1Department of Neurology; and 2Department of Physiology, University of Michigan Medical Center, Ann Arbor, Michigan 48104-1687
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ABSTRACT |
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Kapur, Jaideep, Kevin F. Haas, and Robert L. Macdonald. Physiological properties of GABAA receptors from acutely dissociated rat dentate granule cells. Study of fast, GABAA receptor-mediated, inhibitory postsynaptic currents (IPSCs) in hippocampal dentate granule cells has suggested that properties of GABAA receptors influence the amplitude and time course of the IPSCs. This study describes the physiological properties of GABAA receptors present on hippocampal dentate granule cells acutely isolated from 18- to 35-day-old rats. Rapid application of 1 mM GABA to outside-out macropatches excised from granule cells produced GABAA receptor currents with rapid rise time and biexponential decay of current after removal of GABA. After activation, granule cell GABAA receptor currents desensitized incompletely. During a 400-ms application of 1 mM GABA, peak current only desensitized ~40%. In symmetrical chloride solutions there was no outward rectification of whole cell current. Activation rates and peak currents elicited by rapid application of GABA to macropatches were also similar at positive and negative holding potentials. However, deactivation of GABAA receptor currents was slower at positive holding potentials. When whole cell currents were recorded without ATP in the pipette, current run-down was not apparent for 30 min in 50% of neurons, but run-down appeared to start soon after access was established in the remaining neurons. When 2 mM ATP was included in the recording pipette no run-down was apparent in 30 min of recording. The efficacy and potency of GABA were lower in cells recorded with no ATP in the pipette and during run-down compared with those recorded with 2 mM ATP and no run-down.
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INTRODUCTION |
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GABA mediates inhibitory neurotransmission in the
forebrain by activation of GABAA and GABAB
receptors. Study of fast, GABAA receptor-mediated,
inhibitory postsynaptic currents (IPSCs) in the dentate granule cells
of the hippocampus has produced a model of central inhibitory synaptic
function that postulated quantal transmission and a small number of
postsynaptic receptors activated by a saturating concentration of GABA
(Edwards et al. 1990; Mody et al. 1994
).
This model predicted that properties of GABAA receptors were likely to influence the amplitude and time course of the IPSCs.
IPSC amplitude is regulated by GABAA receptor single
channel conductance, ion selectivity of the channel, desensitization, and rectification, whereas IPSC time course is determined by the processes of activation, desensitization, and deactivation
(Jones and Westbrook 1995
, 1996
). In addition to these
factors, the state of phosphorylation of GABAA receptors
played a role in maintenance of their function. Progressive loss or
"run-down" of GABAA receptor currents occurred during
whole cell recording from many types of neurons, including acutely
isolated CA1 pyramidal neurons (Stelzer et al. 1988
),
cultured chick spinal cord neurons (Gyenes et al. 1994
),
and recombinant receptors expressed in fibroblasts (Lin et al.
1994
). This ATP dependence was likely due to ATP-dependent phosphorylation of GABAA receptors or of a closely
associated protein by a protein kinase (Chen et al.
1990
; Gyenes et al. 1994
).
The identification of multiple subunits and subtypes of
GABAA receptors and the heterogeneous distribution of their
mRNAs suggested the existence of substantial diversity of native
GABAA receptors (Macdonald and Olsen 1994).
Expression of specific GABAA receptor isoforms in different
neurons may result in distinct forms of GABAergic inhibition because
GABAA receptors composed of different subunit subtype
combinations expressed in heterologous expression systems have been
shown to have different pharmacological and biophysical properties
(Burgard et al. 1996
; Gingrich et al. 1995
; Saxena and Macdonald 1994
). In situ
hybridization studies of GABAA receptor subtype mRNAs
suggested that many subtypes with a restricted distribution were
expressed in hippocampal dentate granule cells (Wisden et al.
1992
).
GABAA receptor subunit composition has been shown to
influence the pharmacological properties of GABAA receptors
(Macdonald and Olsen 1994) and activation, deactivation,
desensitization, and voltage dependence (Burgard et al.
