Cell-Specific Alterations in Synaptic Properties of Hippocampal CA1 Interneurons After Kainate Treatment

F. Morin1, 2, C. Beaulieu2, and J.-C. Lacaille1

Centre de Recherche en Sciences Neurologiques et Départements de 1 Physiologie et de 2 Pathologie, Université de Montréal, Montréal, Québec H3C 3J7 Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Morin, F., C. Beaulieu, and J.-C. Lacaille. Cell-specific alterations in synaptic properties of hippocampal CA1 interneurons after kainate treatment. J. Neurophysiol. 80: 2836-2847, 1998. Hippocampal sclerosis and hyperexcitability are neuropathological features of human temporal lobe epilepsy that are reproduced in the kainic acid (KA) model of epilepsy in rats. To assess directly the role of inhibitory interneurons in the KA model, the membrane and synaptic properties of interneurons located in 1) stratum oriens near the alveus (O/A) and 2) at the border of stratum radiatum and stratum lacunosum-moleculare (LM), as well as those of pyramidal cells, were examined with whole cell recordings in slices of control and KA-lesioned rats. In current-clamp recordings, intrinsic cell properties such as action potential amplitude and duration, amplitude of fast and medium duration afterhyperpolarizations, membrane time constant, and input resistance were generally unchanged in all cell types after KA treatment. In voltage-clamp recordings, the amplitude and conductance of pharmacologically isolated excitatory postsynaptic currents (EPSCs) were significantly reduced in LM interneurons of KA-treated animals but were not significantly changed in O/A and pyramidal cells. The rise time of EPSCs was not significantly changed in any cell type after KA treatment. In contrast, the decay time constant of EPSCs was significantly faster in O/A interneurons of KA-treated rats but was unchanged in LM and pyramidal cells. The amplitude and conductance of pharmacologically isolated gamma -aminobutyric acid-A (GABAA) inhibitory postsynaptic currents (IPSCs) were not significantly changed in any cell type of KA-treated rats. The rise time and decay time constant of GABAA IPSCs were significantly faster in pyramidal cells of KA-treated rats but were not significantly changed in O/A and LM interneurons. These results suggest that complex alterations in synaptic currents occur in specific subpopulations of inhibitory interneurons in the CA1 region after KA lesions. A reduction of evoked excitatory drive onto inhibitory cells located at the border of stratum radiatum and stratum lacunosum-moleculare may contribute to disinhibition and polysynaptic epileptiform activity in the CA1 region. Compensatory changes, involving excitatory synaptic transmission on other interneuron subtypes and inhibitory synaptic transmission on pyramidal cells, may also take place and contribute to the residual, functional monosynaptic inhibition observed in principal cells after KA treatment.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Intracerebroventricular injection of kainic acid (KA) in rats produces a selective loss of hippocampal CA3 and hilar cells and renders the surviving CA1 pyramidal cells hyperexcitable (Ben-Ari 1985; Franck and Schwartzkroin 1985; Nadler 1981). Ammon's horn sclerosis and hippocampal hyperexcitability are also major features of human temporal lobe epilepsy (TLE) (Ben-Ari 1985; Lothman et al. 1991), and this makes the KA model useful to investigate the basic mechanisms of human TLE. In the KA model, the hyperexcitability of CA1 pyramidal cells may be due to multiple changes in excitatory and inhibitory synaptic pathways. First, hyperexcitable CA1 pyramidal cells of KA-treated rats show increased N-methyl-D-aspartate (NMDA) excitatory postsynaptic responses (Turner and Wheal 1991; Williams et al. 1993) that may arise in part from aberrant, newly formed, recurrent excitatory synapses among CA1 pyramidal cells (Meier and Dudek 1996; Nadler et al. 1980a,b; Perez et al. 1996). In addition, polysynaptic gamma -aminobutyric acid (GABA)-mediated inhibitory synaptic responses are diminished in CA1 pyramidal cells of KA-treated rats (Ashwood et al. 1986; Franck and Schwartzkroin 1985; Franck et al. 1988). However, direct electrical stimulation of inhibitory cells revealed unimpaired monosynaptic inhibition in hyperexcitable CA1 pyramidal cells (Williams et al. 1993), suggesting that the reduction in polysynaptic inhibition after KA treatment may be due to diminished excitatory drive on inhibitory interneurons. Such reduction of excitatory drive on interneurons is analogous to the dormant cell hypothesis proposed for other models of epilepsy (Bekenstein and Lothman 1993; Lothman et al. 1995; Sloviter 1991, 1994). Impairment in inhibition may also be due to a selective loss of inhibitory cells after KA because the number of parvalbumin- and somatostatin-containing interneurons in the CA1 region is reduced after KA treatment (Best et al. 1993, 1994). Despite their crucial role in generating inhibition (Freund and Buzsáki 1996; Lacaille et al. 1989), the physiological properties of inhibitory cells still remain ill defined in the KA model as well as in human TLE (Isokawa 1996) and other related experimental models (Buhl et al. 1996; Lothman et al. 1995; Mangan et al. 1995; Rice et al. 1996; Sloviter 1994).

GABA inhibition of CA1 pyramidal cells involves multiple subtypes of interneurons that can be differentiated on the basis of their soma location, postsynaptic targets, intrinsic membrane properties, and local circuit connectivity (Buhl et al. 1994; Freund and Buzsáki 1996; Houser 1991; Lacaille et al. 1989; Sik et al. 1995). This study was undertaken to assess directly if impairment in the physiological properties of inhibitory cells contributes to the generation of epileptiform activity in the CA1 region of the KA model of epilepsy (Franck et al. 1988; Nakajima et al. 1991; Williams et al. 1993). We have taken advantage of the laminar organization of the CA1 region and the known distribution of various subtypes of GABA interneurons (Morin et al. 1996) to examine, by using whole cell recordings, the membrane properties as well as the evoked excitatory and inhibitory postsynaptic currents (IPSCs) of inhibitory interneurons in hippocampal slices from KA-treated rats. We focused on interneurons located 1) in stratum oriens near the alveus (O/A) and 2) at the border of stratum radiatum and stratum lacunosum-moleculare (LM) because these populations of interneurons are differentially involved in feedback and feedforward inhibition, respectively (Blasco-Ibanez and Freund 1995; Lacaille and Schwartzkroin 1988a,b; Lacaille et al. 1987; Maccaferri and McBain 1995). Our results suggest that, in hyperexcitable slices of KA-treated animals, the membrane properties of inhibitory interneurons are generally unimpaired, the excitatory synaptic currents are selectively reduced in certain interneurons, and the inhibitory synaptic currents are not diminished in interneurons or pyramidal cells.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Kainate lesions

