1Department of Electronic Engineering, Graduate School of Engineering, Osaka University, Suita 565-0871; 2Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University; and 3Core Research for Evolutional Science and Technology/Murakami Laboratory, Center for Advanced Research Projects, Osaka University, Toyonaka 560-8531, Japan
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ABSTRACT |
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Otsuka, Takeshi,
Fujio Murakami, and
Wen-Jie Song.
Excitatory Postsynaptic Potentials Trigger a Plateau Potential in
Rat Subthalamic Neurons at Hyperpolarized States.
J. Neurophysiol. 86: 1816-1825, 2001.
The subthalamic
nucleus (STN) directly innervates the output structures of the basal
ganglia, playing a key role in basal ganglia function. It is therefore
important to understand the regulatory mechanisms for the activity of
STN neurons. In the present study, we aimed to investigate how the
intrinsic membrane properties of STN neurons interact with their
synaptic inputs, focusing on their generation and the properties of the
long-lasting, plateau potential. Whole cell recordings were obtained
from STN neurons in slices prepared from postnatal day 14 (P14) to P20 rats. We found that activation of glutamate receptor-mediated excitatory synaptic potentials (EPSPs) evoked a plateau potential in a
subpopulation of STN neurons (n = 13/22), in a
voltage-dependent manner. Plateau potentials could be induced only when
the cell was hyperpolarized to more negative than about 75 mV.
Plateau potentials, evoked with a depolarizing current pulse, again
only from a hyperpolarized state, were observed in about half of STN neurons tested (n = 162/327). Only in neurons in which
a plateau potential could be evoked by current injection did EPSPs
evoke plateau potentials. L-type Ca2+ channels,
Ca2+-dependent K+ channels,
and TEA-sensitive K+ channels were found to be
involved in the generation of the potential. The stability of the
plateau potential, tested by the injection of a negative pulse current
during the plateau phase, was found to be robust at the early phase of
the potential, but decreased toward the end. As a result the early part
of the plateau potential was resistant to membrane potential
perturbations and would be able to support a train of action
potentials. We conclude that excitatory postsynaptic potentials, evoked
in a subpopulation of STN neurons at a hyperpolarized state, activate
L-type Ca2+ and other channels, leading to the
generation of a plateau potential. Thus about half of STN neurons can
transform short-lasting synaptic excitation into a long train of output
spikes by voltage-dependent generation of a plateau potential.
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INTRODUCTION |
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The subthalamic nucleus (STN)
acts as a driving force of the basal ganglia by exerting glutamatergic
excitatory effect on the output structures of the basal ganglia, the
globus pallidus and the substantia nigra (see Kitai and Kita
1987 for a review). The significance of the STN in motor
control has been implicated in a clinical observation that pathological
changes in the nucleus causes hemiballism (Whittier
1947
) and in animal experiments in which manipulation of the
activity of the nucleus dramatically affects motor behavior
(Hamada and Hasegawa 1996
; Wichmann et al.
1994b
). These observations indicate the importance of
controlling outputs of the basal ganglia by STN neurons. It is
therefore crucial to know how the activity of STN neurons is regulated
for understanding basal ganglia function in motor control.
The activity of a neuron is determined by extrinsic synaptic inputs and
intrinsic membrane properties. The STN is known to receive excitatory
input from the cortex (Fujimoto and Kita 1993; Hartmann-von Monakow et al. 1978
; Kitai and
Deniau 1981
; Nambu et al. 1996
) and the thalamus
(Féger et al. 1994
; Mouroux and Féger
1993
) and inhibitory inputs from the globus pallidus
(Groenewegen and Berendse 1990
; Kita et al.
1983
; Moriizumi and Hattori 1992
). How STN
neurons respond to these inputs depends on their membrane properties.
In slice studies, STN neurons fire regularly, increasing firing
frequencies linearly with the magnitude of injected currents (Bevan and Wilson 1999
; Nakanishi et al.
1987
). This would suggest that the STN works as a linear
transformer relaying excitatory inputs to its targets. Indeed, a recent
study in anesthetized rat suggests that STN neurons follow the activity
of cortical neurons (Magill et al. 2000
). STN neurons,
however, have intrinsic membrane properties, which can significantly
change neuronal firing pattern. The generation of a plateau potential,
a long-lasting depolarizing potential, for example, has been described
in a subset of STN neurons (Beurrier et al. 1999
;
Nakanishi et al. 1987
; Otsuka et al. 1998
,
1999
; Overton and Greenfield 1995
; Song
et al. 1998
). Because of its slow decay kinetics, the plateau
potential would lead to a long-lasting, high-frequency discharge in the
absence of synaptic inputs. In behaving animals, burst activity lasting several hundred milliseconds has been observed in STN neurons (Matsumura et al. 1992
; Wichmann et al.
