The Neurosteroid 3a-Hydroxy-5a-Pregnan-20-One Induces Cytoarchitectural Regression in Cultured Fetal Hippocampal Neurons

The neurosteroid 3a-hyclroxy-5a-pregnan-20-one (3a,5a-THP) acts as a potent allosteric modulator and a direct activator of the GABA-chloride channel complex. This neurosteroid has also been found to protect against seizures that arise from blockade of the GABA-chloride channel complex. Because 3a,5a-THP protects against excitotoxin-induced seizure activity and because seizure activity has been found to be associated with aberrant hippocampal nerve cell growth, the rapid effect of the neurosteroid 3a,5a-THP upon nerve cell growth was investigated using videomicroscopy of hippocampal neurons in culture. Within 40 min of exposure 3a,5aTHP induced a significant decrease in the area and length of neurites. A concomitant decrement in the number and length of filopodia decorating neuritic extensions also occurred within the 40 min of 3a,5a-THP exposure. Both rapid and slow retrograde movement of intracellular organelles was observed in 3a,Ba-THP-treated neurons. Sa,Ba-THPinduced regression of neuritic extensions occurred only in nerve cells that had not yet established contact with other nerve or glial cells in culture. Established structural connections between neurons or glia did not erode during 3a,5aTHP exposure. Neither the inactive stereoisomer 38-hydroxy-5&pregnan-20-one nor progesterone had a significant effect upon any of the morphological parameters assessed. In approximately 25% of the cells in which 3a,5a-THP had induced regression, subsequent exposure to 17 8-estradiol induced profuse filopodial growth within 60 set of exposure. In cultures similar in age to those used in the morphological studies, 3a,5a-THP induced a significant increase in 3BCIuptake within 10 sec. The magnitude of 36CIuptake was comparable to that induced by exposure to 100 rM GABA. In older, more mature cultures in which the nerve cells had established structural connections, 3a,5a-THP protected cells from picrotoxin-induced nerve cell death. These results demonstrate that 3a,5a-THP can induce regression of neuronal morphology within a relatively rapid time frame. 3a,5aTHP induction of 3BCIuptake within 10 set suggests that