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Responses to ramp current stimulation of the neurons in substantia nigra pars compacta in vitro
Brain Research, 475 (1988) 177-181
177
Elsevier BRE 23237
Responses to ramp current stimulation of the neurons in substantia nigra pars compacta in vitro K. Fujimura and Y. Matsuda Department of Physiology, Nagasaki UniversitySchool of Medicine, Nagasaki (Japan) (Accepted 23 August 1988)
Key words: Accommodation; Hyperpolarization; A-current; Cadmium; Pars compacta neuron; Substantia nigra
The response to ramp current stimulation was studied in the pars compacta neurons in guinea pig substantia nigra slices. Although accommodation was not seen with current duration up to 1000 ms, a threshold peak emerged on the threshold-latency curve at 30-400 ms when the cell was hyperpolarized. The appearance of the peak was closely correlated with an inflection in the potential trajectory before the spike. A voltage-dependent, fast inactivating outward current may underlie these responses. They persisted in Ca2+-free solution, but disappeared when Cd2÷ (0.4 mM) or Co2÷ (5 mM) ions were applied extracellularly.
It is now established that degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNC) is closely related to Parkinson's syndrome in primates 3. The neurons fire at a relatively low rate 8"14, and they adapt readily to direct stimulation 7'1°. The stability of low frequency firing may be of functional importance in the basal supply of dopamine to the striatum. In general, a major factor which controls the rate of neural firing may be the spike afterhyperpolarization (AHP) 9. Indeed, the SNC neuron shows a profound A H P , which is suggested to be due to a Ca2+-activated K + current 1°. However, some other factors could still control the spike discharge. Thus, an increase of spike threshold in response to an increasing depolarization has been observed in several mammalian central neurons 1'2,11'16'19. This response, termed 'accommodation', may be ascribed to the delayed rectifier current and/or partial inactivation of Na ÷ current 2°. It is also suggested that the voltage-dependent transient outward current, Acurrent (IA), can participate in it 4. Nevertheless, it has not yet been shown whether the accommodation is involved in the firing of the SNC neuron. In the present study, we examined the accommodation of the SNC neuron in slice preparations with linearly in-
creasing current (ramp current) stimulation. We found that the response can be modulated greatly by the shift of resting membrane potential. Slices of the substantia nigra obtained from male guinea pigs (250-350 g) were used for the experiment. After decapitation, the brain was quickly removed from the skull, and coronal section of the midbrain containing the SNC was performed at a thickness of 400 ¢tm using a vibrating slicer. The slices were incubated in standard Krebs solution (mM): NaC1 124, KCI 5, KH2PO 4 1.24, MgSO 4 1.3, CaC12 2.4, N a H C O 3 26, glucose 10 (pH 7.4), which was bubbled continuously with a mixed gas of 95% 02/5% CO2. A modified saline, in which KH2PO 4 and MgSO 4 were replaced, respectively, by KC! and MgC12 in an equimolar concentration, was used when Cd 2+ or Co 2+ ions were applied. Calcium-free Krebs solution (containing 5 mM Mg 2+) was also used. The temperature of the superfusate was 30 °C. Intracellular recording was performed with a 4 M potassium acetate-filled microelectrode. Current injection was made through the recording electrode by means of a bridge circuit. The study is based on data obtained from 25 successful recordings from the major type 14 of neurons
Correspondence: K. Fujimura, Department of Physiology, Nagasaki University School of Medicine, Nagasaki 852, Japan. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
178 of the SNC. Resting membrane potentials ranged from - 5 0 to - 7 0 mV (-55 _+ 5 mV, mean _+ S.D.),
Rather, the current amplitude decreased as the latency was prolonged, up to 1000 ms.
when measured at the midpoint between the spontaneously triggering level and the bottom of the AHP.
