Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices.

JOURNALOFNEUROPHYSIOLOGY Vol. 65, No. 4, April 1991. Printed

In L’.S.A.

Physiology and Pharmacology of Epileptiform Activity Induced by 4-Aminopyridine in Rat Hippocampal Slices PERREAULT AND MASSIMO AVOLI Montreal Neurological Institute and Department ofNeurology Montreal, Quebec, H3A 2B4, Canada PAUL

SUMMARY

AND

CONCLUSIONS

1. Conventional intracellular and extracellular recording techniques were used to investigate the physiology and pharmacology of epileptiform bursts induced by 4-aminopyridine (4-AP, 50 /IM) in the CA3 area of rat hippocampal slices maintained in vitro. 2. 4-AP-induced epileptiform bursts, consisting of a 25- to 80ms depolarizing shift of the neuronal membrane associated with three to six fast action potentials, occurred at the frequency of 0.6 1 t 0.29 (SD)/s. The bursts were generated synchronously by CA3 neurons and were triggered by giant excitatory postsynaptic potentials (EPSPs). A second type of spontaneoys activity consisting of a slow depolarization also occurred but at a lower rate

and Neurosurgery,

McGill

University,

smallnonsynchronousEPSPsmediatedby non-NMDA receptors are involved in the generationof the burstsin 4-AP.

INTRODUCTION

The in vitro slice preparation has provided neuroscientists with a very powerful technique to study in detail some of the cellular phenomena underlying focal epilepsy. Throughout the years, various experimental models have emerged based on the ability of certain drugs or ionic media to generate paroxysmal depolarization shifts (PDS) that rep(0.04 t 0.2/s). resent the cellular correlate of interictal spikes seen on the 3. The effectsof 4-AP on EPSPsand inhibitory postsynaptic (EEG) of epileptic patients (Matsupotentials(IPSPs)evokedby mossyfiber stimulation werestudied electroencephalogram moto and Ajmone Marsan 1964). Although the variety of on neuronsimpaled with a mixture of K acetateand 2(triethylamino)-N-(2,6-dimethylphenyl) acetamide(QX-3 14)-filled mi- models that have been used to date do not allow us to design croelectrodes.After the addition of 4-AP, the EPSPbecamepoten- any unifying theory of focal epileptogenesis, it is generally tiated and wasfollowed by the appearanceof a giant EPSP.This accepted that this approach remains very useful for undergiant EPSPcompletely obscuredthe early IPSP recorded under standing some of the mechanisms of neuronal hyperexcitcontrol conditionsand inverted at -32 * 3.9 mV (n = 4), suggest- ability that might be involved in the generation of the epiing that both inhibitory and excitatory conductanceswere in- leptiform discharges. volved in itsgeneration.IPSPsevokedby SchaffercollateralstimuMany factors have been identified as being critical for the lation increasedin amplitudeandduration after 4-AP application. generation of the PDS: I) reduction of inhibition; 2) poten4. The spontaneous field burstsand the stimulus-inducedgiant tiation of excitatory postsynaptic potentials (EPSPs); 3) acEPSPinducedby 4-AP werenot affectedby N-methyl-D-aspartate (NMDA) receptors; and (NMDA) receptor antagonists3-3 (2-carboxy piperazine-4-yl) tivation of N-methyl-D-aspartate propyl- 1-phosphonate(CPP)and DL-2-amino-5-phosphonovaler- 4) mechanisms of synchronization such as recurrent synapate(APV) but wereblockedby quisqualate/kainatereceptorantag- tic excitation, changes in extracellular ionic concentration, onists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7- ephaptic effects, and electrotonic coupling (for review see dinitroquinoxaline-2,3-dione(DNQX). CNQX alsoabolishedthe Delgado-Escueta et al. 1986). The relevance of each of these presenceof smallspontaneouslyoccurringEPSPs,therebydisclos- factors in focal epileptogenesis, however, remains very coning the presenceof bicuculline-sensitive(BMI, 20 PM) IPSPs. troversial because their relative contribution varies greatly 5. Small, nonsynchronousEPSPsplayed an important role in depending on the model being studied. For instance, althe generationof 4-AP-induced epileptiform activity. 1) After the though NMDA receptors have been shown to be involved addition of 4-AP, smallEPSPsappearedrandomly on the baseline activity in some experiand then becameclusteredto producea depolarizingenvelopeof in the production of epileptiform mental models of epilepsy (Meldrum 1987), NMDA antagoirregular shapethat progressivelyformed an epileptiform burst. 2) ThesesmallEPSPsweremore numerousin the 100msperiodthat nists have no effect on the epileptiform discharges seen with precededburst onset. 3) The frequency of occurrence of small other models (Neuman et al. 1988; Thomson and West EPSPswaspositively correlatedwith the frequency of occurrence 1986), suggesting that NMDA receptors are not necessary of synchronousbursts.4) Small EPSPsand burstswere similarly for PDS generation. Similarly, the importance of reduced decreasedafter the addition of different concentrationsof CNQX synaptic inhibition is not yet really clear. On the one hand, (I& in both casesof = 1.2 ELM). there is evidence indicating that many convulsant drugs 6. The postsynapticresponseof neuronsto ionophoresedy- studied experimentally reduce synaptic inhibition meaminobutyric acid (GABA) and glutamatewasnot changedafter diated by y-aminobutyric acid (GABA) (for review see the addition of 4-AP, implying that the potentiation of synaptic Avoli 1988). On the other hand, these data have been chaltransmissionproducedby 4-AP resultsprimarily from a presynaplenged by recent findings indicating that GABAergic inhibitic mechanism. 7. We concludethat 4-AP-induced epileptiform activity isgen- tion is not impaired in other models such as exposure to low erated by giant EPSPsthat occur despitethe presenceof poten- Mg2+ (Tancredi et al. 1990), mast cell degranulating peptide tiated synaptic inhibition. Furthermore, we demonstratedthat (Cherubini et al. 1988), 4-aminopyridine (4-AP; Perreault 0022~3077/9 1 $1.50 Copyright 0 199 1 The American Physiological Society

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P. PERREAULT

and Avoli 1989a; Rutecki et al. 1987), and tetanic stimulation (Higashima 1988). We became interested in 4-AP as a model of focal epilepsy for several reasons. First, 4-AP has been shown in both in vivo (Szente and Baranyi 1987) and in vitro experiments (Chestnut and Swann 1988; Galvan et al. 1982; Rutecki et al. 1987; Voskuyl and Albus 1985) to readily evoke epileptiform activity when applied or injected in low concentrations. The convulsant properties of 4-AP have also been reported clinically in humans (Spyker et al. 1980; Thesleff 1980). Second, unlike other convulsant drugs that act primarily by diminishing the efficiency of GABA-mediated inhibition (Avoli 1988), the evidence available indicates that 4-AP-induced epileptiform discharges occur despite the presence of normal or even enhanced synaptic inhibition (Chestnut and Swann 1988; Perreault and Avoli 1989a; Rutecki et al. 1987). 4-AP may therefore provide a suitable model to investigate the pathophysiological mechanisms involved in the generation of epileptiform activity in conditions where synaptic inhibition is preserved. Third, 4-AP is known primarily as a blocker of K+ currents, mainly of the early transient K+ current or A-current (for review see Rudy 1988), although other evidence suggests it might affect Ca*’ currents as well (Segal and Barker 1986). In the soma-dendrite region, these effects of 4-AP on intrinsic conductances lower the threshold and reduce the latency for action potential generation (Rudy 1988), whereas at presynaptic terminals, the result is a facilitation of neurotransmitter release (Thesleff 1980). It appeared of interest, therefore, to investigate also the relative importance and possible relationship of these pre- and postsynaptic effects of 4-AP in the generation of epileptiform discharges. In this report we show that epileptiform bursts generated by CA3 hippocampal neurons perfused with 4-AP occur while both inhibitory and excitatory synaptic potentials are potentiated. Furthermore, we demonstrate the role played by small nonsynchronous EPSPs mediated by quisqualate/ kainate receptors in the initiation of these spontaneous discharges. Part of these results have been communicated at a recent meeting of the Society for Neuroscience (Perreault and Avoli 1989a).

AND

M.