Procedures for kainate lesions were as previously described (Perez et al. 1996). Briefly, adult male Sprague Dawley rats (150-160 g, n = 74; Charles River) were anesthetized with a mixture of ketamine and xylazine (165 and 10 mg/kg im, respectively) after pretreatment with atropine (0.27 mg/kg ip) to prevent respiratory difficulties during anesthesia. Bilateral intracerebroventricular injections of KA (0.55 µg/µl over 30 min; in 0.9% saline, pH 7.3-7.4) were made with a 10-µl Hamilton syringe at the following stereotaxic coordinates: 0.6 mm posterior to bregma, 2.0 mm lateral to the midline, and 3.5 mm ventral to the dura. Wounds were treated with aerosol antibiotic (Neosporin) and closed with sutures. Animals were returned to their cages and given water and food ad libitum for a period of 2 wk.

Hippocampal slices

Hippocampal slices were obtained as described previously (Morin et al. 1996). Briefly, KA-treated rats (2 wk postlesion) and age-matched unoperated control rats (225-250 g; n = 40) were anesthetized with ether and decapitated. The brain was quickly removed from the skull and rinsed in artificial cerebrospinal fluid (ACSF, 4°C) containing (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 dextrose. Transverse hippocampal slices (300-µm thick) were cut from each hemisphere on a Vibratome (Campden Instruments) and transferred to a container filled with oxygenated ACSF at room temperature. After a recovery period of 1 h, a slice was positioned in the recording chamber, maintained submerged with a U-shaped platinum wire (Edwards et al. 1989), and continuously perfused with oxygenated ACSF (3-4 ml/min) at room temperature (22-24°C). The recording chamber was mounted on an upright microscope (Zeiss Axioskop) equipped with a long-range water immersion objective (×40), differential interference contrast, and an infrared video camera (Cohu 6500), which allowed visual identification of interneurons and pyramidal cells in slices.

Extracellular and whole cell recordings

Field potentials were recorded with patch electrodes (5-7 MOmega ) filled with 2 M NaCl placed in stratum pyramidale and were evoked by stimulating CA1 afferent fibers in stratum radiatum (bipolar electrode, 0.05 ms, 0-1,000 µA). In all experiments, the CA1 and CA3 regions were isolated by a surgical cut. For current-clamp experiments, patch electrodes (5-10 MOmega ) were filled with (in mM) 140 K-methanesulfonate, 5 NaCl, 2 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 0.5 CaCl2, 0.5 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 2 ATPtris, and 0.4 GTPtris (pH 7.4 adjusted with KOH). For voltage-clamp experiments, the internal solution of the patch electrodes contained (in mM) 120 Cs-methanesulfonate, 20 QX314, 8 NaCl, 1 MgCl2, 10 HEPES, 1 EGTA, 2 ATPtris, and 0.4 GTPtris (pH 7.4 adjusted with CsOH). In these experiments, Cs-methanesulfonate and QX314 (bromide salt; Research Biochemicals, RBI) were added to the recording solution to improve the space clamp and block K+ and Na+ currents (Nathan et al. 1990). In all experiments, 0.1% biocytin was added to the internal patch solution to label cell bodies and processes, and allow their subsequent morphological characterization.

After a tight seal (>1 GOmega ) was formed on the cell body of a neuron, whole cell recording was obtained by rupturing the cell membrane with negative pressure (Hamill et al. 1981). Extracellular, current and voltage-clamp recordings were made with an Axopatch 1D amplifier (Axon Instruments) with low-pass filtering at 10 kHz (3 db) and digitized at 22 kHz for storage on a videocassette recorder (Neurocorder DR886). Recordings were also digitized and analyzed with a microcomputer equipped with a data acquisition system (TL125 and pClamp, Axon Instruments). Recordings with unstable holding current or with series resistance and capacitance that could not be properly compensated were discarded. Liquid junction potentials were measured at the end of the experiments after withdrawal from the cell, and membrane potentials were subsequently corrected.

Whole cell postsynaptic responses were evoked by electrical stimulation (constant current pulses, 0.05 ms, 0-300 µA) of nearby afferent fibers with a monopolar tungsten microelectrode (Frederick Haer). For responses evoked in pyramidal cells, the stimulating electrode was positioned in stratum radiatum, whereas for interneurons it was placed 150-250 µm laterally from the soma (within the same layer). In voltage-clamp experiments, the peak amplitude and kinetics (10-90% rise time and decay time constant obtained from a single-exponential fit) of EPSCs and IPSCs were also compared in the different cell types between control and KA-treated rats. The analysis of postsynaptic currents (PSCs) was performed on averaged responses (n = 2).

Pharmacology

Receptor antagonists were prepared in distilled water and stored frozen in 1-ml aliquots: 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 3.1 mM, RBI) for non-NMDA receptors, (±)-2-amino-5-phosphonopentoanoic acid (AP5; 7.7 mM, Sigma) for NMDA receptors, and bicuculline (BIC; 3.3 mM, Sigma) for GABAA receptors. Before each experiment, concentrated stock solutions were diluted in ACSF to their final concentration (CNQX, 20 µM; AP5, 50 µM; BIC, 25 µM) and bath applied.

Statistical analysis

Group differences between cells in control slices and in hyperexcitable slices from KA-treated animals were analyzed with Student's t-tests. All data are given as means ± SE.

