1Department of Electronic Engineering, Graduate School of Engineering, Osaka University, Suita 565-0871; and 2Division of Biophysical Engineering, Graduate School of Engineering Science 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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Song, Wen-Jie,
Yosuke Baba,
Takeshi Otsuka, and
Fujio Murakami.
Characterization of Ca2+ Channels in Rat Subthalamic
Nucleus Neurons.
J. Neurophysiol. 84: 2630-2637, 2000.
The subthalamic
nucleus (STN) plays a key role in motor control. Although previous
studies have suggested that Ca2+ conductances may
be involved in regulating the activity of STN neurons,
Ca2+ channels in this region have not yet been
characterized. We have therefore investigated the subtypes and
functional characteristics of Ca2+ conductances
in STN neurons, in both acutely isolated and slice preparations.
Acutely isolated STN cells were identified by retrograde filling with
the fluorescent dye, Fluoro-Gold. In acutely isolated STN neurons,
Cd2+-sensitive, depolarization-activated
Ba2+ currents were observed in all cells studied.
The current-voltage relationship and current kinetics were
characteristic of high-voltage-activated Ca2+
channels. The steady-state voltage-dependent activation curves and
inactivation curves could both be fitted with a single Boltzmann function. Currents evoked with a prolonged pulse, however, inactivated with multiple time constants, suggesting either the presence of more
than one Ca2+ channel subtype or multiple
inactivation processes with a single channel type in STN neurons.
Experiments using organic Ca2+ channel blockers
revealed that on average, 21% of the current was nifedipine sensitive,
52% was sensitive to -conotoxin GVIA, 16% was blocked by a high
concentration of
-agatoxin IVA (200 nM), and the remainder of the
current (9%) was resistant to the co-application of all blockers.
These currents had similar voltage dependencies, but the
nifedipine-sensitive current and the resistant current activated at
slightly lower voltages.
-Agatoxin IVA at 20 nM was ineffective in
blocking the current. Together, the above results suggest that acutely
isolated STN neurons have all subtypes of high-voltage-activated
Ca2+ channels except for P-type, but have no
low-voltage-activated channels. Although acutely isolated neurons
provide a good preparation for whole cell voltage-clamp study,
dendritic processes are lost during dissociation. To gain information
on Ca2+ channels in dendrites, we thus studied
Ca2+ channels of STN neurons in a slice
preparation, focusing on low-voltage-activated channels. In
current-clamp recordings, a slow spike was always observed following
termination of an injected hyperpolarizing current. The slow spike
occurred at resting membrane potentials and was sensitive to micromolar
concentrations of Ni2+, suggesting that it is a
low-threshold Ca2+ spike. Together, our results
suggest that STN neurons express low-voltage-activated
Ca2+ channels and several high-voltage-activated
subtypes. Our results also suggest the possibility that the
low-voltage-activated channels have a preferential distribution to the
dendritic processes.
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INTRODUCTION |
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The subthalamic nucleus (STN), the only
excitatory nucleus in the basal ganglia, directly excites both output
structures of the basal ganglia: the substantia nigra reticulata and
the internal globus pallidus (Kita et al. 1983;
Kita and Kitai 1987
, 1991
; Van der
Kooy and Hattori 1980
; see Kitai and Kita 1987
for review). Since the discovery of the association of hemiballism with
pathologic changes in the STN (Whittier 1947
), the STN
has been recognized as playing a vital role in voluntary movement
control (see Mink and Thach 1993
; Wichmann and
DeLong 1996
for reviews). This notion is strongly supported by
findings from animal experiments in which blockade of the activity of
STN neurons induces severe motor disorders (Hamada and Hasegawa
1996
; Wichmann et al. 1994
).
Given the importance of the STN in motor control, it is of general
interest to understand how the activity of STN neurons is regulated.
The electrical activity of a neuron is driven by its synaptic inputs
and shaped by the intrinsic properties of the cytoplasmic membrane. It
has been shown that STN neurons receive excitatory inputs from the
cerebral cortex (Bevan et al. 1995; Fujimoto and
Kita 1993
; Hartmann-von Monakow et al. 1978
;
Kitai and Deniau 1981
; Nambu et al. 1996
)
and the thalamus (Bevan et al. 1995
; Feger et al.
