Inhibitory Nature of Tiagabine-Augmented GABAA Receptor-Mediated Depolarizing Responses in Hippocampal Pyramidal Cells

Michael F. Jackson, Barbara Esplin, and Radan Capek

Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Jackson, Michael F., Barbara Esplin, and Radan Capek. Inhibitory nature of tiagabine-augmented GABAA receptor-mediated depolarizing responses in hippocampal pyramidal cells. Tiagabine is a potent GABA uptake inhibitor with demonstrated anticonvulsant activity. GABA uptake inhibitors are believed to produce their anticonvulsant effects by prolonging the postsynaptic actions of GABA, released during episodes of neuronal hyperexcitability. However, tiagabine has recently been reported to facilitate the depolarizing actions of GABA in the CNS of adult rats following the stimulation of inhibitory pathways at a frequency (100 Hz) intended to mimic interneuronal activation during epileptiform activity. In the present study, we performed extracellular and whole cell recordings from CA1 pyramidal neurons in rat hippocampal slices to examine the functional consequences of tiagabine-augmented GABA-mediated depolarizing responses. Orthodromic population spikes (PSs), elicited from the stratum radiatum, were inhibited following the activation of recurrent inhibitory pathways by antidromic conditioning stimulation of the alveus, which consisted of either a single stimulus or a train of stimuli delivered at high-frequency (100 Hz, 200 ms). The inhibition of orthodromic PSs produced by high-frequency conditioning stimulation (HFS), which was always of much greater strength and duration than that produced by a single conditioning stimulus, was greatly enhanced following the bath application of tiagabine (2-100 µM). Thus, in the presence of tiagabine (20 µM), orthodromic PSs, evoked 200 and 800 ms following HFS, were inhibited to 7.8 ± 2.6% (mean ± SE) and 34.4 ± 18.5% of their unconditioned amplitudes compared with only 35.4 ± 12.7% and 98.8 ± 12.4% in control. Whole cell recordings revealed that the bath application of tiagabine (20 µM) either caused the appearance or greatly enhanced the amplitude of GABA-mediated depolarizing responses (DR). Excitatory postsynaptic potentials (EPSPs) evoked from stratum radiatum at time points that coincided with the DR were inhibited to below the threshold for action-potential firing. Independently of the stimulus intensity with which they were evoked, the charge transferred to the soma by excitatory postsynaptic currents (EPSCs), elicited in the presence of tiagabine (20 µM) during the large (1,428 ± 331 pA) inward currents that underlie the DRs, was decreased on the average by 90.8 ± 1.7%. Such inhibition occurred despite the presence of the GABAB receptor antagonist, CGP 52 432 (10 µM), indicating that GABAB heteroreceptors, located on glutamatergic terminals, do not mediate the observed reduction in the amplitude of excitatory postsynaptic responses. The present results suggest that despite facilitating the induction of GABA-mediated depolarizations, tiagabine application may nevertheless increase the effectiveness of synaptic inhibition during the synchronous high-frequency activation of inhibitory interneurons by enhanced shunting.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

gamma -Aminobutyric acid (GABA)-mediated inhibition plays a critical role in the control of CNS excitability as demonstrated by the convulsions resulting from the administration, in both animals and humans, of compounds that decrease the efficacy of this neurotransmitter system (for review see Gale 1992). The development of pharmacological agents capable of potentiating GABAergic neurotransmission has therefore been an important strategy in the search for novel anticonvulsants. Such a mechanism is thought to underlie or contribute to the effectiveness of the barbiturates and benzodiazepines (Rogawski and Porter 1990), both allosteric modulators of GABAA receptor function, as well as to the more recently developed antiepileptic vigabatrin (Grant and Heel 1991), an inhibitor of GABA metabolism.

