Excitatory synaptic involvement in epileptiform bursting in the immature rat neocortex.

1. Neocortical brain slices were prepared from animals 8-15 days of age and maintained in vitro. Intracellular recordings were obtained from neurons in cortical layers 2-3. The role of synaptic activity and excitatory amino acid receptors in generation of picrotoxin-induced ictal-like epileptiform activity in the immature neocortex was investigated. D-2-amino-5-phosphonovaleric acid (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were used as selective antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, respectively. 2. Ictal-like epileptiform discharges were induced by bath application of the GABAA-receptor antagonist picrotoxin. Paroxysmal discharges, 7-25 s in duration, occurred spontaneously or could be evoked by electrical stimulation. These events consisted of an initial paroxysmal depolarizing shift (PDS) followed by a long-duration depolarization (LLD) with superimposed late PDSs. 3. The amplitudes of the initial PDS, LLD, and late PDSs were linearly dependent on membrane potential, increasing with hyperpolarization and diminishing on depolarization. All responses reversed polarity near 0 mV. Under voltage-clamp conditions, both transient and sustained currents were observed, coincident with PDSs and the LLD, respectively. The duration of the ictal-like events was similar under current- and voltage-clamp conditions, suggesting activation of intrinsic membrane currents did not significantly prolong epileptiform discharges. 4. Bath application of D-APV (20 microM) decreased the amplitude and duration of both the initial PDS and LLD without affecting the time-to-onset of epileptiform activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bicuculline-induced epileptogenesis in the human neocortex maintained in vitro

Summary Intracellular and extracellular recordings were made from human neocortical slices of the temporal lobe maintained in vitro. The slices were treated with bicuculline methiodide to reduce synaptic inhibition mediated by tha gamma-aminobutyric acid A (GABA_A) receptor. Spontaneously occurring epileptiform activity was never observed in over 60 slices examined. All epileptiform discharges were elicited by single-shock stimuli delivered in the underlying white matter or within the cortical layers. Intracellularly, the stimulus-induced epileptiform discharge resembled the paroxysmal depolarization shift (PDS). This potential was observed in neurons located between 200 and 2200 m from the pia. It was characterized by a 100–1800 ms long depolarization which triggered burst firing of action potentials, and was at times followed by an afterdischarge. Simultaneous intracellular and extracellular recordings showed that each PDS was reflected by the synchronous discharge of a neuronal aggregate. The voltage behaviour of the PDS and its preceding EPSP was analyzed in cells that were injected with the lidocaine derivative QX-314. The amplitudes of the PDS depolarizing envelope measured at its peak and during its falling phase both behaved as a monotonic function of the membrane potential by increasing in amplitude during hyperpolarization. In addition, the PDS peak amplitude showed a much greater rate of increase than the early EPSP peak amplitude, thus suggesting that the synaptic conductance underlying the PDS was much greater. Perfusion of the neocortical slices with the N-Methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-phosphonovaleric acid (APV) reduced both the duration and the amplitude of the paroxysmal field discharge in a dose related fashion. The effects of APV were reflected intracellularly by an attenuation of the PDS's late phase and a blockade of the afterdischarge. Similar findings were also obtained by using the NMDA receptor antagonist 3-((±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid. These data indicate that reduction or blockade of the GABA_A receptor is sufficient to elicit epileptiform discharges in the human neocortex maintained in vitro. Mechanisms dependent upon the NMDA receptor contribute to this type of epileptiform response mainly by prolonging the stimulus-induced depolarizing potential and the associated burst of firing.     

Initiation of epileptiform activity by excitatory amino acid receptors in the disinhibited rat neocortex.

