Neurosteroid modulation of embryonic neuronal survival in vitro following anoxia

Neurosteroids are synthesized de novo in the brain from cholesterol or peri pheral steroid precursors and modulate inhibitory gamma-aminobutyric acid ( GABA,) and excitatory N-methyl-D-aspartate (NMDA) receptors. Evidence indic ates that neurosteroids are neuroprotective and important during neurodevel opment. We tested the hypothesis that neurosteroids increase embryonic neur onal survival following anoxia in rat embryonic day 18 cerebral cortical cu ltures to examine potential neurosteroid modulation of this insult during e arly development. Twenty-four hours after plating in serum-free medium, cul tures were exposed to DHEA, DHEAS, or allopregnanolone (10(-10), 10(-8), or 10(-6) M), Or vehicle, for 24 h (n =9 per treatment condition). Cultures w ere then subjected to anoxia for 2 h and subsequently reincubated for 24 h prior to neuron immunostaining with microtubule-associated protein 2 (MAP-2 ) antibody. Supernatant from DHEA and DHEAS-exposed cultures was tested for 17 beta-estradiol metabolite formation by radioimmunoassay. DHEA 10(-6) an d 10(-8) M significantly increased neuron survival by 85-87% following anox ia. DHEAS 10(-6) M significantly increased neuron survival by 74% following anoxia, but DHEAS 10(-10) M decreased neuron survival after this insult. A llopregnanolone had modest effects on neuron survival that did not attain s tatistical significance. 17 beta-Estradiol concentrations were below the li mit of detection in all specimens tested (sensitivity 4.7 nM). Our data ind icate that pretreatment with DHEA and DHEAS at physiologically relevant con centrations promotes neuronal survival following anoxia in embryonic rat ce rebral cortical cultures, and that these effects are not secondary to 17 be ta-estradiol metabolite formation. DHEA and DHEAS modulation of anoxia in e mbryonic neurons may be relevant to disorders of neurodevelopment involving this insult. (C) 2000 Elsevier Science B.V. All rights reserved.