Alcohol during brain development leads to the widespread neuronal death observed in fetal alcohol spectrum disorders (FASD). In comparison, the mature brain is less vulnerable to alcohol. Studies into maturation-acquired alcohol resistance uncovered a protective mechanism that reduces alcohol-induced neuronal death through nitric oxide-cGMP-cyclic GMP-dependent protein kinase (NO-cGMP-cGK) signaling. However, the downstream processes underlying this neuroprotection remain unclear. Alcohol can disrupt levels of intracellular calcium ([Ca2+]i) in vulnerable neuronal populations to trigger cell death in both in vivo and in vitro models of FASD. Since cGK has been demonstrated to regulate and inhibit intracellular Ca2+ release, we examined the hypothesis that cGK confers alcohol resistance by preventing [Ca2+]i disruptions. Alcohol resistance, determined by neuronal survival after 24 h of alcohol exposure, was examined in primary cerebellar granule neuron (CGN) cultures derived from 5 to 7 day-old neonatal mice with an activator, 8-Br-cGMP, and/or an inhibitor, Rp-8-pCPT-cGMPS, of cGK signaling. Intracellular Ca2+ responses to alcohol were measured by ratiometric Ca2+ imaging in Fura-2-loaded CGN cultures after 8-Br-cGMP treatment. Our results indicate that activating cGK with 8-Br-cGMP before alcohol administration provided neuroprotection, which the cGK inhibitor, Rp-8-pCPT-cGMPS, blocked. Alcohol exposure elevated [Ca2+]i, whereas 8-Br-cGMP pretreatment reduced both the level of the alcohol-induced rise in [Ca2+]i as well as the number of cells that responded to alcohol by increasing [Ca2+]i. These findings associate alcohol resistance, mediated by cGK signaling, to reduction of the persistent and toxic increase in [Ca2+]i from alcohol exposure.
Keywords: Alcohol; Fetal alcohol spectrum disorder; Intracellular calcium; Neuroprotection; cGMP-dependent protein kinase.
Copyright © 2018 Elsevier B.V. All rights reserved.