1. Intracellular recordings were obtained from neurons in layer II-III of rat frontal cortex maintained in vitro. The role of excitatory amino acid receptors in generation of picrotoxin (PTX)-induced epileptiform activity was investigated with the use of D-2-amino-5-phosphonovaleric acid (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) as selective antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, respectively. 2. Bath application of PTX resulted in a decrease in evoked inhibitory postsynaptic potentials (IPSPs) in neocortical neurons and a concomitant increase in a polysynaptic late excitatory postsynaptic potential (IEPSP). Epileptiform burst responses, termed paroxysmal depolarizing shifts (PDSs), subsequently developed. Based on response duration, two types of PDSs were identified. Long PDSs were greater than 100 ms in duration, whereas short PDSs lasted less than 50 ms. An early depolarizing potential preceded both types of epileptiform burst response. 3. The NMDA receptor antagonist D-APV reduced the peak amplitude and duration of the PDS. D-APV-insensitive portions of the PDS were greatly attenuated or abolished by CNQX. The non-NMDA antagonist also increased the latency to PDS onset and reduced its duration without affecting peak amplitude. CNQX-insensitive components of the PDS, when present, were abolished by D-APV. 4. Short-duration PDSs could be blocked by CNQX. In these neurons, increasing the stimulation strength produced epileptiform responses of reduced amplitude. 5. Under control conditions, PDS amplitude was a linear function of membrane potential, increasing with hyperpolarization and diminishing on depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

We have investigated the effects of a new class of anticonvulsants, the tetronic acid derivatives AO33 (generic name: losigame) and AO78, on field potentials, extracellular calcium concentration changes and intracellular potentials in rat hippocampal slices treated with the non-competitive GABAA antagonist picrotoxin (PTX). The tetronic acid derivatives reduced and eventually blocked spontaneous epileptiform events, induced by 10 to 30 microM PTX. Stimulus induced burst discharges were shortened in duration, but not blocked. Extracellular calcium concentration changes and associated slow negative field potentials were diminished in a dose dependent manner. Intracellular recordings revealed no effect of AO33 on resting membrane potential, little effect on input resistance, a small increase in the threshold of action potentials and an attenuation of stimulus induced paroxysmal depolarisation shifts (PDSs). Spontaneous PDSs initially decreased in duration until they were no longer observable.     

Muscarinic depression of synaptic transmission in the epileptogenic GABA withdrawal syndrome focus

The GABA withdrawal syndrome (GWS) is a model of local status epilepticus consecutive to the interruption of a prolonged GABA infusion into the rat somatomotor cortex. Bursting patterns in slices from GWS rats include intrinsic bursts of action potentials (APs) induced by intracellular depolarizing current injection and/or paroxysmal depolarization shifts (PDSs) induced by white matter stimulation. Possible changes in the effects of cholinergic drugs after in vivo induction of GWS were investigated on bursting cells (n = 30) intracellularly recorded in neocortical slices. In GWS slices, acetylcholine (Ach, 200-1000 microM) or carbachol (Cch, 50 microM) applications increased the number of bursts induced by depolarizing current injection while synaptically induced PDSs were significantly diminished (by 50-60%) or even blocked independently of the cholinergic-induced depolarization. The intrinsic burst facilitation and PDS depression provoked by Ach or Cch were mimicked by methyl-acetylcholine (mAch, 100-400 microM, n = 11), were reversed by atropine application (1-50 microM, n = 3), and were not mimicked by nicotine (50-100 microM, n = 4), indicating the involvement of muscarinic receptors. In contrast, in nonbursting cells from the same epileptic area (n = 42) or from equivalent area in control rats (n = 24), a nonsignificant muscarinic depression of EPSPs was induced by Cch and Ach. The mAch depression of excitatory postsynaptic potential (EPSPs) was significantly lower than that seen for PDSs in GWS rats. None of the cholinergic agonists caused bursting appearance in these cells. Therefore the present study demonstrates a unique implication of muscarinic receptors in exerting opposite effects on intrinsic membrane properties and on synaptic transmission in epileptiform GWS. Muscarinic receptor mechanisms may therefore have a protective role against the development and spread of epileptiform activity from the otherwise-activated epileptic focus.     

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.     

