 turns/rain for 10-20 sec). It was not unusual for these rats toshow signs of repetitive, stereotyped activities, although these varied in frequency and character with the site injected (see Table 1). Prior treatment with a sub-cataleptic dose of haloperidol (0.5 mg/kg i.p.), 30 min beforehand, prevented the appearance of both locomotor and stereotyped behaviours from the CN, NA, PAG and VM (the other areas were not tested). Vehicle injections of equivalent volume (0.2 td) into each of these brain structures in 6-OHDA-treated rats failed to initiate rotational movements. So, too, did apomorphine (5 pg in 0.2 ~ ) expelled into the claustrum, cerebral cortex dorsal or rostral to the NA, cerebral cortex just dorsal to the SC, superficial laminae of lateral SC, medial geniculate nucleus, medial lenmiscus, crus cerebri, zona incerta or Ford's field in lesioned ~mimals (mostly misplaced injections as seen histologically). 6-OHDA h~s proved a useful tool for investigating central DAergic m~.nanisms, since by destroying DA neurones it renders target receptors supersensitive to DA agoni.,its and thereby facilitates studies of their behaviomai effects. In our hands the neurotoxin reduced striatal DA by 36e/0, promoted contraversive circling to s.c. apom,3rphine and increased the animal's sensitivity to intracaudat; apomorphine approximately 5~-fold. These findings agree closely with those of Setler et al. [16] and reinforce the widely held belief that striated DA receptors oc:upy a central role in the regulation 3f posture and lot~motion. The possibility that less prominent DA projections may also mediate locomotor and s,:ereotyped activities has received little attention. Apart from the study by Kozlowski et al. [ 11], which describes the appearance in 6-OHDA-treated rats of a behavioural sen~itl;'ity to intranigral apomorphine not otherwise present in the neurologically intact ~at (i.e. contraversive circling), there has been no systematic attempt to determine if DA agonists can e_icit rotations from structures other t],an the striat ~m or limbi¢ forebrain. Our present findings clearly show that apom~rphine can indeed l~rovoke contraversive circling and repetitive orofac;al movements from a number of extrastriatal brain sites in the unilaterally 6-OHDA-lesioned rat, at a dose level that is completely ineffective in naive rats. Moreover, these responses are antagonized by haloperidol (wher~ this has been tested) and, apart from being shor':er-lived, closely resemble those of striata! origin. All of these areas, save possibly the SC, receive a demonstrable, if small, neuronal input from the D? cell body-rich region of the basal midbrain [1, 5, 14, 21], suggesting :he appearance of these apomorphine~sensitive sites in denervated individuals coincides with the development of supersensitivity in the respective populations of postsynaptic DA receptors (e.g. refs. 7 and 12).
281
In spite of being highly hpophilic, we think it unlikely that apomcrphinc produces these behaviours by diffusing to the striatum from its point of injection elsewhere in the brain, as circling ~as not subject to diffusional delay and was not evoked indiscriminately from neigl:bouring brain areas. On the other hand, diffusional losses might well account for the shorter duration of the circling induced by apomorphine from these extrastriatal sites. We conclude from this study that the characteristic circling motion described b~ unilaterally 6-OHDA-lesioned rats in response to a systemic DA agonist, is the combined effect of the drug's action at multiple supersensitive ,;ites throughout the brain. Such a multiplicity of action might explain the apparent lack of temporal correlation between apomorphine-induced circling and striatal DA receptor changes reported recently by Staunton and coworkers [18]. It is further conceivable that the muscular contractions which occur with L-DOPA in chronically spinalized cats may be mediated by DA receptors situated postsynaptically to ~he newly-discovered nigrospinal DA fibres [2], and not via the descending noradrenergic system as was originally supposed [6]. If future experiments confirm our proposal, by demonstrating a depleted DA content and raised DA binding ~t these different sites, as well as a non-involvement of 5-hydroxytryptamine recel:tors in the action of apomorphine [3, 13], then our views of the therapeutic mode of action of DA agonists employed to alleviate clinical conditions characterised by DA receptor supersensitivity (e.g. Parkinson's disease) may have to be radically revised. Furthermore, the validity of using apomorphine-stimalated circling in the 6-OHDAdenervated rat as a suitable model fiat elucidating the neural outflow pathways mediating striatal influences on skeletal muscle activity, will ha~e to be seriously reconsidered [4, 8, 151. We are grateful to the Medical Research Council for financial support. M. Sur.merha;'-s is in receipt of a grant from the Pharmaceutical Society of Great Britain.
