1Department of Neurological Surgery and 2Department of Physiology 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 Reversal
Potential of GABAB-Mediated IPSPs in Hippocampal Pyramidal
Cells.
J. Neurophysiol. 85: 2381-2387, 2001.
Changes in intracellular
chloride concentration, mediated by chloride influx through
GABAA receptor-gated channels, may modulate GABAB receptor-mediated inhibitory postsynaptic
potentials (GABAB IPSPs) via unknown
mechanisms. Recording from CA3 pyramidal cells in hippocampal
slices, we investigated the impact of chloride influx during
GABAA receptor-mediated IPSPs
(GABAA IPSPs) on the properties of
GABAB IPSPs. At relatively positive membrane
potentials (near 55 mV), mossy fiber-evoked
GABAB IPSPs were reduced (compared with their
magnitude at
60 mV) when preceded by GABAA
receptor-mediated chloride influx. This effect was not associated with
a correlated reduction in membrane permeability during the
GABAB IPSP. The mossy fiber-evoked
GABAB IPSP showed a positive shift in reversal potential (from
99 to
93 mV) when it was preceded by a
GABAA IPSP evoked at cell membrane potential of
55 mV as compared with
60 mV. Similarly, when intracellular
chloride concentration was raised via chloride diffusion from an
intracellular microelectrode, there was a reduction of the
pharmacologically isolated monosynaptic GABAB
IPSP and a concurrent shift of GABAB IPSP
reversal potential from
98 to
90 mV. We conclude that in
hippocampal pyramidal cells, in which "resting" membrane potential
is near action potential threshold, chloride influx via
GABAA IPSPs shifts the reversal potential of
subsequent GABAB receptor-mediated postsynaptic
responses in a positive direction and reduces their magnitude.
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INTRODUCTION |
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The inhibitory effect of
-aminobutyric acid (GABA) is mediated in forebrain primarily by two
different types of postsynaptic receptors, termed
GABAA and GABAB (for review
see Sivilotti and Nistri 1991
). Stimulation of afferents
to hippocampal pyramidal cells results in short-lasting glutamatergic
excitation followed by a fast inhibitory postsynaptic potential (IPSP)
generated by chloride influx though GABAA
receptor-gated ion channels (Ben-Ari et al. 1981
;
Knowles et al. 1984
; Newberry and Nicoll
1984b
). A longer latency long-lasting IPSP often follows the
GABAA receptor-mediated IPSP and is mediated by
postsynaptic GABAB receptors linked to potassium
channels via intracellular G-proteins (Alger 1984
;
Andrade et al. 1986
; Dutar and Nicoll
1988
; Hablitz and Thalmann 1987
; Newberry
and Nicoll 1984a
,b
; Thalmann 1988
). Functional
properties of this GABAB-mediated potential are
not completely understood. For instance, current evoked by the
GABAB receptor agonist, baclofen, exhibits inward
rectification; i.e., a reduction in current amplitude as the cell's
membrane potential is depolarized. This feature has been attributed to
the properties of the potassium channels coupled to
GABAB receptors (Gähwiler and Brown
1985
; Lüscher et al. 1997
;
Sodickson and Bean 1996
). However, both inward
rectification (Knowles et al. 1984
; Newberry and
Nicoll 1985
) and linear voltage dependency (Hablitz and
Thalmann 1987
; Otis et al. 1993
) have been
reported for synaptically activated
GABAB-mediated potentials/currents in hippocampal slices.
Artificial changes in intracellular chloride concentration may modulate
G-protein-linked potassium permeability, including that activated by
GABAB receptors (Lenz et al.
1997). Therefore recently we investigated possible effects of
GABAA receptor-mediated chloride influx on
GABAB-mediated IPSPs in hippocampal pyramidal cells (Lopantsev and Schwartzkroin 1999
). We showed that
reduction of GABAB-mediated IPSPs, at relatively
positive membrane potentials (close to
55 mV), is induced by
GABAA-mediated chloride influx; inward rectifying
properties of the potassium channels did not contribute in this effect
over the range of membrane potentials investigated in our study.
