GABAA-Dependent Chloride Influx Modulates GABAB-Mediated IPSPs in Hippocampal Pyramidal Cells

Valeri Lopantsev1 and Philip A. Schwartzkroin1,2

Departments of  1Neurological Surgery and  2Physiology and Biophysics, University of Washington, Seattle, Washington 98195-6470


    ABSTRACT
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ABSTRACT
INTRODUCTION
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DISCUSSION
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Lopantsev, Valeri and Philip A. Schwartzkroin. GABAA-Dependent Chloride Influx Modulates GABAB-Mediated IPSPs in Hippocampal Pyramidal Cells. J. Neurophysiol. 82: 1218-1223, 1999. The relationship between postsynaptic inhibitory responses [the fast GABAA-mediated inhibitory postsynaptic potential (IPSP) and the slow GABAB-mediated IPSP] were investigated in hippocampal CA3 pyramidal cells. Mossy fiber-evoked GABAB-mediated IPSPs were, paradoxically, of greater amplitude in cells with resting membrane potential of -62 mV (13.6 ± 0.5 mV; mean ± SE) as compared with cells with resting membrane potential of -54 mV (7.0 ± 0.8 mV). In addition, when a cell's membrane potential was artificially manipulated, GABAB-mediated IPSPs were reduced at relatively depolarized levels (-55 mV) and enhanced at relatively hyperpolarized potentials (at least -60 mV). In contrast, the preceding GABAA-mediated IPSPs were larger at the more positive membrane potentials and smaller as the cell was hyperpolarized. Similar voltage dependency was obtained when monosynaptic GABAA- and GABAB-mediated IPSPs were isolated in the presence of glutamatergic receptor antagonists. However, monosynaptic GABAB-mediated IPSPs isolated in the presence of glutamatergic and GABAA receptor antagonists were not reduced at the more positive membrane potentials, and were significantly larger in amplitude than GABAB-mediated IPSPs preceded by a monosynaptic GABAA-mediated IPSP. The amplitude of the isolated monosynaptic GABAB-mediated IPSPs recorded with potassium chloride-containing microelectrodes was significantly smaller than the comparable potential recorded with potassium acetate microelectrodes without chloride. We conclude that voltage-dependent chloride influx, via GABAA receptor-gated channels, modulates postsynaptic GABAB-mediated inhibition in hippocampal CA3 pyramidal cells.


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ABSTRACT
INTRODUCTION
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DISCUSSION
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Inhibition mediated through postsynaptic gamma -aminobutyric acid (GABA) receptors modulates neuronal excitability in the neocortex, hippocampus and others forebrain structures of the adult CNS. Under normal conditions, afferent activation of neurons in these regions induces short-lasting excitatory synaptic drive followed by two inhibitory postsynaptic potentials, mediated by GABAA and GABAB postsynaptic receptors. These fast and slow inhibitory postsynaptic potentials (IPSPs) are closely linked in time, but exhibit very different electrophysiological and pharmacological properties (for review, see Sivilotti and Nistri 1991). Chloride influx through GABAA receptor-gated ion channels leads to an initial fast hyperpolarizing potential, which can be reversed by intracellular chloride injection; this action can be blocked by the GABAA receptor antagonists bicuculline and picrotoxin (Ben-Ari et al. 1981; Knowles et al. 1984; Newberry and Nicoll 1984b). The metabotropic postsynaptic GABAB receptors are linked to an increase in potassium permeability via activation of intracellular G proteins; the consequent slow, late hyperpolarizing potential can be imitated by microapplications of the GABAB receptor agonist baclofen (Dutar and Nicoll 1988a,b; Gähwiler and Brown 1985; Newberry and Nicoll 1984a,b). The potassium current linked to GABAB receptor activation exhibits inward rectification, reflected in a reduction in current amplitude as the cell's membrane potential is moved in a positive direction, further from potassium equilibrium potential (Gähwiler and Brown 1985; Lüscher et al. 1997; Sodickson and Bean 1996, 1998).

