Abstracts

RATIONALE: Neurosteroids have been implicated in the hormonal regulation of seizure susceptibility in catamenial epilepsy and stress. Allopregnanolone (3[alpha],5[alpha]-THP), a powerful positive modulator of GABAergic neurotransmission, has anticonvulsant activity in animal models, whereas pregnenolone sulfate (PS), a GABA[sub]A[/sub] receptor antagonist and positive modulator of NMDA receptors, is proconvulsant. Here we examined the modulatory effects of 3[alpha],5[alpha]-THP (and its less active 5[beta] isomer) and PS on seizure susceptibility in an vitro model system.
METHODS: Extracellular field recordings were carried out with glass micropipettes from the CA3 region of 500-[mu]m rat hippocampal slices continuously perfused with oxygenated ACSF at 2.5-3.0 ml/min. Spontaneous epileptiform bursting was evoked by superfusion with 4-aminopyridine (4-AP; 55-75 [mu]M) or picrotoxin (PTX; 100 [mu]M). The rate of bursting was monitored for 1-2 h. Bursts were detected using Mini Analysis (Synaptsoft).
RESULTS: Superfusion with 4-AP evoked high frequency discharges (up to 50 per min). The frequency of discharges increased gradually to a constant level at approximately 40 min after the addition of 4-AP Washout led to a gradual disappearance of the discharges. Addition of 10 or 100 [mu]M PS 1 h after 4-AP did not alter the discharge frequency (6 slices). However, 100 [mu]M PS alone did induce spontaneous discharges at a low rate (1 per min; 5 of 6 slices). Addition of 90 [mu]M (3[alpha],5[alpha]-THP) 1 h after 4-AP led within 40 min to a cessation in epileptiform activity in all 5 slices tested. Removal of (3[alpha],5[alpha]-THP) from the medium resulted in the reappearance of epileptiform discharges.
Addition of 90 [mu]M (3[alpha],5[alpha]-THP) simultaneously with 4-AP resulted in an 83% suppression of bursting (5 slices). 3[alpha],5[beta]-THP also suppressed bursting by 83% (8 slices). PTX induced epileptiform discharges similar to those produced by 4-AP, but these occurred at a lower frequency (up to 9 per min). The PTX-induced activity began within 10 min of drug application and was maintained at a constant rate. 3[alpha],5[alpha]-THP (90 [mu]M) perfused concurrently with PTX completely inhibited the epileptiform activity (3 slices). In contrast, 3[alpha],5[beta]-THP (100 [mu]M) failed to affect the rate of discharge (3 slices).
CONCLUSIONS: The neurosteroid 3[alpha],5[alpha]-THP suppresses 4-AP and PTX induced epileptiform bursting. However, there was no stereoselectivity of the THP isomers in the 4-AP model suggesting their activity does not occur through an interaction with GABA[sub]A[/sub] receptors. (A similar lack of stereoselectivity is observed in the in vivo maximal electroshock model.) In contrast, the effect on PTX bursting showed stereoselectivity that matches the stereoselective effects of the steroids on GABA[sub]A[/sub] receptors as is the case for the in vivo pentylenetetrazol seizure model. Thus, neurosteroids can protect against seizures by effects on GABA[sub]A[/sub] receptors and also by other mechanisms. PS failed to affect epileptiform discharges induced by 4-AP but did itself weakly induce bursting, consistent with its proconvulsant activity in animals.
[Supported by: NIH]