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Pergamon PII: Neuroscience Vol. 77, No. 4, pp. 1021–1028, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(96)00555-6 ELECTROPHYSIOLOGICAL STUDIES OF RAT SUBSTANTIA NIGRA NEURONS IN AN IN VITRO SLICE PREPARATION AFTER MIDDLE CEREBRAL ARTERY OCCLUSION H. NAKANISHI,*‡ A. TAMURA,† K. KAWAI† and K. YAMAMOTO* *Department of Pharmacology, Faculty of Dentistry, Kyushu University, Fukuoka 812, Japan †Department of Neurosurgery, Teikyo University, School of Medicine, Tokyo 173, Japan Abstract––We studied sequential changes in electrophysiological profiles of the ipsilateral substantia nigra neurons in an in vitro slice preparation obtained from the middle cerebral artery-occluded rats. Histological examination revealed marked atrophy and neurodegeneration in the ipsilateral substantia nigra pars reticulata at 14 days after middle cerebral artery occlusion. Compared with the control group, there was no significant change in electrical membrane properties and synaptic responses of substantia nigra pars reticulata neurons examined at one to two weeks after middle cerebral artery occlusion. On the other hand, there was a significant increase in the input resistance and spontaneous firing rate of substantia nigra pars compacta neurons at 13–16 days after middle cerebral artery occlusion. Furthermore, inhibitory postsynaptic potentials evoked by stimulation of the subthalamus in substantia nigra pars compacta neurons was suppressed at five to eight days after middle cerebral artery occlusion. At the same time excitatory postsynaptic potentials evoked by the subthalamic stimulation was increased. Bath application of bicuculline methiodide (50 µM), a GABAA receptor antagonist, significantly increased the firing rate of substantia nigra pars compacta neurons from intact rats. These results strongly suggest that changes in electrophysiological responses observed in substantia nigra pars compacta neurons is caused by degeneration of GABAergic afferents from the substantia nigra pars reticulata following middle cerebral artery occlusion. While previous studies indirectly suggested that hyperexcitation due to deafferentation from the neostriatum may be a major underlying mechanism in delayed degeneration of substantia nigra pars reticulata neurons after middle cerebral artery occlusion, the present electrophysiological experiments provide evidence of hyperexcitation in substantia nigra pars compacta neurons but not in pars reticulata neurons at the chronic phase of striatal infarction. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: middle cerebral artery occlusion, substantia nigra, electrical membrane property, synaptic response, slice preparation, rat. A permanent or transient occlusion of the middle cerebral artery (MCA) in the rat causes acute neuronal degeneration in the territory of the MCA such as the cortex and the lateral part of the neostriatum. 6,11,25,26 In contrast, delayed degeneration was observed in the substantia nigra which lies outside of the MCA territory.6,12,27 In rats, the neuropathological change in the substantia nigra was mainly localized in the pars reticulata (SNR). Neuronal damages evidenced by appearance of dark neurons were first detected in the ipsilateral SNR approximately one week after MCA occlusion, whereas there was no remarkable change in the substantia nigra pars compacta (SNC). After two weeks following MCA occlusion, neuronal loss, gliosis, and atrophy of the ipsilateral SNR were observed. Furthermore, there was a marked induc‡To whom correspondence should be addressed. Abbreviations: AHP, afterhyperpolarization; EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential; MCA, middle cerebral artery; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; STH, subthalamic nucleus. tion of heat shock protein in the ipsilateral SNR but not in SNC at three days after MCA occlusion.32 Neuronal degeneration of the substantia nigra following cerebral infarction in the striatum was also clinically recognized.2,13,18 Although the precise mechanism of delayed neuronal degeneration in the SNR following MCA occlusion is not known, there is increasing evidence that the excessive excitation induced by a loss of an inhibitory GABAergic input from the neostriatum and/or the globus pallidus plays a major role. The delayed degeneration of SNR neurons resembles the response after excitotoxin application in the striatum. The finding of Saji and Reise that the GABA-agonist muscimol prevents SNR degeneration following excitotoxic lesion of the ipsilateral neostriatum led to a disinhibition