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Evidence that a nigral gabaergic-chrolinergic balance controls posture
European Journal of Pharmacology, 53 (1979) 181--190 © Elsevier/North-Holland Biomedical Press
181
EVIDENCE THAT A NIGRAL GABAERGIC--CHOLINERGIC BALANCE CONTROLS POSTURE G R A Z I E L L A M. DE MONTIS, MARIA C. OLIANAS, GINO SERRA, ALESSANDRO TAGLIAMONTE * and JORGEN SCHEEL-KRUGER
2nd Chair of Pharmacology, University of Cagliari, Italy, and Psychopharmacological Research Laboratory, Dept. E, St. Hans Mental Hospital, DK-4000 Roskilde, Denmark Received 21 March 1978, revised MS received 11 August 1978, accepted 20 September 1978
G.M. DE MONTIS, M.C. OLIANAS, G. SERRA, A. TAGLIAMONTE and J. SCHEEL-KRt)GER, Evidence that a nigral GABAergic--cholinergic balance controls posture, European J. Pharmacol. 53 (1979) 181--190. The intranigral injection of kainic acid (k.a.) (3.5 nM/s.n.) produced a lesion which resulted in a decreased muscarinic receptor binding capacity and in a decreased choline acetyl transferase (CAT) activity confined to the pars reticulata. The unilateral, intranigral injection of carbachol in the substantia nigra (s.n.) produced turning, ipsilateml to the injected side, of dose-related intensity, which was antagonized by scopolamine given either i.p. or intranigrally together with carbachol. The bilateral, intranigral injection of carbachol produced rigid catalepsy, highly resistant to apomorphine administration and antagonized by scopolamine. On the other hand, the catalepsy produced by intranigral picrotoxin was much more sensitive to apomorphine and was disrupted by systemic scopolamine administration. Intranigral scopolamine per se produced either contralateral turning or stereotyped movements consistently, when injected unilaterally or bilaterally, respectively. In addition, scopolamine injected bilaterally in the s.n. but not in the caudate nucleus (c.n.), at the concentration of 64 nM side, was able to antagonize the haloperidol-induced catalepsy and to prevent the tremors and the muscular rigidity produced by arecoline. This effect of scopolamine was surmountable with a higher dose of arecoline. Finally, intranigral muscimol (0.44 nM/s.n.) prevented the occurrence of the parkinsonian syndrome produced by systemic arecoline. It is concluded that the muscarinic receptors present in the s.n. pars reticulata play a role in the control of posture opposite to that of the nigral GABA receptors. Choline acetyltransferase Muscarinic receptors
Acetylcholine Stereotypies
Turning behavior Catalepsy
1. Introduction
The injection of kainic acid (k.a.) within the substantia nigra (s.n.) produces a rather selective degeneration of the neurons of the pars reticulata (Di Chiara et al., 1977; Olianas et al., 1978a). Such a lesion results in chronic turning, contralateral to the injected side, in the animals treated unilaterally and it pro*Please send reprint requests to: M.C. Olianas, Psychopharmacological Research Laboratory, Department E, Sct. Hans Hospital, DK-4000 Roskilde, Denmark.
Dopamine
GABA
duces chronic stereotypies in rats injected bilaterally (Di Chiara et al., 1977; Schwartz and Coyle, 1977; Olianas et al., 1978a). These symptoms are not antagonized by haloperidol administration and are independent of the integrity of dopaminergic nigro-neostriatal neurons (Di Chiara et al., 1977; Olianas et al., 1978a). An analogous syndrome is observed after acute injection of GABA or other gabaergic compounds into the s.n. (ScheelKr/iger et al., 1977; Dray et al., 1975; Olianas et al., 1978b). These findings demonstrate that in t h e s.n. pars reticulata there is present a neuronal
182
system which controls posture in a manner opposite to that of the nigro-striatal dopaminergic neurons (Pars Reticulata Posture Controlling neurons: PRCP neurons). Since lesions of the strio-nigral pathways abolish the effect of apomorphine treatment (Marshall and Ungerstedt, 1977), we proposed that the inhibition of these PRCP neurons via the strionigral gabaergic system mediates the striatal dopaminergic responses (Olianas et al., 1978a, b). The increase of GABA turnover in the s.n. which is produced b y apomorphine (Perez De La Mora et al., 1977) strongly supports this hypothesis. On the other hand, the unilateral, intranigral administration of picrotoxin or bicucullin (Scheel-Kriiger et al., 1977; Olianas et al., 1978b), t w o post-synaptic GABA-receptor blockers, results in turning ipsilateral to the injected side. This fact suggests that, in the absence of GABA inhibition, the PRCP neurons are under the effect of a tonic excitatory activity. The finding that neurons of the pars reticulata were markedly excited b y iontophoretically applied acetylcholine (Ach) and that this effect was a specific one, since scopolamine blocked the Ach-induced excitation b u t had no effect on glutamine excitation in the same cells, prompted us to study muscarinic agonists as the possible physiological stimulators of PRCP neurons. Our interest in Ach was also sustained by the fact that both biochemical (McGeer et al., 1973) and muscarinic binding studies (Kuhar, unpublished data) have shown that the s.n. contained Ach and Ach receptors, although the receptor density is lower than in other areas. In addition, AchE staining techniques suggest the presence of b o t h cholinergic terminals and cells bodies in the s.n. (Olivier et al., 1970; Palkovits and Jacobowitz, 1974). The present report shows that the intranigral injection of carbachol produced postural symptoms opposite to those observed after muscimol and that k.a. lesioning of the s.n. resulted in a decreased binding capacity of the local muscarinic receptors.
G.M. DE MONTIS ET AL.
2. Materials and methods
2.1. Animals Experiments were carried out on male albino Sprague-Dawley rats weighing a b o u t 300 g. Animals were housed 10 to a cage (45 X 80 cm), had free access to food and water and were maintained under controlled experimental conditions, with a 12 h light-dark cycle.
2.2. Surgical procedures Animals were anaesthetized with ethyl ether and placed on a Stoelting-Stellar stereotaxic apparatus. Drugs were injected in the s.n. using stereotaxic coordinates A: --4, L: 2, V: 7.8, according to the Atlas of Pellegrino and Cushman (1971). The injection volume was 0.3 pl of saline, injected at a rate of 1 pl/18 min, through a 0.25 mm stainless steel cannula. Coordinates for the caudate nucleus (c.n.) were A: +2, L: 3, V: 5.5, with an injection volume of I pl given in 6 min. At the end of the acute experiments, animals were killed, the brain rapidly removed, fixed in 10% formalin for 2 h and then stored in 0.1 M phosphate buffer (pH 7.4) containing 5% sucrose, until the mesencephalon was cut into serial 50 pm slices with a cryostat, to determine the position of the cannula tip. Only the results obtained in rats correctly injected are reported.
2.3. Behavior Circling behavior and catalepsy were scored as already described (Olianas et al., 1978a,b). Stereotyped behavior was evaluated, after placing the rats in Perspex cages with a wirenet b o t t o m , as follows: 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; 5, phenomena of self-mutilation. Tremors were evaluated as: 1, a fit tremors in response to an external stimulus; 2, spontaneous fits of tremors or continuous tremors during handling; 3, continuous, spontaneous tremors.
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N I G R A L GABAERGIC--CHOLINERGIC B A L A N C E
2.4. Biochemistry 3H-QNB binding was studied in whole s.n. homogenates according to the method of Yamamura et al. (1974). The tissue was homogenized in 10 volumes of ice-cold 0.32 M sucrose. The whole homogenate was centrifuged for 10 min at 1000 ×g, the pellet discarded and the supernatant was re-homogenized with a Brinkman-Polytron (setting No. 5, for 60 sec). Aliquots of this preparation were incubated at 25°C, with 0.05 M sodium-potassium (Na--K) phosphate buffer, pH 7.4, containing 3H-QNB (s.a. 16 Ci/mM) in a total volume of 2 ml. After a 60 min incubation the reaction was stopped by adding 3 ml of ice-cold Na--K phosphate buffer. The content of the tubes was passed through a glass filter (Whatman GF/B) and the filters washed 3 times, under vacuum, with 3 ml of i c e , o l d buffer. Each determination was performed in triplicate together with triplicate samples containing scopolamine (1 #M), to determine non-specific 3H-QNB binding. The specific binding was obtaining by subtracting from the total binding that obtained in the presence of 1 pM scopolamine. The radioactivity was assayed by liquid scintillation spectrometry in a Packard Tri-Carb. Proteins were determined by the method of Lowry et al. (1951). Choline acetyltransferase (CAT) activity was measured in a pool of whole s.n. (5) as well as in a pool of reticulata (9), in triplicate, according to the method Kobayashi et ai. (1975). The experiment was repeated 3 times. Data were analyzed statistically with the two-tailed Student's t-test (Snedecor, 1969).
3. Results
3.1. CAT activity CAT activity in the s.n. following k.a. lesioning was measured in the whole nucleus as well as in the inferior half of the reticulata.
TABLE 1 Decreased C A T activity in the s.n. pars reticulata 5 days after the unilateral, intranigral injection o f k.a. Each value is the m e a n +-S.E.M. of the figures obtained f r o m 36 rats. Intranigral t r e a t m e n t
Saline K.a. 3.5 nM/s.n., 5 days
C A T activity nM/g/h Whole s.n.
