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Two types of neurons in the substantia nigra pars compacta studied in a slice preparation
172
Neuroscience Research, 5 (1987) 172-l 19
Elsevier Scientific Publishers Ireland Ltd. NSR 00212
Two types of neurons in the substantia nigra PUS compacta studied in a slice preparation Yoshihiro Matsuda, Koichi Fujimura and Shigeru Yoshida Department of Physiology. School of Medicine, Nagasaki University. Nagasaki (Japan)
(Received 25 June 1987; Accepted 13 August 1987) Key words: Substantia nigra pars compacta; Guinea pig; Slice preparation; Depolarizing alter-potential; Low-threshold Ca spike; Small neuron
SUMMARY Two types of neurons were differentiated, on the basis of electrophysiological properties, in the substantia nigra pars compacta of the guinea pig maintained in slices. While the majority of neurons, presumably dopaminergic neurons, produced a broad spike accompanied by a relatively long-lasting after-hyperpolarization, a small number of neurons were characterized by the generation of a depolarizing after-potential (DAP) following a fast spike. The DAP was depressed by a slight depolarizing shiR of the resting membrane potential and by the removal of Ca 2 + from the bathing medium, which suggested that it was a low-threshold Ca2+ -dependent response. Intracellular staining revealed that the neurons producing DAP were smaller than the major neurons.
Although it has been suggested in morphological studies that 2 or 3 categories of neurons could be recognized in the substantia nigra pars compacta6~*0~16,physiological studies have mainly given attention to the cells of origin of the dopaminergic nigrostriatal projection because of their intimate relationship to the Parkinson syndrome in primates1v3. The present study is concerned with the question of whether the neurons in this nigral subdivision can be separated, in terms of their physiological properties, into sub-classes that may correspond to the morphologically differentiated types of neurons. We attempted to characterize the whole species of neurons encountered in the substantia nigra pars compacta on the basis of their electrophysiological properties. For this purpose, we utilized a slice preparation, which permitted relatively stable intracellular recordings even from smaller neurons. We report here that there are 2 distinct classes of neurons in the substantia nigra pars compacta which differ from each other in electrophysiological properties, and that they also differ in their morphological features. Correspondence: Y. Matsuda, Department Nagasaki 852, Japan.
of Physiology, School of Medicine, Nagasaki University,
0168-0102/87/$03.50 0 1987 Elsevier Scientific Publishers Ireland Ltd.
173 Slices of the substantia nigra were obtained from guinea pigs (250-300 g in body weight). After decapitation, the brain was quickly removed and put into ice-cold saline. me midbrain containing the substantia nigra was cut at a thickness of 400 pm by means of a vibrating slicer. Slices were incubated for at least 2 h in saline at 27-30 “C. Electrophysiological recordings were made from slices fixed on the nylon grid of an experimental chamber (1 ml in volume), which was perfused with saline kept at 30-33 oC. The saline had the following composition (in mM): NaCl124, KC1 5, CaCl, 2.4, MgSO, 1.3, KH,PO, 1.24, NaHCO, 26, and glucose 10. When CdCl, was administered, KH,PO, was omitted and KC1 was dissolved in a concentration of 6.24 mM instead of 5 mM. The bathing solutions were saturated with a mixed gas composed of 95% 0, and 5% CO,. Glass microelectrodes filled with 4 M potassium acetate (KAc) were used for intracellular recordings. Neuronal responses were induced by intracellular current injection through a bridge circuit, and were displayed on an oscilloscope and photographed. The resting level of the membrane potential was monitored with a chart recorder. For intracellular staining, neurons were impaled with microelectrodes filled with a 1: 1 mixture of 4 M KAc and cobalt-lysine solution prepared by the method of Lazarr2. After successful recordings, cobalt-lysine was injected into neurons by applying a depolarizing current, 10 nA in intensity and 500 ms in duration, at a rate of 1 Hz for 20 min. Thereafter, slices were immersed for 10 min in a phosphate buffer, pH 7.4, containing 0.5 % ammonium sulfide. The intensification was performed according to the method of Davis2. Each slice was microscopically examined as a “whole mount” preparation. We recognized two types of neurons in the substantia nigra pars compacta, each of which were well characterized by the responses to applied currents. The present report is based on the data obtained from 42 neurons of both types in which recordings were stable, with a spike amplitude of more than 60 mV. The majority of the neurons recorded in the pars compacta, exhibiting essentially the same electrophysiological property, were grouped together into a neuron class. These neurons generated a relatively broad spike accompanied by an after-hyperpolarization lasting approximately 100 ms (118 + 67 ms), with a peak amplitude of 6.7 f 2.9 mV at the resting membrane potential of 55 f 6 mV (mean + S.D., n = 33) (Fig. lA, and A,). Differentiation of the spike revealed that the decay of the spike was composed of two phases (arrowheads in Fig. lA,), which were demarcated in some neurons by an inflection on the falling phase of the spike. These characteristics gave the impression that the action potentials of these neurons were less “sharp” than those of other nigral neurons. The neurons were also characterized by exhibiting a time-dependent inward-going rectification during the application of hyperpolarizing currents (fig. lA,). After the cessation of hyperpolarizing pulses, they usually showed a delay in the return of the membrane potential to the Original level (Fig. lA,, arrow). The neurons sometimes fired spontaneously at rather low rates (0.4-4 spikes/s). These response characteristics were similar to those reported for presumed dopaminergic neurons of the substantia nigra4*5,11,13.