Epileptiform activity induced by changes in extracellular potassium in hippocampus

Using extra- and intracellular recording techniques, we investigated the induction and frequency modulation of spontaneous epileptiform activity produced by changes in the concentration of extracellular potassium ([K+]o). This paper describes a quantitative relationship between [K+]o and the frequency of spontaneously occurring epileptiform events. Recordings were made from the CA3 subfield of the rat in vitro hippocampal slice preparation. Intracellular microelectrodes were filled with 2 M Cs2SO4 and connected to a 3-kHz, time-share, single-electrode current- and voltage-clamp device. The frequency of spontaneous epileptiform (interictal) discharges was determined from extracellular recordings as a function of [K+]o. Current- and voltage-clamp techniques were used to characterize the intracellular correlate of these epileptiform events. The frequency of bicuculline-induced spontaneous epileptiform discharges was dependent on [K+]o. Below 4 mM [K+]o, spontaneous discharges occurred sporadically in the presence of 10 microM bicuculline. Increasing [K+]o from 5 to 10 mM caused a fivefold increase in the rate of spontaneous discharges. Spontaneous epileptiform discharges also occurred in the absence of bicuculline when [K+]o was increased above 6.5 mM. The rate of these discharges was dependent on [K+]o in much the same way as the discharges induced by bicuculline. For any given [K+]o concentration greater than 6.5 mM, however, the resultant discharge rate was faster than that obtained when bicuculline was present in the bathing solution. Simultaneous intra- and extracellular recordings revealed that the spontaneous high-[K+]o-induced interictal discharge was accompanied by a large depolarization of the membrane potential that appeared similar to the paroxysmal depolarizing shift (PDS) seen with other convulsants. The intracellularly recorded event fulfilled the criteria for a synaptically mediated PDS. The waveform of the PDS was complex and dependent on the membrane potential. When the membrane potential was held at 0 mV, spontaneously occurring hyperpolarizing potentials were noted during the inter-PDS interval. These events were blocked by picrotoxin or bicuculline and were probably spontaneous inhibitory postsynaptic potentials. The complexity of the PDS waveform suggested that more than one synaptic conductance was involved in the generation of the PDS. The mean measured reversal potential of the depolarizing phase was -10.7 mV. Voltage-clamp techniques were used to measure the conductance underlying the depolarizing phase of the high-[K+]o-induced PDS. The mean measured conductance was 51.5 nS, with a reversal potential of -7.9 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synaptic triggering of epileptiform discharges in CA1 pyramidal cells in vitro

Intra- and extracellular recordings were made in the transverse hippocampal slice in vitro to study the requirements for the triggering of epileptiform discharges of CA1 cells. Spontaneous and induced epileptiform discharges were produced by adding small amounts of sodium benzyl penicillin. Recorded intracellularly, the epileptiform activity consisted of a burst of action potentials superimposed on a depolarizing wave. Extracellular recordings demonstrated a marked synchronization. The epileptiform activity of the CA1 cells appeared without changes in the passive membrane properties or in the spike generating mechanism. Spontaneous epileptiform discharges of the CA1 cells depended upon a synaptic activation from the CA3 region. Stimulation of afferent fibres evoked an early and a late burst response in the CA1 cells. The long latency burst was caused by a re-excitation from the CA3 region. The early burst response seems to be an intrinsic property of the CA1 cells and may be induced by synaptic activation of either apical or basal dendrites. The findings suggest that synaptic depolarization is necessary for the generation of epileptiform discharges of the CA1 cells.     

Activation of a calcium-activated cation current during epileptiform discharges and its possible role in sustaining seizure-like events in neocortical slices

Epileptic seizures are composed of recurrent bursts of intense firing separated by periods of electrical quiescence. The mechanisms responsible for sustaining seizures and generating recurrent bursts are yet unclear. Using whole cell voltage recordings combined with intracellular calcium fluorescence imaging from bicuculline (BCC)-treated neocortical brain slices, I showed isolated paroxysmal depolarization shift (PDS) discharges were followed by a sustained afterdepolarization waveform (SADW) with an average peak amplitude of 3.3 +/- 0.9 mV and average half-width of 6.2 +/- 0.6 s. The SADW was mediated by the calcium-activated nonspecific cation current (I(can)) as it had a reversal potential of -33.1 +/- 6.8 mV, was unaffected by changing the intracellular chloride concentrations, was markedly diminished by buffering [Ca(2+)](i) with intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), and was reversibly abolished by the I(can) blocker flufenamic acid (FFA). The Ca(2+) influx responsible for activation of I(can) was mediated by both N-methyl-d-aspartate-receptor channels, voltage-gated calcium channels and, to a lesser extent, internal calcium stores. In addition to isolated PDS discharges, BCC-treated brain slices also produced seizure-like events, which were accompanied by a prolonged depolarizing waveform underlying individual ictal bursts. The similarities between the initial part of this waveform and the SADW and the fact it was markedly reduced by buffering [Ca(2+)](i) with BAPTA strongly suggested it was mediated, at least in part, by I(can). Addition of FFA reversibly eliminated recurrent bursting, and transformed seizure-like events into isolated PDS responses. These results indicated I(can) was activated during epileptiform discharges and probably participated in sustaining seizure-like events.     

