Neuroscience Letters, 104 (1989) 53 57 Elsevier Scientific Publishers Ireland Ltd.

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NSL 06288

Autogenous oscillatory potentials in neurons of the guinea pig substantia nigra pars compacta in vitro Koichi Fujimura and Yoshihiro Matsuda Department of Physiology, Nagasaki University School of Medicine, Nagasaki (Japan) (Received 3 March 1989; Revised version received 1 May 1989;Accepted 2 May 1989) Key words. Substantia nigra pars compacta; Slice preparation; Spontaneous firing; Membrane potential oscillation; Tetrodotoxin; Calcium dependency In spontaneously firing neurons of the guinea pig substantia nigra pars compacta (SNC) maintained in slices, blockade of the fast spikes by tetrodotoxin (TTX) revealed a slow oscillatory potential change, which was depressed by Cd2÷, a Ca2÷-channel blocker. The spontaneous firing was suppressed by the application of Ca2÷-free saline, Cd2+ or Co2+, but not by nifedipine. These findings lend support to the view that the spontaneous firing of SNC neurons is produced by an intrinsic, Ca2+-dependent pacemaking process affected by Cd2÷ but not by nifedipine.

N e u r o n s in the substantia nigra pars c o m p a c t a (SNC) in situ exhibit a persistent firing o f low-frequency spikes, interspersed with bursts [1, 6, 7, 14, 15]. This persistent firing m a y be o f significance in providing the tonic release o f d o p a m i n e from S N C to the striatum. S N C neurons exhibit a similar persistent firing, even in slice preparations [9, 10, 13]. Since tonic synaptic drive is unlikely to occur in a slice preparation, the persistent firing m a y result from events intrinsic to the neurons. We report here that the principal (presumably dopaminergic) S N C neurons [7, 10, 13] are capable o f producing autogenous oscillatory potentials, which m a y represent a process underlying the persistent firing. Guinea pigs, weighing 250-300g, were decapitated and the brain was rapidly removed and immersed in ice-cold saline. The midbrain containing the substantia nigra was cut in a frontal plane at a thickness o f 400/~m, using a vibrating slicer. After incubation for at least 2 h, the slice was fixed on a nylon grid in an experimental c h a m b e r (1 ml in volume), and was perfused with saline at 30-33°C. The composition o f the saline was as follows (in mM): N a C I 124, KCI 5, CaC12 2.4, MgSO4 1.3, KH2PO4 1.24, N a H C O 3 26, and glucose 10. W h e n CdCI2 was used as the Ca2+-chan nel blocker, KH2PO4 was omitted and the concentration o f KC1 was increased to 6.24 m M . Nifedipine was stored in the dark as a 10 m M stock solution in ethanol and Correspondence: Y. Matsuda, Department of Physiology, Nagasaki University, School of Medicine, Nagasaki 852, Japan. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.

54 added to the perfusing solution just before use. The perfusing solutions were saturated with 95 % 02/5% CO2. For intracellular recordings, glass microelectrodes filled with 4-M potassium acetate were used. Currents were injected into the neurons, when necessary, through a bridge circuit. Neuronal responses were either photographed on an oscilloscope or preserved on a chart recorder. The resting membrane potential was continuously monitored, in every experiment. Data were obtained on 34 SNC neurons which produced action potentials exceeding 60 mV in amplitude (72.5 _+ 5.7 mV, mean + S.D.). The spike duration measured at the midpoint of spike height was 1.3+_0.4 ms and the maximum rate of rise of spike was 133_+38 V/s (mean _+ S.D.). These neurons were considered the principal (presumably dopaminergic) neurons in the SNC, on the basis of their electrophysiological properties [4, 13]. About half of all the neurons tested exhibited a spontaneous firing of action potentials. The firing was regular and of low frequency (in the range of 0.5 4.3 Hz with a mean of 1.4+_ 1.0 Hz, n = 17), as noted in rat SNC neurons in slices [10]. Few impingements of synaptic potentials were observed. Each spike was accompanied by an afterhyperpolarization and then by a slowly rising depolarization that eventually triggered the next spike. The firing decreased in frequency when the neurons were hyperpolarized by injecting currents and the spontaneous activities were usually abolished at levels of potential negative to - 6 0 mV. However, the level of the resting membrane potential did not seem to be the sole factor determining whether or not the neurons fired spontaneously: some of the selected neurons produced no spontaneous activity although their resting membrane potential was at a relatively depolarized level. When the membrane potential measured at the midpoint between the bottom of the afterhyperpolarization and the triggering level of the spike was taken as 'resting' potential in spontaneously active neurons, the value (50_+8 mV, n = 17) did not differ significantly from the resting membrane potential of the silent neurons (55 +_6 mV, n = 17). Observation of the membrane potential trajectories of the spontaneously active neurons suggested that the slowly growing depolarizing potential preceding the spike would be of prime importance for the rhythmic sequence of neuronal excitation. As synaptic potentials were rarely evident, we assumed that SNC neurons possess an intrinsic mechanism which can induce a slow depolarization of the membrane, to the threshold level required for excitation. We questioned whether this process would be evident in the absence of fast Na spikes. Autogenous potential changes under this condition, if any, would be produced by an intrinsic mechanism, since the synaptic input would have been abolished. When the fast Na spikes were suppressed by tetrodotoxin (TTX, 10 6 g/ml), as shown in Fig. 1, 4 out of 8 tested neurons exhibited a recurring potential fluctuation, mainly composed of slow depolarizing waves. This fluctuation persisted for over l0 min after applying TTX. These changes usually recurred at irregular intervals (Fig. 1A, middle frame), except for a neuron which exhibited a cyclic voltage change occurring at a regular interval (Fig. I B, middle frame). The amplitude of the fluctuation ranged from 7 to 15 mV (12_+4 mV, mean -+ S.D., n = 4 ) and the bottom of the fluctuation was situated at levels of - 3 6 to - 4 8 mV ( - 4 3 _ + 5 mV). Fluctuation of the potential was affected by the Ca2~-channel