174
B
I
L --k
2 ___. _.._
3 ____________
J-J----Firs
-L-k2ooms
Fig. 1. Two types of responses recorded from neurons in the substantia nigra pars compacta. A: action potentials (A, and A,) and responses to hyperpolarizing currents (As) observed in a class of neurons constituting the majority of impaled cells. Arrowheads in A, denote two separate peaks on the differentiated record of the falling phase of the spike. The arrow in A, indicates a delayed return of the membrane potential to the baseline level (dashed line) after cessation of a hyperpolarizing current pulse. B: fast spikes accompanied by a depolarizing after-potential (B, and Ba) and rebound depolarization (B,, arrow) observed in a group of nigral pars compacta neurons. Dashed lines in B, show the reference level of the membrane potential and, in the right trace of B,, the resting membrane potential was shifted to a depolarized level by continuous injection of currents. Calibrations in A, also apply to records in A,, B, and B,. Calibrations for potential, current and sweep speed are common in A, and B,.
Besides the major neurons described above, we noticed other pars compacta neurons which could be grouped together by virtue of the characteristic configuration of their action potential. The spike potentials of these neurons were much faster than those of the aforementioned major neurons. A noticeable finding concerning these neurons was that their spike was followed by a depolarizing after-potential (DAP) (Fig. lB,). The amplitude and duration ofthe DAP was 6.8 + 4.0 mV and 15.0 + 13.2 ms, respectively, at the resting membrane potential of 58 + 10 mV (mean + SD., n = 9). The amplitude of the DAP appeared to be dependent, to some extent, on the level of the resting .membrane potential of the neurons; and it increased when the resting membrane potential was forced to move to a hyperpolarized level by injecting continuous currents. In contrast, if the neurons were depolarized to a certain extent, the DAP was no longer evoked. Thus, as seen in Fig. l.B,, a shift of the resting membrane potential from - 59 mV to - 52 mV suppressed the generation of DAP. Although the DAP was followed by a hyperpolarizing potential in some neurons, its amplitude was negligibly small. In contrast to the neurons of the major cell group, pars compacta neurons producing the DAP exhibited no remarkable time-dependent rectification during the
175 injection of hyperpolarizing currents; and, on cessation of the currents, they produced a depolarizing potential which triggered spikes (Fig. IB,, arrow). The DAP and the “rebound” depolarization observed in this study were apparently similar to the low-threshold Ca” -dependent regenerative response noted, e.g. in thalamic neurons8. We therefore examined the contribution of Ca2 + to these responses by applying cadmium, a Ca” channel blocker, and Ca2 +-free saline. At a concentration of OS-l.0 mM, which is enough to suppress the usual Ca2+ -dependent regenerative responses, cadmium failed to block the DAP and rebound depolarization. In fact, it rather enhanced these responses (Fig. 2, middle column). However, the responses were depressed when Ca2 + was removed from the bathing medium (Fig. 2, right column), which suggested the participation of a Ca2 + permeability mechanism in these phenomena. In Table I, the parameters of the spikes and passive electrical responses of the two classes of pars compacta neurons are presented. In confirmation of the impression obtained through inspection of the spikes, the rates of rise and fall of the spikes were significantly faster, and the spike duration was significantly shorter in the neurons producing the DAP than in the neurons of the major class. The measured values of the membrane time constant were lower in the former than in the latter neurons, although the input resistance did not differ significantly between the two groups of neurons. Intracellular staining with a cobalt-lysine complex further revealed morphological differ-
Cont
__f--
--
Fig. 2. Effects of cadmium and Ca* + -free saline on the depolarizing tier-potential and rebound depolarization in a pars compacta neuron. After recording control responses (tirst column), the neuron was perfused with a saline-containing CdCl, at a concentration of 0.5 mM for 20 min (second column), and then the neuron was bathed for 20 mitt in a Ca 2+-free saline in which the concentration of Mga + was raised to 5 mM (third column). The resting membrane potential of the neuron was - 55 mV throughout. Voltage calibration is common to all records. Time scales of 20 ms and 100 ms apply to the traces in the upper and lower rows, respectively. Current scale indicates 1 nA for the record in the third column, upper row, and 2 nA for the rest.