The T - L curve for the neuron was altered greatly, however, by hyperpolarizing the m e m b r a n e poten-
The input resistance measured with hyperpolarizing
tial. Fig. 1B~ shows the T - L relations for the neurons presented in Fig. 1A~, which are now hyperpolarized
pulse was 71 _+ 19 Mff2 at peak deflection. When the neuron at its "resting potential" was stimulated with
by 12 _+ 2 mV (i.e. m e m b r a n e potential, - 6 6 _+ 7 mV) with continuous current (t). 15-0.38 nA); the sponta-
ramp current, the m e m b r a n e potential depolarized almost linearly with the current, until a spike was triggered (Fig. 1Al). The latency of the firing was
neous discharges were suppressed by polarization. It should be noticed that the amplitude of threshold
prolonged as the stimulus gradient was reduced. Fig.
current increased abruptly at a point in the middle of
1A~ shows the relationship between the spike latency
T - L relations. Thereafter, it decreased again at a
and the threshold current for the stimulations with various gradients (ca. 500 nA/s to 0.05 nA/s) in 4 rep-
high rate along with the latency up to 200-400 ms. A total of 11 cells examined exhibited this pattern, and the threshold peak (1.1 _+ 0.4 nA) emerged at a latency of 33 _+ 5 ms (current gradient 34 _+ 17 hA/s)
resentative cells. If the neuron ~accommodated' itself to the stimulation, the threshold-latency ( T - L ) curve would rise gradually with prolongation of the latency 211. Most of the neurons (16/18 cells), howev-
during a hyperpolarization of 11 _+ 3 mV with a continuous current injection of 0.3 + 0.1 nA. The threshold was 0.7 + 0.4 n A at 100 ms latency (current gra-
er, did not demonstrate any sign of accommodation.
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Fig. 1. AI: potential responses of an SNC neuron to ramp current stimulations. The membrane potential depolarized almost linearly with the current before firing. Span of a sweep is indicated on each record. The scale bar indicates 0.4 nA and 20 mV for the left and the middle, 0.2 nA and 20 mV for the right, respectively. Firing latency was prolonged with reduction of stimulus gradient. Az: threshold-latency (T-L) relations for the responses of 4 cells; each of the 4 symbols represents the data from an individual cell. Stimulus current amplitude at the time of the initial firing is plotted against the latency in logarithmic scale, for different gradient stimulations (ca. 500 nA/s - - 0.05 nA/s). The threshold decreased as the latency was prolonged. Bl: potential responses of a hyperpolarized cell. An inflection was observed in the trajectory of the slow depolarization before firing by ramp current stimulation (arrowheads in the left and the middle). The scale bar indicates 1.0 nA and 20 mV for the left and the middle, 0.5 nA and 20 mV for the right, respectively. Hyperpolarizating current was 0.38 nA. B2: T-L relations for the hyperpolarized cells in A, with ramp current stimulation. The threshold increased in the range of the latency between about 30 and 400 ms. The hyperpolarizing current was 0.24 +_0.1 nA (mean +_S.D.), resulting in a 12 _+2 mV hyperpolarization.
179 dient ca. 7 nA/s), and was 0.3 + 0.2 nA at 300 ms (ca. 1 nA/s). A similar transient deflection of the T - L curve was observed in two other cells without hyperpolarization, where the resting potentials were about -55 mV. It was further noted that the threshold increase in the T - L relation under hyperpolarized conditions was associated with a modification of potential trajectories; the rate of depolarization of the membrane potential was abruptly decreased, showing a distinct inflection, which implies a rapid reduction of input resistance, at a certain point (arrowheads in Fig. 1B1) between 7 and 35 ms after the stimulus onset. The phenomenon was evident when the stimulus gradient was 18-58 nA/s (Fig. 1B 1, left and middle), while smaller gradient stimulations failed to evoke it (Fig. 1B1, right). The inflections occurred earlier with steeper ramp currents, unless action potentials were triggered before them. Besides, at the stimulation with a given gradient, the inflections were empha-
A
2.° 1
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1.5
sized by larger polarizations, and were reduced by repolarization to the original potential, reversibly (cf. inset in Fig. 2At). A similar trajectory has been observed in the leech sensory neurons in relation to its accommodative property TM. The response was ascribed mainly to the delayed rectifier current. The assumption does not hold, however, in the case of the SNC neuron, since the threshold increase demands sufficient hyperpolarization before depolarization, but did not occur by slower depolarizations. It is possible that the responses may be related to some Ca2*-mediated outward currents, since certain types of Ca 2÷ currents may be generated by depolarization under the hyperpolarized condition 13. Substitution of Mg 2+ ions for Ca 2÷ in the perfusate, however, little affected either the T - L curve or the potential trajectories (Fig. 2A1). At that time the adaptation to a rectangular stimulation, which was due to the Ca2*-activated K+ current, was totally removed (not shown).
