Department of Physiology and Pharmacology, State University of New York Health Science Center, Brooklyn, New York 11203
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ABSTRACT |
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Perkins, Katherine L..
Cl Accumulation Does Not Account for the
Depolarizing Phase of the Synaptic GABA Response in Hippocampal
Pyramidal Cells.
J. Neurophysiol. 82: 768-777, 1999.
It has been proposed that the depolarizing
phase of the biphasic synaptic GABA response could be mediated by
HCO3
passing through GABAA channels after
dissipation of the transmembrane Cl
gradient due to
intracellular Cl
accumulation. To test this hypothesis,
giant GABA-mediated postsynaptic currents (GPSCs) were recorded from
pyramidal cells in slices of adult guinea pig hippocampus in the
presence of 4-aminopyridine. GPSCs consisted of an early outward
current (GABAA component) followed by a late inward current
(GABAD component). Spontaneous outward inhibitory
postsynaptic currents (IPSCs) occurred during the GABAD
component of the GPSC. GPSCs that were evoked 1-12 s after the
preceding GPSC (short interval, siGPSCs) showed no GABAD component even though in many cells the amplitude of the siGPSC was
greater than the amplitude of the GABAA component of the
preceding spontaneous GPSC. In addition, the siGPSC evoked during the
GABAD component of a spontaneous GPSC was an outward
current. To test whether the siGPSC lacked a GABAD
component because it was generated predominantly at the soma, where
less of an increase in [Cl
]i would occur,
picrotoxin was applied to the soma of the pyramidal cell. To the
contrary, this focal application of picrotoxin caused less of a
reduction in the amplitude of the siGPSC than in the amplitude of the
GABAA component of the GPSC. Furthermore when a GPSC and
siGPSC were evoked 10 s apart using identical stimuli, the area
under the outward current curve was sometimes greater for the siGPSC
than for the GPSC, and yet the siGPSC had no inward component. This
result indicates that even when the location of Cl
entry
was the same, more Cl
could enter the cell during the
siGPSC than during the outward component of the GPSC and yet not lead
to an inward current. In addition, when the second of two identical
stimuli was applied during the inward GABAD component of
the first evoked GPSC, the GABAA response it generated was
always outward, demonstrating that the equilibrium potential for
GABAA responses did not become more positive than the
holding potential during a GPSC. Finally, evoking GPSCs at a
hyperpolarized potential revealed that the siGPSC actually lacked a
GABAD conductance. These results disprove the
Cl
accumulation hypothesis of the synaptic depolarizing
GABA response and suggest the possibility that a separate channel type
may mediate the GABAD component of the GPSC.
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INTRODUCTION |
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The major inhibitory neurotransmitter in the cerebral cortex is
-aminobutyric acid (GABA). GABAA
receptor-channels mediate fast inhibition of the postsynaptic neuron.
Chloride (Cl
) is the major permeant ion of the
GABAA receptor channel, but the channel is also
permeable to bicarbonate ions (HCO3
), with a
permeability ratio of Cl
to HCO3
of 5:1
(Bormann et al. 1987
; Kaila et al. 1993
).
In addition to mediating an inhibitory, hyperpolarizing response, GABA
sometimes mediates an excitatory, depolarizing response in adult
animals (Andersen et al. 1980
; Michelson and Wong
1991
; Staley et al. 1995
). The synaptic
GABA-mediated depolarizing response originally was described as part of
a biphasic hyperpolarizing-depolarizing response in CA1 pyramidal cells
in response to stimulation of the stratum radiatum in the presence of
pentobarbital (Alger and Nicoll 1979
, 1982a
). Because
the equilibrium potential for HCO3
is much more
depolarized than that of Cl
, some investigators
have hypothesized that the late, depolarizing part of the GABA response
is mediated by HCO3
(Grover et al.
1993
; Staley et al. 1995
; Voipio et al.
1995
). According to one such hypothesis, a net inward current
mediated by HCO3
passes through
GABAA receptor channels after the more permeant ion Cl
has accumulated inside the cell during
the early, hyperpolarizing part of the GABA response
(Cl
accumulation hypothesis) (Staley et
al. 1995
). Recently it has been determined that
HCO3
does carry, at least in part, the inward current
underlying the ligand-mediated (cf. Kaila et al. 1997
)
synaptic depolarizing GABA response in CA3 pyramidal cells
(Perkins and Wong 1996
). At present, however, it has not
been determined whether the depolarizing GABA response is mediated by
the same receptor channels as the hyperpolarizing response. An
alternative hypothesis is that a separate channel with a higher
permeability to HCO3
mediates the depolarizing GABA
response (Perkins and Wong 1996
, 1997
). For these
experiments, the convulsant 4-aminopyridine (4-AP) was used to elicit
giant GABA-mediated postsynaptic currents (GPSCs) in pyramidal cells.
