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Electrophysiological evidence for the existence of excitatory fibres in the caudato-nigral pathway in the cat
Neuroscience Letters, 20 (1980) 301-306
301
© Elsevier/North-Holland Scientific Publishers Ltd.
ELECTROPHYSIOLOGICAL EVIDENCE FOR THE EXISTENCE OF EXCITATORY FIBRES IN THE CAUDATO-NIGRAL PATHWAY IN THE CAT
ICHIRO KANAZAWAand MITSUO YOSHIDA Department of Neurology, Institute of Clinical Medicine, University of Tsukuba, Ibaraki-ken, 305 (Japan)
and Department of Neurology, Jichi Medical School, Tochigi-ken, 329-04 (Japan)
(Received May 21st, 1980; Revised version received August 20th, 1980; Accepted August 29th, 1980)
The electrophysiological effect of stimulation of the caudate nucleus on cat nigral neurones was investigated under conditions of the strong inhibitory influence of GABA being blocked by intravenously administered picrotoxin. At 10-20 min after picrotoxin injection, the caudate-evokedinhibition of nigral neurones was markedly diminished and instead the caudate-evoked excitatory responses of the same neurones appeared. These caudate-evoked excitatory responses were antagonized by intravenously administered baclofen, but not by atropine. These results open up the possibility that the caudate-evoked excitation of the nigral neurones may be mediated by substance P.
The role of the undecapeptide, substance P, in the mammalian spinal cord has been extensively studied by Otsuka and his colleagues (e.g. ref. 14), who have strongly suggested its excitatory transmitter function in primary afferent neurones. Recently, we have proposed the hypothesis, based on both biochemical and immunohistochemical evidence, that substance P might be a transmitter mediating the striato-nigral fibre pathway [9]. Indeed, iontophoreticaUy applied substance P has an excitatory effect on nigral neurones [4, 18]. Electrophysiologically, however, a main striato-nigral fibre pathway has been shown to be inhibitory in function [ 19], mediated by 7-aminobutyric acid (GABA) [16], and it is usually difficult to find an excitatory effect on the nigral neurones by stimulation of the striatum [2, 6, 11, 19]. This is inconsistent with the above hypothesis. It could be possible, however, that the excitatory component of the striato-nigral pathway is cancelled out or overcome by its powerful inhibitory one. In order to investigate this possibility the effect of striatal stimulation on nigral neurones was examined under conditions where the effect o f G A B A was blocked by the systemic application o f the G A B A antagonist, picrotoxin. Twelve cats were anaesthetized with sodium pentobarbital (30 m g / k g body
302 weight), paralyzed with gallamine triethiodide and artificially respired. The parietal cortex was partially removed by suction to facilitate microelectrode insertion. The ipsilateral caudate nucleus was stimulated with 2 - 6 iridium platinum electrodes insulated except for the tip. The ventro-medial nucleus o f the thalamus and superior colliculus were also stimulated to obtain antidromic activation of the nigral neurones [1]. Stimulating positions were marked by electrolytic lesions. For extracellular recording of the nigral neurones, glass microelectrodes filled with 2 M NaC1 saturated with Fast Green FCF were used. Nigral neurones were identified after each experiment by referring to spots of green dye deposited electrophoretically from the recording electrode at a known depth and also by antidromic invasion during each experiment. Extracellular spikes were processed by a histogram data processor DAB 5101 (Nihonkoden). Picrotoxin used to abolish the inhibitory effect of GABA neurones was intravenously administered in amounts of 2.0-2.7 mg/kg body weight. Bacrofen (Daiichi Drug Co.) and atropine sulphate was intravenously administered in amounts of 1-3 mg and 120 gg/kg body weight, respectively. A total of 66 spontaneously firing nigral neurones were used for the following observations. The most commonly encountered responses of the nigral neurones to caudate stimulation were inhibitions with latencies of 10-30 msec (mean 18.5, n = 29) and which lasted for 30-150 msec (mean 50.5, n = 29). As described previously [16, 19], the caudate stimulation evoked positive field potentials in the substantia nigra and activities of the nigral neurones were strongly inhibited during the positivity. In more than 60°7o of the nigral cells tested, the caudate-evoked inhibition was followed by a rebound excitation, as shown in Figs. 1A and 2A. Within 10-20 min after the picrotoxin injection the caudate-evoked inhibition of nigral neurones was markedly diminished, and instead an excitation with a latency of 15-30 msec (mean 21.5, n = 6) appeared upon caudate stimulation (Figs. 1B and 2B). Another significant effect of the picrotoxin injection was that it became easy to find nigral neurones which exhibited an exclusively excitatory response to the caudate stimulation. Under such conditions, atropine sulphate was intravenously administered. Although the latency o f the excitation changed by some 5-10 msec, perhaps due to the different time after the administration of picrotoxin, the size of this excitation was not affected by atropine sulphate at all (Fig. 2C). Baclofen was then administered intravenously. As shown in Fig. 2D, 3 - 1 0 min after the baclofen injection the caudate-evoked excitatory effect on the nigral neurones completely disappeared. This baclofen effect lasted for more than 30 min. Interestingly, after the baclofen injection it became difficult to find any nigral neurones responsive to the caudate stimulation. The present investigation revealed two remarkable results. Firstly, when the powerful GABA-mediated, caudate-evoked effects on nigral neurones were abolished by systemically administered picrotoxin, the caudate-evoked excitatory responses of the same nigral neurones appeared. Many previous reports [2, 6, 11]
303
A
T
B
I
I
I
I
20 msec
' ] 1oo v
Fig. 1. Extracellular responses o f a nigral neurone evoked by stimulation of the head of the caudate nucleus. A: inhibition and rebound excitation o f spontaneous discharges of a nigral neurone following stimulation of the head of the caudate. B: excitatory responses of the same nigral neurone as in A to caudate stimulation 20 min after the systemic injection o f picrotoxin. Upward arrows indicate the stimuli and time constant o f the recording was 0.01 sec.
have revealed that under barbiturate anaesthesia the caudate-evoked responses of nigral neurones are exclusively inhibitory and probably mediated by GABA. On abolishing such GABA effects by using picrotoxin, the present study demonstrated that there are actually caudate-evoked excitatory responses of nigral neurones. Although other experimental conditions reported were different, in that no picrotoxin was used, there are several reports on the existence of caudate-evoked excitatory responses of nigral neurones in monkey [5, 12], rat [3] and cat [8]. Secondly, these caudate-evoked excitatory responses of nigral neurones were antagonized by intravenously administered baclofen, which has been reported to antagonize the excitatory effect of substance P probably released from primary afferent fibres in the spinal cord [17]. In addition, it is noteworthy that this caudateevoked excitation was not affected by a muscarinic cholinergic antagonist, atropine. As far as the action of baclofen is concerned, it is now widely accepted that the effects of iontophoretically applied baclofen and that of systemically applied baclofen would be different [7, 13]. Indeed, although the iontophoretically applied baclofen on the nigral neurones of rat suppressed the cell firing induced not only by substance P but also by glutamate or acetylcholine in a rather non-specific manner [4], Otsuka and co-workers [14, 15, 17] have shown a specific antagonistic action at
304
20
A 0
l
B
12
D T
i
, 128 msec
Fig. 2. Post-stimulus histograms of caudate-evoked activity of a nigral neurone. Stimulation frequency was 2 Hz. Extracellularly recorded spikes of the nigral neurone were processed with a histogram data processor. Ordinate indicates the number of spikes. A: caudate-evoked inhibition of a nigral neurone. Same neurone as in B - D , B: 15 min after the systemic injection of picrotoxin (2.0 mg/kg). Note the marked excitation with a latency of approximately 20 msec. C: 27 min after picrotoxin and 10 rain after atropine sulphate injection (120/zg/kg). Note no apparent effect of atropine on the excitation except a slightly shorter latency than in B (see text). D: 37 rain after picrotoxin and 10 rain after the bactofen injection (3 mg/kg). The caudate-evoked excitation completely disappeared and the caudate-evoked inhibition was still blocked by picrotoxin.
305 relatively low doses o f b a c l o f e n o n the s u b s t a n c e P - e v o k e d d e p o l a r i z a t i o n o f the rat spinal, m o t o n e u r o n e s using the bath a p p l i c a t i o n m e t h o d . I n a d d i t i o n , Kato et al. [10] have p r o v i d e d electrophysiological evidence for a specific action o f i n t r a v e n o u s l y a d m i n i s t e r e d baclofen o n the p r i m a r y a f f e r e n t synapses in the cat spinal cord. Therefore, the present results o b t a i n e d b y systemic a p p l i c a t i o n o f baclofen o p e n u p the possibility that the c a u d a t e - e v o k e d excitation m a y be mediated by substance P.