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).     

Chronic epileptic foci in vitro in hippocampal slices from rats with the tetanus toxin epileptic syndrome

1. Minute doses of tetanus toxin were injected into the hippocampi of rats, under pentobarbitone anesthesia, to induce a chronic experimental epilepsy. The effects of this treatment were studied in vitro in hippocampal slices prepared 1-60 days after injection. 2. Epileptic activity was preserved in these slices in vitro, closely resembling that seen in vivo. Epileptiform afterdischarges were evoked by stimulation after survival times of greater than or equal to 3 days from injection. Spontaneous synchronous epileptic discharges were recorded from 7 days after injection. Both kinds of epileptiform activity were found with survival times up to 36 days but not beyond 44 days. This time course resembles the waxing and waning of the epileptic syndrome in vivo. 3. Two distinct types of spontaneous burst were seen. The first was a simple burst lasting 100-300 ms, reminiscent of the "interictal spike" of the clinical electroencephalogram. The second was much more prolonged, lasting several seconds. It consisted of a simple burst followed by a series of discrete afterbursts at 3-6/s and resembled the early stages of an epileptic seizure. Both types of burst were associated with slow field potentials that were positive at the cell-body layer. 4. Both the interictal and the seizure-like spontaneous epileptic discharges originated in the CA3b/c pyramidal cell region and propagated at 0.1-0.25 m/s along the cell layer toward the CA1 region. They occurred at very variable intervals, ranging from 20 s to 30 min. 5. Spontaneous epileptic bursts occurred in media containing 3 mM [K+]o to 5 mM in one-third of experiments during the period 1-4 wk after injection. Spontaneous bursts could be induced by increasing [K+]o to 5 mM in two-thirds of the remaining slices, which initially had produced evoked afterdischarges. 6. Intracellular recordings revealed that spontaneous field bursts were invariably associated with paroxysmal depolarization shifts (PDSs) and bursts of action potentials, suggesting that almost all the pyramidal cells in the region were recruited into the epileptic discharges. In some cells, smaller abnormal depolarizations were also seen; they were clearly larger than the spontaneous synaptic potentials but were not associated with field potentials. They may have been due to a more limited recruitment of pyramidal cells into partially synchronous bursts. 7. The tetanus toxin experimental epileptic syndrome differs from chronic models described previously in retaining in the hippocampal slice in vitro much of the spontaneous epileptic activity seen in vivo in the freely moving chronically epileptic rat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ondes paroxysmales induites par le métrazol (PTZ) sur les neurones d'Helix p.: Modèle fonctionnel

The convulsant pentylenetetrazol (PTZ), 1% induces paroxysmal depolarization shifts (PDS) on regularly firing neurones of Helix. Synaptic or antidromic stimulation or transmembrane current produces PDSs followed by a refractory period. For 5% of PTZ-treated neurons the membrane depolarizes up to -15 mV and PDS can be obtained by injected inward current. In voltage clamp experiments the steady i equals f(V) characteristic of PTZ-treated neurons is N-shaped with a current flowing inwardly between -45 and -15 mV. The negative conductance is enhanced by warming and disappears at temperatures lower than 15 degrees C. Besides, a slow triangular function (less than or equal to 10 mV/sec) applied to the voltage clamp system indicates an important hysteresis in the i equals f(V) curves according to the direction of sweeping: depolarization or repolarization. From a hyperpolarized state (-70 mV) the stable point of the N-shaped i equals f(V) characteristic is at -15 mV bringing the membrane potential to this level. From a depolarized state congruent to 10 mV) the i equals f(V) characteristic shows no local negative slope and the stable point is at -45 mV bringing the membrane potential down to this level. PDSs correspond to the voltage transition between these two stable points. The amplitude of PDSs is directly related to the amplitude of hysteresis and their frequency to the kinetics of the i equals f(V) characteristics. The different modes of activity under PTZ are explained by the position of the i equals 0 axis in relation with the region of negative resistance. It is suggested that increase of K+ -inactivation by PTZ is the main phenomenon leading to a N-shaped characteristic.     