The mechanism underlying chloride-dependent depression of the GABAB-mediated IPSP is still unknown. Here we have further investigated how GABAA receptor-mediated chloride influx affects the properties of the GABAB-mediated IPSP. We tested two main hypotheses: 1) an increase in intracellular chloride concentration reduces membrane permeability associated with GABAB-mediated IPSPs, and 2) enhanced intracellular chloride influences the properties of the current through potassium channels coupled to GABAB receptors without changing membrane permeability. We found that the GABAA receptor-mediated, chloride-dependent, reduction of GABAB-mediated IPSPs is not associated with a decrease in membrane permeability, but is attributable to an alteration in GABAB-mediated IPSP reversal potential.
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METHODS |
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Male Sprague-Dawley rats 1-1.5 mo old, were used in our experiments. After decapitation under halothane anesthesia, the brain was quickly removed 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). Transverse hippocampal slices (400 µm thick) were cut using a Vibroslicer (Campden Instruments, Sileby, UK) and then transferred to a holding chamber containing gas-saturated ACSF at room temperature (22-24°C) for at least 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 obtained with
glass microelectrodes filled with 3 M potassium acetate (resistance,
80-110 M) or with 3 M potassium chloride (resistance, 50-70 M
).
Pipette solutions were adjusted to pH 7.4 with KOH. Only neurons with a
resting membrane potential and synaptic responses stable for at least
20 min were included in our analysis. Signals were recorded using an
Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) in bridge
mode. Bridge balance was monitored throughout the experiment. Cell
resting membrane potential (RMP) was verified after withdrawal of the
microelectrode from the cell; action potential amplitude was calculated
from RMP; and cell apparent input resistance was obtained from maximum
voltage change in response to a hyperpolarizing current pulse (duration
200 ms, amplitude
0.4 nA). Data were digitized (Neuro-Corder, Neuro
Data Instruments, New York, NY) and acquired using AxoScope software
(Axon Instruments, Foster City, CA) on a pentium-based computer.
A bipolar stainless steel stimulating electrode was placed in the
stratum lucidum to activate the mossy fibers. Stimuli (0.1 ms duration)
were delivered at 0.1 Hz, at an intensity maximal for induction of
GABA-mediated IPSPs. 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.
Amplitude of the GABAB-mediated IPSP was measured from the resting membrane potential, at a latency of 140 ms unless otherwise stated. Changes in membrane resistance during IPSPs were
tested with brief hyperpolarizing current pulses (duration, 4-10 ms;
amplitude, 0.2 to
0.4 nA), while apparent input resistance was
tested at the peak of GABAB-mediated IPSP with
longer hyperpolarizing pulses (duration, 70 ms; amplitude,
0.15 to
0.2 nA). The conductance changes (
G) associated with
the GABAB-mediated IPSP were calculated according
to the relation:
G = 1/RIPSP
1/Rrest, where
RIPSP is the membrane resistance
measured during GABAB-mediated IPSP at 200 ms
after mossy fiber stimulation and
Rrest is the resting membrane
resistance (Hablitz and Thalmann 1987
). Reversal
potentials were obtained from the regression lines plotted for every
cell. Latency of pharmacologically isolated, monosynaptic
GABAB-mediated IPSPs was measured between
artifact of stimulation and the time point on response curve
corresponding to the resting membrane potential. Measurements were
expressed as means ± SE, and compared using Student's
t-test. Data were considered significantly different if
P < 0.05.
Bicuculline methiodide (BMI, 20 µM, Sigma, St. Louis, MO),
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM, Research
Biochemicals International, Natick, MA),
(±)-2-amino-5-phosphonopentanoic acid (AP-5, 50 µM, Research
Biochemicals International, Natick, MA), P-3-aminopropyl-P-diethoxymethyl-phosphinic acid (CGP35348, 700 µM, Ciba Geigy, Basel), and cesium chloride (1 mM) were applied via
bath perfusion.
