1Laboratoire de Neurophysiologie, Centre National de la Recherche Scientifique, Unite Mixte de Recherche 5543, Université Bordeaux II, 33076 Bordeaux Cedex; and 2Institut National de la Santé et de la Recherche Médicale U29, 13273 Marseille Cedex 09, France
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ABSTRACT |
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Beurrier, Corinne,
Bernard Bioulac,
Jacques Audin, and
Constance Hammond.
High-Frequency Stimulation Produces a Transient Blockade of
Voltage-Gated Currents in Subthalamic Neurons.
J. Neurophysiol. 85: 1351-1356, 2001.
The effect of
high-frequency stimulation (HFS) of the subthalamic nucleus (STN) was
analyzed with patch-clamp techniques (whole cell configuration,
current- and voltage-clamp modes) in rat STN slices in vitro. A brief
tetanus, consisting of 100-µs bipolar stimuli at a frequency of
100-250 Hz during 1 min, produced a full blockade of ongoing STN
activity whether it was in the tonic or bursting mode. This HFS-induced
silence lasted around 6 min after the end of stimulation, was frequency
dependent, could be repeated without alteration, and was not
synaptically induced as it was still observed in the presence of
blockers of ionotropic GABA and glutamate receptors or in the presence
of cobalt at a concentration (2 mM) that blocks voltage-gated
Ca2+ channels and synaptic transmission. During
HFS-induced silence, the following alterations were observed: the
persistent Na+ current
(INaP) was totally blocked (by 99%),
the Ca2+-mediated responses were strongly reduced
including the posthyperpolarization rebound (62% in amplitude) and
the plateau potential (
76% in duration), suggesting that T- and
L-type Ca2+ currents are transiently depressed by
HFS, whereas the Cs+-sensitive,
hyperpolarization-activated cationic current
(Ih) was little affected. Thus a
high-frequency tetanus produces a blockade of the spontaneous
activities of STN neurons as a result of a strong depression of
intrinsic voltage-gated currents underlying single-spike and bursting
modes of discharge. These effects of HFS, which are completely
independent of synaptic transmission, provide a mechanism for
interrupting ongoing activities of STN neurons.
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INTRODUCTION |
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The observation that deep brain
stimulation applied at a high-frequency (HFS) in the subthalamic
nucleus (STN) and its surgical destruction, both greatly ameliorate
motor signs of Parkinson's disease in patients, led to the hypothesis
that HFS blocks, partly or completely, the activity of STN neurons. In
keeping with this, HFS in the STN has been shown to significantly
decrease the frequency of extracellularly recorded STN neurons in rats
in vivo (Benazzouz et al. 1997). As STN neurons are
glutamatergic excitatory output neurons (Hammond et al.
1978
; Robledo and Féger 1990
; Smith
and Parent 1988
), the immediate consequence of their reduction
of activity could be the decrease of activity in target nuclei
[substantia nigra pars reticulata (SNr) and entopeduncular
nucleus/globus pallidus internal part (EP/GPi)] as observed in
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys and naive rats (Benazzouz
et al. 1995
; Burbaud et al. 1994
; Hayase
et al. 1996
). It has also been suggested that the consequence
of clinical HFS will be to somehow counteract the abnormal bursting
pattern recorded in the STN in animal models of Parkinson disease
(Bergman et al. 1994
; Hassani et al.
1996
; Hollerman and Grace 1992
; Vila et
al. 2000
).
To understand the contribution of HFS in pathological conditions, it is
clearly essential to determine whether a HFS of the STN could modify or
block the intrinsic activities of STN neurons and to analyze the
underlying mechanisms. This is best achieved in vitro, as slice
preparations enable to better isolate the various effects of a tetanus
on neuronal properties. In the present study, using patch-clamp
recordings of rat STN neurons in slices, we report that HFS of the STN
suppresses the spontaneous activity of both single-spike and bursting
STN neurons. The effects of HFS are synaptic-independent and are
mediated by a blockade of the voltage-gated currents and particularly
the persistent Na+
(INaP) current and the L- and T-type
Ca2+ currents
(ICaL and
ICaT) that are known to generate the
intrinsic spontaneous discharge modes of STN neurons (Beurrier
et al. 1999, 2000
; Bevan and Wilson 1999
).
