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Intranigral kainic acid: Evidence for nigral non-dopaminergic neurons controlling posture and behavior in a manner opposite to the dopaminergic ones
European Journal of Pharrnacology, 49 (1978) 223--232
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© Elsevier/North-Holland Biomedical Press
I N T R A N I G R A L K A I N I C ACID: E V I D E N C E F O R N I G R A L N O N - D O P A M I N E R G I C N E U R O N S C O N T R O L L I N G P O S T U R E A N D B E H A V I O R IN A M A N N E R O P P O S I T E T O T H E DOPAMINERGIC ONES MARIA C. OLIANAS, GRAZIELLA M. DE MONTIS, ALBERTO CONCU t , ALESSANDRO TAGLIAMONTE * and GAETANO DI CHIARA 2nd Chair of Pharmacology, and ~ Institute of Physiology, University of Cagliari, Via Porcell 4, 09100 Cagliari, Italy
Received 26 October 1977, revised MS received 25 January 1978, accepted 1 February 1978
M.C. OLIANAS, G.M. DE MONTIS, A. CONCU, A. TAGLIAMONTE and G. DI CHIARA, Intranigral kainic acid: evidence for nigral non-dopaminergic neurons controlling posture and behavior in a manner opposite to the dopaminergic ones, European J. Pharmacol. 49 (1978) 223--232. The unilateral, intranigral administration of kainic acid (k.a.) produced a syndrome characterized by early sequelae of contra- and ipsilateral circling and by a chronic contralateral turning associated with moderate loss of neurons in the pars reticulata. The acute contralateral circling seems to be related to dopaminergic nigro-neostriatal neuron stimulation, since it was prevented by previous intranigral injections of 6-OHDA. The acute ipsilateral circling and the chronic contralateral turning, on the other hand, seem to be independent of the integrity of the dopaminergic system and may be due to an initial stimulation, followed by destruction, of a nigral neuronal system which mediates turning behavior in a manner opposite to that of nigro-striatal dopamine. Treatment with D-amphetamine or apomorphine changed the contralateral into ipsilateral turning, while haloperidol potentiated the contralateral turning. Bilateral injection of k.a. into the nigra resulted in chronic stereotyped sniffing and gnawing, which were not inhibited by haloperidol. Moreover, haloperidol did not produce catalepsy in these animals. It is suggested that the intranigral k.a. injection destroyed a neuronal system antagonistic to dopamine and resulted in a reduction of the response to DA-receptor stimulation of the c. striatum. Substantia nigra Turning behavior
Kainic acid
Stereotypies
1. I n t r o d u c t i o n Selective, unilateral lesioning o f nigroneostriatal d o p a m i n e r g i c n e u r o n s b y t h e intranigral i n j e c t i o n o f 6 - h y d r o x y d o p a m i n e (6O H D A ) p r o d u c e s a d r a m a t i c p o s t u r a l asymm e t r y , consisting o f a t u r n i n g o f t h e b o d y t o w a r d t h e side o f t h e lesion ( U n g e r s t e d t and Arbuthnott, 1970). H o w e v e r , unilateral e l e c t r o l y t i c lesion o f t h e s u b s t a n t i a nigra (s.n.), while it is as e f f e c t i v e as is 6 - O H D A in d e s t r o y i n g d o p a m i n e r g i c n e u r o n s , causes t u r n i n g t o w a r d t h e side c o n t r a l a t e r a l t o t h e * Correspondence to Prof. A. Tagliamonte at the above address.
Catalepsy
GABA
Dopamine
lesion (Costall et al., 1 9 7 6 ; I w a m o t o et al., 1 9 7 6 ; S c h w a r t z et al., 1 9 7 6 ) . T h e l a t t e r finding has b e e n t a k e n t o indicate t h e exist e n c e o f a n e u r o n a l s y s t e m originating in or transversing t h e s.n. a n d w h i c h c o n t r o l s p o s t u r e in a m a n n e r o p p o s i t e t o t h e nigrostriatal d o p a m i n e r g i c n e u r o n s (Costall et al., 1 9 7 6 ; I w a m o t o et al., 1 9 7 6 ; S c h w a r t z et al., 1976). We n o w r e p o r t t h a t t h e selective destruct i o n o f nigral p e r i k a r y a b y t h e local i n j e c t i o n o f kainic acid (k.a.), p r o d u c e d c h r o n i c contralateral t u r n i n g i n d e p e n d e n t o f t h e p r e s e n c e o f nigral striataI d o p a m i n e r g i c n e u r o n s . In addit i o n , bilateral, intranigral injections o f k.a. resulted in s t e r e o t y p e d sniffing and gnawing
M.C. OLIANAS ET AL.
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which were not abolished b y haloperidol administration.
2. Materials and methods
2.1. Animals Experiments were carried out with male albino Sprague-Dawley rats, weighing about 300 g. Animals were housed 10 to a cage (45 × 80 cm), had free access to food and water and were maintained under controlled environmental conditions, with a 12 h light-dark cycle.
2.2. Surgical procedures Rats were anaesthetized with Equithesin (3.75 ml/kg) or with ethyl ether and placed in a Stoelting Stellar stereotaxic apparatus. Drugs were injected in the s.n. using stereotaxic coordinates A: - - 3 ; L: 2; V: 7.8, according to the Atlas of Pellegrino and Cushman (1971). The total injection volume was 0.3 #l of saline injected at a rate of 1 ttl/18 min through a 0.25 mm stainless-steel cannula.
2.3. Histology 10 days after the intranigral injection of k.a., 24 rats were sacrified, the brain perfused with formalin, imbedded in paraffin and the mesencephalon c u t into serial 20 p m slices which were stained with Luxol fast-cresyl violet in order to ascertain the extent of the lesion. Other rats were sacrified b y cervical dislocation for the determination of striatal levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC). The brain areas remaining after dissection of the striata from these rats were fixed in 10% formalin for 2 h; they were then stored in 0.1 M phosphate buffer (pH 7.4) containing 5% sucrose until the mesencephalon could be cut into serial 50 ~m slices on a cryostat to allow the position of the cannula tip to be determined.
2.4. Behavior 24 h after intranigral k.a., rats were scored for m o t o r asymmetries according to a method similar to that described b y Costall et al. (1976) (table 1). The intensity of the circling was measured b y counting the turns made during each 10 min period after placement of the rats in a large open field area. Results are expressed as "maximal circling intensity", i.e. the number of turns/min performed during the 10 min period of maximal circling. The duration o f the test was 20 min when no drugs were administered or 60 min when the response to drugs was studied. Catalepsy was evaluated b y a test in which animals were placed on a horizontal bar 7.5 cm high and the intensity of catalepsy rated as: 1, when the rat did not move for at least 15 sec; 2 or 3, when the rat remained immobile for at least 30 or 60 sec, respectively. Rats were tested 60 min after haloperidol administration. Stereotyped behavior was evaluated by the scoring system of Costall and Naylor (1974),
TABLE 1 Scoring system for the evaluation of posture and motor asymmetries Description
Score
No difference from the posture and the behavior of a normal rat
0
Head and neck are preferentially turned to one side, the animal shows turning of the body when stimulated, but goes straight when moving spontaneously Forced, unilateral twist of head and neck, with preferential turning of the body toward the same direction, the animal can only move by circling ipsilaterally to the body twist Forced twist of head and body toward one side, with frequent episodes of spontaneous circling toward the direction of the body twist
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after placing the rats in Perspex cages with a wire-net bottom: 1, discontinuous sniffing with hypermotility; 2, continuous sniffing around the cage; 3, sniffing restricted to a small area of the cage with licking and discontinuous gnawing; 4, continuous gnawing in a small area of the cage.