1996
) of the chloride channel. GABAA receptors have
been shown to be regulated by phosphorylation of different sites on
different subunit subtypes. We have reported previously that
GABAA receptors present on dentate granule cells acutely
isolated from 28- to 35-day-old rats have distinct pharmacological properties (Kapur and Macdonald 1996
). Here we describe
physiological properties, including activation, deactivation,
desensitization, rectification, and ATP dependence of GABAA
receptor currents recorded from dentate granule cells acutely isolated
from 18- to 35-day-old rats.
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METHODS |
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Isolation of dentate granule cells
All experiments were performed on dentate granule cells isolated
according to the method described originally by Kay and Wong (1986).
Male or female Sprague-Dawley rats (18-35 days old) were euthanized
and decapitated. The brain was dissected free, and the region
containing the hippocampus was blocked and chilled in an oxygenated
PIPES-buffered medium (4°C) for 1 min. The PIPES buffer solution
contained (in mM) 120 NaCl, 2.5 KCL, 1.5 CaCl2, 1 MgCl2, 25 D-glucose, and 20 PIPES (pH 7.0).
After blot drying, the brain was mounted on a vibratome stage, and
500-µm coronal sections containing the hippocampus were cut. The
sections were allowed to recover in oxygenated (95% O2-5%
CO2) PIPES buffer for 30-60 min. Hippocampal sections were
then incubated in oxygenated SIGMA type XXIII (Sigma Chemical; St.
Louis, MO) protease enzyme in the buffer at 32°C for 30-45 min. The
dentate gyrus was dissected out and cut into 0.5-mm cubes that were
triturated in a cold (4°C) PIPES-buffered medium in fire-polished
glass pipettes to isolate neurons. The isolated neurons were plated on
poly-L-lysine-coated 35-mm polystyrene Petri dishes
(Corning Glass Works; Corning, NY), and the recordings were made within
1 h of isolation.
Recording and analysis of GABAA receptor currents from outside-out macropatches excised from acutely isolated dentate granule cells
Patch-clamp recordings were performed on outside-out membrane
patches (Hamill et al. 1981) pulled from acutely
isolated dentate granule cells bathed in an external solution
consisting of (in mM) 142 NaCl, 8 CsCl, 6 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose (pH 7.4, 330 mosmol) at
room temperature. Intrapipette CsCl was used to block potassium
currents. Glass microelectrodes were formed from thick-walled
borosilicate glass (World Precision Instruments) with a Flaming Brown
electrode puller, fire polished to tip resistances of 10-20 M
, and
then coated with Q-dope. Patch electrodes were filled with an internal
solution consisting of (in mM) 153 CsCl, 1 MgCl2, 2 MgATP,
10 HEPES, and 5 EGTA (pH 7.3, 300 mosmol). This combination of internal
and external solutions produced a chloride equilibrium potential of 0 mV. Outside-out membrane patches were voltage clamped at
50 mV with
an EPC-7 amplifier (List; Darmstadt, Germany).
GABA was applied to outside-out membrane patches with a rapid
application system (Franke et al. 1987) consisting of a
double-barreled theta tube (FHC, Brunswick, ME) connected to a
piezoelectric translator (Burleigh Instruments; Fishers, NY). One
barrel was perfused with the external recording solution, and the other
was perfused with a GABA-containing external solution. Activation of
the translator drove the solution interface rapidly across the patch
surface. The solution exchange time was routinely monitored at the end of each recording by blowing out the patch and stepping a dilute (90%)
external solution across the open electrode tip to measure a liquid
junction current; 10-90% rise times for solution exchange were
consistently <400 µs. The recording chamber was continuously perfused with external solution to prevent accumulation of GABA in the bath. All experiments were performed at room temperature (22-23°C).