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METHODS |
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Slice preparation
Experiments were performed on STN neurons in slices obtained from 20- to 28-day-old male Wistar rats. Rats were anesthetized with ether and decapitated. The brain was quickly removed, and a block of tissue containing the STN was isolated on ice in a 0-5°C oxygenated solution containing (in mM) 1.15 NaH2 PO4, 2 KCl, 26 NaHCO3, 7 MgCl2, 0.5 CaCl2, 11 glucose, and 250 saccharose, equilibrated with 95% O2-5% CO2 (pH 7.4). This cold solution, with a low NaCl and CaCl2 content, improved tissue viability. In the same medium, 300- to 400-µm-thick coronal slices were prepared using a vibratome (Campden Instruments, Loughborough, UK) and were then incubated at room temperature in a Krebs solution containing (in mM) 124 NaCl, 3.6 KCl, 1.25 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), 26 NaHCO3, 1.3 MgCl2, 2.4 CaCl2, and 10 glucose, equilibrated with 95% O2-5% CO2 (pH 7.4). After a 2-h recovery period, STN slices were transferred individually to an interface-type recording chamber, maintained at 30 ± 2°C (mean ± SD) and continuously superfused (1-1.5 ml/min) with the oxygenated Krebs solution.
STN stimulation
The stimulating electrode was positioned in the middle of
the STN identified as an ovoid structure just lying at the border of
the basal part of the cerebral peduncle. Two types of stimulating electrodes were tested: The bipolar concentric electrode measuring 0.5 mm in diameter (NEX-100, Rhodes Medical Instruments) used by Burbaud
(Burbaud et al. 1994) and Benazzouz (Benazzouz et
al. 1995
) for the in vivo stimulation of the rat STN and a much
thinner electrode (0.01 mm in diameter) that we designed to avoid any mechanical lesion of the STN.
Electrophysiological recordings
Slices were visualized using a dissecting microscope and the
recording electrode was precisely positioned in the STN.
Electrophysiological recordings of STN neurons were performed in the
current- or voltage-clamp mode using the blind patch-clamp technique in
the whole cell configuration. Patch electrodes were pulled from
filamented borosilicate thin-wall glass capillaries (GC150F-15, Clarck
Electromedical Instruments, Pangbourne, UK) with a vertical puller
(PP-830, Narishige, Japan) and had a resistance of 10-12 M when
filled with the following (in mM): 120 Kgluconate, 10 KCl, 10 NaCl, 10 ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 HEPES, 1 CaCl2, 2 MgATP, and 0.5 NaGTP, pH 7.25.
Reagents
Drugs were applied by bath. Reagents were procured from Sigma
(St. Louis, MO), except 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), D-()-2-amino-5-phosphopentanoic acid
(D-APV), and bicuculline, which were purchased from Tocris
(Bristol, UK).
Data analysis
Membrane potential was recorded using Axoclamp 2A or Axopatch 1D
amplifier (Axon Instruments, Foster City, CA), displayed simultaneously
on a storage oscilloscope and a four-channel chart recorder (Gould
Instruments, Longjumeau, France), digitized (DR-890, NeuroData
Instruments, New York), and stored on a videotape for subsequent
off-line analysis. During voltage-clamp recordings, membrane currents
were fed into an A/D converter (Digidata 1200, Axon Instruments),
stored, and analyzed on a PC using pCLAMP software (version 6.0.3, Axon
Instruments). Corrections for the liquid junction potential were
performed according to Neher (1992):
6 mV for the
K-gluconate-based pipette solution as estimated with a 3 M KCl ground electrode.
