The overall goal of this project is to investigate strategies for the drug treatment of epilepsy through pharmacological studies in animal models and clinical investigation in human subjects. Research was continued evaluating the role of neuroactive steroids in epilepsy and their possible uses in epilepsy therapy. Neuroactive steroids are endogenous steroid hormones (and their synthetic analogs) that rapidly alter the excitability of neurons by direct actions on membrane ion channels, including GABA-A and NMDA receptors. In prior reporting periods, we confirmed that the reproductive hormone progesterone has powerful anticonvulsant activity and we demonstrated that this results from its conversion to the neuroactive steroid allopregnanolone. We proposed that perimenstrual catamenial epilepsy, the increase in seizure frequency that many women with epilepsy experience near the time of menstruation (when progesterone levels fall) may, in part, be related to withdrawal of allopregnanolone. At present, there is no specific treatment for catamenial epilepsy. To investigate possible therapeutic approaches, we continued studies with an animal model of perimenstrual catamenial epilepsy in which fluctuations in brain neuroactive steroid levels mimic those occurring in woman of reproductive age at the time of menstruation. In this model, a state of chronically high serum progesterone is induced in female rats by sequential treatment with pregnant mares' serum gonadotrophin and human chorionic gonadotropin. Neurosteroid withdrawal is then produced by treatment with finasteride, an inhibitor of the enzymatic conversion of progesterone to allopregnanolone. Following finasteride-induced withdrawal, rats show increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. If perimenstrual catamenial epilepsy is related to neurosteroid withdrawal, then neurosteroid replacement is a reasonable approach to therapy. We previously demonstrated that the anticonvulsant potency of the synthetic neurosteroid ganaxolone is increased three-fold after neurosteroid withdrawal although the plasma concentrations of ganaxolone did not differ significantly in control and withdrawn animals. Unlike ganaxolone, neurosteroid withdrawal was associated with a decrease in the anticonvulsant potency of diazepam and valproate. The enhanced anticonvulsant potency of ganaxolone after neurosteroid withdrawal supports the use of ganaxolone as a specific treatment for perimenstrual catamenial epilepsy. To further investigate the therapeutic potential of neurosteroid replacement, in the present reporting period we examined the anticonvulsant activity of a series of four structurally distinct neuroactive steroids [allopregnanolone, pregnanolone, allotetrahydrodeoxycorticosterone (THDOC) and tetrahydrodeoxycorticosterone] in our catamenial epilepsy model. As was the case for ganaxolone, we found that the anticonvulsant activity of these other neuroactive steroids was enhanced by 34 to 127%. The anticonvulsant activity of phenobarbital was also enhanced by 24%. We conclude that the anticonvulsant activity of a wide range of neuroactive steroids is potentiated following neurosteroid withdrawal, supporting the use of such agents in the treatment of catamenial epilepsy. In the present reporting period we also initiated studies examining the role of neurosteroids in stress-induced alterations in seizure susceptibility, focusing specifically on tetrahydrodeoxycorticosterone (DOC), an adrenal steroid whose synthesis is enhanced during stress. DOC is conventionally considered a mineralocorticoid precursor; however, we have obtained evidence indicating that it is also a mediator of stress effects on seizures. DOC undergoes sequential metabolic reduction by 5alpha-reductase and 3alpha-hydroxysteroid oxidoreductase to form dihydrodeoxycorticosterone (DHDOC) and THDOC, which as noted is a GABA-A receptor modulating neurosteroid with anticonvulsant properties. Acute swim stress in rats significantly elevated plasma THDOC concentrations and raised the PTZ seizure threshold. Small systemic doses of DOC produced comparable increases in plasma THDOC levels and PTZ seizure threshold. Pretreatment with finasteride, which blocks the conversion of DOC to DHDOC, reversed the antiseizure effects of stress. DOC also protected mice against PTZ, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-caboxylate), picrotoxin and amygdala kindled seizures in mice. Finasteride reversed the antiseizure activity of DOC; partial antagonism was also obtained with indomethacin, an inhibitor of 3alpha-hydroxysteroid oxidoreductase. Finasteride had no effect on seizure protection by DHDOC and THDOC whereas indomethacin partially reversed DHDOC but not THDOC. The results with finasteride and indomethacin indicated that THDOC and possibly DHDOC could mediate the anticonvulsant activity of DOC. In whole cell voltage clamp recordings from cultured hippocampal neurons, we found that DHDOC, like THDOC, potentiated GABA-activated chloride currents and directly activated GABA-A receptor currents compatible with a role for DHDOC in the antiseizure activity of DOC. Our results demonstrate that DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABA-A receptors including THDOC and possibly also DHDOC. Our results further suggest a role for neuroactive steroids as a treatment approach for stress-related seizures.
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