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Fig. 2. A: effects of extracellular divalent cations on the responses to ramp current stimulation on hyperpolarized cells. AI: the T - L relations in Ca2+-free perfusate. O, control: +, Ca-free perfusate. Inset: potential responses in the Ca2*-free solution at different hyperpolarizations. A2: the T - L relations in the modified solution containing 0.4 mM Cd 2÷ ions. O, control; +, 0.4 mM Cd 2+. Inset: potential responses in the Cd 2* solution. The scale bars indicate 10 ms, 0.4 nA and 20 mV. B: potential responses in a strongly hyperpolarized cell. Left: control. Hyperpolarizing sags (arrowhead) can be observed in recovery potentials after the hyperpolarization. Middle: responses of the cell in the Ca2+-free perfusate. Right: responses in the solution containing 0.4 mM Cd 2+ ions. The spike is truncated. The scale bar indicates 100 ms, 2.0 nA and 20 mV. The resting potential was -51 _+ 1 mV.
180 Judging from these results, the most likely source of the a b o v e - m e n t i o n e d events in the SNC neuron would be a v o l t a g e - d e p e n d e n t , fast inactivating outward current (IA). A l t h o u g h the effect of 4-aminopyridine 22 could not be examined because of its deteriorative action on the m e m b r a n e of the SNC neuron, this inference may also be s u p p o r t e d by correspondence of the ramp current-induced response with an /A-related reaction. It has been described that a strong hyperpolarization (>1 n A ) of the SNC neuron is followed by a potential sag associated with a reduction of m e m b r a n e impedance l° (Fig. 2B, left), which has been considered an indication of the I A (refs. 6, 10). The potential sag (Fig. 2B, middle) was emphasized by the Ca2+-free solution, which may be due to reduction of slow Ca 2+ potential 1°,~3, but it was suppressed largely by the addition of Cd 2+ ions (0.4 mM) to the perfusate (Fig. 2B, right). Correspondingly, the ramp current-induced responses, both the inflection in the potential trajectory and the threshold increase on the T - L curve, entirely disappeared when Cd 2+ ions (0.4 mM) were applied (Fig. 2A2). Similar results were also obtained when Co 2+ ions (5 mM) were administered. Each of the spikes in the SNC neuron is followed by a long-lasting A H P larger than 10 mV in amplitude I°'14. Therefore, the transient increase in threshold could serve as a factor limiting the rate of spontaneous firings, as suggested for the I A in gastropod neurons 4. The shape of the T - L curve may represent a strong barrier for successive firing at shorter inter-
1 Araki, T. and Otani, T., Accommodation and local response in motoneurons of toad's spinal cord, Jpn. J. Physiol., 9 (1959) 69-83. 2 Bradley, K. and Somjen, G.G., Accommodation in motoneurones of the rat and the cat, J. Physiol. (Lond.), 156 (1961) 75-92. 3 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-4phenyl-l,2,3,6-tetrahydropyridine, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 4546-4550. 4 Connor, J.A. and Stevens, C.F., Prediction of repetitive firing behavior from voltage clamp data on an isolated neurone soma, J. Physiol. (Lond.), 213 (1971) 31-53. 5 Crepel, F. and Penit-Soria, J., Inward rectification and low threshold calcium conductance in rat cerebellar Purkinje cells. An in vitro study, J. Physiol. (Lond.), 372 (1986) 1-23. 6 Galvan, M. and Sedlmeir, C., Outward currents in voltage-
vals. Besides, because the mechanism does not operate with slower depolarization, it would also allow high frequency bursts to generate on slow and large depolarization 7. Modification of the T - L curve with m e m b r a n e potential level was also described in other neurons in vivo 2'12'17, but none of them displayed the T - L curve with a peak as observed in the SNC neuron. Recently, t h e / A - l i k e transient current has been r e p o r t e d in the neocortex neurons in vitro 21. Therefore, it is possible that these neurons would exhibit a threshold peak on the T - L relation if they were stimulated with ramp current at negative holding potentials. The disappearance of threshold peak after application of Ca2+-blockers, Cd z+ and Co 2+, is puzzling. This might occur if the activation curve for I A was shifted by the ions in the depolarizing direction as was reported for Co 2+ in hippocampal neurons 15. The p h e n o m e n o n might also be due to a facilitatory effect of these ions on some inward currents, which may be implicitly shown in previous reports 5'1a. Otherwise, Cd 2+ might directly interfere with the current system responsible for the d e v e l o p m e n t of the threshold peak. A t present, we cannot identify the mechanisms underlying the Cd 2+ effect in this study.
