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

Valeri Lopantsev1 and Philip A. Schwartzkroin1,2

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


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Lopantsev, Valeri and Philip A. Schwartzkroin. GABAA-Dependent Chloride Influx Modulates Reversal Potential of GABAB-Mediated IPSPs in Hippocampal Pyramidal Cells. J. Neurophysiol. 85: 2381-2387, 2001. Changes in intracellular chloride concentration, mediated by chloride influx through GABAA receptor-gated channels, may modulate GABAB receptor-mediated inhibitory postsynaptic potentials (GABAB IPSPs) via unknown mechanisms. Recording from CA3 pyramidal cells in hippocampal slices, we investigated the impact of chloride influx during GABAA receptor-mediated IPSPs (GABAA IPSPs) on the properties of GABAB IPSPs. At relatively positive membrane potentials (near -55 mV), mossy fiber-evoked GABAB IPSPs were reduced (compared with their magnitude at -60 mV) when preceded by GABAA receptor-mediated chloride influx. This effect was not associated with a correlated reduction in membrane permeability during the GABAB IPSP. The mossy fiber-evoked GABAB IPSP showed a positive shift in reversal potential (from -99 to -93 mV) when it was preceded by a GABAA IPSP evoked at cell membrane potential of -55 mV as compared with -60 mV. Similarly, when intracellular chloride concentration was raised via chloride diffusion from an intracellular microelectrode, there was a reduction of the pharmacologically isolated monosynaptic GABAB IPSP and a concurrent shift of GABAB IPSP reversal potential from -98 to -90 mV. We conclude that in hippocampal pyramidal cells, in which "resting" membrane potential is near action potential threshold, chloride influx via GABAA IPSPs shifts the reversal potential of subsequent GABAB receptor-mediated postsynaptic responses in a positive direction and reduces their magnitude.


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INTRODUCTION
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The inhibitory effect of gamma -aminobutyric acid (GABA) is mediated in forebrain primarily by two different types of postsynaptic receptors, termed GABAA and GABAB (for review see Sivilotti and Nistri 1991). Stimulation of afferents to hippocampal pyramidal cells results in short-lasting glutamatergic excitation followed by a fast inhibitory postsynaptic potential (IPSP) generated by chloride influx though GABAA receptor-gated ion channels (Ben-Ari et al. 1981; Knowles et al. 1984; Newberry and Nicoll 1984b). A longer latency long-lasting IPSP often follows the GABAA receptor-mediated IPSP and is mediated by postsynaptic GABAB receptors linked to potassium channels via intracellular G-proteins (Alger 1984; Andrade et al. 1986; Dutar and Nicoll 1988; Hablitz and Thalmann 1987; Newberry and Nicoll 1984a,b; Thalmann 1988). Functional properties of this GABAB-mediated potential are not completely understood. For instance, current evoked by the GABAB receptor agonist, baclofen, exhibits inward rectification; i.e., a reduction in current amplitude as the cell's membrane potential is depolarized. This feature has been attributed to the properties of the potassium channels coupled to GABAB receptors (Gähwiler and Brown 1985; Lüscher et al. 1997; Sodickson and Bean 1996). However, both inward rectification (Knowles et al. 1984; Newberry and Nicoll 1985) and linear voltage dependency (Hablitz and Thalmann 1987; Otis et al. 1993) have been reported for synaptically activated GABAB-mediated potentials/currents in hippocampal slices.

Artificial changes in intracellular chloride concentration may modulate G-protein-linked potassium permeability, including that activated by GABAB receptors (Lenz et al. 1997). Therefore recently we investigated possible effects of GABAA receptor-mediated chloride influx on GABAB-mediated IPSPs in hippocampal pyramidal cells (Lopantsev and Schwartzkroin 1999). We showed that reduction of GABAB-mediated IPSPs, at relatively positive membrane potentials (close to -55 mV), is induced by GABAA-mediated chloride influx; inward rectifying properties of the potassium channels did not contribute in this effect over the range of membrane potentials investigated in our study.

