Distinct GABAB Actions Via Synaptic and Extrasynaptic Receptors in Rat Hippocampus In Vitro

Tri M. Pham, Suzanne Nurse, and Jean-Claude Lacaille

Département de Physiologie, Centre de Recherche en Sciences Neurologiques, Université de Montréal, Montreal, Quebec H3C 3J7, Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Pham, Tri M., Suzanne Nurse, and Jean-Claude Lacaille. Distinct GABAB actions via synaptic and extrasynaptic receptors in rat hippocampus in vitro. J. Neurophysiol. 80: 297-308, 1998. Intracellular recordings were obtained from pyramidal cells to examine gamma -aminobutyric acid-B (GABAB)-mediated synaptic mechanisms in the CA1 region of rat hippocampal slices. To investigate if heterogeneous ionic mechanisms linked to GABAB receptors originate from distinct sets of inhibitory fibers, GABAB-mediated monosynaptic late inhibitory postsynaptic potentials (IPSPs) were elicited in the presence of antagonists of ionotropic glutamate and GABAA receptors and of an inhibitor of GABA uptake and were compared after direct stimulation of inhibitory fibers in three different CA1 layers: stratum oriens, radiatum, and lacunosum-moleculare. No significant differences were found in mean amplitude, rise time, or time to decay to half-amplitude of IPSPs evoked from the three layers. Mean equilibrium potential (Erev) of late IPSPs was similar for all groups and close to the equilibrium potential of K+. Bath application of the GABAB antagonist CGP55845A blocked all monosynaptic late IPSPs. During recordings with micropipettes containing guanosine-5'-O-(3-thiotriphosphate) (GTPgamma S), the mean amplitude of all GABAB IPSPs gradually was reduced. Bath application of Ba2+ completely eliminated monosynaptic late IPSPs evoked from any of the stimulation sites. Late IPSPs were blocked completely during Ba2+ applications that reduced the GABAB-mediated hyperpolarizations elicited by local application of exogenous GABA only by ~50%. These results indicate that heterogenous K+ conductances activated by GABAB receptors do not originate from separate sets of inhibitory fibers in these layers. To examine if synchronous release of GABA from a larger number of inhibitory fibers could activate heterogeneous GABAB mechanisms, giant GABAB IPSPs were induced by 4-aminopyridine (4-AP) in the presence of antagonists of ionotropic glutamate and GABAA receptors. The amplitude and time course 4-AP-induced late IPSPs were approximately double that of evoked monosynaptic late IPSPs, but their voltage sensitivity, Erev, and antagonism by the GABAB antagonist CGP55845A and intracellular GTPgamma S were similar. Ba2+ completely abolished 4-AP-induced late IPSPs, whereas responses elicited by exogenous GABA were only reduced by ~50% in the same cells. These results indicate that synchronous activation of large numbers of inhibitory fibers, as induced by 4-AP, may not activate heterogenous GABAB-mediated conductances. Similarly, Ba2+ almost completely blocked late inhibitory postsynaptic currents evoked by stimulus trains. Overall, our results show that exogenous GABA can activate heterogenous K+ conductances via GABAB receptors, but that GABA released synaptically, either by electrical stimulation or 4-AP application, can only activate K+ conductances homogeneously sensitive to Ba2+. Thus GABAB receptors located at synaptic and extrasynaptic sites on hippocampal pyramidal cells may be linked to distinct K+ conductances.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

gamma -aminobutyric acid (GABA) is the most prevalent inhibitory neurotransmitter in the brain (Nicoll et al. 1990; Sivilotti and Nistri 1991). In the hippocampus, GABA is released primarily from interneurons and interacts with two receptor subtypes on pyramidal cells: GABAA and GABAB (Thompson 1994). GABAA receptors are ionotropic receptors that are antagonized by bicuculline and picrotoxin and insensitive to baclofen. They mediate a well-characterized postsynaptic inhibition via an increase in Cl- conductance and provide most of the fast inhibitory signaling in the CNS (Nicoll et al. 1990; Sivilotti and Nistri 1991). GABAB receptors are metabotropic receptors at which baclofen is an agonist but which are not antagonized by bicuculline (Bowery 1993; Kaupmann et al. 1997). GABAB receptors mediate a slow type of postsynaptic inhibition via G-protein activation and an increase in K+ conductance (Dutar and Nicoll 1988a,b; Nicoll et al. 1990). GABAB receptors also are located presynaptically on GABA and glutamate fibers where they mediate inhibition of transmitter release (Nicoll et al. 1990; Thompson 1994). These presynaptic actions appear to involve both a negative modulation of Ca2+ currents (Dunlap 1981; Wu and Saggau 1997) and activation of K+ currents (Thompson and Gähwiler 1992). Overall, activation of GABAB receptors may result in either a decrease or an increase in pyramidal cell excitability depending on which receptors are being activated (i.e., postsynaptic vs. presynaptic on GABA or glutamate fibers).