55

k

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Cd 8rnln

TTX lOmln

Is

B

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TTX iOmin

200ms

Wash 401~In

400ms

Fig. 1. Spontaneous potential fluctuations in SNC neurons observed after blockade of fast spikes by tetrodotoxin (TTX). Records from two neurons are presented. Note irregular (A) and regular (B) changes in the potential in the presence of TTX. The neurons were perfused with a medium containing TTX (10 6 g/ml) for 20 min, and, thereafter, the bathing medium was switched either ~o a solution containing CdC12 (0.4 mM) (A) or to the standard saline (B). In A, responses were recorded with a chart recorder and fast spikes in the control record were curtailed. Arrowheads at the beginning of the record in the middle frames indicate the membrane potential levels of - 3 0 and - 4 0 mV in A and B, respectively. Time scale of 400 ms also applies to the record to the right in B.

blocker, CdC12. In Fig. 1A, the autogenous fluctuation was maintained for 20 min after initiating the superfusion of TTX. The addition of CdC12 (0.4 mM) depressed the fluctuation (Fig. 1A, record to the right), hence a Ca2+-dependent permeability mechanism may be linked to the phenomenon. Contribution of Ca 2+-dependent mechanisms to the process sustaining the spontaneous activity was also suggested by the observation that the spontaneous firing of SNC neurons was suppressed by the application to the neurons of Ca2÷-channel blockers as well as by removal of Ca ions from the bathing medium. Fig. 2A shows the effect of CdCI2 on a spontaneously active SNC neuron. Immediately after applying cadmium at concentrations of 0.2-1.0 mM, firing of the neurons became irregular and the frequency drastically decreased (Fig. 2A, 2nd trace). Failure of the slowly rising depolarizing potential that usually followed the spike afterhyperpolarizations under control conditions was frequent. At 7-8 min after the administration of CdC12, the spontaneous firings ceased (Fig. 2A, 3rd trace). A prolonged washout was required to restore the spontaneous firing (Fig. 2A, 4th trace). The suppressing effect of cadmium on the spontaneous firings was confirmed in 8 S N C neurons. A similar result was observed in a neuron in which COC12 (5 mM) was used as the Ca2+-chan nel blocker. The spontaneous firing in SNC neurons ceased when the neurons were perfused with a Ca2+-free saline (Fig. 2B). It is worth noting that the spontaneous firing was not affected by nifedipine (10 - 4 M), a potent blocker for the L-type Ca 2+ channel [3]. SNC neurons were capable of producing action potentials on direct stimulation,

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A

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Cd 2min

B

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O-Ca++ 8min

Cd lOmin

O-Ca++ 15min

Wash 20min

Wash lOmin

ls Fig. 2. Suppression of the spontaneous firing of SNC neurons by c a d m i u m (A) and by perfusion with a Ca: ~-free saline (B). Records were obtained with a chart recorder and fast spikes were curtailed. Cadmium was applied at a concentration of 0.4 raM. The washout was started immediately after records in the 3rd column had been obtained. Calibrations are c o m m o n to A and B. Arrowheads at the beginning of the control records indicate the membrane potential level of - 5 0 mV.