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RESULTS |
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Intracellular recordings were obtained from 68 neurons in the
pyramidal layer of CA3 region under different pharmacological conditions using different intracellular electrolytes. The resting membrane potential of the recorded cells varied from 52 to
70 mV, action potential amplitude from 82 to 103 mV, and membrane input resistance from 37 to 77 M
.
Recordings from 10 of 68 neurons were made in normal ACSF with
microelectrodes filled with 3 M potassium acetate. Mossy fiber stimulation induced an initial excitatory postsynaptic potential (often
capped by an action potential) followed by a fast IPSP (termed
"GABAA IPSP" since it was blocked by the
GABAA receptor antagonist, BMI) and a subsequent
slow IPSP (termed "GABAB IPSP" since it was
blocked by the GABAB receptor antagonist,
CGP35348). Postsynaptic responses were induced at different membrane
potential levels, established by passing positive or negative steady
current through the intracellular microelectrode (Fig.
1A). Dependency of the
GABAB IPSPs on membrane potential was monotonic
in the range 60 to
95 mV; these potentials had a maximal amplitude at
60 mV and were reduced as the membrane potential was
hyperpolarized (Fig. 1B). However, at a membrane potential
of
55 mV, the amplitude of the GABAB IPSP was
smaller than that recorded at membrane potentials between
60 and
70
mV, and the monotonic relationship was lost.
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Recently we have shown that reduction of the
GABAB IPSP at membrane potentials close to 55
mV is due to chloride influx mediated by the preceding
GABAA IPSP (Lopantsev and Schwartzkroin
1999
). Decline in membrane permeability could be responsible
for chloride-dependent reduction of the GABAB
IPSP. Therefore membrane resistance was measured during
GABAB IPSPs evoked at membrane potentials of
55 and
60 mV, i.e., at membrane potentials where the
GABAB IPSP amplitude lost its monotonic voltage
dependency. Pulses of negative current (duration, 70 ms; amplitude,
0.15 to
0.2 nA) were passed through the intracellular
microelectrode at 130 ms after mossy fiber stimulation (Fig.
1C). Membrane resistance measured during GABAB IPSPs at these membrane potentials
(35.4 ± 1.9 M
at
55 mV, mean ± SE,
n = 7 and 37.7 ± 2.3 M
at
60 mV,
n = 7) was not significantly different (Fig.
1D). The calculated conductance changes associated with
GABAB IPSPs evoked at
55 mV (7.8 ± 1.2 nS, n = 7) and
60 mV (9.9 ± 1.1 nS,
n = 7) were not significantly different. Also changes
in membrane resistance were monitored during mossy fiber-evoked
GABA-mediated IPSPs by passing brief pulses of negative current
(duration, 10 ms; amplitude,
0.2 to
0.4 nA) through the recording
electrode (not shown). Resistance measures were compared during
GABA-mediated IPSPs evoked at membrane potentials of
55 and
60 mV
in eight neurons. Membrane resistance was not different during
GABAB IPSPs at membrane potentials of
55 and
60 mV, except at the time point of 50 ms after mossy fiber
stimulation, when the GABAA IPSP contributes
significantly to the hyperpolarization (asterisk in Fig.
1E). At this time point, membrane resistance was reduced by
54% during GABA-mediated IPSPs evoked at a membrane potential of
55
mV and was significantly lower than in the cell held at
60 mV
(resistance reduced by 40%). Thus in spite of the reduction of
GABAB IPSP amplitude at a membrane potential of
55 mV (as compared with
60 mV), corresponding membrane conductance
was not affected at peak and throughout most of the time course of the
GABAB IPSP.