The mechanism underlying chloride-dependent depression of the GABAB-mediated IPSP is still unknown. Here we have further investigated how GABAA receptor-mediated chloride influx affects the properties of the GABAB-mediated IPSP. We tested two main hypotheses: 1) an increase in intracellular chloride concentration reduces membrane permeability associated with GABAB-mediated IPSPs, and 2) enhanced intracellular chloride influences the properties of the current through potassium channels coupled to GABAB receptors without changing membrane permeability. We found that the GABAA receptor-mediated, chloride-dependent, reduction of GABAB-mediated IPSPs is not associated with a decrease in membrane permeability, but is attributable to an alteration in GABAB-mediated IPSP reversal potential.


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Male Sprague-Dawley rats 1-1.5 mo old, were used in our experiments. After decapitation under halothane anesthesia, the brain was quickly removed into 2-4°C artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 3 KCl, 2 CaCl2, 2 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, and 10 dextrose, saturated with 95% O2-5% CO2 gas (pH 7.4). Transverse hippocampal slices (400 µm thick) were cut using a Vibroslicer (Campden Instruments, Sileby, UK) and then transferred to a holding chamber containing gas-saturated ACSF at room temperature (22-24°C) for at least 1 h before recording. In the recording chamber, slices were kept at 32°C at an interface between oxygenated ACSF and humidified gas. Rate of perfusion (0.8-1 ml/min) was kept constant throughout the experiment.

Intracellular recordings of CA3 pyramidal cells were obtained with glass microelectrodes filled with 3 M potassium acetate (resistance, 80-110 MOmega ) or with 3 M potassium chloride (resistance, 50-70 MOmega ). Pipette solutions were adjusted to pH 7.4 with KOH. Only neurons with a resting membrane potential and synaptic responses stable for at least 20 min were included in our analysis. Signals were recorded using an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) in bridge mode. Bridge balance was monitored throughout the experiment. Cell resting membrane potential (RMP) was verified after withdrawal of the microelectrode from the cell; action potential amplitude was calculated from RMP; and cell apparent input resistance was obtained from maximum voltage change in response to a hyperpolarizing current pulse (duration 200 ms, amplitude -0.4 nA). Data were digitized (Neuro-Corder, Neuro Data Instruments, New York, NY) and acquired using AxoScope software (Axon Instruments, Foster City, CA) on a pentium-based computer.

A bipolar stainless steel stimulating electrode was placed in the stratum lucidum to activate the mossy fibers. Stimuli (0.1 ms duration) were delivered at 0.1 Hz, at an intensity maximal for induction of GABA-mediated IPSPs. To elicit monosynaptic IPSPs, the stimulating electrode was placed close (<1 mm) to the site of recording, and glutamate receptor antagonists were added to the bathing medium. Amplitude of the GABAB-mediated IPSP was measured from the resting membrane potential, at a latency of 140 ms unless otherwise stated. Changes in membrane resistance during IPSPs were tested with brief hyperpolarizing current pulses (duration, 4-10 ms; amplitude, -0.2 to -0.4 nA), while apparent input resistance was tested at the peak of GABAB-mediated IPSP with longer hyperpolarizing pulses (duration, 70 ms; amplitude, -0.15 to -0.2 nA). The conductance changes (Delta G) associated with the GABAB-mediated IPSP were calculated according to the relation: Delta G = 1/RIPSP - 1/Rrest, where RIPSP is the membrane resistance measured during GABAB-mediated IPSP at 200 ms after mossy fiber stimulation and Rrest is the resting membrane resistance (Hablitz and Thalmann 1987). Reversal potentials were obtained from the regression lines plotted for every cell. Latency of pharmacologically isolated, monosynaptic GABAB-mediated IPSPs was measured between artifact of stimulation and the time point on response curve corresponding to the resting membrane potential. Measurements were expressed as means ± SE, and compared using Student's t-test. Data were considered significantly different if P < 0.05.

Bicuculline methiodide (BMI, 20 µM, Sigma, St. Louis, MO), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM, Research Biochemicals International, Natick, MA), (±)-2-amino-5-phosphonopentanoic acid (AP-5, 50 µM, Research Biochemicals International, Natick, MA), P-alpha 3-aminopropyl-P-diethoxymethyl-phosphinic acid (CGP35348, 700 µM, Ciba Geigy, Basel), and cesium chloride (1 mM) were applied via bath perfusion.