Although baclofen and GABA similarly increase an inwardly rectifying K+ conductance that is blocked by Ba2+ and Cs+, several lines of evidence suggest the existence of some heterogeneity in GABAB receptor subtypes and their associated conductances in hippocampal neurons (Misgeld et al. 1995). First, GABAB receptor antagonists such as CGP35348, phaclofen, and 2-OH-saclofen antagonize less effectively postsynaptic responses to GABA than to baclofen, suggesting the presence of two subtypes of postsynaptic GABAB receptors (Jarolimek et al. 1994; Pham and Lacaille 1996a; Solis and Nicoll 1992). Second, K+ conductances mediating GABAB IPSPs induced by 4-aminopyridine (4-AP) are not sensitive to Cs+, in contrast to those activated by baclofen (Jarolimek et al. 1994). Also GABAB IPSPs evoked by glutamate activation of interneurons in stratum lacunosum-moleculare were not antagonized by Ba2+, in contrast to GABAB-mediated late IPSPs evoked by electrical stimulation (Williams and Lacaille 1992). In addition, exogenous GABA has been shown to activate Ba2+-sensitive and -insensitive K+ conductances, whereas baclofen only activated Ba2+-sensitive conductances (Pham and Lacaille 1996a). Finally, baclofen and GABA have been reported to open K+ channels that display outward rectification in cultured hippocampal neurons (Premkumar et al. 1990). Baclofen- and GABA-activated K+ conductances also have been found to be modulated differentially by muscarinic agonists (Müller and Misgeld 1989) and 4-AP (Inoue et al. 1985; Ogata et al. 1987), but these effects are controversial (Solis and Nicoll 1992). Heterogeneity of GABAB receptors or their associated K+ conductances also has been observed in other CNS neurons. In thalamocortical neurons, two different types of GABAB receptors, with low and high affinities for baclofen, mediate inhibition of Ca2+ currents (Guyon and Leresche 1995). Also in cultured premotor neurons, baclofen activates outwardly rectifying K+ channels that are insensitive to Ba2+ and Cs+ (Wagner and Dekin 1993). Finally, the recent cloning of GABAB receptors has revealed two alternatively spliced variants of GABAB receptors in the CNS (Kaupmann et al. 1997).

Because Ba2+ was shown to be an effective tool to discriminate between heterogeneous K+ conductances activated by exogenous GABA in CA1 pyramidal cells (Pham and Lacaille 1996a), the aim of the present work was to examine if synaptically released GABA could activate multiple K+ conductances in the CA1 region of the hippocampus. We also examined if specific sets of inhibitory fibers could activate such distinct K+ conductances by comparing the properties of GABAB IPSPs evoked by stimulation of distinct hippocampal layers: stratum (s.) oriens (OR), s. radiatum (RAD) and s. lacunosum-moleculare (L-M). Finally, we examined if synchronous release of GABA from a large number of inhibitory fibers was capable of activating these multiple K+ conductances by studying the properties of giant GABAB IPSPs induced by 4-AP as well as late inhibitory postsynaptic currents (IPSCs) evoked by stimulus trains. Our results indicate that synaptically released GABA only activates Ba2+-sensitive K+ conductances, whereas exogenous GABA can activate both Ba2+-sensitive and -insensitive K+ conductances. These results imply that synaptic and extrasynaptic GABAB receptors may be coupled to different K+ conductances. Preliminary reports of this work have been presented in abstract form (Lacaille and Pham 1996; Pham and Lacaille 1995, 1996b).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Conventional hippocampal slices (400-450 µm thick) were obtained as described previously (Lacaille and Williams 1990; Pham and Lacaille 1996a; Samulack et al. 1993). Briefly, male Sprague-Dawley rats (125-250 g) were anesthetized with ether and decapitated. The brain was removed quickly from the skull and placed in ice-cold, artificial cerebrospinal fluid (ACSF) composed of (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 dextrose, saturated with 95% O2-5% CO2. The hippocampus was dissected out, and transverse slices were cut using a McIlwain tissue chopper. Slices were placed in a gas/fluid interface chamber where their upper surface was exposed to a warm, humidified gas mixture (95% O2-5% CO2) and were maintained at 32 ± 0.5°C. They were perfused initially with normal ACSF and subsequently with PO4/SO4-free ACSF (NaH2PO4 and MgSO4 replaced with MgCl2) containing antagonists of GABAA receptors [50 µM bicuculline (BIC) and 50 µM picrotoxin (PIC)], of N-methyl-D-aspartate (NMDA) receptors (100 µM 2-amino-5-phosphonopentanoic acid, AP-5) and non-NMDA receptors (40 µM 6-cyano-7-nitro-quinoxaline-2,3-dione, CNQX) for the duration of the experiments. A surgical cut was placed between CA3 and CA1 fields to synaptically disconnect the two regions.

Intracellular recordings were obtained from CA1 pyramidal cells. Intracellular responses were recorded with an Axoclamp-2A amplifier (Axon Instruments) using glass microelectrodes filled with 4 M potassium acetate (KAc) and 0.01 M KCl (70-110 MOmega ). Signals were stored in digitized format on a VHS video cassette recording system (Neurocorder DR-886) and displayed on a digital oscilloscope (Gould 1604). Signals of interest were further digitized with a microcomputer equipped with a data acquisition board (Axon Instruments TL-1-125) and the commercial software pClamp or Axotape (Axon Instruments). Recordings were considered acceptable if resting membrane potential (Vm) was more than -55 mV, if action potential amplitude was >60 mV, cellular input resistance was >10 MOmega , and if Vm was stable without the injection of a steady hyperpolarizing current. Bridge balance was monitored continuously throughout the experiment and adjusted as necessary. In some experiments, recordings were made with microelectrodes containing 25 mM guanosine-5'-O-(3-thiotriphosphate) (GTPgamma S; RBI) dissolved in 4 M KAc. Resting membrane potential was measured after withdrawal from the cell.

GABAB-mediated monosynaptic late IPSPs were evoked in the presence of the GABA uptake blocker nipecotic acid (1 mM, Sigma) by direct electrical stimulation (0.05 ms, 10-300 µA) of inhibitory cells with a monopolar tungsten microelectrode placed in one of three dendrititic layers: OR, RAD, or L-M. The optimal stimulus strength was determined after a series of graded stimulations to obtain maximal IPSP amplitude, and IPSPs were characterized at Vm just below threshold. Spontaneous late IPSPs were induced by bath application of 100 µM 4-aminopyridine (4-AP) in the presence of GABAA, NMDA, and non-NMDA antagonists. The amplitude of late IPSPs was measured at their peak. Equilibrium potential (Erev) of IPSPs was obtained from a series of IPSPs with the membrane potential (Vm) set at different levels using intracellular current injection. Values for Erev were calculated from the linear regression of response amplitude versus membrane potential.

