Role of synaptic metabotropic glutamate receptors in epileptiform discharges in hippocampal slices

Application of group I metabotropic glutamate receptor (mGluR) agonists elicits seizure discharges in vivo and prolonged ictal-like activity in in vitro brain slices. In this study we examined 1) if group I mGluRs are activated by synaptically released glutamate during epileptiform discharges induced by convulsants in hippocampal slices and, if so, 2) whether the synaptically activated mGluRs contribute to the pattern of the epileptiform discharges. The GABA(A) receptor antagonist bicuculline (50 microM) was applied to induce short synchronized bursts of approximately 250 ms in mouse hippocampal slices. Addition of 4-aminopyridine (4-AP; 100 microM) prolonged these bursts to 0.7-2 s. The mGluR1 antagonist (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY 367385; 25-100 microM) and the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP; 10-50 microM), applied separately, significantly reduced the duration of the synchronized discharges. The effects of these antagonists were additive when applied together, suggesting that mGluR1 and mGluR5 exert independent actions on the epileptiform bursts. In phospholipase C beta1 (PLCbeta1) knockout mice, bicuculline and 4-AP elicited prolonged synchronized discharges of comparable duration as those observed in slices from wild-type littermates. Furthermore, mGluR1 and mGluR5 antagonists reduced the duration of the epileptiform discharges to the same extent as they did in the wild-type preparations. The results suggest that mGluR1 and mGluR5 are activated synaptically during prolonged epileptiform discharges induced by bicuculline and 4-AP. Synaptic activation of these receptors extended the duration of synchronized discharges. In addition, the data indicate that the synaptic effects of the group I mGluRs on the duration of epileptiform discharges were mediated by a PLCbeta1-independent mechanism.     

Epileptiform discharges induced by combined application of bicuculline and 4-aminopyridine are resistant to standard anticonvulsants in slices of rats.

Application of 4-aminopyridine (4AP) has previously been reported to produce different patterns of epileptiform discharges in entorhinal cortex-hippocampal-slices. Here we describe that 4-AP induced epileptiform activity in the EC becomes insensitive to anticonvulsant drugs (phenytoin, carbamazepine, valproic acid, phenobarbital) when GABAergic transmission is blocked by bicuculline. We propose that the activities induced by 4-aminopyridine and bicuculline may provide an in vitro model for the development of new drugs against difficult-to-treat focal epilepsy.     

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)     

Local synaptic circuits and epileptiform activity in slices of neocortex from children with intractable epilepsy.

1. Single and dual intracellular recordings were performed in neocortical slices obtained from tissue samples surgically removed from children (8 mo to 15 yr) for the treatment of intractable epilepsy. Electrical stimulation and glutamate microapplication were used to study local synaptic inputs to pyramidal cells. 2. In recordings with potassium-acetate electrodes, activation of presynaptic neocortical neurons with glutamate microdrops did not elicit a clear increase in postsynaptic potentials (PSPs) but did suppress current-evoked repetitive spike firing in recorded neurons. Bicuculline (10 microM) blocked this effect, suggesting it was caused by the activation of presynaptic gamma-aminobutyric acid (GABA) cells. In recordings with KCl electrodes, glutamate microdrops elicited an increase in the frequency and amplitude of depolarizing PSPs. Bicuculline (5-10 microM) blocked the glutamate-evoked PSPs, suggesting they were reversed GABAA-receptor-mediated inhibitory postsynaptic potentials (IPSPs). In one cell recorded with a KCl electrode (total n = 8), current-evoked spike trains elicited afterdischarges of reversed IPSPs, thus revealing a recurrent inhibitory circuit. Therefore local inhibitory synaptic circuits were robust and could be observed in tissue from patients as young as 11 mo. 3. In addition to short-latency (10-25 ms), monosynaptic excitatory postsynaptic potentials (EPSPs), electrical stimulation at low intensities sometimes elicited delayed EPSPs (20-60 ms). When GABAA-receptor-mediated synaptic inhibition was partially reduced in bicuculline (5-10 microM), electrical stimulation evoked large EPSPs at long and variable latencies (100-300 ms). Glutamate microapplication caused an increase in the frequency and amplitude of EPSPs; preliminary results suggest that glutamate microdrops were less likely to evoke EPSPs in tissue from younger patients (8-12 mo) than in slices from patients greater than 4 yr. Evidence for local excitatory synaptic circuits was thus found when synaptic inhibition was partially reduced. 4. After further reduction of inhibition in bicuculline (5-50 microM), electrical stimulation elicited epileptiform bursts. In pairs of simultaneously recorded neurons, bursts were generated synchronously from long-latency EPSPs (100-300 ms) in slices from patients as young as 8 mo. Reflected EPSPs at very long and variable latencies (500-1,100 ms) and repetitive epileptiform bursts could be evoked synchronously in pairs of cells. Glutamate activation of local presynaptic neurons elicited robust epileptiform events in recorded cells. This was seen in slices from patients as young as 16 mo. 5. These data provide physiological evidence for the presence of local inhibitory and excitatory synaptic circuits in human neocortex at least as early as 11 and 8 mo, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