Histology

After completion of physiological recordings, slices were put in a fixative solution containing 4% paraformaldehyde, 1% glutaraldehyde, 2.5% dimethyl sulfoxide (DMSO), and 3.0 mM CaCl2 in 0.1 M cacodylate buffer (CB) for 4 h. They were then rinsed and left overnight in 0.1 M CB (4°C). To improve visualization of the axonal and dendritic plexus of cells, slices were embedded in 1% agarose and cut on a vibratome in 60-µm-thick sections. Sections were treated with 1% H2O2 for 20 min to eliminate endogenous peroxidases and then washed in phosphate buffer salt (PBS, pH 7.4, 4 × 5 min) and in PBS containing 2.5% DMSO (4 × 5 min). Slices were incubated in the avidin biotin complex (ABC kit, Vector Labs) at a concentration of 1:200 for 24 h and revealed according to a modified protocol with the tetramethylbenzidine (TMB) product (see Llewellyn-Smith et al. 1993). Slices were incubated for 20 min in a solution of phosphate buffer (PB, 0.1 M, pH 6.0) containing 0.4% ammonium chloride (Sigma) and 0.001% TMB (Sigma). The blue reaction was visualized by adding 0.05% H2O2 to the first incubating solution. To stabilize the TMB product, the slices were incubated 15 min in a solution of PB (pH 6.0) containing 0.4% NH4Cl, 1% cobalt chloride (Sigma), 0.1% DAB, and 0.1% H2O2. Sections were cleared in xylene and mounted in DPX (Electron Microscopy Sciences) for light microscopy. The dendritic and axonal arborizations of the cells were then drawn and reconstructed with a camera lucida at a final magnification of ×400.

The presence of KA lesions was verified with Nissl staining in three KA-treated rats and compared with two control animals. Rats were anesthetized with sodium pentobarbital (65 mg/kg ip) and perfused through the ascending aorta with 2% paraformaldehyde, 2.5% glutaraldehyde, and 3.0 mM CaCl2 in 0.1 M CB (pH 7.4, room temperature). Coronal sections of the whole hippocampus were taken and processed for conventional Nissl stains.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Kainate lesions and extracellular recordings

Nissl-stained sections from control animals (n = 2) showed intact principal cells (Fig. 1A) in all hippocampal subfields (dentate gyrus, CA3 and CA1). In contrast, extensive lesions of hippocampal CA3 cells were observed (Fig. 1B, arrows) in KA-treated animals (n = 3).


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FIG. 1. A and B: light photomicrographs of Nissl-stained sections of the hippocampus showing cells located in dentate gyrus, CA3 and CA1 regions. Principal cells of the dentate gyrus and CA3-CA1 areas were intact in control rats (A; CA3 indicated by arrows). In contrast, cells of the CA3 area (B; indicated by arrows) were selectively lost after bilateral intracerebroventricular injections of kainic acid, whereas principal cells of the CA1 area and dentate gyrus were undamaged.

Hyperexcitable slices from KA-treated animals were identified with field potentials evoked by stimulation of stratum radiatum. Slices were considered hyperexcitable if evoked responses consisted of two or more population spikes and were selected for further whole cell recordings. In slices of control rats, field potentials recorded in stratum pyramidale consisted of a single population spike (mean amplitude of maximal population spike, 2.0 ± 0.2 mV; n = 52; Fig. 2A). In hyperexcitable slices from KA-treated animals (Fig. 2B), responses were composed of multiple population spikes (amplitude of largest population spike, 2.4 ± 0.2 mV; n = 48).


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FIG. 2. A and B: schematic drawings of hippocampal slices (left) showing the placement of electrodes for extracellular stimulation and field potentials recordings in slices from control (A) and kainic acid (KA)-treated (B) rats. Field potentials were recorded in stratum pyramidale and evoked by stimulation of afferent fibers in stratum radiatum. Representative examples of field responses (right) from a slice of a control rat (A) showing a single population spike and a hyperexcitable slice of a KA-treated rat (B) consisting of multiple population spikes (stimulation indicated by arrow).

Whole cell recordings and morphology of biocytin-filled cells

Interneurons and pyramidal cells were visually identified with infrared video microscopy in hyperexcitable slices of KA-treated rats and in slices of control animals. Cells were selected for whole cell recordings based on their morphology (soma and primary dendrites) and their soma location in specific CA1 layers. Recordings were obtained from interneurons located 1) in stratum oriens near the alveus (O/A, n = 17 in control and in KA groups) and 2) at the border of stratum radiatum and stratum lacunosum-moleculare (LM, n = 32 and 31 in control and KA groups), respectively, and from pyramidal cells (n = 29 and 26 in control and KA groups, respectively).

The morphological profiles of each cell type were generally similar in control and KA-treated rats. The dendritic and axonal arborizations of biocytin-labeled interneurons in O/A (n = 6 in control; n = 14 in KA-treated group) and in LM (n = 18 in control; n = 17 in KA-treated group) as well as of pyramidal cells (n = 13 in control; n = 6 in KA-treated group) were generally as previously described in normal rats (Morin et al. 1996; data not shown).

Membrane properties and postsynaptic potentials

In whole cell current-clamp recordings, the basic membrane properties of O/A, LM, and pyramidal cells were generally not different in control and KA-treated rats (Table 1). The resting membrane potential was near -50 mV for all cells, except for pyramidal cells of KA-treated animals, which displayed a significantly more hyperpolarized membrane potential (-58 mV). In O/A cells, the action potential amplitude was significantly greater after KA treatment (106 mV), but it was unchanged in other cells. Other intrinsic membrane properties (Table 1) were not significantly different in any cell type and were generally similar to those previously described in normal rats (Morin et al. 1996).

 
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TABLE 1. Membrane properties of interneurons and pyramidal cells from control and kainic acid-treated rats

Stimulation of CA1 afferent fibers elicited postsynaptic potentials that were composed of initial excitatory postsynaptic potentials (EPSPs) followed by inhibitory postsynaptic potentials (IPSPs) in all cells. These responses were similar in all cell types of control (O/A, n = 4; LM, n = 8; pyramidal, n = 8) and KA-treated (O/A, n = 6; LM, n = 11; pyramidal, n = 8) animals, except that more action potentials were usually elicited in interneurons of O/A and pyramidal cells after KA (data not shown).

