Departments of 1Neurological Surgery and 2Physiology and Biophysics, University of Washington, Seattle, Washington 98195-6470
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ABSTRACT |
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Lopantsev, Valeri and
Philip A. Schwartzkroin.
GABAA-Dependent Chloride Influx Modulates
GABAB-Mediated IPSPs in Hippocampal Pyramidal Cells.
J. Neurophysiol. 82: 1218-1223, 1999.
The relationship between postsynaptic inhibitory responses
[the fast GABAA-mediated inhibitory postsynaptic potential
(IPSP) and the slow GABAB-mediated IPSP] were investigated
in hippocampal CA3 pyramidal cells. Mossy fiber-evoked
GABAB-mediated IPSPs were, paradoxically, of greater
amplitude in cells with resting membrane potential of 62 mV
(13.6 ± 0.5 mV; mean ± SE) as compared with cells
with resting membrane potential of
54 mV (7.0 ± 0.8 mV). In
addition, when a cell's membrane potential was artificially manipulated, GABAB-mediated IPSPs were reduced at
relatively depolarized levels (
55 mV) and enhanced at relatively
hyperpolarized potentials (at least
60 mV). In contrast, the
preceding GABAA-mediated IPSPs were larger at the more
positive membrane potentials and smaller as the cell was
hyperpolarized. Similar voltage dependency was obtained when
monosynaptic GABAA- and GABAB-mediated IPSPs
were isolated in the presence of glutamatergic receptor antagonists. However, monosynaptic GABAB-mediated IPSPs isolated in the
presence of glutamatergic and GABAA receptor antagonists
were not reduced at the more positive membrane potentials, and were
significantly larger in amplitude than GABAB-mediated IPSPs
preceded by a monosynaptic GABAA-mediated IPSP. The
amplitude of the isolated monosynaptic GABAB-mediated IPSPs
recorded with potassium chloride-containing microelectrodes was
significantly smaller than the comparable potential recorded with
potassium acetate microelectrodes without chloride. We conclude that
voltage-dependent chloride influx, via GABAA receptor-gated
channels, modulates postsynaptic GABAB-mediated inhibition
in hippocampal CA3 pyramidal cells.
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INTRODUCTION |
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Inhibition mediated through postsynaptic
-aminobutyric acid (GABA) receptors modulates neuronal excitability
in the neocortex, hippocampus and others forebrain structures of the
adult CNS. Under normal conditions, afferent activation of neurons in
these regions induces short-lasting excitatory synaptic drive followed by two inhibitory postsynaptic potentials, mediated by
GABAA and GABAB
postsynaptic receptors. These fast and slow inhibitory postsynaptic potentials (IPSPs) are closely linked in time, but exhibit very different electrophysiological and pharmacological properties (for
review, see Sivilotti and Nistri 1991
). Chloride influx
through GABAA receptor-gated ion channels leads
to an initial fast hyperpolarizing potential, which can be reversed by
intracellular chloride injection; this action can be blocked by the
GABAA receptor antagonists bicuculline and
picrotoxin (Ben-Ari et al. 1981
; Knowles et al.
1984
; Newberry and Nicoll 1984b
). The
metabotropic postsynaptic GABAB receptors are
linked to an increase in potassium permeability via activation of
intracellular G proteins; the consequent slow, late hyperpolarizing potential can be imitated by microapplications of the
GABAB receptor agonist baclofen (Dutar and
Nicoll 1988a
,b
; Gähwiler and Brown 1985
;
Newberry and Nicoll 1984a
,b
). The potassium current
linked to GABAB receptor activation exhibits
inward rectification, reflected in a reduction in current amplitude as
the cell's membrane potential is moved in a positive direction,
further from potassium equilibrium potential (Gähwiler and
Brown 1985
; Lüscher et al. 1997
;
Sodickson and Bean 1996
, 1998
).
Thus far, no clear evidence for the direct impact of
GABAA on GABAB-mediated
IPSPs has been documented. However, recent findings suggest that
intracellular chloride may play an important role in the modulation of
different G-protein-linked permeabilities, including that activated by
GABAB receptors (Lenz et al.