Outside-out patch data were low-pass filtered at 3 kHz, digitized at 10 kHz, and analyzed with the pClamp6 software suite (Axon Instruments;
Foster City, CA) and Origin 4.1 (Microcal; Northhampton, MA). Multiple
GABA-elicited responses (5-20) were acquired for each patch at 30-s
intervals and then were averaged to form ensemble currents for
analysis. Activation of ensemble currents was measured as a 10-90%
rise time to the peak current. The desensitization or deactivation time
courses of ensemble GABAA receptor currents were fit with
the Levenberg-Marquardt least-squares method with one or two component
exponential functions. The number of exponential components was
determined by statistically comparing the sum of squared residuals for
one and two component fits (F test, P < 0.001). The extent of desensitization was measured as (peak
current fitted steady state current)/(peak current). Numerical data were expressed as means ± SE. Statistical significance was determined with paired two-tailed t-tests (P < 0.05).
Recording and analysis of whole cell GABAA receptor currents from acutely isolated dentate granule cells
Whole cell GABAA receptor currents were recorded
from hippocampal dentate granule cells acutely isolated from 18- to
35-day-old rats with the technique described by Hamill et al. (1981).
The extracellular recording solution consisted of (in mM) 142 NaCl, 1.0 CaCl2, 8 KCl, 6 MgCl2, 10 glucose, and 10 HEPES, pH adjusted to 7.4 and osmolarity of 310-320 mosmol (all
reagents from Sigma). For ATP-dependence experiments, glass recording
patch pipettes were filled with a solution consisting of the following
(in mM): 115 Trizma phosphate (dibasic), 30 Trizma base, 11 EGTA, 2 MgCl2, and 0.5 CaCl2, pH 7.35. Recording
pipettes contained ATP as specified in the RESULTS. For
voltage-dependence experiments the pipette solution was altered to
obtain symmetrical intracellular and extracellular chloride
concentration. The pipette solution consisted of (in mM) 153 CsCl, 1 MgCl2, 5 EGTA, 10 HEPES, and 2 ATP, pH 7.4. GABA dissolved
in extracellular solution was applied to neurons with a modified U-tube
"multipuffer" rapid application system (Bormann 1992
; Greenfield and Macdonald 1996
) with the
tip of application pipette placed 100-200 µm from the cell.
Whole cell currents were recorded with an Axopatch 1-D amplifier and
low-pass filtered at 2 kHz with a eight-pole Bessel filter before
digitization, storage, and display. Currents were displayed on a Gould
2400S chart recorder, and peak whole cell currents were measured
manually from the chart paper. Currents were also recorded on a hard
disk (Acer-286 turbo personal computer) with the Axotape program (Axon
Instruments) (digitized at 208 Hz) and on a videotape recorder (Sony
SL-HF360) via a digital audio processor (Sony PCM-501 ES, 14-bit, 44 kHz). Peak GABAA receptor currents at various GABA
concentrations were fitted to a sigmoidal function with a
four-parameter logistic equation (sigmoid concentration response) with
a variable slope. The equation used to fit the concentration-response
relationship was
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RESULTS |
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Activation and deactivation of dentate granule cell GABAA receptor outside-out patch currents
In the synaptic cleft of central GABAergic synapses, the GABA
concentration has been estimated to be high (0.5-1 mM) after release
of GABA from presynaptic terminals (Edwards et al. 1990; Jones and Westbrook 1995
; Maconochie et al.
1994
). To mimic the time course of GABA in the synaptic cleft,
1 mM GABA was applied briefly to outside-out membrane patches excised
from acutely isolated dentate granule cells with a piezoelectric
translator to make rapid switches between solutions. GABA was applied
to individual patches every 20-30 s during each experiment. Open-tip
currents were routinely checked at the end of experiments and indicated a rapid solution exchange time under these experimental conditions (10-90% rise time 360 ± 120 µs, n = 6).
Transient (2 ms) application of 1 mM GABA to outside-out membrane
patches voltage clamped to 50 mV produced rapidly activating and
deactivating currents (Fig. 1). In the
example shown, the current activated with a 10-90% rise time of 0.90 ms (Fig. 1A) and deactivated with a biexponential time
course with time constants of 9.7 (58.1%) and 100 ms (41.9%) (Fig.