I Beckstead, R.M., Domesick, V.B. and Nauta, W.J.H., Efferent connections of the substantia nigra and ventral tegmental area in the rat, Brain Res., 175 (1979) 191-217. 2 Commisiong, J.W., Gentleman, S. and Neff, N.H., Spinal cord and dopaminergic neurones: evidence for an uncrossed nigrospinal pathway, Neuropharmacology, 18 (1979) 565-568. 3 Costali, B., Naylor, R.J. and Pycock, C., Non-specific supersensitivity of striatal dopamine recep:ors after 6-hydraxydopamine lesion of the nigrostriatal pathway, Europ. J. Pharmacol., 35 (1976) 275-283. 4 Di Chiara, G., Morelli, M., Porceddu, M.L. and Gessa, G.L., Evidence tha nigral GABA mediates behavioural responses elicited by striatal dopamine receptor stimulation, Life Sci., 23 (1978) 2045-2052. 5 Fallon, J.H., Koziell, D.A. and Moore, I~.Y., Catecholamine innervatic i of the basal forebrain 11. Amygdala, suprarhinal cortex and entorhinal cortex, J. comp. Neurol., 180 (1978) 509-532. 6 Grillner, S., The influence of DOPA on the static and the dynamic fusimotor activity to the triceps sura¢ of the spinal cat, Acta physiol, scand., 77 (1979) 490-509.
28~. 7 Heikkila, R.E., Shapiro, B.S. and Duvoisin, R.C., The rdat~aship between loss of ~ nerve terminals, striatal [~HlsPiroperidol binding and rotational behaviour in unilaun~y 6-hydroxydo~mine lesioned rats, Brain Res., 211 (1981) 285-292. 8 Kilpatrick, i.C., Collingridge, G.L. and Start', M.S., Evidence for the participation of nigrotecta ! "raminobutyrate-conudning neurones in stri'~_~! and nigral-dedved circling in tile rat. Ncaroscience, 7 (1982) 20?-222. 9 Kizer, J.S., Palkovits, M. and Brownstein, M.J., The projections of the AS, A9 and AI0 dopamincagic cell bodies: evidence for a nigro-hypothalalxl~-median ~finence dopaminergic pathway, Brain Res., 108 (1976) 363-370. 10 Konig, J.F.R. and Klippel, R.A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. I I Kozlowski, M.R., Sawyer, S. and Marshall, J.F., Behavioural effects and SUl>~sensitivity following nigral dopamine receptor stimulation, Nature (Lond.), 287 (1980) 52u54. 12 Marshall, J.F. and Ungerstedt, U., Supersensitivity to apomorphine following destruction of the ascending dopamine neurons: quantification using the rotational model, Europ. J. Pharmacoi., 41 (1977) 361-367. 13 Oudan, N. and Boulu, R.G., Possible 5..hydroxytryptamine component in the effect of apomorphine in isolated cerebral and peripheral arteries, Experientia, 37 (1981) 1096-1097. 14 Phiilipson, O.T. and Pycock, C.J., Dopamine neurones of the ventral tefmentum project to both medial and lateral habenula: some implications for habenular function, Exp. Brain Res., 45 {1982) 89-94. 15 Reavill, C., Jeqner, P., Leigh, N. and Marsden, C.D., The role of nigral projections to the thalamus in drug-induced circling behaviour in the rat, Life Sci., 28 (198t) 145"7-1466. 16 Setler, P.E., Malesky, M., McDevitt, J. and Turner, K., Rotation produced by administration of dopamine related substances directly into the ~'apersensitjve caudate r~uclet~s, LiCe Sci., 23 (1978) 12"77-.1284. 17 Shellenberger, M.K. and Gordon, J.H., A rapid, simplified procedure for the simultaneous assay of norepinephrine, dopamine and 5-hydroxytryptamine from discrete brain areas, Analyt. Biochem., 30 (1971) 356-372. 