176 TABLE I RESPONSE PARAMETERS IN TWO TYPES OF NIGRAL NEURONS Values are espressed as mean f SD. In the lower two rows, numbers of measurements are given in parentheses. Values marked by asterisk are significantly different from those of the other neuron group at P
33 53
+6
12.9 k 6.0 1.3 + 0.4 134 +28 51 + 21b 59 + 16’ 69.9 & 21.7 (32) 18.8 + 7.0 (12)
Neurons generating a fast spike with a depolarizing after-potential 9 58
f. 10
80.4 f 7.5 0.6 + O.l* 243 + 34* 122 i 23* 47.7 + 25.8 (6) 7.9 * 1.9* (4)
a Measured at the mid-point of spike height. b,c Values of the first (b) and second (c) downward peaks in differentiated records of spikes
ences between the two electrophysiologically differentiated classes of neurons. Representative neurons of both groups are illustrated in Fig. 3. The neurons of the major class had a fusiform or elliptical soma measuring 20-40 pm along its largest axis (Fig. 3a), while the neurons producing a sharp spike accompanied by DAP showed a soma nearly round in contour and 15-20 pm in diameter (Fig. 3b). Several dendrites, very thick at their trunk and often invested with spine-like protrusions, arose from the cell body of the former neurons and, usually, one or two of them extended deeply into the pars reticulata (as seen in Fig 3a, where approximately two-thirds of the peripheral extension of the downward running dendrites is in the pars reticulata). In contrast, dendrites of the latter neurons, 4-7 in number, were slender, without any spine-like appendages on them, and they were distributed, without any preferential direction of extension, in a circumferential area around the cell body (Fig. 3b). The axon of the major type neurons, arising from the soma or primary dendrite, ran dorsomedially, or first in a dorsal and then in a medial direction (Fig. 3a, arrow). The axon could not be found in the neurons generating the fast spike and DAP. In light of the above-mentioned findings, it could be said that the nigral pars compacta neurons reported in this study fall into two categories, and that the neurons which appear to constitute the major neuronal populations of the pars compacta correspond to those nigral neurons which were presumed to be dopaminergic in This is indicated by the response characteristics of these previous studies 4,5,7,11*13. neurons, e.g. the generation of slow action potentiaIs4*7, time-dependent rectification
Fig. 3. Cell shapes reconstructed from photomicrographs of neurons stained with the cobalt-lysine method. Neurons representative of the two classes of pars compacta cells are presented. Neurons a and b produced responses such as are shown in A and B of Fig. 1, respectively. Neurons are viewed in a plane approximately perpendicular to the axis ofthe brainstem. The top and letI ofthe figures correspond to the dorsal and medial side of the brain, respectively. The arrow denotes the axon. Scale bar, 100 pm.