hypothesis which proposes that degeneration of SNR neurons after neostriatal lesion is caused by hyperexcitation due to inhibitory GABAergic deafferentation.22 The increased glucose utilization and blood flow12,26,28–30 were observed in the ipsilateral substantia nigra after neostriatal infarction induced by MCA occlusion. Furthermore, 1021 1022 H. Nakahishi et al. there was a long-lasting decrease in the concentration of GABA in the ipsilateral SNR.17 These observations are in accordance with the disinhibitory mechanism of the delayed SNR neuronal degeneration following MCA occlusion. However, little is known about changes in neuronal activities in the substantia nigra after MCA occlusion on the basis of electrophysiological study. The present study is an attempt to determine directly if substantia nigra neurons show hyperexcitability after MCA occlusion. In order to examine changes in electrophysiological profiles, we have made intracellular recordings from both SNR and SNC neurons at one to two weeks after MCA occlusion of the rat. In the present study, we employed an in vitro slice preparation since both the SNR and SNC are small and located deep in the brain, making it difficult to obtain a stable intracellular recording in in vivo preparations. EXPERIMENTAL PROCEDURES Surgical preparation This study was approved by the Animal Research Committee of the Teikyo University School of Medicine and the Kyushu University. The study was carried out using 44 male Sprague–Dawley rats (Shizuoka Lab. Animal Cent., Shizuoka, Japan), aged nine to 12 weeks and weighing 300–420 g. The rats were anaesthetized with 2% halothane, and in 34 rats the proximal portion of the left MCA was exposed by the transretro-orbital approach. In 29 rats the left MCA was then permanently occluded by the microsurgical technique that was modified from our original method reported previously.19,25,33 The stem of the MCA was electrocauterized just medial to the olfactory tract and was cut to ensure a complete vascular occlusion. In five sham-operated rats the MCA was only exposed. The remaining 10 rats were not operated on. Electrophysiology Intracellular recordings were made from SNR and SNC neurons in the slice preparations which were prepared at various times after MCA occlusion. 25 MCA-occluded rats were used for electrophysiological experiments at five to eight days (five days, n=3; seven days, n=2; eight days, n=4), 13–16 days (13 days, n=2; 14 days, n=3; 15 days, n=3; 16 days, n=6) and 21 days (n=3) after the operation. The remaining four MCA-occluded rats were killed before use. Sham-operated animals at seven days (n=2) and 14 days (n =3) after the operation and unoperated animals (n=6) were used as controls. In pharmacological experiments, four intact rats were used. Detailed procedure of slice preparation and methods for recording have been described elsewhere. 14–16 Immediately after decapitation under light ether anaesthesia, the brain was rapidly removed from the skull and trimmed with a razor blade to a block. Parasagittal slices (400 µm thickness) containing the substantia nigra were cut from the block with the use of a Vibratome (Vibroslice 752M, Campden Instrument, Cambridge, UK) and placed in an interface-type recording chamber with the bath temperature maintained at 36)C. The Krebs–Ringer solution for superfusion of the slice was composed of (in mM): NaCl 124, KCl 5.0, KH2PO4 1.24, NaHCO3 26, CaCl2 2.4, MgSO4 1.3 and glucose 10. Glass pipettes filled with 2 M K-citrate were used for intracellular recording. Recording electrodes had d.c. resistance of 100–250 MÙ. Intracellular recordings were obtained through a high input impedance amplifier (Neurodata IR183). Electrical stimula- tion (intensity 5–30 V, duration 200 µs, 0.5 Hz) was applied through a bipolar electrode placed on the subthalamic nucleus (STH). Electrical responses were stored in a videocassette recorder through a PCM data processor (VR-10B, Instrutech Corporation) and plotted on an X–Y recorder. The spike amplitude was measured from a threshold of the action potential. The spike duration was measured at threshold of the action potential. The input resistance was determined from the potential shifts across the membrane during passage of inward and outward rectangular current pulses of known intensity. The membrane time constant was estimated by a conventional method which utilizes a simple semilogarithmic plotting of the membrane potential change against time. The spontaneous firing rates were determined from interspike interval histograms which represented neuronal activities from 1000 sweeps with 0.5–1.0 ms