2.5. Fluorometric DOPAC
estimation
of DA
and
Animals were killed by cervical dislocation, the brain rapidly removed and the striata dissected and stored at --20°C until analyzed. Brain DA and DOPAC were assayed fluorometrically as already described (Olianas et al., 1976).
2.6. Measurement of adenylate cyclase activity Adenylate cyclase activity was determined as described by Zivkovic et al. (1975), by measuring the conversion of 14C-ATP to 14CcAMP in a 3 min incubation. The reaction was stopped by boiling and the cAMP formed was purified by passage through alumina and Dowex 50 × 8 columns. All data were statistically analyzed with the two-tailed Student's t-test (Snedecor, 1969).
3.2. Biochemistry Table 2 shows the immediate and chronic effects of a unilateral, intranigral injection of k.a. on the DA and DOPAC concentration of the corresponding c. striatum, as compared to the control side. 45 min after k.a. administration, striatal DA and DOPAC of the lesioned side were increased by 42% and 232%, respectively. On the other hand, 7 days after surgery, both DA and DOPAC decreased by about 25% in the C. striatum ipsilateral to the injected s.n. Table 3 shows the effect of an injection of k.a. on basal and DA-stimulated adenylate cyclase activity in both the treated s.n. and the ipsilateral c. striatum, as compared with the uninjected side. The experiment was carried out 7 days after surgery. Neither basal nor DA-stimulated adenylate cyclase of either area was significantly affected by the nigral k.a. injection.
3.3. Behavior When they recovered from the anaesthesia, 1--2 h after surgery, the rats showed a marked postural asymmetry with flexion of the ipsilateral and extension of the contralateral limbs; they showed spontaneous ipsilateral
3. Results TABLE 2
3.1. Histology Fig. 1 shows the histological appearance of a s.n. injected with 0.75 pg (3.5 nM) of k.a. 10 days previously, as compared to that injected with saline. The pars reticulata of the lesioned nigra revealed moderate loss of neuronal perikarya and gliosis, characterized by proliferation of non-neuronal elements. The pars compacta seemed less affected than the reticulata and still showed a high density of its typical, large and globular neurons. No signs of demyelinization were present in or around the s.n.
E f f e c t o f k.a. i n j e c t e d in t h e s.n. o n t h e levels o f D A a n d D O P A C o f t h e c o r r e s p o n d i n g c. s t r i a t u m , as comp a r e d t o t h e side i n j e c t e d w i t h saline. Each value is t h e m e a n +- S.E. o f at least 8 d e t e r m i n a tions. K.a. was i n j e c t e d in a dose o f 0.75 /~g/0.3 pl a n d t h e same dose was used in all t h e o t h e r experim e n t s r e p o r t e d , unless specified. Intranigral treatment
D A (/~g/g)
D O P A C (ng/g)
Saline 45 m i n K.a. 45 rain Saline 7 days K.a. 7 days
5.46 7.75 5.51 3.96
430 1,427 426 316
-+ 0 . 0 8 +- 0 . 0 9 2 +_ 0 . 1 0 +- 0.06 2
+ 7 +_ 28 1 +_ 9 +- 8 2
1 p < 0 . 0 0 1 , 2 p < 0.01, c o m p a r e d t o t h e side inject e d w i t h saline.
t ~
I
"..2 °',
A
,B~
'
KAINIC ACID IN THE SUBSTANTIA NIGRA
227
TABLE 3 Failure of intranigral k.a. injection to modify the basal and DA-stimulated adenylate cyclase activity of the s.n. and c. striatum. Each value is the mean + S.E. of 8 determinations. Dopamine
c-AMP formed (pmol/mg prot./min) K.a.
-
-
10 -s M 10 -4 M
Controls
S.n.
C. striatum
S.n.
C. striatum
205 +- I I 298+16t 321+141
144 +- 14 258+121 2 8 9 + - 1 5I
202 -+ 13 288+- 9 i 329+-14 I
186 +- 16 249+13 1 284+ 61
I p < 0.001 with respect to basal values. TABLE 4 Incidence and intensity of contralateral turning after various doses of intranigral k.a. Turning was scored 24 h after intranigral injection of k.a., as described in the text. Kainic acid (l~g) 0.25 0.50 0.75 1.00
Rats injected
I0 15 79 20
Rats with turning
2 I0 68 19
Rats turning with score
Maximal circlingI (turns/rain)
0
1
2
3
8 5 II 1
2 2 2 1
-2 6 4
-6 60 14
-6 +- 2 18 -+3 22 -+4
1 Values are means + S.E.
circling, sometimes i nt er r upt ed by muscular dystonias with twisting o f t h e head and rolling o f t h e b o d y . These s y m p t o m s
Fig. I. Micrographs (10x) of s.n. sections (20 pm, stained with Luxol fast-cresylviolet) of rats injected unilaterally with k.a. (0.75 pg) and killed 10 days thereafter. A and B represent the medial portion of the control and k.a.-injected s.n., respectively; the pars compacta is limited dorsally (top of the figure) by the myelinated fibers of the medial lemniscus. In the k.a.-injected side the typical globular neurons of the pars compacta are fairlywell preserved and so are the myelinated fibers. In addition, a high degree of gliosis is present in this side. C and D represent the pars reticulata of the control and k.a.-injectednigra, respectively. The top of the figure is the dorsal part of the s.n. Gliosis associated with loss of neuronal perikarya is evident in the k.a.-injected nigra, while the myelinated fibers appear fairlynormal.
d o m i n a t e d t h e acute phase, which lasted from 8to 18h. T h e second phase was characterized by i n t e r m i t t e n t ipsilateral-contralateral turning, while t he muscular dystonias slowly subsided. 24 h after k.a. administration, t he animals showed an asymmetric posture with turning contralateral t o t h e lesion. T h e y appeared unable t o go straight and t u r n e d contralaterally whenever t h e y had to move, w h e t h e r t o escape a noxi ous stimulus or t o explore t he environment. T h e contralateral turning was maximal during t h e first 3--4 days after k.a. administration. As shown in table 4, t he intensity and t he incidence o f t h e contralateral turning was related to t h e dose o f k.a., being maximal after 1/~g. All t he histological, biochemical and pharmacological data were obtained with t he 0.75 ~g dose.
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M.C. O L I A N A S ET AL.