These GPSCs reflect the synchronous release of GABA from
presynaptic interneurons (Michelson and Wong 1991
). The
experiments reported here indicate that Cl
accumulation does not account for the synaptic depolarizing GABA response and suggest that the hyperpolarizing and depolarizing GABA
responses may be mediated by two different types of GABA channels.
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METHODS |
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Slice preparation
Experiments were done in adult guinea pig hippocampal slices. Guinea pigs (14-30 days old) were anesthetized with halothane and decapitated with a guillotine. One hippocampus was removed, and 300-µm transverse slices were cut in oxygenated, ice-cold solution (solution composition same as bath solution listed in the next section, except 8 mM MgCl2 and 0.5 mM CaCl2) using a vibratome (Technical Products International, St. Louis, MO). Slices were transferred to the recording chamber (Fine Science Tools, Foster City, CA) where they were maintained at an interface between continuously perfusing oxygenated solution and humidified 95% O2-5% CO2 gas at 31°C. Slices were submerged before recording.
Solutions
The bath solution contained (in mM) 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.6 MgCl2, 2.0 CaCl2, and 11 D-glucose. During recording the bath solution included 4-aminopyridine (4-AP, 50 µM), ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM, Tocris Cookson, Ballwin, MO), and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 20 µM, Tocris Cookson), and the GABAB receptor antagonist CGP 55845A (1 µM; gift from Ciba-Geigy, Basel, Switzerland).
Intracellular solutions containing greater than physiological
concentrations of HCO3 were used to accentuate the
appearance of the GABAD component of the GPSC at
potentials near rest (Perkins and Wong 1996
). The HCO3
equilibrium equation,
CO2(dis) + H2O
H2CO3
H+ + HCO3
, describes the
dependence of [HCO3
] on
[H+] and [CO2]. Because
the cell membrane is freely permeable to CO2(dis)
and because the partial pressure of CO2 is kept
constant, to change the intracellular equilibrium concentration of
HCO3
, the pH of the intracellular solutions also must
be changed. The pH corresponding to a given concentration of
HCO3
was calculated using the Henderson-Hasselbalch
equation as described in Perkins and Wong (1996)
.
The solution used in the majority of the experiments was the 102 mM
HCO3/pH 8.0 solution, which contained (in mM) 102 KHCO3, 28 KOH, 5 NaCl, 5 TEA-Cl, 10 HEPES, 4 EGTA, 5 N-(2,6-dimethyl
phenylcarbamoylmethyl)-triethylammonium bromide (QX-314; RBI, Natick,
MA), and 4 Mg-ATP. The 49 mM HCO3
/pH 7.7 solution
contained (in mM) 49 KHCO3, 76 KOH, 5 KCl, 5 NaCl, 5 TEA-Cl, 10 HEPES, 4 EGTA, 5 QX-314, and 4 Mg-ATP. The pH of
solutions containing HCO3
was adjusted with
methanesulfonic acid while bubbling with 95% O2-5% CO2 gas. In
addition, solutions containing HCO3
were continuously
bubbled with 95% O2-5%
CO2 gas for
1 h immediately before filling the
recording pipette. A third solution, used for only one recording,
contained (in mM) 125 CsOH, 10 NaCl, 10 HEPES, 2 EGTA, 5 QX-314, and 4 Mg-ATP and was adjusted to pH 7.3 using methanesulfonic acid. Chemicals
were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.
As noted above, the recording pipette solutions contained QX-314 and
TEA. Intracellular QX-314 blocks voltage-dependent sodium currents
(Connors and Prince 1982) and the
hyperpolarization-activated current Iq
(Perkins and Wong 1995
). Intracellular TEA blocks
several voltage-dependent K+ currents
(Chen and Wong 1992
). Suppression of these
voltage-dependent conductances facilitated the study of synaptic
events. In addition, QX-314 blocks the GABAB
component of the GPSC (Perkins and Wong 1996
).
Whole cell recording
Electrophysiological recordings were carried out in the whole
cell voltage-clamp configuration (Hamill et al. 1981) on
CA3 and CA1 pyramidal cells using a List EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, Germany) and pClamp software (Axon Instruments, Foster City, CA). Whole cell electrode resistances ranged
from 2 to 5 M
when filled with intracellular recording solution.
Seals were established using the patch-slice method of Blanton
et al. (1989)
. No series resistance or slow capacitance compensation was used during the experiment.
The capacitive current response to a 5-mV voltage step
(V) was recorded in all cells and periodically retested
during the experiment. The access resistance
(Ra) was estimated using the equation
Ra =
V/A,
where A is the amplitude of the capacitive current. Only
recordings with an Ra
12 M
were
included in the analyses.