We are grateful to P r o f . M. O t s u k a , D e p a r t m e n t o f P h a r m a c o l o g y , T o k y o Medical a n d D e n t a l University, for his c o n s t a n t e n c o u r a g e m e n t a n d v a l u a b l e advice, a n d to Miss N. N a k a j i m a , Miss Y. N a g a t s u k a a n d Mr. D. Sutoo for their excellent technical assistance.
1 Anderson, M. and Yoshida, M., Axonal branching patterns and location of nigrothalamic and nigrocollicular neurons in the cat, J. Neurophysiol., in press. 2 Crossman, A.R., Walker, R.J. and Woodruff, G.N., Picrotoxin antagonism of gammaaminobutyric acid inhibitory responses and synaptic inhibition in the rat substantia nigra, Brit. J. Pharmacol., 49 (1973) 696-698. 3 Dray, A., Gonye, T.J. and Oakley, N.R., Caudate stimulation and substantia nigra activity in the rat, J. Physiol. (Lond.), 259 (1976) 825-849. 4 Davies,J. and Dray, A., Substance P in the substantia nigra, Brain Res., 107 (1976) 623-627. 5 Feger, J. and Ohye, C., The unitary activity of the substantia nigra following stimulation of the striatum in the awake monkey, Brain Res., 89 (1975) 155-159. 6 Feltz, P., 3,-Aminobutyric acid and a caudate-nigral inhibition, Canad. J. Physiol. Pharmacol., 49 (1971) 1113-1115. 7 Fox, S., Krnjevi~, K., Morris, M.E., Puil, E. and Werman, R., Action of baclofen on mammalian synaptic transmission. Neuroscience, 3 (1978) 495-515. 8 Frigyesi,T. and Purpura, D.P., Electrophysiological analysis of reciprocal caudate-nigral relations, Brain Res., 6 (1967) 440-456. 9 Kanazawa, I., Emson, P.C. and Cuello, A.C., Evidence for the existence of substance P-containing fibres in striatonigral and pallidonigral pathways in rat brain, Brain Res., 119 (1977) 447-453. 10 Kato, M., Waldmann, U. and Murakami, S., Effects of baclofen on spinal neurons of cats, Neuropharmacology, 17 (1978) 827-833. 11 McNair, J.L., Sutin, J. and Tsubokawa, T., Suppression of cell firing in the substantia nigra by caudate nucleus stimulations, Exp. Neurol., 37 (1972) 395-411. 12 Ohye, C., Feger, J. and Albe-Fessard, D., Study in awake monkeys of connections between the corpus striatum and pallidum with the substantia nigra and subthalamus. In T. Desiraju (Ed.), Mechanisms in Transmission of Signals for Conscious Behaviour, Elsevier, Amsterdam, 1976, pp. 45-59. 13 Olpe, H.R., Koella, W.P., Wolf, P. and Haas, H.L., The action of baclofen on neurons of the substantia nigra and of the ventral tegmental area, Brain Res., 134 (1977) 577-580. 14 Otsuka, M. and Konishi, S., Substance P and excitatory transmitter of primary sensory neurons, Cold Spr. Harb. Symp. quant. Biol., 40 (1975) 135-143. 15 Otsuka, M. and Yanagisawa, M., The effects of substance P and baclofen on motoneurones of isolated spinal cord of the newborn rats, J. exp. Biol., in press.
306 16 Precht, W. and Yoshida, M., Blockage of caudate-evoked inhibition of neurons in the substantia nigra by picrotoxin, Brain Res., 32 (1971) 229-233. 17 Saito, K., Konishi, S. and Otsuka, M., Antagonism between Lioresai and substance P in rat spinal cord, Brain Res., 97 (1975) 177-180. 18 Walker, R.J., Kemp, J.A., Yajima, H., Kitagawa, K. and Woodruff, G.N., The action of substance P on mesencephalic reticular and substantia nigral neurones of the cat, Experientia (Basel), 32 (1976) 214-215. 19 Yoshida, M. and Precht, W., Monosynaptic inhibition of neurons of the substantia nigra by caudatonigral fibers, Brain Res., 32 (1971) 225-228.