bin width. Bicuculline methiodide (Sigma) was bath applied via the perfusing oxygenated Krebs–Ringer solution at a concentration of 50 µM. Numerical data are presented as mean&S.D. After electrophysiological examination, some slices were immersed in 4% paraformaldehyde and kept overnight at 4 )C. After washing, slices were embedded in 5% low melting-point agarose and cut by a Microslicer (DTK-3000, Dosaka EM, Kyoto, Japan) into 60 µm sections parallel to the surface. The sections were then stained with Cresyl Violet. RESULTS Histopathological changes in substantia nigra neurons after middle cerebral artery occlusion Histopathologically, acute ischemic changes were limited to the territory supplied by the MCA, which were the lateral part of the neostriatum and the corresponding frontoparietal cortex as described previously.25 The substantia nigra of the control group did not show any pathological changes. At seven days after MCA occlusion, the SNR became smaller in volume than the control (Fig. 1B). The SNR showed a more marked atrophy after 14 days (Fig. 1C). Neuronal necrosis and gliosis were observed at this stage. Changes in electrical membrane properties of substantia nigra neurons after middle cerebral artery occlusion Intracellular recordings were obtained from electrophysiologically identified SNR GABAergic and SNC dopaminergic neurons from sham, intact and MCA-occluded rats. No significant difference was observed between electrical membrane properties and synaptic responses of substantia nigra neurons from sham and intact rats. SNR GABAergic neurons were electrophysiologically identified by following properties:4,14,34 (i) a short duration action potential (about 1 ms), (ii) a low-threshold Ca spike, (iii) a high frequency spontaneous firing (more than 10 Hz). In SNR neurons from the control group, injection of a depolarizing current pulse generated high frequency repetitive firings (Fig. 2A). A long-lasting afterhyperpolarization (AHP) (range: 66.3–72.3 ms) with a relatively small amplitude was occasionally observed after termination of a current pulse. The inward rectification Electrophysiology of substantia nigra after focal ischemia 1023 ward rectification and (v) a spontaneous oscillation of the membrane potential (about 3 Hz). In some SNC neurons, effect of dopamine was also examined and they responded to induce the membrane hyperpolarization (data not shown). In SNC neurons from the control group, injection of a strong depolarizing current pulse (e.g., 0.6 nA) with relatively long duration (e.g., 450 ms) induced a spike accommodation with a marked decrease in the inter-spike interval (Fig. 3A). The membrane response after termination of a large current pulse was followed by an AHP with a relatively large amplitude (range: 10.5–18.1 mV) and long duration (range: 60.2–141.6 ms). The prominent time-dependent inward rectification was induced by injecting hyperpolarizing current pulses (Fig. 3D). The physiological profiles including the spike duration, spike amplitude, input resistance, firing rate, AHP amplitude and time constant, were not significantly changed in SNC neurons at five to eight days after MCA occlusion (Fig. 3B,E; Table 1). At 13–16 days after MCA occlusion, however, the firing rate and input resistance were significantly increased (Fig. 3F; Table 1). At this stage, some neurons showed a prominent spike accommodation, which finally led to a cessation of spike generation (Fig. 3C). There were no significant difference in other membrane properties examined (Table 1). Changes in synaptic responses of substantia nigra neurons after middle cerebral artery occlusion Fig. 1. Photomicrographs of the left substantia nigra with Cresyl Violet staining. (A) Control. (B) Seven days after MCA occlusion. (C) 14 days after MCA occlusion. Scale bar=300 µm. was also induced by injecting hyperpolarizing current pulses (Fig. 2E). Fig. 2 also shows that strong rebound responses underlain by the low-threshold Ca spike were generated in the SNR neurons at the termination of hyperpolarizing current pulses. Compared with the control group, the mean input resistance was decreased and the mean firing rate was increased at fifth through sixteenth day after MCA occlusion (Table 1). However, the differences did not reach statistical significance. There were also no significant changes in other membrane properties examined in SNR neurons (Fig. 2, Table 1). At 21 days after MCA occlusion, intracellular recordings were very difficult to obtain from SNR neurons. On the other hand, SNC dopaminergic neurons were electrophysiologically identified by following