The liquid junction potential (Vlj)
between the whole cell pipette solution and the bath solution was
determined experimentally using the procedure of Neher
(1992). Series resistance error
(Vs) was calculated after the
experiment using the equation Vs = Ra × I. The I
used in the calculation was the baseline holding current at a given
command potential, Vcom. All
potentials reported in this paper have been corrected for
Vlj and
Vs using the equation Vm = Vcom - Vlj
Vs.
4-AP (50 µM) was used to elicit giant GPSCs in hippocampal pyramidal
cells. 4-AP is particularly useful for this purpose because the GPSCs
it elicits in a given cell type have a consistent time course
(Perkins and Wong 1996); this allows comparison of
particular components of the GPSC across events and across cells. Kaila
and colleagues (Kaila et al. 1997
; Smirnov and
Kaila 1997
) studied giant GABA-mediated events elicited by
40-pulse/100-Hz stimuli trains and reported the presence of a late
component to the event that was a K+-mediated,
nonsynaptic inward current; the GPSCs elicited with 4-AP that are
reported here and in two previous papers (Perkins and Wong 1996
,
1997
) do not contain and are not followed by this late
K+ current.
The GPSCs elicited by 4-AP occurred spontaneously and also could be evoked with a stimulating electrode. When evoking GPSCs, single 50-µs stimuli were delivered using a bipolar tungsten electrode placed 0.3-1.5 mm away from the recording electrode. The response to a stimulus was considered to be a GPSC [or short interval GPSC (siGPSC), see following text] if it was at least half the amplitude of the preceding spontaneous GPSC.
Focal application of picrotoxin
To selectively apply picrotoxin (PiTX) to the soma of the
recorded cell, a puffer pipette was positioned just below the surface of the slice and as near to the recording electrode as possible. Puffer
solution was the bath solution, including CNQX, CPP, 4-AP, and CGP
55845A but containing 0.5 mM CaCl2 instead of 2 mM CaCl2 to avoid forming
CaCO3 precipitate. The puffer solution also
contained 100 µM PiTX and 0.1% fast green dye. This concentration
(100 µM) of PiTX was chosen because bath application of 50 µM PiTX
has been shown to block both the GABAA and
GABAD components of 4-AP-induced giant
GABA-mediated events (Michelson and Wong 1991, 1994
) and because some dilution of the puffer solution occurred when it mixed
with the bath solution. The puffer solution contained fast green to
allow visualization of the affected area. The puffer solution was
delivered by applying a constant pressure of 1-5 psi to the puffer
pipette while the bath solution was superfused at 3-4 ml/min. Puffer
pipettes were pulled from 1.5-mm-diam glass and had resistances of 6-8
M
when filled with puffer solution.
A two-way repeated-measures ANOVA was used to assess the effect of PiTX on the amplitudes of GPSCs and siGPSCs. To reduce variability in the GPSC amplitudes under the same condition between cells and the heterogeneity of variance between treatment groups, a transformation of the data were performed. A log10 transformation was chosen because the standard deviations of the treatment groups tended to be proportional to the means of the treatment groups.
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RESULTS |
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GPSCs and IPSCs
As described previously (Perkins and Wong 1996),
the GPSC recorded in pyramidal cells in the presence of
GABAB blockers (Fig. 1) lasts 1.5-2 s and, at a holding
potential of
50 mV, consists of an early outward current (the
GABAA component) followed by a late inward
current (the GABAD component). The
GABAD component corresponds to the depolarizing
GABA response in current-clamp recordings (Michelson and Wong
1991
). Many small spontaneous inhibitory postsynaptic currents
(IPSCs) appear in these recordings. These IPSCs are outward at
potentials more positive than about
60 mV and inward when the
membrane potential is hyperpolarized to the point that the
GABAA component of the GPSC is inward (Fig.
1A). At potentials at which the GABAA
component of the GPSC is outward, small outward IPSCs even can be
observed during the inward GABAD component of the
GPSC (Fig. 1B; n = 39 cells). The occurrence of these outward GABA-mediated IPSCs during the inward
GABAD component of the GPSC indicates that the
GABAA equilibrium potential remains more negative
than the holding potential during a GPSC, at least at the location
where the IPSCs are generated. This finding suggests that the
transmembrane Cl
gradient may not dissipate
sufficiently during a GPSC to account for its transition to inward
current. Alternatively, the synapses mediating the
GABAD component of the GPSC and those mediating the IPSCs may be localized to different parts of the cell. One might
hypothesize that the synapses mediating the GABAD
component of the GPSC are localized to the dendrites (Alger and
Nicoll 1979
, 1982a
,b
; Andersen et al. 1980
;
Thalmann et al. 1981
) and that the dissipation of the
transmembrane Cl
gradient is localized there.