The therapeutic potential of GABA uptake inhibitors in the treatment of epilepsy has been recognized since the early demonstrations of the ability of small amino acids, such as nipecotic acid and guvacine, to enhance GABA-mediated responses evoked either synaptically (Dingledine and Korn 1985; Matthews et al. 1981) or by iontophoretic application of the neurotransmitter (Curtis et al. 1976; Dingledine and Korn 1985). However, being highly polar, these compounds do not readily cross the blood-brain barrier and therefore can only produce an anticonvulsant effect when administered intracerebroventricularly (Croucher et al. 1983; Frey et al. 1979). The addition of lipophilic anchors to these simple structures increases not only their ability to permeate the blood-brain barrier but also their affinity for the GABA transporter and thus allows them to exert an anticonvulsant effect following their systemic administration (Braestrup et al. 1990; Suzdak et al. 1992; Yunger et al. 1984).

We have previously reported that in hippocampal slices, bath application of two of these novel uptake blockers, SKF 89976A and SKF 100330A, results in a relatively selective and prolonged enhancement in the inhibition produced following the high-frequency stimulation (100 Hz, 200 ms) of recurrent inhibitory pathways (Capek and Esplin 1993). Such a frequency-dependent mechanism of action should result in a selective strengthening of synaptic inhibition during episodes of high-frequency activity and allow these compounds to control seizures while minimizing the occurrence of side effects normally associated with drugs that produce an indiscriminate enhancement of inhibition. Paradoxically, recent evidence has demonstrated that high-frequency activation of inhibitory pathways, similar to that used in our previous study, evokes large (5-20 mV) GABAA receptor-mediated depolarizing responses (DRs) capable of triggering a burst of action potentials (APs) in hippocampal pyramidal cells, suggesting that GABAergic mechanisms may contribute to the generation and/or propagation of seizure activity (Grover et al. 1993; Jackson et al. 1995; Staley et al. 1995). Furthermore, we have recently demonstrated that tiagabine, a novel GABA transport inhibitor with demonstrated anticonvulsant properties in animal models of epilepsy (Faingold et al. 1994) as well as in human partial epilepsy (Ben-Menachem 1995; Schachter 1995), greatly facilitates the depolarizing actions of GABA following the stimulation of hippocampal interneurons at high (100 Hz) frequency (Jackson et al. 1996).

We therefore decided to further investigate the mechanisms by which GABA-uptake blockers produce frequency-dependent enhancement of inhibition by studying the effects of tiagabine-augmented GABA-mediated depolarizations on excitatory responses evoked at various (100-1,600 ms) time intervals following the high-frequency stimulation of hippocampal recurrent inhibitory pathways.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Extracellular field and whole cell recordings were made from the CA1 region of hippocampal slices obtained as follows. Male Sprague-Dawley rats (100-200 g) were decapitated, their brains quickly removed and placed in cooled, oxygenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 124 NaCl, 3.3 KCl, 2.5 CaCl2, 2.4 MgSO4, 25.6 NaHCO3, 1.25 KH2PO4, and 10 glucose. Transverse slices (400-500 µm) were then prepared using either a McIlwain tissue chopper or vibratome and allowed to recover at room temperature for at least 1 h in oxygenated ACSF. For recordings, slices were transferred to a tissue chamber and maintained at 31°C at the interface between humidified 95% O2-5% CO2, and oxygenated ACSF perfused through the chamber at a flow rate of 1.5-3.5 ml/min.

The effects of tiagabine on GABA-mediated inhibition were assessed using the antidromic-orthodromic test of inhibition as previously described (Capek and Esplin 1993; Jackson et al. 1994). Briefly, field potentials were recorded with a 3 M NaCl-filled micropipette (5-15 MOmega ) located in the pyramidal cell layer of the CA1 region. Constant-current stimuli (100 µs) delivered through bipolar stimulating electrodes to the Schaffer collaterals of stratum radiatum elicited orthodromic population spikes (PSs). These were inhibited by preceding antidromic conditioning stimulation delivered to the alveus, which causes the activation of inhibitory interneurons and the subsequent release of GABA onto pyramidal cells. Conditioning alvear stimulation was by either a single stimulus or by a train of 20 stimuli delivered at a high (100 Hz) frequency. The delay between the conditioning stimulus and subsequent orthodromic test response is referred to as the interstimulus interval (ISI) and was in the range of 10-1,600 ms. Orthodromic stimulus intensity was adjusted to produce a half-maximal PS. At an ISI of 10 ms, antidromic stimulus intensity was increased until it produced a 70-90% reduction in the amplitude of the orthodromic PS. In this paradigm, inhibition is measured indirectly as a reduction in the amplitude of the test PS expressed as a percentage of the unconditioned PS. For each ISI, responses were obtained before and after drug treatment. Results were expressed as means ± SE, and the significance of the drug-induced change was evaluated using Student's t-test.