Thus far, no clear evidence for the direct impact of GABAA on GABAB-mediated IPSPs has been documented. However, recent findings suggest that intracellular chloride may play an important role in the modulation of different G-protein-linked permeabilities, including that activated by GABAB receptors (Lenz et al. 1997). Because GABAA-mediated IPSPs precede GABAB-mediated IPSPs in orthodromically activated neurons, we decided to investigate possible effects of chloride influx (via GABAA receptor-gated channels) on GABAB receptor-mediated inhibition. The results of the present study show that GABAA receptor-mediated chloride influx, which depends sensitively on membrane potential, significantly affects GABAB receptor-mediated inhibition in hippocampal pyramidal cells and thus may participate in the phenomenon of inward rectification.


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Hippocampal slices were prepared from brains of 1- to 1.5-mo-old Sprague-Dawley male rats. Animals were decapitated under halothane anesthesia, and their brains were removed quickly into 2-4°C artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 3 KCl, 2 CaCl2, 2 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, and 10 dextrose, saturated with 95% O2-5% CO2 gas (pH 7.4). Horizontal hippocampal slices (400-µm thick) were cut using a Vibroslicer (Campden Instruments) and then transferred to a holding chamber containing ACSF at room temperature for >= 1 h before recording. In the recording chamber, slices were kept at 32°C at an interface between oxygenated ACSF and humidified gas. Rate of perfusion (0.8-1 ml/min) was kept constant throughout the experiment.

Intracellular recordings of CA3 pyramidal cells were performed with sharp glass microelectrodes (resistance = 70-100 MOmega ) filled with one of the following solutions: 4 M potassium acetate; 3 M potassium acetate and 0.2 M ethylene-glycol-bis(beta -aminoethyl ether)-N, N,N',N'-tetraacetic acid (EGTA); 3 M potassium acetate and 1 M potassium chloride. All solutions were adjusted (with KOH) to pH 7.4. Only neurons with a resting membrane potential and synaptic responses stable for >= 30 min were included in our analysis. Signals were recorded using an Axoclamp-2A amplifier (Axon Instruments) in bridge mode. Bridge balance was monitored throughout the experiment. Cell resting membrane potential (RMP) was measured after withdrawal of the microelectrode from the cell; action potential amplitude was calculated from RMP; and cell input resistance was obtained from maximum voltage change in response to a hyperpolarizing current pulse (-0.4 nA, 100 ms). Data were digitized (Neuro-Corder, Neuro Data Instruments) and acquired using AxoScope software (Axon Instruments) on a 486-based computer.

A stimulating bipolar stainless steel electrode was placed in the stratum lucidum to activate the mossy fibers. To elicit monosynaptic IPSPs, the stimulating electrode was placed close (<1 mm) to the site of recording and glutamate receptor antagonists were added to the bathing medium. Stimuli (0.1-ms duration) were delivered at 0.1 Hz, at an intensity maximal for induction of a slow GABAB-mediated IPSP. Amplitudes of the GABAA- and GABAB-mediated IPSP were measured from the RMP, at latencies of 15 and 140 ms, respectively. Measurements were expressed as means ± SE, and compared across experimental conditions using Student's t-test. Data were considered significantly different if P < 0.05.

Bicuculline methiodide (BMI, 20 µM, Sigma), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM, Research Biochemicals), 2-amino-5-phosphonovaleric acid (APV, 50 µM, Research Biochemicals), and P-(3-amino-propyl)-P-diethoxymethyl-phosphinic acid (CGP35348, 700 µM, Ciba Geigy) were applied via bath perfusion.