Significant differences in membrane resistance during GABA-mediated
IPSPs, evoked at the membrane potentials of 55 versus
60 mV, were
detected only at 50 ms after mossy fiber stimulation, when
GABAA and GABAB IPSPs
overlap. We therefore compared the changes in membrane resistance
during pharmacologically isolated monosynaptic
GABAA IPSPs evoked at membrane potentials of
55 and
60 mV in 7 neurons (not shown). GABAA IPSPs
were isolated in the presence of the
N-methyl-D-aspartate (NMDA) receptor antagonist AP-5 (50 µM), the non-NMDA receptor antagonist CNQX (20 µM), and the GABAB receptor antagonist CGP35348 (700 µM). These potentials had a similar duration of 131.8 ± 8.4 ms
(n = 7) at
55 mV and 132.5 ± 9.3 ms
(n = 7) at
60 mV. Brief pulses of the negative current (duration, 4 ms; amplitude,
0.2 to
0.4 nA) were passed through the intracellular microelectrode, as above, to test changes in
membrane resistance. Generally, resistance was not different during
monosynaptic GABAA IPSPs evoked at membrane
potentials of
55 and
60 mV and measured up to 500 ms after
stimulation. However, at 50 ms after stimulation, membrane resistance
decreased by 28% at a membrane potential of
55 mV and by only 15%
(significantly less) at
60 mV (asterisk in Fig. 1F).
Overall, reduction in membrane resistance during the pharmacologically
isolated monosynaptic GABAA IPSP was smaller than
the reduction recorded in response to mossy fiber stimulation (in
normal ACSF). This difference may be explained by the likelihood that
fewer inhibitory synapses were activated under the direct stimulation
protocol (used to isolate monosynaptic GABA-mediated potentials in the
presence of glutamate receptor antagonists) than under conditions of
normal synaptic activation of inhibitory interneurons.
Since the experiments described above indicate that the
chloride-mediated reduction of the GABAB IPSP at
55 mV was not associated with reduction in membrane permeability, we
explored an alternative possibility: that GABAA
receptor-mediated chloride influx affects the properties of the current
through potassium channels coupled to GABAB
receptors and induces a shift of the GABAB IPSP
reversal potential. To evaluate this possibility, monosynaptic
GABAB IPSPs were isolated pharmacologically in
the presence of glutamate receptor antagonists (CNQX, 20 µM and AP-5,
50 µM) and the GABAA receptor antagonist BMI
(20 µM), in eight neurons recorded with potassium acetate-filled
microelectrodes and in seven neurons recorded with potassium
chloride-filled microelectrodes. Cesium (Cs+ 1 mM) was added to the medium to block voltage-dependent potassium conductances, and particularly inward rectification in the
hyperpolarizing direction, that might interfere with evaluation of the
GABAB IPSP (Hablitz and Thalmann
1987
). Extracellular cesium does not block outward
GABAB-mediated currents (Jarolimek et al.
1994
). Hyperpolarizing steady current was passed through the
intracellular microelectrode, and the GABAB IPSP
amplitude was measured (at 140 ms after stimulation) at different
membrane potentials. Reversal potential of the monosynaptic GABAB IPSP was then calculated from the
regression lines for each cell. IPSPs recorded with potassium acetate-
and potassium chloride-filled microelectrodes had similar latencies
(36.9 ± 1.9 ms and 37.1 ± 2.7 ms, respectively), but
hyperpolarizing IPSPs recorded with potassium chloride-filled
microelectrodes had smaller amplitudes (compare examples in Fig.
2, A and C). IPSPs
recorded with potassium acetate-filled microelectrodes had a reversal
potential of
98.1 ± 1.2 mV (n = 8), while
potentials recorded with potassium chloride-filled microelectrodes
reversed at significantly more positive level of
90.3 ± 2.0 mV
(n = 7; Fig. 2, B and D). Similar
values were obtained when the reversal potential of these IPSPs was
measured at a latency of 200 ms after mossy fiber stimulation
(
98.3 ± 1.1 mV with potassium acetate- and
90.0 ± 2.4 mV with potassium chloride-filled microelectrodes). These data show
that elevation of intracellular chloride shifts the monosynaptic
GABAB IPSP reversal potential in a positive
direction
an effect that could cause the observed reduction of these
potentials at membrane potentials more positive than
GABAB IPSP reversal potential.
