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Neuroscience Vol. 77, No. 4, pp. 1021–1028, 1997
Copyright ? 1997 IBRO. Published by Elsevier Science Ltd
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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
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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. We gratefully
acknowledge the excellent technical assistance of Mrs
Noriko Kishino.
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(Accepted 9 October 1996)