during membrane polarization 4~1‘,13, low-frequency spontaneous ftigs4v7, and relatively high input resistance 11*13.These neurons may correspond to “large” and “medium-sized” cells defined in Golgi studie@‘. On the other hand, the neurons generating fast spikes accompanied by DAP appear to be comparable to “small” cells recognized in Golgi preparations 6*‘o*16. A characteristic feature of these neurons is the generation of DAP. The effects of manipulation of the resting membrane potential and removal of extracellular Ca’ + suggest that the potential is a low-threshold regenerative response dependent on Ca2+ (refs. 8,9, 14). The failure of cadmium to suppress the DAP (at concentrations below 1 mM) further indicates that the Ca” permeability system for the potential is different from that for the usual (high-threshold) Ca2 + -depen-
178 dent responses”. The DAP is apparently different from the low-threshold Ca’ + spike reported for (presumably) dopaminergic neurons of the substantia nigra13; the latter response was blocked by the application of 1 mM CdCl,, and a relatively high level of membrane polarization ( - 70 to - 80 mV) was required for its generation. Studies are now in progress to evaluate the apparent difference between the low-threshold Ca2+ spike in nigral dopaminergic neurons and the DAP in small nigral neurons. Although the neurons of the major class are also capable of producing a low-threshold Ca2+ -dependent response, we suggest that the response in the form of a DAP would be produced by certain nigral neurons in particular, which should be grouped separately from the principal (presumably dopaminergic) neurons comprising the majority of the neuronal population in the substantia nigra pars compacta. It remains to be determined whether they are interneurons or non-dopaminergic output neurons.
ACKNOWLEDGEMENTS
We express our thanks to Prof. N. Iwahori for his help with the photography of histological materials, and to Mr. M. Yogata and Mrs. N. Momosaki for their assistance in preparing illustrations.
REFERENCES 1 Burns, R.S., Chiueh, C.C., Markey, S.P., Ebert, M.H., Jacobowitz, D.M. and Kopin, I.J., A primate model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-1,2,3,6-tetrahydropyridine, Proc. Nurl. Acad. Sci. U.S.A., 80 (1983) 4546-4550. 2 Davis, N.T., Improved methods for cobalt filling and silver intensification of insect motor neurons, Stain Technol., 51 (1982) 239-244. 3 Ehringer, H. and Homykiewicz, O., Verteiluag von Noradrenalin und Dopamin (3-Hydroxytyramin) im
Gehim des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems, K&I. Wsch., 38 (1960) 1236-1239. 4 Grace, A.A. and Bunney, B.S., Intracellular and extracellular electrophysiology of nigral dopaminergic neurons 1. Identification and characterization, Neuroscience, 10 (1983) 301-315. 5 Grace, A.A. and Bunney, B.S., Intracellular and extracellular electrophysiology of nigral dopaminergic neurons 2. Action potential generating mechanisms and morphological correlates, Neuroscience, 10 (1983) 317-331. 6 Gulley, R.L. and Wood, R.L., The fine structure of the neurons in the rat substantia nigra, Tim. Cell, 3 (1971) 675-590. 7 Guyenet, P.G. and Aghajanian, G.K., Antidromic identification of doparninergic and other output neurons of the rat substantia nigra, Bruin Rex, 150 (1978) 69-84. 8 Jahnsen, H. and Llinh, R., Electrophysiological properties of guinea-pig thalamic neurones: an in vitro study, J. Physiol. (London), 349 (1984) 205-226. 9 Jahnsen, H. and Llinas, R., Ionic basis for the electroresponsiveness and oscillatory properties of guinea-pig thalamic neurones in vitro, J. Physiol. (London), 349 (1984) 227-247. 10 Juraska, J.M., Wilson, C.J. and Groves, P.M., The substantia nigra of the rat: a Golgi study, J. Cow Neural., 172 (1977) 585-600.
179 11 Kita, T., Kita, H. and Kitai, ST., Electrical membrane properties of rat substantia nigra compacta neurons in an in vitro slice preparation, Brain Rx, 327 (1986) 21-30. 12 Lamar, G., Application of cobalt-tilling technique to show retinal projections in the frog, Neuroscience, 3 (1978) 725-736. 13 Llinls, R., Greentield, S.A. and Jahnsen, H., Electrophysiology of pars compacta cells in the in vitro substantia nigra - a possible mechanism for dendritic release, Bruin Res., 294 (1984) 127-132. 14 LlinBs, R. and Yarom, Y., Electrophysiology of mammalian inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances, J. Physiol. (London), 315 (1981) 549-567. 15 Nowycky, M.C., Fox, A.P. and Tsien, R.W., Three types of neuronal calcium channel with different calcium agonist sensitivity, Nature (London), 316 (1985) 440-443. 16 Schwyn, R.C. and Fox, C.A., The primate substantia nigra: a Go@ and electron microscopic study, J. Himforsch., 15 (1974) 95-126.