1997). Because GABAA-mediated IPSPs
precede GABAB-mediated IPSPs in orthodromically activated neurons, we decided to investigate possible effects of
chloride influx (via GABAA receptor-gated
channels) on GABAB receptor-mediated inhibition.
The results of the present study show that GABAA
receptor-mediated chloride influx, which depends sensitively on
membrane potential, significantly affects GABAB receptor-mediated inhibition in hippocampal pyramidal cells and thus
may participate in the phenomenon of inward rectification.
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METHODS |
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Hippocampal slices were prepared from brains of 1- to 1.5-mo-old
Sprague-Dawley male rats. Animals were decapitated under halothane
anesthesia, and their brains were removed quickly into 2-4°C
artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 3 KCl, 2 CaCl2, 2 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, and 10 dextrose,
saturated with 95% O2-5%
CO2 gas (pH 7.4). Horizontal hippocampal
slices (400-µm thick) were cut using a Vibroslicer (Campden
Instruments) and then transferred to a holding chamber containing ACSF
at room temperature for 1 h before recording. In the recording
chamber, slices were kept at 32°C at an interface between oxygenated
ACSF and humidified gas. Rate of perfusion (0.8-1 ml/min) was kept
constant throughout the experiment.
Intracellular recordings of CA3 pyramidal cells were performed with
sharp glass microelectrodes (resistance = 70-100 M) filled with one of the following solutions: 4 M potassium acetate; 3 M
potassium acetate and 0.2 M ethylene-glycol-bis(
-aminoethyl ether)-N, N,N',N'-tetraacetic acid (EGTA); 3 M potassium
acetate and 1 M potassium chloride. All solutions were adjusted (with KOH) to pH 7.4. Only neurons with a resting membrane potential and
synaptic responses stable for
30 min were included in our analysis.
Signals were recorded using an Axoclamp-2A amplifier (Axon Instruments)
in bridge mode. Bridge balance was monitored throughout the experiment.
Cell resting membrane potential (RMP) was measured after withdrawal of
the microelectrode from the cell; action potential amplitude was
calculated from RMP; and cell input resistance was obtained from
maximum voltage change in response to a hyperpolarizing current pulse
(
0.4 nA, 100 ms). Data were digitized (Neuro-Corder, Neuro Data
Instruments) and acquired using AxoScope software (Axon Instruments) on
a 486-based computer.
A stimulating bipolar stainless steel electrode was placed in the stratum lucidum to activate the mossy fibers. To elicit monosynaptic IPSPs, the stimulating electrode was placed close (<1 mm) to the site of recording and glutamate receptor antagonists were added to the bathing medium. Stimuli (0.1-ms duration) were delivered at 0.1 Hz, at an intensity maximal for induction of a slow GABAB-mediated IPSP. Amplitudes of the GABAA- and GABAB-mediated IPSP were measured from the RMP, at latencies of 15 and 140 ms, respectively. Measurements were expressed as means ± SE, and compared across experimental conditions using Student's t-test. Data were considered significantly different if P < 0.05.
Bicuculline methiodide (BMI, 20 µM, Sigma), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM, Research Biochemicals), 2-amino-5-phosphonovaleric acid (APV, 50 µM, Research Biochemicals), and P-(3-amino-propyl)-P-diethoxymethyl-phosphinic acid (CGP35348, 700 µM, Ciba Geigy) were applied via bath perfusion.
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RESULTS |
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Intracellular recordings were obtained from 42 neurons in the
pyramidal layer of CA3 region with microelectrodes filled with 4 M
potassium acetate. The resting membrane potential of the recorded cells
varied from 51 to
72 mV (mean ± SE = 60.9 ± 0.9 mV, n = 42), action potential amplitude from 80 to 104 mV (93.2 ± 1.1 mV, n = 42), membrane input
resistance from 32 to 74 M
(51.3 ± 2.1 M
, n = 42). In normal ACSF, mossy fiber stimulation typically induced a
sequence of postsynaptic potentials that included a fast initial
excitatory postsynaptic potential (EPSP) (often capped by an action
potential), a fast IPSP (termed "GABAA IPSP"
because it was blocked by the GABAA receptor
antagonist, BMI) and a subsequent slow IPSP (termed
"GABAB IPSP" because it was blocked by the
GABAB receptor antagonist, CGP35348) (Fig.