Pars reticulata
260 +- 12 262 +- 13
146 + 5 80 -+ 6 1
I p < 0.001 with respect to the control side.
The high concentration of the enzyme present in the pars compacta (Kobayashi et al., 1975) is likely to dilute and, hence, to mask a decrease of the activity localized in the pars reticulata. As expected, basal CAT activity (table 1) in the whole s.n. was about twice that of the reticulata and did not change after k.a. treatment (McGeer and McGeer, 1975; Di Chiara et al., 1977). On the other hand, CAT concentration was decreased by more than 40% after k.a. when the enzyme activity was measured in the inferior half of the reticulata.
3.2. 3H-QNB binding Muscarinic receptors in the whole s.n. were assayed in the presence of different 3H-QNB concentrations, varying from 0.125 to 1.0 nM. Since each s.n. weighed about 2 rag, in order to obtain a saturation curve we had to pool together the s.n. of 60 rats injected with k.a. in one side and with saline in the other. Fig. 1 shows the Scatchard analysis of the data obtained. Each point represents the mean of 2 consecutive experiments. The s.n. injected with k.a. showed a significant decrease of 3H-QNB binding capacity, with a 17% decline of the receptor density and a lower value of the apparent aff'mity for the ligand (control kd: 33 nM; k.a. kd: 43 nM).
184
G.M. DE M O N T I S E T AL.
Q8
o,7
o. (16 0.5 rn
Control 0 . 4 ~ 3 nM
oOt o.~
0:1
~ 0:2
0:3
0:4
0:5
0:6
0:7
0'.8
0:9
1:0
1:1
1:2
1:3
Bound/Free:pM/nM Fig. 1. S c a t c h a r d p l o t o f t h e specific 3H-QNB binding in k.a.-lesioned a n d c o n t r o l nigras. R e c e p t o r binding in c o n t r o l (o) a n d l e s i o n e d (o) s.n. was d e t e r m i n e d w i t h c o n c e n t r a t i o n s o f 3H-QNB v a r y i n g b e t w e e n 0 . 1 2 5 a n d 1 nM. T h e results are r e p r e s e n t a t i v e o f t w o s e p a r a t e e x p e r i m e n t s . Regression analysis: cont r o l s k d : 0.33 nM, r: 1, y i n t e r c e p t : 0 . 4 2 ; k.a.-lesioned k d : 0 . 4 3 nM, r: 1, y i n t e r c e p t : 0.35.
3.3. Unilateral, intranigral treatments Table 2 shows the effect of the unilateral, intranigral injection of carbachol in both controls and rats pretreated with k.a. (3.5 nM/s.n., 5 days) in the same s.n. Carbachol
produced a spontaneous, ipsilateral circling of dose-dependent duration in the controls and inverted the turning direction of the k.a.pretreated animals. These effects of intranigral carbachol were abolished by systemic scopolamine (6.4 mM/kg i.p.). Moreover, the intranigral injection of different concentrations of scopolamine together with carbachol, through the same cannula, revealed a reciprocal antagonism between the two compounds. Intranigral scopolamine given alone, at the dose of 16 nM/s.n., produced contralateral turning. A group of 16 animals injected unilaterally with k.a. and selected for their spontaneous, contralateral turning, received apomorphine (1.87 mM/kg s.c.) on day 5 after surgery. As expected (Olianas et al., 1978a), 5--10 min after treatment all animals presented a continuous, strict angle circling, ipsilateral to the injected side. This effect lasted from 70 to 90 min. 15 min after apomorphine administration, half of the animals were injected with scopolamine (6.4mM/kg i.p.) and, 2 days later, the experiment was repeated and scopolamine injected into the remaining half. After scopolamine administration the intensity of the apomorphine-induced ipsilateral turning decreased rapidly in 9 animals (from 18 +- 3 to
TABLE 2 I n c i d e n c e a n d i n t e n s i t y o f t h e ipsilateral t u r n i n g p r o d u c e d b y unilateral, intranigral c a r b a c h o l in n o r m a l rats a n d in rats p r e t r e a t e d w i t h k.a. in t h e same s.n. Unilateral, intranigral carbachol (nM/s.n.) (No. o f animals)
Ipsilateral intranigral k.a. (3.5 nM/s.n.)
Scopolamine 6.4. m M / k g i.p.
10 (5) 17.5 ( 1 8 ) 25 (7) 17.5 (8) None (11) 17.5 (6) 17.5 (5)
None None None None Yes Yes Yes
None None None Yes None None Yes
Direction of turning
Ipsilateral Ipsilateral Ipsilateral None Contralateral Ipsilateral Contralateral
Rats turning w i t h score 1
2
3
4
1 1 --
1 3 1
9 3
3 3
1 ---
3 2 3
5 2 2
2 1 --
Turning 1 duration (rain)
M a x i m a l circling (turns/min)
27 + 12 58 +_ 9 87 +_ 16 -Continuous 44 + 11 Continuous
-9 14 -8 6 8
+ 4 _+ 3 + 3 2 +_ 4
I E a c h value is t h e m e a n 4-- S.D. o f t h e n u m b e r o f e x p e r i m e n t s r e p o r t e d in p a r e n t h e s e s . O b s e r v a t i o n t i m e lasted 2 h f r o m t h e e n d of surgery. S c o p o l a m i n e was i n j e c t e d 10 rain a f t e r c a r b a c h o l . 2 Only o n e a n i m a l s h o w e d s p o n t a n e o u s (score 4 ) ipsilateral t u r n i n g .
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185
TABLE 3 I n c i d e n c e a n d i n t e n s i t y o f catalepsy a f t e r bilateral, intranigral i n j e c t i o n o f c a r b a c h o l a n d a n t a g o n i s m o f carbachol- o r p i c r o t o x i n - i n d u c e d catalepsy b y s c o p o l a m i n e or b y d i f f e r e n t d o s e s o f a p o m o r p h i n e . Bilateral, intranigral t r e a t m e n t (nM/s.n.) (No. o f animals)
Systemic treatment (mM/kg)
Rats s h o w i n g Stereotypies 1
Saline Saline Saline Carbachol 17.5 1 Carbachol 17.5 1 Carbachol 17.5 1 Carbachol 17.5 1 P i c r o t o x i n 0.4 1 P i c r o t o x i n 0.4 I P i c r o t o x i n 0.4 1
(3) (5) (5) (18) (6) (7) (6) (10) (5) (6)
None Apomorhine Apomorphine None Apomorphine Apomorphine Scopolamine Apomorphine Apomorphine Scopolamine
0.37 0.93 . 1.87 3.74 6.40 0.93 1.87 6.40
s.c. s.c. . s.c. s.c. i.p. s.c. s.c. i.p.
2
. --. -1 -----
3
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.
4 .
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4
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2
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2 1
-3 .
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. -.
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3
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1 .
1 . .
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.
5
.
. . 5 . 2 1 .
Catalepsy
. . 1 . .
16 6
5
1 Only animals s h o w i n g score 3 catalepsy were used. A p o m o r p h i n e or s c o p o l a m i n e was i n j e c t e d 10 m i n a f t e r surgery t o t h e animals t r e a t e d w i t h saline or carbachol and 20 rain a f t e r surgery t o t h o s e t r e a t e d w i t h picrotoxin.
8 + 3 turns/min) while in the remaining 7 the basal contralateral circling reappeared within 10 min. All rats treated unilaterally in the s.n. received the same volume of the solvent in the opposite s.n. In fact, the intranigral injection of saline may produce, per se, postural effects which have been characterized as (i) a decreased response to the excitatory effect of apomorphine and (ii) an increased sensitivity to striatal DA-receptor blockade. After unilateral, intranigral saline, apomorphine administration (0.93nM/kg s.c.) produced spontaneous turning, ipsflateral to the injected side, which lasted about one hour. When saline was injected bilaterally, apomorphine was sedative at the 0.37 nM/kg dose and 0.93 nM/kg was necessary to produce stereotypies (table 3). In addition, animals pretreated with a subcataleptic dose of haloperidol (0.066 nM/kg s.c. or 1.35 nM/c.n., bilaterally) presented a score 3 catalepsy lasting up to more than 60 min following bilateral, intranigral saline.
3.4. Bilateral, intranigral treatments Table 3 shows the effect of the bilateral, intranigral injection of saline or carbachol on posture. Saline had no effect per se, while carbachol produced catalepsy in all the animals in which the cannula tip was located in the s.n. 6 out of 18 animals, instead of having the typical muscular rigidity, showed a tendency to stand for several minutes on their hind-limbs and to maintain a forced up-sidedown position. The cataleptic syndrome reached the highest degree of intensity 5--7 min after surgery, decreased gradually 30 min later and disappeared completely within 1 h. Table 3 also shows the effects of scopolamine (6.4 mM/kg i.p.) and of different doses of apomorphine in rats previously injected intranigrally with picrotoxin or carbachol. In this experiment only animals showing a score 3 catalepsy were used. Apomorphine, at the dose of 0.37 mM/kg s.c., produced score 2 stereotypies in controls and ejaculation accompanied with marked
186
G.M. DE MONTIS ET AL.