The authors thank Dr. S. Yoshida for his helpful comments on the manuscript and to Drs. Susan Plant and A . I . McNiven for improving the English.
7
8 9 10 11
12
clamped rat sympathetic neurones, J. Physiol. (Lond.), 356 (1984) 115-133. Grace, A.A. and Bunney, B.S., Intracellular and extracellular electrophysiology of nigral dopaminergic neurons 1. Identification and characterization, Neuroscience, 10 (1983) 301-315. Guyenet, P.G. and Aghajanian, G.K., Antidromic identification of dopaminergic and other output neurons of the rat substantia nigra, Brain Research, 150 (1978) 69-84. Kernell, D., The limits of frequency in cat lumbosacral motoneurones possessing different time course of afterhyperpolarization, Acta Physiol. Scand., 65 (1965) 87-100. Kita, T., Kita, H. and Kitai, S.T., Electrical membrane properties of rat substantia nigra compacta neurons in an in vitro slice preparation, Brain Research, 372 (1986) 21-30. Koike, H., Okada, Y., Oshima, T. and Takahashi, K., Accommodative behavior of cat pyramidal tract cells investigated with intracellular injection of currents, Exp. Brain Res., 5 (1968) 173-188. Koike, H., Okada, Y. and Oshima, T., Accommodative
181
13
properties of fast and slow pyramidal tract cells and their modification by different levels of their membrane potential, Exp. Brain Res., 5 (1968) 189-201. Llin~is, R., Greenfield, S.A. and Jahnsen, H., Electrophysiology of pars compacta cells in the in vitro substantia nigra a possible mechanism for dendritic release, Brain Research, 294 (1984) 127-132. Matsuda, Y., Fujimura, K. and Yoshida, S., Two types of neurons in the substantia nigra pars compacta studied in a slice preparation, Neurosci. Res., 5 (1987) 172-179. Numman, R.E., Wadman, W.J. and Wong, R.K., Outward currents of single hippocampal cells obtained from the adult guinea-pig, J. Physiol. (Lond.), 393 (1987) 331-353. Sasaki, K. and Otani, T., Accommodation in spinal motoneurons of the cat, Jpn. J. Physiol., 11 (1961) 443-456. Sasaki, K. and Oka, H., Accommodation, local response and membrane potential in spinal motoneurons of the cat, Jpn. J. Physiol., 13 (1963) 508-522. Schlue, W.R., Sensory neurons in leech central nervous -
14
15
16 17
18
19
-
20
21
22
system: changes in potassium conductance and excitation threshold, J. Neurophysiol., 39 (1976) 1184-1192. Schlue, W.R., Richter, D.W., Maurits, K.-H. and Nacimiento, A.C., Responses of cat spinal motoneuron somata and axons to linearly rising currents, J. Neurophysiol., 37 (1974a) 303-309. Schlue, W.R., Richter, D.W., Maurits, K.-H. and Nacimiento, A.C., Mechanisms of accommodation to linearly rising currents in cat spinal motoneurons, J. Neurophysiol., 37 (1974b) 310-315. Scwindt, P.C., Spain, W.J., Foehring, R.C., Stafstrom, C.E., Chubb, M.C. and Crill, W.E., Multiple potassium conductances and their functions in neurons from cat sensorimotor cortex in vitro, J. Neurophysiol., 59 (1988) 424-449. Thompson, S.H., Three pharmacologically distinct potassium channels in molluscan neurones, J. Physiol. (Lond.), 265 (1977) 465-488.