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Intracellular recordings were obtained from 42 neurons in the pyramidal layer of CA3 region with microelectrodes filled with 4 M potassium acetate. The resting membrane potential of the recorded cells varied from -51 to -72 mV (mean ± SE = 60.9 ± 0.9 mV, n = 42), action potential amplitude from 80 to 104 mV (93.2 ± 1.1 mV, n = 42), membrane input resistance from 32 to 74 MOmega (51.3 ± 2.1 MOmega , n = 42). In normal ACSF, mossy fiber stimulation typically induced a sequence of postsynaptic potentials that included a fast initial excitatory postsynaptic potential (EPSP) (often capped by an action potential), a fast IPSP (termed "GABAA IPSP" because it was blocked by the GABAA receptor antagonist, BMI) and a subsequent slow IPSP (termed "GABAB IPSP" because it was blocked by the GABAB receptor antagonist, CGP35348) (Fig. 1A). At RMP, shape and size of the GABAA and GABAB IPSPs varied across cells, related largely to the RMP value for a given cell (Fig. 1A). At relatively positive RMPs (i.e., close to -50 mV), neurons had a pronounced GABAA IPSP, but the subsequent GABAB IPSP was small and of short-duration (see example in Fig. 1A). Neurons with the RMP more than or equal to -60 mV demonstrated a low-amplitude GABAA IPSP, but a high-amplitude and long-lasting GABAB IPSP (Fig. 1A).



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Fig. 1. Mossy fiber stimulation-induced postsynaptic potentials in CA3 pyramidal cell. A: postsynaptic potentials, recorded in 3 different cells with different resting membrane potentials (RMP), consisted of an initial fast excitatory postsynaptic potential (EPSP) or EPSP-action potential (Down-arrow ), fast GABAA-mediated inhibitory PSP (IPSP) (black-triangle) and slow GABAB-mediated IPSP (). Note that both the amplitude and duration of the GABAB-mediated IPSPs covaried with the RMP value (and the preceding GABAA-mediated IPSP amplitude). Action potentials are truncated. B: histogram showing the amplitude of the GABAA (black-triangle)-and GABAB ()-mediated IPSPs recorded in cells with RMP = -54 and -62 mV. * P < 0.05, Student's t-test.

The amplitudes of the GABAA and GABAB IPSPs were compared in two groups of cells with RMP = -54 and -62 mV (Fig. 1B). The mean amplitude of the GABAA IPSPs was significantly larger at -54 mV (11.3 ± 0.3 mV, n = 4) than at -62 mV (6.6 ± 0.3 mV, n = 4). In contrast the mean amplitude of the GABAB was significantly smaller at -54 mV (7.0 ± 0.8 mV, n = 4) than at -62 mV (13.6 ± 0.3 mV, n = 4). In 18 neurons, positive or negative DC current was passed through the intracellular microelectrode, and postsynaptic responses were induced at the different membrane potential levels (Fig. 2A). The GABAA IPSPs had a monotonic linear dependence on membrane potential and usually reversed in polarity between -65 and -70 mV (Fig. 2B). Dependency of the GABAB IPSPs on membrane potential was monotonic in the range -60 to -95 mV; these potentials had maximal amplitude at -60 mV and were reduced as the membrane potential was hyperpolarized. However, this monotonic relationship was lost as the cell was depolarized from -60 mV. Indeed, in 15 of 18 recorded neurons at -55 mV (where the fast GABAA IPSPs were large), the GABAB IPSPs were smaller than at -60 mV (Fig. 2, A and B). In three remaining cells, GABAB IPSPs had an equal amplitude at -55 and -60 mV.



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Fig. 2. Voltage dependency of the mossy fiber-evoked GABAA- and GABAB-mediated IPSPs. A: GABAA (black-triangle)- and GABAB ()-mediated IPSPs were recorded at different membrane potential (MP) levels. While the GABAA response increased in amplitude as MP was manipulated from -65 to -55 mV (as expected, given relationship of MP to ECl-), the GABAB-mediated IPSP decrease in amplitude, even though driving force for potassium increased with cell depolarization. B: GABAA (black-triangle)-and GABAB ()-mediated IPSP amplitudes plotted against MP level. Note significant reduction in GABAB-mediated IPSP amplitude at MP = -55 mV.

The smaller amplitude of the GABAA IPSP at more negative membrane potentials is due to reduction in the driving force for chloride ion influx. Surprisingly, GABAB IPSPs, which are a result of increased potassium permeability and have a reversal potential close to -100 mV (Alger 1984; Hablitz and Thalmann 1987; Otis et al. 1993), had a smaller amplitude at relatively positive membrane potentials, where the driving force for potassium ion efflux should be maximal. Similar voltage-dependent reduction in the GABAB IPSP amplitude has been reported previously (Knowles et al. 1984; Newberry and Nicoll 1985).

