Severe alcohol‐induced neuronal deficits in the hippocampus and neocortex of neonatal mice genetically deficient for neuronal nitric oxide synthase (nNOS)

Abstract

Alcohol can severely damage the developing brain, and neuronal loss is a critical component of this injury. Thus, identification of molecular factors that ameliorate alcohol-induced neuronal loss is of great importance. Previous in vitro work has demonstrated that nitric oxide (NO) protects neurons against alcohol toxicity. We tested the hypothesis that neonatal mice carrying a null mutation for neuronal nitric oxide synthase (nNOS), the enzyme that synthesizes NO in neurons, have an increased vulnerability to alcohol-induced neuronal loss in the neocortex and hippocampus. Wildtype mice and nNOS-/- mice received ethanol (0.0, 2.2, 3.3, or 4.4 g/kg) daily over postnatal days (P) 4-9 and were sacrificed on P10. The number of hippocampal CA1 and CA3 pyramidal cells, dentate gyrus granule cells, and neocortical neurons were determined using stereological methods. Alcohol pharmacokinetics did not differ between wildtype and nNOS-/- strains. Alcohol induced dose-dependent reductions in all four neuronal populations, and the losses were substantially more severe in the nNOS-/- mice than in wildtype. Furthermore, the threshold dose of alcohol to induce cell death was lower in the nNOS-/- mice than in the wildtype mice for all neuronal populations. While nNOS deficiency worsened alcohol-induced neuronal losses, the magnitude of this exacerbation varied among brain regions and depended on alcohol dose. These results demonstrate that nNOS deficiency decreases the ability of developing neurons in vivo to survive the toxic effects of alcohol and strengthen the hypothesis that NO exerts a neuroprotective effect against alcohol toxicity in the developing brain.

Nrf2-Mediated Transcriptional Induction of Antioxidant Response in Mouse Embryos Exposed to Ethanolin vivo: Implications for the Prevention of Fetal Alcohol Spectrum Disorders

“…Apoptotic death of selected cell populations is a commonly observed pathogenic feature of ethanol-induced teratogenesis in both in vivo and in vitro model systems (3,7,11,14,37,40). Among the proposed mechanisms underlying ethanol-induced apoptosis and malformation is oxidative stress.…”

Genetic Absence of nNOS Worsens Fetal Alcohol Effects in Mice. II: Microencephaly and Neuronal Losses

“…Several previous studies have shown that neuronal losses induced by alcohol are greater in nNOS−/− mice than in wild type (Bonthius et al, 2002; 2006; de Licona et al, 2009). However, in all of those studies, neuronal numbers were determined in infant mice, immediately following the alcohol exposure.…”

Stimulation of the cAMP pathway protects cultured cerebellar granule neurons against alcohol-induced cell death by activating the neuronal nitric oxide synthase (nNOS) gene

“…We have previously reported that genetic deficiency of nNOS worsens alcohol-induced microencephaly and neuronal loss both in vitro and in developing mice (Bonthius et al, 2002;Bonthius et al, 2006). This suggested that nNOS exerts a neuroprotective effect against alcohol toxicity.…”

“…Based on our previous observation that nNOS protects CGN cultures against alcohol-induced cell death (Bonthius et al, 2002) and that nNOS−/− mice have an enhanced vulnerability to alcohol-induced microencephaly and neuronal loss (Bonthius et al, 2002;Bonthius et al, 2006), we hypothesized that delivery of the nNOS gene into CGN derived from nNOS−/− mice could protect the neurons against alcohol toxicity. The results confirmed this hypothesis.…”