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To evaluate whether voltage-dependent GABAA
receptor-mediated chloride influx is sufficient to affect the mossy
fiber-induced GABAB IPSP reversal potential, we
measured the GABAB IPSP reversal potential
directly after GABAA IPSPs in normal ACSF (in the
presence of 1 mM Cs+) with microelectrodes filled
with potassium acetate. Slices were exposed to
Cs+ only for a brief period of time (no longer
than 10 min) after establishing stable intracellular recording in
normal ACSF to avoid development of Cs+-induced
epileptiform discharges (Janigro et al. 1997). Mossy fiber-evoked synaptic responses were paired with pulses of
hyperpolarizing current (latency, 60 ms after stimulation; duration,
1200 ms) injected through the intracellular microelectrode. With this
protocol, the GABAA IPSP was evoked at membrane
potentials of
55 and
60 mV (determined by steady current control),
while the following GABAB IPSP was examined at
different levels of hyperpolarization (determined by the current
pulses; Fig. 3, A and
C). The amplitude of GABAB IPSPs was
measured at 200 ms after mossy fiber stimulation to avoid interference
with the charging curve evoked by the hyperpolarizing current pulse.
Voltage responses evoked by current pulses alone (at membrane
potentials of
55 and
60 mV) were measured at 140 and 1200 ms after
onset of the current pulse (not shown); measurements at these time
points were not different, indicating that at 140 ms the membrane had
reached a steady-state level and maintained this level throughout the
pulse. GABAB IPSPs (measured at a latency of 200 ms) superimposed on these current-induced hyperpolarizations had a
reversal potential of
92.6 ± 2.1 mV (n = 7)
when they were preceded by GABAA IPSPs evoked at
55 mV (Fig. 3B). GABAB IPSPs preceded by GABAA IPSPs at
60 mV had a
significantly more negative reversal potential of
99.1 ± 1.4 mV
(n = 7; Fig. 3D). Similar results were
obtained when reversal potential of monosynaptic GABAB IPSPs was measured (at a latency of 200 ms)
following monosynaptic GABAA IPSPs (not shown).
In these latter experiments, GABAB IPSPs reversed
at
93.0 ± 1.8 mV (n = 9) when preceding
GABAA IPSPs were evoked at
55 mV, but had a
significantly more negative reversal potential of
98.9 ± 1.4 mV
(n = 9) when preceded by GABAA
IPSPs evoked at
60 mV.
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Mossy fiber-evoked GABAA IPSPs could persist
beyond 140 (or 200) ms after mossy fiber stimulation, and thus
interfere with our measurements of GABAB IPSP
reversal potential. To evaluate this possibility, we measured the
duration of the mossy fiber-evoked GABAA IPSPs
in five neurons, at membrane potentials close to the GABAB IPSP reversal potential, in the presence of
the GABAB receptor antagonist CGP35348 (700 µM)
and Cs+ (1 mM; Fig. 3E). Our
recordings showed that the duration of GABAA IPSPs, at membrane potentials between 90 and
100 mV, did not exceed
130 ms (Fig. 3F). Therefore voltage excursions associated with GABAA IPSPs do not interfere with
measurements of the GABAB IPSP reversal potential.
Pharmacologically isolated monosynaptic GABAB
IPSPs have a long latency (onset at about 37 ms) in response to
stimulation that directly activates interneurons. At this time point
during the mossy fiber response, membrane potential is governed
primarily by the GABAA IPSP and is shifted in a
negative direction from resting membrane potential. We estimated a
membrane potential established by GABAA IPSPs at
the time point corresponding to the onset of the
GABAB IPSP so as to better describe its voltage dependency associated with aberrant behavior. Membrane potential was
measured at 37 ms after mossy fiber stimulation, in responses evoked
with the cell resting potential at 55 and
60 mV (Fig. 4A), i.e., at membrane
potentials where the GABAB IPSP had different properties. The membrane potential values (at a latency of 37 ms,
dictated by the GABAA IPSP) were
64.2 ± 0.4 mV (n = 10) and
67.4 ± 0.3 mV
(n = 10), at
55 and
60 mV, respectively (Fig. 4B). Therefore onset of the GABAB IPSP
evoked by mossy fiber stimulation, in a cell with resting membrane
potential of
55 mV, occurred at a membrane potential close to
64
mV.