We did not investigate postsynaptic responses at membrane potentials more positive than -55 mV because afterhyperpolarizations associated with spontaneous action potential firing occurred at more positive potentials and overlapped with small amplitude GABAB IPSPs. Furthermore, because the calcium-dependent potassium conductance activated by action potentials (Alger and Nicoll 1980b; Hotson and Prince 1980; Schwartzkroin and Stafstrom 1980) or glutamatergic excitatory synaptic transmission (Nicoll and Alger 1981) may reshape postsynaptic responses, we examined GABA-mediated postsynaptic potentials in neurons loaded with EGTA diffused from the intracellular microelectrode (0.2 M). EGTA sufficiently buffered intracellular calcium because short pulses of the depolarizing current induced long-lasting burst discharges in these cells with no afterhyperpolarization (a potential attributable to a calcium-dependent potassium current) (data not shown). Under these conditions, both GABAA and GABAB IPSPs demonstrated voltage dependencies similar to those seen in experiments performed with potassium-acetate-filled microelectrodes (n = 5; data not shown).

Inhibitory effect of the GABAB IPSPs on depolarization-induced action potential generation was measured in six neurons. Pulses of depolarizing current were injected through the intracellular microelectrode, and current intensity was adjusted to threshold for spike discharge with the cell membrane potential maintained at either -55 or -60 mV. Current injection (latency = 140 ms) then was paired with a stimulus-evoked synaptic response, such that current-evoked spiking would start at the peak of the GABAB IPSP. Current amplitude was adjusted so that current-evoked action potential discharges induced at membrane potentials of -55 and -60 mV had the same latency (4.5 ± 0.8 and 4.7 ± 0.9 ms, respectively; n = 6; Fig. 3, A and B). When current injection was paired with the GABAB IPSP, spiking was delayed significantly longer when the cell membrane potential was -60 mV (142.3 ± 25.6, n = 6) compared with a membrane potential of -55 mV (29.7 ± 7.2 ms, n = 6; Fig. 3, A and B). This longer spike delay was consistent with the larger, long duration of the GABAB IPSP at -60 mV compared with -55 mV.



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Fig. 3. Voltage-dependent GABAB IPSP-mediated inhibition of the action potential discharge. A: GABAB-mediated IPSP inhibited current-evoked action potential discharge more efficiently at MP = -60 mV as compared with MP = -55 mV. Pulses of the depolarizing current were injected through intracellular microelectrode and current intensity was adjusted to threshold for spike discharge at MP = -55 and -60 mV (top traces, black-diamond ). Current injection was paired with a stimulus-evoked synaptic response (black-down-triangle ; trace overlapped with pure synaptic response). Action potentials are truncated. B: histogram showing the latency of current-evoked spiking (black-diamond ) and duration of the GABAB IPSP-mediated inhibition of the current-evoked action potential discharge (black-down-triangle ) at MP = -55 and -60 mV. * P < 0.05, Student's t-test.

Monosynaptic GABA-mediated IPSPs were investigated in seven neurons in the presence of the non-N-methyl-D-aspartate (NMDA) receptor antagonist CNQX (20 µM) and the NMDA receptor antagonist APV (50 µM). The stimulating electrode was placed close (<1 mm) to the site of recording, so that inhibitory interneurons located near the recorded pyramidal cell could be directly stimulated. Under these conditions, both GABAA and GABAB IPSPs behaved in a manner similar to that seen when the mossy fibers were stimulated in normal ACSF (Fig. 4, A and B). GABAA IPSPs reversed at a slightly more negative membrane potential (between -70 and -75 mV), probably due to blockade of the initial EPSP which overlaps slightly with the initial phase of the GABAA IPSP. GABAB IPSPs had a maximal amplitude at a membrane potential of -60 mV and still showed an amplitude reduction at more depolarized (e.g., -55 mV) levels (Fig. 4, A and B). When plotted against membrane potential, the GABAB IPSP showed a monotonic dependency as long as the preceding GABAA IPSP (amplitude measured at peak) was depolarizing or of relatively small hyperpolarizing amplitude (between membrane potentials of -95 and -60 mV; Fig. 4C). However, the GABAB IPSP was significantly reduced when the monosynaptic GABAA IPSP exceeded some threshold level (GABAA hyperpolarization of 13.5 ± 0.8 mV, at a membrane potential of -55 mV; n = 7).