Whole cell recordings were obtained as described previously (Ouardouz and Lacaille 1997). Hippocampal slices (300 µm) were cut in cold (4°C) ACSF using a vibratome (Campden Instruments). Slices were transferred to a submerged holding chamber and maintained in ACSF at room temperature for >= 1 h. Before recording, a cut was made between CA1 and CA3 subfields, and a slice was placed in a recording chamber on an upright microscope (Zeiss Axioskop). The slice was maintained submerged and perfused at a rate of 3 ml/min with ACSF at room temperature. Patch pipettes (3.5-6 MOmega ) were filled with (in mM) 140 K-gluconate, 5 NaCl, 2 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.5 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 2 ATP-tris(hydroxymethyl)aminomethane (Tris), 0.4 GTP-Tris, pH adjusted to 7.2-7.3 with KOH. Whole cell voltage-clamp recordings were obtained using an Axopatch 1D amplifier (Axon Instruments) with low-pass filtering at 10 kHz (-3 dB), from CA1 pyramidal cells visually identified using differential interference contrast and infrared video microscopy (Cohu 6500). Mean resting membrane potential was -59.5 ± 1.8 mV and mean series resistance was 13.0 ± 2.5 MOmega (n = 6). GABAB currents were isolated by perfusing the slices with PO4/SO4-free ACSF containing 50 µM bicuculline, 50 µM picrotoxin, 40 µM CNQX, and 100 µM AP-5. To evoke monosynaptic late GABAB IPSCs, trains of stimuli (20 pulses, 100 Hz) were delivered to stratum radiatum using ultra-small concentric bipolar microelectrodes (Frederick Haer, 16-75-3). Late IPSCs were further low-pass filtered at 1 kHz using an eight-pole Bessel filter (Frequency Devices 900 L).

GABA (5 mM) was diluted in ACSF containing GABAA, NMDA, and non-NMDA antagonists and applied locally in stratum radiatum to dendrites of CA1 pyramidal cells by micropressure (drop diameter 50-100 µm). The K+ channel blocker Ba2+ (1 mM) and the GABAB receptor antagonist CGP55845A (1 µM, Ciba-Geigy) were bath applied. Action potential duration was monitored routinely during Ba2+ application to verify antagonist effects on K+ channels. Chemicals were obtained from Sigma unless otherwise noted.

For statistical analyses, a one-way analysis of variance (ANOVA) was used to test for differences between means of three groups (differences between stimulation sites). A one-way ANOVA for repeated measures was performed for assessing drug effects on three groups. A paired Student's t-test was used to compare means of single groups before and after treatment. Significance level was set at P < 0.05. Data are expressed as means ± SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Monosynaptic and 4-AP-induced late IPSPs were examined in 84 pyramidal cells. For these cells, the mean resting Vm was -61.0 ± (SE) 0.6 mV, input resistance was 36.9 ± 1 MOmega , and action potential amplitude was 87.1 ± 0.7 mV.

Properties of monosynaptic late IPSPs

To isolate monosynaptic GABAB-mediated IPSPs, electrical stimulation of inhibitory cells was applied in the presence of antagonists of GABAA, NMDA, and non-NMDA receptors and an inhibitor of GABA uptake (Davies et al. 1990; Isaacson et al. 1993). To examine if postsynaptic GABAB responses originating from different populations of inhibitory cells were similar, we compared the properties of monosynaptic late IPSPs evoked from three different strata: OR, RAD, and L-M. We found that responses evoked from these three sites were generally similar and no significant differences were found in either amplitude or time course between each group (Fig. 1). The overall mean amplitude, rise time, and time to decay to half-amplitude of monosynaptic late IPSPs were -4.5 ± 0.2 mV, 99.6 ± 7.1 ms, and 210.3 ± 21.3 ms, respectively (n = 38). Monosynaptic late IPSPs from each stimulation site appeared to result from outward K+ currents. There were no significant differences between the Erev of monosynaptic late IPSPs evoked from each site (Fig. 1), and the overall mean equilibrium potential was -92.3 ± 2.6 mV (n = 27). Response reversal usually was not seen for these monosynaptic late IPSPs in the range of membrane potentials tested (up to -100 mV).


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FIG. 1. Monosynaptic late inhibitory postsynaptic potentials (IPSPs) evoked by stimulation of different dendritic layers: stratum (s.) oriens, radiatum, or lacunosum-moleculare. A: intracellular recordings from 3 representative CA1 pyramidal cells showing monosynaptic late IPSPs obtained in the presence of 50 µM bicuculline (BIC), 50 µM picrotoxin (PIC), 100 µM 2-amino-5-phosphonopentanoic acid (AP-5), 40 µM 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), and 1 mM nipecotic acid (NIP). IPSPs evoked from the 3 different sites had similar amplitude, rise time, and recovery time. B: in another representative cell, the peak amplitude of monosynaptic late IPSPs (s. oriens stimulation) decreased at hyperpolarized membrane potentials and became null near -94 mV. C: summary histograms for all cells tested. Mean amplitude, risetime, time to decay to half-amplitude, and equilibrium potential were not significantly different between IPSPs evoked from the 3 layers.

To verify that monosynaptic late IPSPs evoked from the three dendritic sites were mediated by GABAB receptors, the GABAB antagonist CGP55845A (1 µM) was bath applied (Davies et al. 1993). All monosynaptic late IPSPs were antagonized by CGP55845A within 10 min of application. The mean IPSP amplitude was reduced by 105.5% for IPSPs evoked from SO (n = 5 cells), by 107.1% for IPSPs evoked from RAD (n = 5 cells), and by 106.9% for IPSPs evoked from L-M (n = 4 cells) (Fig. 2). The effects of CGP55845A were not reversible and IPSP amplitude did not recover after 30-90 min of washout of the antagonist.