1994a
). The generation of a plateau potential may be one
possible underlying mechanism for such long-lasting burst.
Although the plateau potential can profoundly alter the output
properties of STN neurons, no work has focused on how the ability to
generate plateau potentials affects synaptic integration.
Beurrier et al. (1999) reported a plateau potential in
STN neurons that in part supports recurrent membrane oscillations,
leading to rhythmic burst firing of STN neurons. Others have observed
plateau potentials evoked by current injections (Nakanishi et
al. 1987
; Overton and Greenfield 1995
). But it
is not known whether plateau potentials can be triggered by synaptic
potentials. In the present study, we therefore tested whether
activation of excitatory synaptic inputs to STN neurons can trigger a
plateau potential and if so, how the plateau potential would interact
with synaptic potentials. We found that activation of glutamate
receptor-mediated synaptic potentials triggered a plateau potential in
about half of STN neurons, only when the cells were hyperpolarized. Our
results thus suggest that about half of STN neurons transform
excitatory synaptic inputs into either a single spike or a train of
spikes, depending on membrane potential. Part of the results has been published in abstract form (Otsuka et al. 1998
, 1999
;
Song et al. 1998
).
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METHODS |
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Slice preparation
Slice preparation including the STN was obtained from Sprague-Dawley rats (14-20 postnatal days). All experiments were conducted in compliance with the Guidelines for Use of Laboratory Animals of Osaka University. Rats were anesthetized with ether and perfused with a high-sucrose solution containing (in mM): 200 sucrose, 2.5 KCl, 0.5 CaCl2, 10 MgSO4, 1.25 KH2PO4, 26 NaHCO3, 10 glucose, 0.2 ascorbic acid, and 1 pyruvic acid (300 ± 5 mOsm/l; pH, 7.4). The brain was then removed, iced, and blocked for slicing. In the medium described in the preceding text, 250- to 300-µm (mostly 300 µm)-thick horizontal slices were prepared using a Microslicer (Dosaka EM, Japan). The slices were then incubated at room temperature (20°C) in oxygenated Kreb's solution containing (in mM): 126 NaCl, 2.5 KCl, 1.25 KH2PO4, 1 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose (300 ± 5 mOsm/l, pH, 7.4; bubbled with 95% O2-5% CO2). Ascorbic acid (0.2) and 1 pyruvic acid (in mM) were added to the holding solutions for improving tissue viability. After 1-h recovery period, a slice was transferred to a recording chamber mounted on the stage of an upright microscope (Olympus, Tokyo). The recording chamber was continuously superfused with the oxygenated Kreb's solution.
Recordings and data analyses
Whole cell recordings of STN neurons employed standard
techniques (Edwards et al. 1989; Stuart et al.
1993
). Electrodes were pulled from glass capillary tubes
(Narishige, Tokyo) and fire-polished. In most experiments, the
recording pipettes were filled with a solution containing (in mM): 120 KCl, 3 MgCl2, 10 HEPES, 0.2 EGTA, 2 Na2ATP, 0.2 Li2GTP, 12 phosphocreatine, and 0.1 leupeptin (pH, 7.2; 270 ± 5 mOsm/l) and
had resistances of 5-7 M
in the bath. The liquid-junction potential
was estimated to be 4.3 mV. All potentials reported in this paper were
corrected for this potential. In experiments chelating internal
Ca2+, the internal solution consisted of (in mM)
100 KCl, 3 MgCl2, 10 HEPES, 20 BAPTA, 2 Na2ATP, 0.2 Li2GTP, 12 phosphocreatine, 0.1 leupeptin (pH, 7.2; 270 ± 5 mOsm/l), and the
concentration of KCl of the external solution was changed to 1.5 mM to
adjust K+ equilibrium potential. We also recorded
with a low-Cl
internal solution containing (in
mM) 70 K2SO4, 2.5 MgCl2, 1.0 EGTA, 0.1 CaCl2,
35 HEPES, 30 N-methyl-D-glucamine (NMG), 2 Na2ATP, 0.2 Li2GTP, and 0.1 leupeptin (pH 7.2, 270 ± 5 mOsm/l). Recordings were obtained with
an EPC-7 amplifier (List-Medical-Electronics, Darmstadt, Germany)
controlled with a Pentium PC running pCLAMP (version 6.0) or Axoscope
(Axon Instruments, Foster City, CA). Synaptic responses were evoked by
electrical stimulation applied through a bipolar tungsten electrode
using a Master-8 stimulator (AMPI, Jerusalem, Israel). The
electrode was placed in a region rostral to the STN. A single current
pulse of 0.1-0.5 mA in amplitude and 100 µs in duration was used for
stimulation. Data analyses were performed with AxoGraph (Axon
Instruments) and Kaleidagraph (Albeck Software, Reading, PA). Data are
represented as means ± standard deviation (SD) and statistical
difference between samples was tested using Mann-Whitney U
test, unless mentioned otherwise. Significance was accepted when
P < 0.05.