KCNQ/Kv7 channel regulation of hippocampal gamma-frequency firing in the absence of synaptic transmission

Synchronous neuronal firing can be induced in hippocampal slices in the absence of synaptic transmission by lowering extracellular Ca2+ and raising extracellular K+. However, the ionic mechanisms underlying this nonsynaptic synchronous firing are not well understood. In this study we have investigated the role of KCNQ/Kv7 channels in regulating this form of nonsynaptic bursting activity. Incubation of rat hippocampal slices in reduced (<0.2 mM) [Ca2+]o and increased (6.3 mM) [K+]o, blocked synaptic transmission, increased neuronal firing, and led to the development of spontaneous periodic nonsynaptic epileptiform activity. This activity was recorded extracellularly as large (4.7 +/- 1.9 mV) depolarizing envelopes with superimposed high-frequency synchronous population spikes. These intraburst population spikes initially occurred at a high frequency (about 120 Hz), which decayed throughout the burst stabilizing in the gamma-frequency band (30-80 Hz). Further increasing [K+]o resulted in an increase in the interburst frequency without altering the intraburst population spike frequency. Application of retigabine (10 microM), a Kv7 channel modulator, completely abolished the bursts, in an XE-991-sensitive manner. Furthermore, application of the Kv7 channel blockers, linopirdine (10 microM) or XE-991 (10 microM) alone, abolished the gamma frequency, but not the higher-frequency population spike firing observed during low Ca2+/high K+ bursts. These data suggest that Kv7 channels are likely to play a role in the regulation of synchronous population firing activity.     

Engagement of rat striatal neurons by cortical epileptiform activity investigated with paired recordings

The striatum is thought to play an important role in the spreading of epilepsy from cortical areas to deeper brain structures, but this issue has not been addressed with intracellular techniques. Paired recordings were used to assess the impact of cortical epileptiform activity on striatal neurons in brain slices. Bath-application of 4-amynopyridine (100 microM) and bicuculline (20 microM) induced synchronized bursts in all pairs of cortical neurons (< or = 5 mm apart) in coronal, sagittal, and oblique slices (which preserve connections from the medial agranular cortex to the striatum). Under these conditions, striatal medium spiny neurons (MSs) displayed a strong increased spontaneous glutamatergic activity. This activity was not correlated to the cortical bursts and was asynchronous in pairs of MSs. Sporadic, large-amplitude synchronous depolarizations also occurred in MSs. These events were simultaneously detected in glial cells, suggesting that they were accompanied by considerable increases in extracellular potassium. In oblique slices, cortically driven bursts were also observed in MSs. These events were synchronized to cortical epileptiform bursts, depended on non-N-methyl-D-aspartate (NMDA) glutamate receptors, and persisted in the cortex, but not in the striatum, after disconnection of the two structures. During these bursts, MS membrane potential shifted to a depolarized value (59 +/- 4 mV) on which an irregular waveform, occasionally eliciting spikes, was superimposed. Thus synchronous activation of a limited set of corticostriatal afferents can powerfully control MSs. Cholinergic interneurons located < 120 microm from simultaneously recorded MSs, did not display cortically driven bursts, suggesting that these cells are much less easily engaged by cortical epileptiform activity.     