TABLE 5 E f f e c t o f h a l o p e r i d o l , a p o m o r p h i n e and D - a m p h e t a m i n e o n t h e contralateral t u r n i n g p r o d u c e d b y the unilateral injection o f k.a. in the nigra in n o r m a l rats and in rats previously i n j e c t e d bilaterally in the s.n. w i t h 6-OHDA. H a l o p e r i d o l a n d D - a m p h e t a m i n e were i n j e c t e d i.p. and a p o m o r p h i n e s.c. O b s e r v a t i o n s t a r t e d 15 rain after treatm e n t and lasted 1 h. Treatment (mg/kg)
Bilateral 1 6-OHDA
K.a.-treated rats
Rats with turning
Turning direction
Rats t u r n i n g with score
Maximal circling 2 (turns/min)
0
1
2
3
2 --
6 2
60 10
2
7 4
25 _+ 4 s 16 + 4
5 6
18 + 5 28 +_ 4 4
7 4
16 + 3 14 +_ 5
None
-Yes
79 14
68 12
Contralateral Contralateral
11 2
Haloperidol (0.2)
-Yes
8 6
7 6
Contralateral Contralateral
1
Apomorphine (0.5)
-Yes
6 6
5 6
Ipsilateral Ipsilateral
1
D-amphetamine (5)
-Yes
7 8
7 8
Ipsilateral Contralateral
--
1
3
18 + 3 15 _+ 5
1 Only animals s h o w i n g n o m o t o r a s y m m e t r i e s a f t e r a dose o f 2 m g / k g o f D - a m p h e t a m i n e , o n the 5th day a f t e r surgery, were used. 2 Values are m e a n s +_ S.D. 3 p < 0.01 w i t h r e s p e c t t o rats t r e a t e d w i t h k.a. alone. 4 p < 0.01 w i t h r e s p e c t t o rats t r e a t e d with k.a. + a p o m o r p h i n e .
In 10 out of the 12 rats which did not show contralateral turning after 0.75--1 pg k.a., the cannula tip was found positioned outside the s.n. Among 68 rats turning contralaterally, the tip of the cannula was localized in the center of the pars reticulata in 46, at the border between the compacta and the reticulata in 16, in the pars lateralis in 4 and in the medial part of the nigra in 2. 12 rats were injected with k.a. under light ether anaesthesia, so that their behavior could be observed soon after surgery. 9 showed spontaneous circling contralateral to the lesion within the first 20--30 min, which was gradually superseded by the ipsilateral syndrome described for the other set of animals. As shown in table 5, 5 days after the lesion, the administration of D-amphetamine (5 mg/ kg i.p.) or apomorphine (0.5 mg/kg s.c.) caused ipsilateral circling, while haloperidol (0.2 mg/kg i.p.) significantly potentiated the contralateral turning. Similar results were ob-
tained 10 and 20 days after the lesion. The same experiments were repeated in animals bilaterally injected in the s.n. with 6-OHDA (8 #g/s.n. in 0.3 pl of saline) under Equithesin anaesthesia and chosen on the basis of their hypersensitivity to the stereotyping effect of a subthreshold dose of apomorphine (0.05 mg/kg s.c.). When k.a. was injected unilaterally in the s.n. under light ether anaesthesia, 10 days after bilateral intranigral 6-OHDA, none of these rats showed the initial short-lived contralateral circling, but all showed the acute ipsilateral circling behavior which evolved into the chronic contralateral turning. In these rats treated with 6-OHDA plus k.a. apomorphine (0.5 mg/kg s.c.), administered one week after k.a. treatment, still produced ipsilateral circling, but D-amphetamine (5 mg/ kg i.p.) failed to change the direction of the k.a.-induced contralateral turning and haloperidol (0.2 mg/kg i.p.) failed to intensify it
K A I N I C ACID IN T H E S U B S T A N T I A N I G R A
229
(table 5). Two days after the last treatment, the animals were sacrified and striatal DA and DOPAC concentrations were found to be depleted by about 90% as compared to the controls. 10 rats unilaterally injected in the s.n. with k.a. and selected for their contralateral turning behavior, were reinjected with k.a. in the same nigra under ether anaesthesia, 7 days after the first treatment. These animals showed short term potentiation of the contralateral turning, lasting about 20 min and which was replaced in 7 of the 10 by circling ipsilateral to the injected side. The ipsilateral turning, never interrupted by muscular dystonias, completely vanished within 2--3 h. 3 animals did not show the ipsilateral phase. One day after the second treatment all rats had resumed the contralateral turning. Histological control showed that both injections were in the center of the s.n. in all animals. 56 rats were injected bilaterally in the s.n. with k.a. (0.75 pg in each), under Equithesin anaesthesia. Beginning the first day after surgery, most of the animals showed aphagia as well as bleeding from the nose and urinary tracts. They had to be fed by garage and 18 died within the first 5 days after treatment.
TABLE 6 Effect of haloperidol on spontaneous stereotypies a n d o n p o s t u r e o f rats bilaterally i n j e c t e d in t h e s.n. w i t h k.a. Pretreatment (No. o f animals)
Hapoleridol ( m g / k g i.p.)
Rats showing Stereotypies
Catalepsy 1
2
3
C o n t r o l s (12) (11) (10)
0.2 2.0 10.0
----
4 1
-3
-7 10
K.a. (16) 1 (18) I (15) I
0.2 2.0 10.0
14 15 14
2 --
-1
-2 1
1 Only animals s h o w i n g s p o n t a n e o u s s t e r e o t y p e d s n i f f i n g a n d gnawing (score 4) w e r e used.
42 rats showed intense stereotyped gnawing and sniffing; 14 showed self-mutilation and 6 had spontaneous seizures. All the animals were hyper-reactive to external stimuli and would jump out of a 20 cm high box after gentle pressing of the tail. These phenomena were still present 10 days after k.a. administration. 5 days after the bilateral injection, haloperidol (0.2--10 mg/kg i.p.) failed to antagonize the spontaneous stereotypies in 43 out of 49 rats (table 6). 1 h after haloperidol administration, the rats were tested for catalepsy. While all control animals showed different degrees of catalepsy and muscular rigidity, depending on the dose of neuroleptic used, only one out of 15 rats pretreated with k.a. presented clearcut signs of catalepsy even after the 10 mg/kg dose of haloperidol, which produced score 3 catalepsy in all the control animals.