Drugs
All reagents were obtained from Sigma Chemical (St. Louis, MO) except nifedipine and 6,7-dinitroquinoxaline-2,3-dione (DNQX), which were obtained from RBI (Natick, MA). Biocytin was diluted in the internal solution at the concentration of 5 mg/ml. Other drugs were diluted in oxygenated Kreb's solution and applied to the slice. Nifedipine was made up as concentrated stocks in 95% ethanol and diluted immediately before use. DNQX was dissolved in dimethylsulfoxide. When using these drugs, equal concentrations of the solvent were added to all control solutions. For the tetraethylammonium chloride (TEA) experiment, the Kreb's solution was modified by replacing NaCl (50 mM) with NMG (50 mM) and was used as the control solution; TEA solutions were prepared by replacing NMG with an equimolar concentration of TEA. Solutions containing bicuculline or nifedipine were protected from ambient light.
Histology
To verify that recorded cells were in fact STN neurons, biocytin
was always included in the internal solution (Horikawa and Armstrong 1988). The avidin-biotin-horseradish peroxidase
reaction was used to visualize recorded neurons. After recording,
slices were fixed with a solution containing 4% paraformaldehyde in
phosphate buffer (0.1 M; pH, 7.4). After rinsing in Tris-buffered
saline (TBS; 50 mM; pH, 7.4), the slices were treated with a mixture of
10% methanol and 0.3%
H2O2 for 20-30 min, rinsed
again in TBS, and treated with TBS containing 0.5% Triton x-100. After
washes with TBS, the slices were incubated for 2 h in the
avidin-biotin-horseradish peroxidase complex (1%; Vector Laboratories,
Burlingame, CA) at room temperature, washed in TBS, and reacted with a
mixture of 3,3'-diaminobenzidine tetrahydrochloride (0.05%) and
H2O2 (0.003%) in TBS for
5-10 min. The slices were then rinsed several times in TBS and mounted
on gelatin-coated glass slides. The mounted slices were stained with
methylene blue for identification of the nucleus, dehydrated in graded
ethanol series, cleared in xylene, and coverslipped with Entellan New
(Merck, Darmstadt, Germany) for observation with a light microscope.
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RESULTS |
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Recordings were obtained from 327 neurons. Each recorded neuron
was labeled intracellularly with biocytin, and all were found within
the STN (Fig. 1A). The stained
STN neurons had soma diameters of 15-30 µm and two to six primary
dendrites. The resting membrane potential was 62.65 ± 5.10 mV
and the input resistance at rest, estimated with a negative current
pulse (
10 pA), was 0.66 ± 0.22 G
.
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Induction of plateau potentials by synaptic potentials
To examine the response of STN neurons to excitatory synaptic
inputs, recordings were obtained while stimulation at a location rostral to the STN was performed to evoke synaptic potentials. Bicuculline (50 µM) was included in the external solution to block inhibitory synaptic potentials. In response to stimulation,
depolarizing potentials or inward currents were observed in most of the
neurons examined (n = 32/41). Stimulation at sites
rostrolateral to the STN failed to evoke a response (n = 7). Coapplication of DNQX (10 µM), a
non-N-methyl-D-aspartate (non-NMDA) receptor
blocker, and 2-amino-5-phosphonovalerate (APV; 50 µM), an NMDA
receptor blocker, abolished the potentials (n = 4, Fig.
1B), indicating that these potentials are excitatory
postsynaptic potentials (EPSPs) mediated by glutamate
receptors. Application of either DNQX or APV alone revealed that
54-71% of the EPSP amplitude was mediated by non-NMDA receptors at
80 mV (n = 3). At 20°C, the latency of the EPSPs
was 4.4-7.2 ms and changed <0.5 ms with varying stimulus strength.
Assuming a Q10 value of 3.5 (Hirano et al.
1986
), the latencies correspond to latencies of 0.5-0.9 ms and
the latency change corresponds to a value <0.1 ms, at 37°C,
suggesting that the EPSPs are of monosynaptic nature.
With increased stimulus strength, the EPSPs always triggered a single
action potential in STN neurons at resting membrane potentials
(n = 22; Fig. 1C). When the membrane
potential was hyperpolarized to about 75 mV, however, stimulation
with the same strength evoked a long-lasting potential, or a plateau
potential, in a subpopulation of STN neurons (n = 13 of
22, Fig. 1D). The holding current required to hyperpolarize
the membrane potential to
75 to
85 mV ranged from
10 to
30 pA.
