375
Journal of Physiology (1991), 444, pp. 375-396 With 9 figures Printed in Great Britain
PAROXYSMAL INHIBITORY POTENTIALS MEDIATED BY GABAB RECEPTORS IN PARTIALLY DISINHIBITED RAT HIPPOCAMPAL SLICE CULTURES
BY MASSIMO SCANZIANI, BEAT H. GAHWILER AND SCOTT M. THOMPSON From the Brain Research Institute, University of Zurich, August Forel-Strasse 1, CH-8029 Zurich, Switzerland
(Received 13 March 1991) SUMMARY
1. Intracellular recording techniques were used to study synaptic potentials in CA3 pyramidal cells elicited with mossy fibre stimulation in partially disinhibited hippocampal slice cultures. Two experimental protocols were used: (1) high concentrations (20-40 gM) of the A-type y-aminobutyric acid (GABAA) receptor antagonist bicuculline plus low concentrations (2-4 /sM) of the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or (2) low concentrations (1-2 5 ,lM) of bicuculline alone. 2. Under the first condition, stimulation of mossy fibre afferents evoked epileptic bursts alternating with a response consisting of an excitatory postsynaptic potential (EPSP) followed by an unusually large and long-lasting hyperpolarizing potential with a maximal amplitude in the range of -30 mV from the resting membrane potential. 3. This paroxysmal inhibitory potential (PIP) had a reversal potential near that of potassium. The amplitude of the PIP was not dependent on action potentials superimposed on the preceding EPSP, and was present in cells recorded with microelectrodes containing the Ca2" chelator EGTA. These data suggest that the PIP is not a Ca2"-activated K+ potential. 4. The PIP was prolonged by the GABA-uptake blocker nipecotic acid, was reduced by hyperpolarizing interneurons with the opioid agonist FK 33-824, and was abolished by the GABAB-receptor antagonist CGP 35 348. These data indicate that the PIP is mediated by the activation of GABAB receptors following GABA release from interneurons. 5. The NMDA-receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) strongly reduced the amplitude of the PIP, but had no effect on the GABAB receptor-mediated inhibitory postsynaptic potential (IPSP) under control conditions. 6. Under the first condition, regular stimulation elicited a cyclical pattern of evoked responses. There was either an alternation between an epileptic burst and a PIP or, at shorter interstimulus intervals, a sequence of gradually increasing PIPs followed by an epileptic burst, which then reset the cycle. MS 9234
376
M. SCANZIANI, B. H. GAHWILER AND S. M. THOMPSON
7. Under the second condition, in low concentrations of bicuculline alone, the early GABAA-mediated IPSP was little affected, but the late GABAB-mediated IPSP was greatly enhanced. These enhanced late IPSPs were comparable in amplitude and duration to the PIPs seen under the first conditions, could exhibit cyclical behaviour, and were reduced by D-APV. 8. Application of CGP 35 348 abolished the late IPSP under control conditions, but had no effect on hippocampal excitability. In contrast, CGP 35 348 blocked the PIP elicited in low bicuculline, and consequently led to intense epileptic discharge. 9. We suggest that the amplification of GABAB receptor-mediated synaptic potentials after partial disinhibition may be an important endogenous compensatory mechanism preventing epileptogenesis.