Tight seal (>3 GOmega ) whole cell recordings were obtained from CA1 pyramidal cells using patch pipettes pulled from borosilicate glass (WPI) with a Brown-Flaming micropipette puller (Sutter Instruments, P-80). Patch electrodes had a resistance of 2-4 MOmega when filled with an internal solution of the following composition (in mM): 140-150 KMeSO4 (or CsMeSO4 where indicated), 10 N-2-hydroxyethylpiperazine-N''2-ethanesulfonic acid (HEPES), and 2 MgCl2. The solution was buffered with KOH (or CsOH) to a pH of 7.2-7.3 and filtered through a 0.2-µm pore size filter (Nalgene). Total osmolality for all intracellular solutions ranged from 270 to 290 mOsm. Current-clamp recordings were made with an Axoclamp 2A amplifier (Axon Instruments) operated in bridge mode, whereas an Axopatch 200A (Axon Instruments) was used for voltage-clamp recordings. All signals were digitized at 18 kHz (Instrutech, VR-10A) and stored unfiltered on videotape for later retrieval and analysis using a PC-based acquisition and analysis system functionally similar to that described previously (Théorêt et al. 1984).

In all recordings, drugs used were dissolved in ACSF and superfused over slices. Tiagabine ((R)-N-[4,4-di-(3-methylthien-2-yl)but-3-enyl] nipecotic acid hydrochloride) and CGP 52 432 (3-[[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxy-methyl)) were generously supplied by Abbott Laboratories (Chicago, IL) and Novartis Pharma AG (Basel), respectively.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Bath application of tiagabine (20 µM) had no effect on the amplitude of the unconditioned PS (Fig. 1A) and produced no change in the inhibition resulting from a single antidromic conditioning stimulation at short ISIs (10-20 ms) (Fig. 1, A and B). At longer ISIs (40-160 ms), inhibition was, however, increased resulting in a further 34.6% reduction in PS amplitude at an ISI of 160 ms (Fig. 1B). The frequency dependence of drug action was tested by stimulating the recurrent inhibitory pathways with a train of stimuli delivered at 100 Hz. The inhibition produced by such high-frequency stimulation was greater at any ISI and lasted longer than that resulting from a single stimulus. Thus, although the PS had fully recovered by 160 ms following conditioning stimulation by a single pulse, with HFS it was inhibited to <35% of its original amplitude at a more delayed ISI of 200 ms and recovered from inhibition over the next several hundred milliseconds (Fig. 1B). When tiagabine was added to the perfusate, inhibition was increased to the extent that high-frequency antidromic conditioning stimulation could now produce a near complete inhibition of the population spike at an ISI of 800 ms (Fig. 1A). The recovery from inhibition was also greatly prolonged such that the PS recovered to its original amplitude over the course of several thousand milliseconds rather than the hundreds of milliseconds time needed in control (Fig. 1B). The effects of tiagabine on recurrent inhibition were concentration-dependent over a range of 2-100 µM.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Effects of tiagabine (20 µM) on recurrent GABA-mediated inhibition. A: bath-applied tiagabine had no effect on unconditioned orthodromic population spikes (PSs), nor on the recurrent inhibition produced by a single pulse. The recurrent inhibition produced by high-frequency stimulation (HFS; 100 Hz, 20 pulses) was greatly enhanced by tiagabine resulting in a complete inhibition of the PS elicited at 800 ms. B: summary of the effects of tiagabine (20 µM) on recurrent inhibition produced by stimulation with either a single pulse (- - - -) or with HFS (------). Inhibited PSs, expressed as a percentage of the unconditioned PS at various interstimulus intervals (ISI) are shown. Each point represents the mean of values obtained from 5 experiments. *P < 0.05; **P < 0.01 compared with controls.