Evidence for bursting pacemaker neurones in cultured spinal cord cells

Intracellular recordings were made from dissociated mouse spinal cord cells in primary culture. One type of spinal cord neurone, with a large cell body (40-50 micron), 3-5 short neurites, and a mean resting potential of -65 mV, was found to fire rhythmic bursts of action potentials with a phase duration of approximately 1s when the membrane potential was depolarized to -55 mV. These bursts did not arise from spontaneous synaptic input, but appeared to result from endogenous ionic conductance properties of the membrane resembling those observed in molluscan bursting pacemaker neurones. Ionic conductances underlying this bursting activity were studied pharmacologically by local application of ionic conductance blockers. Pacemaker potentials depended on Na+ conductance, since tetrodotoxin and Na-free medium were the most potent agents for blocking spontaneous rhythmic activity. However, a Ca2+ conductance was involved in the depolarizing phase of membrane potential oscillations, since Ba2+ application increased oscillation amplitude. Action potentials observed during the bursts were Na+- and Ca2+-dependent. They did not differ significantly from those observed in other spinal cord neurones in culture. Application of tetraethylammonium, CoCl2, BaCl2 and 4-aminopyridine revealed at least three different potassium conductances which controlled this bursting pacemaker activity. A delayed potassium conductance controlled spike duration, a Ca-dependent potassium conductance controlled the duration of the burst and underlay the hyperpolarizing phase terminating the burst, and finally, a transient potassium conductance appeared to be involved in the control of phase duration. The demonstration that spinal cord neurones growing in monolayer culture display typical bursting pacemaker activity raises the possibility that bursting pacemaker neurones in the mammalian spinal cord may be involved in a phasic pattern generator that could control such activities as walking and the respiratory rhythm.     

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)     

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.     

Carbenoxolone depresses spontaneous epileptiform activity in the CA1 region of rat hippocampal slices

An important contributor to the generation of epileptiform activity is the synchronization of burst firing in a group of neurons. The aim of this study was to investigate whether gap junctions are involved in this synchrony using an in vitro model of epileptiform activity. Hippocampal slices (400 μm) were prepared from female Sprague–Dawley rats (120–170 g). The perfusion of slices with a medium containing no added magnesium and 4-aminopyridine (50 μM) resulted in the generation of spontaneous bursts of population spikes of a fast frequency along with less frequent negative-going bursts. The frequency of the bursts produced was consistent over a 3 h period. Carbenoxolone (100 μM), a gap junction blocker and mineralocorticoid agonist, perfused for 75 min, reduced the frequency of both types of spontaneous burst activity. Perfusion of spironolactone (1 μM), a mineralocorticosteroid antagonist, for 15 min prior to and during carbenoxolone perfusion did not alter the ability of carbenoxolone to depress the frequency of spontaneous activity. The incubation of hippocampal slices in carbenoxolone prior to recording increased the time taken for the spontaneous activity to start on change to the zero magnesium/4-aminopyridine medium and decreased the total number of spontaneous bursts over the first 60 min period.

The ability of carbenoxolone to delay induction of epileptiform activity and reduce established epileptiform activity suggests that gap junctions contribute to the synchronization of neuronal firing in this model.     

Synaptic regulation of the slow Ca2+-activated K+ current in hippocampal CA1 pyramidal neurons: implication in epileptogenesis.

The slow Ca2+-activated K+ current (sI(AHP)) plays a critical role in regulating neuronal excitability, but its modulation during abnormal bursting activity, as in epilepsy, is unknown. Because synaptic transmission is enhanced during epilepsy, we investigated the synaptically mediated regulation of the sI(AHP) and its control of neuronal excitability during epileptiform activity induced by 4-aminopyridine (4AP) or 4AP+Mg2+-free treatment in rat hippocampal slices. We used electrophysiological and photometric Ca2+ techniques to analyze the sI(AHP) modifications that parallel epileptiform activity. Epileptiform activity was characterized by slow, repetitive, spontaneous depolarizations and action potential bursts and was associated with increased frequency and amplitude of spontaneous excitatory postsynaptic currents and a reduced sI(AHP.) The metabotropic glutamate receptor (mGluR) antagonist (S)-alpha-methyl-4-carboxyphenylglycine did not modify synaptic activity enhancement but did prevent sI(AHP) inhibition and epileptiform discharges. The mGluR-dependent regulation of the sI(AHP) was not caused by modulated intracellular Ca2+ signaling. Histamine, isoproterenol, and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid reduced the sI(AHP) but did not increase synaptic activity and failed to evoke epileptiform activity. We conclude that 4AP or 4AP+Mg-free-induced enhancement of synaptic activity reduced the sI(AHP) via activation of postsynaptic group I/II mGluRs. The increased excitability caused by the lack of negative feedback provided by the sI(AHP) contributes to epileptiform activity, which requires the cooperative action of increased synaptic activity.     