Neuroscience Research, 5 (1987) 172-l 19
Elsevier Scientific Publishers Ireland Ltd. NSR 00212
Two types of neurons in the substantia nigra PUS compacta studied in a slice preparation Yoshihiro Matsuda, Koichi Fujimura and Shigeru Yoshida Department of Physiology. School of Medicine, Nagasaki University. Nagasaki (Japan)
(Received 25 June 1987; Accepted 13 August 1987) Key words: Substantia nigra pars compacta; Guinea pig; Slice preparation; Depolarizing alter-potential; Low-threshold Ca spike; Small neuron
SUMMARY Two types of neurons were differentiated, on the basis of electrophysiological properties, in the substantia nigra pars compacta of the guinea pig maintained in slices. While the majority of neurons, presumably dopaminergic neurons, produced a broad spike accompanied by a relatively long-lasting after-hyperpolarization, a small number of neurons were characterized by the generation of a depolarizing after-potential (DAP) following a fast spike. The DAP was depressed by a slight depolarizing shiR of the resting membrane potential and by the removal of Ca 2 + from the bathing medium, which suggested that it was a low-threshold Ca2+ -dependent response. Intracellular staining revealed that the neurons producing DAP were smaller than the major neurons.
Although it has been suggested in morphological studies that 2 or 3 categories of neurons could be recognized in the substantia nigra pars compacta6~*0~16,physiological studies have mainly given attention to the cells of origin of the dopaminergic nigrostriatal projection because of their intimate relationship to the Parkinson syndrome in primates1v3. The present study is concerned with the question of whether the neurons in this nigral subdivision can be separated, in terms of their physiological properties, into sub-classes that may correspond to the morphologically differentiated types of neurons. We attempted to characterize the whole species of neurons encountered in the substantia nigra pars compacta on the basis of their electrophysiological properties. For this purpose, we utilized a slice preparation, which permitted relatively stable intracellular recordings even from smaller neurons. We report here that there are 2 distinct classes of neurons in the substantia nigra pars compacta which differ from each other in electrophysiological properties, and that they also differ in their morphological features. Correspondence: Y. Matsuda, Department Nagasaki 852, Japan.
of Physiology, School of Medicine, Nagasaki University,
0168-0102/87/$03.50 0 1987 Elsevier Scientific Publishers Ireland Ltd.
173 Slices of the substantia nigra were obtained from guinea pigs (250-300 g in body weight). After decapitation, the brain was quickly removed and put into ice-cold saline. me midbrain containing the substantia nigra was cut at a thickness of 400 pm by means of a vibrating slicer. Slices were incubated for at least 2 h in saline at 27-30 “C. Electrophysiological recordings were made from slices fixed on the nylon grid of an experimental chamber (1 ml in volume), which was perfused with saline kept at 30-33 oC. The saline had the following composition (in mM): NaCl124, KC1 5, CaCl, 2.4, MgSO, 1.3, KH,PO, 1.24, NaHCO, 26, and glucose 10. When CdCl, was administered, KH,PO, was omitted and KC1 was dissolved in a concentration of 6.24 mM instead of 5 mM. The bathing solutions were saturated with a mixed gas composed of 95% 0, and 5% CO,. Glass microelectrodes filled with 4 M potassium acetate (KAc) were used for intracellular recordings. Neuronal responses were induced by intracellular current injection through a bridge circuit, and were displayed on an oscilloscope and photographed. The resting level of the membrane potential was monitored with a chart recorder. For intracellular staining, neurons were impaled with microelectrodes filled with a 1: 1 mixture of 4 M KAc and cobalt-lysine solution prepared by the method of Lazarr2. After successful recordings, cobalt-lysine was injected into neurons by applying a depolarizing current, 10 nA in intensity and 500 ms in duration, at a rate of 1 Hz for 20 min. Thereafter, slices were immersed for 10 min in a phosphate buffer, pH 7.4, containing 0.5 % ammonium sulfide. The intensification was performed according to the method of Davis2. Each slice was microscopically examined as a “whole mount” preparation. We recognized two types of neurons in the substantia nigra pars compacta, each of which were well characterized by the responses to applied currents. The present report is based on the data obtained from 42 neurons of both types in which recordings were stable, with a spike amplitude of more than 60 mV. The majority of the neurons recorded in the pars compacta, exhibiting essentially the same electrophysiological property, were grouped together into a neuron class. These neurons generated a relatively broad spike accompanied by an after-hyperpolarization lasting approximately 100 ms (118 + 67 ms), with a peak amplitude of 6.7 f 2.9 mV at the resting membrane potential of 55 f 6 mV (mean + S.D., n = 33) (Fig. lA, and A,). Differentiation of the spike revealed that the decay of the spike was composed of two phases (arrowheads in Fig. lA,), which were demarcated in some neurons by an inflection on the falling phase of the spike. These characteristics gave the impression that the action potentials of these neurons were less “sharp” than those of other nigral neurons. The neurons were also characterized by exhibiting a time-dependent inward-going rectification during the application of hyperpolarizing currents (fig. lA,). After the cessation of hyperpolarizing pulses, they usually showed a delay in the return of the membrane potential to the Original level (Fig. lA,, arrow). The neurons sometimes fired spontaneously at rather low rates (0.4-4 spikes/s). These response characteristics were similar to those reported for presumed dopaminergic neurons of the substantia nigra4*5,11,13.