1994
; Mouroux and Feger 1993
), and inhibitory inputs from the globus pallidus (Groenewegen and Berendse
1990
; Moriizumi and Hattori 1992
). Several
studies have examined the membrane properties of STN neurons. In a
pioneering study, Nakanishi et al. (1987)
studied the
response of STN neurons to current injections in an acutely prepared
slice preparation. Several other studies thereafter also examined the
response properties of STN neurons in acutely prepared slices
(Beurrier et al. 1999
; Bevan and Wilson 1999
; Overton and Greenfield 1995
; Song
et al. 1998
) and in cultured slices (Plenz and Kitai
1999
). In all these works, the importance of
Ca2+ conductances in the regulation of STN neuron
activity was invariably noticed. Ca2+
conductances were suggested to be involved in the generation of a
plateau-like potential (Beurrier et al. 1999
;
Nakanishi et al. 1987
; Song et al. 1998
),
in the generation of rebound activities of STN neurons (Beurrier
et al. 1999
; Overton and Greenfield 1995
; Plenz et al. 1997
; Song et al. 1998
), and
in the regulation of Ca2+-dependent conductances
(Beurrier et al. 1999
; Bevan and Wilson 1999
; Nakanishi et al. 1987
; Song et al.
1998
). Nevertheless, because Ca2+
conductances in STN neurons have not been fully characterized, it
remains unknown how Ca2+ channels are related to
these functions. Ca2+ channels are currently
classified into a low-voltage-activated (low-threshold) subtype
(T-type) and several high-voltage-activated (high-threshold) subtypes
(L-, N-, P-, Q-, and R-type) (Birnbaumer et al. 1994
;
Randall and Tsien 1995
). Although it has recently been
suggested that STN neurons express L-type (Beurrier et al. 1999
) and T-type (Beurrier et al. 1999
;
Overton and Greenfield 1995
; Plenz et al.
1997
; Song et al. 1998
) currents, the functional characteristics of these currents and their subcellular localization remain unknown. It is also unknown whether STN neurons express other
subtypes of Ca2+ channels.
To understand how Ca2+ channels are involved in
the variety of functions of STN neurons, the aim of the present study
was to identify the subtypes and functional characteristics of
Ca2+ conductances in STN neurons. To this end, we
first studied Ca2+ channels in acutely
dissociated, retrogradely labeled STN neurons. Acute dissociation trims
off dendritic processes and thus improves space clamp, at the expense
of dendritic information. To gain information on
Ca2+ conductances in dendrites, we also performed
current-clamp recordings from STN neurons in thin slices. Our results
from acutely dissociated cells revealed the absence of low-threshold
Ca2+ channels and the presence of four subtypes
(N-, L-, Q-, and R-type) of high-threshold Ca2+
channels. A low-threshold Ca2+ spike, however,
was observed in STN neurons in slice. We conclude that STN neurons
express low-threshold Ca2+ channels and several
subtypes of high-threshold channels, with the possibility of a
preferential distribution of the low-threshold channel to the dendritic
processes. Part of these results has appeared as an abstract
(Song et al. 1997).
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METHODS |
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Retrograde labeling
To identify STN neurons after dissociation, STN neurons were
retrogradely labeled by injecting a fluorescent dye, Fluoro-Gold, into
the globus pallidus, before dissociation. Sprague-Dawley rats at the
age of postnatal day 19 (P19) to
P24 were anesthetized with intraperitoneal injections of
ketamine (60 mg/kg) and xylezene (7.5 mg/kg). The adequacy of the
anesthesia was judged by the absence of reflex to ear pinches. All
experiments were conducted in compliance with the Guidelines for Use of
Laboratory Animals of Osaka University. Fluoro-Gold (Fluorochrome,
Englewood, CO; 3% in saline; 0.3 ~ 0.8 µl) was injected
bilaterally into the globus pallidus (AP = 0.2, L = 2.2 ~ 2.4, D = 5.6 ~ 5.9), with a glass micropipette
attached to a Hamilton syringe.
