Synchronous Epileptiform Discharges Research Articles

Activation of muscarinic receptors during blockade of GABAA-mediated inhibition induces synchronous epileptiform activity in immature rat hippocampus

We investigated the effects of the cholinergic agonist carbachol (25μM) on the synaptic potentials recorded extracellularly and intracellularly from the CA3 area of immature hippocampal slices of the rat (postnatal days 10–20). In control conditions, carbachol reduced the amplitude of evoked synaptic responses (n=8) and did not induce any spontaneous synchronous activity (n=12); the depressant effect of carbachol was mimicked by acetylcholine (100μM, in eserine 10μM, n=5) and was reversed by the muscarinic antagonist atropine (1μM, n=2). The GABAA-receptor antagonist bicuculline (10μM) enhanced the amplitude and duration of the evoked synaptic responses and induced infrequent (0.016–0.045Hz) spontaneous synchronous discharges in 23/37 of the slices. Application of carbachol in the presence of bicuculline reduced the amplitude of the evoked synaptic responses (n=21) and in addition induced synchronous discharges with rates of occurrence 0.075–0.225Hz, in 64/68 slices. Both effects were mimicked by acetylcholine and eserine, and antagonized by atropine. The specific muscarinic antagonists pirenzepine (M1-type), tripitramine (M2-type), 4-diphenylacetoxy-N-methylpiperidine methiodide (M3-type) and tropicamide (M4-type) (all tested at 0.1–1μM) reversibly reduced the frequency of synchronous carbachol-induced discharges. In addition, these discharges were reversibly blocked by high Ca2+ perfusion medium (7mM CaCl2, n=4) and by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10μM, n=7). Synchronous epileptiform discharges were recorded from both CA1 and CA3 areas in intact slices (n=3), but only from CA3 following disruption of the CA1–CA3 synaptic connections (n=3).These experiments suggest that activation of muscarinic receptors during blockade of GABAA-mediated potentials, may enhance synchronous epileptiform activity in immature (postnatal days 10–20) hippocampus, through activation of local excitatory circuits and that endogenous acetylcholine may be sufficient to play this role.

Developmental features of 4-aminopyridine induced epileptogenesis

4-Aminopyridine (4-AP, 50 μM), perfused in rat hippocampal slices from postnatal days 2–30 (P2–P30), induced in the CA3 area the appearance of spontaneous epileptiform discharges, short (interictal-like) and sustained (ictal-like), as well as a slow potential. The duration of epileptiform discharges decreased and their rate of occurrence (frequency) increased with maturation: their duration during the 1st postnatal week was 4–6 times longer and their frequency 5 times lower, compared to those of the 4th postnatal week. Ictal discharges gradually disappeared at the end of the 4th postnatal week. Spontaneous synchronous activity — as a whole — often appeared in clusters separated by equal or longer length inactive periods, during the first two postnatal weeks. At the same period, ictal discharges were often followed by repetitive afterdischarges, forming sequences which lasted 0.7–1.5 min. Sectioning experiments showed that epileptiform discharges were generated in CA3, and their presence in CA1 depended on the integrity of CA1-CA3 synaptic connections. In conclusion, these findings demonstrate that (i) immature CA3 can generate synchronous epileptiform discharges as early as P2, (ii) such discharges are longer lasting and more complex during the early developmental stages and (iii) there are two time points (end of 2nd, end of 4th postnatal weeks), when maturational changes alter the epileptogenic properties of immature hippocampus.

Burst characteristics of dentate gyrus granule cells: evidence for endogenous and nonsynaptic properties.

1. Hippocampal slices bathed in 8 mM potassium and 0-added calcium exhibited spontaneous epileptiform activity in the dentate gyrus. Extracellular recording revealed recurrent prolonged bursts of population spikes and an associated negative DC shift. These episodes were very similar to the in vivo phenomenon termed maximal dentate activation (MDA). Therefore this in vitro activity will be referred to as MDA-like activity or events. 2. During the MDA-like activity, the individual granule cells exhibited a sustained depolarization that matched the duration of the negative extracellular DC shift. At the beginning of the MDA-like activity, there was a burst of action potentials. After the burst, most granule cells either continued to fire action potentials regularly or in bursts. Some cells exhibited this initial burst of activity and then a dramatic reduction in firing rate. This reduction in rate was followed by a gradual increase in the amplitude and frequency of the epileptiform activity recorded during the remainder of the MDA-like event. 3. Before and between MDA-like events, spontaneous cellular activity consisted of single action potentials and bursts of action potentials on a depolarizing envelope. In addition, depolarizing potentials, up to 13 mV, were recorded. There were no extracellular field potentials associated with these intracellularly recorded potentials. 4. In the 8 mM potassium, 0-added calcium test solution, the membrane potential threshold for burst production was significantly lower than in normal potassium and calcium medium. 5. The effect of depolarizing and hyperpolarizing current injections on the amplitude and frequency of the epileptiform activity was tested. Current injection had no effect on the frequency of the epileptiform activity recorded during the MDA-like events. However, the frequency of the cellular bursts between MDA-like events was very sensitive to current injection. Depolarizing current increased the frequency, and hyperpolarizing current decreased the frequency of the spontaneous activity. 6. This study has shown that in 8 mM potassium and 0-added calcium the granule cells of the dentate gyrus are capable of generating spontaneous bursts that appear to be mediated by endogenous mechanisms. In addition, synchronized epileptiform discharges were recorded from the granule cells at regular intervals that appear were recorded from the granule cells at regular intervals that appear to be mediated by exogenous nonsynaptic mechanisms.