301
© Elsevier/North-Holland Scientific Publishers Ltd.
ELECTROPHYSIOLOGICAL EVIDENCE FOR THE EXISTENCE OF EXCITATORY FIBRES IN THE CAUDATO-NIGRAL PATHWAY IN THE CAT
ICHIRO KANAZAWAand MITSUO YOSHIDA Department of Neurology, Institute of Clinical Medicine, University of Tsukuba, Ibaraki-ken, 305 (Japan)
and Department of Neurology, Jichi Medical School, Tochigi-ken, 329-04 (Japan)
(Received May 21st, 1980; Revised version received August 20th, 1980; Accepted August 29th, 1980)
The electrophysiological effect of stimulation of the caudate nucleus on cat nigral neurones was investigated under conditions of the strong inhibitory influence of GABA being blocked by intravenously administered picrotoxin. At 10-20 min after picrotoxin injection, the caudate-evokedinhibition of nigral neurones was markedly diminished and instead the caudate-evoked excitatory responses of the same neurones appeared. These caudate-evoked excitatory responses were antagonized by intravenously administered baclofen, but not by atropine. These results open up the possibility that the caudate-evoked excitation of the nigral neurones may be mediated by substance P.
The role of the undecapeptide, substance P, in the mammalian spinal cord has been extensively studied by Otsuka and his colleagues (e.g. ref. 14), who have strongly suggested its excitatory transmitter function in primary afferent neurones. Recently, we have proposed the hypothesis, based on both biochemical and immunohistochemical evidence, that substance P might be a transmitter mediating the striato-nigral fibre pathway [9]. Indeed, iontophoreticaUy applied substance P has an excitatory effect on nigral neurones [4, 18]. Electrophysiologically, however, a main striato-nigral fibre pathway has been shown to be inhibitory in function [ 19], mediated by 7-aminobutyric acid (GABA) [16], and it is usually difficult to find an excitatory effect on the nigral neurones by stimulation of the striatum [2, 6, 11, 19]. This is inconsistent with the above hypothesis. It could be possible, however, that the excitatory component of the striato-nigral pathway is cancelled out or overcome by its powerful inhibitory one. In order to investigate this possibility the effect of striatal stimulation on nigral neurones was examined under conditions where the effect o f G A B A was blocked by the systemic application o f the G A B A antagonist, picrotoxin. Twelve cats were anaesthetized with sodium pentobarbital (30 m g / k g body
302 weight), paralyzed with gallamine triethiodide and artificially respired. The parietal cortex was partially removed by suction to facilitate microelectrode insertion. The ipsilateral caudate nucleus was stimulated with 2 - 6 iridium platinum electrodes insulated except for the tip. The ventro-medial nucleus o f the thalamus and superior colliculus were also stimulated to obtain antidromic activation of the nigral neurones [1]. Stimulating positions were marked by electrolytic lesions. For extracellular recording of the nigral neurones, glass microelectrodes filled with 2 M NaC1 saturated with Fast Green FCF were used. Nigral neurones were identified after each experiment by referring to spots of green dye deposited electrophoretically from the recording electrode at a known depth and also by antidromic invasion during each experiment. Extracellular spikes were processed by a histogram data processor DAB 5101 (Nihonkoden). Picrotoxin used to abolish the inhibitory effect of GABA neurones was intravenously administered in amounts of 2.0-2.7 mg/kg body weight. Bacrofen (Daiichi Drug Co.) and atropine sulphate was intravenously administered in amounts of 1-3 mg and 120 gg/kg body weight, respectively. A total of 66 spontaneously firing nigral neurones were used for the following observations. The most commonly encountered responses of the nigral neurones to caudate stimulation were inhibitions with latencies of 10-30 msec (mean 18.5, n = 29) and which lasted for 30-150 msec (mean 50.5, n = 29). As described previously [16, 19], the caudate stimulation evoked positive field potentials in the substantia nigra and activities of the nigral neurones were strongly inhibited during the positivity. In more than 60°7o of the nigral cells tested, the caudate-evoked inhibition was followed by a rebound excitation, as shown in Figs. 1A and 2A. Within 10-20 min after the picrotoxin injection the caudate-evoked inhibition of nigral neurones was markedly diminished, and instead an excitation with a latency of 15-30 msec (mean 21.5, n = 6) appeared upon caudate stimulation (Figs. 1B and 2B). Another significant effect of the picrotoxin