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Intracellular recordings were obtained from 68 neurons in the pyramidal layer of CA3 region under different pharmacological conditions using different intracellular electrolytes. The resting membrane potential of the recorded cells varied from -52 to -70 mV, action potential amplitude from 82 to 103 mV, and membrane input resistance from 37 to 77 MOmega .

Recordings from 10 of 68 neurons were made in normal ACSF with microelectrodes filled with 3 M potassium acetate. Mossy fiber stimulation induced an initial excitatory postsynaptic potential (often capped by an action potential) followed by a fast IPSP (termed "GABAA IPSP" since it was blocked by the GABAA receptor antagonist, BMI) and a subsequent slow IPSP (termed "GABAB IPSP" since it was blocked by the GABAB receptor antagonist, CGP35348). Postsynaptic responses were induced at different membrane potential levels, established by passing positive or negative steady current through the intracellular microelectrode (Fig. 1A). Dependency of the GABAB IPSPs on membrane potential was monotonic in the range -60 to -95 mV; these potentials had a maximal amplitude at -60 mV and were reduced as the membrane potential was hyperpolarized (Fig. 1B). However, at a membrane potential of -55 mV, the amplitude of the GABAB IPSP was smaller than that recorded at membrane potentials between -60 and -70 mV, and the monotonic relationship was lost.



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Fig. 1. Reduction of mossy fiber-evoked GABAB inhibitory postsynaptic potentials (IPSPs) in CA3 pyramidal cells at relatively positive membrane potentials was not associated with changes in membrane permeability. A: mossy fiber-evoked GABAB IPSPs () were reduced in amplitude as membrane potential was depolarized from -60 to -55 mV. Action potentials are truncated. GABAB IPSPs were measured at a latency of 140 ms after mossy fiber stimulation. B: GABAB IPSP amplitude plotted against membrane potential level. Note deviation from monotonic voltage dependency in GABAB IPSP amplitude at membrane potential of -55 mV. C: membrane resistance measured by passing pulses of negative current (70 ms duration) through the intracellular microelectrode at the peak of mossy fiber-evoked GABAB IPSPs; responses are determined for membrane potentials of -55 and -60 mV. Action potentials are truncated. D: membrane resistance values obtained at the peak of mossy fiber-evoked GABAB IPSPs, in responses evoked at membrane potentials of -55 and -60 mV. E: changes in membrane resistance measured by passing brief pulses of negative current (10 ms duration) through the intracellular microelectrode during mossy fiber-evoked GABA-mediated IPSPs, evoked at membrane potentials of -55 and -60 mV and plotted against time after stimulation. Resistance was different only at the time point of 50 ms after stimulation (asterisk: P < 0.05, Student's t-test) when GABAA and GABAB IPSPs overlap. F: changes in membrane resistance measured by passing brief pulses of negative current (4 ms duration) through the intracellular microelectrode during monosynaptic GABAA IPSPs isolated in the presence of glutamate receptor antagonists [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 20 µM and (±)2-amino-5-phosphonopentanoic acid (AP-5), 50 µM] and the GABAB receptor antagonist CGP35348 (700 µM), evoked at membrane potentials of -55 and -60 mV and plotted against time after stimulation. Resistance was different only at the time point of 50 ms after mossy fiber stimulation (asterisk: P < 0.05, Student's t-test).

