Propagation velocity of epileptiform activity in the hippocampus

Summary The propagation of epileptiform burst activity was investigated in the CA1 area of the in-vitro hippocampal slice preparation of the guinea pig. This activity was provoked by 0.1 mM 4-aminopyridine in the bathing medium and was recorded in the pyramidal layer with an array of eight electrodes. The delay between the first population spike of a burst recorded with different electrodes was calculated using the cross-correlation function. The propagation velocity was estimated from the delays and the electrode intervals. It was found that the velocity of spontaneous and evoked epileptiform bursts varies between 0.15 and 5 m/s and is not confined to the range of conduction velocities of the fibre systems in CA1 (0.3–0.55 and 1.0–1.8 m/s). Different velocities can be present in different parts of the CA1 area and the initiation of spontaneous bursts is not confined to the CA2–3 areas, but can also occur in CA1. Burst activity also propagated in a low calcium-high magnesium medium. Different mechanisms of propagation are discussed and it is argued that the propagation velocity due to ephaptic interaction may vary largely. It is concluded that epileptiform activity can be propagated not only by synaptic connections at or near the pyramidal layer, but also by way of electrical field effects of population spikes.     

Decrease in synaptic transmission can reverse the propagation direction of epileptiform activity in hippocampus in vivo

Most types of epileptiform activity with synaptic transmission have been shown to propagate from the CA3 to CA1 region in hippocampus. However, nonsynaptic epileptiform activity induced in vitro is known to propagate slowly from the caudal end of CA1 toward CA2/CA3. Understanding the propagation modes of epileptiform activity, and their causality is important to revealing the underlying mechanisms of epilepsy and developing new treatments. In this paper, the effect of the synaptic transmission suppression on the propagation of epilepsy in vivo was investigated by using multiple-channel recording probes in CA1. Nonsynaptic epileptiform activity was induced by calcium chelator EGTA with varied concentrations of potassium. For comparison, disinhibition synaptic epileptiform activity was induced by picrotoxin (PTX) with or without partial suppression of excitatory synaptic transmission. The propagation velocity was calculated by measuring the time delay between two electrodes separated by a known distance. The results show that in vivo nonsynaptic epileptiform activity propagates with a direction and velocity comparable to those observed in in vitro preparations. The direction of propagation for nonsynaptic activity is reversed from the PTX-induced synaptic activity. A reversal in propagation direction and change in velocity were also observed dynamically during the process of synaptic transmission suppression. Even a partial suppression of synaptic transmission was sufficient to significantly change the propagation direction and velocity of epileptiform activity. These results suggest the possibility that the measurement of propagation can provide important information about the synaptic mechanism underlying epileptic activity.     

Involvement of non-NMDA receptors in picrotoxin-induced epileptiform activity in the hippocampus

The ability of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to suppress picrotoxin-induced epileptiform burst activity was examined. Intracellular recordings were obtained from hippocampal CA1 and CA3 pyramidal neurons maintained in vitro. Bath application of CNQX (5 microM) significantly reduced or abolished evoked paroxysmal depolarizing shifts (PDSs) in all CA1 and CA3 neurons tested. In cells where a CNQX-insensitive component in the PDS was manifest, this remaining activity was abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovaleric acid (20 microM), suggesting the existence of a NMDA-mediated synaptic potential. Our results indicate that non-NMDA receptor antagonists are capable of markedly reducing picrotoxin-induced epileptiform activity and that these receptors play an important role in generation of PDSs.     

Picrotoxin-induced epileptiform activity in hippocampus: role of endogenous versus synaptic factors