Suppression of epileptiform burst discharges in CA3 neurons of rat hippocampal slices by the organic calcium channel blocker,verapamil

Summary We studied the effects of the organic calcium channel blocker, verapamil, on spontaneous and bicuculline-induced epileptiform burst discharges in CA3 pyramidal cells of hippocampal slices. A transient increase of burst discharge rate was observed in most cells within 30 min after the addition of verapamil (100 M) to the perfusing medium. Prolonged verapamil perfusions gradually reduced the rate and duration of burst discharges, then abolished them in all tested slices (over periods of 50–150 min) without blocking synaptic transmission. Responses to intracellular injections of current pulses were also gradually affected by verapamil: Action potential amplitude was decreased, action potential duration increased, frequency adaptation increased, amplitude of the fast hyperpolarization following a single action potential decreased, and amplitude and duration of the slow afterhyperpolarization markedly reduced. The amplitude of calcium spikes elicited in slices perfused with tetrodotoxin-containing medium was not affected by verapamil, but the mean velocity of depolarization near the peak of the calcium spike was decreased. Membrane resting potential and input resistance were not affected by verapamil. These results confirm that verapamil is able to suppress epileptiform activity, but suggest that this effect is rather non-specific, due to inhibition of both postsynaptic sodium and calcium conductances.     

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)     

Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices

The effect of low extracellular Mg2+ concentration ([Mg2+]o) on neuronal activity was studied in rat hippocampal slices. After 20-40 min of perfusion with Mg2+-free medium, when [Mg2+]o declined to approximately 0.1-0.4 mM, spontaneous field potentials developed in the CA1 and CA3 regions, but not in the dentate gyrus. In the CA3 pyramidal cell layer, these potentials consisted of repetitive (0.3-0.5 Hz), 40- to 120-ms-long positive deflections (2-5 mV) with superimposed population spikes. In the stratum (str.) pyramidale of the CA1 region, positive-negative deflections (less than 3 mV) lasting for 30-80 ms were observed, which occurred with a frequency of 0.3-0.5 Hz. In some cases, longer lasting and rapidly recurring events were also observed. In CA3 pyramidal cells, the intracellular correlates of the field potential transients were 20- to 30-mV paroxysmal depolarization shifts (PDS) with superimposed bursts of action potentials, followed by large (greater than 10 mV), 500- to 1,200-ms-long afterhyperpolarizations (AHP). In contrast, pyramidal neurons of the CA1 area did not show PDSs; instead, sequences of excitatory postsynaptic potentials (EPSPs)/inhibitory postsynaptic potentials (IPSPs) accompanied the transient field potential changes. Occasionally, spontaneous EPSPs/IPSPs, occurring with high frequencies, could also be observed in CA1 without any field potential transients. In both hippocampal regions, the epileptiform activity evolved without significant alterations in the resting membrane potential (RMP) and input resistance (RN) of the neurons, although a 2- to 5-mV reduction in action potential threshold was noted. The spontaneous activity in Mg2+-free medium was readily suppressed by raising the extracellular Ca2+ concentration ([Ca2+]o) from 1.6 to 3.6 mM. The perfusion of 10-30 microns DL-2-amino-5-phosphonovaleric acid (2-APV), an antagonist for the glutamate receptors of the N-methyl-D-aspartate (NMDA) type, also attenuated or reversibly blocked the spontaneous activity. Surgical isolation of area CA1 from CA3 ceased the occurrence of the transients in CA1 but not in CA3. The synaptic input/output curves were shifted to the left in the absence of [Mg2+]o. Threshold intensity for eliciting population spikes was 50-75% of that in normal medium. Paired-pulse facilitation was still present near threshold, but was reduced at higher stimulus intensities. Decreases in [Ca2+]o, produced by repetitive stimulation (20-Hz/5-10 s) of the Schaffer collateral/commissural pathway and monitored with ion-selective microelectrodes in the CA1 region, were enhanced in Mg2+-free medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitory mechanisms in epileptiform activity induced by low magnesium