4. Discussion The unilateral, intranigral injection of k.a., a cyclic analog of glutamic acid that produces degeneration of neuronal soma on intracerebral injection (Olney et al., 1975), caused a moderate neuronal loss in the pars reticulata and resulted in a sequence of acute contralateral and ipsilateral circling followed, within 24 h, by chronic, long-lasting contralateral turning. The rapid onset and the short duration of the acute contra- and ipsilateral circling suggest that such behavioral effects may be due to the powerful stimulant activity of k.a. on neurons located within the s.n. The failure of k.a. to induce the early contxalateral circling in animals previously injected intranigrally with 6-OHDA indicates that this contralateral circling is probably due to stimulation of nigro-striatal DA neurons. This is in agreement with the notion that unilateral stimulation of striatal DA receptors results in contralateral circling (Ungerstedt and Arbuthnott, 1970). The stimulation of dopaminergic neurons was
230
confirmed by the increased concentration of striatal DOPAC, an index of DA release. Under the conditions we used, however, k.a. did not induce massive degeneration of DA neurons. This was indicated by the relative intactness of the pars compacta and by the very slight depletion of striatal DA and DOPAC at 7 days after k.a. administration. The ipsilateral circling occurred in animals previously injected with 6-OHDA and seemed therefore to be independent of the integrity of the DA system; it may have been due to the direct stimulation by k.a. of nigral nondopaminergic neurons whose influence on circling behavior is opposite to that of the dopaminergic ones. If these neurons then degenerated, it would explain the substitution of the acute ipsilateral circling by the chronic contralateral turning. This explanation is supported by the finding that a second nigral injection of k.a. at the time when the contralateral turning was established, either failed to produce ipsilateral circling or produced ipsilateral circling lasting no more that 2--3 h. The hypothesis that k.a. destroys a neuronal system whose function is opposite to that of striatal DA is further supported by the observation of chronic stereotyped sniffing and gnawing and by the severe self-mutilation produced after bilateral, intranigral injection of k.a. This behavior cannot be attributed to increased striatal DA-receptor stimulation, since it was affected by neither previous destruction of DA neurons with 6-OHDA nor the administration of high doses of haloperidol, a rather specific DA-receptor blocker. Administration of DA-receptor agonists (Damphetamine or apomorphine) to rats injected with k.a. unilaterally in the nigra changed the turning from contralateral to ipsilateral. Ipsilateral turning after dopaminergic agonists would suggest that the side ipsilateral to the injection had decreased dopaminergic function as compared to the contralateral side. This suggestion is supported by (i) the fact that in animals treated bilaterally with 6OHDA before the unilateral injection of k.a., apomorphine was still effective but D-amphet-
M.C. OLIANAS ET AL.
amine was ineffective in producing ipsilateral turning; and (ii} the potentiation of the contralateral turning with haloperidol. The failure of haloperidol to produce catalepsy in rats injected bilaterally with k.a. also suggests a loss of dopaminergic function in the caudates ipsilateral to the nigral lesions induced by k.a. This apparent loss of dopaminergic function is difficult to explain at the moment. Hyposensitivity of striatal DA receptors seems unlikely in view of the normal response of DA-sensitive adenylate cyclase in the caudate ipsilateral to the lesion. Another possibility is that the nigral nonDA neurons destroyed by k.a. actually mediate certain effects produced by DA-receptor stimulation. If, for example, they mediate the contralateral turning produced by DA-receptor stimulation, such stimulation in animals where these neurons have been destroyed unilaterally could produce a turning ipsilateral to the destruction; this is the effect observed in the k.a. injected animals. Marshall and Ungerstedt (1977) have shown that unilateral lesions, at a midhypothalamic level, of the internal capsule through which course the strio-nigral fibers result in abolition of the contralateral turning induced by apomorphine. This is consistent with the results of our study if one postulates that a strio-nigral inhibitory pathway, activated by striatal DA-receptor stimulation, is interposed between the dopaminergic and the nigral non
231
KAINIC ACID IN THE SUBSTANTIA NIGRA
Since the chronic effects of intranigral k.a. are very similar to those produced by electrocoagulative lesions of the nigra, it is probable that the effects o f the electrocoagulative lesions are also due to damage to nigral neurons and not to lesioning of fibers crossing or terminating within the nigra. Under our experimental conditions, there was only moderate loss of nigral perikarya, suggesting that the lesion produced by k.a. was limited to a fairly specific neuronal population. The histological procedure adopted in this study did not enable us to determine the morphological characteristics of these neurons. Our data are compatible with the possibility that the behavioral effects were produced by destruction of either some nigral interneurons or a non-DA neuronal system efferent from the nigra. The existence of nonDA nigro-striatal and nigro-reticular connections is well-known (Feltz and De Champlain, 1972; Hattori et al., 1973; Hopkins and Niessen, 1976; Rinvik et al., 1976). A particularly conspicuous non-DA nigral projection is that to the ventro-medial thalamus {rat) (Faull and Carman, 1968); since this nucleus, being part of the striato-pallido-thalamo-cortical pathway (Carpenter, 1976), should mediate extrapyramidal influences orginating from the striatum, the destruction of a nigro-thalamic pathway appears a possible mechanism for the turning induced by intranigral k.a. The existence of nigral non-DA neurons which mediated turning behavior in a manner opposite to the nigro-striatal DA system may provide an explanation for some turning responses which are not readily explained by an action on the nigro-striatal DA system. For example, it has been reported that unilateral, intranigral administration of GABA, muscimol (Scheel-Kriiger et al., 1977) or ethanolamineO:sulphate, a GABA-transaminase blocker, (Dray et al., 1975a; Dray et al., 1976b; Koob et al., 1976) results in spontaneous contralateral turning. This syndrome resembles the chronic one produced by intranigral k.a. The similarity between these effects raises the possibility that GABA is inhibitory on the k.a.
sensitive neurons and that, physiologically, striatal DA-receptor stimulation is mediated via the activation of a strio-nigral GABAergic pathway.