INTRODUCTION
Inhibitory synaptic transmission in the hippocampus is mediated by the release of y-aminobutyric acid (GABA) from local circuit interneurons, which then activates two distinct receptors on pyramidal cells. GABAA receptors are blocked by bicuculline and directly gate Cl- permeable channels (Eccles, Nicoll, Oshima & Rubia, 1977) while GABAB receptors are bicuculline-insensitive and activate K+ channels (Hill & Bowery, 1981; Giihwiler & Brown, 1985; Newberry & Nicoll, 1985). Antagonists of GABAB receptors are reported to have no significant effect on hippocampal excitability (Dutar & Nicoll, 1988; Karlsson & Olpe, 1989; Olpe, Karlsson, Pozza, Brugger, Steinmann, Van Riezen, Fagg, Hall, Froestl & Bittiger, 1990; cf. Malouf, Robbins & Schwartzkroin, 1990). In contrast, in the absence of GABAA receptor-mediated inhibition, unchecked recurrent excitation between pyramidal cells allows activity to spread throughout the population, causing the cells to discharge synchronously (e.g. Dichter & Spencer, 1968; Johnston & Brown, 1981; Traub & Wong, 1982; Miles & Wong, 1987; Traub, Miles & Wong, 1989). Disinhibition with GABAA receptor antagonists thus leads to epileptiform discharge. The primary afferents to the hippocampal CA3 region, the mossy fibres, as well as the recurrent collaterals of pyramidal cells, probably use glutamate as their neurotransmitter (for review see Mayer & Westbrook, 1987). Subtypes of glutamate receptors may be pharmacologically distinguished by their sensitivity to the antagonists D-2-amino-5-phosphonovalerate (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Watkins & Olverman, 1987;, Drejer & Honore, 1988). During the course of experiments on epileptic discharge in CA3 pyramidal neurons of hippocampal slice cultures, we discovered that after blocking the GABAA receptor with high concentrations of bicuculline, partial inhibition of the non-NMDA excitatory amino acid receptor by low concentrations of CNQX altered the normal pattern of epileptic discharge and revealed an uncommon inhibitory potential with maximal amplitudes of as much as 30 mV. We furthermore observed that comparable inhibitory potentials were readily elicited after partial disinhibition with low concentrations of bicuculline alone. Portions of these data have been presented in abstract form to the Union of Swiss Societies for Experimental Biology (USGEB) (Scanziani, Giihwiler & Thompson, 1991).
PAROXYSMAL INHIBITORY POTENTIALS
377
METHODS
Preparation of slice cultures Slice cultures of rat hippocampus were prepared and maintained in vitro as described in detail elsewhere (Gahwiler, 1981). Briefly, 4- to 7-day-old rat pups were killed by decapitation. The hippocampus was isolated, and 400 ,um thick sections were cut on a tissue chopper under sterile conditions. The sections were mounted on glass cover-slips in a droplet of clotted chicken plasma, placed in sealed culture tubes containing 0-75 ml of semi-synthetic culture medium, and incubated on a slowly rotating roller drum at 36 'C. The medium consisted of 25 % horse serum, 50 % Eagle's basal medium, and 25 % of either Hanks' or Earle's balanced salt solution (GIBCO). After 2 weeks in vitro the slices become thinned to 1-2 cell layers, yet retain much of the organotypic cellular and synaptic organization of the intact hippocampus (Giihwiler, 1984).
Electrophysiological techniques Cultures were placed in a temperature-controlled (34 'C) recording chamber fixed to the stage of an inverted microscope, and continuously perfused with a modified Tyrode solution. This extracellular saline contained (in mM): Na+, 149; K+, 2-7; Ca2l, 3-8; Cl-, 151-153; Mg2+, 2-3; HCO3-, 11-6; H2PO4, 0 4; and glucose, 5-6. The pH was adjusted to 7-4 by bubbling with C02, and monitored with Phenol Red (10 mg/l). The chamber had a volume of 0-5 ml, and was perfused at a rate of 0-9 ml/min allowing for bath-applied drugs to reach a steady-state concentration in about 1 min. CA3 pyramidal cells were visually identified and impaled with microelectrodes containing either 1-2 M-potassium methylsulphate or 2 M-potassium chloride. The electrode resistances were 30-80 MQ. Current was passed through the recording electrode via an active bridge circuit. Bridge balance was ensured by periodically injecting hyperpolarizing current pulses. Membrane potential was amplified 100 x (Axoclamp 2, Axon Instruments), filtered at 2 kHz, and digitized at 0-5-2 kHz on-line using a Labmaster analog-digital converter (Tecmar Inc.). Data were acquired, stored on a hard disc, and analysed using pClamp software (Axon Instruments) running on a personal computer (COMPAQ Deskpro 386/20). Data were also recorded on a Gould chart recorder during the experiment. Synaptic