On the rising phase of the plateau potential, a short train of action
potentials (1-5 spikes) was evoked (Fig. 1D). When the
stimulus intensity was decreased, only EPSPs were observed. Gradually
changing the stimulus strength revealed that the amplitude of the EPSPs
right before the occurrence of plateau potentials ranged from
61 to
67 mV (n = 5). Neurons in which plateau potentials
were not observed always discharged a single action potential at the
peak of the EPSPs either at rest or at hyperpolarized states, for
stimulus intensities up to 0.5 mA (n = 9). These
results suggest that EPSPs are capable of triggering a plateau
potential in a subpopulation of STN neurons, in a voltage-dependent manner.
Induction of plateau potentials by injected current pulses
To examine how plateau potentials affect synaptic integration in STN neurons, we next studied the properties of plateau potentials in STN neurons. For this purpose, plateau potentials were evoked with current injection instead of synaptic activation for the ease of experimentation.
At resting membrane potentials, injection of depolarizing current pulses evoked action potentials either during the initial phase of the current (70%, n = 205/294; Fig. 2A, top) or during the entire period of current injection (n = 89; Fig. 2C, top). The membrane potential returned to rest in an exponential manner after termination of the current pulse (Fig. 2, A and C, top).
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At hyperpolarized potentials, however, a long-lasting potential was
induced that far outlasted current injection (Fig. 2C, bottom), again in a subpopulation of STN neurons. Other
cells exhibited an exponential relaxation of membrane potential after termination of current injection (Fig. 2A,
bottom). For a quantitative description of the membrane
potentials after current termination, we measured the half-decay time
defined as the time interval from the pulse end to the time when the
potential had decayed to half-amplitude at the current pulse end. STN
neurons could easily be divided into a population having short
half-decay times (30 ± 0.13 ms, n = 146) and a
population having much longer half-decay times (0.66 ± 0.49 s, range 0.20-3.0 s; n = 148). We defined a potential with a half-decay time 0.2 s as a plateau potential. About half of
STN neurons tested (n = 148/294) generated a plateau
potential at hyperpolarized state.
Plateau potentials were also observed with an internal solution of low
Cl concentration (see METHODS) at
30°C, again in a subset of neurons (n = 14/33). Under
this recording condition, some STN neurons showed spontaneous activity
at resting membrane potentials (2-10 Hz, n = 9/33),
although these activities disappeared with membrane hyperpolarization;
all STN neurons discharged during the entire period of current
injection. Plateau-generating neurons fired 5.1 ± 1.6 (n = 14) spikes during current injection (100 ms in duration, 50 pA in amplitude), which is not significantly different from that of nonplateau-generating neurons (4.5 ± 2.1, n = 19; P > 0.05).
A plateau potential was also generated after termination of a hyperpolarizing current pulse (n = 19; Fig. 2D). Such a potential was observed only in neurons in which a plateau potential could be evoked by a depolarizing current pulse, at hyperpolarized states (compare Fig. 2, B-D).
We next tested to what membrane potentials the cell has to be
hyperpolarized for a depolarizing pulse to evoke a plateau potential. Membrane potentials were gradually hyperpolarized by changing the
amount of injected constant current, while a depolarizing current pulse
(50 ms) was injected to test if a plateau potential could be induced.
As a result, a plateau potential was induced in an all-or-none manner,
across certain membrane potentials (Fig. 3A). The membrane potential at
which a plateau potential was first induced was referred to as
threshold potential. Shown in Fig. 3B is a histogram of the
threshold potentials from 19 neurons. The threshold potential was
74.98 ± 1.96 mV.
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From a hyperpolarized state, the membrane potential had to be
depolarized to a certain potential for plateau potential to occur. This
depolarizing threshold for the plateau potential was difficult to
determine from the rising phase of the potential, but with gradual
change of injected current, the potentials immediately before the
occurrence of plateau potentials ranged from 62 to
67 mV,
corresponding to an injected current of ~10 pA. The amplitude of the
plateau potential was independent of the suprathreshold current
amplitude, ranging from 10 to 100 pA.
To estimate the distribution of plateau-generating neurons in the STN,
we divided the nucleus into three equal parts by length, either along
the rostrocaudal or lateromedial axis. Along the rostrocaudal axis, the
ratio of plateau-generating neurons to nonplateau-generating neurons
was 46.9% (n = 15/32) in the rostral part, 47.6%
(n = 32/68) in the middle part, and 51.2%
(n = 15/32) in the caudal part. Along the lateromedial
axis, however, the ratio in the lateral, middle, and medial part of the
STN was 60.3% (n = 38/63), 44.9% (n = 22/49), and 29.0% (n = 9/31), respectively. Thus
plateau-generating neurons tend to be located in the lateral part of
the nucleus. Although the morphology of plateau-generating neurons did
not appear to differ from that of nonplateau-generating neurons, the
input resistance at resting membrane potentials of plateau-generating
neurons was found to be significantly larger than that of
nonplateau-generating neurons (0.813 ± 0.07 vs. 0.524 ± 0.05 G, n = 148; Student's t-test,
P < 0.005).