EPSCs

To examine whether synaptic currents were altered after KA treatment, compound EPSCs and IPSCs were evoked by stimulating CA1 afferent fibers in normal ACSF and were recorded in voltage clamp with the cell held at -80 mV. PSCs were inward and the amplitude increased with stimulation intensity (0-300 µA) until they reached a maximum. Mean amplitude of PSCs was compared at a stimulation intensity of 200 µA in O/A, LM, and pyramidal cells of control and KA-treated rats. In KA-treated rats, the mean amplitude of PSCs was not significantly different in interneurons of either O/A (655.8 ± 145.9 pA in KA vs. 788.7 ± 160.9 pA in control; n = 11 in each group) or LM (338.5 ± 63.0 pA in KA vs. 465.7 ± 71.4 pA in control; n = 20 and 24, respectively). However, in pyramidal cells, PSC amplitude was significantly increased in KA-treated animals (655.3 ± 88.5 pA in KA vs. 380.8 ± 56.1 pA in control; n = 20 in each group; P < 0.05; Fig. 3).


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FIG. 3. Compound excitatory and inhibitory postsynaptic currents (PSCs) recorded in representative LM (A) and pyramidal (B) cells of control and KA-treated rats. PSCs evoked by stimulation of afferent fibers (200 µA intensity) were inward at a membrane potential of -80 mV. PSCs were smaller in amplitude in LM interneurons (A) of KA-treated rats (right trace) relative to control (left trace). In pyramidal cells (B) of KA-treated rats (right trace), responses were increased in amplitude relative to control animals (left trace). C: summary histogram of mean values of peak amplitude of PSCs evoked in interneurons in stratum oriens near the alveus (O/A), at the border of stratum radiatum and stratum lacunosum-moleculare (LM), and pyramidal cells of control (open bars) and KA-treated (filled bars) rats. Mean amplitude of PSCs was not significantly different in O/A and LM interneurons of KA-treated rats. In contrast, mean amplitude of PSCs was significantly increased in pyramidal cells of KA-treated animals (* P < 0.05).

To examine individually how excitatory and inhibitory synaptic transmission were affected by KA treatment, glutamate and GABAA PSCs were pharmacologically isolated. EPSCs were evoked at different membrane potentials (-80 to 60 mV) in the presence of the GABAA receptor antagonist bicuculline. As previously described in control animals (Morin et al. 1996; Sah et al. 1990), EPSCs were composed of a fast component mediated by non-NMDA receptors and a slow component mediated by NMDA receptors in interneurons and pyramidal cells (Fig. 4). Latencies for measuring both components were determined in each cell at a membrane potential of -80 mV. The fast EPSC was measured at the initial peak current, and the slow EPSC was measured at a latency when the fast EPSC had decayed near baseline (Fig. 4, A and B). The mean amplitude of fast EPSCs (measured at -80 mV) was not significantly changed after KA in O/A (100.9 ± 30.1% of control) and pyramidal (101.1 ± 16.7% of control) cells but was significantly reduced in LM interneurons (51.8 ± 10.8% of control; P < 0.05; Table 2). The mean amplitude of slow EPSCs (measured at 60 mV) was not significantly changed after KA in O/A (223.2 ± 75.5% of control), LM (64.5 ± 13.5% of control), or pyramidal (221.8 ± 97.3% of control) cells (Table 2). From the amplitude of both components at various membrane potentials, current-voltage (I-V) relationships of fast and slow EPSCs were determined (Fig. 4, C and D). The mean conductance of fast EPSCs, obtained from the slope of the linear portion of the I-V relation, was not significantly different after KA in O/A interneurons (131.5 ± 39.1% of control) and pyramidal cells (117.2 ± 34.5% of control). In contrast, the mean conductance of fast EPSCs was significantly reduced in LM interneurons of KA-treated rats (46.9 ± 8.2% of control; P < 0.05; Table 2, Fig. 4). The I-V relation of slow EPSCs demonstrated a region of negative slope for membrane potentials between -80 and -30 mV and a linear portion at more depolarized potentials (Fig. 4). The mean conductance of slow EPSCs, obtained from the linear portion of the I-V relation, was not significantly different after KA in O/A (165.7 ± 50.7% of control) and pyramidal (135.7 ± 42.9% of control) cells, but was significantly reduced in LM interneurons (63.2 ± 10.5% of control; P < 0.05; Table 2, Fig. 4).


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FIG. 4. Changes in pharmacologically isolated excitatory postsynaptic currents (EPSCs) after KA treatment. A: averaged fast (open circle) and slow (open square) EPSCs evoked in the presence of 25 µM bicuculline in a representative LM interneuron from a control rat, at different membrane potentials (-108 to 32 mV). B: in a LM cell of a KA-treated rat, the amplitude of both components of EPSCs (bullet  and black-square) evoked in similar conditions were reduced. C and D: current-voltage (I-V) relationships of fast (C) and slow (D) components of EPSCs for the cells shown in A and B. Slope conductance, obtained from the linear portion (dotted lines) of each I-V relation, was reduced in the cell of the KA-treated rat (filled symbols) relative to the control cell (open symbols). E and F: EPSCs were antagonized by non-N-methyl-D-aspartate (NMDA) and NMDA receptor antagonists [20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 µM (±) - 2 - amino - 5 - phosphonopentoanoic acid (AP5), respectively]. Fast and slow EPSCs from representative LM interneurons were significantly reduced by 92 and 68%, respectively, in the control rat (E), and by 69 and 77%, respectively, in the KA-treated animal (F) (note the difference in calibration bars in E and F). G: summary histogram of mean values for all cells (expressed as fraction of control). In KA-treated rats, the conductance of fast and slow EPSCs (gfast and gslow, respectively) was significantly reduced in LM cells (filled bars) but not in O/A (open bars) or pyramidal (hatched bars) cells. The EPSC decay time constant, obtained from single-exponential fitting of the decay phase, was reduced in all cell types, but this was significant only for O/A cells (* P < 0.05).

