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Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 micro m increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 micro m). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMI-exposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post-synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 micro m), but not its inactive analog, oleanolic acid (100 micro m), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process.

Dr. Carlen reviews the evidence that seizures may cause cell death and discusses possible strategies for preventing seizure-induced brain damage.

It is well known that brain damage can cause epilepsy. Post-traumatic epilepsy (PTE), for instance, is a consequence of traumatic brain injury. The risk of PTE ranges from less than 5 percent of patients following blunt head trauma (e.g., simple skull fracture) to 50 percent in patients following penetrating wounds or those involving intracranial hematoma.11 One of the accepted causes of temporal-lobe epilepsy is perinatal brain injury, which can also lead to other epileptic syndromes, such as West’s syndrome and Lennox-Gastaut syndrome. Epilepsy can also be a presenting symptom of both brain tumors and strokes.

1. Focal cortical epilepsy was investigated by applying tetanic stimulation repeatedly (100 Hz. 2 s in duration, once every 10 min, 10 episodes) to layer III association fibers in rat piriform cortex slices and recording both extracellular and intracellular responses from the endopiriform nucleus. To promote excitability, piriform slices were incubated in artificial cerebrospinal fluid (ACSF) containing 0.9 mM Mg2+ and 5 mM K+, at an initial temperature of 10–12 degrees C, which was allowed to warm passively to room temperature. 2. Responses recorded extracellularly in the endopiriform nucleus consisted of two types: weak stimulation evoked an early-occurring, small-amplitude, negatively deflecting potential; strong stimulation evoked a more complex response comprising both an early potential of maximal amplitude and a later-occurring epileptiform potential of greater amplitude and longer duration. Late-occurring epileptiform potentials were not observed in slices incubated in ACSF at room temperature. 3. Both the early potential and the late-occurring epileptiform responses were abolished by the non-N-methyl-D-aspartic acid (non-NMDA) subtype of glutamate receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM). Application of D(-)-2-amino-5-phosphonopentanoic acid (APV; 50 microM) to block NMDA receptors was without effect on the early potential but diminished the late-occurring epileptiform potential. The late-occurring potential was unable to follow stimulation delivered at a frequency of 1 Hz. These results suggest that the early potential was generated monosynaptically and dependent solely on the activation of non-NMDA receptors, whereas the late-occurring epileptiform potential was polysynaptic in origin and possessed both a CNQX- and an APV-sensitive component. 4. Responses increased progressively in both amplitude and duration after tetanic stimulation. The threshold intensity required to evoke the complex dual-component potential was reduced by tetanic stimulation. An increase in multiunit spiking activity, indicating an increase in synchronous discharges, was also observed. A residual potential could be evoked in the presence of CNQX (10 microM) after the tetanic stimulation procedure. 5. Spontaneous discharges occurred as early as after the first episode of tetanic stimulation and persisted for the duration of the experiment. Spontaneous discharges were abolished by either CNQX or by a fourfold increase in extracellular Mg2+ concentration, the latter reversibly. APV reduced the frequency of spontaneous discharges by 38.6 +/- 9.3% (mean +/- SE). The conventional anticonvulsant drug 5,5-diphenylhydantoin, the benzodiazepine receptor agonist midazolam, and the benzodiazepine receptor antagonist flumazenil were without effect on the frequency of spontaneous discharges. Evoked responses were also unaffected by either 5,5-diphenylhydantoin or midazolam. Slices not exposed to cold ACSF, although demonstrating potentiation of evoked responses after tetanization did not produce spontaneous epileptiform discharges. 6. Intracellular recordings from endopiriform neurons revealed the cellular correlates of the extracellular responses. Weak stimulation evoked a small-amplitude depolarizing potential. Increasing the intensity of stimulation increased the amplitude of this response and also evoked a second depolarizing potential of greater amplitude occurring at variable latencies. Maximal stimulation evoked an action potential. After tetanic stimuli, responses resembling a paroxysmal depolarizing shift consisting of a depolarizing potential with superimposed multiple action potentials were evoked reliably. Passive membrane properties after repeated tetanic stimulation were not different when compared with control. 7. This novel model of in vitro focal cortical epilepsy has many features characteristic of conventional kindling including 1) progressive nature; 2) reduced threshold to evoke discharges; and 3) persist

Summary: Purpose: The antiepileptic effects of benzodiaze-pine-receptor (BZR) agonists have been well documented. Surprisingly, an antiepileptic effect for the BZR antagonist, fluma-zenil, has also been described, the mechanism of which is unknown. We investigated the effects of nanomolar concentrations of flumazenil and a structurally dissimilar BZR antagonist, propyl-β-carboline-3-carboxylate (β-CCP), on normal synaptic responses and epileptiform discharges induced by a variety of methods in the CA1 region of rat hippocampal slices. Methods: Extracellular field potentials were recorded from stratum pyramidale of the CA1 region. Orthodromic stimulation was delivered by a bipolar electrode placed in the stratum radiatum at the border of the CA2/KA3 regions. Drugs were bath applied, and epileptiform discharges were quantified by using the Coastline Bursting Index, which calculates the total length of the discharge waveform of evoked multiple population spikes. For statistical comparisons, we calculated the Coastline Bursting Index for the average of five traces at the end of the control period (20 min), drug application (20 min), and washout (20–40 min). Results: Flumazenil was without effect on normal synaptic responses; however, flumazenil reduced epileptiform discharges evoked in the presence of high [K+]0, leu-enkephalin, the BZR inverse agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), or after a cold-shock procedure. β-CCP exhibited an action similar to that observed for flumazenil, suggesting that the antiepileptic effect is due to properties common to BZR antagonists. Conclusions: We suggest that the antiepileptic effect we observed for flumazenil and (β-CCP is mediated at the BZR and might be due to competition with endogenous BZR inverse agonists released preferentially during epileptiform activity.

It has been reported previously that systemic administration of clonidine, an agonist of alpha-2 noradrenergic receptors, significantly retards amygdaloid kindling by delaying the emergence from partial seizure. We examined the effect of either systemic administration (i.p.) or intra-amygdaloid infusions of clonidine on the kindling of seizures with electrical stimulation of the amygdala. Rats received either low-frequency stimulation of the amygdala, to induce rapid kindling, or conventional high-frequency stimulation. Clonidine and electrical stimulation were administered once every 48 h. We observed a significant retardation of kindling in rats receiving i.p. injections of clonidine (0.1 mg/kg) or infusions of clonidine in concentrations of 10(-7)-10(-4) M, regardless of the stimulation frequency. The prophylactic effect was due to a delay in the progression out of partial seizure. The effect was specific to the amygdala/pyriform region, because infusions of clonidine dorsal to the amygdala were without effect. Intra-amygdaloid clonidine had little effect on established generalized seizures, suggesting that it was producing a genuine prophylactic effect against kindling. We conclude that the subpopulation of alpha-2 adrenoceptors in the amygdala/pyriform region contributes to the antiepileptogenic effect observed after systemic administration of clonidine.