To test whether plateau potentials evoked by synaptic potentials and those evoked by current injection are of the same nature, we tried to evoke plateau potentials in the same neuron both by synaptic activation and by current injection. In such experiments, EPSPs evoked plateau potentials only in neurons in which a plateau potential could be evoked by injection of current pulses at hyperpolarized states (n = 13), suggesting that plateau potentials evoked by EPSPs and those evoked by current injections are attributable to a common membrane mechanism.
Effect of temperature
It was puzzling that action potentials were not evoked during the plateau phase of the plateau potential. We suspected that this might be because the experiment was carried out at room temperature. To verify this hypothesis, we raised the temperature from 20 to 25°C. As shown in Fig. 4, although a plateau potential did not evoke action potentials at 20°C (Fig. 4A), it evoked action potentials even at its late phase at 25°C (Fig. 4B; n = 6). Raising the temperature also appeared to increase the duration of the plateau potential (compare Fig. 4, A to B).
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Stability of plateau
To study how the plateau potentials interact with synaptic inputs,
we tested the stability of the plateau potentials. Because plateau
potentials were not terminated by action potentials (Fig. 4B), they appear to be resistant to perturbations of
depolarizing potentials. To test the effect of inhibitory synaptic
potentials, a negative current pulse, used to represent an inhibitory
synaptic current, was injected at successive timings during the course of the plateau potential induced with a short pulse (50 ms; Fig. 5). The negative current had an amplitude
of 60,
80, or
90 pA. The duration of the current was determined
in reference to inhibitory synaptic currents in STN neurons
(Shen and Johnson 2000
), and a 20-ms duration was used.
The stability of the plateau potential was evaluated by the ratio of
the peak potential after the current pulse to the potential immediately
before the current, referred to as stability index here; a stable
potential would give rise to a stability index of one. Shown in Fig.
5B is the stability index estimated at successive time
intervals during the course of plateau potentials. The stability index
was close to one during the early phase of the potential and then fell
gradually afterwards. During the initial 10% of the plateau, however,
the stability index was always >1 (1.18 ± 0.06;
n = 4). Toward the end of the potential, the stability
index fell abruptly. To compare the effect of the amplitude of the
negative current, data between 30 and 70% of the plateau duration was
fitted with an exponential function in the form
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Subcellular origin for the generation of plateau potentials
Whether plateau potentials in STN neurons are generated in the
soma or in the dendrites is of obvious significance for synaptic integration. To address this question, we tested the effect of voltage-clamping somatic membrane on the ability of EPSPs in evoking plateau potentials (currents). Anatomical evidence suggests that excitatory synaptic inputs end on distal dendrites of STN neurons (Bevan et al. 1995). Thus EPSPs in STN neuron, as those
shown in Fig. 1, are expected to be induced from distal dendrites. If a
plateau potential occurs at the soma, voltage-clamping the soma would
prevent the induction of the plateau by EPSPs; in contrast, considering
the filtering effect of dendrites, a plateau potential occurring at
distal dendrites may not be blocked by voltage-clamping the soma and
would thus give rise to a plateau current at the soma.
To verify that excitatory synaptic inputs occur at sites
electrotonically distant from the soma, we tried to determine the reversal potential of the EPSPs, which is calculated to be +6.4 mV for
an appropriate voltage control, assuming equal permeability of
Na+ and K+ and a
Ca2+ permeability of 1.17 relative to
Na+; intracellular Ca2+
concentration was assumed to be 100 nM (Götz et al.
1997; Mayer and Westbrook 1987
). EPSPs were
evoked in the same way as described in the preceding text. As a result,
the reversal potential was +21.0 ± 10.8 mV (n = 4). In three other tested cells, the EPSPs did not reverse for
potentials up to +20 mV. These results suggest that excitatory synaptic
inputs to STN neurons are electrotonically distant from the soma and
are consistent with previous anatomical observations (Bevan et
al. 1995
).