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FIG. 2. Effects of a gamma -aminobutyric acid-B (GABAB) antagonist on monosynaptic late IPSPs. A: representative example of monosynaptic late IPSPs evoked by s. radiatum stimulation in control condition (top) and after 10 min in 1 µM CGP55845A (middle). Superimposed traces (bottom) show the complete block of IPSPs by CGP55845A. B: summary histogram for all cells tested (n = 14), showing similar antagonistic effects of CGP55845A on the amplitude of monosynaptic late IPSPs evoked by stimulation of s. oriens (or), radiatum (rad), or lacunosum-moleculare (l-m).

To examine the involvement of G proteins in monosynaptic late IPSPs, recordings were made with microelectrodes containing 25 mM GTPgamma S to irreversibly activate G proteins and block their subsequent activation by GABAB receptors (Andrade et al. 1986; Thalmann 1988). Gradual membrane potential hyperpolarization, presumably due to GTPgamma S activation of K+ conductances, was compensated by positive current injection. During recordings with microelectrodes containing GTPgamma S, the amplitude of monosynaptic late IPSPs evoked by stimulation of any of the three sites became smaller with time (Fig. 3). IPSP amplitude, compared at 15 and 60 min after cell impalement, was reduced by 92.6% for IPSPs evoked from SO (n = 3), by 91.7% for IPSPs evoked from RAD (n = 3), and by 95.5% for IPSPs evoked from L-M (n = 3; Fig. 3). In control experiments, without GTPgamma S in the recording pipettes, monosynaptic late IPSP amplitude remained stable throughout similar recording periods (Fig. 3, n = 2).


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FIG. 3. Effects of guanosine-5'-O-(3-thiotriphosphate) (GTPgamma S) on monosynaptic late IPSPs. A: representative monosynaptic late IPSPs evoked by stratum oriens stimulation recorded at 15 (top) and 60 min (middle) after cell impalement with an electrode containing 25 mM GTPgamma S. Superimposed traces (bottom) show that after 60 min of recording with the GTPgamma S-containing electrode, IPSP amplitude was blocked in this cell. B: in control recordings with electrodes containing potassium acetate (KAc), monosynaptic late IPSPs did not show such reduction in amplitude. C: summary histogram for all cells tested (n = 11) illustrating that monosynaptic late IPSPs evoked by stimulation of s. oriens (or), radiatum (rad), and lacunosum moleculare (l-m) were similarly antagonized by intracellular GTPgamma S but were not reduced during recordings with KAc-containing electrodes.

Effects of Ba2+ on monosynaptic late IPSPs and GABA responses

To test if heterogeneous K+ conductances contributed to monosynaptic late IPSPs, the effects of Ba2+ (1 mM) were examined. Complete elimination of monosynaptic late IPSPs was observed within 15-25 min of Ba2+ perfusion (Fig. 4). Monosynaptic late IPSPs, evoked by stimulation in any of the three layers, were similarly blocked, indicating that they were generated by potassium conductances that were uniformly sensitive to Ba2+ (n = 19). The only Ba2+-resistant component of synaptic responses consisted of a depolarizing component with faster rise time and decay (e.g., Fig. 4A) than monosynaptic late IPSPs. The nature of this depolarizing component was not characterized further.


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FIG. 4. Differential effects of Ba2+ on monosynaptic late IPSPs and GABAB responses. A: representative monosynaptic late IPSPs evoked by stratum lacunosum-moleculare stimulation in control (top), after 15 min in the presence of 1 mM Ba2+ (middle), and after a 60 min washout period (bottom). Monosynaptic IPSP (bullet ) was blocked completely and reversibly by Ba2+. The only residual response in Ba2+ was a depolarizing response (sount-west-arrow sount-west-arrow ) with faster onset, rise time, and decay than the late IPSP (bullet ). B: summary histogram for all cells tested (n = 19), illustrating that monosynaptic late IPSPs evoked by stimulation of s. oriens (or), radiatum (rad), and lacunosum-moleculare (l-m) were all antagonized by Ba2+ (*significant difference from control). Effects of Ba2+ were reversible for all these stimulation sites (**significant recovery from Ba2+). C: sample traces of GABA-induced hyperpolarizations in another cell. After 25 min of bath application of Ba2+ (1 mM), the amplitude of GABAB responses were reduced to 60% of control (middle). Response at the end of the GABA response in Ba2+ is a rebound action potential that is truncated. GABA-induced hyperpolarizations (bottom) partially recovered after washout of Ba2+. D: summary histograms for all cells tested (n = 7), illustrating that, in the same cells, monosynaptic late IPSPs were blocked completely by Ba2+, whereas GABA-induced hyperpolarizations were only partially, but significantly, reduced during the same period of application (*significantly different from control). Ba2+ effects on monosynaptic late IPSPs and GABAB responses were partially reversible (**significantly different from Ba2+).

Because no Ba2+-resistant component was found in monosynaptic late IPSPs, GABA was applied locally to verify that Ba2+-resistant GABA responses were present in these cells. As previously shown (Pham and Lacaille 1996a), local application of GABA to CA1 pyramidal cells elicited GABAB hyperpolarizing responses that were reduced in amplitude by ~50% in Ba2+ (Fig. 4C). In the same cells (n = 7), whereas GABAB responses were only partially blocked after 25 min of perfusion with Ba2+, monosynaptic late IPSPs were completely blocked (Fig. 4D). Therefore whereas monosynaptic late IPSPs were mediated by Ba2+-sensitive K+ conductances, GABAB responses, in the same cells, consisted of Ba2+-sensitive and -insensitive components (Pham and Lacaille 1996a).

To verify that Ba2+ effects on monosynaptic late IPSPs did not arise from presynaptic effects on transmitter release, control experiments were conducted on biphasic GABAA and GABAB monosynaptic IPSPs in the absence of the GABAA antagonists bicuculline and picrotoxin. Under these conditions, electrical stimulation of inhibitory cells and fibers (in the presence of AP-5 and CNQX) gave rise to biphasic IPSPs, consisting of an early GABAA and a late GABAB component (Fig. 5A). Bath application of 1 mM Ba2+ suppressed only the late GABAB component of monosynaptic IPSPs while the early GABAA component remained intact (Fig. 5, n = 2 cells). These results indicate that Ba2+ effects on monosynaptic late IPSPs did not result from effects on presynaptic Ca2+ processes that could have led to diminished GABA release.