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RESULTS |
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HFS-induced arrest of single-spike or bursting activity
STN activity was recorded in current-clamp mode (whole cell configuration) for at least 1 min before the HFS was applied. Using a bipolar concentric stimulating electrode similar to that used in rat in vivo (see METHODS), a brief (1 min) HFS consisting of 100 µs stimuli of 5-8 V amplitude, produced a blockade of ongoing activity whether it was in single-spike (Fig. 1) or bursting (Fig. 2) mode. This effect was frequency dependent (Figs. 1A and 2) with an optimal frequency of 166 up to 250 Hz that produced a full blockade of the activity (n = 17). The latency of the HFS-induced silence could not be determined in detail as during the 1-min stimulation period, artifacts prevented analysis of the activity. Nevertheless as shown in Figs. 1B and 2, above a certain frequency, the onset of the blockade was immediately obvious by the end of the train. Interestingly, HFS blocked both single spike (Fig. 1) and burst firing (Fig. 2) modes, suggesting that its mechanisms do not involve a current(s) that is expressed only in one type of discharge.
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The suppression of STN spontaneous activity was observed for 5.8 ± 0.7 min (range: 1.1-18.0, n = 31) after HFS. At the
end of the silence period, spontaneous activity slowly recovered in the
same mode as before stimulation (Figs. 1B to 6). During cell silence, membrane potential remained stable at 52.2 ± 0.8 mV (range:
40 to
68, n = 45) for tonic cells and at
56.2 ± 1.4 mV (range:
48 to
61 mV, n = 8)
for bursting cells. These membrane potentials were significantly more
depolarized than the potentials at which cells were silent in control
conditions: before HFS, cells tested in the tonic mode were silent at
60.2 ± 0.6 mV (range:
49 to
68 mV, n = 45, P < 0.001, paired t-test) and cells tested in the bursting mode were silent at
63.5 ± 1.3 mV (range:
56 to
68 mV, n = 8, P = 0.015, paired
t-test). This suggested that HFS did not stop STN cell
activity simply by transiently hyperpolarizing the membrane.
Spikes could still be evoked during the silence period in all tested
neurons (n = 60). However, in half of the cells, spike threshold was significantly higher during the silence period
(39.3 ± 1.6 mV, n = 30) compared with the
control (
47.5 ± 0.6 mV, n = 30, P < 0.001; Fig. 3). So
was also input membrane resistance, which was significantly increased
during HFS-induced silence, when tested at
Vm =
65 mV by applying
hyperpolarizing current pulses of
100/-200 pA amplitude (247.2 ± 21.1 vs. 226.1 ± 16.3 M
, P = 0.035, n = 20). A second tetanus, applied after the cell recovered from the first one, reversibly silenced the cell again (n = 15). This could be repeated as long as patch
recording could last. Therefore HFS does depress neuronal activity in
slices, and this effect is short lasting and can be repeated. In
subsequent experiments, we used a thinner electrode designed to avoid
any mechanical lesion of the STN. We chose to use the same parameters of train duration (1 min) and of bipolar stimuli intensity (5-8 V) and
duration (100 µs) but to vary their frequency in the train (range
100-500 Hz) to obtain a clear-cut suppression of activity during which
a long-lasting analysis of currents or specific responses could be
performed.
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HFS-induced suppression of activity is independent of synaptic activity
An important issue was to determine whether effects of the train
were mediated by synaptic transmission. Bath applications of ionotropic
glutamate and GABAA receptor antagonists, CNQX
(20 µM), D-APV (40 µM), and bicuculline (10 µM)
failed to prevent the effects of HFS (n = 6, Fig.
4). Furthermore HFS still suppressed single-spike activity when synaptic transmission was blocked by 2 mM
Co2+ (n = 16, Fig.