injection was that it became easy to find nigral neurones which exhibited an exclusively excitatory response to the caudate stimulation. Under such conditions, atropine sulphate was intravenously administered. Although the latency o f the excitation changed by some 5-10 msec, perhaps due to the different time after the administration of picrotoxin, the size of this excitation was not affected by atropine sulphate at all (Fig. 2C). Baclofen was then administered intravenously. As shown in Fig. 2D, 3 - 1 0 min after the baclofen injection the caudate-evoked excitatory effect on the nigral neurones completely disappeared. This baclofen effect lasted for more than 30 min. Interestingly, after the baclofen injection it became difficult to find any nigral neurones responsive to the caudate stimulation. The present investigation revealed two remarkable results. Firstly, when the powerful GABA-mediated, caudate-evoked effects on nigral neurones were abolished by systemically administered picrotoxin, the caudate-evoked excitatory responses of the same nigral neurones appeared. Many previous reports [2, 6, 11]
303
A
T
B
I
I
I
I
20 msec
' ] 1oo v
Fig. 1. Extracellular responses o f a nigral neurone evoked by stimulation of the head of the caudate nucleus. A: inhibition and rebound excitation o f spontaneous discharges of a nigral neurone following stimulation of the head of the caudate. B: excitatory responses of the same nigral neurone as in A to caudate stimulation 20 min after the systemic injection o f picrotoxin. Upward arrows indicate the stimuli and time constant o f the recording was 0.01 sec.
have revealed that under barbiturate anaesthesia the caudate-evoked responses of nigral neurones are exclusively inhibitory and probably mediated by GABA. On abolishing such GABA effects by using picrotoxin, the present study demonstrated that there are actually caudate-evoked excitatory responses of nigral neurones. Although other experimental conditions reported were different, in that no picrotoxin was used, there are several reports on the existence of caudate-evoked excitatory responses of nigral neurones in monkey [5, 12], rat [3] and cat [8]. Secondly, these caudate-evoked excitatory responses of nigral neurones were antagonized by intravenously administered baclofen, which has been reported to antagonize the excitatory effect of substance P probably released from primary afferent fibres in the spinal cord [17]. In addition, it is noteworthy that this caudateevoked excitation was not affected by a muscarinic cholinergic antagonist, atropine. As far as the action of baclofen is concerned, it is now widely accepted that the effects of iontophoretically applied baclofen and that of systemically applied baclofen would be different [7, 13]. Indeed, although the iontophoretically applied baclofen on the nigral neurones of rat suppressed the cell firing induced not only by substance P but also by glutamate or acetylcholine in a rather non-specific manner [4], Otsuka and co-workers [14, 15, 17] have shown a specific antagonistic action at
304
20
A 0
l
B
12
D T
i
, 128 msec
Fig. 2. Post-stimulus histograms of caudate-evoked activity of a nigral neurone. Stimulation frequency was 2 Hz. Extracellularly recorded spikes of the nigral neurone were processed with a histogram data processor. Ordinate indicates the number of spikes. A: caudate-evoked inhibition of a nigral neurone. Same neurone as in B - D , B: 15 min after the systemic injection of picrotoxin (2.0 mg/kg). Note the marked excitation with a latency of approximately 20 msec. C: 27 min after picrotoxin and 10 rain after atropine sulphate injection (120/zg/kg). Note no apparent effect of atropine on the excitation except a slightly shorter latency than in B (see text). D: 37 rain after picrotoxin and 10 rain after the bactofen injection (3 mg/kg). The caudate-evoked excitation completely disappeared and the caudate-evoked inhibition was still blocked by picrotoxin.
305 relatively low doses o f b a c l o f e n o n the s u b s t a n c e P - e v o k e d d e p o l a r i z a t i o n o f the rat spinal, m o t o n e u r o n e s using the bath a p p l i c a t i o n m e t h o d . I n a d d i t i o n , Kato et al. [10] have p r o v i d e d electrophysiological evidence for a specific action o f i n t r a v e n o u s l y a d m i n i s t e r e d baclofen o n the p r i m a r y a f f e r e n t synapses in the cat spinal cord. Therefore, the present results o b t a i n e d b y systemic a p p l i c a t i o n o f baclofen o p e n u p the possibility that the c a u d a t e - e v o k e d excitation m a y be mediated by substance P.
