Clminus Accumulation Does Not Account for the Depolarizing Phase of the Synaptic GABA Response in Hippocampal Pyramidal Cells

Katherine L. Perkins

Department of Physiology and Pharmacology, State University of New York Health Science Center, Brooklyn, New York 11203


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Perkins, Katherine L.. Clminus 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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major inhibitory neurotransmitter in the cerebral cortex is gamma -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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 left-right-harpoons  H2CO3 left-right-harpoons  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 MOmega 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 (Delta V) was recorded in all cells and periodically retested during the experiment. The access resistance (Ra) was estimated using the equation Ra = Delta V/A, where A is the amplitude of the capacitive current. Only recordings with an Ra <=  12 MOmega 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 MOmega 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Outward inhibitory postsynaptic currents (IPSCs) occur during the inward GABAD component of the GABA-mediated postsynaptic current (GPSC) at potentials at which the GABAA component of the GPSC is outward. A: at a holding potential of -55 mV, the spontaneous GPSC was composed of an outward current (GABAA component) followed by an inward current (GABAD component), and the spontaneous IPSCs were outward. At -72 mV, both the GABAA and GABAD components of the spontaneous GPSC were inward and the spontaneous IPSCs were inward. B: same GPSC as that in A shown here at increased gain to emphasize that outward IPSCs occurred during the inward GABAD component of the GPSC (up-arrow ). Recordings were from a single CA3 pyramidal cell using the 102 mM HCO3-/pH 8.0 intracellular solution.

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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Fig. 2. GPSC evoked after a short interval (siGPSC) has no late inward component. siGPSC evoked ~7 s after a spontaneous GPSC (top ) had no late inward component; whereas the GPSC evoked 35 s after a spontaneous GPSC (bottom ) had a late inward component. Recordings were from a single CA3 pyramidal cell held at -49 mV and were made using the 102 mM HCO3-/pH 8.0 intracellular solution.

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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Fig. 3. siGPSC evoked during the GABAD component of a spontaneous GPSC is an outward current. Recording was from a CA3 pyramidal cell held at -48 mV. Stimulus artifact is visible as a vertical line. Intracellular solution was the 102 mM HCO3-/pH 8.0 solution.

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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Fig. 4. siGPSC can be larger than the preceding spontaneous GPSC and still have no late inward current component. A: siGPSC evoked after an ~10-s interval had a greater amplitude than the GABAA component of the preceding spontaneous GPSC and yet had no late inward current component. B: GPSC and siGPSC shown in A are shown here overlapped to emphasize that a greater net charge flowed during the siGPSC than during the outward component of the spontaneous GPSC. Recording was from a CA3 pyramidal cell held at -45 mV. Intracellular solution was the 102 mM HCO3-/pH 8.0 solution.

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 used---see METHODS).



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Fig. 5. Focal application of picrotoxin (PiTX) to the soma reveals that the siGPSC is not generated more somatically than the GABAA component of the GPSC. The siGPSCs were evoked 9-11 s after a spontaneous GPSC. In control solution (top ), the siGPSC had a greater outward amplitude than the GABAA component of the preceding spontaneous GPSC. While PiTX was being applied from the puffer pipette to the cell soma (middle ), the GABAA component of the GPSC and the siGPSC were reduced in amplitude, whereas the GABAD component was unaffected. PiTX application to the soma reduced the amplitude of the GABAA component of the spontaneous GPSC more than the amplitude of the siGPSC. Pressure to the puffer pipette then was turned off and the PiTX was washed out. Bottom: recording taken ~5 min after washout commenced shows partial recovery from PiTX. Puffer pipette was positioned very close to the recording electrode in the cell body layer. Stimulating electrode was ~0.5 mm from the recording electrode and straddled the CA3 pyramidal cell layer. Cell was held at -45 mV. Intracellular solution was the 102 mM HCO3-/pH 8.0 solution.

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.