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FIG. 5. Effects of Ba2+ on monosynaptic early and late IPSPs. A: sample trace of early (bullet ) and late (black-triangle) monosynaptic IPSPs, evoked by stimulation of s. lacunosum-moleculare, in the presence of N-methyl-D-aspartate (NMDA) and non-NMDA antagonists. B: bath application of 1 mM Ba2+ for 15 min suppressed the late but not the early component of monosynaptic IPSPs. Because Ba2+ did not block the early component of monosynaptic IPSPs, the disappearance of the late IPSP was not likely due to reduced GABA release.

Properties of 4-AP-induced late IPSPs

Because electrical stimulation of discrete CA1 layers did not elicit Ba2+-resistant monosynaptic late IPSPs, we next examined if a greater presynaptic release of GABA would activate Ba2+-resistant K+ conductances. 4-AP is a blocker of voltage-activated K+ channels; this causes rhythmic and synchronized bursting activity in GABAergic interneurons (Michelson and Wong 1994), increases GABA release from axon terminals (Buckle and Haas 1982), and induces giant spontaneous GABA IPSPs in hippocampal neurons (Jarolimek and Misgeld 1993; Jarolimek et al. 1994; Michelson and Wong 1994; Müller and Misgeld 1991; Perreault and Avoli 1989; Segal 1987). Thus 4-AP (100 µM) was bath-applied in the presence of GABAA, NMDA, and non-NMDA antagonists to induce giant spontaneous GABAB IPSPs. Within 5 min of application of 4-AP, spontaneously occurring slow IPSPs of large amplitude were recorded in all pyramidal cells (Fig. 6). These 4-AP-induced IPSPs appeared at an average frequency of 1.5 ± 0.2 events per min (0.025 ± 0.004 Hz; n = 20 cells). The mean amplitude, rise time, and time to decay to half-amplitude of 4-AP-induced IPSPs were -7.9 ± 0.4 mV, 233.7 ± 17.9 ms, and 588.6 ± 48.4 ms, respectively (n = 22 cells). 4-AP-induced IPSPs were reduced in amplitude at hyperpolarized membrane potentials (Fig. 6). The mean Erev of 4-AP-induced late IPSPs was -103.8 ± 3.7 mV (n = 8 cells). Response reversal was usually not seen for 4-AP-induced late IPSPs in the range of membrane potentials tested (up to -110 mV).


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FIG. 6. Properties of 4-aminopyridine (4-AP)-induced late IPSPs. A: representative example of 4-AP-induced late IPSPs (*) recorded at resting membrane potential in the presence of 50 µM BIC, 50 µM PIC, 100 µM AP-5, and 40 µM CNQX. B: example of 4-AP-induced late IPSPs recorded at different membrane potentials (indicated at left) in the same cell. Top: corresponds to the event marked 1 in A. IPSPs were reduced at hyperpolarized membrane potentials and became null near -86 mV for this cell. C: graph of peak response amplitude vs. membrane potential for traces shown in B. Linear regression gave an Erev of -87 mV for this cell.

To verify that 4-AP-induced late IPSPs were mediated by GABAB receptors, the GABAB antagonist CGP55845A (1 µM) was bath applied. 4-AP-induced late IPSPs diminished in amplitude usually within 15 min of application of CGP55845A and gradually disappeared. (Fig. 7, n = 3 cells). The block of IPSPs by CGP55845A was not reversible with 45-60 min of washout (n = 3 cells). These data demonstrate that the 4-AP-induced late IPSPs in our recording conditions were GABAB receptor mediated.


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FIG. 7. Effects of GABAB receptor antagonist on 4-AP-induced late IPSPs. A: traces showing 2 consecutive 4-AP-induced late IPSPs (left) in control. * Onset of IPSPs. Bar shows the portion of the trace with an IPSP, which is enlarged at right. B: in the same cell, after 7 min of bath application of 1 µM CGP55845A, 4-AP-induced late IPSPs were still present but their amplitude was diminished. C: after 15 min of CGP55845A perfusion, the 4-AP-induced late IPSPs completely disappeared. D: summary histogram of all cells tested (n = 3), showing the gradual block by CGP55845A of 4-AP-induced late IPSPs. IPSP amplitude was significantly reduced after 7 min (*) and completely blocked after 15 min.

To confirm the involvement of G proteins in the 4-AP-induced late IPSPs, recordings were made with electrodes containing 25 mM GTPgamma S. Irreversible activation of G-proteins by the nonhydrolyzable GTP analogue produced a gradual decrease in the amplitude of 4-AP-induced late IPSPs. IPSP amplitude was compared at 15, 30, and 60 min after cell impalement. After 30 min of recording, the mean amplitude of 4-AP-induced late IPSPs was reduced to 35.4 ± 5.9% compared with responses at 15 min. After 60 min of recording, the mean amplitude of IPSPs was further reduced to 6.9 ± 3.8% of initial responses (Fig. 8, n = 5 cells). In contrast, in control recordings with KAc-filled electrodes, 4-AP-induced late IPSPs showed no such reduction in amplitude after 60 min of recording (Fig. 8, n = 3 cells).


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FIG. 8. Effects of intracellular GTPgamma S on 4-AP-induced late IPSPs. A-D: representative examples of 4-AP-induced late IPSPs recorded at 15, 30, and 60 min after cell impalement with an electrode containing 25 mM GTPgamma S (A and B) or with 4 M KAc only (C and D). 4-AP-induced late IPSPs diminished in amplitude during recording with GTPgamma S-containing electrodes (A), but not during recordings with KAc-containing electrodes (C). Superimposed traces in B and D are enlarged portions of traces indicated by bars in A and C showing the gradual block of IPSPs by GTPgamma S (B) but not in control recordings (D). E: summary histogram for all cells tested, showing the significant decrease in amplitude of 4-AP-induced late IPSPs at 30 and 60 min after cell impalement relative to initial responses (at 15 min; n = 5 cells). No significant change in IPSP amplitude was seen during control recordings of similar duration with KAc (n = 3 cells).