To test the effect of voltage-clamping somatic membrane on the ability of EPSPs in evoking plateau potentials, a plateau potential was first elicited in current-clamp mode by EPSPs (Fig. 6A). In voltage-clamp mode, however, only a transient inward current was evoked in the same neuron, in response to the same stimulation (Fig. 6B). To study the nature of the current, we compared its time course to that of currents recorded in the same way from nonplateau-generating neurons. Shown in Fig. 6, C and D, is an example of recordings from a nonplateau-generating neuron. In both plateau- and nonplateau-generating cells, the current recorded in voltage-clamp mode decayed in an exponential manner. The time constant was 12.0 ± 2.0 ms (n = 7) in plateau-generating neurons and 14.5 ± 3.9 ms (n = 6) in nonplateau-generating neurons, which is not significantly different from that of plateau-generating cells (P > 0.05). This result suggests that the current recorded in plateau generating cells was not related to the plateau potential but was likely a pure synaptic current. In plateau-generating cells, doubling the stimulus intensity increased the amplitude of the current, but did not change its kinetics (decay time constant, 12.7 ± 2.2 ms, n = 7; P > 0.05). Taken together, these results suggest the possibility that plateau potentials in STN neurons occur at a region where membrane potential is controlled by an electrode in the soma. This region is likely to be the soma and/or proximal dendrites.
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Ionic mechanisms of a plateau potential
The observation that plateau potentials can be induced only at
hyperpolarized membrane potentials suggests that voltage-dependent conductances are involved in the generation of the plateau potentials. Because a plateau potential could be induced only from a hyperpolarized state, it is natural to consider channels deinactivated at
hyperpolarized potentials to be involved in the generation of plateau
potentials. An obvious candidate is the low-threshold
Ca2+ channel (or T channel). The existence of T
channels in STN neurons has been documented before (Beurrier et
al. 1999; Nakanishi et al. 1987
; Song et
al. 2000
), and its possible involvement in the generation of
plateau potentials in STN neurons has been suggested (Beurrier
et al. 1999
). Because of the transient nature of the T channels
(Carbone and Lux 1984
), it is unlikely that a plateau potential is maintained by T channels. To search for conductances involved in the maintenance of plateau potentials, we tested the effect
of blocking channels that inactivate or deactivate slowly. Previous
studies have suggested that Ca2+
(Nakanishi et al. 1987
) and L-type
Ca2+ channels (Beurrier et al.
1999
) are required for the generation of plateau potentials in
STN neurons. These suggestions were confirmed in our study: the
generation of a plateau potential was blocked by removing
Ca2+ from the external solution
(n = 3; data not shown), and nifedipine (10 µM), an
L-type channel blocker, strongly reduced the duration of plateau
potentials (Fig. 7A;
n = 5), shortening the half decay time from 0.808 ± 0.185 s in control condition to 0.135 ± 0.026 s
(P < 0.05).
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It is conceivable that the current through L-type channels is involved
in the maintenance of the plateau potential, but it is also possible
that it is the increase in intracellular Ca2+
concentration that is important for the maintenance of plateau potentials because intracellular Ca2+ activates a
number of ion channels of slow kinetics. Actually, a
Ca2+-dependent cation conductance has been
suggested to be involved in the plateau potential (Beurrier et
al. 1999). We examined the effect of intracellular
Ca2+ by including a high concentration (20 mM) of
the Ca2+ chelating reagent
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA) in the internal recording solution. Immediately after going
from the cell-attached to the whole cell configuration, plateau
potentials elicited with a depolarizing pulse had half decay times of
0.34 ± 0.01 s (Fig. 7B, top). Over
time, the duration of plateau potentials gradually increased, reaching
a stable value at ~10 min of whole cell recording (Fig.
7B, bottom; n = 5). The half
decay time at 10 min of whole cell recording was 0.65 ± 0.01 s, which is significantly longer than the value on establishment of the
whole cell configuration (P < 0.05). In addition to
the elongation of the duration of the plateau potential, a reduction in
plateau amplitude was also noticed (Fig. 7B). These results suggest that a Ca2+-dependent outward current and
possibly inward currents are involved in the maintenance of the plateau potential.
To search for conductances involved in the repolarization of plateau
potentials, we tested the effect of TEA. As shown in Fig. 7C
(top), a plateau potential was induced in the presence of
TTX (1 µM), a result in agreement with a previous finding
(Beurrier et al. 1999); addition of TEA (10 mM) to the
bath solution increased the duration of the plateau potential (Fig.
7C, bottom). The half decay time was
significantly increased by TEA from 0.49 ± 0.05 to 0.79 ± 0.08 s (P < 0.05, n = 5). Taken
together with the effects of intracellular Ca2+
chelation, these results suggest that the repolarization of plateau potentials is mediated by both Ca2+-dependent
K+ channels and TEA-sensitive
K+ channels.