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Fig. 6. siGPSC can be larger than the preceding GPSC evoked with an identical stimulus, and yet it has no late inward current component. A: siGPSC evoked after an ~10-s interval had a greater integral than the GABAA component of the preceding evoked GPSC and yet had no late inward current component. Stimulus artifacts were clipped for display. B: GPSC and siGPSC shown in A are shown here overlapped to emphasize that a greater net charge flowed during the siGPSC than during the outward component of the GPSC. Stimulus artifacts were clipped for display. C: illustration of the stimulus artifact and initial phase of the evoked GPSC (left), the siGPSC (middle), and the 2 overlapped (right). Note the time scale. Recording was from a CA3 pyramidal cell held at -43 mV. Intracellular solution was the 102 mM HCO3-/pH 8.0 solution.

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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Fig. 7. GABA-mediated postsynaptic current evoked during the GABAD component of an evoked GPSC is an outward current. The two stimuli were identical. Recording was from a CA3 pyramidal cell held at -46 mV. Stimulus artifacts, visible as vertical lines, were clipped for display. Intracellular solution was the 102 mM HCO3-/pH 8.0 solution.

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.



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Fig. 8. Hyperpolarization reveals that the siGPSC has no GABAD component. A, inset: reconstruction of current traces used to depict the 2 protocols. Inset, top: siGPSC was evoked 8 s after the spontaneous GPSC; bottom: GPSC was evoked 60 s after the spontaneous GPSC. In both protocols, the cell membrane potential was hyperpolarized from -50 to -84 mV 600 ms before the GPSC (or siGPSC) was evoked. Main part of the figure compares the siGPSC and evoked GPSC. Traces show the capacitive current and sustained inward current in response to the hyperpolarization followed by the stimulus artifact (up-arrow ) and the siGPSC and evoked GPSC overlapped. siGPSC and evoked GPSC were of comparable amplitudes, yet the siGPSC lasted ~730 ms whereas the GPSC evoked after a 60-s interval lasted ~1550 ms. The dotted trace is a siGPSC that was evoked at -50 mV in the same cell. Comparison of the traces reveals that the siGPSC at both -50 and -84 mV had a shorter time course than the GPSC evoked after 60 s, indicating that the GABA-mediated increase in conductance lasted a shorter time for the siGPSC than for the GPSC evoked after a longer interval. B: siGPSC in A was subtracted from the GPSC in A to reveal the time course of the GABAD component of the GPSC. The siGPSC (labeled A) and the subtracted trace (labeled D) were plotted together to facilitate comparison of the time courses of the GABAA and GABAD components of the GPSC. Subtracted trace begins above baseline due to the fact that the amplitude of the siGPSC was slightly greater than the amplitude of the GABAA component of the GPSC evoked after a longer interval. Recording was from a single CA3 pyramidal cell. Intracellular solution was the 49 mM HCO3-/pH 7.7 solution.

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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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
<IT>E</IT><SUB><IT>rev</IT></SUB><IT>=26.2 mV×ln </IT>[(<IT>5</IT>[<IT>Cl</IT><SUP><IT>−</IT></SUP>]<SUB><IT>i</IT></SUB><IT>+</IT>[<IT>HCO</IT><SUP><IT>−</IT></SUP><SUB><IT>3</IT></SUB>]<SUB><IT>i</IT></SUB>)<IT>/</IT>(<IT>5</IT>[<IT>Cl</IT><SUP><IT>−</IT></SUP>]<SUB><IT>e</IT></SUB><IT>+</IT>[<IT>HCO</IT><SUP><IT>−</IT></SUP><SUB><IT>3</IT></SUB>]<SUB><IT>e</IT></SUB>)]
Note that the denominator stays constant because the extracellular solution is constant. The lower the [Cl-]i is, the more the value of [HCO3-]i dominates the numerator and the more effect changing [HCO3-]i has on Erev. For example, if compartment A has a [Cl-] of 5 mM and compartment D has a [Cl-] of 15 mM, at a [HCO3-]i of 19 mM, the reversal potential of the GABAA response would be -72 mV and the reversal potential of the GABAD response would be -52 mV. If then the [HCO3-]i was changed to 80 mM, the GABAA response would reverse at -49 mV and the GABAD response would reverse at -39 mV. Thus in this example, changing the [HCO3-]i from 19 to 80 mM caused a 23-mV change in the GABAA reversal potential and only a 13-mV change in the GABAD reversal potential.

A third hypothesis is that GABAA channels may be modified during a GABA response---perhaps 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).


    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.


    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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