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Fig. 4. Voltage dependency of the monosynaptic GABAA- and GABAB-mediated IPSPs isolated in the presence of the glutamate receptor antagonists [ 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 20 µM; 2-amino-5-phosphonovaleric acid (APV), 50 µM). A: monosynaptic GABAA (black-triangle)- and GABAB ()-mediated IPSPs recorded at different MP levels. Note the increase in GABAA- and reduction in GABAB-mediated IPSP amplitude at MP = -55 mV as compared with MP = -65 mV. B: GABAA (black-triangle)- and GABAB ()-mediated monosynaptic IPSPs amplitudes plotted against membrane potential level. These monosynaptically evoked IPSPs showed the same voltage dependency as the mossy fiber-evoked IPSPs (cf. Fig. 2). C: histogram showing the amplitude of monosynaptic GABAA-mediated IPSP (measured at the peak of amplitude) and GABAB-mediated IPSP plotted against MP. GABAB-mediated IPSP was reduced as preceding GABAA-mediated IPSP amplitude reached 13.5 ± 0.8 mV (n = 7) at MP = -55 mV.

Application of the GABAA receptor antagonist BMI (20 µM) concomitantly with CNQX (20 µM) and APV (50 µM) blocked fast monosynaptic IPSPs and isolated the monosynaptic GABAB IPSPs. Pharmacologically isolated GABAB IPSPs had a maximal amplitude at -55 mV and showed a monotonic change in amplitude as the membrane was hyperpolarized (n = 8; Fig. 5, A and B). The amplitude of the isolated monosynaptic GABAB IPSP at a membrane potential of -55 mV was 13.7 ± 0.6 mV (n = 5), significantly larger than the amplitude of the comparable GABAB IPSP preceded by a GABAA IPSP (8.6 ± 0.8 mV, n = 5) (i.e., recorded in the presence of only the glutamatergic receptor antagonists; Fig. 5C). No differences in GABAB IPSP amplitude were found between these conditions when cell membrane potential was varied between -60 and -95 mV. Bath application of the GABAB receptor antagonist CGP35348 (700 µM) blocked the isolated slow IPSP (n = 6; Fig. 5A).



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Fig. 5. Voltage dependency of the monosynaptic GABAB-mediated IPSP isolated in the presence of both glutamate receptor antagonists (CNQX, 20 µM; APV, 50 µM) and the GABAA receptor blocker [bicuculline methiodide (BMI), 20 µM]. A: isolated monosynaptic GABAB-mediated IPSPs recorded at different MP levels. Under these conditions, the GABAB-mediated IPSP was at maximal amplitude at MP = -55 mV. GABAB receptor antagonist CGP35348 (700 µM) blocked this IPSP. B: isolated monosynaptic GABAB-mediated IPSP () amplitude plotted against MP level. IPSP had a monotonic voltage dependency across the whole range of MPs investigated. C: amplitudes of the isolated monosynaptic GABAB-mediated IPSP (), and of the GABAB-mediated IPSP preceded by a monosynaptic GABAA-mediated IPSP (), plotted against MP level. * P < 0.05, Student's t-test.

In eight neurons, isolated monosynaptic GABAB IPSPs were recorded with microelectrodes filled with 3 M potassium acetate plus 1 M potassium chloride. IPSP amplitudes were measured 15 min after penetrating the cell, thus allowing chloride ions to diffuse from the micropipette into the cell. The average input resistance of cells recorded with chloride-containing electrodes was 60.4 ± 4.7 MOmega (comparable to 51.3 ± 2.1 MOmega in cells recorded with potassium acetate-filled microelectrodes). The amplitude of IPSPs recorded under these conditions, at a membrane potential of -55 mV was 7.7 ± 0.9 mV (n = 6), significantly smaller than the amplitude of isolated monosynaptic GABAB IPSPs recorded with microelectrodes containing no chloride (13.7 ± 0.6 mV, n = 5) (Fig. 6, A and B). However, this isolated GABAB potential was comparable in amplitude to that of GABAB IPSPs preceded by monosynaptic GABAA IPSPs (recorded in the presence of the glutamatergic receptor antagonists; 8.6 ± 0.8 mV, n = 5) and to the amplitude of the GABAB IPSPs recorded in normal ACSF (7.0 ± 0.8 mV, n = 4).