Effects of Ba2+ on 4-AP-induced late IPSPs and on GABA responses

To examine if K+ conductances underlying 4-AP-induced late IPSPs were heterogeneous in terms of Ba2+ sensitivity, 1 mM Ba2+ was bath-applied. In the presence of Ba2+, 4-AP-induced late IPSPs gradually diminished in amplitude to a mean of 60.3 ± 11.0% of control after 15 min (Fig. 9, n = 9 cells). After 25 min in Ba2+, 4-AP-induced late IPSPs were blocked completely (Fig. 9, n = 8 cells). Mean amplitude of 4-AP-induced late IPSPs recovered to 67.1 ± 4.1% of control after 60 min of wash (Fig. 9, n = 8 cells). These data indicate that 4-AP-induced late IPSPs were mediated only by Ba2+-sensitive K+ conductances.


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FIG. 9. Differential effects of Ba2+ on 4-AP-induced late IPSPs and on GABAB responses elicited by GABA. A: in control conditions (top), large 4-AP-induced late IPSPs were present (*). Bath application of 1 mM Ba2+ gradually blocked these IPSPs (middle). After 15 min in Ba2+, 4-AP-induced late IPSPs were reduced in amplitude and after 25 min they were absent. 4-AP-induced late IPSPs reappeared after washout of Ba2+ (bottom). B: summary histogram of all cells tested (n = 9). Mean amplitude of 4-AP-induced late IPSPs was reduced significantly (*) after 15 min in Ba2+ and completely blocked after 25 min. Mean IPSP amplitude partially recovered after Ba2+ wash. C: in another cell, focal application of GABA to s. radiatum elicited GABAB responses (control). Fifteen and 30 min after 1 mM Ba2+ application, GABAB responses were only partially blocked and a residual response remained. C: summary histograms of all cells (n = 3) showing the partial block by Ba2+ of GABAB responses elicited by GABA and the full block of 4-AP-induced late IPSPs in the same cells (*significant difference from control).

To verify that Ba2+-resistant GABAB conductances were present in cells showing no Ba2+-resistant 4-AP-induced late IPSPs, the effects of Ba2+ were tested at the same time on GABAB responses elicited by GABA and on 4-AP-induced late IPSPs in the same cells. After 15 min in Ba2+, GABA responses were depressed to 62.1 ± 8.6% of control amplitude, whereas 4-AP-induced late IPSPs were reduced to 31.2 ± 4.7% of control amplitude (Fig. 9, n = 3 cells). After 30 min in Ba2+, GABA responses were further reduced to 43.1 ± 6.9% of control amplitude (Fig. 9), whereas 4-AP-induced late IPSPs completely disappeared. The 4-AP-induced late IPSPs recovered to 79.7 ± 3.1% of control amplitude after 60 min of washout, whereas GABA responses recovered to 56.9 ± 13.8% of control amplitude (n = 3 cells). These results demonstrate that Ba2+-resistant GABAB responses were elicited by GABA in cells showing no Ba2+-resistant component in 4-AP-induced late IPSPs.

Effects of Ba2+ on monosynaptic late IPSCs evoked by repetitive stimulation

To examine if Ba2+-resistant K+ conductances could be activated in conditions when GABA release was increased such that GABA could spill over to extrasynaptic sites (Isaacson et al. 1993), we tested the effects of Ba2+ on monosynaptic late IPSCs evoked by stimulus trains. Monosynaptic late IPSCs were evoked during whole cell voltage-clamp recordings of CA1 pyramidal cells by repetitive stimulation (20 pulses at 100 Hz), in ACSF containing CNQX, AP-5, BIC, and PIC. Late IPSCs were recorded as outward currents at holding potentials of -40 mV (Fig. 10) and were blocked by CGP55845A (data not shown). Bath application of 1 mM Ba2+ blocked these monosynaptic late IPSCs by ~90% (Fig. 10, n = 6 cells). These effects of Ba2+ were partially reversible on washout (n = 4 cells). These results suggest that synaptically released GABA does not activate GABAB receptors linked to Ba2+-resistant K+ conductances even when GABA release is increased by such stimulus trains.


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FIG. 10. Effects of Ba2+ on monosynaptic late IPSCs evoked by stimulus trains. A: representative examples of monosynaptic late IPSCs evoked by repetitive electrical stimulation (20 pulses at 100 Hz at time indicate by bar; stimulation artifacts have been truncated) during whole cell voltage-clamp recordings at a holding potential of -40 mV in artificial cerebrospinal fluid containing CNQX, AP-5, BIC, and PIC (top, control). After the addition of 1 mM Ba2+, train-evoked late IPSCs were reduced to 11% of control amplitude in this cell (middle). IPSCs recovered partially to 43% of control amplitude after washout of Ba2+ (bottom). B: summary histogram for all cells tested (n = 6), showing the near complete, and partially reversible, block by Ba2+ (filled bar) of monosynaptic late IPSCs evoked by stimulus trains (* mean amplitude is significantly different from control at P < 0.05 2-tailed test; ** significantly different from Ba2+ at P < 0.05 1-tailed test).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The major findings of the present study were that electrical stimulation of inhibitory fibers in various dendritic layers of CA1 pyramidal cells, or 4-AP-induced synchronous release of GABA from multiple inhibitory fibers, produced GABAB-mediated hyperpolarizations involving K+ conductances that were homogeneously sensitive to Ba2+. In contrast, exogenous GABA hyperpolarized CA1 pyramidal cells via the activation of GABAB receptors linked to heterogeneous K+ conductances differentially sensitive to Ba2+. These differences in responses evoked by exogenous and synaptically released GABA suggest the presence of GABAB receptors located at synaptic and extrasynaptic sites that are coupled to distinct K+ conductances in hippocampal CA1 pyramidal cells.