TABLE 4 Effect of intranigral scopolamine on posture and on catalepsy produced by intranigral carbachol. Observation time lasted a total of 90 rain after surgery. Scopolamine was injected in the s.n. together with carbachol. Bilateral, intranigral treatments (nM/s.n. -- No. of animals)
Rats showing Stereotypies
Carbachol
None None 17.5 17.5 17.5
(4) (5) (6) (6) (7)
Catalepsy
Scopolamine
6.4 64.0 1.6 3.2 6.4
1
2
3
2 1 . . 1
1 2
. 2
. .
4 . .
sedation and sleep in the saline-injected rats. From 0.93 to 3 . 7 4 m M / k g apomorphine produced stereotyped movements of the same intensity in the controls and in the saline-injected animals. The catalepsy which followed intranigral carbachol was more resistant to apomorphine than was that produced by picrotoxin. In fact, apomorphine (0.93 mM/kg) given after intranigral picrotoxin, antagonized the rigidity in 4 out or 10 rats and 1.87 mM produced stereotypies like those observed in the controls. On the other hand, in rats treated with carbachol a dose of 3.74 mM/kg of apomorphine was necessary to antagonize the catalepsy and to transform it into a score 2 degree of stereotyped movements. Scopolamine (6.4 mM/kg i.p.) rapidly antagonized the catalepsy produced by both of the compounds. Moreover, scopolamine given soon before surgery t o 6 animals treated with picrotoxin and to 5 injected with carbachol prevented the catalepsy from occurring; in all these animals the cannula tip was found positioned in the s.n. Finally, the bilateral, intranigral injection of scopolamine together with carbachol, completely antagonized the catalepsy when the 2 compounds were administered in a molar ratio of about 1 to 3, respectively (table 4).
.
. .
1
. .
.
. . 2
5 . .
.
. . 2 3
1 .
3
. .
.
. .
2
.
4 1 .
3.5. Arecoline and haloperidol catalepsy Systemic treatment whith cholinomimetics or neuroleptics are well known to produce parkinsonian symptoms such as muscular rigidity, tremors and akinesia. The localization in the s.n. of a neuronal system which, under cholinergic stimulation produces catalepsy and which, following GABA-receptot blockade, appears to be under tonic cholinergic control, therefore prompted us to study the role of this system in the syndrome produced by haloperidol or arecoline. Haloperidol was given in a dose of 5.32 mM/kg i.p., which produces rigid catalepsy in 100% of our animals. Arecoline (32.2 mM/kg s.n.) given to rats pretreated with n-butyl-scopolamine bromide (2 mg/kg i.p., 10 min before), produced intense tremors, muscular rigidity and catalepsy which began 2--4 min after treatment and lasted about 20 min. Table 5 reports the effect of different intranigral treatments on these syndromes. The bilateral, intranigral injection of saline did not modify the duration and the intensity of the two syndromes. It has been verified that intranigral muscimol produced stereotyped movements of dose-related intensity and, given to animals pretreated with haloperidol,
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187
TABLE 5 E f f e c t s p r o d u c e d b y t h e bilateral, intranigral i n j e c t i o n o f m u s c i m o l or s c o p o l a m i n e o n t h e catalepsy p r o d u c e d b y h a l o p e r i d o l a n d o n t h e p a r k i n s o n i a n s y n d r o m e p r o d u c e d b y arecoline. A r e c o l i n e was i n j e c t e d s.c. 10 m i n after t h e i.p. injection o f 2 m g / k g o f n - b u t y l - s c o p o l a m i n e b r o m i d e . A r e c o l i n e was a d m i n i s t e r e d 15 m i n a f t e r surgery. Haloperidol was given 1 h b e f o r e surgery: o n l y animals s h o w i n g s c o r e 3 catalepsy w e r e used. Bilateral, intranigral t r e a t m e n t (nM/s.n.) ( No. o f animals )
Systemic treatment (mM/kg)
Rats s h o w i n g
1 M u s c i m o l 0.044 M u s c i m o l 0.044 Muscimol 0.44 S c o p o l a m i n e 16 S c o p o l a m i n e 64 S c o p o l a m i n e 16 S c o p o l a m i n e 64 S c o p o l a m i n e 64 Saline Saline
(12) (6) (7) (5) (7) (5) (8) (4) (5) (6)
Haloperidol Arecoline Arecoline Haloperidol Haloperidol Arecoline Arecoline Arecoline Haloperidol Arecoline
5.32 32.20 32.20 5.32 5.32 32.20 32.20 64.40 5.32 32.20
-. . . -. 2 . . .
completely antagonized the catalepsy and produced stereotypies in a dose as low as 0.044 nM/s.n. However, only the 0.44 nM/s.n. dose of muscimol, which per se produced serious self-mutilation phenomena, was able to prevent the occurrence of the tremors and the rigidity produced by arecoline.
TABLE 6 Failure o f s c o p o l a m i n e to m o d i f y t h e catalepsy prod u c e d b y intranigral c a r b a c h o l or b y arecoline or h a l o p e r i d o l a d m i n i s t r a t i o n , w h e n i n j e c t e d bilaterally in t h e c.n. A r e c o l i n e was i n j e c t e d s.c. 10 rain a f t e r n-butyls c o p o l a m i n e b r o m i d e , 15 m i n u t e s a f t e r surgery. Bilateral, intrastriatal t r e a t m e n t ( n M / c . n . ) (No. o f animals )
Catalepsy produced by
S c o p o l a m i n e 64 (6)
C a r b a c h o l 17.5
S c o p o l a m i n e 64 (7)
2
3
4
6
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2 .
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2
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7 .
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1
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2
1 . . .
5
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2 .
4
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. . 2
Tremors 3
1
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. -2 -. 1 . ----
2
3
1 ---
5 ---
--
--
--2
4 -4
.
. .
Intranigral scopolamine, injected at the dose of 16 nM/s.n., prevented the tremors but not the catalepsy produced by arecoline. The 64 nM/s.n, dose of scopolamine produced per se low score stereotypies, completely antagonized the catalepsy produced by haloperidol and prevented the tremors and the catalepsy induced by 32 mM/kg of arecoline. However, the intensity of both tremors and catalepsy were not modified by intranigral scopolamine when arecoline was administered at the dose of 64 mM/kg s.c. Finally, table 6 shows that scopolamine, injected bilaterally in the c.n., failed to affect the syndromes produced by either haloperidol or arecoline even at the dose of 64 mM/c.n.
Rats s h o w ing catalepsy
4. Discussion 1
2
3
--
1
5
nM/s.n. S c o p o l a m i n e 64 (6)
Catalepsy
Stereotypies
H a l o p e r i d o l 5.32 m M / k g i.p. A r e c o l i n e 32.20 m M / k g s.c.
6 7
The intranigral injection of k.a. produced a lesion which resulted in a significant decrease of the binding capacity for 3H-QNB of the whole s.n. and in a lowering of the CAT activity confined to the pars reticulata. In addition, the acute administration of carba-
188
chol within the s.n. produced ipsilateral turning on unilateral injection and rigid catalepsy on bilateral injection. These s y m p t o m s were dose-related and rather resistant to apomorphine administration. Opposite effects were observed after intranigral scopolamine. Moreover, the simultaneous injection of carbachol and scopolamine at different dose ratios, through the same cannula, revealed the antagonism between the t w o compounds. Finally, the bilateral, intranigral injection of scopolamine antagonized the catalepsy produced by haloperidol as well as the arecolineinduced parkinsonian syndrome. The latter effect could only be observed after a fairly high dose of scopolamine and could have been due either to diffusion of the drug to other brain areas or to an unspecific, stabilizing effect on the PRCP neurons. However, the failure of scopolamine injected in the c.n. to antagonize drug-induced catalepsy argues against the first possibility, while the tremors and catalepsy which m a y be produced b y using a higher dose of arecoline suggest that scopolamine blocked nigral receptors even at this high dose. The finding that systemic scopolamine antagonized the catalepsy produced b y intranigral picrotoxin indicates that this s y m p t o m was: sustained b y a cholinergic mechanism otherwise masked b y the gabaergic tonus and demonstrates that posture is controlled in the s.n. b y a balance between muscarinic, excitatory activity and GABAergic, inhibitory activity. These data strongly suggest that the neurons of the pars reticulata sensitive to iontophoretically applied Ach are the same as those which are destroyed b y k.a. and inhibited b y GABA. In addition, the decreased CAT activity in the pars reticulata demonstrates that k.a.-sensitive neurons in this area are, at least partially, cholinergic in nature. Although ascending, cholinergic projections to the s.n. have n o t been excluded, we have been led to. postulate that k.a. destroys the perikarya contalning the mnscarinic receptors as well as cholinergic interneurons which impinge on
G.M. DE M O N T I S E T AL.
them and that the strio-nigral GABA terminals are inhibitory on this cholinergic interneuron. In fact, the inhibition b y GABA of such an interneuron would prevent the release of Ach and the consequent catalepsy. This mechanism is sufficient to explain the. symptoms produced by intranigral muscimol or by k.a. lesions as well as the effect of systemic apomorphine, which is known to increase GABA turnover in the s.n. (Perez De La Mora et al., 1977). Conversely, the blockade of nigral GABA receptors as obtained b y the local injection of picrotoxin or bicucullin, will allow more Ach to be released and, hence, catalepsy to be produced (Scheel-Kri]ger et al., 1977). On the other hand, this neuronal model cannot explain the finding that a high dose of apomorphine antagonized the carbacholinduced catalepsy and that 0.44 nM/s.n, of muscimol prevented the arecoline syndrome from occurring. It is, in fact, difficult to accept that endogenous GABA, no matter which concentration it reaches in the s.n. after 3.74 mM/kg of apomorphine, may block the muscarinic receptors. Maximal GABA turnover stimulation or a high concentration of muscimol seems, therefore, to inhibit directly the same neurons which are stimulated by Ach and to antagonize muscarinic activity at this level. These data led us to the hypothesis that GABA exerts a double effect on the PRCP neurons, according to the scheme of fig. 2: the terminals o f the strionigral GABA neurons impinge and are inhibitory on both the cholinergic interneuron located between them and the PRCP neuron and on the PRCP neuron itself. In such a case, GABA can easily control endogenous cholinergic activity, while its capacity to inhibit the PRCP neurons is markedly decreased if the muscarinic agonist is administered exogenously. According to this neuronal model, the term PRCP neurons is restricted to the neurons projecting outside of the s.n. A variable intensity of the basal cholinergic activity m a y explain the frequent failure of picrotoxin to produce catalepsy.