Acute dissociation
Five to 7 days later, STN neurons were acutely dissociated using
procedures similar to those described previously (Song and Surmeier 1996). The rats that had received Fluoro-Gold
injection, were anesthetized at P26-30 with ethyle-ether
and decapitated; brains were quickly removed, iced, and then blocked
for slicing. The blocked midbrain region was cut into 400-µm-thick
slices in horizontal plane with a Microslicer (Dosaka, Kyoto, Japan)
while bathed in a low Ca2+ (100 µM),
N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic
acid] (HEPES)-buffered salt solution (in mM: 140 Na isethionate, 2 KCl, 4 MgCl2, 0.1 CaCl2, 23 glucose, and 15 HEPES; pH 7.4, 300-305 mOsm/l). Retrograde labeling
was then examined under an epifluorescent microscope (Olympus, Tokyo),
to make sure that the STN was labeled, but that the zona incerta was
not (Fig. 1, A-C). Slices
were then incubated for 1-6 h at room temperature (20-22°C) in
NaHCO3-buffered saline bubbled with 95%
O2-5% CO2 (in mM: 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 1 pyruvic acid,
0.2 ascorbic acid, 0.1 N
-nitro-L-arginine, 1 kynurenic
acid, and 10 glucose; pH 7.4 with NaOH, 300-305 mOsm/l). Slices were
then removed into the low Ca2+ buffer, and, with
the aid of a dissecting microscope, regions of the STN were dissected
with a pair of thin tungsten needles. Dissection at the medial side of
the STN was done at a distance <1 mm from the lateral border. The
dissected STN regions were placed in an oxygenated beaker containing
pronase (1-3 mg/ml) in HEPES-buffered Hank's balanced salt solution
(in mM: 140 NaCl, 2 KCl, 1 CaCl2, 2 MgCl2, 10 glucose, and 15 HEPES; pH 7.4 with NaOH, 300-305 mOsm/l) at 35°C.
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After 30-35 min of enzyme digestion, tissue was rinsed three times in the low Ca2+, HEPES-buffered saline and mechanically dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension was then plated into a 35-mm Lux Petri dish mounted on the stage of an inverted microscope.
Whole cell recordings from dissociated cells
Whole cell recordings employed standard techniques
(Hamill et al. 1981). The internal solution consisted of
(in mM) 170 N-methyl-D-glucamine (NMG), 40 HEPES, 4 MgCl2, 0.1 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA), 12 phosphocreatine, 2 Na2ATP, 0.2 Li3GTP, and 0.1 leupeptin; pH 7.2-3 with
H2SO4, 265-270 mOsm/l. The
external solution consisted of (in mM) 127 NaCl, 20 CsCl, 1 MgCl2, 10 HEPES, 0.0005 TTX, 5 BaCl2, and 10 glucose; pH 7.4 with NaOH, 300-305
mOsm/l.
Nifedipine and ()Bay K 8644 (RBI, Natick, MA) were made up as
concentrated stock solutions in 95% ethanol and diluted immediately before use. These solutions were protected from ambient light. Final
ethanol concentrations never exceeded 0.05% (vol/vol; equal solvent
concentrations were added to all control solutions).
-Conotoxin GVIA
(
-CgTx) and
-agatoxin IVA (
-AgTx; all from Peptide Institute, Osaka, Japan) were made up as concentrated stock solutions in water,
aliquoted, and frozen; aliquots were thawed and diluted on the day of
use. Final dilutions were made in external media containing 0.1%
cytochrome C. Drugs were applied through a gravity-fed manifold system.
Solution changes were effected by electronic valves controlling the
inflow to a manifold feeding a single outlet capillary. The application
capillary (~500 µm ID) was positioned about 1 mm from the cell
under study.
Recordings were obtained with an Axon Instruments 200B patch-clamp
amplifier and controlled and monitored with a Pentium PC running pCLAMP
(v. 6.0) with a 125-kHz interface (Axon Instruments, Foster City, CA).
Electrode resistances were typically 3-6 M in the bath. After seal
rupture, series resistance (7-15 M
) was compensated (80-90%) and
periodically monitored. The adequacy of voltage control was assessed by
examining the tail currents following strong depolarizations. Cells in
which tail currents were broad or unstable at subthreshold potentials
were excluded from the analyses. Potentials were not corrected for the
liquid junction potential. Recordings were made only from retrogradely labeled neurons.
Whole cell recordings in slice
Sprague-Dawley rats at the age of P21-27 were used. The rats were anesthetized with ethyl ether and decapitated. Brains were quickly removed into an ice-cold Ringer solution and kept in the solution for at least 5 min. The solution consisted of (in mM) 126 NaCl, 2.5 KCl, 1 MgSO4, 2 CaCl2, 1.25 KH2PO4, 26 NaHCO3, and 10 glucose, pH 7.4 with HCl, 300-305 mOsm/l. The region of the brain stem containing the STN was cut into 200-µm-thick slices, in a horizontal plane. The slices were then kept in the Ringer solution at room temperature for at least 1 h, before being transferred to a recording chamber mounted on an upright microscope (Olympus, Tokyo). The slice was continuously perfused with the saline during recording.