properties:3,4,7,8,34 (i) a long duration action potential (about 2.5 ms), (ii) a prominent spike AHP, (iii) a ramp-like potential upon injecting hyperpolarizing current pulse, (iv) a prominent time-dependent in- In SNR neurons from the control group, depolarizing potentials with short duration (amplitude: 1.4&0.2 mV; duration: 1.6&0.5 ms, n=4) followed by hyperpolarizing potentials (amplitude: 6.7& 1.3 mV; duration: 38.8&11.3 ms, n=4) were evoked after stimulation of the STH (Fig. 4A). The amplitude of the depolarizing potentials was increased by an injection of hyperpolarizing current, while the amplitude of the hyperpolarizing potentials was decreased and the polarity was finally reversed in depolarizing direction by an injection of hyperpolarizing current. These data indicate that the depolarizing and hyperpolarizing potentials are excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs), respectively. IPSPs evoked by STH stimulation in SNR neurons were not affected by MCA occlusion and IPSPs were still observed in most of SNR neurons at five to eight days (Fig. 4B) and 13–16 days (Fig. 4C) after MCA occlusion. The mean amplitude of IPSPs at 13–16 days after MCA occlusion was 7.2 mV (range: 6.3– 7.8 mV, n=4) and the mean duration was 42.5 ms (range: 28.8–65.0 ms, n=3) (Fig. 4C). In SNC neurons from the control group, depolarizing potentials with short duration (amplitude: 1.3&0.5 mV; duration: 1.5&0.8 ms, n=4) followed by hyperpolarizing potentials (amplitude: 5.8& 0.9 mV; duration: 26.0&5.3 ms, n=4) were evoked after stimulation of the STH (Fig. 5A). The hyper- 1024 H. Nakahishi et al. Fig. 2. Membrane responses recorded from SNR neurons in the control and MCA-occluded rats to intracellularly injected hyperpolarizing and depolarizing currents of various intensities. (A, D) Control. (B, E) Five days after MCA occlusion. (C, F) 14 days after MAC occlusion. Calibrations in A also apply to B and C. Calibrations in D also apply to E and F. Table 1. Physiological profiles of substania nigra pars reticula and subtantia nigra pars compacta neurons in control and middle artery occuded rats SNR Spike duration, ms Spike amplitude, mV Input resistance, MÙ Firing rate, Hz AHP amplitude, mV Time constant, ms SNC Spike duration, ms Spike amplitude, mV Input resistance, MÙ Firing rate, Hz AHP amplitude, mV Time constant, ms Control 5–8 days 13–16 days 1.0&0.2(4) 68.5&4.9(4) 97.3&42.8(4) 16.4&7.0(10) 13.4&1.5(4) 8.0&2.4(4) 1.1&0.2(7) 66.7&5.6(6) 90.7&41.5(5) 19.1&8.3(18) 12.7&2.3(6) 8.2&2.7(6) 1.1&0.1(5) 66.1&12.9(4) 71.7&14.7(4) 22.1&9.2(10) 13.3&2.7(6) 7.8&1.6(4) 2.6&0.5(6) 58.3&6.7(6) 107.9&21.1(11) 2.6&0.4(10) 14.6&2.4(6) 16.8&6.6(5) 2.3&0.3(7) 55.1&5.7(9) 110.2&24.4(7) 2.7&0.7(15) 15.2&1.6(10) 15.1&5.2(8) 2.4&0.4(8) 61.7&2.6(6) 257.2&75.7(5)** 3.3&0.6(14)** 15.4&1.9(10) 16.9&5.5(70) Values represent mean&S.D. **P<0.01 as compared with control (Student’s t-test). polarizing potentials were usually clearly observed when the overlapping depolarizng potentials were minimized by injecting depolarizing current pulses. The amplitude of the depolarizing potentials was increased by injecting hyperpolarizing current pulses, while the polarity of the hyperpolarizing potentials was reversed in depolarizing direction by injecting hyperpolarizing current pulses (Fig. 5A). These data indicate that the depolarizing and hyperpolarizing potentials are EPSPs and IPSPs, respectively. At five to eight days after MCA occlusion, the amplitude and duration of IPSPs were markedly decreased. At the same time the amplitude of EPSPs was significantly increased (amplitude: 4.7&1.4 mV, P<0.01, Student’s t-test; duration: 18.0&8.8 ms, n=7) (Fig. 5B). The most prominent increase in EPSPs was usually observed in SNC neurons after 13–16 days of MCA occlusion. At this stage, the mean amplitude of EPSPs was 7.6 mV (range: 6.3–9.4 mV, n=5) and the mean duration was 45.8 ms (range: 38–50 ms, n=5) (Fig. 5C). Electrophysiology of substantia nigra after focal ischemia 1025 Fig. 3. Membrane responses recorded from SNC neurons in the control and MCA-occluded rats to intracellularly injected hyperpolarizing and depolarizing currents of various intensities. (A, D) control. (B, E) eight days after MCA occlusion. (C, F) 13 days after MCA occlusion. Square waves at the bottom of oscillographic records indicate the intensities and duration of injected currents in this and all subsequent figures. Calibrations in A also apply to B and C. Calibrations in D also apply to E and F. Fig. 4. Synaptic responses evoked by STH stimulation recorded from SNR neurons in the control and MCA-occluded rats. Injection of