Recently we have shown that reduction of the GABAB IPSP at membrane potentials close to -55 mV is due to chloride influx mediated by the preceding GABAA IPSP (Lopantsev and Schwartzkroin 1999). Decline in membrane permeability could be responsible for chloride-dependent reduction of the GABAB IPSP. Therefore membrane resistance was measured during GABAB IPSPs evoked at membrane potentials of -55 and -60 mV, i.e., at membrane potentials where the GABAB IPSP amplitude lost its monotonic voltage dependency. Pulses of negative current (duration, 70 ms; amplitude, -0.15 to -0.2 nA) were passed through the intracellular microelectrode at 130 ms after mossy fiber stimulation (Fig. 1C). Membrane resistance measured during GABAB IPSPs at these membrane potentials (35.4 ± 1.9 MOmega at -55 mV, mean ± SE, n = 7 and 37.7 ± 2.3 MOmega at -60 mV, n = 7) was not significantly different (Fig. 1D). The calculated conductance changes associated with GABAB IPSPs evoked at -55 mV (7.8 ± 1.2 nS, n = 7) and -60 mV (9.9 ± 1.1 nS, n = 7) were not significantly different. Also changes in membrane resistance were monitored during mossy fiber-evoked GABA-mediated IPSPs by passing brief pulses of negative current (duration, 10 ms; amplitude, -0.2 to -0.4 nA) through the recording electrode (not shown). Resistance measures were compared during GABA-mediated IPSPs evoked at membrane potentials of -55 and -60 mV in eight neurons. Membrane resistance was not different during GABAB IPSPs at membrane potentials of -55 and -60 mV, except at the time point of 50 ms after mossy fiber stimulation, when the GABAA IPSP contributes significantly to the hyperpolarization (asterisk in Fig. 1E). At this time point, membrane resistance was reduced by 54% during GABA-mediated IPSPs evoked at a membrane potential of -55 mV and was significantly lower than in the cell held at -60 mV (resistance reduced by 40%). Thus in spite of the reduction of GABAB IPSP amplitude at a membrane potential of -55 mV (as compared with -60 mV), corresponding membrane conductance was not affected at peak and throughout most of the time course of the GABAB IPSP.

Significant differences in membrane resistance during GABA-mediated IPSPs, evoked at the membrane potentials of -55 versus -60 mV, were detected only at 50 ms after mossy fiber stimulation, when GABAA and GABAB IPSPs overlap. We therefore compared the changes in membrane resistance during pharmacologically isolated monosynaptic GABAA IPSPs evoked at membrane potentials of -55 and -60 mV in 7 neurons (not shown). GABAA IPSPs were isolated in the presence of the N-methyl-D-aspartate (NMDA) receptor antagonist AP-5 (50 µM), the non-NMDA receptor antagonist CNQX (20 µM), and the GABAB receptor antagonist CGP35348 (700 µM). These potentials had a similar duration of 131.8 ± 8.4 ms (n = 7) at -55 mV and 132.5 ± 9.3 ms (n = 7) at -60 mV. Brief pulses of the negative current (duration, 4 ms; amplitude, -0.2 to -0.4 nA) were passed through the intracellular microelectrode, as above, to test changes in membrane resistance. Generally, resistance was not different during monosynaptic GABAA IPSPs evoked at membrane potentials of -55 and -60 mV and measured up to 500 ms after stimulation. However, at 50 ms after stimulation, membrane resistance decreased by 28% at a membrane potential of -55 mV and by only 15% (significantly less) at -60 mV (asterisk in Fig. 1F). Overall, reduction in membrane resistance during the pharmacologically isolated monosynaptic GABAA IPSP was smaller than the reduction recorded in response to mossy fiber stimulation (in normal ACSF). This difference may be explained by the likelihood that fewer inhibitory synapses were activated under the direct stimulation protocol (used to isolate monosynaptic GABA-mediated potentials in the presence of glutamate receptor antagonists) than under conditions of normal synaptic activation of inhibitory interneurons.