 
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TABLE 2. Properties of evoked EPSCs in interneurons and pyramidal cells from control and kainic acid-treated rats

To determine whether KA treatment caused changes in the kinetics of EPSCs, the rise time and decay time constant of fast EPSCs (at -80 mV) were also compared between cells of control and KA-treated rats. The 10-90% rise time was not significantly different in all cell types after KA (O/A, 67.0 ± 40.8% of control; LM, 57.1 ± 5.4% of control; pyramidal cells, 88.8 ± 15.5% of control; Table 2). The decay time constant of fast EPSCs, obtained from single-exponential fit of the decay phase, was significantly faster in O/A interneurons of KA-treated rats (29.4 ± 7.4% of control; P < 0.05) but was not significantly changed in LM (59.9 ± 17.1% of control) and pyramidal (83.6 ± 19.3% of control) cells (Table 2, Fig. 4). Therefore the kinetics of fast EPSCs were in general not significantly changed between control and KA-treated rats, except for a faster decay in O/A interneurons of KA-treated animals.

To verify that fast and slow EPSCs were mediated by non-NMDA and NMDA receptors, their antagonists (CNQX and AP5, respectively) were bath applied (Fig. 4). Fast EPSCs, recorded at -80 mV, were significantly reduced in all cell types: in O/A cells by 98.3 ± 1.8% (n  = 6) and 97.5 ± 0.6% (n = 3), in control and KA groups, respectively; in LM cells by 88.4 ± 2.8% (n = 9) and 92.8 ± 2.9% (n = 7), in control and KA groups, respectively; and in pyramidal cells by 94.9 ± 4.3% (n = 10) and 93.3 ± 4.8% (n = 8), in control and KA groups, respectively. Slow EPSCs recorded at positive membrane potentials (60 mV) in the same cells were also significantly reduced during application of these antagonists: in O/A cells by 94.7 ± 1.0% (n = 5) and 90.6 ± 2.1% (n = 3), in control and KA groups, respectively; in LM cells by 78.3 ± 3.4% (n = 9) and 86.1 ± 2.3% (n = 7), in control and KA groups, respectively; and in pyramidal cells by 75.8 ± 5.1% (n = 9) and 86.6 ± 5.1% (n = 7), in control and KA groups, respectively. In all cell types of either control or KA-treated groups, the effects of these antagonists on fast and slow EPSCs were partially reversible after washout of the antagonists (range 13.0 ± 6.1 to 66.8 ± 8.6%, data not shown).

IPSCs

To examine GABA synaptic responses in isolation, monosynaptic GABAA IPSCs were evoked in the presence of non-NMDA and NMDA receptor antagonists (CNQX and AP5, respectively) (Fig. 5). Monosynaptic GABAA IPSCs, recorded at -80 mV, were inward, and their mean peak amplitude was not significantly different after KA in interneurons of O/A (126.8 ± 48.6% of control) and LM (123.1 ± 32.8% of control) or in pyramidal cells (159.4 ± 44.7% of control) (Table 3). The I-V relations of GABAA IPSCs, obtained from series of responses recorded at different membrane potentials (-80 to 60 mV; Fig. 5), were linear. The mean reversal potentials of GABAA IPSCs were not significantly different in KA-treated rats for interneurons in O/A (85.5 ± 7.7% of control) and LM (94.1 ± 6.8% of control) and in pyramidal cells (95.9 ± 7.9% of control) (Table 3). The mean conductance of GABAA IPSCs, obtained from the slope of the linear I-V relation, was generally increased after KA in interneurons of O/A (113.1 ± 36.5% of control) and LM (123.1 ± 37.4% of control) and in pyramidal cells (152.6 ± 39.1% of control), but this increase was not significant (Table 3, Fig. 5, C and F).


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FIG. 5. Changes in monosynaptic gamma -aminobutyric acid-A (GABAA) IPSCs after KA treatment. A and B: series of averaged IPSCs recorded at different membrane potentials (-80 to 60 mV) from representative pyramidal cells of control (A) and KA-treated (B) rats in the presence of non-NMDA and NMDA antagonists (20 µM CNQX and 50 µM AP5, respectively). The peak IPSC amplitude was larger in the cell of KA-treated rats (B, filled circle) relative to control (A, open circle). C: I-V relations of IPSCs shown in A and B. The IPSC conductance, obtained from the slope of the linear regression (dotted lines), was larger in the cell from the KA-treated animal. D and E: reversible antagonism of IPSCs by the GABAA antagonist, bicuculline in representative LM interneurons from control (D) and KA-treated (E) rats. After bath application of 25 µM bicuculline, IPSCs were reduced by 88 and 93% in the LM cell of control and KA-treated rats, respectively (+BIC, middle trace). After the washout of the antagonist (CNQX/AP5, right traces), IPSC amplitude partially recovered in both cells. F: summary histogram of conductance (gIPSC), rise time, and decay time constant of GABAA IPSCs in cells of KA-treated rats, expressed as fraction of control, for all cell types (O/A, open bars; LM, filled bars; pyramidal cells, oblique bars). The IPSC conductance was not significantly different in any cell type after KA treatment. The IPSC rise time and decay time constant were significantly faster in pyramidal cells of KA-treated rats (* P < 0.05).

 
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TABLE 3. Properties of evoked inhibitory postsynaptic currents in interneurons and pyramidal cells of control and KA-treated rats

The 10-90% rise time and decay time constant of GABAA IPSCs were also compared in cells of control and KA-treated rats. After KA treatment, the mean rise time of GABAA IPSCs recorded at -80 mV was not significantly different in interneurons of O/A (108.9 ± 44.4% of control) and LM (113.8 ± 11.7% of control) but was significantly faster in pyramidal cells (69.9 ± 4.9% of control, P < 0.05; Table 3, Fig. 5F). The mean decay time constant of GABAA IPSCs recorded at -80 mV, obtained from a single-exponential fit of the decay phase, was not significantly different in KA-treated rats for interneurons of O/A (74.0 ± 15.3% of control) and LM (103.0 ± 9.7% of control) but was significantly faster for pyramidal cells (68.8 ± 7.4% of control, P < 0.05; Table 3, Fig. 5F). These results indicate that the kinetics of monosynaptic GABAA IPSCs evoked in pyramidal cells were significantly faster after KA treatment. Despite these faster kinetics, the total charge transfer during IPSCs was not significantly changed in CA1 pyramidal cells of KA-treated animals (148.3 ± 26.6% of control), likely because of the nonsignificant increase in IPSC amplitude.