continuous hyperpolarizing current ("0.1 and "0.2 nA) changed the amplitude of postsynaptic potentials. (A) Control. (B) Seven days after MCA occlusion. (C) 14 days after MCA occlusion. Arrowhead in this and the next figures indicate an onset of STH stimulation. Calibrations in A also apply to B and C. Effects of bicuculline methiodide on the input resistance and firing rate of substantia nigra pars compacta neurons from normal rats Bath application of bicuculline methiodide (50 µM) significantly increased the firing rate of sub- stantia nigra pars compacta neurons from normal rats. The mean input resistance was also increased after an application of bicuculline methiodide, while the difference did not reach statistical significance (Table 2). During application of bicuculline methiodide, IPSPs evoked by STH stimulation were 1026 H. Nakahishi et al. Fig. 5. Synaptic responses evoked by STH stimulation recorded from SNC neurons in the control and MCA-occluded rats. Injection of depolarizing and hyperpolarizing current pulses changed the amplitude of postsynaptic potentials. (A) Control. (B) Eight days after MCA occlusion. (C) 16 days after MCA occlusion. Calibrations in A also apply to B and C. Table 2. Effects of bicuculline methodide (50 µM) on the input resistance and firing rate of substantia nigra pars compacta neurons in normal rats Input resistance, MÙ Firing rate, Hz Control Bicucilline methiodide 107.0&25.0(5) 2.5&0.8(5) 126.2&7.6(5) 3.9&1.0(5)* Values represent mean&S.D.*P<0.05 as compared with Control (Student’s t-test). markedly suppressed and EPSPs were increased in the amplitude and duration as reported previously (data not shown). DISCUSSION No significant alteration of electrophysiological profiles of substantia nigra pars reticulata neurons after middle cerebral artery occlusion On the basis of our hypothesis that delayed degeneration of SNR neurons after MCA occlusion results from excessive excitation due to loss of inhibitory GABAergic inputs,27 we expected marked changes in electrophysiological profiles of SNR neurons after MCA occlusion. In the present experiments, however, neither the electrical membrane properties nor synaptic responses of SNR neurons was changed significantly from fifth through sixteenth day after MCA occlusion. We have previously suggested that IPSPs evoked by STH stimulation in SNR neurons were mediated through GABAA receptors activated by the GABAergic strionigral fibres and/or pallidonigral fibres since IPSPs were markedly suppressed by bicuculline methiodide and there was a large reduction in the amplitude of IPSPs after chronic transection of the internal capsule at the level of the entopeduncular nucleus.14 Thus the present results strongly suggest that GABAergic strionigral fibres and/or pallidonigral fibres are still preserved in SNR neurons which survived even after 14 days of MCA occlusion. In the 4-vessel occlusion model of the rat, Saji et al. have recently demonstrated that delayed degeneration of SNR neurons may depend on the interruption of the inhibitory afferents from the globus pallidus to the STH,21,23 which sends excitatory glutamatergic fibres to the SNR.14,20,24 The EPSP evoked by STH stimulation in SNR neurons was considered to be mediated by glutamatergic afferents from the STH,14,16 whereas neither amplitude nor duration of EPSPs was affected after MCA occlusion in the present study. The increased local cerebral blood flow and local cerebral glucose utilization in the ipsilateral SNR was observed from the 24 h through the seventh day after MCA occlusion.28–30 These early increase in the local cerebral blood flow and glucose utilization likely reflect the hypermetabolic state of SNR neurons. Furthermore, Nakayama et al. showed that the GABA content of the ipsilateral SNR increased slightly one day after MCA occlusion in the rat but decreased remarkably from the third through 28th day.17 It has been also reported that there was an increased expression of heat shock proteins32 and 45 Ca accumulation12 in SNR neurons at three days after MCA occlusion. These observations consistently suggest the hyperexcitation of SNR neurons due to disinhibition after neostriatal infarction even in the acute phase after MCA occlusion. There are at least four possible explanations for the present conflicting results: (i) the essential hyperexcitation which might be detected electrophysiologically occurs only in the acute phase after MCA occlusion, (ii) the afferent fibres which may cause hyperexcitability of Electrophysiology of substantia nigra after focal ischemia SNR neurons in vivo have been transected during