Monosynaptic activation of GABAB-mediated K+ conductances by electrical stimulation

Local application of GABA activates GABAB receptors linked to two types of K+ conductances that can be distinguished on the basis of their sensitivity to Ba2+ in CA1 pyramidal cells (Pham and Lacaille 1996a). In contrast, local application of baclofen was found to open K+ conductances homogeneously sensitive to Ba2+ (Gähwiler and Brown 1985; Newberry and Nicoll 1985; Pham and Lacaille 1996a). Thus exogenous GABA may activate two types of GABAB receptors linked to two types of K+ conductances (Pham and Lacaille 1996a). These results were consistent with previous findings that selective activation of interneurons in s. lacunosum-moleculare by local injection of glutamate produced GABAB-mediated hyperpolarizations in CA1 pyramidal cells that were resistant to Ba2+, whereas in the same cells, the polysynaptic late GABAB-mediated IPSPs were antagonized (Williams and Lacaille 1992). Thus release of endogenous GABA may be coupled to both Ba2+-sensitive and -insensitive K+ conductances and specific inhibitory fibers may be involved in the activation of these distinct K+ conductances.

The present results showed that similar GABAB-mediated monosynaptic late IPSPs were elicited by stimulation of inhibitory fibers in any dendritic layers of CA1 pyramidal cells: s. oriens, radiatum, or lacunosum-moleculare (Andrade et al. 1986; Dutar and Nicoll 1988a,b; Jarolimek et al. 1993; Pham and Lacaille 1996a; Thalmann 1988). In addition, these monosynaptic late IPSPs were completely blocked by Ba2+. Thus synaptic release of endogenous GABA arising from single electrical stimulation of inhibitory fibers in any dendritic layer of the CA1 region activated GABAB receptors coupled only to Ba2+-sensitive K+ conductances. The absence of a Ba2+-resistant component in monosynaptic late IPSPs was not due to an absence of Ba2+-resistant K+ conductances because the latter were elicited by exogenous GABA in the same cells. The block of monosynaptic late IPSPs could not be attributed to presynaptic actions of Ba2+ on GABA release because Ba2+ selectively suppressed the late component of biphasic monosynaptic IPSPs without blocking the early component (Knowles et al. 1984; Lambert et al. 1991; Williams and Lacaille 1992). Other presynaptic actions of Ba2+ that could have influenced GABA release were its blocking actions on K+ conductances, however, these would have led to increases in transmitter release. Previous studies of Ba2+ effects on GABAB-mediated IPSPs reported variable effects with partial to complete block of polysynaptic late IPSPs by bath application of 1-2 mM Ba2+ (Alger 1984; Knowles et al. 1984). However, monosynaptic late IPSPs in CA3 pyramidal cells evoked by s. lucidum stimulation were completely blocked by bath application of 300 µM Ba2+ (Xie and Smart 1993).

Giant synchronous GABAB IPSPs induced by 4-AP

The complete block of single-pulse evoked monosynaptic late IPSPs by Ba2+ suggested that perhaps more GABA needed to be released to activate GABAB receptors coupled to Ba2+-insensitive K+ conductances. Low concentrations of 4-AP are known to enhance transmitter release at excitatory and inhibitory synapses (Buckle and Haas 1982; Perreault and Avoli 1989; Thesleff 1980), to induce a synchronization of hippocampal inhibitory interneurons (Michelson and Wong 1994; Segal 1987), and to produce giant synchronous GABAB IPSPs (Jarolimek and Misgeld 1993; Perreault and Avoli 1989; Segal 1987). Bath application of 4-AP generated spontaneous late IPSPs with an amplitude and time course double that of monosynaptic late IPSPs, indicating that more GABA was being released (Jarolimek and Misgeld 1993; Jarolimek et al. 1993, 1994; Misgeld et al. 1992; Perreault and Avoli 1989). The equilibrium potential of 4-AP-induced late IPSPs was close to EK but more hyperpolarized (about -100 mV) than that of monosynaptic late IPSPs (~ -90 mV). Other investigators have observed similar Erev of 4-AP-induced late IPSPs (Jarolimek et al. 1993, 1994; Misgeld et al. 1992) and attributed them to the blocking actions of 4-AP on K+ conductances (Misgeld et al. 1992). However, in some of the present experiments, a depolarizing response partially overlapped with monosynaptic late IPSPs (Fig. 4A in Ba2+), and this component may have resulted in a shift of the Erev of monosynaptic IPSPs in a more depolarized direction. Despite the enhanced GABA release, 4-AP-induced late IPSPs were blocked completely and reversibly by Ba2+. Similarly, when synaptic release of GABA was increased by using 20 pulses stimulus trains (Isaacson et al. 1993), late IPSCs were blocked almost completely by Ba2+. Thus even under conditions of increased release induced either by 4-AP or by repetitive stimulation, synaptically released GABA activated only GABAB receptors coupled to Ba2+-sensitive K+ conductances.

Synaptic and extrasynaptic GABAB receptors

The absence of coupling of synaptically released GABA to Ba2+-insensitive K+ conductances is in contrast to the responses of pyramidal cells to exogenous GABA that are coupled to both Ba2+-sensitive and -insensitive K+ conductances (Pham and Lacaille 1996a). Overall, these results argue for the presence of GABAB receptors localized at synaptic and extrasynaptic sites and for a coupling of synaptic and extrasynaptic GABAB receptors to different K+ conductances. Thus synaptic GABAB receptors, activated by synaptically released GABA, appear to be homogeneously coupled to Ba2+-sensitive K+ conductances, whereas extrasynaptic GABAB receptors, activated by exogenous GABA, may be linked to Ba2+-insensitive K+ conductances. Extrasynaptic GABAB receptors also could be coupled to Ba2+-sensitive K+ conductances. Estimates of single channel conductance of K+ channels activated by synaptically released GABA and by exogenous agonists are also consistent with such a presence of synaptic and extrasynaptic GABAB receptors coupled to distinct K+ channels in hippocampal neurons. Using nonstationary variance analysis, synaptically released GABA has been reported to activate small conductance channels (~8 pS) (De Koninck and Mody 1997), whereas during single channel recordings exogenous GABA has been found to open large conductance K+ channels (~65 pS) (Premkumar and Gage 1994). Thus GABAB receptors or K+ channels activated by synaptically released GABA may be distinct from those stimulated by exogenous agonists (De Koninck and Mody 1997). Similarly, in hippocampal CA3 pyramidal neurons, synaptic and extrasynaptic GABAB receptors coupled to different K+ channels have been proposed to account for the different sensitivity of baclofen responses and of 4-AP-induced late IPSPs to the K+ channels blocker Cs+ (Jarolimek et al. 1994).

