The overall goal of this project is to investigate new strategies for the treatment of epilepsy. Research was continued evaluating the role of neurosteroids in epilepsy and the possible uses of neurosteroids (and their synthetic analogs) in epilepsy therapy. Neurosteroids are endogenous steroid hormones 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. Studies with progesterone receptor knockout (PRKO) mice demonstrated that the effects of progesterone on seizure susceptibility are not mediated by its cognate nuclear hormone receptors. Instead, we found that progesterone is anticonvulsant as a result of its conversion to the neurosteroid 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 be related to withdrawal of allopregnanolone. At present, there is no specific treatment for catamenial epilepsy. However, our studies with an animal model of catamenial epilepsy suggested that neurosteroid replacement could be useful. In addition, we have investigated the role of neurosteroids in stress-induced alterations in seizure susceptibility, focusing specifically on deoxycorticosterone (DOC), an adrenal steroid whose synthesis is enhanced during stress. Our results demonstrated 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 tetrahydrodeoxycorticosterone (THDOC). Our results further suggest a role for neuroactive steroids as a treatment approach for stress-related seizures. Recent studies have focused on the role of androgen-related steroids in the regulation of seizure susceptibility. Men with epilepsy often have sexual or reproductive abnormalities that are attributed to alterations in androgen levels, including subnormal free testosterone. Levels of the major metabolites of testosterone--androsterone (5alpha-androstan-3alpha-ol-17-one), a neurosteroid that acts as a positive allosteric modulator of GABA-A receptors, and its 5beta-epimer etiocholanolone (5beta-androstan-3alpha-ol-17-one)--also may be reduced in epilepsy. We previously reported that androsterone and etiocholanolone have anticonvulsant properties and we proposed that they could represent endogenous modulators of seizure susceptibility. Thus, low levels of these metabolites in men with epilepsy could lead to enhanced seizure susceptibility and poor seizure control. As in catamenial epilepsy, neuroactive steroid replacement is a potential therapeutic strategy. In the present reporting period, we continued to investigate the role of androgen-related steroids in the regulation of seizure susceptibility through a study of the androgen-related steroid androstenol, which is a steroidal compound belonging to the group of odorous 16-androstenes, first isolated from boar testes and also found in humans. Androstenol has pheromone-like properties in both animals and humans, but the molecular targets of its pheromonal activity are unknown. Recognizing that androstenol is structurally similar to endogenous A-ring reduced neurosteroids, we hypothesized that it could serve as a GABA-A receptor positive modulator and that it would have anticonvulsant properties. This hypothesis was confirmed in whole-cell recordings from cerebellar granule cells, where androstenol (but not its 3beta-epimer) caused a concentration-dependent enhancement of GABA-activated currents and prolonged the duration of spontaneous and miniature inhibitory postsynaptic currents. Androstenol also potentiated the amplitude of GABA-activated currents in human embryonic kidney 293 cells transfected with recombinant alpha1beta2gamma2 and alpha2beta2gamma2 GABA-A receptors and, at high concentrations, directly activated currents in these cells. Systemic administration of androstenol caused anxiolytic-like effects in mice in the open-field test and elevated zero-maze and antidepressant-like effects in the forced swim test. Androstenol, but not its 3beta-epimer, conferred seizure protection in the 6-Hz electroshock and pentylenetetrazol models. The various actions of androstenol in the whole-animal models are consistent with its activity as a GABA-A receptor modulator. Like other androgen-related neurosteroids, androstenol could represent an endogenous modulator of seizure susceptibility. During this reporting period we initiated a new direction of research on neurosteroids in which we examined the possibility that neurosteroids may modulate the development of epilepsy in a model of temporal lobe epileptogenesis. In these studies, rats experiencing pilocarpine-induce status epilepticus exhibited spontaneous recurrent seizures following a latent period. We found that P450scc, the rate-limiting enzyme in steroid synthesis, is upregulated in hippocampal glia during the latent period. More prolonged status epilepticus was associated with greater P450scc expression and longer latencies to the development of seizures, suggesting that enhanced steroid synthesis retards epileptogenesis. The 5alpha-reductase inhibitor finasteride, which blocks neurosteroid synthesis, reduced the latent period, indicating that neurosteroids may be antiepileptogenic. These studies raise the possibility that neurosteroids may have utility in preventing the development of epilepsy in susceptible individuals.
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