Picrotoxin-(PTX) induced epileptiform activity was studied in guinea pig hippocampal slices maintained in vitro, using intra- and extracellular recording techniques. The observed pattern of spontaneous and evoked epileptiform activity was quite complex. Spontaneous epileptiform events originated in the CA3 region and subsequently spread or propagated to CA1. Activation of CA1 could then reactivate CA3. This reverberation of activity was seen also following stimulation of the mossy fiber afferents from the dentate gyrus to CA3. Stimulation of fibers in the stratum radiatum of the CA1 region could trigger, at short latency, epileptiform activity that either was localized in CA1 or also occurred in CA3, with a late secondary discharge in CA1. This is attributed to a backfiring of the Schaffer collaterals and illustrates the ability of a variety of CA3 inputs to trigger epileptiform activity. Bath-applied PTX, at concentrations of 50-200 microM, had no apparent effect on the resting membrane potential or input resistance of the CA3 cells tested. Depolarizing current pulses elicited characteristic endogenous-burst responses that were not altered by PTX. Synaptic activity evoked by mossy fiber stimulation was altered markedly by PTX. The pattern of observed changes indicated that PTX reduced inhibitory postsynaptic potential (IPSP) amplitudes, resulting in the appearance of repetitive (presumably recurrent) excitatory inputs. Paroxysmal depolarizing shifts ( PDSs ) were generated by the coalescence of these excitatory inputs. Two types of spontaneous bursting were observed after PTX application. The first type was nonepileptiform , all or none in nature, and its frequency was voltage dependent. The second type of spontaneous burst was the PDS. It was epileptiform in character because it was associated with the synchronous discharge of many neurons. It was graded in nature, and its frequency was voltage independent. The graded nature of the PDS was demonstrated by varying the duration and intensity of the orthodromic stimulation. Trains of stimulation could produce PDSs that lasted 500-800 ms. A refractory period was observed following a PDS. By varying the strength of the orthodromic stimulation, it was possible to demonstrate that for the intervals tested this was a relative, not absolute, refractory period. Intracellular recordings in CA3 neurons indicated that each spontaneous PDS was followed by an afterhyperpolarization (AHP).     

Cesium induces spontaneous epileptiform activity without changing extracellular potassium regulation in rat hippocampus

Cesium has been widely used to study the roles of the hyperpolarization-activated (I(h)) and inwardly rectifying potassium (K(IR)) channels in many neuronal and nonneuronal cell types. Recently, extracellular application of cesium has been shown to produce epileptiform activity in brain slices, but the mechanisms for this are not known. It has been proposed that cesium blocks the K(IR) in glia, resulting in an abnormal accumulation of potassium in the extracellular space and inducing epileptiform activity. This hypothesis has been tested in hippocampal slices and cultured hippocampal neurons using potassium-sensitive microelectrodes. In the present study, application of cesium produced spontaneous epileptiform discharges at physiological extracellular potassium concentration ([K(+)](o)) in the CA1 and CA3 regions of hippocampal slices. This epileptiform activity was not mimicked by increasing the [K(+)](o). The epileptiform discharges induced by cesium were not blocked by the N-methyl-D- aspartate (NMDA) receptor antagonist AP-5, but were blocked by the non-NMDA receptor antagonist CNQX. In the dentate gyrus, cesium induced the appearance of spontaneous nonsynaptic field bursts in 0 added calcium and 3 mM potassium. Moreover, cesium increased the frequency of field bursts already present. In contrast, ZD-7288, a specific I(h) blocker, did not cause spontaneous epileptiform activity in CA1 and CA3, nor did it affect the field bursts in the dentate gyrus, suggesting that cesium induced epileptiform activity is not directly related to blockade of the I(h). When potassium-sensitive microelectrodes were used to measure [K(+)](o), there was no significant increase in [K(+)](o) in CA1 and CA3 after cesium application. In the dentate gyrus, cesium did not change the baseline level of [K(+)](o) or the rate of [K(+)](o) clearance after the field bursts. In cultured hippocampal neurons, which have a large and relatively unrestricted extracellular space, cesium also produced cellular burst activity without significantly changing the resting membrane potential, which might indicate an increase in [K(+)](o). Our results suggest that cesium causes epileptiform activity by a mechanism unrelated to an alteration in [K(+)](o) regulation.     

Block of glutamate-glutamine cycle between astrocytes and neurons inhibits epileptiform activity in hippocampus

Recurrent epileptiform activity occurs spontaneously in cultured CNS neurons and in brain slices in which GABA inhibition has been blocked. We demonstrate here that pharmacological treatments resulting in either the block of glutamine production by astrocytes or the inhibition of glutamine uptake by neurons suppress or markedly decrease the frequency of spontaneous epileptiform discharges both in primary hippocampal cultures and in disinhibited hippocampal slices. These data point to an important role for the neuron-astrocyte metabolic interaction in sustaining episodes of intense rhythmic activity in the CNS, and thereby reveal a new potential target for antiepileptic treatments.     