Whole cell recordings from slices employed standard techniques
(Edwards et al. 1989). The STN was visually identified
as an cell-dense structure anterior to the substantia nigra and
surrounded by the internal capsule from the rostral, lateral, and
caudal sides. For voltage-clamp recordings, the internal consisted of (in mM) 140 NMG, 15 NaCl, 4 MgCl2, 40 HEPES, 0.1 BAPTA, 12 phosphocreatine, 2 Na2ATP, 0.2 Li3GTP, and 0.1 leupeptin; pH 7.2 with
H2SO4, 265-270 mOsm/l; the
external solution consisted of (in mM) 150 TEACl, 2 MgCl2, 5 HEPES, 0.001 TTX, 5 BaCl2, and 20 glucose; pH 7.4 with NaOH, 300 ± 5 mOsm/l. For current-clamp recordings, the internal solution
consisted of (in mM) 120 KCl, 3 MgCl2, 10 HEPES,
0.2 EGTA, 12 phosphocreatine, 2 Na2ATP, 0.2 Li3GTP, and 0.1 leupeptin, pH 7.2-3 with
H2SO4, 265-270 mOsm/l; the
external solution consisted of (in mM) 126 NaCl, 2.5 KCl, 1 MgSO4, 2 CaCl2, 1.25 KH2PO4, 26 NaHCO3, and 10 glucose, pH 7.4 with NaOH,
300-305 mOsm/l.
Recordings were obtained with an EPC-7 patch-clamp amplifier (List Instruments, Germany) and controlled and monitored with a Pentium PC running pCLAMP (v. 6.0) with a 125-kHz interface (Axon Instruments, Foster City, CA).
Statistical methods
Sample statistics are given either as medians or as means with
standard error of the mean. Box plots were used for graphic presentation of the data because of the small sample sizes
(Tukey 1977). Wilcoxon's signed-ranks test was used to
test the difference between samples.
In some experiments, membrane permeability was estimated as a function
of membrane potential using the Goldman-Hodgkin-Katz constant current
equation (Hille 1992; Song and Surmeier
1996
) I(Vm) = g(Vm)P(Vm),
where g(Vm) = z2(VmF2/RT){[[Ca]i
[Ca]o
exp(
zVmF/RT)]/[1
exp(
zVmF/RT)]};
I(Vm) is the measured
membrane current density (A/cm2);
Vm is the membrane potential (mV);
z = 2; and F, R, and T have their usual meanings.
Membrane area was calculated from whole cell capacitance assuming 1 µF/cm2. [Ca]i and
[Ca]o are Ca2+
concentrations inside and outside the cell, respectively.
[Ca]i was assumed to be 100 nM. The external
Ba2+ concentration was taken as
[Ca]o.
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RESULTS |
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Cell identification after dissociation
The STN is located anterior to the substantia nigra, medial to the internal capsule, and lateral to the zona incerta. Because axon bundles are less transparent under translucent illumination compared with soma-rich regions, the STN, under a dissecting microscope, can be identified as a brighter region surrounded by the darker internal capsule in a horizontal section (Fig. 1A). In this way, the caudal, rostral, and lateral edges of the nucleus could be identified, but not the medial edge (Fig. 1A). Thus dissecting the STN from untreated slices is problematic, as contamination from the zona incerta, which lies immediately medial to the STN, is unavoidable. To circumvent this problem, we labeled STN neurons retrogradely by injecting Fluoro-Gold into the globus pallidus. Shown in Fig. 1, B and C, are retrogradely labeled STN neurons before dissociation. While the substantia nigra was also labeled, zona incerta was not (Fig. 1, B and C). The STN region was dissected out with a mediolateral width <1 mm (Fig. 1A). Although cells from the zona incerta may be included in such tissue blocks, they could be distinguished from STN neurons by the absence of fluorescence. Thus the landmark of the internal capsule, together with retrograde labeling, provided us with an unambiguous identification of the STN. Shown in Fig. 1, D and E, are an STN neuron after dissociation under normal and epifluorescent illumination, respectively. In most experiments, only one or two cells could be identified and recorded. All recordings were made in retrogradely labeled neurons. These neurons had whole cell capacitances of 4-8 pF.