Since the experiments described above indicate that the chloride-mediated reduction of the GABAB IPSP at -55 mV was not associated with reduction in membrane permeability, we explored an alternative possibility: that GABAA receptor-mediated chloride influx affects the properties of the current through potassium channels coupled to GABAB receptors and induces a shift of the GABAB IPSP reversal potential. To evaluate this possibility, monosynaptic GABAB IPSPs were isolated pharmacologically in the presence of glutamate receptor antagonists (CNQX, 20 µM and AP-5, 50 µM) and the GABAA receptor antagonist BMI (20 µM), in eight neurons recorded with potassium acetate-filled microelectrodes and in seven neurons recorded with potassium chloride-filled microelectrodes. Cesium (Cs+ 1 mM) was added to the medium to block voltage-dependent potassium conductances, and particularly inward rectification in the hyperpolarizing direction, that might interfere with evaluation of the GABAB IPSP (Hablitz and Thalmann 1987). Extracellular cesium does not block outward GABAB-mediated currents (Jarolimek et al. 1994). Hyperpolarizing steady current was passed through the intracellular microelectrode, and the GABAB IPSP amplitude was measured (at 140 ms after stimulation) at different membrane potentials. Reversal potential of the monosynaptic GABAB IPSP was then calculated from the regression lines for each cell. IPSPs recorded with potassium acetate- and potassium chloride-filled microelectrodes had similar latencies (36.9 ± 1.9 ms and 37.1 ± 2.7 ms, respectively), but hyperpolarizing IPSPs recorded with potassium chloride-filled microelectrodes had smaller amplitudes (compare examples in Fig. 2, A and C). IPSPs recorded with potassium acetate-filled microelectrodes had a reversal potential of -98.1 ± 1.2 mV (n = 8), while potentials recorded with potassium chloride-filled microelectrodes reversed at significantly more positive level of -90.3 ± 2.0 mV (n = 7; Fig. 2, B and D). Similar values were obtained when the reversal potential of these IPSPs was measured at a latency of 200 ms after mossy fiber stimulation (-98.3 ± 1.1 mV with potassium acetate- and -90.0 ± 2.4 mV with potassium chloride-filled microelectrodes). These data show that elevation of intracellular chloride shifts the monosynaptic GABAB IPSP reversal potential in a positive direction---an effect that could cause the observed reduction of these potentials at membrane potentials more positive than GABAB IPSP reversal potential.



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Fig. 2. Increase in intracellular chloride concentration, due to diffusion from the intracellular microelectrode, shifted the reversal potential of the pharmacologically isolated monosynaptic GABAB IPSPs in a positive direction. A: monosynaptic GABAB IPSPs isolated in the presence of glutamate receptor antagonists (CNQX, 20 µM and AP-5, 50 µM) and the GABAA receptor antagonist bicuculline methiodide (BMI, 20 µM), recorded at different membrane potential levels with potassium acetate-filled microelectrode, in the presence of Cs+ (1 mM). B: voltage dependency of monosynaptic GABAB IPSPs recorded with potassium acetate-filled microelectrodes. The reversal potential was close to -98 mV. C: isolated monosynaptic GABAB IPSPs, recorded at different membrane potential levels with potassium chloride-filled microelectrode in the presence of Cs+ (1 mM). These potentials had smaller amplitude than those recorded with potassium acetate-filled microelectrodes. D: voltage dependency of monosynaptic GABAB IPSPs, recorded with potassium chloride-filled microelectrodes. The reversal potential was close to -90 mV.

To evaluate whether voltage-dependent GABAA receptor-mediated chloride influx is sufficient to affect the mossy fiber-induced GABAB IPSP reversal potential, we measured the GABAB IPSP reversal potential directly after GABAA IPSPs in normal ACSF (in the presence of 1 mM Cs+) with microelectrodes filled with potassium acetate. Slices were exposed to Cs+ only for a brief period of time (no longer than 10 min) after establishing stable intracellular recording in normal ACSF to avoid development of Cs+-induced epileptiform discharges (Janigro et al. 1997). Mossy fiber-evoked synaptic responses were paired with pulses of hyperpolarizing current (latency, 60 ms after stimulation; duration, 1200 ms) injected through the intracellular microelectrode. With this protocol, the GABAA IPSP was evoked at membrane potentials of -55 and -60 mV (determined by steady current control), while the following GABAB IPSP was examined at different levels of hyperpolarization (determined by the current pulses; Fig. 3, A and C). The amplitude of GABAB IPSPs was measured at 200 ms after mossy fiber stimulation to avoid interference with the charging curve evoked by the hyperpolarizing current pulse. Voltage responses evoked by current pulses alone (at membrane potentials of -55 and -60 mV) were measured at 140 and 1200 ms after onset of the current pulse (not shown); measurements at these time points were not different, indicating that at 140 ms the membrane had reached a steady-state level and maintained this level throughout the pulse. GABAB IPSPs (measured at a latency of 200 ms) superimposed on these current-induced hyperpolarizations had a reversal potential of -92.6 ± 2.1 mV (n = 7) when they were preceded by GABAA IPSPs evoked at -55 mV (Fig. 3B). GABAB IPSPs preceded by GABAA IPSPs at -60 mV had a significantly more negative reversal potential of -99.1 ± 1.4 mV (n = 7; Fig. 3D). Similar results were obtained when reversal potential of monosynaptic GABAB IPSPs was measured (at a latency of 200 ms) following monosynaptic GABAA IPSPs (not shown). In these latter experiments, GABAB IPSPs reversed at -93.0 ± 1.8 mV (n = 9) when preceding GABAA IPSPs were evoked at -55 mV, but had a significantly more negative reversal potential of -98.9 ± 1.4 mV (n = 9) when preceded by GABAA IPSPs evoked at -60 mV.