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Evoking siGPSCs
The data discussed above demonstrated that the transmembrane
Cl gradient does not dissipate over the entire
cell during a GPSC to the point that all GABA-mediated synaptic
responses are inward. To test whether the transmembrane
Cl
gradient dissipates sufficiently at the site
of generation of the GPSCs to cause the late component to be inward,
GPSCs were evoked at short intervals after a spontaneous GPSC. The
GPSCs evoked after a short interval will be referred to as siGPSCs. Assuming for now that the spontaneous GPSC and the siGPSC are generated
at the same cellular location, the Cl
accumulation hypothesis predicts that the siGPSC will develop into an
inward current more quickly due to residual Cl
accumulation from the previous GPSC. To test this prediction, CA3 and
CA1 pyramidal cells were held at a potential at which the GPSC
consisted of an outward current followed by an inward current, and the
spontaneous IPSCs were outward, usually
40 to
50 mV. A siGPSC was
evoked with a stimulating electrode 5-12 s after a spontaneous GPSC.
Instead of converting from outward current to inward current more
quickly than usual, the siGPSC consisted of only an outward current
(n = 20 of 20 cells; Fig. 2, top). GPSCs evoked after a
30- to 60-s interval had normal outward and inward components
(n = 20 of 21 cells, Fig. 2, bottom).
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Considering only the data of the type illustrated in Fig. 2, it could
be hypothesized that the cell was able to extrude the Cl that entered during the preceding GPSC by
the time of the siGPSC and that the siGPSC was not big enough to cause
sufficient Cl
accumulation for the development
of an inward current. However, in some cases a siGPSC was evoked 1-2 s
after the onset of the preceding spontaneous GPSC. In these cases
(n = 6 cells), the siGPSC still consisted of only
outward current. Figure 3 illustrates that even when a siGPSC was evoked during the
GABAD component of a spontaneous GPSC, the siGPSC
was outward. If dissipation of the Cl
driving
force were responsible for the late inward current, then the siGPSC
stimulated during the GABAD component of the GPSC
would have been inward.
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Another result also sheds doubt on the Cl
accumulation hypothesis: the siGPSC evoked 1-12 s after a spontaneous
GPSC was sometimes of greater amplitude than the preceding spontaneous
GPSC (Fig. 4; n = 11 of
24 cells) and yet still had no inward current component. Integration of
the outward current revealed that more net charge flowed during the
siGPSC than during the outward current component of the preceding
spontaneous GPSC. For example Q = 4.6 × 10
10C for the siGPSC shown in Fig. 4, and
Q = 2.7 × 10
10C for the
outward component of the spontaneous GPSC shown in Fig. 4. Overlap of
the spontaneous GPSC with its corresponding siGPSC in Fig.
4B illustrates that more negative charge entered the cell during the siGPSC than during the GABAA component
of the GPSC. This finding is counter to the Cl
accumulation hypothesis. If sufficient Cl
had
accumulated during the outward component of the spontaneous GPSC to
cause it to turn inward, then the siGPSC also should have turned inward
after a similar (or even lesser) amount of Cl
current flow, and it did not.
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Effect of focal somatic picrotoxin
Although the above findings suggest that
Cl accumulation does not account for the late
inward component of the GPSC, there is an explanation that could
reconcile these data with the Cl
accumulation
hypothesis: perhaps the synapses responsible for the siGPSC are largely
somatic whereas the synapses responsible for the spontaneous GPSC are
largely dendritic.
This hypothesis of a differential site of generation proposes, first,
that the spontaneous GPSC would involve either dendritic synapses only
or both somatic and dendritic synapses with Cl
accumulation in the dendrites leading to the appearance of the late
inward component. Second, the siGPSC instead would involve primarily
synapses on the soma, where less of an increase in
[Cl
]i would occur as a
result of Cl
entry, due to the lower
surface/volume ratio of the soma as compared with the dendrites (and
due to the reservoir of solution in the patch electrode); thus no net
inward current would develop. This somatic location would explain how
the siGPSC evoked during the GABAD component of
the spontaneous GPSC (Fig. 3) could be outward (less
[Cl
]i increase in the
soma than in the dendrites during the spontaneous GPSC) and would
explain the lack of a GABAD component to the
siGPSC even though more Cl
entered the cell
during the siGPSC (Fig. 4; less of an increase in
[Cl
]i because the
active synapses are somatic). If this hypothesis is correct, blocking
somatic GABA synapses should cause a greater reduction in the amplitude
of the siGPSC than in the amplitude of the
GABAA component of the spontaneous GPSC.
To test the possibility that, in contrast to the GPSC, the siGPSC reflects the activation of predominantly somatic synapses, a puffer pipette containing 100 µM PiTX was situated in the CA3 cell body layer near the recording electrode (see METHODS). For this experiment, CA3 pyramidal cells were chosen in which the siGPSC evoked after a 5- to 12-s interval was of greater amplitude than the GABAA component of the preceding spontaneous GPSC. After recording control spontaneous GPSCs and siGPSCs, PiTX was delivered to the soma of the recorded cell. The presence of fast green in the puffer solution allowed visualization of the spread of PiTX. The affected area included the cell body layer around the electrodes, the local stratum oriens at least two-thirds of the way to the fimbria, the local stratum lucidum, and sometimes the proximal edge of the local stratum radiatum.