AVOLI

in the CA3 region than thosethat were slicedfrom hippocampi manipulatedwith the alvear sidefacing up and wherea goodpart of the CA3 regionwasin contact with the filter paperduring dissectionand slicing. 4-AP was added to the perfusing solution to achieve a final concentration of 50 PM. In some experiments, tetrodotoxin (TTX, 1 PM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 0.5-4.0 PM), 6,7-dinitroquinoxaline-2,3-dione (DNQX, 0.5-5 ELM),3-3 (2-carboxy piperazine-4-yl)propyl- 1-phosphonate(CPP, l-5 PM), DL-2-amino-5-phosphonovalerate (APV, IO- 100PM), and bicuculline methiodide(BMI, 50 PM) were alsoaddedto the bathing medium. Drugs usedfor ionophoresisincluded GABA and glutamate. GABA (1 mM) wasdissolvedin distilled water and the pH of the solution adjustedto 3.0 with 1 M HCl, whereasL-glutamate(200 mM) wasdissolvedin distilled water and the pH adjustedto 8.0 with 1M NaOH. Selectiveionophoreticapplication of GABA was achievedby usingsolution-filled micropipettes(2- to 5-pm diam) positionedin the stratum (s.) pyramidale, closeto the recording site,to obtain somatichyperpolarization (H-response)or in the s. radiatum to evokedendritic depolarization(D-response)(cf. Alger and Nicoll 1982;Andersenet al. 1980).Glutamate responses were obtained by positioning the ionophoretic pipette in the s. radiaturn (proximal one-third, usually)and by moving it a few micrometersup or down to find a “hot spot” where the amplitude of the responsewasmaximal (Schwartzkroin and Andersen 1975).Experimentswerestartedafter stabilization of that response.All the chemicalsusedwere acquiredfrom Sigmawith the exception of CPP and CNQX, which were obtained from Tocris Neuramine, and QX-3 14,which wasobtained from Astra.

Recording and stimulation

Conventional extra- and intracellular recording techniques were employedin the experimentsreported here.Electrodesused for intracellular recordingswere pulled from capillary tubes(1.5 mm OD) and filled with one of the following solutions: 4 M K acetate,2 M CsCl,or a mixture of K acetate(4 M) and 2(triethylamino)-N-(2,6=dimethylphenyl)acetamide (QX-3 14; 50 mM). After the cell had stabilized from impalement, QX-3 14 was ejectedby passingbrief depolarizingcurrent pulses(0.8- 1.5 nA) for severalminutes until action potentials were abolished.The final resistanceof the electrodesvaried between35 and 70 MR For extracellular recording, low-impedanceelectrodes(2- 10Mfi) were filled with 2 M NaCl. Intra- and extracellular signalswere fed to a high-impedance negative-capacitanceamplifier with standardbridge circuit that METHODS allowedcurrent to be passedthrough the intracellular recording electrode.The bridge wasmonitored carefully throughout the exPreparation and incubation ofthe slices . perimentsand adjustedasnecessary.The signalsweredisplayed Male Sprague-Dawleyrats weighing200-300 g were anesthe- on a Gould chart recorder and oscilloscopeand in somecases tized with ether and decapitated,and their brains were quickly recordedon tape for later analysis.Intracellular recordingswere removedfrom the skulls.The two hemispheres wereseparatedand obtainedfrom cellslocatedin CA3b and CA3c regions(Lorente de placedin cold (8- 10°C)artificial cerebrospinalfluid (ACSF). The No 1934).The intracellular resultsshownin this study weretaken hippocampusof each hemispherewasdissectedfree and sliced from a total of 70 neuronsimpaledfor periodslastingfrom 20 min transverselyinto 400-pm-thick sectionsusing a McIlwain tissue to 4 h and displaying resting membranepotential (vm) greater chopper. After cutting, the sliceswere transferred to a tissue than -55 mV (65.4 t 9.5 mV, mean t SD; n = 44), input resischamberwherethey were kept at the interface betweenthe oxy- tance > 15 MS2(35.2 t 16.1 Mfi, y1= 43), and action potential genatedACSF (34-35°C) and humidified atmospheregassed with amplitude ~80 mV (95.3 t 10.7mV, n = 30). 95%0,5% C02. The sliceswereperfusedcontinuously at a rate of Mossy fiber stimulationswere delivered through bipolar stain0.3-0.5 ml/min with an oxygenatedACSF of the following com- lesssteelelectrodesplacedin the upper bladeof the dentategranposition (in mM): 124NaCl, 2 KCl, 1.25 KH,PO,, 2 MgS04, 2 ule cell body layer or in the hilar region of the dentate area, CaCl,, 26 NaHCO,, and 10 glucose(pH 7.4). To improve the whereasstimulation of the Schaffercollateralswascarried out by viability of the CA3 neurons,specialprecautionswere taken dur- placing the electrodein the s. radiatum of the CA1 region. CA3 ing the manipulationsof the excised hippocampi to keep their neuronswerealsoactivated by stimulatingthe outer portion of the alvear sideunderneath,facing the wet filter paper.The slicesob- white matter forming the fimbria. Stimulation intensity varied tained in this way usuallydisplayedbetter field potential responses between 10and 200 PA (duration 50-90 ps).


4-AP

AND

EPILEPTIFORM

RESULTS

Characteristics

of 4-AP-induced

epileptiform

bursts

Addition of 4-AP (50 PM) to the ACSF led to the appearante of spontaneous synchronous epileptiform bursts in the CA3 area of the hippocampus that were similar to those described in previous studies (Rutecki et al. 1987; Voskuyl and Albus 1985). As shown in Fig. IA, 4-AP-induced bursts were rather brief (55.4 t 19.2 ms, n = 11; range 25-80 ms) and appeared at the frequency of 0.6 1 t 0.29/s (n = 38; range 0.2- 1.4/s), which was quite fast compared with other convulsant models (Miles and Wong 1983; Neuman et al. 1989; Swann et al. 1986). In addition, 4-AP induced the appearance of a second type of spontaneous activity that corresponded to a slow and long-lasting depolarization often preceded by a burst (Fig. lA, right expanded trace). This second type of activity occurred at a slower rate than the epileptiform bursts (Fig. IA, *) and could be recorded simultaneously in all regions of the slice (not shown). It corresponds to a GABA-mediated depolarization similar to the one reported in CA 1 (Perreault and Avoli 1989b) and will be discussed in a separate paper (as-yet-un-

773

ACTIVITY

published data). These effects of 4-AP could be washed within 30-90 min after the reintroduction of normal ACSF. When recorded extracellularly in the cell body layer, the epileptiform bursts consisted of a series of three to eight population spikes of variable amplitude, riding on a positive wave (Fig. lB, bottom trace). Population spikes, however, were not always as sharp and clearly defined as those seen with other models of epilepsy such as penicillin or low Mg2+ (Swann et al. 1986; Tancredi et al. 1990). Simultaneous intracellular and extracellular recordings revealed that field bursts were correlated with a slow depolarization of the neuron’s membrane with overriding fast action potentials that corresponded to population spikes on the extracellular record (Fig. 1B). As shown in the example of Fig. 1B, in = 10% of the cells studied, intracellular bursts were preceded by one to three action potentials that were not synchronous, as indicated by the absence of correlated population spikes ( L ). These action potentials were triggered by brief depolarizations, presumably EPSPs. Similar bursts could be evoked by stimulating the mossy fibers, the fimbria, and in most slices the Schaffer collaterals. This activity usually propagated to the CA 1 region where small bursts or EPSP-inhibitory postsynaptic potential (IPSP) sequences

* LL

P

I20 mV

2s

,

200 ms

FIG. 1. Characteristics of 4-AP-induced epileptiform bursts in the CA3 area of the hippocampus. A, top: a low-speed chart record showing the spontaneous activity elicited by 4-AP application in a CA3c neuron. In addition to the brief bursts that occurred at a fast rate, a less frequent slow depolarization also occurred (e). Examples of these 2 types of activity are shown below at faster time base. B: simultaneous intracellular (top) and extracellular (bottom) recordings obtained from the CA3c s. pyramidale of a slice bathed in the presence of 4-AP showing that the intracellularly recorded burst coincides with an extracellular burst. However, the initial action potentials generated by the cell ( I) were not synchronous, as indicated by the lack of corresponding population spike on the extracellular trace. C: examples of spontaneous bursts occurring at rest (top) and at postsynaptic membrane potential (vm) of -22 mV (middle) and -46 mV (bottom) relative to rest demonstrate the presence of a high amplitude and prolonged EPSP mediating the epileptiform discharge. Neuron was hyperpolarized by passing negative DC current through the recording electrode. Action potential amplitude is reduced in the top trace in A because of the slow time response of the pen. Action potentials are cut in C( -22 and -46 mV traces). Rest vm of the cell in B and C was -66 and -77 mV, respectively. vm of the cell in A is not available. Concentration of 4-AP used for experiments shown in this figure was 50 PM; the same applies for all other figures as well.