1A). At RMP, shape and size of
the GABAA and GABAB IPSPs
varied across cells, related largely to the RMP value for a given cell
(Fig. 1A). At relatively positive RMPs (i.e., close to
50
mV), neurons had a pronounced GABAA IPSP, but the
subsequent GABAB IPSP was small and of
short-duration (see example in Fig. 1A). Neurons with the
RMP more than or equal to
60 mV demonstrated a low-amplitude
GABAA IPSP, but a high-amplitude and long-lasting
GABAB IPSP (Fig. 1A).
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The amplitudes of the GABAA and
GABAB IPSPs were compared in two groups of cells
with RMP = 54 and
62 mV (Fig. 1B). The mean
amplitude of the GABAA IPSPs was significantly
larger at
54 mV (11.3 ± 0.3 mV, n = 4) than at
62 mV (6.6 ± 0.3 mV, n = 4). In contrast the
mean amplitude of the GABAB was significantly smaller at
54 mV (7.0 ± 0.8 mV, n = 4) than at
62 mV (13.6 ± 0.3 mV, n = 4). In 18 neurons,
positive or negative DC current was passed through the intracellular
microelectrode, and postsynaptic responses were induced at the
different membrane potential levels (Fig.
2A). The
GABAA IPSPs had a monotonic linear dependence on membrane potential and usually reversed in polarity between
65 and
70 mV (Fig. 2B). Dependency of the
GABAB IPSPs on membrane potential was monotonic
in the range
60 to
95 mV; these potentials had maximal amplitude at
60 mV and were reduced as the membrane potential was hyperpolarized.
However, this monotonic relationship was lost as the cell was
depolarized from
60 mV. Indeed, in 15 of 18 recorded neurons at
55
mV (where the fast GABAA IPSPs were large), the
GABAB IPSPs were smaller than at
60 mV (Fig. 2,
A and B). In three remaining cells,
GABAB IPSPs had an equal amplitude at
55 and
60 mV.
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The smaller amplitude of the GABAA IPSP at more
negative membrane potentials is due to reduction in the driving force
for chloride ion influx. Surprisingly, GABAB
IPSPs, which are a result of increased potassium permeability and have
a reversal potential close to 100 mV (Alger 1984
;
Hablitz and Thalmann 1987
; Otis et al.
1993
), had a smaller amplitude at relatively positive membrane potentials, where the driving force for potassium ion efflux should be
maximal. Similar voltage-dependent reduction in the
GABAB IPSP amplitude has been reported previously
(Knowles et al. 1984
; Newberry and Nicoll
1985
).
We did not investigate postsynaptic responses at membrane potentials
more positive than 55 mV because afterhyperpolarizations associated
with spontaneous action potential firing occurred at more positive
potentials and overlapped with small amplitude
GABAB IPSPs. Furthermore, because the
calcium-dependent potassium conductance activated by action potentials
(Alger and Nicoll 1980b
; Hotson and Prince
1980
; Schwartzkroin and Stafstrom 1980
) or
glutamatergic excitatory synaptic transmission (Nicoll and Alger
1981
) may reshape postsynaptic responses, we examined
GABA-mediated postsynaptic potentials in neurons loaded with EGTA
diffused from the intracellular microelectrode (0.2 M). EGTA
sufficiently buffered intracellular calcium because short pulses of the
depolarizing current induced long-lasting burst discharges in these
cells with no afterhyperpolarization (a potential attributable to a
calcium-dependent potassium current) (data not shown). Under these
conditions, both GABAA and
GABAB IPSPs demonstrated voltage dependencies
similar to those seen in experiments performed with
potassium-acetate-filled microelectrodes (n = 5; data
not shown).