N I G R A L GABAERGIC--CHOLINERGIC BALANCE
~
C
Acetylcholine /('~~CAUD~ATE ate ~ , ~
NUCLEUS
Dopa~ne G/BA
_
_
-..................L
~
F?COMPACTA P.RETICULATA Fig. 2. This scheme for a neuronal model summarizes the recent findings of several laboratories. The stimulation of striatal DA receptors inhibits a cholinergic interneuron (B) which, in turn, inhibits a GABA neuron (D) projecting to the s.n. (Mao et al., 1977). D is excited by glutamate (Mao et al., 1977), so that striatal DA receptor stimulation results in an increase of nigral GABA turnover (Perez De La Mora et al., 1977). In fact, a lesion of the strio-nigral pathways prevents the effects of apomorphine administration from occurring (Marshall and Ungerstedt, 1977). On the other thand, either the destruction or the inhibition b y GABA of a neuronal system contained in the pars reticulata (F), mimics striatal DA receptor stimulation (Olianas et al., 1978a,b). Finally, the present data led to the hypothesis that the GABA terminals in the pars reticulata control the function of the PRCP neurons by a dual mechanism: (1) they impinge directly on and inhibit the PRCP neurons (F) and (ii) they inhibit a cholinergic interneuron (E) which, in turn, is excitatory on the PRCP neurons. The direct interaction of GABA terminals with the DA cell bodies is, in our opinion, presently uncertain and is thus not shown in the figure.
We have no explanation, at the moment, for the decreased affinity of the residual muscarinic receptors for the ligand after k.a.
189
lesioning. A possible interpretation is that a new population of receptors with altered characteristics spreads on the remaining PRCP perikarya. The finding that the intranigral injection of carbachol very well antagonized the k.a.-induced contralateral turning suggests that the dose of k.a. used only partially destroys the perikarya of the neurons present in the pars reticulata. It is, indeed, more difficult to explain the decreased response to apomorphine and the increased sensitivity to haloperidol produced by intranigral saline. The lesion per se seemed to be responsible for these effects; in fact (i) apomorphine, at adose which inverts the direction of the contralateral turning produced by intranigral muscimol (Olianas et al., 1978b), produced only symmetric stereotypies when the control s.n. was injected with saline and (ii) scopolamine had no effect on the apomorphine-induced ipsilateral turning following intranigral saline (our unpublished observations). On the other hand, a different mechanism, cholinergic in nature, underlies the ipsilateral turning produced by apomorphine in rats unilaterally lesioned with k.a., as was seen from the scopolamine antagonism on this effect. We have no explanation, at the moment, for this finding. In conclusion, the present data indicate that posture is controlled, in the s.n. pars reticulata, by a balance between Ach and GABA activity. This model can be seen as an alternative to the DA-Ach balance proposed by several authors (Fog et al., 1966; Pedersen, 1967; Arnfred and Randrup, 1968; ScheelKri]ger, 1970). In our opinion, a thorough characterization of the nigral Ach and GABA receptors may lead to results of practical relevance for the management of extrapyramidal diseases. References Aghajanian, G.K. and S.B. Bunney, 1973, Central dopaminergic neurons: Neurophysiological identification and responses to drugs, in: Frontiers in Catecholamine Research, III International
190 Catecholamine Symposium, eds. E. Usdin and S. Snyder (Pergamon Press, Oxford) p. 643. Arnfred, T. and A. Randrup, 1968, Cholinergic mechanism in brain inhibiting amphetamineinduced stereotyped behavior, Acta Pharmacol. Toxicol. 26, 384. Di Chiara, G., M. Olianas, M. Del Fiacco, P.F. Spano and A. Tagliamonte, 1977, Intranigral kainic acid is evidence that nigral non-dopaminergic neurons control posture, Nature 268, 743. Dray, A., N.R. Oakley and N.A. Simmonds, 1975, Rotational behavior following inhibition of GABA metabolism unilaterally in the rat substantia nigra, J. Pharm. Pharmacol. 27,627. Fog, R., A. Randrup and H. Pakkenberg, 1966, Amines in the corpus striatum associated with the effect of both amphetamine and antipsychotic drugs, Excerpta Medica Intern. Congress Series No. 150, Proc. IV World Congress of Psychiatry, p. 2580. Kobayashi, R.M., M. Brownstein, J.M. Saavedra and M. Palkovits, 1975, Choline acetyltransferase content in discrete regions of the rat brain stem, J. Neurochem. 24, 637. Lowry, O.H., N.J. Rosenbrogh, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin Phenol reagent, J. Biol. Chem. 193, 265. Mao, C.C., E. Marco, A. Revuelta, L. Bertilsson and E. Costa, 1977, The turnover rate of -/-aminobutyric acid in the nuclei of the telencephalon: implications in the pharmacology of antipsychotic and of minor tranquilizer, Biol. Psychiatry 13 (3), 359. Marshall, J.F. and U. Ungerstedt, 1977, Supersensitivity to apomorphine following destruction of the sscending doparnine neurons: quantification using the rotational model, European J. Pharmacol. 41, 361. McGeer, E.G., H.C. Fibiger, P.L. McGeer and S. Brooke, 1973, Temporal changes in amine synthesizing enzymes of rat extrapyramidal structure after hemitransections or 6-hydroxydopamine administration, Brain Res. 82, 289. McGeer, E.G. and P.L. McGeer, 1975, Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kalnic acid, Nature 263, 517.
G.M. DE MONTIS ET AL. Olianas, M.C., G.M. De Montis, A. Concu, A. Tagliamonte and G. Di Chiara, 1978a, 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, 223. Olianas, M.C., G.M. De Montis, G. Mulas and A. Tagliamonte, 1978b, The striatal'dopaminergic function is mediate by the inhbition of a nigral, non-dopaminergic neuronal system via a strionigral gabaergic pathway, European J. Pharmacol. 49,233. Olivier, A., A. Parent, H. Simard and L.J. Poirier, 1970, Cholinesterasic striopallidal and striatonigral efferents in the cat and monkey, Brain Res. 18, 273. Palkovits, M. and D.M. Jacobowitz, 1974, Topographic Atlas of catecholamine and acetylcholinesterasecontaining neurons in the rats brain. II. Hindbrain (mesencephalon, rombencephalon), J. Comp. Neurol. 157, 29. Pedersen, V., 1967, Potentiation of apomorphine effect (compulsive gnawing behavior) in mice, Acta Pharm. Toxicol. 25, Suppl. 4, 63. Pellegrino, L.J. and A.J. Cushman, 1971, A Stereotaxic Atlas of the Rat Brain (Meredith, New York). Perez De La Mora, L., K. Fuxe, T. HSkfelt and A. Ljungdal, 1977, Evidence for a nerve impulsedependent GABA accumulation in the substantia nigra after treatment with gamma-glutamylhydrazine, Neurosci. Lett. 5, (1--2)/75. Scheel-Kriiger, J., 1970, Central effects of anticholinergic drugs measured by the apomorphine gnawing test in mice, Acta Pharmacol. Toxicol. 28,1. Scheel-Kriiger, J., J. Arnt and G. Magelund, 1977, Behavioral stimulation induced by muscimol and other GABA agonists injected into the substantia nigra, Neurosci. Lett. 4,351. Schwartz, R. and J.T. Coyle, 1977, Neurochemical sequelae of kaninate injections in corpus striatum and substantia nigra of the rat, Life Sci. 20, 431. Snedecor, J.W., 1969, Statistical Methods, 5th edn. (Iowa S~:: ~'~ College Press, Ames, Iowa). Yamamura, H.I. and S.H. Snyder, 1974, Muscarinic cholinergic binding in rat brain, Proc. Nat. Acad. Sci. 71, 1725.