1B). The average rise time of the outside-out patch
GABAA receptor currents, although rapid (10-90% rise
time, 0.90 ± 0.09 ms, n = 6), was two to three times slower than the solution exchange time. This rise time was similar to reported dentate granule cell IPSC rise times
(Edwards et al. 1990
) but was clearly slower than that
reported by De Koninck and Mody (1994)
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Deactivation of dentate granule cell GABAA receptor
currents was studied after brief (2 ms), rapid application of 1 mM
GABA. The current decay time course was fitted best by the sum of two exponential functions (fast = 9.9 ± 1.8 ms and
slow = 95.8 ± 10.3 ms) (Table
1). The fast component contributed 63.1%
and the slow component contributed 36.9% of the peak current decay. The biphasic deactivation time course of outside-out patch currents after transient application of GABA was similar to the biexponential IPSC decay in the dentate granule cells reported by Edwards et al. (1990)
but differed from the monoexponential decay reported for
dentate granule cell mIPSCs (DeKonnick and Mody 1994
).
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Desensitization of dentate granule cell GABAA receptor outside-out patch currents
Desensitization of GABAA receptor currents was studied
after rapid application of 1 mM GABA for 400 ms to outside-out patches excised from acutely isolated dentate granule cells. In the example shown, the current desensitized with two exponential components with
time constants (and relative proportions) of 12.4 ms (26.0%) and 218 ms (74.0%) (Fig. 2). During the 400-ms
period of application, average GABAA receptor currents
desensitized with a biexponential time course to 60 ± 4.9% of
the peak response (n = 7). The fast phase of
desensitization (fast 16.4 ± 2.7 ms) contributed
to 76% of the total desensitization, whereas the slow phase
(
slow 211 ± 31.6 ms) contributed the remainder of
the desensitization (Table 1).
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Voltage dependence of whole cell dentate granule cell GABAA receptor currents
The voltage dependence of dentate granule cell GABAA
receptor currents was studied with symmetrical transmembrane chloride concentration. Under these conditions, reversal of the currents occurred at 0 mV, and identical amplitude outward and inward currents were recorded in response to 10 µM GABA applied to cells voltage clamped to +50 mV and 50 mV (Fig.
3A). The current-voltage
relationship of dentate granule cell GABAA receptor
currents elicited by 10 µM GABA with symmetrical transmembrane
chloride concentrations was linear (n = 5 neurons)
(Fig. 3B).
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Voltage dependence of dentate granule cell GABAA receptor outside-out patch currents
The effects of transmembrane potential on rectification,
activation, and deactivation of GABAA receptor currents in
outside-out patches were determined. GABA (1 mM) was rapidly applied
for 2 ms to outside-out patches excised from acutely isolated dentate granule cells when the same membrane patch was voltage clamped to 50
and +50 mV (Fig. 4). The +50/
50-mV peak
current ratio of 1.05 ± 0.11:1 (n = 5)
demonstrated no difference in the peak currents at +50 and
50 mV.
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Although peak current amplitudes were identical at positive and
negative holding potentials, the currents deactivated at different rates when patches were voltage clamped to +50 mV or to 50 mV. In the
example shown, the outside-out patch currents deactivated with two
similar time constants at both positive (
f = 11.3 and
s = 85.7 ms) and negative (
f = 12.4 ms and
s = 103.4 ms) holding potentials (Fig.