Inhibitory effect of the GABAB IPSPs on
depolarization-induced action potential generation was measured in six
neurons. Pulses of depolarizing current were injected through the
intracellular microelectrode, and current intensity was adjusted to
threshold for spike discharge with the cell membrane potential
maintained at either 55 or
60 mV. Current injection (latency = 140 ms) then was paired with a stimulus-evoked synaptic response, such that current-evoked spiking would start at the peak of the
GABAB IPSP. Current amplitude was adjusted so
that current-evoked action potential discharges induced at membrane
potentials of
55 and
60 mV had the same latency (4.5 ± 0.8 and 4.7 ± 0.9 ms, respectively; n = 6; Fig.
3, A and B). When
current injection was paired with the GABAB IPSP,
spiking was delayed significantly longer when the cell membrane
potential was
60 mV (142.3 ± 25.6, n = 6)
compared with a membrane potential of
55 mV (29.7 ± 7.2 ms,
n = 6; Fig. 3, A and B). This
longer spike delay was consistent with the larger, long duration of the
GABAB IPSP at
60 mV compared with
55 mV.
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Monosynaptic GABA-mediated IPSPs were investigated in seven neurons in
the presence of the non-N-methyl-D-aspartate
(NMDA) receptor antagonist CNQX (20 µM) and the NMDA receptor
antagonist APV (50 µM). The stimulating electrode was placed close
(<1 mm) to the site of recording, so that inhibitory interneurons
located near the recorded pyramidal cell could be directly stimulated. Under these conditions, both GABAA and
GABAB IPSPs behaved in a manner similar to that
seen when the mossy fibers were stimulated in normal ACSF (Fig.
4, A and B).
GABAA IPSPs reversed at a slightly more negative
membrane potential (between 70 and
75 mV), probably due to blockade
of the initial EPSP which overlaps slightly with the initial phase of
the GABAA IPSP. GABAB IPSPs
had a maximal amplitude at a membrane potential of
60 mV and still
showed an amplitude reduction at more depolarized (e.g.,
55 mV)
levels (Fig. 4, A and B). When plotted against
membrane potential, the GABAB IPSP showed a
monotonic dependency as long as the preceding GABAA IPSP (amplitude measured at peak) was
depolarizing or of relatively small hyperpolarizing amplitude (between
membrane potentials of
95 and
60 mV; Fig. 4C). However,
the GABAB IPSP was significantly reduced when the
monosynaptic GABAA IPSP exceeded some threshold level (GABAA hyperpolarization of 13.5 ± 0.8 mV, at a membrane potential of
55 mV; n = 7).
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Application of the GABAA receptor antagonist BMI
(20 µM) concomitantly with CNQX (20 µM) and APV (50 µM) blocked
fast monosynaptic IPSPs and isolated the monosynaptic
GABAB IPSPs. Pharmacologically isolated
GABAB IPSPs had a maximal amplitude at 55 mV
and showed a monotonic change in amplitude as the membrane was
hyperpolarized (n = 8; Fig.
5, A and B). The
amplitude of the isolated monosynaptic GABAB IPSP
at a membrane potential of
55 mV was 13.7 ± 0.6 mV (n = 5), significantly larger than the amplitude of the
comparable GABAB IPSP preceded by a
GABAA IPSP (8.6 ± 0.8 mV, n = 5) (i.e., recorded in the presence of only the glutamatergic receptor
antagonists; Fig. 5C). No differences in
GABAB IPSP amplitude were found between these
conditions when cell membrane potential was varied between
60 and
95 mV. Bath application of the GABAB receptor
antagonist CGP35348 (700 µM) blocked the isolated slow IPSP
(n = 6; Fig. 5A).