References Carpenter, M.B., 1976, Anatomy of the Basal Ganglia and Rela~ed Nuclei: A Review, Advances in Neurology, 14, eds. R. Eldridge and S. Fahn (Raven Press, New York). Costall, B., C.D. Marsden, R.J. Naylor and C.J. Picock, 1976, The relationship between striatal and mesolimbic dopamine disfunction and the nature of circling responses following 6-hydroxydopamine and electrolytic lesions of the ascending dopamine system of rat brain, Brain Res. 118, 87. Costail, B. and R.J. Naylor, 1974, Stereotyped and circling behavior induced by dopaminergic agonists after lesions of the midbrain raphe nuclei, European J. Pharmacol. 29,206. Coyle, J.T. and R. Scwartz, 1976, Lesions of striatal neurons with k.a. provides a model for Huntington's chorea, Nature 263,244. Dray, A., L.J. Fowler, N.R. Oakley, N.A. Simmonds and T. Tanner, 1975a, Comparison of circling behavior following unilateral inhibition of GABAtransaminase or discrete electrolytic lesioning in the rat substantia nigra, Brit. J. Pharmacol. 55, 288P. Dray, A., N.R. Oakley and N.A. Simmonds, 1975b, Rotational behavior following inhibition of GABA metabolism unilaterally in the rat substantia nigra, J. Pharm. Pharmacol. 27,627. Faull, R.L. and J.B. Carman, 1968, Ascending projections of the substantia nigra in the rat, J. Comp. Neurol. 132, 73. Feltz, P. and J. De Champlain, 1972, Persistence of caudate unitary responses to nigral stimulation after destruction and functional impairment of the striatai dopaminergic terminals, Brain Res. 43,595. Hattori, T., H.C. Fibiger, P.L. Mc Geer and L. Maler, 1973, Analysis of the fine structure of the dopaminergic nigro-striatal projection by electron microscopic autoradiography, Exp. Neurol. 41,599. Hopkins, D.A. and L.W. Niessen, 1976, Substantia nigra projections to the reticular formation, superior colliculus and central gray in the rat, cat and monkey, Neurosci. Letters 2,253. Iwamoto, E.T., H.H. Loh and E.L. Way, 1976, Circling behavior in rats with 6-hydroxydopamine or electrolytic nigral lesions, European J. Pharmacol. 37,339. Koob, G., M. Del Fiacco and S.D. Iversen, 1976, T h e behavioral effects of EOS-induced changes in sub-
232 stantia nigra GABA levels, Proc. Brit. Pharmac. Soc., Abstr. C 60, Oxford. Marshall, J.F. and U. Ungerstedt, 1977, Supersensivity to apomorphine following destruction of the ascending dopamine neurons: quantification using the rotational model, European J. Pharmacol. 41, 361. Mc Geer, E.G. and P.L. Mc Geer 1975, Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kainic acid, Nature 263, 517. Olianas, M.C., M.G. De Montis, G. Mulas and A. Tagliamonte, 1976, Synergistic potentiation of haloperidol induced catalepsy by 7-butyrolactone, J. Neurochem. 27,821. Olney, J.W., L.G. Sharpe and T. De Gubareff, 1975, Excitotoxic aminoacids, Neurosc. Abstr. 1,371. Pellegrino, L.J. and A.J. Cushman, 1971, A Stereotaxic Atlas of the Rat Brain (Mredith, New York). Premont, J., A.M. Thierry, J.P. Tassin, J. Glowinski, G. Blanc and J. Bockaert, 1976, Is the dopamine sensitive adenylate cyclase in the rat substantia nigra coupled with "Autoreceptors"?, FEBS Letters 68, 99. Rinvik, E., I. Grofova and O.P. Ottersen, 1976, Demonstration of nigro-tectal and nigro-reticular
M.C. OLIANAS ET AL. projections in the cat by axonal transport of proteins, Brain Res. 112,388. Scheel-Krtiger, J., J. Arnt and G. Magelund, 1977, Behavioural stimulation induced by muscimol and other GABA agonists injected into the substantia nigra, Neurosci. Letters 4,351. Schwartz, W.J., R.H. Gunn, F.R. Sharp and E.V. Everts, 1976, Unilateral electrolytic lesions of the substantia nigra cause contralateral cyrcling in rats, Brain Res. 105,358. Snedecor, J.W., 1969, Statistical Methods, 5th edn. (Iowa State College Press, Ames, Iowa). Spano, P.F., M. Trabucchi and G. Di Chiara, 1977, Localization of DA-sensitive adenylate cyclase on neurons originating from the corpus striatum, Science 196, 1343. Ungerstedt, U. and G.W. Arbuthnott, 1970, Quantitative recording of rotational behavior in rats after 6-hydroxydopamine lesions of the nigro-striatal dopamine system, Brain Res. 24,485. Zivkovic, B., A. Guidotti, A. Revuelta and E. Costa, 1975, Effect of thioridazine, clozapine and other antipsychotic drugs on the kinetic state of tyrosine hydroxylase and on the turnover rate of dopamine in striatum and nucleus accumbens, J. Pharmacol. Exptl. Therap. 194, 37.
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© Elsevier/North-Holland Biomedical Press
I N T R A N I G R A L K A I N I C ACID: E V I D E N C E F O R N I G R A L N O N - D O P A M I N E R G I C N E U R O N S C O N T R O L L I N G P O S T U R E A N D B E H A V I O R IN A M A N N E R O P P O S I T E T O T H E DOPAMINERGIC ONES MARIA C. OLIANAS, GRAZIELLA M. DE MONTIS, ALBERTO CONCU t , ALESSANDRO TAGLIAMONTE * and GAETANO DI CHIARA 2nd Chair of Pharmacology, and ~ Institute of Physiology, University of Cagliari, Via Porcell 4, 09100 Cagliari, Italy
Received 26 October 1977, revised MS received 25 January 1978, accepted 1 February 1978
M.C. OLIANAS, G.M. DE MONTIS, A. CONCU, A. TAGLIAMONTE and G. DI CHIARA, Intranigral kainic acid: evidence for nigral non-dopaminergic neurons controlling posture and behavior in a manner opposite to the dopaminergic ones, European J. Pharmacol. 49 (1978) 223--232. The unilateral, intranigral administration of kainic acid (k.a.) produced a syndrome characterized by early sequelae of contra- and ipsilateral circling and by a chronic contralateral turning associated with moderate loss of neurons in the pars reticulata. The acute contralateral circling seems to be related to dopaminergic nigro-neostriatal neuron stimulation, since it was prevented by previous intranigral injections of 6-OHDA. The acute ipsilateral circling and the chronic contralateral turning, on the other hand, seem to be independent of the integrity of the dopaminergic system and may be due to an initial stimulation, followed by destruction, of a nigral neuronal system which mediates turning behavior in a manner opposite to that of nigro-striatal dopamine. Treatment with D-amphetamine or apomorphine changed the contralateral into ipsilateral turning, while haloperidol potentiated the contralateral turning. Bilateral injection of k.a. into the nigra resulted in chronic stereotyped sniffing and gnawing, which were not inhibited by haloperidol. Moreover, haloperidol did not produce catalepsy in these animals. It is suggested that the intranigral k.a. injection destroyed a neuronal system antagonistic to dopamine and resulted in a reduction of the response to DA-receptor stimulation of the c. striatum. Substantia nigra Turning behavior
Kainic acid
Stereotypies
1. I n t r o d u c t i o n Selective, unilateral lesioning o f nigroneostriatal d o p a m i n e r g i c n e u r o n s b y t h e intranigral i n j e c t i o n o f 6 - h y d r o x y d o p a m i n e (6O H D A ) p r o d u c e s a d r a m a t i c p o s t u r a l asymm e t r y , consisting o f a t u r n i n g o f t h e b o d y t o w a r d t h e side o f t h e lesion ( U n g e r s t e d t and Arbuthnott, 1970). H o w e v e r , unilateral e l e c t r o l y t i c lesion o f t h e s u b s t a n t i a nigra (s.n.), while it is as e f f e c t i v e as is 6 - O H D A in d e s t r o y i n g d o p a m i n e r g i c n e u r o n s , causes t u r n i n g t o w a r d t h e side c o n t r a l a t e r a l t o t h e * Correspondence to Prof. A. Tagliamonte at the above address.
Catalepsy
GABA
Dopamine
lesion (Costall et al., 1 9 7 6 ; I w a m o t o et al., 1 9 7 6 ; S c h w a r t z et al., 1 9 7 6 ) . T h e l a t t e r finding has b e e n t a k e n t o indicate t h e exist e n c e o f a n e u r o n a l s y s t e m originating in or transversing t h e s.n. a n d w h i c h c o n t r o l s p o s t u r e in a m a n n e r o p p o s i t e t o t h e nigrostriatal d o p a m i n e r g i c n e u r o n s (Costall et al., 1 9 7 6 ; I w a m o t o et al., 1 9 7 6 ; S c h w a r t z et al., 1976). We n o w r e p o r t t h a t t h e selective destruct i o n o f nigral p e r i k a r y a b y t h e local i n j e c t i o n o f kainic acid (k.a.), p r o d u c e d c h r o n i c contralateral t u r n i n g i n d e p e n d e n t o f t h e p r e s e n c e o f nigral striataI d o p a m i n e r g i c n e u r o n s . In addit i o n , bilateral, intranigral injections o f k.a. resulted in s t e r e o t y p e d sniffing and gnawing
M.C. OLIANAS ET AL.
224
which were not abolished b y haloperidol administration.