774

P. PERREAULT

AND M. AVOLI

shown in Fig. 2A, responses to mossy fiber stimulations were evoked at various membrane potentials obtained by passing negative and positive DC current through the intracellular recording electrode. In control, mossy fiber stimulations generated a small-amplitude EPSP followed by an IPSP that inverted near rest (Fig. 2A, fe#). After 4-AP application, the amplitude of the EPSP increased and was followed by a high amplitude and prolonged depolarization that completely obscured the IPSP seen in control, indicating the latter was either blocked by 4-AP application or simply obscured by the overlying, prolonged depolarizing envelope. These changes can be further appreciated graphiEficts oS4-AP on evoked EPSPs and IPSPs . cally in Fig. 2B, where the responses were measured at 8 ms Previous evidence has indicated that 4-AP application to (0, 0) and 25 ms (1, 0) after stimulation. In control these the hippocampus produces a facilitation of both inhibitory time latencies corresponded to the peak of the EPSP and and excitatory synaptic pathways (Buckle and Haas 1982; the peak of the early IPSP, respectively. The extrapolated meaPerreault and Avoli 1989b; Rutecki et al. 1987). To assess reversal potential of the prolonged depolarization further the changes in synaptic potentials produced by 4- sured at peak (Fig. 2A, $) was -29 mV (-32.8 t 3.9 mV, n = 4 cells), indicating therefore a 4 I-mV depolarizing shift of AP at the time when epileptiform activity occurs, we monitored the orthodromic responses evoked in individual CA3 the reversal potential of the IPSP. Its amplitude also displayed a linear relationship with the membrane potential, neurons before and after 4-AP application. To obtain more reliable measures of synaptic potentials, we impaled cells indicating the whole depolarizing envelope corresponded with QX-3 14 microelectrodes, preventing the generation of to a synaptic potential or a giant EPSP. The reversal potential of this giant EPSP was indeed more negative than what action potentials and associated afterhyperpolarizations (Connors and Prince 1982). These currents overlap nor- would be expected from a pure EPSP that inverts near zero mally with the synaptic potentials and make their measurein CA3 pyramidal cells (Brown and Johnston 1983), suggesting that inhibitory synaptic conductances are also inment and interpretation more difficult. In the example

were recorded. Note also that the long-lasting depolarization mentioned above followed the burst evoked by low-frequency (co.5 Hz) mossy fiber stimulations. Hyperpolarization of the neuron’s membrane by intracellular injection of DC current revealed that the prolonged depolarization underlying the burst increased progressively in amplitude as the membrane was hyperpolarized (Fig. 1C). Therefore the 4-AP-induced epileptiform discharges were triggered by giant EPSPs (Rutecki et al. 1987). The frequency of occurrence of the spontaneous bursts was insensitive to the membrane potential (not shown).

A

= 4AP $

Control

A

40 ms oControl

25 mV

(8 ms)

l 4AP (8 ms) OControl

(25 ms)

n 4AP (25 ms)

Membrane Control

Potential

(mV)

FIG. 2. Effects of 4-AP on mossy fiber- and Schaffer collateral-evoked responses. .4: responses to mossy fiber stimulation were evoked at various I$, in a CA3c neuron impaled with an electrode filled with a mixture of K acetate (4 M) and QX-314 (50 mM). In control (left), note the presence of an EPSP followed by an IPSP that inverted near resting level (RL). After 4-AP application (r&$rt), evoked responses consisted of high amplitude and prolonged depolarizations that completely obscured the IPSP recorded in control. B: graph of the relationship between the amplitude of the responses measured at 8 ms (o,o) and 25 ms (m,n)after the stimulus and the membrane potential of the cell before (0.0) and after 4-AP (0-m). Extrapolated reversal potential of the response measured at the peak of the giant EPSP was -29 mV (J), indicating a 4 1-mV positive shift of the reversal potential of the early IPSP measured in the control. Resting F’mof the cell was -68 mV. C: stimulation of the Schaffer collaterals in control evoked an IPSP that lasted -60 ms (/@). In the presence of 4-AP (riglzr) the same stimulation produced an initial IPSP followed by a slow depolarization and a longlasting IPSP, indicating a clear potentiation of inhibitory synaptic potentials. Arrow indicates a truncated late action potential that arose abruptly from the peak of the early IPSP. Resting I/m of the cell was -60 mV.

4AP


4-AP

AND

EPILEPTIFORM

volved in the generation of the stimulus-induced bursts. This characteristic of the bursts induced by 4-AP is not shared by other convulsants such as picrotoxin (Johnston and Brown 198 1) or NMDA-containing (Neuman et al. 1989) or kainate-containing ACSF (Ben Ari and Gho 1988), where the PDS was shown to invert near 0 mV. Findings similar to ours were obtained by Rutecki et al. (1987) with the voltage-clamp technique. We analyzed in greater detail the effects of 4-AP on synaptic inhibition by monitoring the IPSP evoked by stimulating the Schaffer collaterals (n = 5 cells). As shown in Fig. 2C, after 4-AP application the IPSP increased in amplitude and duration and acquired a complex triphasic shape similar to the sequence of GABA-mediated synaptic potentials recorded in CA1 under similar conditions (Perreault and Avoli 1989b). Typically, the responses were composed of an early IPSP followed by a late depolarization and a late IPSP. Schaffer collateral stimulations could also evoke bursts that preceded this sequence of inhibitory potentials. These findings provided an additional indication that bursting activity occurred despite the presence of a potentiated synaptic inhibition.

A

ACTIVITY

775

Eflects of excitatory amino acid receptor antagonists on epileptiform activity induced by 4-AP Results obtained from several models of epilepsy, both in vivo and in vitro, have emphasized the important role played by NMDA receptors in the generation of epileptiform discharges. To test the possibility that NMDA receptors are involved in the 4-AP model, we analyzed the effects of NMDA receptor antagonists CPP (1-5 PM) and APV (lo- 100 PM) on spontaneous and evoked bursts. The involvement of other excitatory amino acid receptors was also assessedby use of CNQX and DNQX, which are antagonists of quisqualate and kainate receptors (Honor-e et al. 1988). Figure 3A illustrates the effects of CPP and CNQX on spontaneously occurring epileptiform bursts recorded with an extracellular electrode positioned in the s. pyramidale of CA3c. Positive-going deflections that appear in those traces correspond to spontaneous bursts, whereas negativegoing deflections (3 in each sample) correspond to the slow GABAergic potential mentioned above. When CPP (5 PM) was added to the perfusing solution, the frequency of the spontaneous activity remained unchanged (n = 5 slices).

Control

(4AP 50 pM)

,...

.,..I ...., .

-

.,...

CPP (5 FM)

Wash CPP

-

0.8

HP< 0.01

tii =

FIG. 3. Effects of excitatory amino acid receptor antagonists on 4-AP-induced spontaneous bursts. A: lowspeed chart records of spontaneously occurring activity recorded extracellularly in the s. pyramidale of the CA3c area of a hippocampal slice. Upward-going potentials in each record correspond to spontaneous bursts, whereas each downward bar (3 in each record) reflects the occurrence of a slow GABA-mediated depolarization (see text). Records on the rig& show examples of single bursts displayed at low chart speed that corresponded to the trace on the kifi. As shown in the 2nd truck’,CPP application had basically no effect on the spontaneous burst. After CPP washout (rid&) the application of CNQX (4 PM) produced a complete blockage of the spontaneous epileptiform activity. Slow potentials, however, were not sensitive to CNQX, as indicated by the persistence of the downward deflections. CNQX effects were partially washable (hottorn). B: graph of the effects of CPP and CNQX on the frequency of occurrence of the epileptiform bursts.