Channel activation and inactivation properties
When the membrane potential was depolarized from a holding
potential of 80 mV, inward currents were evoked (Fig.
2A). These currents could be
blocked by Cd2+ in a dose-dependent manner, with
an IC50 near 1 µM (average of 0.9 µM in 3 cells; data not shown). The amplitude of the currents decreased when
the concentration of extracellular Ba2+ was
changed from 5 to 2 mM (n = 5, data not shown). These
results suggest that STN neurons express Ca2+
channels, and that the currents recorded are Ba2+
currents through Ca2+ channels.
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The amplitude of the current was voltage dependent. Shown in Fig.
2B is the relationship between voltage and the amplitude of
the current 5 ms after initiation of the depolarizing pulse (Fig.
2A, arrow). Current-voltage curves exhibited an inverted bell shape, with the peak at either 0 or 10 mV when examined with
voltages changing by a 10-mV step (Fig. 2B;
n = 25). Currents began to appear at approximately
50
mV. This was also true when the holding potential was shifted to
100
mV (n = 9). These results suggest the absence of T-type
channels in dissociated STN neurons. The kinetics of the current also
showed some voltage dependence. Currents activated progressively faster
with stronger depolarization (Fig. 2A). Some inactivation
occurred at voltages more positive than
20 mV during the 30-ms pulse,
but no inactivation was noticed for currents evoked at lower voltages
(Fig. 2A). To examine the voltage dependence of the
Ca2+ conductances, tail current amplitudes were
normalized and plotted against voltage (Fig. 2C). This plot
should reflect the steady-state activation of
Ca2+ channels in STN neurons, with a minor error
caused by the moderate inactivation occurred during the 30-ms pulse.
The data could be well fitted with a single Boltzmann function, with a
half-activation voltage of
13.6 mV and a slope factor of 6.8 mV (Fig.
2C). In a sample of 12 cells, the average half-activation
voltage was
12.6 ± 1.3 (SE) mV, and the average slope factor
was 7.0 ± 0.6 mV. These results suggest that most, if not all, of
the Ca2+ channels in acutely dissociated STN
neurons are of the high-voltage-activated subtype.
The fact that the voltage dependence of activation could be described
by a single Boltzmann function suggests that there is either a single
subtype of channel in STN neurons or there are multiple subtypes having
similar voltage dependence. To test these possibilities, we first
examined the voltage dependence of inactivation of the channels, using
a conventional prepulse protocol. As shown in Fig.
3A, prepulses of 3 s
suppressed the amplitude of the current evoked by a subsequent pulse to
0 mV, in a voltage-dependent manner. The prepulse only partially
inactivated the current, as shown in Fig. 3B (60.8 ± 0.1%, mean ± SE, n = 8). The voltage-dependent inactivation could be approximated with a single Boltzmann function, with a constant term expressing the residual current. The average half-inactivation voltage was 30.0 ± 2.1 mV (n = 8), and the slope factor was 8.1 ± 0.9 mV. Because the voltage
dependence of both activation and inactivation of the currents did not
show clear heterogeneity, we next examined the kinetics of
inactivation. For this, currents evoked by a pulse to 0 mV for 10 s were recorded (Fig. 3C). In agreement with the prepulse
experiments, on average 38.3% of the current (n = 6)
did not inactivate during the pulse. Of interest is the decay phase of
the current. Shown in Fig. 3D is the log plot of the current
against time. It is clear from the figure that the current decayed with
more than one time constant: three time constants could be dissected in
all cases tested (n = 6), with the longest being
41.7 ± 15.3 s, the middle being 1.9 ± 0.3 s, and
the shortest being 217.6 ± 62.0 ms (mean ± SE; Fig. 3D). These results raise the possibility that STN neurons
have either multiple subtypes of high-threshold
Ca2+ channels or multiple inactivation processes
with a single channel type.
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Channel subtypes
To test whether acutely dissociated STN neurons have multiple
subtypes of channels, organic Ca2+ channel
blockers were applied at saturating concentrations (Randall and
Tsien 1995). Currents here were evoked by a slow voltage ramp, to estimate at the same time the voltage dependence of channel activation (Bargas et al. 1994
). Ramps with a rate of
0.3 mV/ms were found to produce current-voltage curves similar to those produced with voltage steps (n = 6; data not shown) and
were therefore used throughout the experiments. As shown in Fig.