177
Elsevier BRE 23237
Responses to ramp current stimulation of the neurons in substantia nigra pars compacta in vitro K. Fujimura and Y. Matsuda Department of Physiology, Nagasaki UniversitySchool of Medicine, Nagasaki (Japan) (Accepted 23 August 1988)
Key words: Accommodation; Hyperpolarization; A-current; Cadmium; Pars compacta neuron; Substantia nigra
The response to ramp current stimulation was studied in the pars compacta neurons in guinea pig substantia nigra slices. Although accommodation was not seen with current duration up to 1000 ms, a threshold peak emerged on the threshold-latency curve at 30-400 ms when the cell was hyperpolarized. The appearance of the peak was closely correlated with an inflection in the potential trajectory before the spike. A voltage-dependent, fast inactivating outward current may underlie these responses. They persisted in Ca2+-free solution, but disappeared when Cd2÷ (0.4 mM) or Co2÷ (5 mM) ions were applied extracellularly.
It is now established that degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNC) is closely related to Parkinson's syndrome in primates 3. The neurons fire at a relatively low rate 8"14, and they adapt readily to direct stimulation 7'1°. The stability of low frequency firing may be of functional importance in the basal supply of dopamine to the striatum. In general, a major factor which controls the rate of neural firing may be the spike afterhyperpolarization (AHP) 9. Indeed, the SNC neuron shows a profound A H P , which is suggested to be due to a Ca2+-activated K + current 1°. However, some other factors could still control the spike discharge. Thus, an increase of spike threshold in response to an increasing depolarization has been observed in several mammalian central neurons 1'2,11'16'19. This response, termed 'accommodation', may be ascribed to the delayed rectifier current and/or partial inactivation of Na ÷ current 2°. It is also suggested that the voltage-dependent transient outward current, Acurrent (IA), can participate in it 4. Nevertheless, it has not yet been shown whether the accommodation is involved in the firing of the SNC neuron. In the present study, we examined the accommodation of the SNC neuron in slice preparations with linearly in-
creasing current (ramp current) stimulation. We found that the response can be modulated greatly by the shift of resting membrane potential. Slices of the substantia nigra obtained from male guinea pigs (250-350 g) were used for the experiment. After decapitation, the brain was quickly removed from the skull, and coronal section of the midbrain containing the SNC was performed at a thickness of 400 ¢tm using a vibrating slicer. The slices were incubated in standard Krebs solution (mM): NaC1 124, KCI 5, KH2PO 4 1.24, MgSO 4 1.3, CaC12 2.4, N a H C O 3 26, glucose 10 (pH 7.4), which was bubbled continuously with a mixed gas of 95% 02/5% CO2. A modified saline, in which KH2PO 4 and MgSO 4 were replaced, respectively, by KC! and MgC12 in an equimolar concentration, was used when Cd 2+ or Co 2+ ions were applied. Calcium-free Krebs solution (containing 5 mM Mg 2+) was also used. The temperature of the superfusate was 30 °C. Intracellular recording was performed with a 4 M potassium acetate-filled microelectrode. Current injection was made through the recording electrode by means of a bridge circuit. The study is based on data obtained from 25 successful recordings from the major type 14 of neurons
Correspondence: K. Fujimura, Department of Physiology, Nagasaki University School of Medicine, Nagasaki 852, Japan. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
178 of the SNC. Resting membrane potentials ranged from - 5 0 to - 7 0 mV (-55 _+ 5 mV, mean _+ S.D.),
Rather, the current amplitude decreased as the latency was prolonged, up to 1000 ms.
when measured at the midpoint between the spontaneously triggering level and the bottom of the AHP.
The T - L curve for the neuron was altered greatly, however, by hyperpolarizing the m e m b r a n e poten-
The input resistance measured with hyperpolarizing
tial. Fig. 1B~ shows the T - L relations for the neurons presented in Fig. 1A~, which are now hyperpolarized
pulse was 71 _+ 19 Mff2 at peak deflection. When the neuron at its "resting potential" was stimulated with
by 12 _+ 2 mV (i.e. m e m b r a n e potential, - 6 6 _+ 7 mV) with continuous current (t). 15-0.38 nA); the sponta-
ramp current, the m e m b r a n e potential depolarized almost linearly with the current, until a spike was triggered (Fig. 1Al). The latency of the firing was
neous discharges were suppressed by polarization. It should be noticed that the amplitude of threshold
prolonged as the stimulus gradient was reduced. Fig.