4-Aminopyridine produces epileptiform activity in hippocampus and enhances synaptic excitation and inhibition

Using extra- and intracellular recording techniques, we investigated the epileptiform activity induced by low concentrations (5 and 10 microM) of bath-applied 4-aminopyridine (4-AP) in the CA3 subfield of rat hippocampal slices. We also studied the effects of 4-AP on the excitatory and inhibitory synaptic conductance changes in CA3 neurons produced by mossy fiber stimulation. Low concentrations of 4-AP induced spontaneously occurring epileptiform discharges at extracellular potassium concentrations between 1 and 10 mM. In contrast, picrotoxin and bicuculline produced spontaneous epileptiform discharges at extracellular potassium concentrations between 5 and 10 mM. The paroxysmal depolarizing shift (PDS) induced by 4-AP was also investigated. At potentials between -40 and -10 mV, the waveform of the PDS consisted of a depolarizing component enveloped by a hyperpolarizing component. The amplitude of the depolarizing component of the PDS was a monotonic function of the membrane potential, and the mean measured reversal potential was -25.7 mV. Under voltage-clamp conditions, the measured conductance associated with the depolarizing component of the PDS averaged 110 nS, with a reversal potential of -14.1 mV. Application of 5 microM 4-AP produced an increase in the inhibitory synaptic conductance change calculated from currents measured 15 ms following mossy fiber stimulation. The mean value increased from 35.2 to 58.1 nS (P less than 0.05) without a significant change in reversal potential. A concentration of 10 microM 4-AP also produced an increase in this inhibitory synaptic conductance change (from 53.3 to 66.3 nS, P less than 0.05) but caused a significant depolarization of the reversal potential (from -66.5 to -61.6 mV, P less than 0.05). This change in reversal potential may reflect a prolongation of the excitatory synaptic currents produced by 4-AP that contributes to the current measured 15 ms from the stimulus. Following application of either 5 or 10 microM 4-AP, there were no significant changes in the resting potential or input resistance of the neurons studied. Application of 5 microM 4-AP also significantly increased the amplitude of the measured excitatory synaptic conductance change produced by mossy fiber stimulation (from 27.9 to 44.1 nS, P less than 0.05) without producing a change in the reversal potential. In 5 of 21 neurons studied, a long-lasting outward synaptic current was present at holding potentials near rest following mossy fiber stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Repeated blockade of GABAA receptors in the medial septal region induces epileptiform activity in the hippocampus

Changes in the activity of putative interneurons of the stratum oriens of the hippocampus and hippocampal EEG after the delivery of the GABAA receptor antagonist bicuculline (1.0nmol/1microl) to the medial septal region were investigated in awake rabbits. The injection of bicuculline produced a sharp increase in the firing rate in 94.3% of hippocampal cells. The effect of bicuculline on the neuronal theta rhythmicity depended on the number of injections. The first five daily infusions decreased the theta activity in 85.7% of cells. On the fourth to fifth experimental days paroxysmal discharges and 8-15Hz oscillations were recorded in the hippocampal EEG. Six to seven further daily bicuculline injections following a brief diminution of theta activity produced a sharp augmentation of theta oscillations in 78.9% of cells and provoked seizures. Immediately before seizures, stabilization of theta bursts and an increase in burst frequency was usually observed in putative interneurons. During seizures, neuronal rhythmic activity was either disordered and then turned into seizure discharges or was inhibited, partially or completely. In the hippocampal EEG, the power of theta rhythm before seizures usually strongly increased compared with controls. Injection of the GABAA agonist muscimol (30nmol/1microl) 15min before bicuculline infusion prevented the development of seizures. These findings suggest that the interplay between septal neurons via GABAA receptors is critical in the tuning of septal output signals that insure generation of natural theta rhythm as well as adequate functioning of the hippocampus.     

Involvement of electrical coupling in the in vivo ictal epileptiform activity induced by 4-aminopyridine in the neocortex