181
EVIDENCE THAT A NIGRAL GABAERGIC--CHOLINERGIC BALANCE CONTROLS POSTURE G R A Z I E L L A M. DE MONTIS, MARIA C. OLIANAS, GINO SERRA, ALESSANDRO TAGLIAMONTE * and JORGEN SCHEEL-KRUGER
2nd Chair of Pharmacology, University of Cagliari, Italy, and Psychopharmacological Research Laboratory, Dept. E, St. Hans Mental Hospital, DK-4000 Roskilde, Denmark Received 21 March 1978, revised MS received 11 August 1978, accepted 20 September 1978
G.M. DE MONTIS, M.C. OLIANAS, G. SERRA, A. TAGLIAMONTE and J. SCHEEL-KRt)GER, Evidence that a nigral GABAergic--cholinergic balance controls posture, European J. Pharmacol. 53 (1979) 181--190. The intranigral injection of kainic acid (k.a.) (3.5 nM/s.n.) produced a lesion which resulted in a decreased muscarinic receptor binding capacity and in a decreased choline acetyl transferase (CAT) activity confined to the pars reticulata. The unilateral, intranigral injection of carbachol in the substantia nigra (s.n.) produced turning, ipsilateml to the injected side, of dose-related intensity, which was antagonized by scopolamine given either i.p. or intranigrally together with carbachol. The bilateral, intranigral injection of carbachol produced rigid catalepsy, highly resistant to apomorphine administration and antagonized by scopolamine. On the other hand, the catalepsy produced by intranigral picrotoxin was much more sensitive to apomorphine and was disrupted by systemic scopolamine administration. Intranigral scopolamine per se produced either contralateral turning or stereotyped movements consistently, when injected unilaterally or bilaterally, respectively. In addition, scopolamine injected bilaterally in the s.n. but not in the caudate nucleus (c.n.), at the concentration of 64 nM side, was able to antagonize the haloperidol-induced catalepsy and to prevent the tremors and the muscular rigidity produced by arecoline. This effect of scopolamine was surmountable with a higher dose of arecoline. Finally, intranigral muscimol (0.44 nM/s.n.) prevented the occurrence of the parkinsonian syndrome produced by systemic arecoline. It is concluded that the muscarinic receptors present in the s.n. pars reticulata play a role in the control of posture opposite to that of the nigral GABA receptors. Choline acetyltransferase Muscarinic receptors
Acetylcholine Stereotypies
Turning behavior Catalepsy
1. Introduction
The injection of kainic acid (k.a.) within the substantia nigra (s.n.) produces a rather selective degeneration of the neurons of the pars reticulata (Di Chiara et al., 1977; Olianas et al., 1978a). Such a lesion results in chronic turning, contralateral to the injected side, in the animals treated unilaterally and it pro*Please send reprint requests to: M.C. Olianas, Psychopharmacological Research Laboratory, Department E, Sct. Hans Hospital, DK-4000 Roskilde, Denmark.
Dopamine
GABA
duces chronic stereotypies in rats injected bilaterally (Di Chiara et al., 1977; Schwartz and Coyle, 1977; Olianas et al., 1978a). These symptoms are not antagonized by haloperidol administration and are independent of the integrity of dopaminergic nigro-neostriatal neurons (Di Chiara et al., 1977; Olianas et al., 1978a). An analogous syndrome is observed after acute injection of GABA or other gabaergic compounds into the s.n. (ScheelKr/iger et al., 1977; Dray et al., 1975; Olianas et al., 1978b). These findings demonstrate that in t h e s.n. pars reticulata there is present a neuronal
182
system which controls posture in a manner opposite to that of the nigro-striatal dopaminergic neurons (Pars Reticulata Posture Controlling neurons: PRCP neurons). Since lesions of the strio-nigral pathways abolish the effect of apomorphine treatment (Marshall and Ungerstedt, 1977), we proposed that the inhibition of these PRCP neurons via the strionigral gabaergic system mediates the striatal dopaminergic responses (Olianas et al., 1978a, b). The increase of GABA turnover in the s.n. which is produced b y apomorphine (Perez De La Mora et al., 1977) strongly supports this hypothesis. On the other hand, the unilateral, intranigral administration of picrotoxin or bicucullin (Scheel-Kriiger et al., 1977; Olianas et al., 1978b), t w o post-synaptic GABA-receptor blockers, results in turning ipsilateral to the injected side. This fact suggests that, in the absence of GABA inhibition, the PRCP neurons are under the effect of a tonic excitatory activity. The finding that neurons of the pars reticulata were markedly excited b y iontophoretically applied acetylcholine (Ach) and that this effect was a specific one, since scopolamine blocked the Ach-induced excitation b u t had no effect on glutamine excitation in the same cells, prompted us to study muscarinic agonists as the possible physiological stimulators of PRCP neurons. Our interest in Ach was also sustained by the fact that both biochemical (McGeer et al., 1973) and muscarinic binding studies (Kuhar, unpublished data) have shown that the s.n. contained Ach and Ach receptors, although the receptor density is lower than in other areas. In addition, AchE staining techniques suggest the presence of b o t h cholinergic terminals and cells bodies in the s.n. (Olivier et al., 1970; Palkovits and Jacobowitz, 1974). The present report shows that the intranigral injection of carbachol produced postural symptoms opposite to those observed after muscimol and that k.a. lesioning of the s.n. resulted in a decreased binding capacity of the local muscarinic receptors.
G.M. DE MONTIS ET AL.
2. Materials and methods
2.1. Animals Experiments were carried out on male albino Sprague-Dawley rats weighing a b o u t 300 g. Animals were housed 10 to a cage (45 X 80 cm), had free access to food and water and were maintained under controlled experimental conditions, with a 12 h light-dark cycle.
2.2. Surgical procedures Animals were anaesthetized with ethyl ether and placed on a Stoelting-Stellar stereotaxic apparatus. Drugs were injected in the s.n. using stereotaxic coordinates A: --4, L: 2, V: 7.8, according to the Atlas of Pellegrino and Cushman (1971). The injection volume was 0.3 pl of saline, injected at a rate of 1 pl/18 min, through a 0.25 mm stainless steel cannula. Coordinates for the caudate nucleus (c.n.) were A: +2, L: 3, V: 5.5, with an injection volume of I pl given in 6 min. At the end of the acute experiments, animals were killed, the brain rapidly removed, fixed in 10% formalin for 2 h and then stored in 0.1 M phosphate buffer (pH 7.4) containing 5% sucrose, until the mesencephalon was cut into serial 50 pm slices with a cryostat, to determine the position of the cannula tip. Only the results obtained in rats correctly injected are reported.
2.3. Behavior Circling behavior and catalepsy were scored as already described (Olianas et al., 1978a,b). Stereotyped behavior was evaluated, after placing the rats in Perspex cages with a wirenet b o t t o m , as follows: 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; 5, phenomena of self-mutilation. Tremors were evaluated as: 1, a fit tremors in response to an external stimulus; 2, spontaneous fits of tremors or continuous tremors during handling; 3, continuous, spontaneous tremors.
183
N I G R A L GABAERGIC--CHOLINERGIC B A L A N C E
2.4. Biochemistry 3H-QNB binding was studied in whole s.n. homogenates according to the method of Yamamura et al. (1974). The tissue was homogenized in 10 volumes of ice-cold 0.32 M sucrose. The whole homogenate was centrifuged for 10 min at 1000 ×g, the pellet discarded and the supernatant was re-homogenized with a Brinkman-Polytron (setting No. 5, for 60 sec). Aliquots of this preparation were incubated at 25°C, with 0.05 M sodium-potassium (Na--K) phosphate buffer, pH 7.4, containing 3H-QNB (s.a. 16 Ci/mM) in a total volume of 2 ml. After a 60 min incubation the reaction was stopped by adding 3 ml of ice-cold Na--K phosphate buffer. The content of the tubes was passed through a glass filter (Whatman GF/B) and the filters washed 3 times, under vacuum, with 3 ml of i c e , o l d buffer. Each determination was performed in triplicate together with triplicate samples containing scopolamine (1 #M), to determine non-specific 3H-QNB binding. The specific binding was obtaining by subtracting from the total binding that obtained in the presence of 1 pM scopolamine. The radioactivity was assayed by liquid scintillation spectrometry in a Packard Tri-Carb. Proteins were determined by the method of Lowry et al. (1951). Choline acetyltransferase (CAT) activity was measured in a pool of whole s.n. (5) as well as in a pool of reticulata (9), in triplicate, according to the method Kobayashi et ai. (1975). The experiment was repeated 3 times. Data were analyzed statistically with the two-tailed Student's t-test (Snedecor, 1969).
3. Results
3.1. CAT activity CAT activity in the s.n. following k.a. lesioning was measured in the whole nucleus as well as in the inferior half of the reticulata.
TABLE 1 Decreased C A T activity in the s.n. pars reticulata 5 days after the unilateral, intranigral injection o f k.a. Each value is the m e a n +-S.E.M. of the figures obtained f r o m 36 rats. Intranigral t r e a t m e n t
Saline K.a. 3.5 nM/s.n., 5 days
C A T activity nM/g/h Whole s.n.
Pars reticulata
260 +- 12 262 +- 13
146 + 5 80 -+ 6 1
I p < 0.001 with respect to the control side.