Given its ability to increase the depolarizing actions of GABA released following the HFS of inhibitory interneurons (Jackson et al. 1996), tiagabine could easily have been expected to produce an increase in pyramidal cell excitability. However, consistent with our previous findings with the SKF series of GABA uptake blockers (Capek and Esplin 1993), bath application of tiagabine resulted in a large increase in the functional inhibition resulting from the HFS of recurrent inhibitory pathways. Using the same stimulation paradigm, whole cell recordings were made from CA1 pyramidal cells to investigate the basis for enhanced inhibition following the application of tiagabine. In the absence of preceding alvear HFS, excitatory postsynaptic potential-inhibitory postsynaptic potential (EPSP-IPSP) sequences were evoked following the single stimulation of Schaffer collaterals in stratum radiatum (Fig. 2, A1 and B1). The evoked IPSPs were comprised of both fast and slow hyperpolarizing components (fIPSPs and sIPSPs, respectively). As illustrated in the insets of Fig. 2, A1 and B1, a current intensity for Schaffer-collateral stimulation was chosen, which could reliably elicit unconditioned EPSPs capable of triggering the firing of a single AP. When evoked 400 ms following the high-frequency activation of recurrent inhibitory pathways, the resulting EPSPs could no longer trigger an AP (Fig. 2A2). This occurred in spite of the fact that the amplitude and duration of the EPSPs appeared enhanced due to a reduction in the amplitude of the overlapping fIPSP. Such a reduction most likely occurred as a result of a decrease in the driving force for Cl- following its intracellular accumulation during the sustained activation of postsynaptic GABAA receptors resulting from high-frequency alvear stimulation (McCarren and Alger 1985; Thompson and Gähwiler 1989). In contrast, when the interstimulus interval was 800 ms, preceding high-frequency alvear stimulation no longer prevented the firing of APs following orthodromic stimulation (Fig. 2B2). Consistent with previous reports of the effects of GABA uptake inhibitors on evoked IPSPs (Roepstorff and Lambert 1992; Thompson and Gähwiler 1992), bath application of tiagabine greatly prolonged the duration of IPSPs while producing little or no effect on their amplitude (Fig. 2, A1 and B1). Rather than producing a qualitatively similar change in the duration of hyperpolarization following HFS, tiagabine either caused the appearance or greatly increased the amplitude and duration of a GABA-mediated depolarization. The presence of the DR coincided with a prolonged period of inhibition as demonstrated by the suppression of AP firing and large reduction in the amplitude of EPSPs superimposed at various time points along the different phases of the DR (Fig. 2, A2 and B2). Thus, in control, inhibition of AP firing was observed in only 4 of 9 and 4 of 10 cells at ISIs of 200 and 400 ms, respectively. Following the addition of tiagabine to the superfusate, inhibition could now be seen in all cells at these same ISIs, with AP firing now observed in only 1 of 10 cells at 800 ms and in only 1 of 7 at 1,600 ms following HFS of the alveus (Table 1). It is interesting to note that, under control conditions, inhibition of cell firing was observed in only those cells from which a DR could be elicited. Furthermore, following the application of tiagabine, the change in the time course of the inhibition of EPSP-induced AP firing was similar to that of the inhibition of stratum radiatum evoked PSs (compare with Fig. 1).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2. Effects of the tiagabine-induced increase of depolarizing responses (DRs) on superimposed evoked excitatory postsynaptic potentials (EPSPs) in a typical CA1 pyramidal neuron. EPSP-inhibitory postsynaptic potential (IPSP) sequences were evoked by a single stimulation in stratum radiatum. Although tiagabine produced no effect on evoked EPSPs, an increase in the duration of IPSPs was observed after its application (A1 and B1). With HFS of the alveus, action-potential (AP) firing in response to evoked EPSPs was inhibited at 400 ms (A2 and inset), but not at 800 ms (B2 and inset). Application of tiagabine resulted in the appearance of DRs following HFS (A2 and B2). EPSPs, superimposed on the DRs, were of reduced amplitude, and the AP firing was inhibited at both 400 and 800 ms. In this and the subsequent figure the bars below or above traces indicate timing and duration of HFS in the alveus, whereas filled triangles show timing of stratum radiatum stimulation.