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Fig. 3. Reversal potential of GABAB IPSPs was more positive when preceded by GABAA IPSPs evoked at relatively positive membrane potentials. A: mossy fiber-evoked postsynaptic responses were paired with pulses of hyperpolarizing current (latency, 60 ms; duration, 1200 ms) such that GABAA IPSPs were evoked at a membrane potential of -55 mV, while subsequent GABAB IPSPs developed at different hyperpolarizing levels. GABAB IPSPs were measured at a latency of 200 ms after mossy fiber stimulation (140 ms after hyperpolarizing pulse onset). Responses were recorded in the presence of Cs+ (1 mM). Action potentials are truncated. B: GABAB IPSP amplitudes, plotted against membrane potential level, when preceded by GABAA IPSPs evoked at a membrane potential of -55 mV. The reversal potential was close to -93 mV. C: mossy fiber-evoked postsynaptic responses were paired with the pulses of hyperpolarizing current such that GABAA IPSPs were evoked at a membrane potential of -60 mV, while subsequent GABAB IPSPs developed at different hyperpolarizing levels. Responses were recorded in the presence of Cs+ (1 mM). Action potentials are truncated. D: GABAB IPSP amplitudes, plotted against membrane potential level, when preceded by GABAA IPSPs evoked at a membrane potential of -60 mV. The reversal potential was close to -99 mV. E: mossy fiber-evoked GABAA IPSPs were recorded at different membrane potential levels in the presence of the GABAB receptor antagonist CGP35348 (700 µM) and Cs+ (1 mM). Action potentials are truncated. F: GABAA IPSP duration, plotted against membrane potential level. Note that GABAA IPSP duration did not exceed 130 ms when measured at membrane potentials between -90 and -100 mV.

Mossy fiber-evoked GABAA IPSPs could persist beyond 140 (or 200) ms after mossy fiber stimulation, and thus interfere with our measurements of GABAB IPSP reversal potential. To evaluate this possibility, we measured the duration of the mossy fiber-evoked GABAA IPSPs in five neurons, at membrane potentials close to the GABAB IPSP reversal potential, in the presence of the GABAB receptor antagonist CGP35348 (700 µM) and Cs+ (1 mM; Fig. 3E). Our recordings showed that the duration of GABAA IPSPs, at membrane potentials between -90 and -100 mV, did not exceed 130 ms (Fig. 3F). Therefore voltage excursions associated with GABAA IPSPs do not interfere with measurements of the GABAB IPSP reversal potential.

Pharmacologically isolated monosynaptic GABAB IPSPs have a long latency (onset at about 37 ms) in response to stimulation that directly activates interneurons. At this time point during the mossy fiber response, membrane potential is governed primarily by the GABAA IPSP and is shifted in a negative direction from resting membrane potential. We estimated a membrane potential established by GABAA IPSPs at the time point corresponding to the onset of the GABAB IPSP so as to better describe its voltage dependency associated with aberrant behavior. Membrane potential was measured at 37 ms after mossy fiber stimulation, in responses evoked with the cell resting potential at -55 and -60 mV (Fig. 4A), i.e., at membrane potentials where the GABAB IPSP had different properties. The membrane potential values (at a latency of 37 ms, dictated by the GABAA IPSP) were -64.2 ± 0.4 mV (n = 10) and -67.4 ± 0.3 mV (n = 10), at -55 and -60 mV, respectively (Fig. 4B). Therefore onset of the GABAB IPSP evoked by mossy fiber stimulation, in a cell with resting membrane potential of -55 mV, occurred at a membrane potential close to -64 mV.