To verify that monosynaptic IPSCs were GABAA mediated, the effects of the GABAA antagonist bicuculline were examined (Fig. 5). The mean amplitude of GABAA IPSCs (recorded at -80 mV) was significantly reduced in cell types: in O/A cells by 60.0 ± 4.9% (n = 3) and 95.5 ± 3.7% (n = 2), in control and KA groups, respectively; in LM cells by 96.9 ± 1.0% (n = 9) and 94.8 ± 2.4% (n = 6), in control and KA groups, respectively; and in pyramidal cells by 62.3 ± 7.7% (n = 7) and 79.9 ± 5.2% (n = 5), in control and KA groups, respectively. The bicuculline effects were reversible with washout in all cell types of control and KA groups (range 31.4 ± 7.0 to 81.5 ± 7.2%, e.g., Fig. 5).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The major findings of this study were that multiple changes take place in inhibitory circuits after KA treatment and that these alterations occur selectively in certain interneuron subtypes. First, morphological characteristics of biocytin-labeled interneurons were similar in control and KA-treated animals. Second, in current-clamp experiments, basic intrinsic membrane properties of interneurons and pyramidal cells were generally not changed after KA treatment, except for action potentials of larger amplitude in O/A interneurons, and more hyperpolarized resting membrane potentials in pyramidal cells. Third, EPSPs and IPSPs could be evoked in all cell types of KA-treated rats. Fourth, in voltage-clamp experiments, the amplitude and conductance of pharmacologically isolated EPSCs were significantly reduced but specifically in LM interneurons of KA-treated animals. The rise time of EPSCs was unchanged in all cell types, but the decay time constant was significantly faster in O/A interneurons after KA. Fifth, the conductance and amplitude of pharmacologically isolated IPSCs were not significantly changed in any cell type after KA. However, the rise time and decay time constant of IPSCs were significantly faster in pyramidal cells of KA-treated rats.

Morphology of biocytin-filled cells

Overall, the morphology of biocytin-labeled interneurons and pyramidal cells was similar in control and KA-treated groups, indicating that recordings were obtained from similar types of interneurons in both groups. Interneurons in O/A were similar to the previously described vertical (Lacaille and Williams 1990; Lacaille et al. 1987) and horizontal (Maccaferri and McBain 1995; McBain et al. 1994) cells, whereas LM interneurons were like stellate cells (Kawaguchi and Hama 1988; Lacaille and Schwartzkroin 1988a; Williams et al. 1994). A quantitative analysis of glutamic acid decarboxylase-immunostained cells in the CA1 region demonstrated that 50% of interneurons located in stratum oriens and the alveus were lost after KA treatment (Morin et al. 1998). Others have shown that somatostatin- and parvalbumin-immunoreactive cells were lost after KA treatment (Best et al. 1993, 1994). Because of their soma location and axonal arborization patterns, these may correspond to the horizontal interneurons in O/A (Maccaferri and McBain 1995). In this study, some of our biocytin-labeled O/A interneurons of KA-treated animals displayed axonal projections typical of horizontal cells, which suggests that the loss of these cells may only be partial after KA treatment.

Intrinsic membrane properties

Intrinsic properties were generally unaltered in interneurons and pyramidal cells after KA treatment. Resting membrane potential, membrane time constant, cell input resistance, action potential duration, and amplitude of afterhyperpolarizations were unchanged. However, in KA-treated animals, action potential amplitude was larger in O/A cells, and resting membrane potential was more hyperpolarized in pyramidal cells. Our results indicate that the hyperexcitability in the CA1 region after KA treatment may not be due to an impairment of the basic intrinsic properties of interneurons that could have led to their loss of excitability. This is consistent with a previous report using intracellular recordings with sharp electrodes that the input resistance of interneurons located within stratum pyramidale was not significantly different after KA treatment (Franck et al. 1988). Similarly, in a model of status epilepticus, membrane properties of interneurons located in stratum oriens, stratum radiatum, or stratum LM were not significantly altered after chronic seizures (Rempe et al. 1997). As previously found with intracellular recordings (Perez et al. 1996; Williams et al. 1993), our results also suggest that the CA1 hyperexcitability does not arise from alterations in intrinsic properties of pyramidal cells because these were unchanged after KA treatment. However, we have not observed the increases in input resistance of CA1 pyramidal cells that were described after KA treatment by others (Franck and Schwartzkroin 1985; Franck et al. 1988; Nakajima et al. 1991). Basic properties of hippocampal CA1 pyramidal cells and dentate gyrus granule cells also appear unchanged in the chronic model of status epilepticus (Bekenstein and Lothman 1993; Rempe et al. 1995), after kindling (Mody et al. 1988; Olivier and Miller 1985), and in human TLE (Isokawa 1996).