This focal application of PiTX (Fig. 5)
reduced the amplitude of the GABAA component of
the spontaneous GPSC by 56 ± 6% (mean ± SE,
n = 3 cells) and the amplitude of the siGPSC by 37 ± 8% (n = 3 cells). The amplitude of the
GABAD component of the spontaneous GPSC showed no
consistent change as a result of PiTX application to the soma (0 ± 8%, n = 3 cells). The siGPSC still had no inward component in PiTX. The effect of PiTX was largely reversible (Fig. 5).
After washout, the amplitude of the GABAA
component of the spontaneous GPSC recovered to 89 ± 9% of
control, the siGPSC amplitude was 91 ± 7% of control, and the
amplitude of the GABAD component of the
spontaneous GPSC was 95 ± 11% of control. Notably, rather than
reducing the amplitude of the siGPSC to a greater extent than the
amplitude of the GABAA component of the GPSC, the
focal application of PiTX actually reduced the amplitude of the
GABAA component of the spontaneous GPSC to a
greater extent. A two-way repeated-measures ANOVA revealed that PiTX
significantly reduced the amplitudes of the outward component of the
spontaneous GPSC and the siGPSC (P < 0.05) and that
the PiTX had a greater effect on the amplitude of the outward component
of the spontaneous GPSC than on the amplitude of the siGPSC
(P < 0.05; a log10
transformation of the data were usedsee METHODS).
|
These results indicate that the siGPSC was not generated more
somatically than the GABAA component of the
spontaneous GPSC. Notably, in the presence of focal somatic PiTX, the
siGPSC was still greater in amplitude than the
GABAA component of the preceding spontaneous
GPSC, and yet had no late inward current. This result indicates that
even when GABA-mediated Cl entry is occurring
exclusively in dendrites, which should be the site with a greater
potential for [Cl
]i
increase, more Cl
can enter the cell during a
siGPSC than during the preceding spontaneous GPSC and still not result
in an inward current.
Evoking pairs of GPSCs with identical stimuli
The preceding results suggest that Cl
accumulation cannot account for the depolarizing phase of the biphasic
synaptic GABA response. However, one might imagine a scenario in which
these data still could be reconciled with the
Cl
accumulation hypothesis: even though the
spontaneous GPSC and the evoked siGPSC both are generated partly in the
dendrites, perhaps they are segregated to different areas of the
dendrite or to different dendritic branches. In this case, one might
propose that the siGPSC evoked during the GABAD
component of a spontaneous GPSC was outward because it was generated in
a different location where no Cl
had
accumulated. In addition, to explain why a greater amount of
Cl
can enter during a siGPSC than during the
previous spontaneous GPSC and yet not result in an inward current, one
might propose that a greater amount of Cl
entry
is required to cause a sufficient rise in
[Cl
]i at those
dendritic sites involved in the siGPSC. Furthermore one would have to
propose that a lesser amount of Cl
flows into
the cell during a siGPSC (because the siGPSC does not have an inward
component) than during a GPSC evoked with an identical stimulus (which
does have an inward component, see Fig. 2).
As a final test of the Cl accumulation
hypothesis, pairs of GPSCs were evoked at varying intervals with
identical stimuli and recorded from a CA3 pyramidal cell. The
stimulating electrode was placed 0.3-0.6 mm away from the recording
electrode. To assess whether the stimuli directly activated the
interneurons generating the GPSC, the delay between the beginning of
the stimulus artifact and the beginning of the GPSC (and siGPSC) was
measured. The delay was measured for six stimuli in each of the three
cells. The values in the three cells were 1.02 ± 0.04 ms
(mean ± SD), 1.08 ± 0.06 ms, and 1.8 ± 0.1 ms. The
delay values for GPSCs and siGPSCs were pooled. An illustration of the
short delay is in Fig. 6C.