18 Staunton, D.A., Wolfe, B.B., Groven, P.M. and Molinoff, P.B., Dupamine receptor change~ following destruction of the nigrostriatal pathway: lack of a relationship to rotational behaviour, Brain Res., 211 (1981) 315-327. 19 Thierry, A.M., Blanc, G., Sobel, A., S~inus, L. and Glowinski, J., Dopaminergic termiltals in the rat cortex, Science, 182 (1973) 499-501. 20 Ungerstedt, U., Postsynaptic supersensitivity a"ter 6-hydroxydopamin¢ induced degeneration of the nigro-striatal dopamine system in the rat brain, Acta physiol., scand., 82, Suppl. 367 (1971) 69-93. 21 Wright, A.K. and A~butlmott, G.W., Non-doj)amine containing efferents of substantia nigra: the pathway to :he lower bt ~in stem, J. Neural Transm., 47 (1980) 221-226.
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M U L ~ ~ S ~ FOR APOMORPmNE-INDUCED CIRCLING AND,: ~ , ~ ~ ' . n MOTORBEHAVIOUR IN THE 6-HYDRO.X ~ A M I N E - L F ~ ! O N E D RAT _
M.S. STA]RR and. M.. SUMMERHAYES Depw~r, ent of P&~nacology, The School of Pharmacy, 29/39 Brunswick Square, Lond~,n W C I N lAX
(u~.) (Received August 20th, 1982; Revised version received October 26th, 1982; Accepted November 8th, 1982)
Key words: stereotaxic injection - 6-hydroxydopamine - apomorphine - circling - stereotypy
Rats pre~reated with 6-hydroxydopamine (6-OHDA, 8~g in 4 ~) in one medial forebrain bundle exhibited an ~ { of stereotyped activities and pronounced circling towards the unlesioned side when al~omorphine was administered either subcutaneously (0.5 mg/kg), or by discrete stereotaxic injection (5 pg in 0.2 ~1) into the caudate nucleus, nucleus nccumbens, amygdala, lateral habenula, ventromedial thalamus, sub~Jmtta nigra zona reticulata, periaqueductal grey or superior colliculus (but not a variety of other areas) ipsilateral to the lesion. These rotational responses were absent in unlesioned animals and, where tetled, were attenuated by intraperitoneal haloperidol (0.5 mg/kg). It is suggested that multiple brain sites become sensitive to apomorphine followin$ dopamine depletion by 6-OHDA.
Lesk ning the dopamine (DA) neurones on one side of the brain produces an animal that moves in circles when administered a DA agonist [20]° Originally emplo3 ed to distinguish between DA agonists acting at pre- and post-synaptic central sites, more recently this behavioural model has gained wider application as a means for investigating the neural substrates of DA-mediated behaviours (e.g. refs. 4, 8 and 15). These have generally been regarded to be principally of striatal origin, such that secondary lesions may attenuate the contraversive circling elicited by systemic apomorphine in the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat by impainag striatal o~-~tflow.However, the observation that intranigral apomorphine will eficit locomotor asymmetry in 6-OHDA-denervated, but not in intact, rats suggests that ~his pretreatment generates new sites outside the striatum via which DA agonists may modify motor behaviour [11]. We should therefore not ignore the possiblecontribution to circfing of supersensitive DA receptors in other brain areas normslly receiving a spar.~er DAergic innervafion, such as the frontal cortex [19], h ~ h a l a m u s [9], amygdala [5], habenuia [14], pefiaqueductal grey [21], spinal 0304-3940/82/0000-0000/$ 02 75 © 1.982 Elsevier Scientific Publishers Ireland Ltd.