Isoflurane hyperpolarizes neurones in rat and human cerebral cortex

The effect of the anaesthetic gas isoflurane was studied by intracellular recordings in neurones from rat hippocampal cortex and neurones from human neocortex in vitro. Anaesthetic concentrations of isoflurane abolished spontaneous activity and reduced synaptically evoked activity without rendering individual cells inexcitable or preventing evoked synaptic activity to increased afferent input. Induced epileptiform activity was not observed. Isoflurane reversibly hyperpolarized the cell membrane in a dose-dependent manner, isoflurane 1.5, 3 and 5% causing 4 +/- 1, 6 +/- 2 and 8 +/- 2 mV (mean +/- SD) hyperpolarization, respectively. The hyperpolarization was accompanied by a reduction in the input resistance, 18 +/- 3% for 3% isoflurane. The effects remained unchanged after synaptic transmission was blocked. Five experiments with intracellular recordings from human cortical neurones in vitro showed identical results.     

Epileptiform activity in hippocampal slice cultures with normal inhibitory synaptic drive

The synaptic events responsible for epileptiform burst discharge are often difficult to define. Blockade of inhibition has been used to produce epileptiform events, but it is unclear whether increased excitatory activity in the presence of normal inhibition can also result in burst discharge. In the hippocampal slice culture preparation, a small percentage of cultures exhibit spontaneous bursts. To determine whether the absence of inhibitory postsynaptic potentials (IPSPs) is responsible for these spontaneous bursts, we applied the glutamate antagonist, kynurenic acid (KYN) to block burst activity, and unmask any underlying IPSPs. KYN (10 mM) quickly reduced synaptic activity with concomitant loss of burst discharge. Washout of KYN resulted in a gradual return of synaptic activity, during which time both fast and slow IPSPs were clearly observed. As burst activity returned to control levels, excitatory postsynaptic potentials (EPSPs) were increasingly superimposed within the inhibitory events, obscuring (but not eliminating) the IPSPs. In these hippocampal slice cultures, therefore, epileptiform bursts appear to be the result of an abnormally high level of excitatory synaptic drive, not a reduction in inhibition.     

Strong, reliable and precise synaptic connections between thalamic relay cells and neurones of the nucleus reticularis in juvenile rats

The thalamic reticular nucleus (nRT) is composed entirely of GABAergic inhibitory neurones that receive input from pyramidal cortical neurones and excitatory relay cells of the ventrobasal complex of the thalamus (VB). It plays a major role in the synchrony of thalamic networks, yet the synaptic connections it receives from VB cells have never been fully physiologically characterised. Here, whole-cell current-clamp recordings were obtained from 22 synaptically connected VB-nRT cell pairs in slices of juvenile (P14-20) rats. At 34-36 °C, single presynaptic APs evoked unitary EPSPs in nRT cells with a peak amplitude of 7.4 ± 1.5 mV (mean ± s.e.m .) and a decay time constant of 15.1 ± 0.9 ms. Only four out of 22 pairs showed transmission failures at a mean rate of 6.8 ± 1.1 %. An NMDA receptor (NMDAR)-mediated component was significant at rest and subsequent EPSPs in a train were depressed. Only one out of 14 pairs tested was reciprocally connected; the observed IPSPs in the VB cell had a peak amplitude of 0.8 mV and were completely abolished in the presence of 10 μ m bicuculline. Thus, synaptic connections from VB cells to nRT neurones are mainly 'drivers', while a small subset of cells form closed disynaptic loops.     

Calcium-activated afterhyperpolarizations regulate synchronization and timing of epileptiform bursts in hippocampal CA3 pyramidal neurons