The present results appear to be inconsistent with reports of GABAB-mediated IPSPs elicited by glutamate stimulation of interneurons, which were not blocked by Ba2+ (Williams and Lacaille 1992), i.e., synaptically activated Ba2+-insensitive K+ conductances. Multiple factors may account for this apparent discrepancy. First, the differences in Ba2+ effects in the two studies may be due to the different methods of application of Ba2+. In the present study, bath application of Ba2+ ensured a homogeneous application to the slice, whereas in the previous study, local drop application was used. Thus in the latter case, the preferential block of electrically evoked, and not of glutamate-evoked, late IPSPs (Williams and Lacaille 1992) could have been due to a restricted regional block of K+ conductances. However, this possibility is difficult to reconcile with the observation of similar blocking effects by local drop application of a GABAB antagonist (2-OH-saclofen) on both electrically and glutamate-evoked late IPSPs in the same study (Williams and Lacaille 1992). Alternatively, these differences may be due to the different modes of stimulation. In the present study, single and 20 pulses of presynaptic stimulation, as well as 4-AP-induced synchronous activation, were used to elicit release of GABA, whereas glutamate stimulation of interneurons, which probably involved a more sustained presynaptic activation of inhibitory cells (Samulack et al. 1993), was used in the previous study (Williams and Lacaille 1992). Such sustained activation of interneurons with glutamate may have resulted in more GABA release than in the present study and possibly more spillover of transmitter to extrasynaptic sites. However, the almost complete block by Ba2+ of train-evoked late IPSCs would suggest that GABA release needs to exceed that evoked by 20 pulses stimulation for spillover to reach extrasynaptic GABAB receptors.

GABAB receptor heterogeneity

Bonnano and Raiteri (1993a,b) have proposed a classification for heterogeneous GABAB receptors, based primarily on GABAB inhibition of transmitter release in the CNS. In this classification, two main subtypes of GABAB receptors have been differentiated: GABAB1 receptors that are baclofen sensitive and GABAB2 receptors that are baclofen insensitive. We have shown previously that Ba2+-sensitive and -insensitive K+ conductances appear to be coupled to different GABAB receptors because the former are activated by baclofen and GABA but the latter only by GABA (Pham and Lacaille 1996a). Thus according to the classification of Bonnano and Raiteri (1993a,b), Ba2+-sensitive K+ conductances may be coupled to GABAB1 receptors and Ba2+-insensitive K+ conductances to GABAB2 receptors (Pham and Lacaille 1996a). The present results suggest that Ba2+-sensitive K+ conductances may be linked to synaptic GABAB1 receptors, whereas Ba2+-insensitive K+ conductances may be coupled to extrasynaptic GABAB2 receptors. The correspondence of this classification to the structurally different types of GABAB receptors identified recently (Kaupmann et al. 1997) remains to be determined.

Physiological implications

The presence of GABAB receptors localized at synaptic and extrasynaptic sites suggests that their activation may be differentially regulated. Receptors at synaptic sites may be activated readily by GABA released during single activation of inhibitory fibers (Dutar and Nicoll 1988a). In contrast, extrasynaptic receptors may only be activated when sufficient GABA is released to spill over to more remote sites. Activation of GABAB receptors by spillover of synaptically released GABA has been shown to occur in heterosynaptic depression of excitatory synaptic transmission induced by repetitive stimulation in the hippocampus (Isaacson et al. 1993). Although in this case, the spillover of GABA is to presynaptic receptors, a similar phenomenon also could take place at postsynaptic receptors. The situation may be analogous in part to the activation of metabotropic receptors at glutamate synapses where these receptors have been localized at perisynaptic membrane sites (Baude et al. 1993). Single-pulse stimulus, or low-frequency stimuli, may not release enough neurotransmitter to reach these perisynaptic receptors and may only activate receptors located at synaptic sites. In contrast, agonist application, or sustained higher frequency stimuli that release more transmitter, could activate these extrasynaptic receptors. Such a stimulus-dependent activation of extrasynaptic GABAB receptors would likely play a role during periods of intense presynaptic stimulation and thus may become relevant during conditions of hyperexcitability such as during epileptiform activity. Our results indicate, however, that these conditions would need to exceed those encountered during 4-AP-induced hyperactivity or after short trains of repetitive stimulation.

    ACKNOWLEDGEMENTS

  The authors thank Ciba-Geigy for the generous gift of CGP-55845A.

  This research was supported by a grant from the Medical Research Council of Canada to J.-C. Lacaille, who was a Senior Scholar of the Fonds de la Recherche en Santé du Québec (FRSQ), a member of the Research Group on the CNS [Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR)], and a member of a FCAR-Équipe de Recherche. T. M. Pham was supported by a studentship from the Savoy Foundation and S. Nurse by a postdoctoral fellowship from the FRSQ.

    FOOTNOTES

  Address for reprint requests: J.-C. Lacaille, Département de Physiologie, Faculté de Médecine, Université de Montréal, PO Box 6128, Station Centre-Ville, Montréal, Quebec H3C 3J7, Canada.

  Received 18 November 1997; accepted in final form 31 March 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society