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DISCUSSION |
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In the present study, we investigated how intrinsic membrane properties of STN neurons may interact with synaptic inputs. We have found that activation of excitatory synaptic inputs evoked a plateau potential, in a voltage-dependent manner. Plateau potentials, evoked with a depolarizing pulse, were observed in about half of STN neurons. L-type Ca2+ channels, Ca2+-dependent K+ channels, and TEA-sensitive K+ channels were found to be involved in the generation of the potential. We conclude that EPSPs, evoked at a hyperpolarized state, activate L-type Ca2+ channels and other channels, leading to the generation of a plateau potential. Thus about half of STN neurons can transform short-lasting synaptic excitation into a long train of output spikes, in a voltage-dependent manner.
Ionic mechanisms
Although the occurrence of a plateau potential in STN neurons has
been reported before (Beurrier et al. 1999;
Nakanishi et al. 1987
; Overton and Greenfield
1995
), we have shown for the first time that a plateau
potential in STN neurons can be induced by activation of synaptic
inputs from hyperpolarized membrane potentials. The EPSPs recorded here
were evoked by stimulation of a site rostral to the STN. Because axon
collaterals of fibers descending in the cerebral peduncle enter the STN
from the rostral aspect (Iwahori 1978
), the EPSPs are
possibly of cortical origin, although we cannot exclude the
contribution of parafascicular thalamic origin. Previous in vivo
studies have found that STN neurons exhibit prolonged depolarizations
in response to stimulation of the cortex (Fujimoto and Kita
1993
; Kitai and Deniau 1981
). These
depolarizations, however, appear different from plateau potentials in
nature, because the duration of the depolarizations is only ~20 ms.
We have reported in abstract form that plateau potentials in STN
neurons can be evoked only when the membrane potential is hyperpolarized (Otsuka et al. 1998, 1999
; Song et
al. 1998
). Similar results were reported by Beurrier et
al. (1999)
, although there was a quantitative difference: in
Beurrier et al. (1999)
plateau potentials were observed
in a voltage range from
50 to
70 mV, while in the present study,
STN neurons had to be hyperpolarized to about
75 mV for the plateau
potential to occur. Why then does the membrane potential have to be
hyperpolarized for plateau potentials to occur? One possibility is that
hyperpolarization deinactivates voltage-dependent channels that are
involved in the generation of plateau potentials. T-type
Ca2+ channels, which are present in STN neurons
(Beurrier et al. 1999
, 2000
; Nakanishi et al.
1987
; Song et al. 2000
), are well known to be
inactivated at resting membrane potentials and deinactivated at
hyperpolarized potentials (Carbone and Lux 1984
;
Mouginot et al. 1997
). However, because of the transient
nature of T-type current, it is expected that T-type channels may play
a role in the induction, but not the maintenance, of plateau potential. What channels are then activated to produce a plateau potential? In
principle, the occurrence of a plateau potential requires the steady-state current-voltage (I-V) curve to cross zero
current with a negative slope. Thus in STN neurons, synaptic potentials have to activate an inward current that decays only slowly for plateau
potentials to occur. The channel giving rise to this current has to be
again inactivated (or partially inactivated) at the resting membrane
potential and deinactivated at hyperpolarized potentials. Possible
candidates for such channels include high-threshold Ca2+ channels and probably noninactivating
Na+ channels.
Ca2+-dependent cation channels may also be
involved if Ca2+ channels are activated. Although
a persistent, TTX-sensitive Na+ channel has been
reported in STN neurons (Beurrier et al. 2000
; Bevan and Wilson 1999
), it does not appear to be
essential for plateau potentials to occur because plateau potentials in
STN neurons could be evoked in the presence of TTX (see Fig. 7)
(Beurrier et al. 1999
). Plateau potentials have also
been found and well studied in motoneurons and interneurons of the
spinal cord (reviewed in Kiehn 1991
). In the spinal
cord, L-type Ca2+ channels can impart a negative
slope region to the steady-state I-V curve
(Hounsgaard and Kiehn 1989
; Svirskis and
Hounsgaard 1997
). Nakanishi et al. first showed that plateau
potentials in STN neurons were Ca2+ dependent
(Nakanishi et al. 1987
). After that, Beurrier et al. identified the Ca2+ channel to be of the L type
(Beurrier et al. 1999
), and this was confirmed in the
present study. In a computer simulation study, we found that the
voltage dependence of plateau potential induction can be solely
attributed to voltage-dependent inactivation of L-type
Ca2+ channel (Otsuka et al. 2000
).
We, therefore propose that the voltage dependence of L-type channels
plays an important role in the voltage-dependent induction of plateau
potentials in STN neurons.
Besides carrying an inward current, Ca2+ influx
may also cause activation of Ca2+-dependent
inward currents. Actually, Beurrier et al. have found that chelating
intracellular Ca2+ greatly shortened the duration
of STN plateau potentials and concluded that a
Ca2+-dependent cation channel is activated and is
essential for the maintenance of plateau potentials (Beurrier et
al. 1999). Chelating intracellular Ca2+,
however, enhanced the duration of plateaus in the present study (see
Fig. 7). Because a Ca2+-dependent
K+ current is obvious in STN neurons (e.g., Fig.