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Fig. 6. Monosynaptic GABAB-mediated IPSPs, isolated in the presence of the glutamate (CNQX, 20 µM; APV, 50 µM) and GABAA (BMI, 20 µM) receptor antagonists, were of smaller amplitude when intracellular chloride concentration was increased. A: isolated monosynaptic GABAB-mediated IPSPs from 2 different cells, 1 recorded with a potassium-acetate (K-acetate)-filled microelectrode and the other with a potassium-chloride (KCl)-filled microelectrode. Even after adjusting membrane potential to -55 mV the GABAB-mediated IPSP was smaller in the KCl-recorded cell. B: histogram showing the amplitude of the monosynaptic GABAB-mediated IPSP recorded (at the MP = -55 mV) with potassium acetate and potassium acetate-filled microelectrodes. * P < 0.05, Student's t-test.


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For both fast GABAA and slow GABAB IPSPs, the reversal potential is negative to the usual CA3 resting membrane potential of -60 mV. Thus depolarizing the cell should increase IPSP amplitudes because there is an increased driving force for both chloride (ECl- approx  -70 mV) and potassium (EK+ approx  -90 mV), responsible for the fast GABAA and slow GABAB IPSPs, respectively. Our results, however, show that mossy fiber-evoked GABAB IPSPs were reduced in amplitude when the cell's membrane potential is depolarized from -60 to -55 mV. During the same manipulations, the earlier GABAA IPSP increases in amplitude. This relationship was observed both as a function of resting membrane potential and as a function of experimentally manipulated membrane potential (through intracellular current injection). Monosynaptically evoked GABA IPSPs (i.e., in the presence of the glutamatergic receptor antagonists) showed the same relationship as afferent-evoked IPSPs. Such depolarization-dependent reduction of the GABAB IPSPs has been reported previously (Knowles et al. 1984; Newberry and Nicoll 1985), but the underlying basis for this unexpected relationship has not been explained.

Previous investigations (Gähwiler and Brown 1985; Lüscher et al. 1997; Sodickson and Bean 1996, 1998) identified an inwardly rectifying potassium current linked to the GABAB receptor (as well as to other G-protein-coupled receptors) but did not explore its chloride sensitivity. This current is likely to contribute to the reduction of the GABAB-mediated IPSPs at positive membrane potentials. In our experiments (also see Otis et al. 1993), pharmacologically isolated GABAB IPSPs did not show "anomalous" voltage dependence but rather exhibited a monotonic increase in amplitude as the cell was depolarized to -55 mV. We did not examine the chloride sensitivity of inwardly rectifying potassium current at more positive membrane potentials.

However, Lenz et al. (1997) showed that G-protein-linked permeabilities, including the potassium conductance associated with GABAB receptor-mediated inhibition, can be depressed by high intracellular chloride concentrations. This finding is consistent with the results of our experiments, in which the amplitude of the GABAB IPSP decreased under conditions in which the preceding GABAA chloride-dependent IPSP increased. Given these results, we postulate that GABAB IPSP amplitude is modulated by chloride influx associated with GABAA receptor activation. Consistent with this hypothesis is the observation that the GABAB IPSP amplitude was reduced significantly when chloride ions diffused into the CA3 cell from a penetrating microelectrode. We do not think that this effect can be attributed to increased intracellular levels of calcium (e.g., associated with calcium-dependent afterhyperpolarization) because EGTA injection did not affect the anomalous voltage sensitivity of the GABAB IPSP.