0.6

Control

CPP 5 PM

CPP Wash

CNQX 4 ctM

CNQX Wash


776

P. PERREAULT

AND M. AVOLI

CNQX (9-15 PM; n = 4 cells) to be blocked completely. These findings indicated, therefore, that a similar receptor type was involved in both the early and the late part of the response, but also raised the possibility that they were mediated by different mechanisms. The higher sensitivity of the evoked giant EPSP to antagonists, combined with their longer latency, suggested that they were mediated polysynaptically. Moreover, the fact that spontaneously occurring bursts were equally sensitive to low concentrations of CNQX suggests that polysynaptic connections might be critical for the generation of spontaneous activity as well. Blocking the late part of the giant EPSP also uncovered an IPSP, indicating this potential clearly overlapped with the depolarizing envelope seen in control (Fig. 4A). With higher concentrations of CNQX (9- 15 PM), an IPSP without any visible EPSP could be elicited (Fig. 4A), presumably as a result of direct activation of inhibitory interneurons (Davies and Collingridge 1989; Neuman et al. 1988). These data confirm the results obtained in the previous section, indicating that both inhibitory and excitatory synaptic inputs participate in the generation of the bursts in 4-AP. A further indication that inhibition was increased in the presence of 4-AP was obtained by analyzing the effects of CNQX on spontaneous, baseline synaptic activity (n = 8 cells, Fig. 5). As shown in the intracellular recordings of Fig. 5A, synaptic potentials were small in amplitude (~2 mV) in control ACSF. However, after the application of 4-AP, a pronounced, nonsynchronous, baseline activity composed

Furthermore, the shape of individual bursts was not affected either, as demonstrated by the example shown on the right expanded trace. On the contrary, application of CNQX (4 PM) produced a complete blockage of epileptiform bursts, an effect that was only partially washable (n = 8 slices). The effects of these antagonists on burst frequency are summarized graphically in Fig. 3B. In all the experiments analyzed, the frequency of spontaneous slow potentials was not affected by either CPP or CNQX. To characterize further the effects of excitatory amino acid antagonists on the epileptiform bursts induced by 4AP, we investigated the action of CPP, APV, and CNQX on the high-amplitude EPSP that triggers the burst. Cells were impaled with QX-3 14-filled microelectrodes and responses were evoked by stimulating the mossy fibers. Bath applicationofC P(1-5~M,n=6cells)orAPV(10-100~M,n= 3 cells) had no significant effect on the amplitude and the duration of this depolarizing potential (Fig. 4A). In comparison, low concentrations of CNQX (~5 PM), which readily abolished spontaneous events, caused a complete blockage of the late part of the response corresponding to the giant EPSP. The early EPSP that appeared on the initial part of the response was not affected by low concentrations of CNQX or DNQX (n = 9 cells, Fig. 4A). This can be further appreciated graphically in Fig. 4, B and C, where the EPSP amplitude was measured as a function of membrane potential at 5 and 20 ms time latency after the stimulus. As can be seen, the early EPSP required higher concentrations of

A

Control (4AP 50 pM)

CPP 2.5 pM

CNQX 3.0 pM

CNQX 9.0 pM

A

A

I

20 ms

C

Measured at 5 ms

I

1

I

-160

-120

-80

9 E

100

A CNQX

3.0 pM

;;;

80

A CNQX

9.0 pM

2.-c,

60

1

$

Measured at 20 ms

120

0 Control l CPP 2.5 pM

-40

Membrane Potential (mV)

A

-160

-120

-80

A> -40

Membrane Potential (mV)

FIG. 4. Effects of excitatory amino acid receptor antagonists on mossy fiber-induced giant EPSPs. A: neuron was impaled with a microelectrode filled with 4 M K acetate/50 mM QX-3 14 to block fast action potentials that are generated during the bursts. Traces shown in each part of the figure display the neuron’s responses to mossy fiber stimulation at 3 different ym. lMidci/c trace in each set shows the response obtained at rest. In control (left) mossy fiber stim: elation evoked a giant EPSP that increased in amplitude as the membrane was hyperpolarized. After the addition of CPP (2.5 PM) to the perfusing medium, the responses remained unchanged or appeared slightly depressed at depolarized levels. However, when CNQX (3.0 PM) was added after CPP washout, the late part of the depolarizing response was blocked, thus revealing the presence of an underlying IPSP. Higher concentration of CNQX (9.0 PM) produced a complete blockage of the response (right). B: relationship between the amplitude of the giant EPSP, measured at 5 ms after stimulus, and the I’m of the neuron in control (0) and after the addition of 2.5 PM CPP (o), 3.0 PM CNQX (A) and 9.0 PM CNQX (A). Cis similar to B, although the amplitude of the responses was measured at 25 ms latency, which corresponded approximately to the peak amplitude of the giant EPSP. Note the higher sensitivity of the late part of the giant EPSP to low concentration of CNQX compared with the initial part of the response. Resting level of the cell was -55 mV.


4-AP

A

AND

EPILEPTIFORM

Control

ACTIVITY

D

ji

777

4AP + CNQX + BMI

4AP

4AP + CNQX

4AP + TTX

FIG. 5. Spontaneously occurring small synaptic potentials in the presence of 4-AP are composed of both EPSPs and IPSPs. A: spontaneous activity recorded intracellularly in control medium. B: small spontaneous potentials in 4-AP. The 2 traces show activity that occurred during 2 successive interburst intervals. C: addition of CNQX (3.0 PM) to the bathing medium abolished the small EPSPs and uncovered numerous IPSPs. D: IPSPs shown in C were blocked by the addition of BMI (20 PM), indicating they were mediated by GABA, receptors. E: spontaneous activity recovered partially after the washout of CNQX and BMI but was completely blocked after the application of 1 ,uM TTX (F). All the traces shown in this figure were taken from the same CA3 neuron, which had a rest Vm of -56 mV.

of EPSPs and IPSPs ( 1- 10 mV in amplitude) was recorded during the interburst intervals (Fig. 5B). The addition of CNQX to the perfusing medium caused a complete blockage of the EPSPs, allowing small IPSPs to be more clearly appreciated. By comparing Fig. 5, A and c‘, one can appreciate that this tonic inhibition was much more prominent in the presence of 4-AP than in control. As expected, the amplitude of these IPSPs increased as the membrane was depolarized and decreased at hyperpolarized levels, confirming their synaptic origin (not shown). Furthermore, they were blocked by the application of 20 PM BMI (Fig. 5D), indicating GABA, receptors were primarily involved in their generation. The remaining small hyperpolarizations left after BMI application could involve GABA, receptors or the receptors of some other putative inhibitory neurotransmitters. Figure 5E shows the partial recovery of the spontaneous potentials after CNQX and BMI washout. The spontaneous activity recorded in the presence of 4-AP was blocked by adding TTX (0.5-l .O PM) to the perfusing medium, indicating that it was caused by the spontaneous firing of neurons (Fig. 5F). Small nonsynchronous EPSPs and the initiation spontaneous epileptiform bursts

of

The contribution of EPSPs arising from newly revealed polysynaptic excitatory connections to the development of

synchronous firing has been demonstrated by Miles and Wong (1987) in the hippocampal slice treated with picrotoxin, a convulsant drug that suppresses inhibition. The possibility that small spontaneous EPSPs could play a role in the development and occurrence of 4-AP-induced epileptiform bursts was therefore investigated in a series of experiments in which we examined the changes in spontaneously occurring EPSPs after 4-AP application and the relationship between spontaneous EPSPs and synchronous bursts. We also investigated the influence of single cell firing on synchronous activity. In 12 cells analyzed, epileptiform activity did not appear abruptly after 4-AP application, but rather developed progressively after a gradual series of changes in the amplitude, number, and distribution of spontaneous EPSPs (Fig. 6). Initial changes recorded after the application of 4-AP consisted of the appearance of random spontaneous potentials, mainly EPSPs, although in some cells IPSPs were more prominent than EPSPs (Fig. 6A, 10 min). Later, EPSPs that were sparsely distributed at the beginning gradually appeared in clusters of three to four single events (Fig. 6, A, 12 min, and Ba). As more EPSPs became recruited and summated within each cluster, an irregular and more prolonged depolarizing envelope was recorded (Fig. 6, A, 15 min, and Bb). This activity became progressively better organized and led eventually to the appearance of typical bursts triggered by a high-amplitude EPSP where unitary events were


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A Control 4AP

15

I JlOmV

Ic

C

v

I\

-I

10mV

100ms

FIG. 6. Appearance of epileptiform bursts in 4-AP follows gradual changes in the amplitude and distribution of EPSPs. Traces shown in .4 were taken from the same neuron in control (top) and at various times after the exposure to 50 PM 4-AP. B shows the events labeled a-c on traces in A but expanded for clarity. A, top: small baseline spontaneous activity recorded before adding 4-AP. After 10 min ( 10’) 4-AP, small random fluctuations of membrane potential indicate the presence of small EPSPs. At 12 min (12’), spontaneous EPSPs were increased in amplitude and appeared in small clusters, as can be further appreciated in Ba. After 15 min (15’), higher amplitude and more prolonged depolarizations were recorded. As shown in the expanded trace in Bb, the depolarizing envelope displayed a very irregular morphology where contributing single events could be easily distinguished. This activity became progressively more organized and at 20 min (20’), when epileptiform bursts appeared. Note in Bc that, compared with the depolarizations seen at 15 min, the depolarizing envelope in this case had a higher amplitude but was slightly reduced in duration. Rest vm of the cell was -76 mV.