                              
View this table:
[in this window]
[in a new window]
 
Table 1. Summary of tiagabine effects on the inhibition of AP firing in response to Schaffer-collateral stimulation following HFS of recurrent-inhibitory pathways

The observed reduction in the amplitude of EPSPs following the HFS of recurrent inhibitory pathways could be explained by two possible mechanisms: 1) reduced activation of postsynaptic excitatory amino acid receptors due to a decrease in transmitter release caused by the activation of presynaptic GABAB heteroreceptors present at glutamatergic terminals, and 2) shunting of the dendritically evoked EPSP by the concurrently active conductance responsible for the DR. These possibilities were investigated in cells voltage clamped near the reversal potential for GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) and superfused with ACSF containing 20 µM tiagabine and 10 µM of the specific GABAB receptor antagonist CGP 52 432, a concentration that prevents the inhibition of glutamate release by GABA (Waldmeier et al. 1994). Under these conditions, relatively pure excitatory postsynaptic currents (EPSCs) could be elicited following the single stimulation of Schaffer collaterals. HFS of the alveus evoked large (1,428 ± 331 pA, n = 5) inward currents with a time course similar to that of the DRs recorded in current clamp and which reversed at a membrane potential of -51 mV (Fig. 3A), a value identical to the previously reported reversal potential of GABA-mediated DRs (Perkins and Wong 1996; Staley et al. 1995). As with the EPSPs, EPSCs evoked 800 ms following HFS of the alveus (Fig. 3B, conditioned) were of much reduced amplitude, this in spite of the presence of CGP 52 432, indicating a lack of involvement of presynaptic GABAB receptors in the reduction of excitatory responses. This effect was quantified by measuring the charge transferred to the soma during EPSCs (Staley and Mody 1992) that were superimposed on the large HFS-evoked inward currents (Fig. 3C). The inhibition produced following HFS of the alveus was independent of the stimulus intensity used to evoke the EPSCs and reduced the charge delivered to the soma by evoked EPSCs to only 9.2 ± 1.7% (n = 5) of unconditioned responses across all stimulus intensities used. In an attempt to improve the quality of the space clamp, and thus record a larger fraction of the distally generated EPSC, recordings were made using CsMeSO4-based electrode solutions. Under these recording conditions, the inhibitory influence of the HFS-evoked inward current on evoked EPSCs was still, nevertheless, observed. Thus, across an identical range of stimulus intensities, the mean charge transferred was reduced to only 12.7 ± 2.9% (n = 4) of unconditioned evoked EPSCs, a similar value to that which we observed in the absence of intracellular Cs+. For this reason, the data obtained under both recording conditions were pooled to generate the graph in Fig. 3C. In separate recordings, we determined that the membrane resistivity during the large GABAA receptor-mediated inward current, as estimated from a 10-mV, 200-ms hyperpolarizing step, was reduced to only 16.6 ± 1.2% (n = 2) of its resting value (data not shown).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3. Large proximally evoked inward currents underlying GABA-mediated depolarizations inhibit distally evoked excitatory postsynaptic currents (EPSCs). Recordings were made in the presence of 20 µM tiagabine and 10 µM CGP 52 432. A: reversal potential of GABAA receptor-mediated currents evoked by HFS in the alveus was determined from responses generated at membrane potentials of -30 to -70 mV. Dashed line indicates the time point at which the amplitude of evoked inwards was measured (1,100 ms following HFS). Amplitudes (means ± SE, n = 2) were plotted vs. membrane potential, and the data points were fitted with a straight line obtained by linear regression. B: influence of concurrently active inward GABAA receptor currents on EPSCs evoked from stratum radiatum was tested 800 ms after HFS. Traces show EPSCs evoked with (conditioned) or without (unconditioned) preceding HFS in the alveus. The region of the traces selected by the dashed box are shown to the right on an expanded time base. C: the charge carried by EPSCs evoked using a range of stimulus intensities (70-310 µA) was determined by measuring the time integral of recorded currents. The charge carried by EPSCs evoked 800 ms after HFS in the alveus was expressed as a percentage of the charge transferred during unconditioned responses (mean ± SE, n = 7-9) and plotted against the intensity of stratum radiatum stimulation used to evoke EPSCs.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the adult rat, biphasic GABAA receptor-mediated postsynaptic responses consisting of an initial hyperpolarization followed by a depolarization have been observed under a number of circumstances that have in common the prolonged activation of postsynaptic GABAA receptors. These include the following: 4-aminopyridine-induced epileptiform activity (Perkins and Wong 1996; Perreault and Avoli 1992; Traub et al. 1995), iontophoretic application of GABA (Alger and Nicoll 1982a; Andersen et al. 1980; Thalmann et al. 1981), evoked synaptic responses in the presence of barbiturates (Alger and Nicoll 1982b; Thalmann et al. 1981) or zinc (Xie and Smart 1991), and responses evoked by high-frequency stimulation of inhibitory interneurons (Grover et al. 1993; Jackson et al. 1995; Staley et al. 1995). In neurons from immature animals, GABA also produces depolarizing responses that appear to be due to the maintenance of a Cl- gradient that is positive to the cells resting membrane potential (RMP) (Cherubini et al. 1990; Mueller et al. 1984). In the adult, however, the DR appears to be mediated by bicarbonate (HCO3-) anions, which in addition to Cl- are known to permeate the GABAA ionophore (Kaila 1994). However, given the fact the Cl- reversal potential in CNS neurons is typically more negative than the RMP (with dorsal root ganglion and hippocampal granule cells being exceptions) and that GABAA receptors are approximately five times more permeable to Cl- anions than to HCO3- (Bormann et al. 1987), the inward flow of Cl- normally dominates postsynaptic currents leading to a hyperpolarization of the neuronal membrane. As mentioned above, DRs typically occur following the decay of an initial hyperpolarization of variable amplitude and duration. A recently proposed model (Staley et al. 1995) suggests that during the sustained activation of GABAA receptors the inward flow of Cl-, which underlies the initial hyperpolarization, causes a partial collapse of the gradient for this anion resulting in a diminished flow of Cl-. This allows a greater net flow of HCO3-, whose reversal potential is more positive than the RMP, leading to the generation of a depolarizing potential.