In rat hippocampal slices epileptiform activity was induced by superfusion with Mg²⁺-free artificial cerebrospinal fluid (ACSF). Paroxysmal depolarization shifts (PDS) were evoked by electrical stimulation of Schaffer collaterals. To investigate the afterpotentials that follow PDS, intracellular recordings were made from CA1 pyramidal cells. The experiments revealed that several components are engaged in the generation of PDS afterpotentials in Mg²⁺-free ACSF. A long lasting component which determined the overall duration of the PDS afterhyperpolarization was blocked by intracellular application of ethylenebis(oxonitrilo)-tetraacetate (EGTA); concomitantly, the afterhyperpolarizations following depolarizing current injections were blocked. This indicated that the long lasting component was due to a slow Ca²⁺-activated K⁺ current. The block of Ca²⁺-activated K⁺ current uncovered a depolarizing PDS afterpotential with an N-shaped voltage dependence, suggesting that this depolarizing afterpotential component may be due to an N-methyl d-aspartate (NMDA) conductance. Intracellular injection of Cl^– revealed that the PDS were followed by Cl^– currents lasting about 500 ms. This component could be blocked by application of bicuculline suggesting that it is due to a synaptically GABA-mediated (i.e. -aminobutyric acid) Cl^– current. A comparison of PDS afterpotentials in Mg²⁺-free ACSF and those in other models of epileptiform activity suggests that similar sequences of inhibitory components are activated in spite of different pharmacological alterations of membrane conductances which induce the epileptiform discharges.     

The involvement of excitatory amino acids in neocortical epileptogenesis: NMDA and non-NMDA receptors

Summary Conventional intracellular recording techniques were used to investigate the N-methyl-D-aspartate (NMDA) and non-NMDA mediated synaptic mechanisms underlying the stimulus-induced paroxysmal depolarization shift (PDS) generated by cells in rat neocortical slices treated with bicuculline methiodide (BMI). The NMDA receptor antagonists CPP or MK-801 were ineffective in abolishing the PDS. However, both drugs were able to attenuate the late phase of the PDS and delay its time of onset. In contrast, the non-NMDA receptor blocker CNQX demonstrated potent anticonvulsant property by reducing the PDS into a depolarizing potential that was graded in nature. This CNQX-resistant depolarizing potential was readily blocked by CPP. Voltage-response analysis of the PDS indicated that the entire response (including its NMDA-mediated phase) displayed conventional voltage characteristics reminiscent of an excitatory postsynaptic potential that is mediated by non-NMDA receptors. We conclude that the activation of non-NMDA receptors is necessary and sufficient to induce epileptiform activity in the neocortex when the GABAergic inhibitory mechanism is compromised. The NMDA receptors contribute to the process of PDS amplification by prolonging the duration and reducing the latency of each epileptiform discharge. However, the participation of NMDA receptors is not essential for BMI-induced epileptogenesis, and their partial involvement in the PDS is dependent upon the integrity of the non-NMDA mediated input. The lack of NMDA-like voltage dependency observed in the PDS's late phase might reflect an uneven distribution of NMDA receptors along the cell and/or an association of this excitatory amino acid receptor subtype in the polysynaptic pathways within the neocortex.     

Low-magnesium medium induces epileptiform activity in mouse olfactory bulb slices