In the present study we have investigated the possible role of gap junctions in the induction and manifestation of 4-aminopyridine-induced acute seizure activity both at the primary focus and at the mirror focus in anaesthetized rats by combining electrophysiological, pharmacological and molecular biological techniques. In the course of the intracellular recordings, unusual firing patterns that are assumed to be mediated by electrical coupling and appearing either randomly or in close time-locked manner with the ictal discharges were observed. In another series of experiments, a significant decrease in the intensity of seizure activity of the already active epileptic foci was detected when electrical synaptic transmission was blocked by carbenoxolone either at the primary focus or at the mirror focus. When electrical synaptic transmission was depressed relative to the initial baseline prior to the induction of epileptic focus, only a mild influence on the induction of seizure discharges occurred. The role of the gap junctional communication in the epileptiform activity was further investigated by following the expression pattern of two connexin genes. Both, connexin-32 and connexin-43 mRNA levels were significantly elevated at the primary focus as well as at the mirror focus, after 60 min of repeated ictal discharges.We conclude that gap junction communication probably became a part of the neuronal synchronization both in the primary and in the secondarily-induced acute epileptiform activity in the neocortex in vivo. These results, together with earlier observations, indicate a direction for the development of new drugs targeting gap junctions for therapeutic intervention.     

Cyclothiazide induces robust epileptiform activity in rat hippocampal neurons both in vitro and in vivo

Cyclothiazide (CTZ) is a potent blocker of AMPA receptor desensitization. We have recently demonstrated that CTZ also inhibits GABA_A receptors. Here we report that CTZ induces robust epileptiform activity in hippocampal neurons both in vitro and in vivo . We first found that chronic treatment of hippocampal cultures with CTZ (5 μ m , 48 h) results in epileptiform activity in the majority of neurons (80%). The epileptiform activity lasts more than 48 h after washing off CTZ, suggesting a permanent change of the neural network properties after CTZ treatment. We then demonstrated in in vivo recordings that injection of CTZ (5 μmol in 5 μl) into the lateral ventricles of anaesthetized rats also induces spontaneous epileptiform activity in the hippocampal CA1 region. The epileptogenic effect of CTZ is probably due to its enhancing glutamatergic neurotransmission as shown by increasing the frequency and decay time of mEPSCs, and simultaneously inhibiting GABAergic neurotransmission by reducing the frequency of mIPSCs. Comparing to a well-known epileptogenic agent kainic acid (KA), CTZ affects neuronal activity mainly through modulating synaptic transmission without significant change of the intrinsic membrane excitability. Unlike KA, which induces significant cell death in hippocampal cultures, CTZ treatment does not result in any apparent neuronal death. Therefore, the CTZ-induced epilepsy model may provide a novel research tool to elucidate the molecular and cellular mechanisms of epileptogenesis without any complication from drug-induced cell death. The long-lasting epileptiform activity after CTZ washout may also make it a very useful model in screening antiepileptic drugs.     

Action potential fidelity during normal and epileptiform activity in paired soma–axon recordings from rat hippocampus

Although action potential initiation and propagation are fundamental to nervous system function, there are few direct electrophysiological observations of propagating action potentials in small unmyelinated fibres, such as the axons within mammalian hippocampus. To circumvent limitations of previous studies that relied on extracellular stimulation, we performed dual recordings: whole-cell recordings from hippocampal CA3 pyramidal cell somas and extracellular recordings from their axons, up to 800 μm away. During brief spike trains under normal conditions, axonal spikes were more resistant to amplitude reduction than somatic spikes. Axonal amplitude depression was greatest at the axon initial segment < 150 μm from the soma, and initiation occurred ∼75 μm from the soma. Although prior studies, which failed to verify spike initiation, suggested substantial axonal depression during seizure-associated extracellular K⁺ ([K⁺]_o) rises, we found that 8 m m [K⁺]_o caused relatively small decreases in axonal spike amplitude during brief spike trains. However, during sustained, epileptiform spiking induced in 8 m m [K⁺]_o, axonal waveforms decreased significantly in peak amplitude. During epileptiform spiking, bursts of two or more action potentials > 20 Hz failed to propagate in most cases. In normal [K⁺]_o at 25 and 32°C, spiking superimposed on sustained somatic depolarization, but not spiking alone, produced similar axonal changes as the epileptiform activity. These results highlight the likely importance of steady-state inactivation of axonal channels in maintaining action potential fidelity. Such changes in axonal propagation properties could encode information and/or serve as an endogenous brake on seizure propagation.     