current increased abruptly at a point in the middle of
1A~ shows the relationship between the spike latency
T - L relations. Thereafter, it decreased again at a
and the threshold current for the stimulations with various gradients (ca. 500 nA/s to 0.05 nA/s) in 4 rep-
high rate along with the latency up to 200-400 ms. A total of 11 cells examined exhibited this pattern, and the threshold peak (1.1 _+ 0.4 nA) emerged at a latency of 33 _+ 5 ms (current gradient 34 _+ 17 hA/s)
resentative cells. If the neuron ~accommodated' itself to the stimulation, the threshold-latency ( T - L ) curve would rise gradually with prolongation of the latency 211. Most of the neurons (16/18 cells), howev-
during a hyperpolarization of 11 _+ 3 mV with a continuous current injection of 0.3 + 0.1 nA. The threshold was 0.7 + 0.4 n A at 100 ms latency (current gra-
er, did not demonstrate any sign of accommodation.
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Fig. 1. AI: potential responses of an SNC neuron to ramp current stimulations. The membrane potential depolarized almost linearly with the current before firing. Span of a sweep is indicated on each record. The scale bar indicates 0.4 nA and 20 mV for the left and the middle, 0.2 nA and 20 mV for the right, respectively. Firing latency was prolonged with reduction of stimulus gradient. Az: threshold-latency (T-L) relations for the responses of 4 cells; each of the 4 symbols represents the data from an individual cell. Stimulus current amplitude at the time of the initial firing is plotted against the latency in logarithmic scale, for different gradient stimulations (ca. 500 nA/s - - 0.05 nA/s). The threshold decreased as the latency was prolonged. Bl: potential responses of a hyperpolarized cell. An inflection was observed in the trajectory of the slow depolarization before firing by ramp current stimulation (arrowheads in the left and the middle). The scale bar indicates 1.0 nA and 20 mV for the left and the middle, 0.5 nA and 20 mV for the right, respectively. Hyperpolarizating current was 0.38 nA. B2: T-L relations for the hyperpolarized cells in A, with ramp current stimulation. The threshold increased in the range of the latency between about 30 and 400 ms. The hyperpolarizing current was 0.24 +_0.1 nA (mean +_S.D.), resulting in a 12 _+2 mV hyperpolarization.
179 dient ca. 7 nA/s), and was 0.3 + 0.2 nA at 300 ms (ca. 1 nA/s). A similar transient deflection of the T - L curve was observed in two other cells without hyperpolarization, where the resting potentials were about -55 mV. It was further noted that the threshold increase in the T - L relation under hyperpolarized conditions was associated with a modification of potential trajectories; the rate of depolarization of the membrane potential was abruptly decreased, showing a distinct inflection, which implies a rapid reduction of input resistance, at a certain point (arrowheads in Fig. 1B1) between 7 and 35 ms after the stimulus onset. The phenomenon was evident when the stimulus gradient was 18-58 nA/s (Fig. 1B 1, left and middle), while smaller gradient stimulations failed to evoke it (Fig. 1B1, right). The inflections occurred earlier with steeper ramp currents, unless action potentials were triggered before them. Besides, at the stimulation with a given gradient, the inflections were empha-
A
2.° 1
•,
1.5
sized by larger polarizations, and were reduced by repolarization to the original potential, reversibly (cf. inset in Fig. 2At). A similar trajectory has been observed in the leech sensory neurons in relation to its accommodative property TM. The response was ascribed mainly to the delayed rectifier current. The assumption does not hold, however, in the case of the SNC neuron, since the threshold increase demands sufficient hyperpolarization before depolarization, but did not occur by slower depolarizations. It is possible that the responses may be related to some Ca2*-mediated outward currents, since certain types of Ca 2÷ currents may be generated by depolarization under the hyperpolarized condition 13. Substitution of Mg 2+ ions for Ca 2÷ in the perfusate, however, little affected either the T - L curve or the potential trajectories (Fig. 2A1). At that time the adaptation to a rectangular stimulation, which was due to the Ca2*-activated K+ current, was totally removed (not shown).
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!