Calcium-activated potassium conductances regulate neuronal excitability, but their role in epileptogenesis remains elusive. We investigated in rat CA3 pyramidal neurons the contribution of the Ca(2+)-activated K(+)-mediated afterhyperpolarizations (AHPs) in the genesis and regulation of epileptiform activity induced in vitro by 4-aminopyridine (4-AP) in Mg(2+)-free Ringer. Recurring spike bursts terminated by prolonged AHPs were generated. Burst synchronization between CA3 pyramidal neurons in paired recordings typified this interictal-like activity. A downregulation of the medium afterhyperpolarization (mAHP) paralleled the emergence of the interictal-like activity. When the mAHP was reduced or enhanced by apamin and EBIO bursts induced by 4-AP were increased or blocked, respectively. Inhibition of the slow afterhyperpolarization (sAHP) with carbachol, t-ACPD, or isoproterenol increased bursting frequency and disrupted burst regularity and synchronization between pyramidal neuron pairs. In contrast, enhancing the sAHP by intracellular dialysis with KMeSO(4) reduced burst frequency. Block of GABA(A-B) inhibitions did not modify the abnormal activity. We describe novel cellular mechanisms where 1) the inhibition of the mAHP plays an essential role in the genesis and regulation of the bursting activity by reducing negative feedback, 2) the sAHP sets the interburst interval by decreasing excitability, and 3) bursting was synchronized by excitatory synaptic interactions that increased in advance and during bursts and decreased throughout the subsequent sAHP. These cellular mechanisms are active in the CA3 region, where epileptiform activity is initiated, and cooperatively regulate the timing of the synchronized rhythmic interictal-like network activity.     

Horizontal spread of synchronized activity in neocortex and its control by GABA-mediated inhibition

1. Suppression of GABAA receptor-mediated inhibition disrupts the neural activity of neocortex and can lead to synchronized discharges that mimic those of partial epilepsy. We have studied the role of GABAA-mediated inhibition in controlling the synchronization and horizontal (tangential) spread of cortical activity. 2. Slices of rat SmI were maintained in vitro and focally stimulated in layer VI while recording with a horizontal array of extracellular electrodes. Inhibition was slightly suppressed by adding low concentrations of the GABAA antagonists bicuculline or bicuculline methiodide to the bathing medium. Under control conditions neural activity was narrowly confined to a vertical strip of cortex. The horizontal spread of activity expanded about twofold in the presence of antagonist concentrations (less than or equal to 0.5 microM) that were expected to suppress GABAA function by no more than 10-20%. 3. At antagonist concentrations between 0.4 and 1.0 microM, evoked epileptiform activity appeared. These threshold-dose epileptiform events showed wide variations in size and duration (even at the same recording site), very variable distances of horizontal propagation, specific sites of propagation failure, reversals of propagation direction, and directional asymmetries in their probability of propagation. This contrasts with activity observed previously (Ref. 9) in high bicuculline concentrations (greater than or equal to 10 microM): large, stereotyped events that propagate reliably without decrement or reflection. 4. Intracellular recordings were obtained from pyramidal neurons in layers II/III in the presence of less than or equal to 1 microM bicuculline. Inhibitory postsynaptic potentials (IPSPs) were observed during both primary evoked responses and propagating epileptiform events and were often comparable in size and duration to those in untreated cortex. Epileptiform field potentials were always correlated with synaptic activity in single cells, but the pattern and type of PSPs varied with the form of the field potentials. Large amplitude epileptiform events coincided with an overwhelming inhibition of upper layer neurons. 5. We conclude that 1) the horizontal spread of normal cortical activity is strongly constrained by GABAA-mediated IPSPs, 2) a relatively small reduction in the efficacy of inhibition leads to a large increase in the spread of excitation, 3) initiation and propagation of synchronized epileptiform activity can occur even in the presence of robust cortical inhibition, and 4) the character of epileptiform activity is strongly affected by the influences of inhibition.     

