THE NEUROSTEROID 3-ALPHA-HYDROXY-5-ALPHA-PREGNAN-20-ONE INDUCES CYTOARCHITECTURAL REGRESSION IN CULTURED FETAL HIPPOCAMPAL-NEURONS

The neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alp ha-THP) acts as a potent allosteric modulator and a direct activator o f the GABA-chloride channel complex. This neurosteroid has also been f ound to protect against seizures that arise from blockade of the GABA- chloride channel complex. Because 3 alpha,5 alpha-THP protects against excitotoxin-induced seizure activity and because seizure activity has been found to be associated with aberrant hippocampal nerve cell grow th, the rapid effect of the neurosteroid 3 alpha,5 alpha-THP upon nerv e cell growth was investigated using videomicroscopy of hippocampal ne urons in culture. Within 40 min of exposure 3(alpha,5 alpha-THP) induc ed a significant decrease in the area and length of neurites. A concom itant decrement in the number and length of filopodia decorating neuri tic extensions also occurred within the 40 min of 3 alpha,5 alpha-THp exposure. Both rapid and slow retrograde movement of intracellular org anelles was observed in 3 alpha,5 alpha-THP-treated neurons. 3 alpha,5 alpha-THPinduced regression of neuritic extensions occurred only in n erve cells that had not yet established contact with other nerve or gl ial cells in culture. Established structural connections between neuro ns or glia did not erode during 3 alpha,5 alpha-THP exposure. Neither the inactive stereoisomer 3 beta-hydroxy-5 beta-pregnan-20-one nor pro gesterone had a significant effect upon any of the morphological param eters assessed. In approximately 25% of the cells in which 3 alpha,5 a lpha-THP had induced regression, subsequent exposure to 17 beta-estrad iol induced profuse filopodial growth within 60 sec of exposure. In cu ltures similar in age to those used in the morphological studies, 3 al pha,5 alpha-THP induced a significant increase in Cl-36-uptake within 10 sec. The magnitude of Cl-36- uptake was comparable to that induced by exposure to 100 mu M GABA. In older, more mature cultures in which the nerve cells had established structural connections, 3 alpha,5 alph a-THP protected cells from picrotoxin-induced nerve cell death. These results demonstrate that 3 alpha,5 alpha-THP can induce regression of neuronal morphology within a relatively rapid time frame. 3 alpha,5 al pha-THP induction of Cl-36- uptake within 10 sec suggests that activat ion of neurosteroid-regulated chloride channels is an initial step in the biochemical mechanism underlying the retraction induced by this pr ogesterone metabolite steroid. In select instances, 17 beta-estradiol induced an extremely rapid reversal of the filopodial regression produ ced by 3 alpha,5 alpha-THP. Collectively, these findings indicate that steroid factors acting singly and in combination can induce significa nt changes in nerve cell morphology within a time frame that is consis tent with 3 alpha,5(alpha-THP and 17 beta-estradiol regulation of exci tability.