responses were elicited with monopolar anodal stimulation (0-1 ms duration, 10-100 ,uA in amplitude) delivered, as selectively as possible, to the mossy fibre pathway by a microelectrode (filled with 155 mM-NaCl, 1-2 ,sm tip diameter) placed in the region of the hilus. Numerical data are presented as means + standard deviation, with n referring to the number of cells in which a given experiment was repeated. D-2-Amino-5-phosphonovalerate (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were purchased from Tocris Neuramin. Bicuculline methochloride (referred to simply as bicuculline in the text) was prepared from Fluka bicuculline. CGP 35 348 and FK 33-824 were gifts of CibaGeigy AG and Sandoz AG, respectively. RESULTS
In hippocampal slice cultures, stimulation of mossy fibre afferents to CA3 pyramidal neurons results in a triphasic postsynaptic potential consisting of a shortlatency EPSP followed by an early Cl--mediated and a late K+-mediated inhibitory postsynaptic potential (IPSP) (Thompson & Giihwiler, 1989; Maloufet al. 1990). The late IPSP was typically 5-10 mV in amplitude and had a duration of about 0-5 s (Fig. 3A). Complete block of the fast IPSP with high concentrations (40 /M) of the GABAA receptor antagonist bicuculline resulted in both spontaneous and stimulus-evoked, synchronized epileptic burst activity. In hippocampal slice cultures, epileptic discharges were 40-50 mV in amplitude, and had durations of 300-1000 ms, comparable to previous descriptions of the disinhibited hippocampus in situ and in vitro (Fig. lBa) (e.g. Johnston & Brown, 1981; Traub & Wong, 1982). Epileptic bursts were followed by a pronounced after-hyperpolarization of 5-15 mV in
M. SCANZIANI, B. H. GAHWILER AND S. M. THOMPSON
378
amplitude lasting for 20-40 s. Bath application of low concentrations (2-4 #M) of the non-NMDA excitatory amino acid receptor antagonist CNQX, produced slight changes in the waveform of the epileptic burst discharge (Fig. lBb) (see also Chamberlain, Traub & Dingledine, 1990). Unexpectedly, however, low concen-
A
Bicuculline, 40 uM a
b
c
10 mV
60 s
CNQX, 2 PM B a
b
c
10 mV
1400 m
Fig. 1. Effects of the glutamate receptor antagonist CNQX (2 /M) on bicuculline-induced epileptiform activity in hippocampal slice cultures. A, chart recording of membrane potential from a CA3 neuron. Responses elicited every 10 s with a stimulation electrode placed in the hilus. In 40 uM-bicudulline, before application of CNQX, every stimulation elicits an epileptiform burst discharge. Perfusion with CNQX reversibly increased the inter-burst interval and led to the appearance of stimulation-induced paroxysmal inhibitory potentials (PIPs) (narrow downward deflections). Note the regular alternation between bursts and gradually increasing PIPs. The depolarization of the cell upon perfusion of CNQX was due to the larger inter-burst interval. Summation of burst afterhyperpolarizations occurred before application of CNQX and caused a tonic hyperpolarization because the inter-burst interval was shorter than the duration of a burst after-hyperpolarization (V-shaped downward deflections). During perfusion of CNQX the membrane potential recovered due to the increased inter-btrst interval. B, same cell. Pairs of digitally recorded responses at enlarged time scale, corresponding to the labelled periods in A. Each pair of superimposed sweeps illustrates the response of the cell to two consecutive stimuli. The membrane potential before application of CNQX was -73 mV; KCI electrode. Action potentials truncated in this and subsequent figures.
trations of CNQX also resulted in the appearance of evoked responses which had not previously been observed under such conditions (Fig. 1). Whereas epileptic bursts were elicited with each stimulus in the presence of bicuculline, addition of CNQX resulted in the regular alternation of epileptic discharges and events consisting of an EPSP followed by a large, sustained hyperpolarization (n = 39). The
PAROXYSMAL INHIBITORY POTENTIALS
379
EPSPs were typically about 10 mV in amplitude and 100-200 ms in duration. The hyperpolarizations had maximal amplitudes of as large as 30 mV from the resting membrane potential (about -60 mV) and a duration of 0-5-2 s, i.e. considerably larger and longer than the control late IPSP (Fig. 3A), and were accompanied by a decrease in cell input resistance (not shown). B -55 mV
-5 20
~~~~~~~~~~~E
_
~~
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0
0
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0-
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00 0
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0a
0-
a~~~~~~~~~~~a o
z
0
13
PHY 444
384
M. SCANZIANI, B. H. GAHWILER AND
A t=
6
0
ISI 6 s
12
18
S.