2. Materials and methods
2.1. Animals Experiments were carried out with male albino Sprague-Dawley rats, weighing about 300 g. Animals were housed 10 to a cage (45 × 80 cm), had free access to food and water and were maintained under controlled environmental conditions, with a 12 h light-dark cycle.
2.2. Surgical procedures Rats were anaesthetized with Equithesin (3.75 ml/kg) or with ethyl ether and placed in a Stoelting Stellar stereotaxic apparatus. Drugs were injected in the s.n. using stereotaxic coordinates A: - - 3 ; L: 2; V: 7.8, according to the Atlas of Pellegrino and Cushman (1971). The total injection volume was 0.3 #l of saline injected at a rate of 1 ttl/18 min through a 0.25 mm stainless-steel cannula.
2.3. Histology 10 days after the intranigral injection of k.a., 24 rats were sacrified, the brain perfused with formalin, imbedded in paraffin and the mesencephalon c u t into serial 20 p m slices which were stained with Luxol fast-cresyl violet in order to ascertain the extent of the lesion. Other rats were sacrified b y cervical dislocation for the determination of striatal levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC). The brain areas remaining after dissection of the striata from these rats were fixed in 10% formalin for 2 h; they were then stored in 0.1 M phosphate buffer (pH 7.4) containing 5% sucrose until the mesencephalon could be cut into serial 50 ~m slices on a cryostat to allow the position of the cannula tip to be determined.
2.4. Behavior 24 h after intranigral k.a., rats were scored for m o t o r asymmetries according to a method similar to that described b y Costall et al. (1976) (table 1). The intensity of the circling was measured b y counting the turns made during each 10 min period after placement of the rats in a large open field area. Results are expressed as "maximal circling intensity", i.e. the number of turns/min performed during the 10 min period of maximal circling. The duration o f the test was 20 min when no drugs were administered or 60 min when the response to drugs was studied. Catalepsy was evaluated b y a test in which animals were placed on a horizontal bar 7.5 cm high and the intensity of catalepsy rated as: 1, when the rat did not move for at least 15 sec; 2 or 3, when the rat remained immobile for at least 30 or 60 sec, respectively. Rats were tested 60 min after haloperidol administration. Stereotyped behavior was evaluated by the scoring system of Costall and Naylor (1974),
TABLE 1 Scoring system for the evaluation of posture and motor asymmetries Description
Score
No difference from the posture and the behavior of a normal rat
0
Head and neck are preferentially turned to one side, the animal shows turning of the body when stimulated, but goes straight when moving spontaneously Forced, unilateral twist of head and neck, with preferential turning of the body toward the same direction, the animal can only move by circling ipsilaterally to the body twist Forced twist of head and body toward one side, with frequent episodes of spontaneous circling toward the direction of the body twist
K A I N I C A C I D IN T H E S U B S T A N T I A N I G R A
225
after placing the rats in Perspex cages with a wire-net bottom: 1, discontinuous sniffing with hypermotility; 2, continuous sniffing around the cage; 3, sniffing restricted to a small area of the cage with licking and discontinuous gnawing; 4, continuous gnawing in a small area of the cage.
2.5. Fluorometric DOPAC
estimation
of DA
and
Animals were killed by cervical dislocation, the brain rapidly removed and the striata dissected and stored at --20°C until analyzed. Brain DA and DOPAC were assayed fluorometrically as already described (Olianas et al., 1976).
2.6. Measurement of adenylate cyclase activity Adenylate cyclase activity was determined as described by Zivkovic et al. (1975), by measuring the conversion of 14C-ATP to 14CcAMP in a 3 min incubation. The reaction was stopped by boiling and the cAMP formed was purified by passage through alumina and Dowex 50 × 8 columns. All data were statistically analyzed with the two-tailed Student's t-test (Snedecor, 1969).
3.2. Biochemistry Table 2 shows the immediate and chronic effects of a unilateral, intranigral injection of k.a. on the DA and DOPAC concentration of the corresponding c. striatum, as compared to the control side. 45 min after k.a. administration, striatal DA and DOPAC of the lesioned side were increased by 42% and 232%, respectively. On the other hand, 7 days after surgery, both DA and DOPAC decreased by about 25% in the C. striatum ipsilateral to the injected s.n. Table 3 shows the effect of an injection of k.a. on basal and DA-stimulated adenylate cyclase activity in both the treated s.n. and the ipsilateral c. striatum, as compared with the uninjected side. The experiment was carried out 7 days after surgery. Neither basal nor DA-stimulated adenylate cyclase of either area was significantly affected by the nigral k.a. injection.
3.3. Behavior When they recovered from the anaesthesia, 1--2 h after surgery, the rats showed a marked postural asymmetry with flexion of the ipsilateral and extension of the contralateral limbs; they showed spontaneous ipsilateral
3. Results TABLE 2
3.1. Histology Fig. 1 shows the histological appearance of a s.n. injected with 0.75 pg (3.5 nM) of k.a. 10 days previously, as compared to that injected with saline. The pars reticulata of the lesioned nigra revealed moderate loss of neuronal perikarya and gliosis, characterized by proliferation of non-neuronal elements. The pars compacta seemed less affected than the reticulata and still showed a high density of its typical, large and globular neurons. No signs of demyelinization were present in or around the s.n.
E f f e c t o f k.a. i n j e c t e d in t h e s.n. o n t h e levels o f D A a n d D O P A C o f t h e c o r r e s p o n d i n g c. s t r i a t u m , as comp a r e d t o t h e side i n j e c t e d w i t h saline. Each value is t h e m e a n +- S.E. o f at least 8 d e t e r m i n a tions. K.a. was i n j e c t e d in a dose o f 0.75 /~g/0.3 pl a n d t h e same dose was used in all t h e o t h e r experim e n t s r e p o r t e d , unless specified. Intranigral treatment
D A (/~g/g)
D O P A C (ng/g)
Saline 45 m i n K.a. 45 rain Saline 7 days K.a. 7 days
5.46 7.75 5.51 3.96
430 1,427 426 316
-+ 0 . 0 8 +- 0 . 0 9 2 +_ 0 . 1 0 +- 0.06 2
+ 7 +_ 28 1 +_ 9 +- 8 2
1 p < 0 . 0 0 1 , 2 p < 0.01, c o m p a r e d t o t h e side inject e d w i t h saline.
t ~
I
"..2 °',
A
,B~
'
KAINIC ACID IN THE SUBSTANTIA NIGRA
227
TABLE 3 Failure of intranigral k.a. injection to modify the basal and DA-stimulated adenylate cyclase activity of the s.n. and c. striatum. Each value is the mean + S.E. of 8 determinations. Dopamine
c-AMP formed (pmol/mg prot./min) K.a.
-
-
10 -s M 10 -4 M
Controls
S.n.
C. striatum
S.n.