174
B
I
L --k
2 ___. _.._
3 ____________
J-J----Firs
-L-k2ooms
Fig. 1. Two types of responses recorded from neurons in the substantia nigra pars compacta. A: action potentials (A, and A,) and responses to hyperpolarizing currents (As) observed in a class of neurons constituting the majority of impaled cells. Arrowheads in A, denote two separate peaks on the differentiated record of the falling phase of the spike. The arrow in A, indicates a delayed return of the membrane potential to the baseline level (dashed line) after cessation of a hyperpolarizing current pulse. B: fast spikes accompanied by a depolarizing after-potential (B, and Ba) and rebound depolarization (B,, arrow) observed in a group of nigral pars compacta neurons. Dashed lines in B, show the reference level of the membrane potential and, in the right trace of B,, the resting membrane potential was shifted to a depolarized level by continuous injection of currents. Calibrations in A, also apply to records in A,, B, and B,. Calibrations for potential, current and sweep speed are common in A, and B,.
Besides the major neurons described above, we noticed other pars compacta neurons which could be grouped together by virtue of the characteristic configuration of their action potential. The spike potentials of these neurons were much faster than those of the aforementioned major neurons. A noticeable finding concerning these neurons was that their spike was followed by a depolarizing after-potential (DAP) (Fig. lB,). The amplitude and duration ofthe DAP was 6.8 + 4.0 mV and 15.0 + 13.2 ms, respectively, at the resting membrane potential of 58 + 10 mV (mean + SD., n = 9). The amplitude of the DAP appeared to be dependent, to some extent, on the level of the resting .membrane potential of the neurons; and it increased when the resting membrane potential was forced to move to a hyperpolarized level by injecting continuous currents. In contrast, if the neurons were depolarized to a certain extent, the DAP was no longer evoked. Thus, as seen in Fig. l.B,, a shift of the resting membrane potential from - 59 mV to - 52 mV suppressed the generation of DAP. Although the DAP was followed by a hyperpolarizing potential in some neurons, its amplitude was negligibly small. In contrast to the neurons of the major cell group, pars compacta neurons producing the DAP exhibited no remarkable time-dependent rectification during the
175 injection of hyperpolarizing currents; and, on cessation of the currents, they produced a depolarizing potential which triggered spikes (Fig. IB,, arrow). The DAP and the “rebound” depolarization observed in this study were apparently similar to the low-threshold Ca” -dependent regenerative response noted, e.g. in thalamic neurons8. We therefore examined the contribution of Ca2 + to these responses by applying cadmium, a Ca” channel blocker, and Ca2 +-free saline. At a concentration of OS-l.0 mM, which is enough to suppress the usual Ca2+ -dependent regenerative responses, cadmium failed to block the DAP and rebound depolarization. In fact, it rather enhanced these responses (Fig. 2, middle column). However, the responses were depressed when Ca2 + was removed from the bathing medium (Fig. 2, right column), which suggested the participation of a Ca2 + permeability mechanism in these phenomena. In Table I, the parameters of the spikes and passive electrical responses of the two classes of pars compacta neurons are presented. In confirmation of the impression obtained through inspection of the spikes, the rates of rise and fall of the spikes were significantly faster, and the spike duration was significantly shorter in the neurons producing the DAP than in the neurons of the major class. The measured values of the membrane time constant were lower in the former than in the latter neurons, although the input resistance did not differ significantly between the two groups of neurons. Intracellular staining with a cobalt-lysine complex further revealed morphological differ-
Cont
__f--
--
Fig. 2. Effects of cadmium and Ca* + -free saline on the depolarizing tier-potential and rebound depolarization in a pars compacta neuron. After recording control responses (tirst column), the neuron was perfused with a saline-containing CdCl, at a concentration of 0.5 mM for 20 min (second column), and then the neuron was bathed for 20 mitt in a Ca 2+-free saline in which the concentration of Mga + was raised to 5 mM (third column). The resting membrane potential of the neuron was - 55 mV throughout. Voltage calibration is common to all records. Time scales of 20 ms and 100 ms apply to the traces in the upper and lower rows, respectively. Current scale indicates 1 nA for the record in the third column, upper row, and 2 nA for the rest.