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Fig. 4. Mossy fiber-evoked GABAB IPSP is generated at a membrane potential more negative than resting membrane potential. A: membrane potential set by GABAA IPSP was 8-10 mV more negative than resting membrane potential of -60 and -55 mV at the onset of mossy fiber-evoked GABAB IPSP (37 ms after mossy fiber stimulation). B: membrane potential recorded at a latency of 37 ms after mossy fiber stimulation in responses evoked at membrane potentials of -55 and -60 mV.


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ABSTRACT
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In hippocampal pyramidal cells recorded in vitro, stimulus-evoked GABAB IPSPs are reduced when the cell is depolarized positive to -60 mV (Knowles et al. 1984; Newberry and Nicoll 1985). This behavior has been explained by inwardly rectifying properties of the potassium channels coupled to GABAB receptors (Gähwiler and Brown 1985; Lüscher et al. 1997; Sodickson and Bean 1996). However, this explanation is not entirely satisfying, since GABAB receptor-mediated potentials/currents pharmacologically isolated from GABAA receptor-mediated potentials/currents show monotonic voltage dependency (Hablitz and Thalmann 1987; Otis et al. 1993). Further, monosynaptic GABAB IPSPs demonstrate long latency---close to 37 ms in our experiments. At this time point, membrane potential is primarily governed by the preceding GABAA IPSP evoked by mossy fiber activation. Our calculations show that GABAB receptor-coupled potassium channels are activated when the GABAA IPSP sets the membrane potential at 7-9 mV below resting membrane potential for CA3 pyramidal cell. These results indicate that at the resting membrane potential at which GABAB IPSPs were reduced (i.e., at -55 mV), the onset of GABAB IPSPs occurred at approximately -64 mV. Rectification of GABAB receptor-mediated responses at this membrane potential has not been reported.

Recently we found that reduction of the GABAB IPSPs at relatively positive membrane potentials (close to -55 mV) is due to chloride influx associated with the preceding GABAA IPSP (Lopantsev and Schwartzkroin 1999). In the present study, we have found that membrane depolarization (from -60 to -55 mV) enhances the GABAA receptor-mediated conductance during mossy fiber-evoked IPSPs or during monosynaptically evoked GABAA IPSPs. This observation suggests that a more intensive chloride influx occurs during GABAA receptor-mediated events evoked at more positive membrane potentials.

Changes in intracellular chloride concentrations also could be mediated by a voltage-activated chloride conductance described in hippocampal pyramidal cells (Madison et al. 1986; Staley 1994). However, we may rule out its possible impact on GABAB IPSPs for a number of reasons. First, this conductivity operates at membrane potentials close to (or more negative than) resting level, while we described chloride sensitivity of the GABAB IPSP during membrane depolarization. Second, chloride-dependent modulation of GABAB IPSPs was completely blocked by application of a GABAA receptor antagonist (Lopantsev and Schwartzkroin 1999). Also, measurements of membrane resistance during pharmacologically isolated monosynaptic GABAA IPSPs have shown that at a membrane potential of -55 mV, enhanced chloride influx does not activate any additional long-lasting membrane permeability, which could overlap with GABAB IPSPs and affect their magnitude.