even when the spontaneous firing was suppressed by divalent cations or by removal of Ca ions from the perfusing medium. Shape of the action potentials changed with these procedures; the spike was narrowed due to disappearance of the slow component of its falling phase [13] and the spike afterhyperpolarization was shortened, probably due to depression of the Ca 2+-activated K + currents [9]. Our finding that some of the principal (presumably dopaminergic) SNC neurons [7, 10, 13] are capable of producing autogenous potential changes in the presence of TTX lends support to the view that the neurons are endowed with an intrinsic mechanism for the generation of spontaneous firing. The suppression of spontaneous activities by Ca2+-channel blockers suggests the participation of a Ca 2~ permeability system. Putative intrinsic mechanisms may act in depolarizing the neuronal membrane to the threshold for spike generation, thereby playing the role of pacemaker. In the general scheme of autogenous excitation, a non-inactivating or slowly inactivating inward current is considered to underlie such a 'pacemaker' potential and the current may be carried by Ca ions, in certain cases [2, 5, 12, 17]. The current system sustaining the spontaneous firing of SNC neurons appears different from the L-type Ca 2. channel that is sensitive to dihydropyridines [3]. The possibility that the low-threshold Ca2+-dependent responses in SNC neurons [9, 11] participate in the generation of autogenous potential fluctuation in the presence of TTX can be ruled out since they should be inactivated in the membrane potential range in which fluctuation of the potential occurred [8, 16]; the bottom of the potential fluctuation was at the range from - 4 8 to - 3 6 i n V . The autogenous potential changes of SNC neurons observed in the presence of TTX were often irregular, presumably because in the absence of fast spikes after application of TTX, SNC neurons failed to produce the well-developed afterhyperpolarization which contributes to activation of'pacemaker' potential. Other subtle conditions seem to be required for elicitation of intrinsic activity of SNC neurons, since

57 n o t all t h e n e u r o n s w e r e s p o n t a n e o u s l y

a c t i v e , e i t h e r i n t h e s t a n d a r d p e r f u s i n g so-

lution or after the application of TTX. We thank M. Ohara for comments and M. Yogata for assistance with the illustrations. 1 Deniau, J.M., Hammond, C., Riszk, A. and Feger, J., Electrophysiological properties of identified output neurons of the rat substantia nigra (pars compacta and pars reticulata): evidence for the existence of branched neurons, Exp. Brain. Res., 32 (1978) 409~,22. 2 Eckert, R. and Lux, H.D., A voltage-sensitive persistent calcium conductance in neuronal somata of Helix, J. Physiol. (Lond.), 254 (1976) 129 151. 3 Fox, A.P., Nowycky, M.C. and Tsien, R.W., Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones, J. Physiol. (Lond.), 394 (1987) 14%172. 4 Fujimura, K. and Matsuda, Y., Responses to ramp current stimulation of the neurons in substantia nigra pars compacta in vitro, Brain Res., 475 (1988) 177-181. 5 Gorman. A.L.F., Hermann, A. and Thomas, M.V., Ionic requirements for membrane oscillations and their dependence on the calcium concentration in a molluscan pace-maker neurone, J. Physiol. (Lond.), 327 (1982) 185 217. 6 Grace, A.A. and Bunney, B.S., Intracellular and extracellular electrophysiology of nigral dopaminergic neurons. 1. Identification and characterization, Neuroscience, I0 (1983) 301-315. 7 Guyenet, P.G. and Aghajanian, G.K., Antidromic identification of dopaminergic and other output neurons of the rat substantia nigra, Brain Res., 150 (1978) 69-84. 8 Jahnsen, H. and Llin~s, R., Ionic basis for the electroresponsiveness and oscillatory properties of guinea-pig thalamic neurones in vitro, J. Physiol. (Lond.), 349 (1984) 227 247. 9 Kita, T., Kita, H. and Kitai, S.T., Electrical membrane properties of rat substantia nigra compacta neurons in an in vitro slice preparation, Brain Res., 372 (1986) 21-30. 10 Lacey, M.G., Mercuri, N.B. and North, R.A., Dopamine acts on D2 receptors to increase potassium conductance in neurones of the rat substantia nigra zona compacta, J. Physiol. (Lond.), 392 (1987) 397-416. 11 Llinfis, 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 Res., 294 (1984) 127 132. 12 Elinb.s, R. and Sugimori, M., Electrophysiological properties of in vitro purkinje cell somata in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 171 -195. 13 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. 14 Sanghera, M.K., Trulson, M.E. and German, D.C., Electrophysiological properties of mouse dopamine neurons: in vivo and in vitro studies, Neuroscience, 12 (1984) 793 801. 15 Strecker, R.E. and Jacobs, B.L., Substantia nigra dopaminergic unit activity in behaving cats: effect of arousal on spontaneous discharge and sensory evoked activity, Brain Res., 361 (1985) 33%350. 16 Wilcox, K.S., Gutnick, M.J. and Christoph, G.R., Electrophysiological properties of neurons in the lateral habenula nucleus: an in vitro study, J. Neurophysiol., 59 (1988) 212-225. 17 Williams, J.T., North, R.A., Shefner, S.A., Nishi, S. and Egan, T.M., Membrane properties of rat locus coeruleus neurons, Neuroscience, 13 (1984) 136~156.