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DISCUSSION |
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In hippocampal pyramidal cells recorded in vitro, stimulus-evoked
GABAB IPSPs are reduced when the cell is
depolarized positive to 60 mV (Knowles et al. 1984
;
Newberry and Nicoll 1985
). This behavior has been
explained by inwardly rectifying properties of the potassium channels
coupled to GABAB receptors (Gähwiler and Brown 1985
; Lüscher et al. 1997
;
Sodickson and Bean 1996
). However, this explanation is
not entirely satisfying, since GABAB receptor-mediated potentials/currents pharmacologically isolated from
GABAA receptor-mediated potentials/currents show
monotonic voltage dependency (Hablitz and Thalmann 1987
;
Otis et al. 1993
). Further, monosynaptic
GABAB IPSPs demonstrate long latency
close to 37 ms in our experiments. At this time point, membrane potential is
primarily governed by the preceding GABAA IPSP
evoked by mossy fiber activation. Our calculations show that
GABAB receptor-coupled potassium channels are
activated when the GABAA IPSP sets the membrane
potential at 7-9 mV below resting membrane potential for CA3 pyramidal
cell. These results indicate that at the resting membrane potential at
which GABAB IPSPs were reduced (i.e., at
55
mV), the onset of GABAB IPSPs occurred at
approximately
64 mV. Rectification of GABAB
receptor-mediated responses at this membrane potential has not been reported.
Recently we found that reduction of the GABAB
IPSPs at relatively positive membrane potentials (close to 55 mV) is
due to chloride influx associated with the preceding
GABAA IPSP (Lopantsev and Schwartzkroin
1999
). In the present study, we have found that membrane
depolarization (from
60 to
55 mV) enhances the
GABAA receptor-mediated conductance during mossy
fiber-evoked IPSPs or during monosynaptically evoked
GABAA IPSPs. This observation suggests that a
more intensive chloride influx occurs during
GABAA receptor-mediated events evoked at more
positive membrane potentials.
Changes in intracellular chloride concentrations also could be mediated
by a voltage-activated chloride conductance described in hippocampal
pyramidal cells (Madison et al. 1986; Staley
1994
). However, we may rule out its possible impact on
GABAB IPSPs for a number of reasons. First, this
conductivity operates at membrane potentials close to (or more negative
than) resting level, while we described chloride sensitivity of the
GABAB IPSP during membrane depolarization.
Second, chloride-dependent modulation of GABAB IPSPs was completely blocked by application of a
GABAA receptor antagonist (Lopantsev and
Schwartzkroin 1999
). Also, measurements of membrane resistance
during pharmacologically isolated monosynaptic GABAA IPSPs have shown that at a membrane
potential of
55 mV, enhanced chloride influx does not activate any
additional long-lasting membrane permeability, which could overlap with
GABAB IPSPs and affect their magnitude.
One possible explanation for chloride-dependent reduction of the
GABAB IPSP is that higher intracellular chloride
interacts with the intracellular G-protein signaling mechanism and/or
the coupled potassium channel to reduce membrane permeability. However, we found that membrane permeability was not affected during reduced GABAB IPSPs at positive membrane potentials.