Magnesium-free medium can be used in brain slice studies to enhance glutamate receptor function, but this manipulation causes seizure-like activity in many cortical areas. The rodent olfactory bulb (OB) slice is a popular preparation, and potentially ictogenic ionic conditions have often been used to study odor processing. We studied low Mg(2+)-induced epileptiform discharges in mouse OB slices using extracellular and whole cell electrophysiological recordings. Low-Mg(2+) medium induced two distinct types of epileptiform activity: an intraglomerular delta-frequency oscillation resembling slow sniff-induced activity and minute-long seizure-like events (SLEs) consisting of large negative-going field potentials accompanied by sustained depolarization of output neurons. SLEs were dependent on N-methyl-D-aspartate receptors and sodium currents and were facilitated by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors. The events were initiated in the glomerular layer and propagated laterally through the external plexiform layer at a slow time scale. Our findings confirm that low-Mg(2+) medium should be used with caution in OB slices. Furthermore, the SLEs resembled the so-called slow direct current (DC) shift of clinical and experimental seizures, which has recently been recognized as being of great clinical importance. The OB slice may therefore provide a robust and unique in vitro model of acute seizures in which mechanisms of epileptiform DC shifts can be studied in isolation from fast oscillations.     

Intracellular study of excitability in the seizure-prone neocortex in vivo

The excitability of neocortical neurons from cat association areas 5-7 was investigated during spontaneously occurring seizures with spike-wave (SW) complexes at 2-3 Hz. We tested the antidromic and orthodromic responsiveness of neocortical neurons during the "spike" and "wave" components of SW complexes, and we placed emphasis on the dynamics of excitability changes from sleeplike patterns to seizures. At the resting membrane potential, an overwhelming majority of neurons displayed seizures over a depolarizing envelope. Cortical as well as thalamic stimuli triggered isolated paroxysmal depolarizing shifts (PDSs) that eventually developed into SW seizures. PDSs could also be elicited by cortical or thalamic volleys during the wave-related hyperpolarization of neurons, but not during the spike-related depolarization. The latencies of evoked excitatory postsynaptic potentials (EPSPs) progressively decreased, and their slope and depolarization surface increased, from the control period preceding the seizure to the climax of paroxysm. Before the occurrence of full-blown seizures, thalamic stimuli evoked PDSs arising from the postinhibitory rebound excitation, whereas cortical stimuli triggered PDSs immediately after the early EPSP. These data shed light on the differential excitability of cortical neurons during the spike and wave components of SW seizures, and on the differential effects of cortical and thalamic volleys leading to such paroxysms. We conclude that the wave-related hyperpolarization does not represent GABA-mediated inhibitory postsynaptic potentials (IPSPs), and we suggest that it is a mixture of disfacilitation and Ca(2+)-dependent K(+) currents, similar to the prolonged hyperpolarization of the slow sleep oscillation.     

Involvement of amygdala networks in epileptiform synchronization in vitro

We used field potential and intracellular recordings in rat brain slices that included the hippocampus, a portion of the basolateral/lateral nuclei of the amygdala (BLA) and the entorhinal cortex (EC). Bath application of the convulsant 4-aminopyridine (50 microM) to slices (n=12) with reciprocally connected areas, induced short-lasting interictal-like epileptiform discharges that (i) occurred at intervals of 1.2-2.8 s, (ii) originated in CA3, and (iii) spread to EC and BLA. Cutting the Schaffer collaterals abolished them in both parahippocampal areas where slower interictal-like (interval of occurrence=4-17 s) and prolonged ictal-like discharges (duration=15+/-6.9 s, mean+/-S.D., n=7) appeared. These new types of epileptiform activity originated in either EC or BLA. Similar findings were obtained in slices (n=19) in which the hippocampus outputs were not connected with the EC and BLA under control conditions. Cutting the EC-BLA connections made independent slow interictal- and ictal-like activities appear in both areas (n=5). NMDA receptor antagonism (n=6) abolished ictal-like discharges and reduced the duration of the slow interictal-like events. Repetitive stimulation of BLA at 0.5-1 Hz in Schaffer collateral cut slices, induced interictal-like epileptiform depolarizations in EC and reversibly blocked ictal-like activity (n=14). Thus, CA3 outputs in intact slices entrain EC and BLA networks into an interictal-like pattern that inhibits the propensity of these parahippocampal areas to generate prolonged ictal-like paroxysms. Accordingly, NMDA receptor-dependent ictal-like events are initiated in BLA or EC once the propagation of CA3-driven interictal-like discharges to these areas is abated by cutting the Schaffer collaterals. Similar inhibitory effects also occur by activating BLA outputs directed to EC at rates that mimic the CA3-driven interictal-like pattern.