These short delay times indicate that monosynaptic connections were evoked by the stimuli, suggesting that the second of two identical stimuli will result in activation of the same synapses, or a subset of
those synapses, on the recorded cell.
|
When the stimuli were 8-12 s apart, the second evoked GPSC of
the pair (the siGPSC) was of the same or shorter amplitude as the
preceding evoked GPSC and never had an inward component
(n = 3 cells, Fig. 6). The integral of the siGPSC was
compared with the integral of the outward current component of the
first evoked GPSC of a pair (the GPSC) to compare
Cl entry between the two. In at least one case
in every cell (n = 3 cells), the area under the siGPSC
was
5% larger than the area under the GPSC, and yet the siGPSC had
no inward component. For example, Q = 3.0 × 10
10C for the siGPSC shown in Fig. 6, and
Q = 2.5 × 10
10C for the
outward component of the GPSC shown in Fig. 6. Overlapping the two
traces (Fig. 6B) illustrates that the integral for the siGPSC was greater than the integral of the outward component of the
GPSC because the siGPSC was longer in duration, perhaps because no
GABAD component interrupted it. Assuming that
Cl
entry was occurring at the same sites on the cell
following each stimulus, if Cl
accumulation had
been the cause of the reversal to inward current during the first
evoked GPSC, then the siGPSC should have also become inward, but did
not. When the interval between the two GPSCs was 45-60 s, the second
GPSC of a pair had a normal GABAD component
(n = 3 cells).
A second set of experiments was carried out using pairs of identical
stimuli. In this case, the second stimulus was delivered < 2 s after the first, which was during the GABAD
component of the first evoked GPSC. In every case (n = 14 trials in 3 cells), the evoked current, while smaller than a GPSC,
was always outward (Fig. 7). If
Cl accumulation had been the cause of the
reversal to inward current during the first GPSC, then an additional
GABA-mediated current at that same site also should have been inward.
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Time course of conductance change revealed by hyperpolarization
The previous experiments indicate that Cl
accumulation cannot account for the GABAD
component of the GPSC. The data also suggest that evoking a GPSC after
a short interval may selectively activate the
GABAA component of the GPSC to the exclusion of
the GABAD component. From the data illustrated in
Fig. 6B, it is apparent that while the duration of the
siGPSC was longer than that of the outward component of the GPSC,
overall, the duration of the siGPSC was shorter. Possible explanations
for the apparent lack of a GABAD component to the
siGPSC are that the siGPSC had no GABAD
conductance or that the conductance underlying the
GABAD component of the GPSC was present but
either the cell was at the GABAD reversal
potential or the GABAD current was masked by an outward current. To determine whether the siGPSC has a
GABAD conductance, the time course of the
conductance change associated with the siGPSC and the GPSC was compared.
Recordings from both CA3 and CA1 pyramidal cells were used in this set
of experiments. Either 5-10 s or 30-60 s after a spontaneous GPSC at
50 mV, the membrane potential was hyperpolarized by 15-35 mV. Six
hundred milliseconds after the hyperpolarization, a GPSC (or siGPSC)
was evoked using a stimulating electrode (Fig.
8). The evoked GPSCs
and siGPSCs were entirely inward, as expected. The GPSC evoked after
30-60 s lasted 1.5-2 s, the duration of a normal GPSC. The siGPSC
evoked after 5-10 s had a shorter duration (0.7
0.9 s)
than the normal GPSC, even though the amplitudes of the
GABAA components of the GPSC and the siGPSC were
comparable (Fig. 8, n = 8). The duration of the siGPSC
evoked at the hyperpolarized potential was comparable with the duration
of the siGPSC evoked at
50 mV (Fig. 8, dotted trace). The fact that
the duration of the siGPSC was shorter both at
50 mV and at more
hyperpolarized potentials indicates that the conductance underlying the
siGPSC is also shorter in duration than that underlying the GPSC. The siGPSC has no inward component at
50 mV because the siGPSC is actually missing the GABAD conductance.
|
Subtraction of the siGPSC evoked at a hyperpolarized potential (which is missing the GABAD component) from the GPSC evoked at the same hyperpolarized potential after a longer interval (which has a GABAD component) revealed the time course of the GABAD component of the GPSC. The siGPSC and the subtracted trace are plotted together in Fig. 8B for comparison. The siGPSC should show the time course of the GABAA component, and the subtracted trace should show the time course of the GABAD component. The peak of the GABAA component occurred at just under 100 ms; whereas the peak of the GABAD component did not occur until 350-400 ms after the stimulus. There was significant overlap between the GABAD component and the falling phase of the GABAA component.
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DISCUSSION |
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Previous experiments showed that changing
[HCO3]i has a much greater
effect on the reversal potential of the GABAD
component of the GPSC than on the reversal potential of the
GABAA component (Perkins and Wong
1996
). Those experiments did not determine the reason for this
difference. At that time, the two hypotheses proposed to explain the
greater effect of [HCO3
] on the reversal potential
of the GABAD component were that the Cl
driving force had dissipated during the
GABAA component of the GPSC, leaving a net
HCO3
current through GABAA
channels (Cl
accumulation hypothesis), or that
the GABAD response was mediated by a separate set
of channels which showed a greater permeability to
HCO3
. The data presented in this paper favor the
latter hypothesis.