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In eight neurons, isolated monosynaptic GABAB
IPSPs were recorded with microelectrodes filled with 3 M potassium
acetate plus 1 M potassium chloride. IPSP amplitudes were measured 15 min after penetrating the cell, thus allowing chloride ions to diffuse
from the micropipette into the cell. The average input resistance of cells recorded with chloride-containing electrodes was 60.4 ± 4.7 M (comparable to 51.3 ± 2.1 M
in cells recorded with
potassium acetate-filled microelectrodes). The amplitude of IPSPs
recorded under these conditions, at a membrane potential of
55 mV was 7.7 ± 0.9 mV (n = 6), significantly smaller than
the amplitude of isolated monosynaptic GABAB
IPSPs recorded with microelectrodes containing no chloride (13.7 ± 0.6 mV, n = 5) (Fig.
6, A and B). However, this isolated GABAB potential was
comparable in amplitude to that of GABAB IPSPs
preceded by monosynaptic GABAA IPSPs (recorded in
the presence of the glutamatergic receptor antagonists; 8.6 ± 0.8 mV, n = 5) and to the amplitude of the
GABAB IPSPs recorded in normal ACSF (7.0 ± 0.8 mV, n = 4).
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DISCUSSION |
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For both fast GABAA and slow
GABAB IPSPs, the reversal potential is negative
to the usual CA3 resting membrane potential of 60 mV. Thus
depolarizing the cell should increase IPSP amplitudes because there is
an increased driving force for both chloride (ECl
70 mV) and potassium
(EK+
90 mV), responsible for the fast
GABAA and slow GABAB IPSPs, respectively. Our results, however, show that mossy fiber-evoked GABAB IPSPs were reduced in amplitude when the
cell's membrane potential is depolarized from
60 to
55 mV. During
the same manipulations, the earlier GABAA IPSP
increases in amplitude. This relationship was observed both as a
function of resting membrane potential and as a function of
experimentally manipulated membrane potential (through intracellular
current injection). Monosynaptically evoked GABA IPSPs (i.e., in the
presence of the glutamatergic receptor antagonists) showed the same
relationship as afferent-evoked IPSPs. Such depolarization-dependent
reduction of the GABAB IPSPs has been reported
previously (Knowles et al. 1984
; Newberry and
Nicoll 1985
), but the underlying basis for this unexpected
relationship has not been explained.
Previous investigations (Gähwiler and Brown 1985;
Lüscher et al. 1997
; Sodickson and Bean
1996
, 1998
) identified an inwardly rectifying potassium current
linked to the GABAB receptor (as well as to other
G-protein-coupled receptors) but did not explore its chloride
sensitivity. This current is likely to contribute to the reduction of
the GABAB-mediated IPSPs at positive membrane potentials. In our experiments (also see Otis et al.
1993
), pharmacologically isolated GABAB
IPSPs did not show "anomalous" voltage dependence but rather
exhibited a monotonic increase in amplitude as the cell was depolarized
to
55 mV. We did not examine the chloride sensitivity of inwardly
rectifying potassium current at more positive membrane potentials.
However, Lenz et al. (1997) showed that G-protein-linked
permeabilities, including the potassium conductance associated with GABAB receptor-mediated inhibition, can be
depressed by high intracellular chloride concentrations. This finding
is consistent with the results of our experiments, in which the
amplitude of the GABAB IPSP decreased under
conditions in which the preceding GABAA
chloride-dependent IPSP increased. Given these results, we postulate
that GABAB IPSP amplitude is modulated by
chloride influx associated with GABAA receptor
activation. Consistent with this hypothesis is the observation that the
GABAB IPSP amplitude was reduced significantly
when chloride ions diffused into the CA3 cell from a penetrating
microelectrode. We do not think that this effect can be attributed to
increased intracellular levels of calcium (e.g., associated with
calcium-dependent afterhyperpolarization) because EGTA injection did
not affect the anomalous voltage sensitivity of the
GABAB IPSP.
These results suggest that the driving force of the
GABAA receptor-mediated chloride influx, which
depends on membrane potential level, may affect
GABAB-mediated inhibition. Because anomalous effects of membrane potential on GABAB-mediated
inhibition were observed when membrane potential was manipulated
between 55 and
60 mV, it seems likely that membrane potential
fluctuations in a physiological range may lead to the changes in
intracellular chloride concentrations that are sufficient to modulate
GABAB-mediated inhibition. In fact,
GABAB IPSP-mediated inhibition of action potential discharge was more efficient at a cell membrane potential of
60 mV than at
55 mV.