not easily distinguishable (Fig. 6, A, 20 min, and Bc). These observations are compatible with the view that, as synaptic transmission is potentiated, EPSPs grow in amplitude and cellular activity spreads more efficiently within the network, allowing synchronization of firing to occur (Miles and Wong 1987). According to that scheme, small EPSPs would be key factors involved in the recruitment of individual neurons within the network that participate in the generation of the PDS. In an attempt to demonstrate more clearly the role of small EPSPs in the initiation of synchronous discharge, we analyzed the temporal relationship between these two events in eight neurons. EPSPs selected for the computation displayed an amplitude at least two times larger than the baseline noise, determined by looking at a low-speed chart recording. In most cases this corresponded to an amplitude >2-2.5 mV. Their occurrence was calculated relative to the onset of synchronous burst, which was determined from a simultaneous extracellular recording obtained from an electrode positioned in the s. pyramidale, next to the intracellular electrode (Fig. 7). Spontaneous nonsynchronous EPSPs were not evenly distributed during the interburst interval but were more numerous in the period immediately preceding the onset of a burst (Fig. 7A, 0). This tendency is clearly demonstrated in Fig. 7B, where the distribution of EPSPs was quantified in 104 successive interburst intervals (hatched bars). As can be seen, the frequency of occurrence of EPSPs in the lOO-ms period preceding burst onset was more than three times higher than that observed at any other time during the interval. On average this corresponded to a 4.4 times higher probability of occurrence than what would be expected on

the basis of random occurrence (n = 8 cells). The distribution of EPSPs in all cells was bimodal, with an additional, although smaller, peak between 400-600 ms. The latter finding was more obvious when we plotted EPSPs distribution occurring after burst onset (not shown). In three cells, when summated EPSPs failed to trigger a burst, a lower amplitude and presumably partially synchronized field potential was correlated with the intracellularly recorded EPSPs (Fig. 7C), further supporting the idea that synchronous events are produced by temporally summating small EPSPs. Because small EPSPs appeared to be directly involved in burst initiation, we postulated that the burst rate in a slice could be related to the rate of occurrence of spontaneous EPSPs. In 10 different slices, the mean frequency of small EPSPs was estimated from the intracellular recordings of one to two cells and compared with the mean rate of synchronous burst measured with a simultaneous extracellular electrode. As shown in Fig. 8, the frequency of synchronous burst was positively correlated with the frequency of occurrence of nonsynchronous EPSPs measured from individual cells in the slices. The frequency of EPSPs appears, therefore, to be a good indicator of the level of excitability of the net/work and of the propensity of the slices to produce spontaneous bursts. To characterize further the relation between spontaneous EPSPs and synchronous bursts, we performed experiments in the presence of diverse concentrations of CNQX. Shown in Fig. 9A is the gradual decrease in both EPSP and burst frequency as the concentration of CNQX in the bathing medium was progressively increased. This gradual change was monitored in six different neurons, and the results ob-


4-AP AND EPILEPTIFORM

ACTIVITY

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Intra. Extra.

q n

60 cn E $ 50

EPSPs Bursts N=

104

W

gj 40 0 t!i m 30 E 8 0 20 10

200

500 800 1100 1400 Time Latency before the Bursts (ms)

1700

FIG. 7. Temporal relationship between spontaneous nonsynchronous EPSPs and synchronous epileptiform bursts. A: simultaneous intracellular (WD) and extracellular (h~~t~m) recordings from the s. pyramidale of the CA3c area of a hippocampal slice bathed in 4-AP. Traces show a representative example of 1 interburst interval during which small EPSPs occurred (a). Dashed vertical lines indicate 1 interburst interval delimited by the onset of 2 synchronous bursts. Distribution of time latenties of EPSPs and bursts was measured relative to the onset of synchronized events and computed from 104 successive interburst periods to construct the graph shown in B. B: hatched bars show the latency distribution of small nonsynchronous EPSPs measured before a synchronized burst, whereas black bars indicate the latency distribution of the preceding bursts. Regression shows the computer’s best fit of EPSPs’ distribution. CI:simultaneous intracellular (top) and extracellular (h~om) recording from the s pyramidale of another slice showing the occurrence of a partly synchronized potential between 2 synchronized bursts. Action potentials occurring during the intracellular bursts in A and C‘are cut. Rest I’m of the cell in A is -66 mV; in

C, -65.

rd

?

(25mV .

500 ms

burst frequency. Note also the similarities in the numeric values of the burst to EPSP ratio obtained under these conditions (Fig. 9B) to that obtained in Fig. 8. The origin of the small EPSPs cannot be assessed directly in these experiments. However, the fact that the effects induced by 4-AP on CA3 neurons are not modified after 0.8 complete surgical separation from the remainder of the slice (as-yet-unpublished data) indicates that the anatomic ‘;substrate of 4-AP actions responsible for EPSP occurrence i 0.6 and burst generation must be found within CA3 itself. In cn addition, the blocking effect of TTX described above demi/, 5 0.4 onstrates the role played by spontaneously active cells or m nerve terminals in their origin. We propose, therefore, that recurrent excitatory connections that have been previously 0.2 described in CA3 (Christian and Dudek 1988; MacVicar 0.5 1.0 1.5 2.0 2.5 3.0 and Dudek 1980; Miles and Wong 1986, 1987) play a maEPSPs (set -1) jor role in this respect. FIG. 8. Frequency of occurrence of nonsynchronous EPSPs is posiCA3 recurrent excitatory synapses studied with dual intively correlated to the frequency of synchronous bursts. In 10 different tracellular recordings (Miles and Wong 1983, 1987) have slices, the mean frequency of EPSPs was estimated from the intracellular recordings of 1 or 2 cells. This value was compared with the rate of occur- been shown to provide a very effective mechanism for rerence of field bursts recorded simultaneously with an extracellular elec- cruitment of neurons and synchronization of firing. The trode positioned in the s. pyramidale. Graph shows the positive relation“strength” of the recurrent monosynaptic or polysynaptic ship between the frequency of occurrence of bursts and that of the EPSPs in 10 different slices. synapses is such that, under conditions where inhibition is

tained were plotted in the graph in Fig. 9B. These results demonstrate a similar sensitivity of both EPSPs and bursts to CNQX (I& of = 1.2 PM for both). The decrease in EPSP frequency occurred in parallel with the decrease in


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3.0

=: $J

-

2.0

z E , t

0.2 3.0 pM

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1 5 mV 250

0.0

ms CNQX

(FM)

9. Similar blockage produced by CNQX on small non-synchronous EPSPs and synchronous bursts. A: recordings from 1 CA3c neuron showing the spontaneous potentials that were recorded in the presence of 50 PM 4-AP (control) and after the addition of progressively higher concentrations of CNQX. In control (top), note the presence of 2 spontaneous bursts and numerous small EPSPs that sometimes triggered action potentials. In the presence of 1.OPM CNQX, bursts were less frequent and EPSPs reduced in number. After the application of 2.0 PM CNQX, spontaneous bursts failed to occur and EPSPs were rare. Increasing the concentration of CNQX to 3.0 PM (bottom) almost completely blocked EPSPs. B: graph representing the relationship between the frequency of bursts (M) and nonsynchronous EPSPs (0) as a function of CNQX concentration. Data computed from the mean value obtained from simultaneous intracellular and extracellular recordings in 6 different slices. In the control trace in A, action potentials occurring during the bursts are cut whereas the amplitude of action potentials triggered from 3 EPSPs is reduced because of the slow time constant of the pen. Rest I/mof the cell in A was -66 mV. FIG.

suppressed (Miles and Wong 1983), or when slices are exposed to NMDA or 0 Mg*+ (Neuman et al. 1989), the firing of a single neuron can influence the synchronous activity of the population of cells. In a series of experiments we tested the role of this mechanism in 4-AP-induced epileptiform discharges by simultaneously recording intracellular and extracellular activity. Firing was induced in neurons by injection of depolarizing current pulses (50-200 ms, 1.7-6.5 nA), and their influence on population activity was monitored by comparing the average burst frequency during the pulse period with that observed during the prepulse and the postpulse period. During the whole procedure, the membrane potential of the neurons was hyperpolarized by a few millivolts by negative DC current injection to ensure that firing occurred only during periods of depolarizing current pulse injections or epileptiform bursts. Two different protocols were employed for current injections. In a first series of experiments, a train of depolarizing current pulses was injected at a different frequency from that of the bursts for a period of l-4 min. Up to five different pulse frequencies corresponding to 1.3-6.5 times the mean burst frequency of the population were studied in each cell. In a second series of experiments, a window discriminator was used to trigger an intracellular pulse of depolarizing current after the occurrence of a burst at intervals corresponding to 23-80% of the mean burst interval. This second strategy allowed us to avoid injecting current during the postburst hyperpolarization, a period where failures to activate the neuron occurred during the first protocol. Out of a total of 25 neurons studied, no significant entraining effect could be detected by the use of either of the two protocols ( 19 neurons were impaled with K acetate- and 6 with CsCl,-filled electrodes to further promote firing). There was no significant change in burst frequency detected with either protocol 1 (control: 0.57 t 0.24-s; pulse period: 0.58 t 0.24/s; y2= 20 cells) or protocol 2 (control: 0.6 1 t 0.25/s; pulse period: 0.60 t 0.26/s; n = 15