These results suggest that the driving force of the GABAA receptor-mediated chloride influx, which depends on membrane potential level, may affect GABAB-mediated inhibition. Because anomalous effects of membrane potential on GABAB-mediated inhibition were observed when membrane potential was manipulated between -55 and -60 mV, it seems likely that membrane potential fluctuations in a physiological range may lead to the changes in intracellular chloride concentrations that are sufficient to modulate GABAB-mediated inhibition. In fact, GABAB IPSP-mediated inhibition of action potential discharge was more efficient at a cell membrane potential of -60 mV than at -55 mV.

It is noteworthy, however, that high-amplitude GABAB IPSPs were recorded at membrane potentials between -60 and -70 mV---membrane potentials at which the GABAA IPSP was still hyperpolarizing and at which GABAA receptors still mediated chloride influx. These chloride currents apparently did not lead to an intracellular chloride concentration sufficient to interfere with GABAB-mediated IPSPs. Indeed, our measurements have shown that monosynaptic GABAB-mediated IPSPs were reduced significantly only when the preceding monosynaptic GABAA IPSP exceeded some threshold level. This result suggests that chloride influx (associated with high-amplitude GABAA IPSPs) must establish some minimal level of intracellular chloride concentration, perhaps at sites remote from the region of chloride influx, to affect GABAB-mediated inhibition. Lenz et al. (1997) suggest that intracellular chloride reduces GABAB-mediated inhibition by targeting either potassium channels or G proteins (to which the channels are tightly coupled) (Andrade et al. 1986). The need for a relatively high intracellular chloride concentration to produce effects on GABAB-mediated inhibition may be a function of the low sensitivity of these targets to chloride. Alternatively, potassium channel-linked G proteins may be spatially distant from chloride influx, thus requiring diffusion of chloride to a cell site "remote" from the GABAA receptor-gated channels.

That such a separation might exist is suggested by the finding that distinct types of inhibitory interneurons, with separate synaptic target sites, may be responsible for GABAA- and GABAB-mediated responses in the hippocampus (Nurse and Lacaille 1997). However, distal GABAB-mediated responses, even those in CA1 dendrites induced by activation of interneurons in stratum lacunosum-moleculare (Williams and Lacaille 1982), may still be subject to chloride-dependent modulation. These neurons receive a high level of spontaneous inhibitory synaptic input, mediated primarily by postsynaptic GABAA receptors (Alger and Nicoll 1980a; Collingridge et al. 1984). The chloride flux associated with this spontaneous GABAA-mediated input may help explain the "anomalous" behavior of GABAB-mediated responses induced by baclofen microapplications to CA1 cells at relatively positive membrane potentials (Newberry and Nicoll 1985a); these GABAB-mediated hyperpolarizations were reduced in amplitude, much as seen with afferent stimulation.

Chloride-dependent modulation of GABAB-mediated inhibition may play a significant role in some physiological and pathological phenomena. For instance, GABAB-mediated IPSPs have not been detected at early postnatal periods of development (Gaiarsa et al. 1995; McLean et al. 1996), when intracellular chloride concentrations are high (Owens et al. 1996). The absence of GABAB-mediated IPSPs at this age may be due to strong chloride-dependent inhibition of GABAB-mediated events rather than to the absence of GABAB receptors. Indeed, high levels of GABAB receptors may be seen at early postnatal times; binding experiments suggest that these receptors peak at postnatal day 3 and then decline into adulthood (Turgeon and Albin 1994). Also, it has been shown, that GABAB-mediated IPSPs may control the duration of interictal epileptiform discharges (deCurts et al. 1999; Karlsson et al. 1992) as well as the transitions from interictal to ictal-like activity in the hippocampus (Malouf et al. 1990; Scanziani et al. 1991). If GABAB-mediated inhibition is sensitive to intracellular chloride concentration, it is possible that intracellular chloride oscillations (mediated by GABAA receptors or other mechanisms) may modulate the strength of GABAB-mediated inhibition and thus help determine the pattern of epileptiform activity.


    ACKNOWLEDGMENTS

This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-18895.


    FOOTNOTES

Address for reprint requests: P. A. Schwartzkroin, University of Washington, Department of Neurological Surgery, Box 356470, Seattle, WA 98195.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 February 1999; accepted in final form 12 May 1999.


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DISCUSSION
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