It is noteworthy, however, that high-amplitude
GABAB IPSPs were recorded at membrane
potentials between 60 and
70 mV
membrane potentials at which the
GABAA IPSP was still hyperpolarizing and at which
GABAA receptors still mediated chloride influx.
These chloride currents apparently did not lead to an intracellular chloride concentration sufficient to interfere with
GABAB-mediated IPSPs. Indeed, our measurements
have shown that monosynaptic GABAB-mediated IPSPs
were reduced significantly only when the preceding monosynaptic GABAA IPSP exceeded some threshold level. This
result suggests that chloride influx (associated with high-amplitude
GABAA IPSPs) must establish some minimal level of
intracellular chloride concentration, perhaps at sites remote from the
region of chloride influx, to affect
GABAB-mediated inhibition. Lenz et al.
(1997)
suggest that intracellular chloride reduces
GABAB-mediated inhibition by targeting either
potassium channels or G proteins (to which the channels are tightly
coupled) (Andrade et al. 1986
). The need for a
relatively high intracellular chloride concentration to produce effects
on GABAB-mediated inhibition may be a function of
the low sensitivity of these targets to chloride. Alternatively,
potassium channel-linked G proteins may be spatially distant from
chloride influx, thus requiring diffusion of chloride to a cell site
"remote" from the GABAA receptor-gated channels.
That such a separation might exist is suggested by the finding that
distinct types of inhibitory interneurons, with separate synaptic
target sites, may be responsible for GABAA- and
GABAB-mediated responses in the hippocampus
(Nurse and Lacaille 1997). However, distal
GABAB-mediated responses, even those in CA1
dendrites induced by activation of interneurons in stratum
lacunosum-moleculare (Williams and Lacaille 1982
), may
still be subject to chloride-dependent modulation. These neurons
receive a high level of spontaneous inhibitory synaptic input, mediated
primarily by postsynaptic GABAA receptors
(Alger and Nicoll 1980a
; Collingridge et al.
1984
). The chloride flux associated with this spontaneous
GABAA-mediated input may help explain the
"anomalous" behavior of GABAB-mediated responses induced by baclofen microapplications to CA1 cells at relatively positive membrane potentials (Newberry and Nicoll
1985a
); these GABAB-mediated
hyperpolarizations were reduced in amplitude, much as seen with
afferent stimulation.
Chloride-dependent modulation of GABAB-mediated
inhibition may play a significant role in some physiological and
pathological phenomena. For instance,
GABAB-mediated IPSPs have not been detected at early postnatal periods of development (Gaiarsa et al.
1995; McLean et al. 1996
), when intracellular
chloride concentrations are high (Owens et al. 1996
).
The absence of GABAB-mediated IPSPs at this age
may be due to strong chloride-dependent inhibition of
GABAB-mediated events rather than to the absence
of GABAB receptors. Indeed, high levels of
GABAB receptors may be seen at early postnatal times; binding experiments suggest that these receptors peak at postnatal day 3 and then decline into adulthood (Turgeon and
Albin 1994
). Also, it has been shown, that
GABAB-mediated IPSPs may control the duration of
interictal epileptiform discharges (deCurts et al. 1999
;
Karlsson et al. 1992
) as well as the transitions from
interictal to ictal-like activity in the hippocampus (Malouf et
al. 1990
; Scanziani et al. 1991
). If
GABAB-mediated inhibition is sensitive to
intracellular chloride concentration, it is possible that intracellular
chloride oscillations (mediated by GABAA
receptors or other mechanisms) may modulate the strength of
GABAB-mediated inhibition and thus help determine
the pattern of epileptiform activity.
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ACKNOWLEDGMENTS |
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This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-18895.
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FOOTNOTES |
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Address for reprint requests: P. A. Schwartzkroin, University of Washington, Department of Neurological Surgery, Box 356470, Seattle, WA 98195.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 February 1999; accepted in final form 12 May 1999.
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REFERENCES |
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