The high concentration of the enzyme present in the pars compacta (Kobayashi et al., 1975) is likely to dilute and, hence, to mask a decrease of the activity localized in the pars reticulata. As expected, basal CAT activity (table 1) in the whole s.n. was about twice that of the reticulata and did not change after k.a. treatment (McGeer and McGeer, 1975; Di Chiara et al., 1977). On the other hand, CAT concentration was decreased by more than 40% after k.a. when the enzyme activity was measured in the inferior half of the reticulata.
3.2. 3H-QNB binding Muscarinic receptors in the whole s.n. were assayed in the presence of different 3H-QNB concentrations, varying from 0.125 to 1.0 nM. Since each s.n. weighed about 2 rag, in order to obtain a saturation curve we had to pool together the s.n. of 60 rats injected with k.a. in one side and with saline in the other. Fig. 1 shows the Scatchard analysis of the data obtained. Each point represents the mean of 2 consecutive experiments. The s.n. injected with k.a. showed a significant decrease of 3H-QNB binding capacity, with a 17% decline of the receptor density and a lower value of the apparent aff'mity for the ligand (control kd: 33 nM; k.a. kd: 43 nM).
184
G.M. DE M O N T I S E T AL.
Q8
o,7
o. (16 0.5 rn
Control 0 . 4 ~ 3 nM
oOt o.~
0:1
~ 0:2
0:3
0:4
0:5
0:6
0:7
0'.8
0:9
1:0
1:1
1:2
1:3
Bound/Free:pM/nM Fig. 1. S c a t c h a r d p l o t o f t h e specific 3H-QNB binding in k.a.-lesioned a n d c o n t r o l nigras. R e c e p t o r binding in c o n t r o l (o) a n d l e s i o n e d (o) s.n. was d e t e r m i n e d w i t h c o n c e n t r a t i o n s o f 3H-QNB v a r y i n g b e t w e e n 0 . 1 2 5 a n d 1 nM. T h e results are r e p r e s e n t a t i v e o f t w o s e p a r a t e e x p e r i m e n t s . Regression analysis: cont r o l s k d : 0.33 nM, r: 1, y i n t e r c e p t : 0 . 4 2 ; k.a.-lesioned k d : 0 . 4 3 nM, r: 1, y i n t e r c e p t : 0.35.
3.3. Unilateral, intranigral treatments Table 2 shows the effect of the unilateral, intranigral injection of carbachol in both controls and rats pretreated with k.a. (3.5 nM/s.n., 5 days) in the same s.n. Carbachol
produced a spontaneous, ipsilateral circling of dose-dependent duration in the controls and inverted the turning direction of the k.a.pretreated animals. These effects of intranigral carbachol were abolished by systemic scopolamine (6.4 mM/kg i.p.). Moreover, the intranigral injection of different concentrations of scopolamine together with carbachol, through the same cannula, revealed a reciprocal antagonism between the two compounds. Intranigral scopolamine given alone, at the dose of 16 nM/s.n., produced contralateral turning. A group of 16 animals injected unilaterally with k.a. and selected for their spontaneous, contralateral turning, received apomorphine (1.87 mM/kg s.c.) on day 5 after surgery. As expected (Olianas et al., 1978a), 5--10 min after treatment all animals presented a continuous, strict angle circling, ipsilateral to the injected side. This effect lasted from 70 to 90 min. 15 min after apomorphine administration, half of the animals were injected with scopolamine (6.4mM/kg i.p.) and, 2 days later, the experiment was repeated and scopolamine injected into the remaining half. After scopolamine administration the intensity of the apomorphine-induced ipsilateral turning decreased rapidly in 9 animals (from 18 +- 3 to
TABLE 2 I n c i d e n c e a n d i n t e n s i t y o f t h e ipsilateral t u r n i n g p r o d u c e d b y unilateral, intranigral c a r b a c h o l in n o r m a l rats a n d in rats p r e t r e a t e d w i t h k.a. in t h e same s.n. Unilateral, intranigral carbachol (nM/s.n.) (No. o f animals)
Ipsilateral intranigral k.a. (3.5 nM/s.n.)
Scopolamine 6.4. m M / k g i.p.
10 (5) 17.5 ( 1 8 ) 25 (7) 17.5 (8) None (11) 17.5 (6) 17.5 (5)
None None None None Yes Yes Yes
None None None Yes None None Yes
Direction of turning
Ipsilateral Ipsilateral Ipsilateral None Contralateral Ipsilateral Contralateral
Rats turning w i t h score 1
2
3
4
1 1 --
1 3 1
9 3
3 3
1 ---
3 2 3
5 2 2
2 1 --
Turning 1 duration (rain)
M a x i m a l circling (turns/min)
27 + 12 58 +_ 9 87 +_ 16 -Continuous 44 + 11 Continuous
-9 14 -8 6 8
+ 4 _+ 3 + 3 2 +_ 4
I E a c h value is t h e m e a n 4-- S.D. o f t h e n u m b e r o f e x p e r i m e n t s r e p o r t e d in p a r e n t h e s e s . O b s e r v a t i o n t i m e lasted 2 h f r o m t h e e n d of surgery. S c o p o l a m i n e was i n j e c t e d 10 rain a f t e r c a r b a c h o l . 2 Only o n e a n i m a l s h o w e d s p o n t a n e o u s (score 4 ) ipsilateral t u r n i n g .
NIGRAL GABAERGIC--CHOLINERGIC BALANCE
185
TABLE 3 I n c i d e n c e a n d i n t e n s i t y o f catalepsy a f t e r bilateral, intranigral i n j e c t i o n o f c a r b a c h o l a n d a n t a g o n i s m o f carbachol- o r p i c r o t o x i n - i n d u c e d catalepsy b y s c o p o l a m i n e or b y d i f f e r e n t d o s e s o f a p o m o r p h i n e . Bilateral, intranigral t r e a t m e n t (nM/s.n.) (No. o f animals)
Systemic treatment (mM/kg)
Rats s h o w i n g Stereotypies 1
Saline Saline Saline Carbachol 17.5 1 Carbachol 17.5 1 Carbachol 17.5 1 Carbachol 17.5 1 P i c r o t o x i n 0.4 1 P i c r o t o x i n 0.4 I P i c r o t o x i n 0.4 1
(3) (5) (5) (18) (6) (7) (6) (10) (5) (6)
None Apomorhine Apomorphine None Apomorphine Apomorphine Scopolamine Apomorphine Apomorphine Scopolamine
0.37 0.93 . 1.87 3.74 6.40 0.93 1.87 6.40
s.c. s.c. . s.c. s.c. i.p. s.c. s.c. i.p.
2
. --. -1 -----
3
.
. . 1
.
4 .
.
.
4
.
.
2
. . .
.
.
.
. .
. 2
2 1
-3 .
.
.
.
. -. .
.
.
. -.
.
. .
3
. .
.
1 .
1 . .
.
. .
.
.
5
.
. . 5 . 2 1 .
Catalepsy
. . 1 . .
16 6
5
1 Only animals s h o w i n g score 3 catalepsy were used. A p o m o r p h i n e or s c o p o l a m i n e was i n j e c t e d 10 m i n a f t e r surgery t o t h e animals t r e a t e d w i t h saline or carbachol and 20 rain a f t e r surgery t o t h o s e t r e a t e d w i t h picrotoxin.
8 + 3 turns/min) while in the remaining 7 the basal contralateral circling reappeared within 10 min. All rats treated unilaterally in the s.n. received the same volume of the solvent in the opposite s.n. In fact, the intranigral injection of saline may produce, per se, postural effects which have been characterized as (i) a decreased response to the excitatory effect of apomorphine and (ii) an increased sensitivity to striatal DA-receptor blockade. After unilateral, intranigral saline, apomorphine administration (0.93nM/kg s.c.) produced spontaneous turning, ipsflateral to the injected side, which lasted about one hour. When saline was injected bilaterally, apomorphine was sedative at the 0.37 nM/kg dose and 0.93 nM/kg was necessary to produce stereotypies (table 3). In addition, animals pretreated with a subcataleptic dose of haloperidol (0.066 nM/kg s.c. or 1.35 nM/c.n., bilaterally) presented a score 3 catalepsy lasting up to more than 60 min following bilateral, intranigral saline.
3.4. Bilateral, intranigral treatments Table 3 shows the effect of the bilateral, intranigral injection of saline or carbachol on posture. Saline had no effect per se, while carbachol produced catalepsy in all the animals in which the cannula tip was located in the s.n. 6 out of 18 animals, instead of having the typical muscular rigidity, showed a tendency to stand for several minutes on their hind-limbs and to maintain a forced up-sidedown position. The cataleptic syndrome reached the highest degree of intensity 5--7 min after surgery, decreased gradually 30 min later and disappeared completely within 1 h. Table 3 also shows the effects of scopolamine (6.4 mM/kg i.p.) and of different doses of apomorphine in rats previously injected intranigrally with picrotoxin or carbachol. In this experiment only animals showing a score 3 catalepsy were used. Apomorphine, at the dose of 0.37 mM/kg s.c., produced score 2 stereotypies in controls and ejaculation accompanied with marked
186
G.M. DE MONTIS ET AL.