176 TABLE I RESPONSE PARAMETERS IN TWO TYPES OF NIGRAL NEURONS Values are espressed as mean f SD. In the lower two rows, numbers of measurements are given in parentheses. Values marked by asterisk are significantly different from those of the other neuron group at P
33 53
+6
12.9 k 6.0 1.3 + 0.4 134 +28 51 + 21b 59 + 16’ 69.9 & 21.7 (32) 18.8 + 7.0 (12)
Neurons generating a fast spike with a depolarizing after-potential 9 58
f. 10
80.4 f 7.5 0.6 + O.l* 243 + 34* 122 i 23* 47.7 + 25.8 (6) 7.9 * 1.9* (4)
a Measured at the mid-point of spike height. b,c Values of the first (b) and second (c) downward peaks in differentiated records of spikes
ences between the two electrophysiologically differentiated classes of neurons. Representative neurons of both groups are illustrated in Fig. 3. The neurons of the major class had a fusiform or elliptical soma measuring 20-40 pm along its largest axis (Fig. 3a), while the neurons producing a sharp spike accompanied by DAP showed a soma nearly round in contour and 15-20 pm in diameter (Fig. 3b). Several dendrites, very thick at their trunk and often invested with spine-like protrusions, arose from the cell body of the former neurons and, usually, one or two of them extended deeply into the pars reticulata (as seen in Fig 3a, where approximately two-thirds of the peripheral extension of the downward running dendrites is in the pars reticulata). In contrast, dendrites of the latter neurons, 4-7 in number, were slender, without any spine-like appendages on them, and they were distributed, without any preferential direction of extension, in a circumferential area around the cell body (Fig. 3b). The axon of the major type neurons, arising from the soma or primary dendrite, ran dorsomedially, or first in a dorsal and then in a medial direction (Fig. 3a, arrow). The axon could not be found in the neurons generating the fast spike and DAP. In light of the above-mentioned findings, it could be said that the nigral pars compacta neurons reported in this study fall into two categories, and that the neurons which appear to constitute the major neuronal populations of the pars compacta correspond to those nigral neurons which were presumed to be dopaminergic in This is indicated by the response characteristics of these previous studies 4,5,7,11*13. neurons, e.g. the generation of slow action potentiaIs4*7, time-dependent rectification
Fig. 3. Cell shapes reconstructed from photomicrographs of neurons stained with the cobalt-lysine method. Neurons representative of the two classes of pars compacta cells are presented. Neurons a and b produced responses such as are shown in A and B of Fig. 1, respectively. Neurons are viewed in a plane approximately perpendicular to the axis ofthe brainstem. The top and letI ofthe figures correspond to the dorsal and medial side of the brain, respectively. The arrow denotes the axon. Scale bar, 100 pm.