4A). However, at +50 mV current deactivation occurred
equally with both components (af = 51.2% and
as = 48.8%), whereas at
50 mV current
deactivation occurred primarily with the fast component
(af = 72.9% and as = 27.1%). The net result was that the current deactivated more rapidly
at
50 mV (average deactivation time constant of 47.6 ms) than at +50
mV (average deactivation time constant of 36.3 ms at
50 mV). On
average (n = 5 patches), when outside-out patches were
clamped to
50 mV, the fast phase of deactivation occurred with a time
constant 10.1 ± 1.9 ms and contributed 66 ± 3.9% of total
desensitization. When patches were clamped to +50 mV the fast phase of
deactivation was 6.9 ± 1.2 ms but only contributed 47 ± 6.2% of total deactivation. When patches were clamped to
50 mV, the
slow phase of deactivation occurred with a time constant of 88.7 ± 13.6 ms and contributed 34 ± 3.9% of total deactivation,
whereas when patches were clamped to +50 mV the slow phase of
deactivation occurred with a time constant of 73.9 ± ms and
contributed 56.0 + 8.0% of total deactivation. The weighted sum of
deactivation time constants when macropatches were voltage clamped to
50 mV was 35.9 ± 6.8 ms and at +50 mV was 42.8 ± 6.8 ms
(P < 0.05, paired t-test) (Fig.
4B).
ATP dependence of currents
We determined the ATP dependence of dentate granule cell
GABAA receptor currents. When whole cell GABAA
receptor currents were recorded from acutely isolated dentate granule
cells with pipettes that did not contain ATP (n = 8),
two response patterns were noted. In one-half of the neurons studied,
stable GABAA receptor currents in response to repeated
application of 30 µM GABA were recorded for 30 min (Figs.
5, top panel, and
6,
), whereas in the remaining
neurons GABAA receptor current began to decline (run-down)
within 10 min of establishing access and continued to so steadily
thereafter (Figs. 5, bottom panel, and 6,
). There were
no obvious morphological or physiological differences between the
neurons in which currents ran down and those they did not. When 2 mM
ATP was included in the recording pipette, steady GABAA receptor currents were recorded for 30 min (n = 7),
(Fig. 6,
).
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To determine if the run-down in GABAA receptor currents during the recording period with recording pipettes that did not contain ATP resulted from a reduction in the affinity and/or efficacy of GABA for GABAA receptors, two GABA concentration-response relationships were determined in a single neuron before (Fig. 7A, top traces) and after (Fig. 7A, bottom traces) the onset of run-down. In this dentate granule cell, run-down resulted in a decrease in maximal GABAA receptor current and increase in EC50 (Fig. 7B). However, because it took ~20 min to complete a concentration-response relationship, this experiment could not be repeated a sufficient number of times to quantify the effect. Therefore to determine the effect of run-down in GABAA receptor an alternate approach was used. GABA concentration-GABAA receptor-current relationships were determined in 13 dentate granule cells recorded with 2 mM ATP in the pipette (Fig. 8A) and in another 9 cells were recorded without ATP (Fig. 8B). In the neurons recorded with ATP in the pipette (Fig. 8A), the concentration-response data were recorded after two currents evoked by 10 µM GABA were identical. In neurons recorded without ATP in the pipette (Fig. 8B), if there was an initial increase in current data collection was started after two applications of 10 µM GABA elicited identical currents. Data collection was started immediately if run-down was apparent. Because run-down occurred as the concentration-response relationship was being studied, application of GABA in ascending concentrations might have resulted in the currents elicited by high GABA concentration that were smaller than if they had been recorded earlier. To compensate for this potential run-down, GABA was applied in ascending or descending concentration steps ranging from 1 to 1,000 µM in alternate neurons. Compared with granule cells recorded with 2 mM ATP, in granule cells recorded without ATP the threshold for evoking GABAA receptor currents was higher, and smaller peak GABAA receptor currents were recorded at all GABA concentrations (Fig. 8, A and B).
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In dentate granule cells recorded with ATP in the pipette, the peak
currents obtained in response to GABA concentrations ranging from 1 to
1,000 µM were fitted to a sigmoid function, and the equation for the
best fit was used to derive EC50 and maximal GABAA receptor currents elicited by GABA (Fig.