Lateral inhibition and granule cell synchrony in the rat hippocampal dentate gyrus

Studies of patients with temporal lobe epilepsy and of experimental models of this disorder suggest that the hippocampal dentate gyrus may be a common site of seizure onset and propagation. However, the nature of the dentate "network defect" that could give rise to spontaneous, intermittent, and synchronous population discharges is poorly understood. We have hypothesized that large expanses of the dentate granule cell layer have an underlying tendency to discharge synchronously in response to afferent excitation, but do not do so normally because vulnerable dentate hilar neurons establish lateral inhibition in the granule cell layer and thereby prevent focal discharges from spreading to surrounding segments. To address this hypothesis, we (1) identified functionally independent segments of the granule cell layer; (2) determined whether discharges in one segment evoke lateral inhibition in surrounding segments; and, (3) determined if disinhibition induces normally independent segments of the granule cell layer to discharge synchronously. Simultaneous extracellular recordings were made from two locations along the longitudinal or transverse axes of the granule cell layer using saline- and bicuculline-filled electrodes that were glued together. Leakage of 10 mM bicuculline from the electrode tip produced no detectable spontaneous activity. However, single perforant path stimuli evoked multiple population spikes at the bicuculline electrode and simultaneous normal responses at the nearby saline electrode. The multiple spikes evoked at the bicuculline electrode did not propagate to, and were not detected by, the adjacent saline electrode, indicating functional separation between neighboring subgroups of granule cells. Paired-pulse stimulation revealed that multiple discharges were not only restricted to one segment of the granule cell layer, but strongly inhibited surrounding segments. This lateral inhibition in surrounding segments often lasted longer than 150 msec. Finally, we evaluated granule cell activity at two normally independent sites within the granule cell layer both before and after disinhibition was induced by high frequency stimulus trains or bicuculline injection. Following a 10 sec, 20 Hz perforant path stimulus train, 2 Hz stimulation evoked virtually identical synchronized epileptiform discharges from normally separated sites. Similarly, intrahippocampal or intravenous bicuculline injection produced spontaneous synchronous epileptiform discharges throughout the granule cell layer. These results indicate that lateral or "surround" inhibition is an operant physiological mechanism in the normal dentate gyrus and suggest that afferent stimuli to a disinhibited dentate network evoke highly synchronized discharges from large expanses of the granule cell layer that are normally kept functionally separated by GABA-mediated inhibition.

Low-magnesium epilepsy in rat hippocampal slices: Inhibitory postsynaptic potentials in the CA1 subfield

Spontaneous, synchronous epileptiform discharges were recorded in both CA3 and CA1 subfields of rat hippocampal slices perfused with Mg2+-free medium. Surgical separation of the two areas abolished the spontaneous discharges only in the CA1 subfield. However, epileptiform responses in the isolated CA1 subfield could still be evoked by orthodromic stimulation. Intracellularly these stimulus-induced responses were characterized by a depolarization associated with a burst of action potentials. Stimulation of the alveus still evoked a hyperpolarizing potential, presumably a recurrent inhibitory postsynaptic potential (IPSP) in CA1 pyramidal cells. Both spontaneous and stimulus-induced epileptiform discharges were blocked by the selective antagonist of N-methyl-D-aspartate (NMDA) receptors DL-2-amino-phosphonovalerate (APV). APV also reduced the amplitude and duration of the IPSP induced by alveus stimulation. Thus, epileptiform discharges evoked by lowering Mg2+ in the CA1 subfield are associated with a preservation of inhibitory mechanisms. Furthermore the effects exerted by APV upon the IPSP implicate that NMDA receptors might be involved in the neuronal circuit responsible for the hyperpolarizing IPSP generated by CA1 pyramidal neurons.