The amplitude and duration of GABA-mediated DRs are critically dependent on both the duration of HFS as well as on the stimulus intensity used (Jackson et al. 1995). Furthermore, they are typically much larger when evoked monosynaptically following the blockade of excitatory amino acid receptors with a combination of 6-cyano-7-nitroquinoxaline-2,3-dione and 3-((R)-carboxypiperazin-4-yl-propyl-1-phosphonic acid, which antagonizes alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate and N-methyl-d-aspartate receptors (unpublished observation). The activation of a greater number of inhibitory interneurons due to the necessity, under these conditions, of positioning the stimulating electrodes much closer to the cell from which monosynaptic GABAergic responses are to be recorded, is most likely responsible for this. Given the fact that the DRs are generated only after such strong, sustained activation of postsynaptic GABAA receptors, which is unlikely to occur during normal synaptic transmission, the exact physiological role of GABA-mediated depolarizations is unclear. Evidence has been presented suggesting that the transient transformation of the postsynaptic actions of GABA from mainly hyperpolarizing to depolarizing may contribute to synaptic plasticity by relieving the voltage-dependent Mg2+ block of NMDA receptors (Staley et al. 1995). Although such an interaction may occur in dendritic compartments in which both GABAA and NMDA receptors are activated simultaneously, our results suggest that a large proximal increase in input conductance resulting from the activation of postsynaptic GABAA receptors, such as would be expected to occur following the intense activation of inhibitory interneurons during epileptiform activity, results in a transient suppression in the ability of excitatory transmission to depolarize the soma following the activation of distal excitatory amino acid receptors.