Consequences of prolonged afferent stimulation of the rat fascia dentata: epileptiform activity in area CA3 of hippocampus

Following prolonged stimulation of the perforant path input to the dentate gyrus, long-lasting changes occur in the synaptic responses and cell properties of cells in the fascia dentata. The present study describes the effects of sustained stimulation on the major population of cells innervated by the dentate granule cells: are CA3 pyramidal cells of hippocampus. In 46% of slices from rat, sustained stimulation of perforant path was followed by spontaneous, synchronized, rhythmic bursting activity in area CA3 pyramidal cells that was evident for several hours. These bursts could be recorded extracellularly in the pyramidal cell layer, throughout the hilar region, and even in the granule cell layer. With intracellular recording, all of the cells of the fascia dentata were found to be affected by the pyramidal cell bursts. Hyperpolarizing, inhibitory postsynaptic potential (IPSP)-like events occurred in all granule cells tested during the CA3 pyramidal cell burst. In contrast, spiny hilar "mossy" cells discharged synchronously with the pyramidal cells, as did some of the "fast spiking" interneurons. However, most interneurons only depolarized a few millivolts during the pyramidal cell burst. These results show that sustained stimulation of the perforant path is followed by a period of hyperexcitability in area CA3 of the hippocampus, and that hyperexcitability in area CA3 influences the activity of the cells in the fascia dentata.     

Kindling-like stimulus patterns induce epileptiform discharges in the guinea pig in vitro hippocampus

In the guinea pig in vitro hippocampal slice preparation, we have demonstrated that the repeated tetanic stimulation of the Schaffer collateral-commissural input to CA1 pyramidal neurones produces a progressive increase in the amplitude and duration of postsynaptic potentials, and stimulus-induced and spontaneous paroxysmal depolarization shifts (PDSs). Both the enhancement of synaptic transmission and the genesis of PDSs were reversibly blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)-2-amino-5-phosphonovalerate (APV). These results provide evidence that progressive, stimulation-induced long-term potentiation may serve as the trigger for kindling-induced epileptogenesis, and this process is dependent on the repeated activation of an APV-sensitive receptor.     

Neuropeptide Y suppresses epileptiform activity in rat frontal cortex and hippocampus in vitro via different NPY receptor subtypes.

Neuropeptide Y (NPY) and different NPY receptor (Y) subtype-selective agonists were tested for their effects on spontaneous epileptiform discharges which developed in rat cortical and hippocampal slices in Mg(2+)-free medium. Epileptiform activity, recorded extracellularly, was attenuated by NPY (0.5-1 microM) in both the frontal cortex and hippocampal CA3/CA1 pyramidal cell layers. In the cortex the Y1/5 selective agonist [Leu31 Pro34] NPY was more effective than the Y2 preferring agonist NPY13-36 and the Y2/5 preferring agonist NPY3-36. The suppression of epileptiform discharges induced by NPY in cortical slices was blocked by the selective Y1 receptor antagonist (R)-N2-(diphenylacetyl)-N-((4-hydroxyphenyl)methyl] argininamide (BIBP 3226). In the hippocampus, NPY13-36 and NPY3-36 were more effective than [Leu31 Pro34] NPY. In conclusion, the antiepileptic activity of NPY is mediated predominantly by the Y1 receptor subtype in the frontal cortex and by Y2 and probably Y5 receptors in the hippocampal CA3/CA1 areas.     

Initiation of spontaneous epileptiform events in the rat neocortex in vivo

We used voltage-sensitive dye imaging to visualize the distribution of initiation sites of the spontaneous interictal-like spikes (sISs) in rat neocortex, in vivo, induced by bicuculline or picrotoxin over the exposed cortex. The initiation site was small (approximately 200 microm diam). On average each initiation site initiated 2.0 +/- 0.8 sISs (9 animals, 499 sISs, 251 sites). This is significantly different from that in neocortical slices, where each initiation site initiated 30-100 sISs. The initiation sites were not randomly distributed. The distance between two consecutive sites tended to be either <800 or >1200 microm, suggesting a temporal "suppression annulus" surrounding each initiation site. Within the annulus, the likelihood for initiating the next sIS was reduced. Suppression annulus did not have a noticeable change in the presence of GABA(b) antagonist, suggesting it did not depend on the GABA(b) inhibition. We also applied bicuculline locally to a spot of 800 x 800 microm(2) for approximately 45 min. During this period approximately 1000 sISs occurred within the spot. Bicuculline or picrotoxin was then applied to the entire craniotomy window. The pretreatment created an obvious cluster of initiation sites. Around this cluster, the suppression annulus became obvious in individual animals. Our results suggest that, in disinhibited cortex, epileptiform events were initiated from small sites. The initiation sites may cluster in an area with increased local activity. Surrounding each initiation site there may be a temporal suppression annulus.