Another possibility is that a rise in intracellular chloride modulates
properties of the current through the potassium channels coupled to
GABAB receptors. This latter explanation is
consistent with the results of our experiments. First, we found that
the reversal potential of the pharmacologically isolated monosynaptic
GABAB IPSP shifted in a positive direction (from
98 to
90 mV) in cells loaded with chloride ions from the
intracellular microelectrode. This finding is in line with previous
studies showing that GABA- and baclofen-induced currents, recorded in
cultured hippocampal neurons with low resistance chloride-filled
intracellular microelectrodes, reversed at relatively positive membrane
potentials (close to
72 mV) (Gähwiler and Brown
1985
). Second, direct measurements of the mossy fiber-evoked GABAB IPSP revealed a more positive reversal
potential when the preceding GABAA IPSP was
evoked at
55 mV than at
60 mV.
Interestingly, GABAA receptor-mediated chloride
influx still exists in the cell soma at membrane potentials between
60 and
70 mV. However, this level of chloride influx was not
sufficient to affect GABAB IPSP reversal
potential. Only at membrane potentials more positive than
60 mV was
the GABAA receptor-mediated chloride influx
sufficient to induce the positive shift in reversal potential of the
GABAB IPSP
i.e., to alter the properties of the
current through GABAB receptor-coupled potassium
channels. This apparent paradox may be due to a spatial separation of
chloride channels mediating GABAA IPSPs and the
GABAB receptor-linked channels. Predominant
dendritic location of GABAB receptors
(Newberry and Nicoll 1985
), where transmembrane
distribution of chloride may be different from that in the cell soma
(Jarolimek et al. 1999
; Misgeld et al.
1986
), makes it difficult to estimate exactly what direction
and strength of transmembrane chloride flow is able to affect
significantly the functioning of GABAB
receptor-linked potassium channels.
It is interesting to note that spontaneous GABAA-
but not GABAB-mediated events have been recorded
in hippocampal slices in normal ACSF (Alger and Nicoll
1980; Collingridge et al. 1984
; Miles and
Wong 1984
; Otis and Mody 1992
). However, recent
study has revealed mixed fast/slow IPSPs, evoked in CA1 pyramidal cell when a single presynaptic interneuron generated a burst of action potentials (Thomson and Destexhe 1999
). Blockade of the
fast GABAA-mediated IPSP (by bicuculline) was
necessary to uncover the GABAB receptor-mediated response. This result suggests that the same interneuron can activate both GABAA and GABAB
postsynaptic receptors, but that concomitant activation of these
receptors may evoke a "pure" GABAA IPSP;
i.e., the GABAB IPSP is suppressed. This scenario
can be explained by the interaction demonstrated in our study,
involving a GABAA-mediated chloride-dependent
reduction of the GABAB receptor-mediated component.
Does this experimentally identified chloride modulation of
GABAB IPSPs have any real physiological
consequences? It is perhaps relevant that GABAB
receptor-mediated currents evoked by activity of inhibitory
interneurons contribute to the rhythmic activity (8-15 Hz) induced by
a muscarinic receptor agonist in hippocampal slice culture
(Scanziani 1999). Similar rhythmic activities in hippocampus in vivo (e.g., theta-rhythm) are also characterized by
intense discharges of the inhibitory interneurons (Freund and Buzsaki 1996
; Ylinen et al. 1995
) that may
provide a significant drive for evoking both
GABAA- and GABAB
receptor-mediated events in postsynaptic pyramidal cells. It seems
likely that under these conditions, interaction between spontaneously
occurring GABAA- and GABAB
IPSPs may involve chloride-dependent modulation of
GABAB IPSPs since resting membrane potential of
pyramidal cells in vivo is close to values investigated in our
experiments (
55 and
60 mV). We can speculate that
GABAA receptor-mediated fluctuations in
intracellular chloride concentration may affect
GABAB IPSP reversal potential, and thus modulate
strength of GABAB-mediated postsynaptic
potentials or even mask their appearance.
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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, Dept. of Neurological Surgery, University of Washington, Box 356470, Seattle, WA 98195-6470 (E-mail: pas{at}u.washington.edu).
Received 13 June 2000; accepted in final form 12 February 2001.
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REFERENCES |
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