9, ). The mean EC50 for
GABA for these neurons was 39 ± 6 µM, and the maximal current
was 961 ± 102 pA (n = 13). In dentate granule
cells recorded without ATP in the pipette, the mean GABA
EC50 was 117 ± 30 µM, and the maximal current was
377 ± 115 pA (n = 9, Fig. 9,
). The EC50 (P < 0.05) was significantly higher
in neurons recorded with no ATP in the pipette compared with those
recorded with 2 mM ATP in the pipette. Maximal currents
(P < 0.05) were significantly smaller when obtained
with no ATP in the recording in the pipette compared with those
obtained with 2 mM ATP in the recording pipette. These findings were
similar to the changes in GABA EC50 and maximal current
caused by run-down observed in a neuron recorded with no ATP in the
pipette.
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DISCUSSION |
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Activation and deactivation properties
Current models of GABAergic synapses terminating on the somata of
dentate granule cells postulate that mIPSCs result from saturation of a
small number of postsynaptic receptors by a high concentration of GABA
(Busch and Sakmann 1990; De Koninck and Mody
1994
; Edwards et al. 1990
). The rapid rise time
of mIPSCs on dentate granule cell somata was only partially replicated
by application of 1 mM GABA for 2 ms to macropatches containing small numbers of receptors because the 10-90% rise time of
GABAA receptor activation current was submillisecond but
slower than the 290-µs rise time reported for mIPSCs (De
Koninck and Mody 1994
). One explanation of faster rise time
mIPScs on soma and proximal dendrites of dentate granule cells compared
with the activation rate of GABAA receptors on macropatches
was that our application system was not sufficiently fast to replicate
conditions in the synaptic cleft and test the saturation hypothesis.
Alternately, rapid rise times of mIPSCs are possible even for
nonsaturating GABA concentrations if the time course of GABA in the
synaptic cleft is rate limiting (Galarreta and Hestrin
1997
). The deactivation of GABAA receptor currents
on removal of saturating concentration of GABA was quantitatively and
qualitatively distinct from the decay of mIPSCs recorded from the
somata of dentate granule cells. Edwards et al. (1990)
fitted the decay
of mIPSCs with the sum of two exponential functions, with time
constants of 2.0 ± 0.38 ms and 54 ± 18 ms. Several other studies (De Koninck and Mody 1994
; Otis and Mody
1992
; Soltesz et al. 1995
) described the decay
of mIPSCs by a single exponential function with a time constant of
3.7-7.2 ms. The deactivation of GABAA receptor currents
from macropatches excised from dentate granule cells was fitted best to
two exponential functions with time constants of 9.9 ± 1.8 and
95.8 ± 4.5 ms. There are several possible explanations for the
discrepancy between the deactivation of macropatch GABAA
receptor currents and mIPSC decay. In addition to the deactivation
kinetic properties of the GABAA receptors, the mIPSC decay
is likely to be effected by cable filtering of dendritic mIPSCs
(Soltesz et al. 1995
), rate of reuptake of GABA in the
synaptic cleft, and unbinding and rebinding of GABA to the postsynaptic
receptors. Additionally, we determined the deactivation properties in
outside-out membrane patches pulled from the somata of dentate granule
cells that may have included synaptic and extrasynaptic GABAA receptors; in contrast mIPSC decay largely depends on
the properties of subsynaptic receptors.
Desensitization and rectification
Compared with CA1 pyramidal neuron GABAA receptor
currents, dentate granule cell GABAA receptor currents had
a slow desensitization rate and large residual current
(Celentano and Wong 1994; Jones and Westbrook
1995
). One possible explanation for this slow rate of
desensitization may have been the presence of a
subunit in these
receptors. The mRNA for the
subunit is well expressed in dentate
granule cells, and
subunit incorporation into recombinant GABAA receptors was shown to slow desensitization of the
receptor currents (Fisher and Macdonald 1997
;
Haas and Macdonald 1998
; Saxena and Macdonald
1994
). Other possibilities include the potential contribution
of
4 subtype-containing isoforms to the currents and the modulation
of desensitization kinetics by phosphorylation.