during membrane polarization 4~1‘,13, low-frequency spontaneous ftigs4v7, and relatively high input resistance 11*13.These neurons may correspond to “large” and “medium-sized” cells defined in Golgi studie@‘. On the other hand, the neurons generating fast spikes accompanied by DAP appear to be comparable to “small” cells recognized in Golgi preparations 6*‘o*16. A characteristic feature of these neurons is the generation of DAP. The effects of manipulation of the resting membrane potential and removal of extracellular Ca’ + suggest that the potential is a low-threshold regenerative response dependent on Ca2+ (refs. 8,9, 14). The failure of cadmium to suppress the DAP (at concentrations below 1 mM) further indicates that the Ca” permeability system for the potential is different from that for the usual (high-threshold) Ca2 + -depen-
178 dent responses”. The DAP is apparently different from the low-threshold Ca’ + spike reported for (presumably) dopaminergic neurons of the substantia nigra13; the latter response was blocked by the application of 1 mM CdCl,, and a relatively high level of membrane polarization ( - 70 to - 80 mV) was required for its generation. Studies are now in progress to evaluate the apparent difference between the low-threshold Ca2+ spike in nigral dopaminergic neurons and the DAP in small nigral neurons. Although the neurons of the major class are also capable of producing a low-threshold Ca2+ -dependent response, we suggest that the response in the form of a DAP would be produced by certain nigral neurons in particular, which should be grouped separately from the principal (presumably dopaminergic) neurons comprising the majority of the neuronal population in the substantia nigra pars compacta. It remains to be determined whether they are interneurons or non-dopaminergic output neurons.
ACKNOWLEDGEMENTS
We express our thanks to Prof. N. Iwahori for his help with the photography of histological materials, and to Mr. M. Yogata and Mrs. N. Momosaki for their assistance in preparing illustrations.
REFERENCES 1 Burns, R.S., Chiueh, C.C., Markey, S.P., Ebert, M.H., Jacobowitz, D.M. and Kopin, I.J., A primate model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-1,2,3,6-tetrahydropyridine, Proc. Nurl. Acad. Sci. U.S.A., 80 (1983) 4546-4550. 2 Davis, N.T., Improved methods for cobalt filling and silver intensification of insect motor neurons, Stain Technol., 51 (1982) 239-244. 3 Ehringer, H. and Homykiewicz, O., Verteiluag von Noradrenalin und Dopamin (3-Hydroxytyramin) im
Gehim des Menschen und ihr Verhalten bei Erkrankungen des extrapyramidalen Systems, K&I. Wsch., 38 (1960) 1236-1239. 4 Grace, A.A. and Bunney, B.S., Intracellular and extracellular electrophysiology of nigral dopaminergic neurons 1. Identification and characterization, Neuroscience, 10 (1983) 301-315. 5 Grace, A.A. and Bunney, B.S., Intracellular and extracellular electrophysiology of nigral dopaminergic neurons 2. Action potential generating mechanisms and morphological correlates, Neuroscience, 10 (1983) 317-331. 6 Gulley, R.L. and Wood, R.L., The fine structure of the neurons in the rat substantia nigra, Tim. Cell, 3 (1971) 675-590. 7 Guyenet, P.G. and Aghajanian, G.K., Antidromic identification of doparninergic and other output neurons of the rat substantia nigra, Bruin Rex, 150 (1978) 69-84. 8 Jahnsen, H. and Llinh, R., Electrophysiological properties of guinea-pig thalamic neurones: an in vitro study, J. Physiol. (London), 349 (1984) 205-226. 9 Jahnsen, H. and Llinas, R., Ionic basis for the electroresponsiveness and oscillatory properties of guinea-pig thalamic neurones in vitro, J. Physiol. (London), 349 (1984) 227-247. 10 Juraska, J.M., Wilson, C.J. and Groves, P.M., The substantia nigra of the rat: a Golgi study, J. Cow Neural., 172 (1977) 585-600.
179 11 Kita, T., Kita, H. and Kitai, ST., Electrical membrane properties of rat substantia nigra compacta neurons in an in vitro slice preparation, Brain Rx, 327 (1986) 21-30. 12 Lamar, G., Application of cobalt-tilling technique to show retinal projections in the frog, Neuroscience, 3 (1978) 725-736. 13 Llinls, R., Greentield, S.A. and Jahnsen, H., Electrophysiology of pars compacta cells in the in vitro substantia nigra - a possible mechanism for dendritic release, Bruin Res., 294 (1984) 127-132. 14 LlinBs, R. and Yarom, Y., Electrophysiology of mammalian inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances, J. Physiol. (London), 315 (1981) 549-567. 15 Nowycky, M.C., Fox, A.P. and Tsien, R.W., Three types of neuronal calcium channel with different calcium agonist sensitivity, Nature (London), 316 (1985) 440-443. 16 Schwyn, R.C. and Fox, C.A., The primate substantia nigra: a Go@ and electron microscopic study, J. Himforsch., 15 (1974) 95-126.