M. THOMPSON
24
K----
ISI 12 s
ISI 24 s
A
Journal of Physiology (1991), 444, pp. 375-396 With 9 figures Printed in Great Britain
PAROXYSMAL INHIBITORY POTENTIALS MEDIATED BY GABAB RECEPTORS IN PARTIALLY DISINHIBITED RAT HIPPOCAMPAL SLICE CULTURES
BY MASSIMO SCANZIANI, BEAT H. GAHWILER AND SCOTT M. THOMPSON From the Brain Research Institute, University of Zurich, August Forel-Strasse 1, CH-8029 Zurich, Switzerland
(Received 13 March 1991) SUMMARY
1. Intracellular recording techniques were used to study synaptic potentials in CA3 pyramidal cells elicited with mossy fibre stimulation in partially disinhibited hippocampal slice cultures. Two experimental protocols were used: (1) high concentrations (20-40 gM) of the A-type y-aminobutyric acid (GABAA) receptor antagonist bicuculline plus low concentrations (2-4 /sM) of the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or (2) low concentrations (1-2 5 ,lM) of bicuculline alone. 2. Under the first condition, stimulation of mossy fibre afferents evoked epileptic bursts alternating with a response consisting of an excitatory postsynaptic potential (EPSP) followed by an unusually large and long-lasting hyperpolarizing potential with a maximal amplitude in the range of -30 mV from the resting membrane potential. 3. This paroxysmal inhibitory potential (PIP) had a reversal potential near that of potassium. The amplitude of the PIP was not dependent on action potentials superimposed on the preceding EPSP, and was present in cells recorded with microelectrodes containing the Ca2" chelator EGTA. These data suggest that the PIP is not a Ca2"-activated K+ potential. 4. The PIP was prolonged by the GABA-uptake blocker nipecotic acid, was reduced by hyperpolarizing interneurons with the opioid agonist FK 33-824, and was abolished by the GABAB-receptor antagonist CGP 35 348. These data indicate that the PIP is mediated by the activation of GABAB receptors following GABA release from interneurons. 5. The NMDA-receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) strongly reduced the amplitude of the PIP, but had no effect on the GABAB receptor-mediated inhibitory postsynaptic potential (IPSP) under control conditions. 6. Under the first condition, regular stimulation elicited a cyclical pattern of evoked responses. There was either an alternation between an epileptic burst and a PIP or, at shorter interstimulus intervals, a sequence of gradually increasing PIPs followed by an epileptic burst, which then reset the cycle. MS 9234
376
M. SCANZIANI, B. H. GAHWILER AND S. M. THOMPSON
7. Under the second condition, in low concentrations of bicuculline alone, the early GABAA-mediated IPSP was little affected, but the late GABAB-mediated IPSP was greatly enhanced. These enhanced late IPSPs were comparable in amplitude and duration to the PIPs seen under the first conditions, could exhibit cyclical behaviour, and were reduced by D-APV. 8. Application of CGP 35 348 abolished the late IPSP under control conditions, but had no effect on hippocampal excitability. In contrast, CGP 35 348 blocked the PIP elicited in low bicuculline, and consequently led to intense epileptic discharge. 9. We suggest that the amplification of GABAB receptor-mediated synaptic potentials after partial disinhibition may be an important endogenous compensatory mechanism preventing epileptogenesis.