4, A and B, application of the L-type channel blocker, nifedipine (5 µM), partially blocked the current. Bay K 8644 (1 µM) increased the current amplitude and slowed down the tail current decay
(n = 5, data not shown). Application of
-CgTx (1 µM) in the presence of nifedipine greatly reduced the current (Fig.
4A).
-AgTX had no effect on the current at a
concentration of 20 nM (n = 14), but blocked part of
the current at 200 nM (Fig. 4A). Co-application of all drugs
did not completely block the current (Fig. 4A). In a sample
of six neurons from six animals, the residual current, or R-type
current, consisted 8.7 ± 3.1% of the total current;
-CgTx-sensitive current, or N-type current, was the major
Ca2+ current, comprising 52.1 ± 2.4% of
the total; currents sensitive to high concentrations of
-AgTX,
defined as Q-type current (Randall and Tsien 1995
),
comprised 16.1 ± 4.1%, while L-type current was 21.0 ± 2.9% of the total (Fig. 4B, inset).
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Currents of each subtype were isolated by subtraction and are shown in
Fig. 4C. In agreement with the results shown in Fig. 2, the
current-voltage relationship of the subtypes had similar shapes.
However, some differences in channel voltage dependence were also
noticed. Shown in Fig. 4D are the voltage dependencies of
channel activation curves, calculated from the currents shown in Fig.
4C, using the Goldman-Hodgkin-Katz current equation (see METHODS). The activation of each current subtype could be
well fitted with a single Boltzmann function. For clarity, the fitted Boltzmann curves are shown in the inset of Fig.
4D. Although each subtype had similar voltage dependencies,
R-type current had the lowest threshold, and the voltage dependence of
L-type current was shifted ~5 mV toward hyperpolarization, as
compared with that of the Q- and N-types. Similar observations were
obtained in all cells tested (n = 6). The median
half-activation voltage was 19.8 mV for L-type,
16.3 mV for N-type,
15.9 mV for Q-type, and
18.6 mV for R-type current. The
half-activation voltage of L-type current was significantly lower than
both N- and Q-type (P < 0.05, Wilcoxon's signed-ranks test).
Low-threshold Ca2+ spikes in a slice preparation
The absence of T-type channels in acutely dissociated STN neurons
seems to be at odds with previous reports that suggest the presence of
T-type channels in STN neurons in slice (Beurrier et al.
1999; Overton and Greenfield 1995
) and in
culture (Plenz et al. 1997
). Results obtained from
acutely isolated STN neurons, however, should primarily reflect the
channel subtype composition in the somatic membrane. To gain
information on Ca2+ channels in dendritic
processes, we studied Ca2+ channel expression in
STN neurons in a slice preparation, focusing on low-threshold channels.
When the membrane potential was depolarized from
80 to
50 mV in
voltage-clamp mode, a transient current was observed with a delay. This
delay was voltage dependent, indicating a lack of space clamp
(n = 3, data not shown). The current thus could not be
characterized in voltage-clamp mode. Because T-type channels give rise
to a characteristic slow spike potential in current-clamp mode
(Bal and McCormick 1996
; Deschenes et al.
1982
; Llinás and Yarom 1981
), we did
current clamp recordings from STN slices. As shown in Fig.
5A, in response to a
hyperpolarizing current injection, the membrane potential
hyperpolarized and then exhibited a slow return to the resting
potential; on termination of the current pulse, a rebound action
potential was observed. Application of the Na+
channel blocker, tetrodotoxin (TTX, 1 µM) blocked the action potential, revealing an underlying, broader spike of much smaller amplitude (Fig. 5B, arrow). This spike was blocked by the
addition of Ni2+ (40 µM) to the perfusion
solution (Fig. 5C). Application of Cs+
(3 mM) blocked the return of the membrane potential toward the resting
potential during current injection (Fig. 5D). Although the
number of action potentials on the rebound differed between cells, an
Ni2+-sensitive spike was always observed
(n = 5).
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DISCUSSION |
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By recording from both acutely dissociated STN neurons and STN neurons in slice, we found that rat STN neurons express both low- and high-voltage-activated Ca2+ currents. All known high-voltage-activated channels were found in STN neurons except the P-type. We also obtained evidence suggesting the possibility that low-threshold, or T-type, channels are preferentially distributed to dendritic processes.