Cl accumulation does not account for the late inward
current component of the GPSC
Cl accumulation has been shown to occur in
hippocampal pyramidal cells due to repeated applications of exogenous
GABA (Huguenard and Alger 1986
), due to repetitively
evoked IPSCs (Ling and Benardo 1995
) and due to a rise
in extracellular [K+] as a result of a
10-pulse/100-Hz train of stimuli (inhibition of
Cl
/K+ cotransport) (see
Fig. 7 in Kaila et al. 1997
). The data in this paper
demonstrate, however, that Cl
accumulation
cannot account for the GABAD component of the
GPSC in hippocampal pyramidal cells. If the GABAA
equilibrium potential (EGABA) had
shifted during the GABAA component of the GPSC to the point where EGABA was more
positive than the holding potential, either due to
Cl
entry through GABAA
channels or due to an inhibition of
Cl
/K+ cotransport (see
Kaila et al. 1997
), then the GABA-mediated synaptic current evoked during the GABAD component of a
GPSC should have been an inward current instead of the outward current
which was observed (Figs. 3 and 7) (see also Alger and Nicoll
1979
, 1982a
). In addition, if the Cl
accumulation hypothesis were correct, then siGPSCs that reached the
magnitude of a normal GPSC would then have turned inward; instead,
siGPSCs reached a greater magnitude than the preceding GPSC without
exhibiting an inward current (Figs. 4 and 6). The experiments using
pairs of identical stimuli demonstrated that Cl
does not accumulate during a GPSC to the point that
EGABA is more positive than holding
potential even right at the site of GPSC generation:
Cl
accumulation cannot account for the late
inward current component of the GPSC.
Why is the GABAD component of the GPSC an inward current?
Cl accumulation cannot account for the
late inward current component of the GPSC. The most obvious remaining
possibility is that the GABAA and
GABAD components of the GPSC are mediated by two
different channels, the GABAA channel and the
hypothetical GABAD channel (which may simply be a
particular isoform of the GABAA channel). On the
basis of ion permeability experiments (Perkins and Wong
1996
), the GABAD channel would have a
higher permeability ratio of HCO3
:
Cl
than the GABAA channel.
Initially, it seems that a second viable hypothesis is that the
GABAA component and the
GABAD component of the GPSC are mediated by
synapses localized to different parts of the cell that are maintained
at different levels of Cl (Misgeld et
al. 1986
; Müller et al. 1989
). However,
this hypothesis cannot account for earlier experiments, which showed
that changing the HCO3
concentration inside the cell
([HCO3
]i) had a greater
effect on the reversal potential of the GABAD component of the GPSC than on the reversal potential of the
GABAA component of the GPSC (Perkins and
Wong 1996
). The Goldman equation predicts that the opposite
result would have been obtained if the steady-state level of
Cl
were higher at the part of the cell where
the GABAD component of the GPSC was initiated.
The reasoning is as follows. Call the site where the
GABAD response originates the D compartment and the site where the GABAA response originates the
A compartment. Because the GABAD component of the
GPSC reverses at a more depolarized potential than the
GABAA component of the GPSC, the D compartment would be maintained at a higher level of Cl
.
The Goldman equation can be written as follows for a
Cl
:HCO3
of 5:1 at 31°C
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A third hypothesis is that GABAA channels may be
modified during a GABA responseperhaps as a result of prolonged
contact with GABA
so that they become more permeable to
HCO3
. To explain why the duration of the
GABA-mediated conductance is much longer when a
GABAD component is present, the process that is
responsible for conversion to a more HCO3
-permeable
channel also would change the channel kinetics. A similar hypothesis
has been proposed to explain the ability of neuroactive steroids to
induce a depolarizing GABA response (Burg et al. 1998
). To explain the lack of an inward current component to the siGPSC, this
hypothesis would further require that the process, whatever it is,
takes several seconds to recover after a previous period of activation.
If this hypothesis were correct, however, and the same channels were
responsible for both the GABAA and
GABAD components of the GPSC, one would have
expected that when PiTX blocked a portion of the
GABAA component of the spontaneous GPSC, it also would have blocked a portion of the accompanying
GABAD component, but it did not (Fig. 5). It is
not sufficient to propose that the hypothetical conversion renders the
channel insensitive to PiTX, because it has been shown that the
depolarizing response to GABA may actually be more sensitive to PiTX
than the hyperpolarizing response (Alger and Nicoll
1982b
).
Why does the siGPSC lack the GABAD component?
The GABAD component of the GPSC is absent or
greatly reduced in the siGPSCs (Figs. 2, 4, 6, and 8). The experiment
in which GPSCs were evoked at hyperpolarized potentials after different intervals was particularly informative in this regard (Fig. 8). It
demonstrated that the apparent absence of a GABAD
component to the siGPSC at 50 mV was not due to the fact that the
GABAD current was at its reversal potential and
was also not due to its being masked by an outward GABA-mediated
current. That experiment revealed the time course of the GABA-mediated
conductance change and demonstrated that the
GABAD component of the GPSC was actually absent
from the siGPSC.