TABLE 4 Effect of intranigral scopolamine on posture and on catalepsy produced by intranigral carbachol. Observation time lasted a total of 90 rain after surgery. Scopolamine was injected in the s.n. together with carbachol. Bilateral, intranigral treatments (nM/s.n. -- No. of animals)
Rats showing Stereotypies
Carbachol
None None 17.5 17.5 17.5
(4) (5) (6) (6) (7)
Catalepsy
Scopolamine
6.4 64.0 1.6 3.2 6.4
1
2
3
2 1 . . 1
1 2
. 2
. .
4 . .
sedation and sleep in the saline-injected rats. From 0.93 to 3 . 7 4 m M / k g apomorphine produced stereotyped movements of the same intensity in the controls and in the saline-injected animals. The catalepsy which followed intranigral carbachol was more resistant to apomorphine than was that produced by picrotoxin. In fact, apomorphine (0.93 mM/kg) given after intranigral picrotoxin, antagonized the rigidity in 4 out or 10 rats and 1.87 mM produced stereotypies like those observed in the controls. On the other hand, in rats treated with carbachol a dose of 3.74 mM/kg of apomorphine was necessary to antagonize the catalepsy and to transform it into a score 2 degree of stereotyped movements. Scopolamine (6.4 mM/kg i.p.) rapidly antagonized the catalepsy produced by both of the compounds. Moreover, scopolamine given soon before surgery t o 6 animals treated with picrotoxin and to 5 injected with carbachol prevented the catalepsy from occurring; in all these animals the cannula tip was found positioned in the s.n. Finally, the bilateral, intranigral injection of scopolamine together with carbachol, completely antagonized the catalepsy when the 2 compounds were administered in a molar ratio of about 1 to 3, respectively (table 4).
.
. .
1
. .
.
. . 2
5 . .
.
. . 2 3
1 .
3
. .
.
. .
2
.
4 1 .
3.5. Arecoline and haloperidol catalepsy Systemic treatment whith cholinomimetics or neuroleptics are well known to produce parkinsonian symptoms such as muscular rigidity, tremors and akinesia. The localization in the s.n. of a neuronal system which, under cholinergic stimulation produces catalepsy and which, following GABA-receptot blockade, appears to be under tonic cholinergic control, therefore prompted us to study the role of this system in the syndrome produced by haloperidol or arecoline. Haloperidol was given in a dose of 5.32 mM/kg i.p., which produces rigid catalepsy in 100% of our animals. Arecoline (32.2 mM/kg s.n.) given to rats pretreated with n-butyl-scopolamine bromide (2 mg/kg i.p., 10 min before), produced intense tremors, muscular rigidity and catalepsy which began 2--4 min after treatment and lasted about 20 min. Table 5 reports the effect of different intranigral treatments on these syndromes. The bilateral, intranigral injection of saline did not modify the duration and the intensity of the two syndromes. It has been verified that intranigral muscimol produced stereotyped movements of dose-related intensity and, given to animals pretreated with haloperidol,
NIGRAL GABAERGIC--CHOLINERGIC BALANCE
187
TABLE 5 E f f e c t s p r o d u c e d b y t h e bilateral, intranigral i n j e c t i o n o f m u s c i m o l or s c o p o l a m i n e o n t h e catalepsy p r o d u c e d b y h a l o p e r i d o l a n d o n t h e p a r k i n s o n i a n s y n d r o m e p r o d u c e d b y arecoline. A r e c o l i n e was i n j e c t e d s.c. 10 m i n after t h e i.p. injection o f 2 m g / k g o f n - b u t y l - s c o p o l a m i n e b r o m i d e . A r e c o l i n e was a d m i n i s t e r e d 15 m i n a f t e r surgery. Haloperidol was given 1 h b e f o r e surgery: o n l y animals s h o w i n g s c o r e 3 catalepsy w e r e used. Bilateral, intranigral t r e a t m e n t (nM/s.n.) ( No. o f animals )
Systemic treatment (mM/kg)
Rats s h o w i n g
1 M u s c i m o l 0.044 M u s c i m o l 0.044 Muscimol 0.44 S c o p o l a m i n e 16 S c o p o l a m i n e 64 S c o p o l a m i n e 16 S c o p o l a m i n e 64 S c o p o l a m i n e 64 Saline Saline
(12) (6) (7) (5) (7) (5) (8) (4) (5) (6)
Haloperidol Arecoline Arecoline Haloperidol Haloperidol Arecoline Arecoline Arecoline Haloperidol Arecoline
5.32 32.20 32.20 5.32 5.32 32.20 32.20 64.40 5.32 32.20
-. . . -. 2 . . .
completely antagonized the catalepsy and produced stereotypies in a dose as low as 0.044 nM/s.n. However, only the 0.44 nM/s.n. dose of muscimol, which per se produced serious self-mutilation phenomena, was able to prevent the occurrence of the tremors and the rigidity produced by arecoline.
TABLE 6 Failure o f s c o p o l a m i n e to m o d i f y t h e catalepsy prod u c e d b y intranigral c a r b a c h o l or b y arecoline or h a l o p e r i d o l a d m i n i s t r a t i o n , w h e n i n j e c t e d bilaterally in t h e c.n. A r e c o l i n e was i n j e c t e d s.c. 10 rain a f t e r n-butyls c o p o l a m i n e b r o m i d e , 15 m i n u t e s a f t e r surgery. Bilateral, intrastriatal t r e a t m e n t ( n M / c . n . ) (No. o f animals )
Catalepsy produced by
S c o p o l a m i n e 64 (6)
C a r b a c h o l 17.5
S c o p o l a m i n e 64 (7)
2
3
4
6
.
.
.
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2 .
.
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.
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. .
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.
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.
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--
.
.
2
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7 .
.
.
1
.
.
2
1 . . .
5
.
2 .
4
.
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Tremors 3
1
. 5 -5 . 5 . 4 5 4
. -2 -. 1 . ----
2
3
1 ---
5 ---
--
--
--2
4 -4
.
. .
Intranigral scopolamine, injected at the dose of 16 nM/s.n., prevented the tremors but not the catalepsy produced by arecoline. The 64 nM/s.n, dose of scopolamine produced per se low score stereotypies, completely antagonized the catalepsy produced by haloperidol and prevented the tremors and the catalepsy induced by 32 mM/kg of arecoline. However, the intensity of both tremors and catalepsy were not modified by intranigral scopolamine when arecoline was administered at the dose of 64 mM/kg s.c. Finally, table 6 shows that scopolamine, injected bilaterally in the c.n., failed to affect the syndromes produced by either haloperidol or arecoline even at the dose of 64 mM/c.n.
Rats s h o w ing catalepsy
4. Discussion 1
2
3
--
1
5
nM/s.n. S c o p o l a m i n e 64 (6)
Catalepsy
Stereotypies
H a l o p e r i d o l 5.32 m M / k g i.p. A r e c o l i n e 32.20 m M / k g s.c.
6 7
The intranigral injection of k.a. produced a lesion which resulted in a significant decrease of the binding capacity for 3H-QNB of the whole s.n. and in a lowering of the CAT activity confined to the pars reticulata. In addition, the acute administration of carba-
188
chol within the s.n. produced ipsilateral turning on unilateral injection and rigid catalepsy on bilateral injection. These s y m p t o m s were dose-related and rather resistant to apomorphine administration. Opposite effects were observed after intranigral scopolamine. Moreover, the simultaneous injection of carbachol and scopolamine at different dose ratios, through the same cannula, revealed the antagonism between the t w o compounds. Finally, the bilateral, intranigral injection of scopolamine antagonized the catalepsy produced by haloperidol as well as the arecolineinduced parkinsonian syndrome. The latter effect could only be observed after a fairly high dose of scopolamine and could have been due either to diffusion of the drug to other brain areas or to an unspecific, stabilizing effect on the PRCP neurons. However, the failure of scopolamine injected in the c.n. to antagonize drug-induced catalepsy argues against the first possibility, while the tremors and catalepsy which m a y be produced b y using a higher dose of arecoline suggest that scopolamine blocked nigral receptors even at this high dose. The finding that systemic scopolamine antagonized the catalepsy produced b y intranigral picrotoxin indicates that this s y m p t o m was: sustained b y a cholinergic mechanism otherwise masked b y the gabaergic tonus and demonstrates that posture is controlled in the s.n. b y a balance between muscarinic, excitatory activity and GABAergic, inhibitory activity. These data strongly suggest that the neurons of the pars reticulata sensitive to iontophoretically applied Ach are the same as those which are destroyed b y k.a. and inhibited b y GABA. In addition, the decreased CAT activity in the pars reticulata demonstrates that k.a.-sensitive neurons in this area are, at least partially, cholinergic in nature. Although ascending, cholinergic projections to the s.n. have n o t been excluded, we have been led to. postulate that k.a. destroys the perikarya contalning the mnscarinic receptors as well as cholinergic interneurons which impinge on
G.M. DE M O N T I S E T AL.