5A, top). Since the silencing
effect of HFS did not require Ca2+-dependent
transmitter release, we tested whether it was possible to mimic this
effect with intracellular stimulation of the recorded cell. When
comparing the two types of HFS (extracellular and intracellular) in the
same tonically active STN neurons (n = 8), it appeared that both HFS resulted in a silence of the cell. However, intracellular HFS had a different effect on membrane potential: there was a strong
hyperpolarization of the membrane at the break of the intracellular pulses (to 63.2 ± 3.1 mV) that declined in about 20 s to
48.1 ± 4.1 mV, a potential at which tonic activity recovered
(n = 8, data not shown). Such an after
hyperpolarization and slow membrane repolarization were never observed
after extracellular HFS where membrane potential remained stable during
cell silence (Figs. 1-6).
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HFS-induced decrease of voltage-gated currents
We hypothesized that HFS induced a modification of
voltage-sensitive currents essential for the expression of tonic and
burst-firing modes (Beurrier et al. 2000; Bevan
and Wilson 1999
). In the tonic mode, the silencing effect of
HFS did not require Ca2+ influx since it was
still observed in the presence of 2 mM Co2+ nor
increase of intracellular Ca2+ concentration
since it was present in BAPTA-loaded cells (n = 4, data
not shown). We therefore tested the effect of HFS on spontaneous tonic
activity and INaP recorded from the
same STN neurons by shifting from current- to voltage-clamp mode
before, during, and after HFS-induced silence. In voltage-clamp mode,
in response to a voltage ramp and in the continuous presence of
Co2+, a TTX-sensitive inward current that
had the characteristics of a persistent Na+
current was recorded. It was strongly reduced during HFS-induced silence (Fig. 5). I-V relationships before and during
HFS-induced silence showed that peak amplitude of
INaP was reduced by 99% during cell
silence as compared with the control (from
122.2 ± 13.1 to
1.1 ± 1.1 pA, n = 9; Fig. 5, B and
C). This effect reversed to 78% of control (to
92.5 ± 9.9 pA, n = 8) once cell activity recovered. When
applied at the end of the experiment, TTX (1 µM) totally blocked this
current, confirming that it was INaP
(Fig. 5A).
Spontaneous bursting mode and ICa were
then analyzed. However, since the recording of
Ca2+ currents requires the presence of
K+ channel blockers, a procedure incompatible
with the recording of burst firing in current-clamp mode, the amplitude
of Ca2+ currents was therefore evaluated from the
evoked potentials they underlie: the rebound depolarization, also
called low-threshold Ca2+ spike (LTS), that
results from the activation of a T-type Ca2+
current and the plateau potential that results from the combined action
of the nifedipine-sensitive L-type Ca2+ current
and a Ca2+-activated inward current
(Beurrier et al. 1999). Following HFS, during minutes of
silence, plateau duration was reduced by 62% (from 1119.4 ± 150.6 to 425.6 ± 111.4 ms, n = 32) sometimes with a total suppression of the after spike depolarization (Fig.
6, A, top and
middle, and B, left). Concomitantly, the
amplitude of the rebound potential was reduced by 75.9% (from 8.8 ± 0.4 to 2.1 ± 0.5 mV, n = 23; Fig. 6, A,
top and bottom, and B, right). Once cell
activity recovered, the effects on plateau potential duration and on
the amplitude of rebound potential reversed to 66% of control (to
739.4 ± 217.9 ms, n = 18) and to 39% of control (to 3.4 ± 0.8 mV, n = 14), respectively.
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In contrast, the Cs+-sensitive,
hyperpolarization-activated cation current
(Ih) was not affected by HFS at
potentials normally traversed by the membrane during tonic firing. It
was reduced between 80 and
110 mV (by 26.5% at
90 mV,
n = 5, Fig. 7).
Consistent with these findings on Ih,
the amplitude of the depolarizing sag observed during a hyperpolarizing
current pulse was not significantly affected (it was reduced by 4.8%,
from 5.21 ± 0.82 to 4.99 ± 0.80 mV, P = 0.69, n = 12; Fig. 6A, bottom).