We are grateful to P r o f . M. O t s u k a , D e p a r t m e n t o f P h a r m a c o l o g y , T o k y o Medical a n d D e n t a l University, for his c o n s t a n t e n c o u r a g e m e n t a n d v a l u a b l e advice, a n d to Miss N. N a k a j i m a , Miss Y. N a g a t s u k a a n d Mr. D. Sutoo for their excellent technical assistance.
1 Anderson, M. and Yoshida, M., Axonal branching patterns and location of nigrothalamic and nigrocollicular neurons in the cat, J. Neurophysiol., in press. 2 Crossman, A.R., Walker, R.J. and Woodruff, G.N., Picrotoxin antagonism of gammaaminobutyric acid inhibitory responses and synaptic inhibition in the rat substantia nigra, Brit. J. Pharmacol., 49 (1973) 696-698. 3 Dray, A., Gonye, T.J. and Oakley, N.R., Caudate stimulation and substantia nigra activity in the rat, J. Physiol. (Lond.), 259 (1976) 825-849. 4 Davies,J. and Dray, A., Substance P in the substantia nigra, Brain Res., 107 (1976) 623-627. 5 Feger, J. and Ohye, C., The unitary activity of the substantia nigra following stimulation of the striatum in the awake monkey, Brain Res., 89 (1975) 155-159. 6 Feltz, P., 3,-Aminobutyric acid and a caudate-nigral inhibition, Canad. J. Physiol. Pharmacol., 49 (1971) 1113-1115. 7 Fox, S., Krnjevi~, K., Morris, M.E., Puil, E. and Werman, R., Action of baclofen on mammalian synaptic transmission. Neuroscience, 3 (1978) 495-515. 8 Frigyesi,T. and Purpura, D.P., Electrophysiological analysis of reciprocal caudate-nigral relations, Brain Res., 6 (1967) 440-456. 9 Kanazawa, I., Emson, P.C. and Cuello, A.C., Evidence for the existence of substance P-containing fibres in striatonigral and pallidonigral pathways in rat brain, Brain Res., 119 (1977) 447-453. 10 Kato, M., Waldmann, U. and Murakami, S., Effects of baclofen on spinal neurons of cats, Neuropharmacology, 17 (1978) 827-833. 11 McNair, J.L., Sutin, J. and Tsubokawa, T., Suppression of cell firing in the substantia nigra by caudate nucleus stimulations, Exp. Neurol., 37 (1972) 395-411. 12 Ohye, C., Feger, J. and Albe-Fessard, D., Study in awake monkeys of connections between the corpus striatum and pallidum with the substantia nigra and subthalamus. In T. Desiraju (Ed.), Mechanisms in Transmission of Signals for Conscious Behaviour, Elsevier, Amsterdam, 1976, pp. 45-59. 13 Olpe, H.R., Koella, W.P., Wolf, P. and Haas, H.L., The action of baclofen on neurons of the substantia nigra and of the ventral tegmental area, Brain Res., 134 (1977) 577-580. 14 Otsuka, M. and Konishi, S., Substance P and excitatory transmitter of primary sensory neurons, Cold Spr. Harb. Symp. quant. Biol., 40 (1975) 135-143. 15 Otsuka, M. and Yanagisawa, M., The effects of substance P and baclofen on motoneurones of isolated spinal cord of the newborn rats, J. exp. Biol., in press.
306 16 Precht, W. and Yoshida, M., Blockage of caudate-evoked inhibition of neurons in the substantia nigra by picrotoxin, Brain Res., 32 (1971) 229-233. 17 Saito, K., Konishi, S. and Otsuka, M., Antagonism between Lioresai and substance P in rat spinal cord, Brain Res., 97 (1975) 177-180. 18 Walker, R.J., Kemp, J.A., Yajima, H., Kitagawa, K. and Woodruff, G.N., The action of substance P on mesencephalic reticular and substantia nigral neurones of the cat, Experientia (Basel), 32 (1976) 214-215. 19 Yoshida, M. and Precht, W., Monosynaptic inhibition of neurons of the substantia nigra by caudatonigral fibers, Brain Res., 32 (1971) 225-228.