One possible explanation for chloride-dependent reduction of the GABAB IPSP is that higher intracellular chloride interacts with the intracellular G-protein signaling mechanism and/or the coupled potassium channel to reduce membrane permeability. However, we found that membrane permeability was not affected during reduced GABAB IPSPs at positive membrane potentials. Another possibility is that a rise in intracellular chloride modulates properties of the current through the potassium channels coupled to GABAB receptors. This latter explanation is consistent with the results of our experiments. First, we found that the reversal potential of the pharmacologically isolated monosynaptic GABAB IPSP shifted in a positive direction (from -98 to -90 mV) in cells loaded with chloride ions from the intracellular microelectrode. This finding is in line with previous studies showing that GABA- and baclofen-induced currents, recorded in cultured hippocampal neurons with low resistance chloride-filled intracellular microelectrodes, reversed at relatively positive membrane potentials (close to -72 mV) (Gähwiler and Brown 1985). Second, direct measurements of the mossy fiber-evoked GABAB IPSP revealed a more positive reversal potential when the preceding GABAA IPSP was evoked at -55 mV than at -60 mV.

Interestingly, GABAA receptor-mediated chloride influx still exists in the cell soma at membrane potentials between -60 and -70 mV. However, this level of chloride influx was not sufficient to affect GABAB IPSP reversal potential. Only at membrane potentials more positive than -60 mV was the GABAA receptor-mediated chloride influx sufficient to induce the positive shift in reversal potential of the GABAB IPSP---i.e., to alter the properties of the current through GABAB receptor-coupled potassium channels. This apparent paradox may be due to a spatial separation of chloride channels mediating GABAA IPSPs and the GABAB receptor-linked channels. Predominant dendritic location of GABAB receptors (Newberry and Nicoll 1985), where transmembrane distribution of chloride may be different from that in the cell soma (Jarolimek et al. 1999; Misgeld et al. 1986), makes it difficult to estimate exactly what direction and strength of transmembrane chloride flow is able to affect significantly the functioning of GABAB receptor-linked potassium channels.

It is interesting to note that spontaneous GABAA- but not GABAB-mediated events have been recorded in hippocampal slices in normal ACSF (Alger and Nicoll 1980; Collingridge et al. 1984; Miles and Wong 1984; Otis and Mody 1992). However, recent study has revealed mixed fast/slow IPSPs, evoked in CA1 pyramidal cell when a single presynaptic interneuron generated a burst of action potentials (Thomson and Destexhe 1999). Blockade of the fast GABAA-mediated IPSP (by bicuculline) was necessary to uncover the GABAB receptor-mediated response. This result suggests that the same interneuron can activate both GABAA and GABAB postsynaptic receptors, but that concomitant activation of these receptors may evoke a "pure" GABAA IPSP; i.e., the GABAB IPSP is suppressed. This scenario can be explained by the interaction demonstrated in our study, involving a GABAA-mediated chloride-dependent reduction of the GABAB receptor-mediated component.

Does this experimentally identified chloride modulation of GABAB IPSPs have any real physiological consequences? It is perhaps relevant that GABAB receptor-mediated currents evoked by activity of inhibitory interneurons contribute to the rhythmic activity (8-15 Hz) induced by a muscarinic receptor agonist in hippocampal slice culture (Scanziani 1999). Similar rhythmic activities in hippocampus in vivo (e.g., theta-rhythm) are also characterized by intense discharges of the inhibitory interneurons (Freund and Buzsaki 1996; Ylinen et al. 1995) that may provide a significant drive for evoking both GABAA- and GABAB receptor-mediated events in postsynaptic pyramidal cells. It seems likely that under these conditions, interaction between spontaneously occurring GABAA- and GABAB IPSPs may involve chloride-dependent modulation of GABAB IPSPs since resting membrane potential of pyramidal cells in vivo is close to values investigated in our experiments (-55 and -60 mV). We can speculate that GABAA receptor-mediated fluctuations in intracellular chloride concentration may affect GABAB IPSP reversal potential, and thus modulate strength of GABAB-mediated postsynaptic potentials or even mask their appearance.


    ACKNOWLEDGMENTS

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


    FOOTNOTES

Address for reprint requests: P. A. Schwartzkroin, Dept. of Neurological Surgery, University of Washington, Box 356470, Seattle, WA 98195-6470 (E-mail: pas{at}u.washington.edu).

Received 13 June 2000; accepted in final form 12 February 2001.


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ABSTRACT
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society