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

Abstract

Background Prenatal alcohol exposure can kill developing neurons, leading to microencephaly and mental retardation. However, not all fetuses are equally vulnerable to alcohol’s neurotoxic effects. While some fetuses are severely affected and are ultimately diagnosed with fetal alcohol syndrome (FAS), others have no evidence of neuropathology and are behaviorally normal. These widely different outcomes among alcohol-exposed fetuses are likely due, in part, to genetic differences. Some fetuses possess genotypes that make them much more vulnerable than others to alcohol’s teratogenic effects. However, to date, only one gene has been identified whose mutation can worsen alcohol-induced behavioral deficits in an animal model of FAS. That gene is neuronal nitric oxide synthase (nNOS). The purpose of this study was to determine whether mutation of nNOS can likewise worsen alcohol-induced microencephaly and lead to permanent neuronal deficits. Methods Wild type and nNOS−/− mice received alcohol (0.0, 2.2, or 4.4 mg/g) daily over postnatal days (PD) 4–9. Beginning on PD 85, the mice underwent a series of behavioral tests, the results of which are reported in the companion paper. The brains were then weighed, and stereological cell counts were performed on the cerebral cortex and hippocampal formation, which are the brain regions that mediate the aforementioned behavioral tasks. Results Alcohol caused dose-dependent microencephaly, but only in the nNOS−/− mice and not in wild type mice. Alcohol-induced neuronal losses were more severe in the nNOS−/− mice than in the wild type mice in all of the brain regions examined, including the cerebral cortex, hippocampal CA3 subregion, hippocampal CA1 subregion, and dentate gyrus. Conclusions Targeted mutation of the nNOS gene increases the vulnerability of the developing brain to alcohol-induced growth restriction and neuronal losses. This increased neuropathology is associated with worsened behavioral dysfunction. The results demonstrate the critical importance of genotype in determining the outcome of developmental alcohol exposure.

Effects of prenatal alcohol exposure (PAE): insights into FASD using mouse models of PAE

“…Karacay et al used alcohol doses of 2.2 or 4.4 g/kg, i.p, on PN4–9, in either nNos −/− knockout transgenic offspring (on a C57BL/6 × 129S6 background) or WT controls. PAE nNos −/− offspring showed a significant decrease in adult (PN110–120) brain weight and increased incidence of microcephaly (Karacay et al 2015). Moreover, nNos −/− mice demonstrated severe PAE-induced cerebral cortex pyramidal neuronal loss compared with the WT controls, suggesting abnormalities in brain wiring/connection (Karacay et al 2015).…”

“…PAE nNos −/− offspring showed a significant decrease in adult (PN110–120) brain weight and increased incidence of microcephaly (Karacay et al 2015). Moreover, nNos −/− mice demonstrated severe PAE-induced cerebral cortex pyramidal neuronal loss compared with the WT controls, suggesting abnormalities in brain wiring/connection (Karacay et al 2015). Furthermore, there was a significant increase in anxiety-like behavior, an impaired startle response, and impaired learning and memory (Morris Water Maze) in PN85–90 mice; behavior outcomes that have previously been observed in other PAE mouse models (Brady et al 2012; Allan et al 2014).…”

“…Increased anxiety and deficits in learning and memory have also been observed in children with FASD (Clarke and Gibbard 2003). In comparison, only the 4.4 g/kg cohort of WT mice experienced a decrease in body weight at PN9–10 and had learning and memory deficits (Karacay et al 2015). Taken together, normal nNos activity appears to have a protective role, while nNos mutations worsen brain abnormalities and behavioral outcomes in the presence of PAE when compared with the PAE WT controls.…”

“…The link between brain abnormalities and behavioral deficits may lie in changes at the neural cell level (Caldwell et al 2008; Allan et al 2014; Smiley et al 2015). Changes in developing neural cells can lead to changes in brain wiring and connections that are associated with behavioral outcomes (Kleiber et al 2011; El Shawa et al 2013; Allan et al 2014; Karacay et al 2015). …”

Genetic Absence of nNOS Worsens Fetal Alcohol Effects in Mice. I: Behavioral Deficits

“…Compared to wild type, mice that were genetically deficient for nNOS had worsened behavioral deficits following developmental alcohol exposure. These mutant mice also have worsened microencephaly and neuronal losses, as shown in the companion paper (Karacay et al, 2014). Some of these alcohol-induced differences in outcome between wild type and nNOS−/− mice were not only quantitative , but qualitative .…”