cells). The maximum increase observed in burst frequency was 17% in one cell, a value that, however, did not reach the level of significance. Efects of 4-AP on postsynaptic responses to GABA and glutamate In principle the potentiation of synaptic transmission by 4-AP can result from either presynaptic and/or postsynaptic changes. These possibilities were investigated by analyzing the effects of 4-AP on the postsynaptic response of neurons to the ionophoresis of glutamate and GABA. All experiments were performed in the presence of TTX (0.5- 1.O PM) to block synaptic transmission. After the responses to the ionophoresed agonists had stabilized, 4-AP was applied for 30 min (a period sufficient in normal conditions to produce full effects) and then washed for an additional 30-60 min. Because 4-AP-sensitive current(s) requires a hyperpolarization of the neuron’s membrane to be fully activated (Gustafsson et al. 1982; Storm 1988), responses were monitored over a broad range of membrane potentials by passing DC current inside the cell. Voltage-response plots were generated on the basis of these measurements made in control and after the application of 4-AP. This procedure was employed to discriminate postsynaptic effects resulting from direct alterations of receptor properties from indirect changes produced by modifications of the postsynaptic membrane excitability. As shown in Table 1, the amplitude and the duration of the glutamate responses were not modified after 4-AP application. 4-AP also failed to modify the voltage-response curve of the cells to glutamate application, except in two cells, where a small (= 10%) increase in the early part of the response was observed (data not shown). This increase was most prominent at hyperpolarized levels, a finding that was reminiscent of the effect produced by 4-AP on the response to depolarizing current pulses (Storm 1988).


4-AP AND EPILEPTIFORM TABLE 1. lZf/<.sq/*4-AP on the neuronalresponse to GABA and glutamate ionophoresis Control Glutamate ampl., mV GABA H ampl., mV GABA H rev. pot., mV GABA D ampl., mV GABA D rev. pot., mV

14.1 2 7.5 -5.9 + 4.3 -70.0 Ik 4.0 6.4 + 3.3 -53.5 -t 5.7

4-AP 14.2 -5.5 -69.3 5.7 -53.3

& 8.4 + 3.7 + 4.1 -+ 2.6 -+ 6.1

n

P"

11 5 3 6 3

NS NS NS NS NS

Values are means * SD; n, number of cells. Response amplitudes were measured at a fixed latency, near the peak. 4-AP, 4-aminopyridine; ampl., measured response amplitude; GABA, y-aminobutyric acid; GABA H, hyperpolarizing response to GABA application near the soma; rev. pot., measured reversal potential; GABA D, depolarizing response to dendritic GABA application. *Significance measured by paired Student’s t test; NS at P> 0.05.

The effect of 4-AP on the neuronal response to GABA application was monitored for both somatic hyperpolarization and dendritic depolarization (see METHODS). The appearance of a GABA-mediated long-lasting depolarization in 4-AP (see also Perreault and Avoli 1989) and the depolarizing shift of the equilibrium potential of the early IPSP that was observed prompted us to investigate also the changes induced on the reversal potential of both responses. These were measured from the voltage-response plots generated. As shown in Table 1, the amplitude and the reversal potential of the responses to both somatic and dendritic application of GABA remained unchanged during 4-AP application. We conclude, therefore, that 4-AP effects on synaptic transmission involve primarily presynaptic mechanisms (Thesleff 1980). DISCUSSION

In this study we investigated the physiological and pharmacological basis of 4-AP-induced epileptiform activity in the CA3 area of rat hippocampal slices. Our results indicate that a decrease in the efficacy of synaptic inhibition and the activation of NMDA receptors are not necessary for the generation of synchronous epileptiform bursts by 4-AP. Furthermore, these data suggest a prominent role played by nonsynchronous CNQX-sensitive EPSPs in the generation of synchronous discharges. Synaptic inhibition

and 4-AP

A general effect of 4-AP on neuronal tissue is the enhancement of neurotransmitter release at both inhibitory and excitatory synapses (Thesleff 1980). Our findings are in agreement with this view. IPSPs evoked by stimulating the Schaffer collaterals markedly increased in amplitude and duration after 4-AP application. The initial response that consisted of a single hyperpolarization acquired a typical triphasic shape composed of an early hyperpolarization, a slow depolarization, and a late hyperpolarization. All these features are similar to the results observed previously in the CA 1 region (Perreault and Avoli 1989b), where we demonstrated that this response resulted from the following sequence of receptors being activated: somatic GABA, (early hyperpolarization), dendritic GABA, (slow depolarization), and presumably GABA, receptors (late hyperpolar-

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ization). This kind of response was also recorded in CA3 pyramidal cells after stimulating mossy fibers or fimbria where they followed the evoked bursts. After 4-AP application, the evoked EPSP increased in amplitude and became markedly prolonged because of the appearance of a high-amplitude or giant EPSP. At the same time, the early IPSP, measured at 25 ms after mossy fiber stimulation, could no longer be detected. Two pieces of evidence indicate that the evoked IPSP was not blocked but simply masked by the overlying EPSP. First, the reversal potential of the giant EPSP (that peaked at a time latency corresponding to the early IPSP in control) was -32 mV, i.e., a value that is below what would be expected from a “pure” EPSP (Brown and Johnston 1983). Second, blockage of the EPSP after the application of CNQX (~5 PM) revealed the presence of an underlying IPSP clearly overlapping in time with the prolonged EPSP seen in control. These data provide evidence for the involvement of both inhibitory and excitatory Tonductances in generating the 4-AP-induced epileptiform bursts and confirm previous results obtained with the voltage-clamp technique by Rutecki et al. (1987). Numerous spontaneous small IPSPs were also present during the interburst interval, providing a further indication that epileptiform activity recorded in the presence of 4-AP was not associated with an impairment of synaptic inhibition. These small IPSPs were more prominent after the blockage of EPSPs with CNQX and were blocked by BMI or TTX, indicating they were generated by activation of GABA, receptors by GABA released from spontaneously active interneurons. Brief, nonsynchronous IPSPs have also been reported previously at the time 4-APinduced epileptiform activity occurs (Rutecki et al. 1987; Chestnut and Swann 1988). The importance attributed to disinhibition in epileptogenesis stems from the fact that GABAergic inhibition has been shown to be suppressed in several models of experimental epilepsy (Avoli 1988). The relevance of these experimental findings to the actual disease as it occurs in a human brain, however, is not clear. For example, analysis of brain slices obtained from human epileptogenic neocortex resected from patients suffering from intractable seizures indicate that synaptic inhibition is present (Avoli and Olivier 1989; Schwartzkroin and Haglund 1986). Recently, singleunit recordings of human hippocampal neurons performed in vivo have confirmed these findings (Isokawa-Akesson et al. 1989). Neuroanatomic and biochemical data obtained from excised human epileptic tissue are rather controversial, although most of the reports indicate no significant difference in GABA levels, glutamic acid decarboxylase (GAD), GABA aminotransferase activity (Sherwin and van Gelder 1986), or in GAD-immunoreactive neurons (Babb et al. 1989). Moreover, GABAergic inhibition does not appear to be impaired in many in vitro models of epilepsy such as exposure to low Mg2+ (Tancredi et al. 1990), mast cell degranulating peptide (Cherubini et al. 1988), tetanic stimulation in the CA3 area (Higashima 1988), and 4-AP (Rutecki et al. 1987; Chestnut and Swann 1989). Synaptic excitation and 4-AP The use of QX-3 14-filled microelectrodes that block fast sodium-dependent action potentials (Connors and Prince