the process of slicing, (iii) intracellular recording method may create a sampling bias which may skew the data toward more accessible healthy neurons than affected ones, (iv) MCA occlusion leads to a hypermetabolic state in the SNR without affecting electrophysiological profiles of SNR neurons. The first and the second explanations are unlikely since Asai et al. have recently examined the changes in spontaneous single-unit activities in the ipsilateral SNR after MCA occlusion in chloral hydrate-anaesthetized rats and found that the firing rates of SNR neurons measured at 1 h and one day after MCA occlusion were not significantly altered.1 In their study, the firing rates of SNR neurons measured at seven and 14 days after MCA occlusion were rather significantly decreased. Further studies will be needed to clarify the mechanism of delayed degeneration of SNR neurons following MCA occlusion. Hyperexcitabilty of substantia nigra pars compacta neurons after middle cerebral artery occlusion In contrast to the SNR neurons, there was a significant increase in both the input resistance and the spontaneous firing rate of SNC neurons at around 14 days after neostriatal infarction induced by MCA occlusion. Furthermore IPSPs recorded from SNC neurons were largely reduced at around seven days after MCA occlusion. At the same time, the amplitude and duration of EPSPs in SNC neurons were significantly increased. Recently, connections between GABAergic SNR and dopaminergic SNC neurons were demonstrated.5 Furthermore, Moore et al. have reported that IPSPs recorded in SNC dopaminergic neurons after stimulation of the STH arose through activation of the axon collaterals of SNR GABAergic neurons.10 Thus the marked reduction of IPSPs evoked by STH stimulation in SNC neurons is likely due to degeneration of SNR neurons following MCA occlusion since a marked neuronal necrosis was observed in the SNR at this stage. The significant increase in both the input resistance and the spontaneous firing rate of SNC neurons following MCA occlusion can be also explained by a reduction of tonic GABAergic inputs from the SNR since a tonic activation of GABAA receptors is known to exhibit a shunting effect on synaptic inputs due to the decreased membrane input resistance. This deduction was further supported by the present pharmacological finding that an applica- 1027 tion of bicuculline methiodide, a GABAA receptor antagonist, significantly increased the firing rate of SNC neurons in slice preparation from normal rats. Although the difference did not reach statistical significance, the mean input resistance of SNC neurons was also increased after an application of bicuculline methiodide. EPSPs evoked by STH stimulation in SNC neurons was also considered to be originated in the STH.10 It has been reported that there was a significant loss of tyrosine hydroxylaseimmunoreactive neurons in the SNC and dendritic arborization in the SNR at three weeks after reperfusion in the 4-vessel occlusion model of the rat demonstrating the delayed degeneration of SNC dopaminergic neurons after neostriatal infarction.31 Therefore, it is conceivable that subthalamic glutamatergic afferents in the SNC are normally regulated by GABAergic afferents from the SNR, whereas the degeneration of SNR neurons following MCA occlusion allows glutamatergic afferents from the STH to dominate resulting in hyperexcitation possibly culminating in degeneration of SNC neurons. The decrease in number of SNC neurons has been reported at seven days after MCA occlusion,9 while delayed loss of tyrosine hydroxylase-positive dopaminergic neurons in the SNC remains to be determined. CONCLUSIONS Histological examination revealed marked atrophy and neurodegeneration in the SNR, whereas the present in vitro electrophysiological study provides no evidence of hyperexcitation in SNR neurons at one to two weeks after MCA occlusion. On the other hand, SNC neurons showed hyperexcitation evidenced by the significant increase in the spontaneous firing rate and the amplitude of EPSPs evoked by STH stimulation at 13–16 days after MCA occlusion. Furthermore, the input resistance of SNC neurons was significantly increased. The increase in the spontaneous firing rate and the amplitude of EPSPs in SNC neurons after MCA occlusion was mimicked by an application of bicuculline methiodide. Based on these results, it is likely that there are differential pathophysiological processes occurring in the SNR and SNC after MCA occlusion. Acknowledgements—This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. 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