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DISCUSSION |
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Our results show that HFS blocks the spontaneous activity of tonic
and bursting STN neurons with a mechanism that does not require
Ca2+-dependent transmitter release. The silencing
effect of HFS has a short latency, is brief, reversible, can be
repeated several times with little change, and is frequency dependent.
It is mediated by a dramatic reduction of Na+ and
Ca2+ voltage-gated currents leading to an
interruption of the spontaneous activities of the neurons. In fact, in
single-spike activity, a TTX-sensitive, persistent
Na+ current
(INaP), underlies the slow pacemaker
depolarization that spontaneously depolarizes the membrane from the
peak of the after spike hyperpolarization to the threshold potential
for spike initiation (Beurrier et al. 2000; Bevan
and Wilson 1999
). In contrast, in burst-firing mode, the
interplay between a T-type Ca2+ current
(ICaT), an L-type
Ca2+ current
(ICaL), and a
Ca2+-activated inward current, all insensitive to
TTX, underlie recurrent membrane oscillations (Beurrier et al.
1999
). The blockade of these subliminal currents can also
explain the increase of membrane resistance observed during HFS-induced silence.
The silencing effect of HFS does not result from the activation of a
local network and is not mediated by the stimulation of afferents to
the STN, since it was still observed in the presence of blockers of
glutamatergic and GABAergic ionotropic synaptic transmission and in the
presence of cobalt at a concentration that totally blocked synaptic
transmission in the STN. It was in fact reproduced by direct
stimulation of the recorded STN cell as previously tested by
Borde et al. (2000) in hippocampal CA1 pyramidal
neurons. In this preparation, a low-frequency intracellular stimulation
induced a depression of activity that developed rapidly, was
reversible, persisted up to 3 min and was still observed when synaptic
transmission was strongly reduced by the P-type
Ca2+ channel blocker
-agatoxin IVA or enhanced
by 4-aminopyridine. The insensitivity of depression to synaptic
blockade indicates little if any involvement of synaptic mechanisms and
implies that postsynaptic mechanisms are key factors as observed in the
present study with extracellular HFS. However, mechanisms underlying
intracellular stimulation may be different from those underlying
extracellular HFS. The silencing effect of intracellular stimulation is
Ca2+-dependent since it requires
Ca2+ influx and intracellular
Ca2+ increase in the stimulated cell
(Borde et al. 2000
), whereas that of extracellular HFS
is Ca2+-independent (the present study).
As the pattern of discharge of STN neurons may play an important role
in the physio-pathology of parkinsonism (Bergman et al.
1994; Hollerman and Grace 1992
), it is tempting
to correlate the present effects of in vitro HFS on the spontaneous STN
activity, to the beneficial effects of high-frequency deep brain
stimulation in the STN of MPTP-treated monkeys (Benazzouz et al.
1992
; Hayase et al. 1996
) or parkinsonian
patients (Benabid et al. 1994
; Limousin et al.
1998
). However, such a direct correlation needs further experiments. First, clinical HFS is performed in vivo where it could
affect the whole basal ganglia network, at least at the onset of
stimulation. Second, clinical HFS is efficient at lower frequencies
(125-185 Hz) than sometimes in vitro HFS does. This could be explained
by the differences in the characteristics of the stimulating electrode.
Finally, beneficial clinical effects are observed during the continuous
application of the stimulation and only for a short while after the
stimulation, whereas in the present study, only events that followed
the stimulation have been studied. Nevertheless, the present results
give some insights in the way intrinsic activity of STN neurons can be depressed.
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ACKNOWLEDGMENTS |
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Present address of C. Beurrier: Dept. of Psychiatry and Behavioral Sciences, School of Medicine, Stanford University, 1201 Welch Rd., Palo Alto, CA 94304-5485.
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FOOTNOTES |
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Address for reprint requests: C. Hammond, INSERM U29, Route de Luminy, 13273 Marseille Cedex 09, France (E-mail: hammond{at}inmed.univ-mrs.fr).
Received 8 June 2000; accepted in final form 22 December 2000.
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REFERENCES |
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