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1982) allowed us to study more clearly the changes in the EPSPs after 4-AP application. The results obtained indicate that, after the addition of 4-AP to the perfusing medium, the mossy fiber-evoked EPSP increased in amplitude and became markedly prolonged. This potentiation could be observed when the amplitude of evoked EPSPs was measured 5- 10 ms after stimulation, i.e., a time that corresponded approximately to its peak amplitude in control; the more prominent changes occurred between 10 and 60 ms after stimulation. This was a result of the appearance of a second depolarizing component, the giant EPSP, which prolonged the initial EPSP and masked completely the underlying early IPSP recorded in control. The giant EPSP produced by 4-AP is likely to arise from a combination of two factors: I) an increased release of neurotransmitters from hyperexcitable terminals or simply from terminals invaded by broader action potentials (Shimara 1983; Targ and Kocsis 1986) and 2) the activation of recurrent excitatory collaterals (MacVicar and Dudek 1980; Miles and Wong 1986). However, the longer delay of occurrence and the higher sensitivity to CNQX of the giant EPSP compared with the evoked initial EPSP suggests the involvement of polysynaptic connections in its generation. Pharmacology c!f’4-AP-induced bursts NMDA receptor antagonists have been shown to exert potent anticonvulsant activity in several experimental models of epilepsy (for reviews see Chapman 1988; Meldrum 1987). In this respect, one important feature of the epileptiform bursts recorded in the presence of 4-AP is its lack of sensitivity to NMDA receptor antagonists APV and CPP. In the presence of these antagonists, there was no change in the frequency of occurrence of spontaneous bursts or in the number of population spikes associated with each single discharge. The amplitude of the giant EPSP evoked by mossy fiber stimulation remained unaffected as well. The concentrations of APV (lo- 100 PM) and CPP (l-5 PM) used in this study were well within or above their reported range of pharmacological activity, ruling out the possibility that these negative findings are attributable to insufficient amount of drugs added to the perfusing medium. The application of non-NMDA receptor antagonists CNQX and DNQX produced, however, a complete blockage of the epileptiform bursts. The disappearance of the extracellularly recorded field responses was correlated with a blockage of the giant EPSP recorded with intracellular electrodes. The IC,, of these effects was - 1.2 PM, a value that is below the range where these compounds display nonspecific antagonism to all excitatory amino acid receptor subtypes (Honore et al. 1988). A complete block of the evoked EPSP required higher concentrations between 9 and 15 PM, presumably because of the competitive nature of these antagonists. As quinoxalinediones have been shown in binding studies (Honore et al. 1988) and electrophysiological experiments (Fletcher et al. 1988) to be equally effective antagonists on both quisqualate and kainate receptor types, we conclude that one or both of these receptors is involved in generating 4-AP-induced bursts. Anatomic studies indicate that binding for all the three major receptor subtypes can be found in the CA3 area of the hippocampus. Although

AND

M.

AVOLI

NMDA and quisqualate binding sites are found in rather low levels in CA3 (Monaghan et al. 1984; Monaghan and Cotman 1985), kainate binding sites in comparison are present in particularly high levels and are mainly located in the s. lucidum, where the mossy fiber pathway terminates (Patel et al. 1986; Represa et al. 1987). Our results obtained with 4-AP are consistent with this anatomic distribution and are in agreement with a series of other studies indicating the lack of effect of NMDA antagonists on epileptiform bursts elicited in CA3 by kainate, mast cell degranulating peptide, anoxia, high K+ (Neuman et al. 1988), and penicillin on slices of immature animals (Brady and Swann 1988). NMDA receptors are not devoid of anticonvulsant properties in the CA3 area, because NMDA antagonists have been shown to block epileptiform discharges induced by low Mg2+ (Mody et al. 1987; Neuman et al. 1988; Tancredi et al. 1990), picrotoxin (Stone 1988), and NMDA application (Neuman et al. 1988) and to prevent the induction of electrographic seizures elicited by successive stimulus trains (Stasheff et al. 1989). NMDA antagonists also prevent the induction of long-term epileptiform bursting by electrical stimulation and bath-applied NMDA (Anderson et al. 1980; Stasheff et al. 1989). These findings indicate, therefore, the existence of important differences in the role played by NMDA receptor in epileptogenesis in the CA3 area of the rat hippocampus, depending on the model being studied. Role ofnonsynchronous EPSPs in the initiation of. synchronous bursts Experimental evidence suggests that reduction or loss of synaptic inhibition is critical for epileptiform synchronization, because it allows excitation to spread through the recurrent excitatory connections (Christian and Dudek 1988; Miles and Wong 1987). In the light of these results, how then does synchronization of firing occur in the presence of 4-AP, because synaptic inhibition is potentiated? Several converging lines of evidence indicate that small nonsynchronous EPSPs play a crucial role in the initiation of epileptiform burst in 4-AP. First, spontaneous EPSPs are more numerous in the period immediately preceding synchronous burst onset than at any other time during the interburst interval. Second, the frequency of occurrence of small EPSPs was positively correlated with the frequency of occurrence of synchronous bursts. Third, the depression in the frequency of spontaneous EPSPs th at occurred after CNQX application was accompanied by a similar decrease in burst frequency. Fourth, synchronous epileptiform discharges did not develop abruptly after 4-AP application but instead followed the appearance of spontaneous EPSPs, which increased in number over time and summated temporally to form clusters that appeared as irregular depolarizations. These depolari zatio Ins progressively became larger in amplitude and smoother and shorter in duration, finally giving rise to epileptiform bursts. We suggest, therefore, that bursting activity in 4-AP follows a progressive facilitation of excitatory synaptic transmission through the CA3 neuronal circuit. According to that scheme, small single EPSPs that are barely visible in control can reach firing threshold after the addition of 4-AP and allow excitation to cascade rapidly from one neuron to another, thereby lead-


4-AP

AND

EPILEPTIFORM

ing to a growing level of synchronization among individual cells of the network. In any single cell, the temporal summation of small EPSPs produced by many neighboring neurons will generate a giant EPSP and cause bursting activity. The blocking effect of TTX on spontaneous EPSPs indicates that actively firing neurons are required for their generation. Furthermore, the persistence of spontaneous EPSPs and bursts in surgically isolated CA3 sections suggests that these cells are located in the CA3 region itself (as-yet-unpublished data). Pyramidal neurons that can spontaneously generate intrinsic bursts have been described in the CA2CA3 area and would appear good candidates, considering that bursts are more effective at eliciting EPSPs in synaptically connected cells (Miles and Wong 1987). We suggest, therefore, that spontaneous EPSPs recorded in the presence of 4-AP are caused by a facilitation of transmitter release at presynaptic terminals of these normally active cells. However, we did not determine whether the blockage of K+ current(s) by 4-AP (which is known to raise the level of intrinsic excitability of neurons) was also reflected by an increased firing of individual cells or by a greater number of cells being active. The high level of synaptic activity recorded in CA3 neurons bathed in 4-AP during our study did not allow detailed investigation of intrinsic parameters of the cells, although numerous cells recorded fired readily during the interburst interval. These intrinsic effects of 4AP on the soma-dendrite area of neurons would act as a further amplification mechanism and increase the security of the propagation of synaptic potentials through multisynaptic pathways. Direct evidence for the role of recurrent excitatory polysynaptic connections in synchronization of firing has been obtained by Miles and Wong while recording from pairs of CA3 neurons ( 1987). Christian and Dudek ( 1988), by use of glutamate microapplication, also studied local interactions in CA3. They have also shown that picrotoxin treatment allows glutamate to induce an increase in EPSP frequency, which sometimes appeared as long-lasting barrage of reverberating EPSPs. Both studies emphasize that activity cannot spread through the recurrent excitatory pathways unless inhibition has been suppressed. On the contrary, our results suggests that disinhibition is not a sine qua non condition to trigger the sequential activation of excitatory connections. Paired recordings or glutamate microapplication could both be used successfully with 4-AP to test some of the predictions raised by our findings. It is interesting to note that Traub et al. ( 1987), using computer models that displayed realistic features of CA3 neuronal cells, found that considerable synchronization can occur over a wide range of inhibitory strength, provided excitatory connections are powerful enough. In the high K+ model of epilepsy, small CNQX-sensitive EPSPs have also been shown to be involved in the generation of spontaneous epileptiform bursts (Chamberlin and Dingledine 1988b). In this case, however, the efficacy of synaptic inhibition is reduced after a depolarizing shift of the equilibrium potential of the IPSP (Chamberlin and Dingledine 1988a). A recent computer simulation of the high-K+ model also indicates that small EPSPs are key factors in the initiation of bursts and the synchronization of activity when inhibition is partially removed (Traub et al. 1989).