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

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 microM) in the CA3 area of rat hippocampal slices maintained in vitro. 2. 4-AP-induced epileptiform bursts, consisting of a 25-to 80-ms depolarizing shift of the neuronal membrane associated with three to six fast action potentials, occurred at the frequency of 0.61 +/- 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 spontaneous activity consisting of a slow depolarization also occurred but at a lower rate (0.04 +/- 0.2/s). 3. The effects of 4-AP on EPSPs and inhibitory postsynaptic potentials (IPSPs) evoked by mossy fiber stimulation were studied on neurons impaled with a mixture of K acetate and 2(triethyl-amino)-N-(2,6-dimethylphenyl) acetamide (QX-314)-filled microelectrodes. After the addition of 4-AP, the EPSP became potentiated and was followed by the appearance of a giant EPSP. This giant EPSP completely obscured the early IPSP recorded under control conditions and inverted at -32 +/- 3.9 mV (n = 4), suggesting that both inhibitory and excitatory conductances were involved in its generation. IPSPs evoked by Schaffer collateral stimulation increased in amplitude and duration after 4-AP application. 4. The spontaneous field bursts and the stimulus-induced giant EPSP induced by 4-AP were not affected by N-methyl-D-aspartate (NMDA) receptor antagonists 3-3 (2-carboxy piperazine-4-yl) propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV) but were blocked by quisqualate/kainate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). CNQX also abolished the presence of small spontaneously occurring EPSPs, thereby disclosing the presence of bicuculline-sensitive (BMI, 20 microM) IPSPs. 5. Small, nonsynchronous EPSPs played an important role in the generation of 4-AP-induced epileptiform activity. 1) After the addition of 4-AP, small EPSPs appeared randomly on the baseline and then became clustered to produce a depolarizing envelope of irregular shape that progressively formed an epileptiform burst, 2) These small EPSPs were more numerous in the 100 ms period that preceded burst onset. 3) The frequency of occurrence of small EPSPs was positively correlated with the frequency of occurrence of synchronous bursts. 4) Small EPSPs and bursts were similarly decreased after the addition of different concentrations of CNQX (IC50 in both cases of approximately 1.2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Synchronized neuronal activities in neocortical explant cultures

Summary Intracellular recordings revealed that in neocortical expiant cultures prepared on the day of birth and examined 3–6 weeks later, neurons mature and establish complex synaptic relationships that lead to spontaneous and triggered synchronous discharge. The spontaneous synchronous activity took several forms, including periodic generation of epileptiform depolarizing waves, prolonged periods of seizure-like discharge, and periodic, intense barrages of IPSPs. Synchronous depolarizations were associated with a marked increase in membrane conductance. Intracellular injection of currents of varying polarity and intensity affected their amplitudes and polarities without influencing the probability of their occurance, indicating that the discharge reflected the synchronous activities of a neuronal population. This conclusion was confirmed with simultaneous recordings from pairs of neurons. Effects of the GABAa receptor antagonist, bicuculline, and the NMDA receptor antgonist, 2-aminophosphonvalerate (2APV), were used to assess the contributions of impairment of inhibition and enhancement of excitation to the initiation of synchronous discharge. The frequency with which spontaneous depolarizations were generated in normal medium was markedly reduced by 2APV. Moreover, seizure-like activity was induced by removing Mg⁺⁺ from the medium, a condition that enhances conductance through NMDA receptor-coupled channels. This behavior was also attenuated by 2APV. Perfusion of bicuculline was potently epileptogenic. 2APV cut short the late, voltage-dependent phase of bicuculline-induced paroxysmal depolarizations, indicating a role of NMDA receptors in generating this component of the wave. Epileptiform activities induced by withdrawal of Mg⁺⁺ were greatly augmented by bicuculline, indicating that blockade of inhibition was not a prerequisite for seizure-like activity. This conclusion is supported by the finding that in many neurons in untreated cultures, paroxysmal generation of trains of IPSPs was the primary manifestation of spontaneous, synchronous population discharge.     

Synchrony levels during evoked seizure-like bursts in mouse neocortical slices

Slices (n = 45) from the somatosensory cortex of mouse (P8-13) generated spontaneous bursts of activity (0.10 +/- 0.05 Hz) that were recorded extracellularly. Multiunit action potential (AP) activity was integrated and used as an index of population activity. In this experimental model, seizure-like activity (SLA) was evoked with bicuculline (5-10 microM) or N-methyl-d-aspartate (NMDA, 5 microM). SLA was an episode with repetitive bursting at a frequency of 0.50 +/- 0.06 Hz. To evaluate whether SLA was associated with a change in synchrony, we obtained simultaneous intracellular and extracellular recordings (n = 40) and quantified the relationship between individual cells and the surrounding population of neurons. During the SLA there was an increase in population activity and bursting activity was observed in neurons and areas that were previously silent. We defined synchrony as cellular activity that is consistently locked with the population bursts. Signal-averaging techniques were used to determine this component. To quantitatively assess change in synchronous activity at SLA onset, we estimated the entropy of the single cell's spike trains and subdivided this measure into network burst-related information and noise-related entropy. The burst-related information was not significantly altered at the onset of NMDA-evoked SLA and slightly increased when evoked with bicuculline. The signal-to-noise ratio determined from the entropy estimates showed a significant decrease (instead of an expected increase) during SLA. We conclude that the increased population activity during the SLA is attributed to recruitment of neurons rather than to increased synchrony of each of the individual elements.