278
cord [2], etc., and perhaps even the ventromedial nucleus of the thal~unus [1]. To investigate this proposal we studied the behavioural consequences of injecting apomorphine stereotaxically ~nto various brain regions of control rats and rats pretreated unilaterally with 643HDA, O u r findinp ~point to the d e v e l o p m ~ t of multiple apomorphine sensitive sitesin the 6,OHDA d e n ~ rat, that are Other wise absent in the intact animal. We therefore suggest that the striatocentric view of apomorphine-evoked motor activity in this behavioural model should be revised. For this study male Wistar albino rats weighing 140-160 g were anaesthetized with 1.5% halothane in oxygen, secured in a Kopf stereotaxic frame and injected with 6-OHDA hydrobromide (8 ~g base in 4 ~ physiological saline) into the medial forebrain bundle CA 4.1, L 1.4 and V - 3 . 1 ) [10]. One week later the rats were screened for supersensitive DA receptors by noting the occurrence and frequency of contraversive circling co systemic apomorphine hydrochloride (0.5 mg/kg s.c. as base) when placed singly in a flat-bottomed test box, 0.7 m diameter and 0.3 m high. The responders received a second, stereotaxic injection of apomorphine (0.1-5 ~g in 0.2 ~1, or 25 ~g in 2 ~d saline) in the denervated hemisphere 7 days later under light halothane anaesthesia, into either the caudate nucleus (A 7 4, L 2.5 and V 0.5), nucleus accumbens CA 9.4, L 1.2 and V -1.0), amygdaia CA 4.6, L 3.8 and V -2.95), lateral habenula (A 3.3, L 0.5 and V 1.1), ventromedial thalamus (A 4.6, L. I. 15 and V - 1.2), substantia nigra zona reticulata (A 1.8, L 2.0 and V -2.2), superior colliculus (A 1.0, L 1.5 and V 0.8) or periaqueductal grey (A 1.0, L 0.6 and V 0.0). A separate group of previously untreated rats were similarly injected and served as controls. Circling was scored as turns/rain whilst stereotyped activity was noted but not quantified. All injection sites were verified histologically in 20 ~ n thick coronal brain sections stained with cresyl violet. In a separate experiment brains fcom 6-OHDA or vehicle-injected rats were dropped into solid CO2/acetone mixture and, using a razor blade, samples of rostral striatum Capprox. 20 rag) were dissected from a frozen coronal section and assayed for DA [17]. Microinjection of up to 5 ~g apomorphh~-, (in 0.2 ~d saline) into the caudate nucleus (CN) of control rats failed to modify their motor behaviour. A 5-fold higher dose (25 ~g in 2 ~l saline) elicited a pronounced contralateral posture of the head and trunk immediat(.ly ur)on recovery from the anaesthetic (approximately 5 rain), ,'hich then graduated into weak contraversive circling, reaching a peak at 15-20 rain (7.2 ± 2.8 turns/rain, n = 7) and disappearing after 30--40 rain. Administration of the smaller dose (5 ~g) of apomorphine into the nucleus accumbens (NA), amygdala (~MY), ventromedial thalamus (VM), substantia nigra zona reticulata (SNR)~ superior c6Iliculus (SC), periaqueductal grey (PAG) or lateral habe~ula (LH), or of ~..2 v~ saline into any of the~e regions in control rats, neither affec:ed the animal's posture or locomotion, nor induced stereotyped movements. The nigher dose (25 #g) delivered into the NA p?oduced low intensity stereotypies (sniffing and head bobbing) over the period 20-35 rain post-injection, but not actual circling. At 14 days post-lesion with 6-OHDA the DA concentration in denervated striata