Cell identification
These conclusions clearly depend on reliable identification of the
neurons recorded. Acutely dissociated cells provide a good preparation
for patch-clamp recording, but dissociation also makes it difficult to
identify the neuronal phenotype. Cell identification is especially
important here, as the border between STN and the zona incerta is not
discernible. Our method of identification of the STN, using a
combination of landmark identification and retrograde labeling, leaves
little ambiguity. Our identification method also limits the neurons
analyzed in this study to STN neurons that project to the globus
pallidus. Such neurons, however, should represent the majority of STN
neurons, because it has been shown that more than 90% of STN neurons
project to the globus pallidus (Van der Kooy and Hattori
1980).
Channel subtypes expressed in STN neurons
Although the presence of L-type Ca2+
conductance in STN neurons has been reported recently (Beurrier
et al. 1999), expression of other subtypes has remained
unknown. Here we have shown that all high-voltage-activated subtypes,
except P-type, are expressed at different levels in STN neurons. It is
well established that the L-type current can be identified by its
sensitivity to dihydropyridines and Q-type current can be identified by
its sensitivity to high concentrations of
-AgTx (Randall and
Tsien 1995
). But the identification of R-type current can be
problematic, as it is only defined by its resistance to all known
organic blockers. The presence of an R-type current in STN neurons is
based on the fact that we used saturating concentrations of blockers to
other subtypes of current. Nevertheless, the proportion of R-type
current might have been overestimated, because of the slow time course
of
-AgTx block. The presence of R-type current in STN neurons is
also evidenced by the biophysical differences between R- and Q-type
currents shown in this study. Furthermore, class E
1 subunit mRNA,
which is suggested to encode R-type channels (Zhang et al.
1993
), has been shown to be expressed in STN neurons
(Yokoyama et al. 1995
).
Because currents recorded from acutely dissociated cells began to
activate at around 50 mV, Ca2+ channels in
acutely dissociated STN neurons are exclusively of the
high-voltage-activated variety. The absence of low-threshold channels
in dissociated cells is also evidenced by the inactivation kinetics of
the current. In addition to the low threshold of activation (approximately
60 mV), fast inactivation is another characteristic of
T-type current (Carbone and Lux 1984
). T current
inactivates with a time constant of 10-25 ms at room temperature
(Mouginot et al. 1997
; Tarasenko et al.
1998
). The fact that the shortest inactivation time constant in
STN neurons was longer than 200 ms (see Fig. 3D) supports
the view that dissociated STN neurons has little, if any, T-type current.
The TTX-resistant broad spike observed on the rebound potential in
slice is likely to be a low-threshold Ca2+ spike
(Llinás and Yarom 1981). First, the spike occurred
at a low membrane potential close to
60 mV. Second, the shape of the
spike resembled that of low-threshold Ca2+ spikes
reported in other neuron types (Bal and McCormick 1996
; Deschenes et al. 1982
). And third, the spike could be
blocked by micromolar concentration of Ni2+
(Fox et al. 1987
). Previous electrophysiological studies
have also suggested the presence of a low-threshold
Ca2+ spike in STN neurons (Beurrier et al.
1999
; Overton and Greenfield 1995
; Plenz
et al. 1997
). These observations are consistent with the recent
demonstration that STN neurons express mRNAs coding for T-type channels
(Perez-Reyes et al. 1998
; Talley et al.
1999
). However, T-type channels were not observed in acutely
dissociated STN neurons. This discrepancy might be attributable to the
age difference between animals used for the slice experiments
(P21-27) and those used for dissociated cell experiments
(P26-30), because T-type channels often disappear during
maturation (Bargas et al. 1994
; Chameau et al.
1999
). In STN neurons, however, mRNAs coding for T-type
channels are expressed even in adults (Perez-Reyes et al.
1998
; Talley et al. 1999
), although mRNA
expression may not necessarily mean expression of functional proteins.
Another possibility is that T-type channels may not survive the
enzymatic treatment used for dissociation, but T-type currents have
been successfully recorded in neostriatal neurons treated with the same enzyme as in the present experiment, under the same condition (Bargas et al. 1994
). Thus the absence of detectable
level of T-type currents in dissociated STN neurons may be attributable to loss of dendritic processes during dissociation, and suggests the
possibility that STN neurons express T-type channels preferentially in
dendrites. This notion, however, is based on the negative finding of
T-type currents in dissociated neurons, and thus needs to be further tested.