INTRODUCTION
Inhibitory synaptic transmission in the hippocampus is mediated by the release of y-aminobutyric acid (GABA) from local circuit interneurons, which then activates two distinct receptors on pyramidal cells. GABAA receptors are blocked by bicuculline and directly gate Cl- permeable channels (Eccles, Nicoll, Oshima & Rubia, 1977) while GABAB receptors are bicuculline-insensitive and activate K+ channels (Hill & Bowery, 1981; Giihwiler & Brown, 1985; Newberry & Nicoll, 1985). Antagonists of GABAB receptors are reported to have no significant effect on hippocampal excitability (Dutar & Nicoll, 1988; Karlsson & Olpe, 1989; Olpe, Karlsson, Pozza, Brugger, Steinmann, Van Riezen, Fagg, Hall, Froestl & Bittiger, 1990; cf. Malouf, Robbins & Schwartzkroin, 1990). In contrast, in the absence of GABAA receptor-mediated inhibition, unchecked recurrent excitation between pyramidal cells allows activity to spread throughout the population, causing the cells to discharge synchronously (e.g. Dichter & Spencer, 1968; Johnston & Brown, 1981; Traub & Wong, 1982; Miles & Wong, 1987; Traub, Miles & Wong, 1989). Disinhibition with GABAA receptor antagonists thus leads to epileptiform discharge. The primary afferents to the hippocampal CA3 region, the mossy fibres, as well as the recurrent collaterals of pyramidal cells, probably use glutamate as their neurotransmitter (for review see Mayer & Westbrook, 1987). Subtypes of glutamate receptors may be pharmacologically distinguished by their sensitivity to the antagonists D-2-amino-5-phosphonovalerate (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Watkins & Olverman, 1987;, Drejer & Honore, 1988). During the course of experiments on epileptic discharge in CA3 pyramidal neurons of hippocampal slice cultures, we discovered that after blocking the GABAA receptor with high concentrations of bicuculline, partial inhibition of the non-NMDA excitatory amino acid receptor by low concentrations of CNQX altered the normal pattern of epileptic discharge and revealed an uncommon inhibitory potential with maximal amplitudes of as much as 30 mV. We furthermore observed that comparable inhibitory potentials were readily elicited after partial disinhibition with low concentrations of bicuculline alone. Portions of these data have been presented in abstract form to the Union of Swiss Societies for Experimental Biology (USGEB) (Scanziani, Giihwiler & Thompson, 1991).
PAROXYSMAL INHIBITORY POTENTIALS
377
METHODS
Preparation of slice cultures Slice cultures of rat hippocampus were prepared and maintained in vitro as described in detail elsewhere (Gahwiler, 1981). Briefly, 4- to 7-day-old rat pups were killed by decapitation. The hippocampus was isolated, and 400 ,um thick sections were cut on a tissue chopper under sterile conditions. The sections were mounted on glass cover-slips in a droplet of clotted chicken plasma, placed in sealed culture tubes containing 0-75 ml of semi-synthetic culture medium, and incubated on a slowly rotating roller drum at 36 'C. The medium consisted of 25 % horse serum, 50 % Eagle's basal medium, and 25 % of either Hanks' or Earle's balanced salt solution (GIBCO). After 2 weeks in vitro the slices become thinned to 1-2 cell layers, yet retain much of the organotypic cellular and synaptic organization of the intact hippocampus (Giihwiler, 1984).
Electrophysiological techniques Cultures were placed in a temperature-controlled (34 'C) recording chamber fixed to the stage of an inverted microscope, and continuously perfused with a modified Tyrode solution. This extracellular saline contained (in mM): Na+, 149; K+, 2-7; Ca2l, 3-8; Cl-, 151-153; Mg2+, 2-3; HCO3-, 11-6; H2PO4, 0 4; and glucose, 5-6. The pH was adjusted to 7-4 by bubbling with C02, and monitored with Phenol Red (10 mg/l). The chamber had a volume of 0-5 ml, and was perfused at a rate of 0-9 ml/min allowing for bath-applied drugs to reach a steady-state concentration in about 1 min. CA3 pyramidal cells were visually identified and impaled with microelectrodes containing either 1-2 M-potassium methylsulphate or 2 M-potassium chloride. The electrode resistances were 30-80 MQ. Current was passed through the recording electrode via an active bridge circuit. Bridge balance was ensured by periodically injecting hyperpolarizing current pulses. Membrane potential was amplified 100 x (Axoclamp 2, Axon Instruments), filtered at 2 kHz, and digitized at 0-5-2 kHz on-line using a Labmaster analog-digital converter (Tecmar Inc.). Data were acquired, stored on a hard disc, and analysed using pClamp software (Axon Instruments) running on a personal computer (COMPAQ Deskpro 386/20). Data were also recorded on a