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It has been shown that the frequency of synchronous epileptiform discharges in the CA3 area of the hippocampus can be influenced by the activity of a single neuron within the population. These results have been obtained on disinhibited slices exposed to picrotoxin (Miles and Wong 1983) and also on slices bathed in the presence of NMDA or lowMg2+ medium (Neuman et al. 1989). We tested this hypothesis for 4-AP-induced epileptiform discharges; our results indicate that single-cell activation does not suffice to entrain the whole population in this model. Because robust firing appeared to be required for population entrainment (Miles and Wong 1983; Neuman et al. 1989), very large amounts of currents (up to 5 nA) were injected to evoke maximal firing (up to 14 action potentials). Furthermore, because pulses applied during the postburst AHP failed to elicit firing, we designed a second protocol whereby neurons were stimulated at a given time after burst onset, thus avoiding this refractory period (see RESULTS). However, the findings obtained by employing this other approach were also negative. The most parsimonious interpretation of our results implies, then, that synchronous epileptiform discharges in the presence of 4-AP result from the nearly simultaneous activation of two or more neurons. This cooperative mechanism differs from previous models where activity was shown to spread like a chain reaction from one single neuron (Miles and Wong 1983; Neuman et al. 1989). Recent data obtained from computer simulation also suggested that a similar mode of neuronal recruitment is probably operating in the high-K+ model of epilepsy (Traub et al. 1989). A direct demonstration of this hypothesis would require the simultaneous recording of two or more neurons. An additional factor that could also play a synchronizing role in 4-AP-induced epileptiform discharges is the potentiation of GABAergic inhibition. As the GABA-mediated IPSPs that follow each burst become stronger, more cells are simultaneously experiencing an inhibitory or refractory state. Therefore, as the period of inhibition recedes, more neurons will simultaneously approach the firing threshold and a greater number of them will then simultaneously fire in response to incoming excitatory inputs. A similar role for increased GABA-mediated inhibition has been proposed recently by Babb et al. (1989) to explain the apparent GABA hyperinnervation of pyramidal cells of human epileptic hippocampus. 4-AP eficts on GABA and glutamate .

responses The evidence obtained from several preparations indicates that low micromolar concentrations of 4-AP produces a facilitation of synaptic transmission by presynaptic mechanisms (Thesleff 1980). Our demonstration that 4-AP application had no significant effect on the responses evoked by ionophoresis of GABA and glutamate confirms this hypothesis. It could be argued that the potentiating effect of 4-AP was activity dependent and therefore failed to occur because of the presence of TTX that was added to ensure that responses monitored resulted from a direct action of the agonists on the pyramidal cell membrane. This is unlikely, however, because the enhancement of synaptic potentials produced by 4-AP clearly preceded the increase in neuronal bursting activity. It is also possible that the responses obtained were not a good reflection of 4-AP effects


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on excitatory synapses. For instance, glutamate application might have activated both extrasynaptic and synaptic receptors or a receptor subtype different from the CNQXsensitive one that is involved in 4-AP actions. We do not believe, however, that these factors are major concerns for two reasons. First, glutamate responses were not easily evoked, even with high intensity of ionophoretic currents, but instead required careful manipulation of the ionophoresis electrode to localize hot spots where responses arose abruptly (Schwartzkroin and Andersen 1975). This finding is contradictory to what would be expected if glutamate responses originated primarily from extrasynaptic sites and suggests, therefore, a synaptic origin of the responses evoked. Second, as with the potentiated EPSP recorded in 4-AP, the glutamate responses were, at least in part, also CNQX-sensitive (unpublished data). Our data also provide evidence that the depolarizing shift of the early IPSP is attributable merely to the overlapping EPSPs, because the reversal potential of the hyperpolarizing response to GABA remained unchanged after 4-AP application. We expected the intrinsic postsynaptic effects induced by 4-AP (Gustafsson et al. 1982; Storm 1988) to be reflected by changes in the amplitude of the depolarizations produced by glutamate application. The reason that we failed to record any significant change consistent with these actions is unclear. In each cell, glutamate responses were studied over a large range of membrane levels, including hyperpolarized levels that are required to activate IA or ID, thus eliminating any possible bias resulting from, for example, a lower resting level of our cells that would not have allowed the expression of these currents. In two neurons, however, we noted a small increase in the amplitude of the early part of the responses to glutamate, an effect that was more prominent at hyperpolarized levels, as would be expected from typical blockage of IA or ID (Storm 1988). This suggests that perhaps 4-AP-sensitive channels are not evenly distributed among CA3 cells. The potentiating effect of 4-AP has been attributed to an enhanced amount of Ca2+ entering nerve terminals during the depolarizing phase of the action potential (Thesleff 1980). This effect is presumably caused by a prolongation of the time course of the action potential through a blockage of K+ currents (Llinas et al. 1976; Kocsis 1986) or through direct effects of 4-AP on Ca2’ channels (Segal and Barker 1986). Others have also demonstrated the ability of 4-AP-treated nerve fibers to generate bursts of action potentials (Kocsis 1986) a mechanism that could further promote transmitter release. Although we have previously described the occurrence of ectopic and presumably axonal bursts of action potentials in the CA 1 area (Perreault and Avoli 1989b), we did not find any direct evidence for their involvement in the bursting activity recorded in CA3. This type of activity appears to be primarily linked with the occurrence of GABA-mediated long-lasting depolarizations (Perreault and Avoli 1989b). In summary, our results indicate that 4-AP-induced epileptiform discharges are caused by the appearance of giant EPSPs involving both inhibitory and excitatory synaptic conductances. Bursts were not sensitive to NMDA antagonists but were blocked by the quisqualatelkainate receptor antagonists CNQX and DNQX. We showed also that small

AND

M.

AVOLI

CNQX-sensitive nonsynchronous EPSPs play a critical role in the generation of spontaneous discharges. Because inhibition has not been shown conclusively to be impaired in human epileptic focus, the 4-AP model may therefore provide an interesting approach that can be used on a simple system such as the hippocampal slice to elucidate some of the mechanisms by which synchronous epileptiform activity can occur in a neuronal network where synaptic inhibition is not impaired. We thank S. Schiller and A. Topaczewski for technical assistance. This work was supported by a grant from the Medical Research Council (MRC) of Canada (MA-8 109) to M. Avoli. P. Perreault is an MRC Fellow; M. Avoli is a Fonds de la Recherche en Sante du Quebec Scholar. Address for reprint reauests: M. Avoli, Montreal Neurological Institute. I 380 1 University St., Montreal, Quebec H3A 2B4, Canada. Received 10 May 1990; accepted in final form 12 November 1990. REFERENCES

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Anticonvulsant effects of tetronic acid derivatives on picrotoxin induced epileptiform activity in rat hippocampal slices.

Chaotic activity during iron-induced "epileptiform" discharge in rat hippocampal slices.

Transition from normal to epileptiform activity in kindled rat hippocampal slices.

Effects of anti-epileptic drugs on spreading depolarization-induced epileptiform activity in mouse hippocampal slices.

Methohexitone antagonises kainate and epileptiform activity in rat neocortical slices.

The change of picrotoxin-induced epileptiform discharges to the beta oscillation by carbachol in rat hippocampal slices.

Induction of epileptiform activity by temperature elevation in hippocampal slices from young rats: an in vitro model for febrile seizures?

Potassium-induced long-term potentiation in rat hippocampal slices.

Low magnesium-induced epileptiform discharges in guinea pig hippocampal slices: depression by the organic calcium antagonist verapamil.

Ethanol inhibits epileptiform activity and NMDA receptor-mediated synaptic transmission in rat amygdaloid slices.

Hippocampal epileptiform activity induced by magnesium-free medium: differences between areas CA1 and CA2-3.

CPP, an NMDA-receptor antagonist, blocks 4-aminopyridine-induced spreading depression episodes but not epileptiform activity in immature rat hippocampal slices.

Stimulation of estradiol biosynthesis by tributyltin in rat hippocampal slices.

Effects of non-opioid antitussives on epileptiform activity and NMDA responses in hippocampal and olfactory cortex slices.

Selective inhibition of KCC2 leads to hyperexcitability and epileptiform discharges in hippocampal slices and in vivo.

Late low magnesium-induced epileptiform activity in rat entorhinal cortex slices becomes insensitive to the anticonvulsant valproic acid.

Adrenalectomy increases phosphoinositide hydrolysis induced by norepinephrine or excitatory amino acids in rat hippocampal slices.

Effects of the triazole derivative loreclezole (R72063) on stimulus induced ionic and field potential responses and on different patterns of epileptiform activity induced by low magnesium in rat entorhinal cortex-hippocampal slices.

Epileptiform activity in microcultures containing small numbers of hippocampal neurons.

Long-lasting potentiation and epileptiform activity produced by GABAB receptor activation in the dentate gyrus of rat hippocampal slice.

Bursting-induced epileptiform EPSPs in slices of piriform cortex are generated by deep cells.

Monitoring of sympathetic activity in man: physiology and pharmacology.

Activation of extrasynaptic GABA(A) receptors inhibits cyclothiazide-induced epileptiform activity in hippocampal CA1 neurons.

Epileptiform activity in microcultures containing one excitatory hippocampal neuron.