Fig. 2. A: effects of extracellular divalent cations on the responses to ramp current stimulation on hyperpolarized cells. AI: the T - L relations in Ca2+-free perfusate. O, control: +, Ca-free perfusate. Inset: potential responses in the Ca2*-free solution at different hyperpolarizations. A2: the T - L relations in the modified solution containing 0.4 mM Cd 2÷ ions. O, control; +, 0.4 mM Cd 2+. Inset: potential responses in the Cd 2* solution. The scale bars indicate 10 ms, 0.4 nA and 20 mV. B: potential responses in a strongly hyperpolarized cell. Left: control. Hyperpolarizing sags (arrowhead) can be observed in recovery potentials after the hyperpolarization. Middle: responses of the cell in the Ca2+-free perfusate. Right: responses in the solution containing 0.4 mM Cd 2+ ions. The spike is truncated. The scale bar indicates 100 ms, 2.0 nA and 20 mV. The resting potential was -51 _+ 1 mV.
180 Judging from these results, the most likely source of the a b o v e - m e n t i o n e d events in the SNC neuron would be a v o l t a g e - d e p e n d e n t , fast inactivating outward current (IA). A l t h o u g h the effect of 4-aminopyridine 22 could not be examined because of its deteriorative action on the m e m b r a n e of the SNC neuron, this inference may also be s u p p o r t e d by correspondence of the ramp current-induced response with an /A-related reaction. It has been described that a strong hyperpolarization (>1 n A ) of the SNC neuron is followed by a potential sag associated with a reduction of m e m b r a n e impedance l° (Fig. 2B, left), which has been considered an indication of the I A (refs. 6, 10). The potential sag (Fig. 2B, middle) was emphasized by the Ca2+-free solution, which may be due to reduction of slow Ca 2+ potential 1°,~3, but it was suppressed largely by the addition of Cd 2+ ions (0.4 mM) to the perfusate (Fig. 2B, right). Correspondingly, the ramp current-induced responses, both the inflection in the potential trajectory and the threshold increase on the T - L curve, entirely disappeared when Cd 2+ ions (0.4 mM) were applied (Fig. 2A2). Similar results were also obtained when Co 2+ ions (5 mM) were administered. Each of the spikes in the SNC neuron is followed by a long-lasting A H P larger than 10 mV in amplitude I°'14. Therefore, the transient increase in threshold could serve as a factor limiting the rate of spontaneous firings, as suggested for the I A in gastropod neurons 4. The shape of the T - L curve may represent a strong barrier for successive firing at shorter inter-
1 Araki, T. and Otani, T., Accommodation and local response in motoneurons of toad's spinal cord, Jpn. J. Physiol., 9 (1959) 69-83. 2 Bradley, K. and Somjen, G.G., Accommodation in motoneurones of the rat and the cat, J. Physiol. (Lond.), 156 (1961) 75-92. 3 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-4phenyl-l,2,3,6-tetrahydropyridine, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 4546-4550. 4 Connor, J.A. and Stevens, C.F., Prediction of repetitive firing behavior from voltage clamp data on an isolated neurone soma, J. Physiol. (Lond.), 213 (1971) 31-53. 5 Crepel, F. and Penit-Soria, J., Inward rectification and low threshold calcium conductance in rat cerebellar Purkinje cells. An in vitro study, J. Physiol. (Lond.), 372 (1986) 1-23. 6 Galvan, M. and Sedlmeir, C., Outward currents in voltage-
vals. Besides, because the mechanism does not operate with slower depolarization, it would also allow high frequency bursts to generate on slow and large depolarization 7. Modification of the T - L curve with m e m b r a n e potential level was also described in other neurons in vivo 2'12'17, but none of them displayed the T - L curve with a peak as observed in the SNC neuron. Recently, t h e / A - l i k e transient current has been r e p o r t e d in the neocortex neurons in vitro 21. Therefore, it is possible that these neurons would exhibit a threshold peak on the T - L relation if they were stimulated with ramp current at negative holding potentials. The disappearance of threshold peak after application of Ca2+-blockers, Cd z+ and Co 2+, is puzzling. This might occur if the activation curve for I A was shifted by the ions in the depolarizing direction as was reported for Co 2+ in hippocampal neurons 15. The p h e n o m e n o n might also be due to a facilitatory effect of these ions on some inward currents, which may be implicitly shown in previous reports 5'1a. Otherwise, Cd 2+ might directly interfere with the current system responsible for the d e v e l o p m e n t of the threshold peak. A t present, we cannot identify the mechanisms underlying the Cd 2+ effect in this study.
The authors thank Dr. S. Yoshida for his helpful comments on the manuscript and to Drs. Susan Plant and A . I . McNiven for improving the English.
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