them and that the strio-nigral GABA terminals are inhibitory on this cholinergic interneuron. In fact, the inhibition b y GABA of such an interneuron would prevent the release of Ach and the consequent catalepsy. This mechanism is sufficient to explain the. symptoms produced by intranigral muscimol or by k.a. lesions as well as the effect of systemic apomorphine, which is known to increase GABA turnover in the s.n. (Perez De La Mora et al., 1977). Conversely, the blockade of nigral GABA receptors as obtained b y the local injection of picrotoxin or bicucullin, will allow more Ach to be released and, hence, catalepsy to be produced (Scheel-Kri]ger et al., 1977). On the other hand, this neuronal model cannot explain the finding that a high dose of apomorphine antagonized the carbacholinduced catalepsy and that 0.44 nM/s.n, of muscimol prevented the arecoline syndrome from occurring. It is, in fact, difficult to accept that endogenous GABA, no matter which concentration it reaches in the s.n. after 3.74 mM/kg of apomorphine, may block the muscarinic receptors. Maximal GABA turnover stimulation or a high concentration of muscimol seems, therefore, to inhibit directly the same neurons which are stimulated by Ach and to antagonize muscarinic activity at this level. These data led us to the hypothesis that GABA exerts a double effect on the PRCP neurons, according to the scheme of fig. 2: the terminals o f the strionigral GABA neurons impinge and are inhibitory on both the cholinergic interneuron located between them and the PRCP neuron and on the PRCP neuron itself. In such a case, GABA can easily control endogenous cholinergic activity, while its capacity to inhibit the PRCP neurons is markedly decreased if the muscarinic agonist is administered exogenously. According to this neuronal model, the term PRCP neurons is restricted to the neurons projecting outside of the s.n. A variable intensity of the basal cholinergic activity m a y explain the frequent failure of picrotoxin to produce catalepsy.
N I G R A L GABAERGIC--CHOLINERGIC BALANCE
~
C
Acetylcholine /('~~CAUD~ATE ate ~ , ~
NUCLEUS
Dopa~ne G/BA
_
_
-..................L
~
F?COMPACTA P.RETICULATA Fig. 2. This scheme for a neuronal model summarizes the recent findings of several laboratories. The stimulation of striatal DA receptors inhibits a cholinergic interneuron (B) which, in turn, inhibits a GABA neuron (D) projecting to the s.n. (Mao et al., 1977). D is excited by glutamate (Mao et al., 1977), so that striatal DA receptor stimulation results in an increase of nigral GABA turnover (Perez De La Mora et al., 1977). In fact, a lesion of the strio-nigral pathways prevents the effects of apomorphine administration from occurring (Marshall and Ungerstedt, 1977). On the other thand, either the destruction or the inhibition b y GABA of a neuronal system contained in the pars reticulata (F), mimics striatal DA receptor stimulation (Olianas et al., 1978a,b). Finally, the present data led to the hypothesis that the GABA terminals in the pars reticulata control the function of the PRCP neurons by a dual mechanism: (1) they impinge directly on and inhibit the PRCP neurons (F) and (ii) they inhibit a cholinergic interneuron (E) which, in turn, is excitatory on the PRCP neurons. The direct interaction of GABA terminals with the DA cell bodies is, in our opinion, presently uncertain and is thus not shown in the figure.
We have no explanation, at the moment, for the decreased affinity of the residual muscarinic receptors for the ligand after k.a.
189
lesioning. A possible interpretation is that a new population of receptors with altered characteristics spreads on the remaining PRCP perikarya. The finding that the intranigral injection of carbachol very well antagonized the k.a.-induced contralateral turning suggests that the dose of k.a. used only partially destroys the perikarya of the neurons present in the pars reticulata. It is, indeed, more difficult to explain the decreased response to apomorphine and the increased sensitivity to haloperidol produced by intranigral saline. The lesion per se seemed to be responsible for these effects; in fact (i) apomorphine, at adose which inverts the direction of the contralateral turning produced by intranigral muscimol (Olianas et al., 1978b), produced only symmetric stereotypies when the control s.n. was injected with saline and (ii) scopolamine had no effect on the apomorphine-induced ipsilateral turning following intranigral saline (our unpublished observations). On the other hand, a different mechanism, cholinergic in nature, underlies the ipsilateral turning produced by apomorphine in rats unilaterally lesioned with k.a., as was seen from the scopolamine antagonism on this effect. We have no explanation, at the moment, for this finding. In conclusion, the present data indicate that posture is controlled, in the s.n. pars reticulata, by a balance between Ach and GABA activity. This model can be seen as an alternative to the DA-Ach balance proposed by several authors (Fog et al., 1966; Pedersen, 1967; Arnfred and Randrup, 1968; ScheelKri]ger, 1970). In our opinion, a thorough characterization of the nigral Ach and GABA receptors may lead to results of practical relevance for the management of extrapyramidal diseases. References Aghajanian, G.K. and S.B. Bunney, 1973, Central dopaminergic neurons: Neurophysiological identification and responses to drugs, in: Frontiers in Catecholamine Research, III International
190 Catecholamine Symposium, eds. E. Usdin and S. Snyder (Pergamon Press, Oxford) p. 643. Arnfred, T. and A. Randrup, 1968, Cholinergic mechanism in brain inhibiting amphetamineinduced stereotyped behavior, Acta Pharmacol. Toxicol. 26, 384. Di Chiara, G., M. Olianas, M. Del Fiacco, P.F. Spano and A. Tagliamonte, 1977, Intranigral kainic acid is evidence that nigral non-dopaminergic neurons control posture, Nature 268, 743. Dray, A., N.R. Oakley and N.A. Simmonds, 1975, Rotational behavior following inhibition of GABA metabolism unilaterally in the rat substantia nigra, J. Pharm. Pharmacol. 27,627. Fog, R., A. Randrup and H. Pakkenberg, 1966, Amines in the corpus striatum associated with the effect of both amphetamine and antipsychotic drugs, Excerpta Medica Intern. Congress Series No. 150, Proc. IV World Congress of Psychiatry, p. 2580. Kobayashi, R.M., M. Brownstein, J.M. Saavedra and M. Palkovits, 1975, Choline acetyltransferase content in discrete regions of the rat brain stem, J. Neurochem. 24, 637. Lowry, O.H., N.J. Rosenbrogh, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin Phenol reagent, J. Biol. Chem. 193, 265. Mao, C.C., E. Marco, A. Revuelta, L. Bertilsson and E. Costa, 1977, The turnover rate of -/-aminobutyric acid in the nuclei of the telencephalon: implications in the pharmacology of antipsychotic and of minor tranquilizer, Biol. Psychiatry 13 (3), 359. Marshall, J.F. and U. Ungerstedt, 1977, Supersensitivity to apomorphine following destruction of the sscending doparnine neurons: quantification using the rotational model, European J. Pharmacol. 41, 361. McGeer, E.G., H.C. Fibiger, P.L. McGeer and S. Brooke, 1973, Temporal changes in amine synthesizing enzymes of rat extrapyramidal structure after hemitransections or 6-hydroxydopamine administration, Brain Res. 82, 289. McGeer, E.G. and P.L. McGeer, 1975, Duplication of biochemical changes of Huntington's chorea by intrastriatal injections of glutamic and kalnic acid, Nature 263, 517.
G.M. DE MONTIS ET AL. Olianas, M.C., G.M. De Montis, A. Concu, A. Tagliamonte and G. Di Chiara, 1978a, 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, 223. Olianas, M.C., G.M. De Montis, G. Mulas and A. Tagliamonte, 1978b, The striatal'dopaminergic function is mediate by the inhbition of a nigral, non-dopaminergic neuronal system via a strionigral gabaergic pathway, European J. Pharmacol. 49,233. Olivier, A., A. Parent, H. Simard and L.J. Poirier, 1970, Cholinesterasic striopallidal and striatonigral efferents in the cat and monkey, Brain Res. 18, 273. Palkovits, M. and D.M. Jacobowitz, 1974, Topographic Atlas of catecholamine and acetylcholinesterasecontaining neurons in the rats brain. II. Hindbrain (mesencephalon, rombencephalon), J. Comp. Neurol. 157, 29. Pedersen, V., 1967, Potentiation of apomorphine effect (compulsive gnawing behavior) in mice, Acta Pharm. Toxicol. 25, Suppl. 4, 63. Pellegrino, L.J. and A.J. Cushman, 1971, A Stereotaxic Atlas of the Rat Brain (Meredith, New York). Perez De La Mora, L., K. Fuxe, T. HSkfelt and A. Ljungdal, 1977, Evidence for a nerve impulsedependent GABA accumulation in the substantia nigra after treatment with gamma-glutamylhydrazine, Neurosci. Lett. 5, (1--2)/75. Scheel-Kriiger, J., 1970, Central effects of anticholinergic drugs measured by the apomorphine gnawing test in mice, Acta Pharmacol. Toxicol. 28,1. Scheel-Kriiger, J., J. Arnt and G. Magelund, 1977, Behavioral stimulation induced by muscimol and other GABA agonists injected into the substantia nigra, Neurosci. Lett. 4,351. Schwartz, R. and J.T. Coyle, 1977, Neurochemical sequelae of kaninate injections in corpus striatum and substantia nigra of the rat, Life Sci. 20, 431. Snedecor, J.W., 1969, Statistical Methods, 5th edn. (Iowa S~:: ~'~ College Press, Ames, Iowa). Yamamura, H.I. and S.H. Snyder, 1974, Muscarinic cholinergic binding in rat brain, Proc. Nat. Acad. Sci. 71, 1725.