C. striatum
205 +- I I 298+16t 321+141
144 +- 14 258+121 2 8 9 + - 1 5I
202 -+ 13 288+- 9 i 329+-14 I
186 +- 16 249+13 1 284+ 61
I p < 0.001 with respect to basal values. TABLE 4 Incidence and intensity of contralateral turning after various doses of intranigral k.a. Turning was scored 24 h after intranigral injection of k.a., as described in the text. Kainic acid (l~g) 0.25 0.50 0.75 1.00
Rats injected
I0 15 79 20
Rats with turning
2 I0 68 19
Rats turning with score
Maximal circlingI (turns/rain)
0
1
2
3
8 5 II 1
2 2 2 1
-2 6 4
-6 60 14
-6 +- 2 18 -+3 22 -+4
1 Values are means + S.E.
circling, sometimes i nt er r upt ed by muscular dystonias with twisting o f t h e head and rolling o f t h e b o d y . These s y m p t o m s
Fig. I. Micrographs (10x) of s.n. sections (20 pm, stained with Luxol fast-cresylviolet) of rats injected unilaterally with k.a. (0.75 pg) and killed 10 days thereafter. A and B represent the medial portion of the control and k.a.-injected s.n., respectively; the pars compacta is limited dorsally (top of the figure) by the myelinated fibers of the medial lemniscus. In the k.a.-injected side the typical globular neurons of the pars compacta are fairlywell preserved and so are the myelinated fibers. In addition, a high degree of gliosis is present in this side. C and D represent the pars reticulata of the control and k.a.-injectednigra, respectively. The top of the figure is the dorsal part of the s.n. Gliosis associated with loss of neuronal perikarya is evident in the k.a.-injected nigra, while the myelinated fibers appear fairlynormal.
d o m i n a t e d t h e acute phase, which lasted from 8to 18h. T h e second phase was characterized by i n t e r m i t t e n t ipsilateral-contralateral turning, while t he muscular dystonias slowly subsided. 24 h after k.a. administration, t he animals showed an asymmetric posture with turning contralateral t o t h e lesion. T h e y appeared unable t o go straight and t u r n e d contralaterally whenever t h e y had to move, w h e t h e r t o escape a noxi ous stimulus or t o explore t he environment. T h e contralateral turning was maximal during t h e first 3--4 days after k.a. administration. As shown in table 4, t he intensity and t he incidence o f t h e contralateral turning was related to t h e dose o f k.a., being maximal after 1/~g. All t he histological, biochemical and pharmacological data were obtained with t he 0.75 ~g dose.
228
M.C. O L I A N A S ET AL.
TABLE 5 E f f e c t o f h a l o p e r i d o l , a p o m o r p h i n e and D - a m p h e t a m i n e o n t h e contralateral t u r n i n g p r o d u c e d b y the unilateral injection o f k.a. in the nigra in n o r m a l rats and in rats previously i n j e c t e d bilaterally in the s.n. w i t h 6-OHDA. H a l o p e r i d o l a n d D - a m p h e t a m i n e were i n j e c t e d i.p. and a p o m o r p h i n e s.c. O b s e r v a t i o n s t a r t e d 15 rain after treatm e n t and lasted 1 h. Treatment (mg/kg)
Bilateral 1 6-OHDA
K.a.-treated rats
Rats with turning
Turning direction
Rats t u r n i n g with score
Maximal circling 2 (turns/min)
0
1
2
3
2 --
6 2
60 10
2
7 4
25 _+ 4 s 16 + 4
5 6
18 + 5 28 +_ 4 4
7 4
16 + 3 14 +_ 5
None
-Yes
79 14
68 12
Contralateral Contralateral
11 2
Haloperidol (0.2)
-Yes
8 6
7 6
Contralateral Contralateral
1
Apomorphine (0.5)
-Yes
6 6
5 6
Ipsilateral Ipsilateral
1
D-amphetamine (5)
-Yes
7 8
7 8
Ipsilateral Contralateral
--
1
3
18 + 3 15 _+ 5
1 Only animals s h o w i n g n o m o t o r a s y m m e t r i e s a f t e r a dose o f 2 m g / k g o f D - a m p h e t a m i n e , o n the 5th day a f t e r surgery, were used. 2 Values are m e a n s +_ S.D. 3 p < 0.01 w i t h r e s p e c t t o rats t r e a t e d w i t h k.a. alone. 4 p < 0.01 w i t h r e s p e c t t o rats t r e a t e d with k.a. + a p o m o r p h i n e .
In 10 out of the 12 rats which did not show contralateral turning after 0.75--1 pg k.a., the cannula tip was found positioned outside the s.n. Among 68 rats turning contralaterally, the tip of the cannula was localized in the center of the pars reticulata in 46, at the border between the compacta and the reticulata in 16, in the pars lateralis in 4 and in the medial part of the nigra in 2. 12 rats were injected with k.a. under light ether anaesthesia, so that their behavior could be observed soon after surgery. 9 showed spontaneous circling contralateral to the lesion within the first 20--30 min, which was gradually superseded by the ipsilateral syndrome described for the other set of animals. As shown in table 5, 5 days after the lesion, the administration of D-amphetamine (5 mg/ kg i.p.) or apomorphine (0.5 mg/kg s.c.) caused ipsilateral circling, while haloperidol (0.2 mg/kg i.p.) significantly potentiated the contralateral turning. Similar results were ob-
tained 10 and 20 days after the lesion. The same experiments were repeated in animals bilaterally injected in the s.n. with 6-OHDA (8 #g/s.n. in 0.3 pl of saline) under Equithesin anaesthesia and chosen on the basis of their hypersensitivity to the stereotyping effect of a subthreshold dose of apomorphine (0.05 mg/kg s.c.). When k.a. was injected unilaterally in the s.n. under light ether anaesthesia, 10 days after bilateral intranigral 6-OHDA, none of these rats showed the initial short-lived contralateral circling, but all showed the acute ipsilateral circling behavior which evolved into the chronic contralateral turning. In these rats treated with 6-OHDA plus k.a. apomorphine (0.5 mg/kg s.c.), administered one week after k.a. treatment, still produced ipsilateral circling, but D-amphetamine (5 mg/ kg i.p.) failed to change the direction of the k.a.-induced contralateral turning and haloperidol (0.2 mg/kg i.p.) failed to intensify it
K A I N I C ACID IN T H E S U B S T A N T I A N I G R A
229
(table 5). Two days after the last treatment, the animals were sacrified and striatal DA and DOPAC concentrations were found to be depleted by about 90% as compared to the controls. 10 rats unilaterally injected in the s.n. with k.a. and selected for their contralateral turning behavior, were reinjected with k.a. in the same nigra under ether anaesthesia, 7 days after the first treatment. These animals showed short term potentiation of the contralateral turning, lasting about 20 min and which was replaced in 7 of the 10 by circling ipsilateral to the injected side. The ipsilateral turning, never interrupted by muscular dystonias, completely vanished within 2--3 h. 3 animals did not show the ipsilateral phase. One day after the second treatment all rats had resumed the contralateral turning. Histological control showed that both injections were in the center of the s.n. in all animals. 56 rats were injected bilaterally in the s.n. with k.a. (0.75 pg in each), under Equithesin anaesthesia. Beginning the first day after surgery, most of the animals showed aphagia as well as bleeding from the nose and urinary tracts. They had to be fed by garage and 18 died within the first 5 days after treatment.
TABLE 6 Effect of haloperidol on spontaneous stereotypies a n d o n p o s t u r e o f rats bilaterally i n j e c t e d in t h e s.n. w i t h k.a. Pretreatment (No. o f animals)
Hapoleridol ( m g / k g i.p.)