1), enhancement of plateau duration by chelating intracellular
Ca2+ is expected. The reduction in plateau
amplitude by intracellular BAPTA observed in the present study,
however, might be attributable to the blocking of
Ca2+-dependent cation channels.
Subcellular origin of the plateau potential
Although plateau potentials have been reported in STN neurons for
a long time, it is not known whether the potential is generated in the
dendrites or soma. If dendrites are the origin of plateau potentials,
then plateau potentials of different durations may be generated in
several dendrites of the same neuron. The integrated potential at the
soma could thus be a step-wise potential (but this may not occur in
slice preparation, where dendrites are not all preserved)
(Reuveni et al. 1993) and would produce complex spiking
behavior. In contrast, if the soma is the origin, STN neurons would
show simpler discharge patterns. Thus the site of origin of plateau
potentials has significance in synaptic integration and in regulating
firing behavior.
Our strategy for addressing this issue took advantage of the fact that
excitatory synaptic inputs to STN neurons end on distal dendrites. Thus
if a plateau potential occurs in dendrites, voltage-clamping the soma
would still allow the plateau potential to be evoked in dendrites by
the synaptic input. In this case, an inward current similar in shape to
the plateau is expected to be recorded at the soma. Because such
currents were never observed, plateau potentials in STN neurons appear
to be generated in the soma. This inference is consistent with previous
findings that L-type Ca2+ channels, which are
essential for plateau potentials, tend to be localized in the soma
(Hell et al. 1993). Nevertheless other possibilities
cannot be excluded at this time because our current argument is based
on negative findings.
Functional significance
The STN receives excitatory inputs from the cortex
(Fujimoto and Kita 1993; Hartmann-von Monakow et
al. 1978
; Kitai and Deniau 1981
; Nambu et
al. 1996
). Because of the voltage dependence of the generation
of plateau potential, cortical input to the STN will be transformed
into either a single or a train of action potentials, depending on the
membrane potential of STN neurons at the time of the input. What may
hyperpolarize the cell for a plateau potential to occur? Opening of
K+ channels by metabolic signaling pathways is
one possibility. High-frequency inhibitory input from, for example, the
globus pallidus, would also help hyperpolarize the cell. It is an
important task to identify the pathway that hyperpolarizes STN neurons
in future studies.
Because a plateau potential is more or less a long-lasting, stereotyped
potential, generation of a plateau potential would lead to a reduction
in spatiotemporal integrative ability of the cell. The benefit of using
a plateau potential, in addition to transforming a single input volley
into a train of output spikes, may lie in the generation of rhythmic
bursting in STN-related neuronal networks. A previous study has shown
that STN neurons "switch from single-spike activity to burst-firing
mode" and suggested plateau potentials are involved in the generation
of rhythmic burst activity (Beurrier et al. 1999). In
our experiments, rhythmic burst activity was seldom observed. This is
consistent with the recent observation in explant culture that STN
neurons exhibit pacemaker activity when STN is in the STN-GP network
but not when STN is alone (Plenz and Kitai 1999
). Our
observation that plateau potentials were observed in about half of STN
neurons is in coincidence with the observation by Plenz and Kitai that
about half of STN neurons exhibits pacemaker activity (Plenz and
Kitai 1999
). This coincidence suggests the possibility that
plateau potentials in STN neurons are involved in the generation of
oscillatory bursting in the STN-GP network. Two features of STN plateau
potentials may be relevant. First, because a plateau potential can be
evoked as a rebound potential (see Fig. 2) (Beurrier et al.
1999
; Overton and Greenfield 1995
), a short
train of spikes in GP neurons would hyperpolarize STN neurons and a
plateau potential would then occur as a rebound potential, evoking a
train of spikes in STN neurons. Second, STN activity would cause
immediate feedback inhibition from the GP, but this inhibition might
not immediately terminate STN spiking activity because the early part
of plateau potentials appears to be resistant to inhibitory perturbations.
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ACKNOWLEDGMENTS |
---|
We thank S. Maeda for technical assistance in data analysis.
This work was supported by the Uehara Foundation, Grants-in-Aid for Scientific Research on Priority Areas (Grants 12210099, 12053247, and 11170232) from the Ministry of Education, Science, and Culture, Japan to W.-J. Song, and a grant from Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation to F. Murakami.
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FOOTNOTES |
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Address for reprint requests: W.-J. Song, Dept. of Electronic Engineering, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan (E-mail: song{at}ele.eng.osaka-u.ac.jp).
Received 5 March 2001; accepted in final form 26 June 2001.
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REFERENCES |
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