279 averaged 0.SZ~ =l: 0.04 ~ g / g wet wt. (n = 10), c o m p a r e d to 6.03 ± 0.22 ~ g / g wet wt. in the:contralateral hemisphere ( P < 0 . 0 0 1 , n = 10) a n d 6.39 =1:0.27 ~ g / g wet wt. in vehicle-injectcdcontrols ( P < 0.001,, n = 10), The assay pcocedure employed here was not sufficiently sensitive t o detect claanges in D A levels in extrastriatal tissues. Seven d a d prior t o t h e biochemical d e t e l m i n a t i o n the 6 - O H D A - t r e a t e d g r o u p responded to 0.5 m g / k g a p o m o r p h i n e s.c. with a mean turning score of 15.6 =l= 2.9 t u r n s / m i n , recorded at 20 rain after injection. Other 6-OHDA-injected rats were accordingly screened with s.c. a p o m o r p h i n e and selected for f u ~ h e r behavioural testing if they circled with a similar intensity (i.e. within the range 10-26 t u r n s / m i n ) . Stereotaxic injectio~ o f 5 ~g a p o m o r p h i n e into the CN ipsilateral to a 14 day-old 6 - O H D A lesion evoke.t a p r o n o u n c e d contralateral posture and circling that lasted 6 0 - 8 0 rain (Table I). "l~ne f, equency of rotation was maximal over the period 2 0 - 4 0 min and was clearly dc se-related as similar, though less intense circling was recorded after injections o f 0.5 ~g (5.7 =t: 1.9 turns/rain, n = 5) and 2 ~,g a p o m o r p h i n e (18.1 ± 3.3 t u r n ~ / m i n , n = 5) directly into a supersensitive CN. Some rats which responded only weakly to systemic a p o m o r p h i n e often exhibited robust circling following intracaudate administration of the c o m p o u n d . Stereotyped sniffing, g r o o m i n g and teeth chattering were also observed intermittently.
TABLE I BEHAVIOURAL F:.ESPONSESTO APOMORPHINE INJECTED STEREOTAXICALLY INTO DIFFERENT BRAIN AREAS OF RATS PRETREATED UNILATERALLY WITH 6-OHDA Rats were prepared by injecting 6-OHDA (8/~g in 4 tA) into one medial forebrain bundle and screened for contraversive c rcling to s.c apomorphine (0.5 mg/kg) one week later, l~'.espondersthen r"o:ived 5 st apomorphine (in 0.2 ~,1).aereotaxically into one of 8 brain areas on the same side and the animal's bchaviour noted, Results show average circling scores (turns/min at 20 min post-injection) and range of response (in parenthesis), and the nature of any other stereotyped motor behaviour. All circling reported here was in the contralateral direction. Brain area
n
Circling (turns/min)
Posture
Stereotypy SniffT'~g,glooming, biting Sniffing, biting Sniffing, biting, licking Sniffing, biting, grooming Occasional suiffing and b:,t.ing Occasional sniffing Occasional sniffing ann biting Uccasional sniffing and biting
Candat¢ nucleus
44 27.9 (12-52)
Very tight
Naclcus accumhens Amygdala
9 9
13.6(5-21) 16.0(9-24)
Very tight Very tight
Ventromedial thalamu~ Substaa:2a nigra zona reticulata Superior colliculus Periaqueducml grey
8
21.0 (9-44)
Tight
$
14.5 (4-18)
Tight
8 7
20.4 (I 5-=,2) 23.2 (6-~,6)
Tight Very tight
Lateral habenula
8
18.8(12-30)
Very tight
280 As can be seen from Table I, tightly postured contraversive rotations were also elicited by 5 jt~ apomorl~hine applied discretely to the NA, AMY, VM, SNR, SC, PAG and LH on the side bearing the 6-OHDA lesion. In these experiments the circling was less well sustained and usually subsided after 2 0 - 4 0 min, though at: this lime stroking the animal's fur was often sufficient to reinstate short bursts of intense circling (up to 28 turns/rain for 10-20 sec). It was not unusual for these rats toshow signs of repetitive, stereotyped activities, although these varied in frequency and character with the site injected (see Table 1). Prior treatment with a sub-cataleptic dose of haloperidol (0.5 mg/kg i.p.), 30 min beforehand, prevented the appearance of both locomotor and stereotyped behaviours from the CN, NA, PAG and VM (the other areas were not tested). Vehicle injections of equivalent volume (0.2 td) into each of these brain structures in 6-OHDA-treated rats failed to initiate rotational movements. So, too, did apomorphine (5 pg in 0.2 ~ ) expelled into the claustrum, cerebral cortex dorsal or rostral to the NA, cerebral cortex just dorsal to the SC, superficial laminae of lateral SC, medial geniculate nucleus, medial lenmiscus, crus cerebri, zona incerta or Ford's field in lesioned ~mimals (mostly misplaced injections as seen histologically). 