The lack of a GABAD component to the siGPSCs can
be attributed to the short interval after which they are evoked because
GPSCs evoked with an identical stimulus, but after 30-60 s, have a
normal GABAD component. Other than the
above-described hypothesis of a refractory channel conversion process,
there are three possible explanations for this lack of a
GABAD conductance to the siGPSC. The first is
that the hypothetical GABAD channels are
desensitized after the GPSC and take some time to recover from
desensitization. This explanation may be unlikely: earlier experiments
showed that the depolarizing component of biphasic responses to a brief
application of 1 mM GABA solution to a CA1 pyramidal cell could last
>4 s (Wong and Watkins 1982), which is much longer than
the duration of the GABAD component of the GPSC.
The other two explanations involve a presynaptic mechanism. One
possibility is that less GABA is released in response to the second of
two closely timed stimuli and that the hypothetical
GABAD channels are extrasynaptic (Alger
and Nicoll 1982b
) or located at the periphery of synapses. In
this case, less GABA release would result in a failure of GABA to
diffuse to extrasynaptic sites and thus in a failure to activate the
GABA receptor channels which are more permeable to
HCO3
. The second explanation involving a presynaptic
mechanism is that separate classes of presynaptic interneurons (or
separate axon collaterals from the same presynaptic neurons) innervate exclusively either GABAA channels or
GABAD channels on pyramidal cells. A similar
segregation of receptors has been proposed for GABAA and GABAB receptors
(reviewed in Nurse and Lacaille 1997
) and for
GABAA,fast and GABAA,slow
receptors (Banks et al. 1998
). In this scenario, the
class of presynaptic neurons (or axon collaterals) innervating
GABAD channels would take longer to recover after a GPSC than the class innervating GABAA channels.
Location of the synaptic GABAD response
The results of focal application of PiTX to the somata of CA3
pyramidal cells demonstrated that both the GABAA
and GABAD components of the GPSC can be elicited
in the dendrites, confirming an earlier study that used current source
density analysis in the CA1 region (Lambert et al.
1991). Exposing the soma and proximal dendrites to PiTX reduced
the GABAA response while having little effect on
the GABAD response, suggesting that synaptic
GABAD responses are generated preferentially on
the distal dendrites of CA3 pyramidal cells. A dendritic origin of
GABAD responses in CA1 pyramidal cells has been
suggested by many earlier studies (e.g., Alger and Nicoll
1979
; Andersen et al. 1980
; Thalmann et
al. 1981
). Because the GABAD response to
exogenously applied GABA is more sensitive to PiTX than the
GABAA response (Alger and Nicoll
1982b
), PiTX presumably would have blocked the
GABAD component of the GPSC if it had originated
on the soma. Thus the results presented here suggest that synapses
containing GABAA channels exist on both the
somata and the dendrites of CA3 pyramidal cells; whereas synapses
containing the hypothetical GABAD channels exist
predominantly, or even exclusively, on the dendrites.
Hypothetical GABAD channel
These experiments disprove the Cl
accumulation hypothesis and suggest that the
GABAD response could be mediated by a different channel than the GABAA response. The data
illustrated in Fig. 8B demonstrate that the
GABAD response has slower rise and decay times
than the GABAA response; thus if the GPSC is
mediated by two different channels, the hypothetical
GABAD channel might be predicted to have slower
channel kinetics. Earlier ion permeability experiments (Perkins
and Wong 1996
) indicate that this hypothetical GABAD channel should have a greater permeability
to HCO3
than the GABAA channel.
In addition, the GABAD receptor channel would be
expected to have a lower affinity for GABA (Wong and Watkins
1982
) and perhaps a greater sensitivity to PiTX and bicuculline (Alger and Nicoll 1982b
). Furthermore
GABAD channels on pyramidal cells would be
expected to be localized to the dendrites. The fact that giant
GABAA-mediated responses (siGPSCs) can occur
independent of GABAD responses suggests, in
addition, that if two different channel types exist, they would be
segregated either to different synapses or to different subsynaptic
sites on hippocampal pyramidal cells. The hypothetical
GABAD channel would very likely not only mediate
the GABAD component of the GPSC in pyramidal
cells but also the HCO3
-dependent (Lamsa and
Kaila 1997
), GABA-mediated excitatory transmission among
interneurons (Michelson and Wong 1991
).
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ACKNOWLEDGMENTS |
---|
The author thanks S. Young, R.K.S. Wong, and R. Bianchi for helpful discussion and R. Margolis for help with statistical analysis.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-24517 to R.K.S. Wong.
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FOOTNOTES |
---|
Address for reprint requests: SUNY HSCB, Box 29, 450 Clarkson Ave., Brooklyn, NY 11203.
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 19 January 1999; accepted in final form 16 March 1999.
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REFERENCES |
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