Dentate granule cell GABAA receptor currents did not
rectify at positive holding potentials when intracellular and
extracellular chloride concentrations were symmetrical. There was a
linear current-voltage relationship for unitary mIPSCs recorded from
the somata of dentate granule cells (De Koninck and Mody
1994). In contrast outward rectification of GABAA
receptor single channel currents present on cultured hippocampal
neurons (Segal and Barker 1984
), hippocampal neurons in
slices (Gray and Johnston 1985
), and whole cell currents recorded from acutely isolated CA1 neurons (Burgard et al.
1996
) have been reported. Burgard et al. (1996)
demonstrated
that the degree of rectification depended on the subunit composition of GABAA receptors; for example,
1
3
2 receptor
currents rectified far less than
5
3
2 receptor currents. It was
likely that the linear current-voltage relationship of the dentate
granule cell GABAA receptor currents was a consequence of
its distinct subunit composition.
The deactivation of GABAA receptor currents from
macropatches after brief, rapid application was slower at +50 mV than
at 50 mV. This was primarily due to greater participation of the slower phase of deactivation at +50 mV compared with
50 mV. This might explain the voltage dependence of mIPSC decay in dentate granule
cells (Otis and Mody 1992
). Slower deactivation of
GABAA receptor currents at depolarized potentials was
described in cultured cerebellar granule cells (Mellor and
Randall 1998
).
Run-down of dentate granule cell GABAA receptor current
Progressive loss or run-down of GABAA receptor
currents under whole cell recording conditions has been reported in
many types of neurons, including acutely isolated CA1 pyramidal neurons
(Stelzer et al. 1988), cultured chick spinal cord
neurons (Gyenes et al. 1994
), and recombinant receptors
expressed in fibroblasts (Lin et al. 1994
). Run-down of
currents became apparent within 10 min in native neurons, and a large
reduction of current occurred within 30 min. In contrast, in acutely
isolated dentate granule cells in the absence of ATP in the recording
pipette, either no run-down occurred for 30 min or run-down started in
first 10 min of recording and then proceeded slowly. Clearly run-down
and ATP dependence of acutely dissociated dentate granule cells and CA1
neurons was different where GABAA receptor currents ran
down rapidly in the absence of ATP in the recording pipette
(Chen et al. 1990
). The greater susceptibility of
GABAA receptor currents to run-down in acutely dissociated
CA1 pyramidal neurons may have been due to their larger volume and
greater metabolic needs, greater susceptibility to trauma associated
with dissociation, or different GABAA receptor isoform
expressed by these cells.
This study suggested that the reduction in GABAA receptor
currents observed (when recorded with no ATP in the pipette) was due to
reductions in both potency and efficacy of GABA. However this
conclusion was limited because the current data were obtained during
the development of run-down. Additionally, because the concentration-response curve recorded within the pipettes containing no ATP was not generated with sufficiently high GABA concentration to
obtain a plateau current amplitude, the reported maximal current was
likely underestimated, and a large reduction in GABA potency may have
falsely appeared as a reduction in GABA efficacy. In another study a
reduction in efficacy but increased potency of GABA in cultured chick
neuron GABAA receptors was reported (Gyenes et al.
1994). This probably reflected differences in isoforms or
neurons used for the study.
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ACKNOWLEDGMENTS |
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We thank N. Esmaiel for assistance with the experiments.
This work was supported by a grant from the Epilepsy Foundation of America, by National Institute of Neurological Disorders and Stroke Grants KO8 NS-01748 and KO2 NS-02081 to J. Kapur and RO1 NS-33300 to R. L. Macdonald, and by a grant from the Lucille P Markey Charitable Trust Fund.
Present address of J. Kapur: Dept. of Neurology, University of Virginia Health Sciences Center, Charlottesville, VA 22908.
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FOOTNOTES |
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Address for reprint requests: R. L. Macdonald, Neuroscience Laboratory Building, 1103 East Huron, Ann Arbor, MI 48104-1687.
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 29 May 1998; accepted in final form 29 January 1999.
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REFERENCES |
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