Although the inhibition of EPSPs could have been due to the activation of GABAB heteroreceptors located at glutamatergic terminals, the suppression of EPSCs, which occurred despite the presence of the GABAB receptor blocker CGP 52 432, argues against the involvement of such a mechanism. The evidence presented here suggests that shunting inhibition of evoked EPSPs most likely underlies the much stronger inhibition of stratum radiatum-evoked population spikes resulting from the activation of recurrent inhibitory pathways with HFS compared with that produced following the activation with a single stimulus (Fig. 1). Shunting inhibition of excitatory responses can be defined as a reduction in the depolarizing effects of the EPSC due to a drop in membrane resistance. Because the magnitude of the EPSC is independent of the extrasynaptic membrane resistance, it would have been impossible for us to observe shunting inhibition had the entire dendritic membrane voltage been perfectly controlled by the somatically applied voltage clamp. However, due to the extended geometry of hippocampal pyramidal neurons, especially those of the CA1 region (Carnevale et al. 1997), space clamp of the distal dendritic compartments is incomplete. Following HFS of the recurrent inhibitory pathway, these distal compartments become even more electrotonically remote, due to the associated decrease in membrane resistivity and dendritic space constant. Thus less of the charge that crosses the subsynaptic membrane at the excitatory synapses is transferred to the soma. Changes in the charge transfer allow then to assess shunting inhibition even under voltage-clamp conditions.

For a conductance to effectively shunt the potential generated by the activation of another, it should be sufficiently large and generated more proximally (Koch et al. 1983; Qian and Sejnowski 1990; Vu and Krasne 1992). The conductance increase resulting from the activation of GABAA receptors following HFS stimulation was large, as demonstrated by the substantial drop in input resistance (to only 16.6% of rest) that was observed in our experiments. Furthermore, either directly or through the recurrent axon collaterals of pyramidal cells, the electrical stimulation of the alveus that we used will have caused the activation of interneurons predominantly located within the alveus and stratum oriens, and to a lesser extent, stratum pyramidale. Among the different types of interneurons responding to such stimulation, GABAergic basket and axo-axonic cells have been shown to preferentially make synaptic contacts with the soma, proximal dendrites, and initial segment of CA1 pyramidal cells. The electrical stimulation of these interneuronal populations from an electrode positioned in the alveus is therefore likely to have caused a proximal increase in conductance following the activation of GABAA receptors. Furthermore, our demonstration that the percent reduction in charge transferred to the soma was independent of the strength of the excitatory input is consistent with predictions of the consequences of proximal shunting inhibition on distally evoked EPSPs (Vu and Krasne 1992). The present findings are entirely in line with our previous results suggesting that GABA-mediated DRs generated near the soma and proximal dendrites are inhibitory in nature whereas those elicited more distally, following HFS in stratum radiatum, are more likely to initiate AP discharges (Jackson et al. 1997).

Due to their ability to directly trigger the firing of bursts of APs, DRs have also been implicated in the generation of epileptiform activity. This is further supported by experiments demonstrating a reduction in the duration of afterdischarges following the blockade of GABAA receptors by the specific antagonist bicuculline (Higashima et al. 1996; Traub et al. 1995). In light of this, the large increase in GABA-mediated depolarizations, observed following the application of tiagabine, would seem contrary to the expected actions of an anticonvulsant. However, strong inhibition was always observed in response to the HFS of recurrent inhibitory pathways in the presence of tiagabine (Fig. 1). Furthermore, our results suggest that during the synchronous, high-frequency activation of proximally located interneurons, tiagabine increases the GABAA receptor-mediated conductance to such a large extent that the ability of cells to fire APs in response to dendritically evoked EPSPs is suppressed, indicating that tiagabine-facilitated DRs can retain the inhibitory character typical of GABA action. Therefore, despite producing a facilitation of GABA-mediated depolarizations, the potentiation of GABA-mediated shunting inhibition following tiagabine administration may allow this drug to produce an anticonvulsant effect by suppressing the generation of burst discharges during epileptiform activity.


    ACKNOWLEDGMENTS

We thank A. Constantin for competent technical assistance and Dr. Yves De Koninck for helpful advice as well as for the use of recording equipment.

This work was supported by the Medical Research Council of Canada. M. Jackson was supported by a Fonds pour la Formation de Chercheurs et l'Aide à la Recherche studentship, and by a McGill Faculty of Medicine Internal Studentship.


    FOOTNOTES

Address for reprint requests: R. Capek, Dept. of Pharmacology and Therapeutics, McGill University, 3655 Drummond St., Montreal, Quebec H3G lY6, Canada.

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 9 June 1998; accepted in final form November 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society