Epileptiform activity induced by 4-aminopyridine in immature hippocampus

Bath application of 4-aminopyridine (4-AP) to hippocampal slices taken from rats on postnatal days 10–15 produced prolonged synchronized epileptiform discharges in the CA3 subfield. Extracellular field recordings obtained from the pyramidal cell body layer recorded repetitive synchronized afterdischarges which were often 30 sec in duration. These ictal-like events were interspersed with variable amplitude positive-going interjetal burst-like discharges. The afterdischarges consisted of a sustained negative field potential, upon which were superimposed negative-going population spikes. Simultaneous recordings from areas CA3 and CA1 indicated that the afterdischarge activity originated in CA3 since population spikes recorded there preceded and were time locked to spikes in CA1 pyramidal neurons. The burst-like interictal events recorded in CA3 were not all-or-none and had 2 clearly identifiable phases, the first being a smooth positive wave of relatively constant amplitude and duration. A second and subsequent excitatory phase was also positive going but more variable in size. This latter phase was accompanied by multiple population spikes. Intracellular events recorded simultaneously were most often excitatory, depolarized potentials. These varied in size and duration with coincident field potentials. Thus variations of the extracellular burst-like discharges recorded are more likely to be produced by changes from time to time in excitatory synaptic drive to CA3 pyramidal neurons than by alterations in the number of these pyramidal cells discharging in an all-or-none manner. The 4-AP-induced epileptiform discharge occurred in the presence of inhibition. Spontaneous IPSPs were recorded in all cells and in 20% of cells, novel prolonged large hyperpolarizing potentials capable of inhibiting action potentials were recorded simultaneously with the extracellular interictal burst-like discharge. These potentials were readily reversed when a cell was hyperpolarized by intracellular current injection. Our recordings from immature hippocampus bathed in 4-AP thus support the notion that alterations in neuronal function other than a marked decrease of inhibitory processes can contribute to the propensity of immature hippocampus for seizure.

Functional microsurgical partial callosotomy in patients with secondary generalized epilepsies. I. Disruption of bilateral synchrony of spike and wave discharges.

Fourteen patients with secondary generalized epilepsy suffering from multiform seizures (MS) not amenable to medication were submitted to partial section of the corpus callosum. In all patients, there was a partial disruption of the previous generalized bilateral synchronous epileptiform discharges (GBSD). The electroencephalographic findings after callosal section are discussed with respect to their implications in furthering our understanding of the mechanisms subserving the organization of GBSD.

Control of spontaneous epileptiform discharges by extracellular potassium: an ?in vitro? study in the CA1 subfield of the hippocampal slice

1. The effects evoked by changing [K+]o upon the synchronous epileptiform discharges (SEDs) generated in the presence of GABA antagonists were studied in the "in vitro" hippocampal slice with extra- and intracellular recordings. [K+] in the artificial cerebrospinal fluid (ACSF) was varied in steps of 1 or 2 mM between 3.25 and 10.25 mM. 2. Spontaneous SEDs occurred rarely at [K+]o lower than 5.25 mM. Augmenting [K+]o from 5.25 to 10.25 mM caused a four to five fold increase in the frequency of occurrence of SEDs while the duration of each SED was inversely related to the rate of occurrence. 3. Similar findings were observed when the CA1 subfield had been surgically disconnected from the CA2-CA3 subfields. In these experiments SEDs occurred independently in the two regions, but at any given [K+]o SEDs in the CA3 subfield displayed a frequency two to three times higher than that of SEDs generated in the CA1 area. 4. The intracellular correlate of the SEDs in the CA1 subfield either intact or isolated from the CA2-CA3 ones was a large amplitude depolarization (duration 100-600 ms) associated with a burst of action potentials. This intracellular event, which was similar to the paroxysmal depolarizing shift (PDS) recorded in focal models of epilepsy "in vivo", behaved largely like a synaptic phenomenon when the resting membrane potential (Vm) was changed with intracellularly injected current. A long lasting (half-width: 0.3-2 s in 6.25 mM [K+]o) hyperpolarizing potential usually followed the PDS and could be inverted by hyperpolarizing the Vm by 15-25 mV. When [K+] in the ACSF was raised from 7.25 to 10.25 mM, pyramidal cells depolarized in a dose related fashion. At the same time the post-PDS hyperpolarization decreased in duration and peaked earlier, thus curtailing the depolarizing envelope of the PDS. Consequently, the effect of increasing [K+]o was that of evoking more frequent, but shorter PDSs. 6. These findings demonstrate that the appearance of spontaneous SEDs in the presence of GABA antagonists is dependent upon [K+]o. The effects of evoked by increasing [K+]o are presumably mediated through: (i) a decreased strength of K+ repolarizing conductances; (ii) an increased efficacy of synaptic potentials; (iii) a steady depolarization of the neuronal membrane. The modulation of the frequency of occurrence of SEDs appears to be related to a decreased duration of the hyperpolarization which follows the PDS, a potential which is largely mediated by a K+ conductance.