Rats showing Stereotypies
Catalepsy 1
2
3
C o n t r o l s (12) (11) (10)
0.2 2.0 10.0
----
4 1
-3
-7 10
K.a. (16) 1 (18) I (15) I
0.2 2.0 10.0
14 15 14
2 --
-1
-2 1
1 Only animals s h o w i n g s p o n t a n e o u s s t e r e o t y p e d s n i f f i n g a n d gnawing (score 4) w e r e used.
42 rats showed intense stereotyped gnawing and sniffing; 14 showed self-mutilation and 6 had spontaneous seizures. All the animals were hyper-reactive to external stimuli and would jump out of a 20 cm high box after gentle pressing of the tail. These phenomena were still present 10 days after k.a. administration. 5 days after the bilateral injection, haloperidol (0.2--10 mg/kg i.p.) failed to antagonize the spontaneous stereotypies in 43 out of 49 rats (table 6). 1 h after haloperidol administration, the rats were tested for catalepsy. While all control animals showed different degrees of catalepsy and muscular rigidity, depending on the dose of neuroleptic used, only one out of 15 rats pretreated with k.a. presented clearcut signs of catalepsy even after the 10 mg/kg dose of haloperidol, which produced score 3 catalepsy in all the control animals.
4. Discussion The unilateral, intranigral injection of k.a., a cyclic analog of glutamic acid that produces degeneration of neuronal soma on intracerebral injection (Olney et al., 1975), caused a moderate neuronal loss in the pars reticulata and resulted in a sequence of acute contralateral and ipsilateral circling followed, within 24 h, by chronic, long-lasting contralateral turning. The rapid onset and the short duration of the acute contra- and ipsilateral circling suggest that such behavioral effects may be due to the powerful stimulant activity of k.a. on neurons located within the s.n. The failure of k.a. to induce the early contxalateral circling in animals previously injected intranigrally with 6-OHDA indicates that this contralateral circling is probably due to stimulation of nigro-striatal DA neurons. This is in agreement with the notion that unilateral stimulation of striatal DA receptors results in contralateral circling (Ungerstedt and Arbuthnott, 1970). The stimulation of dopaminergic neurons was
230
confirmed by the increased concentration of striatal DOPAC, an index of DA release. Under the conditions we used, however, k.a. did not induce massive degeneration of DA neurons. This was indicated by the relative intactness of the pars compacta and by the very slight depletion of striatal DA and DOPAC at 7 days after k.a. administration. The ipsilateral circling occurred in animals previously injected with 6-OHDA and seemed therefore to be independent of the integrity of the DA system; it may have been due to the direct stimulation by k.a. of nigral nondopaminergic neurons whose influence on circling behavior is opposite to that of the dopaminergic ones. If these neurons then degenerated, it would explain the substitution of the acute ipsilateral circling by the chronic contralateral turning. This explanation is supported by the finding that a second nigral injection of k.a. at the time when the contralateral turning was established, either failed to produce ipsilateral circling or produced ipsilateral circling lasting no more that 2--3 h. The hypothesis that k.a. destroys a neuronal system whose function is opposite to that of striatal DA is further supported by the observation of chronic stereotyped sniffing and gnawing and by the severe self-mutilation produced after bilateral, intranigral injection of k.a. This behavior cannot be attributed to increased striatal DA-receptor stimulation, since it was affected by neither previous destruction of DA neurons with 6-OHDA nor the administration of high doses of haloperidol, a rather specific DA-receptor blocker. Administration of DA-receptor agonists (Damphetamine or apomorphine) to rats injected with k.a. unilaterally in the nigra changed the turning from contralateral to ipsilateral. Ipsilateral turning after dopaminergic agonists would suggest that the side ipsilateral to the injection had decreased dopaminergic function as compared to the contralateral side. This suggestion is supported by (i) the fact that in animals treated bilaterally with 6OHDA before the unilateral injection of k.a., apomorphine was still effective but D-amphet-
M.C. OLIANAS ET AL.
amine was ineffective in producing ipsilateral turning; and (ii} the potentiation of the contralateral turning with haloperidol. The failure of haloperidol to produce catalepsy in rats injected bilaterally with k.a. also suggests a loss of dopaminergic function in the caudates ipsilateral to the nigral lesions induced by k.a. This apparent loss of dopaminergic function is difficult to explain at the moment. Hyposensitivity of striatal DA receptors seems unlikely in view of the normal response of DA-sensitive adenylate cyclase in the caudate ipsilateral to the lesion. Another possibility is that the nigral nonDA neurons destroyed by k.a. actually mediate certain effects produced by DA-receptor stimulation. If, for example, they mediate the contralateral turning produced by DA-receptor stimulation, such stimulation in animals where these neurons have been destroyed unilaterally could produce a turning ipsilateral to the destruction; this is the effect observed in the k.a. injected animals. Marshall and Ungerstedt (1977) have shown that unilateral lesions, at a midhypothalamic level, of the internal capsule through which course the strio-nigral fibers result in abolition of the contralateral turning induced by apomorphine. This is consistent with the results of our study if one postulates that a strio-nigral inhibitory pathway, activated by striatal DA-receptor stimulation, is interposed between the dopaminergic and the nigral non
231
KAINIC ACID IN THE SUBSTANTIA NIGRA
Since the chronic effects of intranigral k.a. are very similar to those produced by electrocoagulative lesions of the nigra, it is probable that the effects o f the electrocoagulative lesions are also due to damage to nigral neurons and not to lesioning of fibers crossing or terminating within the nigra. Under our experimental conditions, there was only moderate loss of nigral perikarya, suggesting that the lesion produced by k.a. was limited to a fairly specific neuronal population. The histological procedure adopted in this study did not enable us to determine the morphological characteristics of these neurons. Our data are compatible with the possibility that the behavioral effects were produced by destruction of either some nigral interneurons or a non-DA neuronal system efferent from the nigra. The existence of nonDA nigro-striatal and nigro-reticular connections is well-known (Feltz and De Champlain, 1972; Hattori et al., 1973; Hopkins and Niessen, 1976; Rinvik et al., 1976). A particularly conspicuous non-DA nigral projection is that to the ventro-medial thalamus {rat) (Faull and Carman, 1968); since this nucleus, being part of the striato-pallido-thalamo-cortical pathway (Carpenter, 1976), should mediate extrapyramidal influences orginating from the striatum, the destruction of a nigro-thalamic pathway appears a possible mechanism for the turning induced by intranigral k.a. The existence of nigral non-DA neurons which mediated turning behavior in a manner opposite to the nigro-striatal DA system may provide an explanation for some turning responses which are not readily explained by an action on the nigro-striatal DA system. For example, it has been reported that unilateral, intranigral administration of GABA, muscimol (Scheel-Kriiger et al., 1977) or ethanolamineO:sulphate, a GABA-transaminase blocker, (Dray et al., 1975a; Dray et al., 1976b; Koob et al., 1976) results in spontaneous contralateral turning. This syndrome resembles the chronic one produced by intranigral k.a. The similarity between these effects raises the possibility that GABA is inhibitory on the k.a.
sensitive neurons and that, physiologically, striatal DA-receptor stimulation is mediated via the activation of a strio-nigral GABAergic pathway.
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