Functional significance
Our results demonstrate that STN neurons express T-, L-, N-, Q-, and R-type Ca2+ channels. A question arises then of what is the functional significance of each subtype. This is especially puzzling for the high-voltage-activated subtypes, because they share similar biophysical properties.
The function of T-type current seems to be well established. With a
sufficient channel density, T-type currents can generate a spike: T
spike (Llinás and Yarom 1981) (see Fig.
5B). This spike is broader than sodium spikes, because of
the slower kinetics of the T-type channels, as compared with
Na+ channels. Therefore on top of the T spike, a
short train of action potentials is often elicited (Bal and
McCormick 1996
). Furthermore, by interacting with other ion
channels, T-type channels are thought to be important in the generation
of oscillatory activity (Bal and McCormick 1996
). The
functional significance of T-type channels in STN neurons, however,
remains obscure. Although STN neurons express many key elements for
oscillatory bursting, including T-type channels, H channels (Fig. 5)
(Beurrier et al. 1999
; Bevan and Wilson
1999
; Plenz et al. 1997
), and
Ca2+-dependent K+ channels
(Beurrier et al. 1999
; Bevan and Wilson
1999
; Nakanishi et al. 1987
), STN neurons do not
appear to generate rhythmic bursting by themselves (Georgopoulos
et al. 1983
; Magill et al. 2000
; but see
Beurrier et al. 1999
). Provided that T-type channels in
STN neurons have a preferential distribution in dendritic processes, they may play a role in synaptic integration in STN neurons.
In the present experiment, among the high-voltage-activated subtypes,
N-type current comprised more than half of the current. N-type current
is expressed in most central neurons, but the proportion of this
current to the total is often between 20 and 30% (Bargas et al.
1994; Cardozo and Bean 1995
; Lorenzon and
Foehring 1995
). Because it is now well established that N-type
current is subject to neuromodulation by a number of receptors coupled
to trimeric guanine-binding proteins (Hille 1994
), the
predominance of N-type current in STN neurons would make
Ca2+ entry into these neurons highly modifiable.
A variety of neurotransmitters are known to be released within the STN,
including GABA (Bevan and Bolam 1995
), acetylcholine
(Bevan and Bolam 1995
), serotonin (Pompeiano et
al. 1994
), glutamate (Bevan and Bolam 1995
;
Mouroux and Feger 1993
) and probably dopamine
(Canteras et al. 1990
). All these neurotransmitters may
modulate Ca2+ entry through N-type channels into
STN neurons, depending on the receptors expressed. It thus would be
interesting to test how Ca2+ currents in STN
neurons are modulated by these neurotransmitters. In addition to the
difference in susceptibility to neuromodulation, different subtypes of
the high-voltage-activated channels may be differentially coupled to
other signaling mechanisms. For example, N-type channels, but not
L-type channels, are coupled to Ca2+-dependent
K+ channels in motor neurons (Viana et al.
1993
), while in hippocampal neurons L-type channels are known
to be coupled to the K+ channel (Moyer et
al. 1992
). Modulation of Ca2+ currents in
STN neurons may in turn change the activity of
Ca2+-dependent processes as well.
The biophysical differences between the high-voltage-activated
channels, although small, may also have a significant impact on the
contribution of each channel subtype to neuronal activity. The slightly
lower activation voltage of the L- and R-type channels may result in
them serving unique functions. A long-lasting plateau potential
generated in STN neurons has been shown to be predominantly mediated by
L-type currents (Beurrier et al. 1999; Song et
al. 1998
). Because the plateau is at a low voltage
(approximately
40 mV) (Beurrier et al. 1999
), it is
likely that this unique function of L-type channels is attributable to
its lower activation-voltage demonstrated in the present study.
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ACKNOWLEDGMENTS |
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We thank Dr. P. Mermelstein for reading an early version of the manuscript. F. Murakami is an investigator of Core Research for Evolutional Science and Technology.
W.-J. Song was supported by grants from the Ministry of Education, Science, and Culture, Japan (9280217, 11170232, 9780769, and 12053247), the Naito Foundation, and the Uehara Memorial Foundation.
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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 26 April 2000; accepted in final form 28 July 2000.
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REFERENCES |
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