Alteration in glutamate synaptic transmission

Current-clamp experiments showed that excitatory and inhibitory postsynaptic potentials were evoked in all cell types in KA-treated rats. However, voltage-clamp analysis of pharmacologically isolated EPSCs and IPSCs indicated that evoked currents were altered after KA treatment, but only in specific cell types. In interneurons, evoked EPSCs were composed of a fast component mediated by non-NMDA receptors and a slow component mediated by NMDA receptors. Our results demonstrated that the conductance of both non-NMDA and NMDA components of EPSCs was significantly reduced after KA treatment in LM interneurons but was intact in O/A interneurons and pyramidal cells. These results suggest a reduction of evoked excitatory drive specific to LM interneurons after KA. Because degenerating terminals were identified on LM interneurons after KA treatment (Kunkel et al. 1998), the decrease in excitatory drive likely reflects the partial deafferentation of LM cells. This observation of a reduced excitatory drive onto some CA1 inhibitory interneurons provides direct support to the dormant cell hypothesis (Sloviter 1991). According to this hypothesis, the disinhibition that contributes to epileptogenesis is due to a loss of afferent excitatory drive onto inhibitory interneurons resulting in a reduction of inhibition of principal cells (Sloviter 1987, 1991). In the CA1 region of the KA model, disinhibition is seen as a decrease in polysynaptic inhibition of pyramidal cells (Franck et al. 1988; Perez et al. 1996; Williams et al. 1993). However, direct electrical stimulation of inhibitory interneurons in slices of KA-treated rats demonstrated that monosynaptic inhibition was still intact in CA1 pyramidal cells after KA treatment (Nakajima et al. 1991; Williams et al. 1993), suggesting that inhibitory circuits are still functional after KA. Our results are consistent with these findings and further indicate that the reduced excitatory drive on LM interneurons may contribute to the impairment of polysynaptic inhibition in CA1 pyramidal cells and to the development of epileptiform activity in the CA1 area after KA treatment. In a model of chronic epilepsy, whole cell current-clamp recordings of interneurons in stratum oriens and alveus, in stratum radiatum, and in stratum lacunosum-moleculare found that the excitatory drive of CA1 interneurons was still functional (Rempe et al. 1997). However, this model of status epilepticus produces a more generalized hippocampal cell loss (Rempe et al. 1997), suggesting that a specific loss of afferents from the CA3 region may be the key factor for the reduction of excitatory drive on LM interneurons after KA.

In this study, no deficit was observed in the amplitude and conductance of non-NMDA and NMDA EPSCs in O/A interneurons after KA treatment. Thus this reduction in evoked excitatory drive occurs selectively in some subtypes of interneurons. This loss of excitatory afferents restricted to certain specific types of interneurons may explain why previous investigations did not observe a reduction of excitatory drive in interneurons after KA (Esclapez et al. 1997). Although the amplitude and conductance of evoked responses were unchanged, the faster decay time constant of EPSCs in O/A interneurons after KA treatment suggests that alterations have taken place at excitatory inputs on O/A cells. Indeed, preliminary findings indicated that the kinetics of spontaneous EPSCs in O/A interneurons were modified after KA treatment (Perez and Lacaille 1997). Interneurons in O/A may also become partially deafferented after KA treatment because they are normally contacted by CA3 afferents (Lacaille et al. 1987). The presence of excitatory synaptic currents with intact conductance but altered kinetics suggests that compensatory changes may have taken place in these interneurons after KA. Thus the increase in local axonal arborizations of CA1 pyramidal cells after KA (Perez et al. 1996) may have led to the formation of new excitatory synapses onto O/A interneurons to compensate for the KA-induced loss. These new synapses may restore the amplitude and conductance of EPSCs to control levels, but they may have different properties from the original ones, resulting in different kinetics from control EPSCs. These ongoing compensatory changes involving O/A interneurons may contribute to the recovery of polysynaptic IPSPs that was reported at longer interval (2-4 mo) after KA treatment (Franck and Schwartzkroin 1985). Finally, a determinant factor in the interneuron response to KA treatment appears to be its hippocampal connections. LM interneurons, which do not receive excitatory inputs from CA1 pyramidal cells (Lacaille and Schwartzkroin 1988a,b), remain partially deafferented and show reduced excitatory drive after KA treatment. In contrast, O/A interneurons that receive excitatory inputs from CA1 pyramidal cells (Blasco-Ibanez and Freund 1995; Lacaille et al. 1987; Maccaferri and McBain 1995) may undergo compensatory changes and do not show reduced excitatory drive.

Changes at GABAergic synapses

Our results suggest that inhibitory synapses in interneurons and pyramidal cells were unimpaired in KA-treated rats. Inhibitory synaptic responses were recorded in all cell types, and the amplitude and conductance of GABAA IPSCs were not significantly changed in either interneurons or pyramidal cells after KA treatment. Therefore, in the KA model of epilepsy, increased inhibition of inhibitory interneurons does not appear to be the cause of polysynaptic epileptiform activity in the CA1 region, in contrast to the seizure-sensitive gerbil (Peterson et al. 1985). Moreover, the tendency for the peak conductance of monosynaptic GABAA IPSCs to be increased in pyramidal cells after KA treatment indicated that a reduction of monosynaptic GABAA inhibition in pyramidal cells was not responsible for their polysynaptic disinhibition. These results are consistent with previous reports of functional monosynaptic inhibition in the CA1 region of KA (Esclapez et al. 1997; Williams et al. 1993) and other models (Mangan and Lothman 1996; Mangan et al. 1995) of human TLE. However, the faster rise time and decay time constant of evoked monosynaptic GABAA IPSCs in pyramidal cells after KA would suggest that some alterations occurred at GABAergic synapses. In addition, the unimpaired synaptic conductance of GABAA IPSCs in pyramidal cells despite a partial loss of interneurons in the CA1 region after KA treatment (Best et al. 1993, 1994; Morin et al. 1998) suggests that compensatory changes occurred at GABAergic synapses. It remains to be clarified whether these compensatory changes involve the formation of new synapses caused by the sprouting of axons of KA-resistant inhibitory interneurons, as found in the dentate gyrus (Davenport et al. 1990), or because of a functional alteration of existing synapses of KA-resistant interneurons. Region-specific alterations in GABAA receptor function caused by changes in receptor subunit expression were found in other models of human TLE (Gibbs et al. 1997; Kapur and Coulter 1995; Kapur and Macdonald 1997). In the pilocarpine model, the mRNA expression for alpha 2 and alpha 5 subunits of GABAA receptors was decreased in CA1 hippocampal cells, whereas that of the alpha 1, beta 2, or gamma 2 subunits was unchanged (Houser and Esclapez 1996; Rice et al. 1996; Vick et al. 1996). Additionally, complex changes in GABAA receptor function were described in the CA1 region after status epilepticus (Gibbs et al. 1997; Kapur and Coulter 1995). The efficacy of GABA in activating GABA currents was decreased in acutely isolated CA1 pyramidal cells, and the potency of GABA was enhanced (Gibbs et al. 1997). Furthermore, the benzodiazepine augmentation of GABA currents was reduced in these neurons (Gibbs et al. 1997). Overall, these data clearly indicate that GABA receptor function may be modified but remain functional in these experimental models of TLE.