6-OHDA h~s proved a useful tool for investigating central DAergic m~.nanisms, since by destroying DA neurones it renders target receptors supersensitive to DA agoni.,its and thereby facilitates studies of their behaviomai effects. In our hands the neurotoxin reduced striatal DA by 36e/0, promoted contraversive circling to s.c. apom,3rphine and increased the animal's sensitivity to intracaudat; apomorphine approximately 5~-fold. These findings agree closely with those of Setler et al. [16] and reinforce the widely held belief that striated DA receptors oc:upy a central role in the regulation 3f posture and lot~motion. The possibility that less prominent DA projections may also mediate locomotor and s,:ereotyped activities has received little attention. Apart from the study by Kozlowski et al. [ 11], which describes the appearance in 6-OHDA-treated rats of a behavioural sen~itl;'ity to intranigral apomorphine not otherwise present in the neurologically intact ~at (i.e. contraversive circling), there has been no systematic attempt to determine if DA agonists can e_icit rotations from structures other t],an the striat ~m or limbi¢ forebrain. Our present findings clearly show that apom~rphine can indeed l~rovoke contraversive circling and repetitive orofac;al movements from a number of extrastriatal brain sites in the unilaterally 6-OHDA-lesioned rat, at a dose level that is completely ineffective in naive rats. Moreover, these responses are antagonized by haloperidol (wher~ this has been tested) and, apart from being shor':er-lived, closely resemble those of striata! origin. All of these areas, save possibly the SC, receive a demonstrable, if small, neuronal input from the D? cell body-rich region of the basal midbrain [1, 5, 14, 21], suggesting :he appearance of these apomorphine~sensitive sites in denervated individuals coincides with the development of supersensitivity in the respective populations of postsynaptic DA receptors (e.g. refs. 7 and 12).
281
In spite of being highly hpophilic, we think it unlikely that apomcrphinc produces these behaviours by diffusing to the striatum from its point of injection elsewhere in the brain, as circling ~as not subject to diffusional delay and was not evoked indiscriminately from neigl:bouring brain areas. On the other hand, diffusional losses might well account for the shorter duration of the circling induced by apomorphine from these extrastriatal sites. We conclude from this study that the characteristic circling motion described b~ unilaterally 6-OHDA-lesioned rats in response to a systemic DA agonist, is the combined effect of the drug's action at multiple supersensitive ,;ites throughout the brain. Such a multiplicity of action might explain the apparent lack of temporal correlation between apomorphine-induced circling and striatal DA receptor changes reported recently by Staunton and coworkers [18]. It is further conceivable that the muscular contractions which occur with L-DOPA in chronically spinalized cats may be mediated by DA receptors situated postsynaptically to ~he newly-discovered nigrospinal DA fibres [2], and not via the descending noradrenergic system as was originally supposed [6]. If future experiments confirm our proposal, by demonstrating a depleted DA content and raised DA binding ~t these different sites, as well as a non-involvement of 5-hydroxytryptamine recel:tors in the action of apomorphine [3, 13], then our views of the therapeutic mode of action of DA agonists employed to alleviate clinical conditions characterised by DA receptor supersensitivity (e.g. Parkinson's disease) may have to be radically revised. Furthermore, the validity of using apomorphine-stimalated circling in the 6-OHDAdenervated rat as a suitable model fiat elucidating the neural outflow pathways mediating striatal influences on skeletal muscle activity, will ha~e to be seriously reconsidered [4, 8, 151. We are grateful to the Medical Research Council for financial support. M. Sur.merha;'-s is in receipt of a grant from the Pharmaceutical Society of Great Britain.
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