Role of afferent input of subcortical origin in the genesis of bilaterally synchronous epileptic discharges of feline generalized penicillin epilepsy

The effects of (i) neural isolation of large bilateral cortical slabs with intact callosal connections, and (ii) of large unilateral subtotal thalamectomies associated with transection of the interhemispheric commissures on the generalized epileptiform discharges of generalized penicillin epilepsy of the cat were studied. Cortex devoid of subcortical, particularly thalamic, inputs was incapable of generating the characteristic 3.5- to 5-cycles/s epileptiform bursts of feline generalized penicillin epilepsy either in response to the i.m. injection of a large dose of penicillin or to the diffuse direct cortical application of a dilute penicillin solution. Such deafferented cortex, however, produced, even without penicillin, widespread often highly synchronized epileptiform discharges of a different kind consisting at times of slow, 1- to 2.5-cycles/s spike and wave complexes. This epileptiform activity increased in response to i.m. penicillin injection or to widespread direct cortical application of a dilute penicillin solution, but never assumed the characteristics of the typical discharges of feline generalized penicillin epilepsy as seen in cortex which has retained its subcortical connections. The discharges differed from those of intact cortex by being slower in frequency and less prone to become organized in bursts. In the bilateral slab preparation with preserved callosal connections, these atypical discharges occurred, however, in a bilaterally synchronous manner, as did the typical discharges in the intact cat with generalized penicillin epilepsy. This suggests that the corpus callosum is necessary for the bilateral synchrony of both types of epileptiform discharges.

Pathophysiology of generalized penicillin epilepsy in the cat: the role of cortical and subcortical structures. II. Topical application of penicillin to the cerebral cortex and to subcortical structures

In the cat, topical applications of a weak penicillin solution to large areas of cortex of both hemispheres (50–250 I.U./hemisphere) induces bilaterally synchronous epileptiform discharges, often of a characteristic spike and wave type, which in all respects are similar to those obtained with intramuscular injection of high doses of penicillin. The only difference is that with diffuse cortical application no synchronous paroxysmal high voltage discharges are seen in subcortical structures. This cortically induced generalized epileptiform condition responds in the same manner to single shock and low frequency stimulation of a variety of brain structures as that induced by intramuscular injection of penicillin: bilaterally synchronous epileptiform discharges are precipitated with a high probability by stimulations in those subcortical structures which in the normal cat easily induce spindle waves or recruiting responses (intralaminar and midline nuclei of the thalamus and some association nuclei); in contrast, stimulation of other cortical and subcortical structures which are much less effective in inducing spindles and recruiting responses, are also less effective in precipitating bilaterally synchronous epileptiform discharges. Topical application of penicillin to subcortical sites, particularly to the thalamus, including the intralaminar and midline nuclei, fails to elicit any form of epileptic discharge (except for penicillin application to the amygdala). It is concluded that the epileptogenic alteration of neuronal behavior in generalized penicillin epilepsy of the cat resides in the cortex, but that subcortical structures involved in spindle generation and recruiting responses are the most potent triggers for precipitating bilaterally synchronous epileptic discharges. A functional alteration of thalamic structures is clearly insufficient for precipitating bilaterally synchronous epileptic discharges. The findings of this and of the preceding paper (Quesney et al. 1977) indicate that the basic pathophysiological mechanism of feline generalized penicillin epilepsy, and by extraplation, perhaps that of human generalized corticoreticular (“centrecephalic”) epilepsy, is based on a mild, diffuse cortical epileptogenic state, with subcortical structures involved in spindle generation and recruiting responses, (particularly the “thalamic reticular system”) acting as the most potent triggers of generalized bilaterally synchronous spike and wave discharge. Thus both cortical and subcortical mechanisms are important for the elaboration of generalized bilaterally synchronous spike and wave discharge.

Generalized penicillin epilepsy in the cat: Effect of midbrain cooling

The hypothesis that a reduction in desynchronizing reticular drive exerted upon the cortex by the brain-stem reticular formation is an important factor in promoting the occurrence of bilaterally synchronous epileptiform discharges in generalized penicillin epilepsy in the cat was tested in immobilized and in freely moving animals by non-pharmacological means using local cooling of the midbrain reticular formation through implanted thermodes perfused with supercooled methyl-butane. Reversible blockade of the reticular formation, as evidenced by the appearance of a highly synchronized EEG pattern and abolition of the desynchronizing response to electrical stimulation of the reticular formation, was associated with a very consistent and often dramatic increase in bilaterally synchronous generalized epileptiform discharge. Subsequent rebound arousal with EEG desynchronization conversely was associated with a diminution or arrest of bilaterally synchronous epileptiform discharge. These observations are compatible with the hypothesis that reduction of desynchronizing drive exerted by the reticular formation upon the cortex may represent an important factor in the genesis of bilaterally synchronous generalized spike and wave discharge in animals and man.