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FIG. 6. Schematic diagram of alterations in hippocampal CA1 inhibitory circuits after KA treatment. A: in the normal hippocampus, 2 types of inhibitory interneurons are represented in stratum oriens near the alveus, the vertical (V) and the horizontal (H) cells. Both cell types receive an excitatory input (open terminals) from pyramidal cells (P) and subsequently make inhibitory contacts (filled terminals) onto the principal cells, thus mediating feedback inhibition. The axonal projections of CA3 pyramidal cells, the Schaffer collaterals (Sch), make excitatory connections onto CA1 pyramidal cells but also on vertical cells and on other interneurons located at the border of stratum radiatum and stratum lacunosum-moleculare (LM), the stellate cells (S). Through these pathways, these interneurons mediate feed-forward inhibition. B: 2 wk after KA treatment, there is a partial loss of CA3 afferents to CA1 (broken lines) and consequently a decrease of excitatory drive from CA3 onto interneurons involved in feed-forward inhibition (V and S cells). However, only stellate cells remain partially deafferented and have reduced excitatory drive. Sprouting of CA1 pyramidal cell axons takes place, and new excitatory terminals (marked by asterisks) may be formed onto neighboring pyramidal cells and onto interneurons. These latter contacts may compensate for the partial deafferentation of interneurons in stratum oriens induced by KA. A partial loss of somatostatin- and parvalbumin-immunopositive interneurons (H cells) is also observed after KA treatment (broken outline). Because no decrease in monosynaptic inhibition of pyramidal cells was observed, compensatory mechanisms such as axonal sprouting of inhibitory inputs from KA-resistant interneurons may take place (inhibitory terminals marked by question marks and asterisks), or other functional changes may occur at inhibitory synapses of KA-resistant interneurons.

Functional implications

Overall, our results suggest that multiple changes occur in hippocampal inhibitory circuits after intraventricular kainate injection and that these changes do not affect inhibitory interneurons uniformly. In addition, some of these changes may contribute to disinhibition of CA1 pyramidal cells, whereas others may tend to reestablish inhibition. These alterations in CA1 hippocampal circuits are illustrated in Fig. 6. In this diagram of the hippocampal CA1 region, the only inhibitory interneurons represented are those that were studied in the current experiments. These are the interneurons located in stratum oriens and the alveus, vertical (Lacaille et al. 1987; Lacaille and Williams 1990) and horizontal (Maccaferri and McBain 1995) cells, and those found at the border of stratum radiatum and stratum lacunosum-moleculare, stellate cells (Lacaille and Schwartzkroin 1988a,b). The vertical cells are involved in both feed-forward and feedback inhibition (Lacaille et al. 1987; Maccaferri and McBain 1996), whereas the horizontal cells are involved in feedback inhibition (Maccaferri and McBain 1995, 1996). These inhibitory interneurons receive excitatory inputs from CA1 pyramidal cells and in turn make inhibitory contacts onto pyramidal cells. In contrast, interneurons located at the border of stratum radiatum and stratum lacunosum-moleculare (stellate cells) are mostly responsible for feed-forward inhibition (Lacaille and Schwartzkroin 1988a,b). These cells receive excitatory inputs from CA3 pyramidal cells and make inhibitory contacts onto dendrites of CA1 pyramidal cells (Kunkel et al. 1988). Two weeks after KA treatment, there is a partial loss of Schaffer collaterals; however, excitatory inputs to CA1 pyramidal cells remain unimpaired because of synaptic replacement caused by sprouting of CA1 afferents (spared Schaffer collaterals and commissural fibers) (Nadler et al. 1980b). In addition, two types of changes in the inhibitory circuitry promote disinhibition and epileptiform activity in the CA1 region. First, there is a significant loss of GABA interneurons in stratum oriens and the alveus (Best et al. 1993; Morin et al. 1998). Because these vulnerable interneurons are immunopositive for parvalbumin and somatostatin (Best et al. 1993, 1994), they may correspond to horizontal cells (Blasco-Ibanez and Freund 1995). Second, a partial deafferentation of interneurons results in a partial loss of evoked excitatory drive in stellate cells that may contribute to the decrease in polysynaptic inhibition of pyramidal cells. However, other alterations in the circuitry appear compensatory and may restore inhibition. First, because KA-resistant interneurons in O/A have unimpaired excitatory synaptic currents despite their partial deafferentation, axonal sprouting of pyramidal cells may have restored their excitatory inputs. Second, monosynaptic GABAA IPSCs were unimpaired in pyramidal cells despite a partial loss of interneurons, suggesting that some compensatory changes must have taken place at inhibitory synapses. These latter changes may involve sprouting by KA-resistant interneurons and the formation of new inhibitory terminals or functional alterations of synapses of KA-resistant interneurons.

    ACKNOWLEDGEMENTS

  We thank I. Jutras for assistance with kainic acid lesions and G. Lambert for photographic work.

  This work was supported by the Fonds de la Recherche en Santé du Québec to J.-C. Lacaille, the Medical Research Council of Canada to J.-C. Lacaille and C. Beaulieu, the Savoy Foundation to F. Morin, a Research Center grant from the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR) to the Groupe de Recherche sur le Système Nerveux Central, and an Équipe de Recherche grant from the FCAR to J.-C. Lacaille.

    FOOTNOTES

  Address for reprint requests: J.-C. Lacaille, Dép. de Physiologie-124, Université de Montréal, C. P. 6128, Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada.

  Received 18 March 1998; accepted in final form 13 August 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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