Gould chart recorder during the experiment. Synaptic responses were elicited with monopolar anodal stimulation (0-1 ms duration, 10-100 ,uA in amplitude) delivered, as selectively as possible, to the mossy fibre pathway by a microelectrode (filled with 155 mM-NaCl, 1-2 ,sm tip diameter) placed in the region of the hilus. Numerical data are presented as means + standard deviation, with n referring to the number of cells in which a given experiment was repeated. D-2-Amino-5-phosphonovalerate (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were purchased from Tocris Neuramin. Bicuculline methochloride (referred to simply as bicuculline in the text) was prepared from Fluka bicuculline. CGP 35 348 and FK 33-824 were gifts of CibaGeigy AG and Sandoz AG, respectively. RESULTS
In hippocampal slice cultures, stimulation of mossy fibre afferents to CA3 pyramidal neurons results in a triphasic postsynaptic potential consisting of a shortlatency EPSP followed by an early Cl--mediated and a late K+-mediated inhibitory postsynaptic potential (IPSP) (Thompson & Giihwiler, 1989; Maloufet al. 1990). The late IPSP was typically 5-10 mV in amplitude and had a duration of about 0-5 s (Fig. 3A). Complete block of the fast IPSP with high concentrations (40 /M) of the GABAA receptor antagonist bicuculline resulted in both spontaneous and stimulus-evoked, synchronized epileptic burst activity. In hippocampal slice cultures, epileptic discharges were 40-50 mV in amplitude, and had durations of 300-1000 ms, comparable to previous descriptions of the disinhibited hippocampus in situ and in vitro (Fig. lBa) (e.g. Johnston & Brown, 1981; Traub & Wong, 1982). Epileptic bursts were followed by a pronounced after-hyperpolarization of 5-15 mV in
M. SCANZIANI, B. H. GAHWILER AND S. M. THOMPSON
378
amplitude lasting for 20-40 s. Bath application of low concentrations (2-4 #M) of the non-NMDA excitatory amino acid receptor antagonist CNQX, produced slight changes in the waveform of the epileptic burst discharge (Fig. lBb) (see also Chamberlain, Traub & Dingledine, 1990). Unexpectedly, however, low concen-
A
Bicuculline, 40 uM a
b
c
10 mV
60 s
CNQX, 2 PM B a
b
c
10 mV
1400 m
Fig. 1. Effects of the glutamate receptor antagonist CNQX (2 /M) on bicuculline-induced epileptiform activity in hippocampal slice cultures. A, chart recording of membrane potential from a CA3 neuron. Responses elicited every 10 s with a stimulation electrode placed in the hilus. In 40 uM-bicudulline, before application of CNQX, every stimulation elicits an epileptiform burst discharge. Perfusion with CNQX reversibly increased the inter-burst interval and led to the appearance of stimulation-induced paroxysmal inhibitory potentials (PIPs) (narrow downward deflections). Note the regular alternation between bursts and gradually increasing PIPs. The depolarization of the cell upon perfusion of CNQX was due to the larger inter-burst interval. Summation of burst afterhyperpolarizations occurred before application of CNQX and caused a tonic hyperpolarization because the inter-burst interval was shorter than the duration of a burst after-hyperpolarization (V-shaped downward deflections). During perfusion of CNQX the membrane potential recovered due to the increased inter-btrst interval. B, same cell. Pairs of digitally recorded responses at enlarged time scale, corresponding to the labelled periods in A. Each pair of superimposed sweeps illustrates the response of the cell to two consecutive stimuli. The membrane potential before application of CNQX was -73 mV; KCI electrode. Action potentials truncated in this and subsequent figures.
trations of CNQX also resulted in the appearance of evoked responses which had not previously been observed under such conditions (Fig. 1). Whereas epileptic bursts were elicited with each stimulus in the presence of bicuculline, addition of CNQX resulted in the regular alternation of epileptic discharges and events consisting of an EPSP followed by a large, sustained hyperpolarization (n = 39). The
PAROXYSMAL INHIBITORY POTENTIALS
379
EPSPs were typically about 10 mV in amplitude and 100-200 ms in duration. The hyperpolarizations had maximal amplitudes of as large as 30 mV from the resting membrane potential (about -60 mV) and a duration of 0-5-2 s, i.e. considerably larger and longer than the control late IPSP (Fig. 3A), and were accompanied by a decrease in cell input resistance (not shown). B -55 mV
-5 20
~~~~~~~~~~~E
_
~~
o~~~~~~~~~c 0
0
0
o~~~~~~~~~~~
0-
0~~~~~~~~~~~~~~~~ C
0
00 0
C.)~~~~~~~~~~~~
0a
0-
a~~~~~~~~~~~a o
z
0
13
PHY 444
384
M. SCANZIANI, B. H. GAHWILER AND
A t=
6
0
ISI 6 s
12
18
S.
M. THOMPSON
24
K----
ISI 12 s
ISI 24 s
A
