• Energy Research

AbstractBackgroundAbnormal diffusion within white matter (WM) tracts has been linked to cognitive impairment in children with fetal alcohol spectrum disorder. Whether changes to myelin organization and structure underlie the observed abnormal diffusion patterns remains unknown. Using a third trimester‐equivalent mouse model of alcohol exposure, we previously demonstrated acute loss of oligodendrocyte lineage cells with persistent loss of myelin basic protein and lower fractional anisotropy (FA) in the corpus callosum (CC). Here, we tested whether these WM deficits are accompanied by changes in: (i) axial diffusion (AD) and radial diffusion (RD), (ii) myelin ultrastructure, or (iii) structural components of the node of Ranvier.MethodsMouse pups were exposed to alcohol or air vapor for 4 h daily from postnatal day (P)3 to P15 (BEC: 160.4 ± 12.0 mg/dl; range = 128.2 to 185.6 mg/dl). Diffusion tensor imaging (DTI) and histological analyses were performed on brain tissue isolated at P50. Diffusion parameters were measured with Paravision™ 5.1 software (Bruker) following ex vivo scanning in a 7.0 T MRI. Nodes of Ranvier were identified using high‐resolution confocal imaging of immunofluorescence for Nav1.6 (nodes) and Caspr (paranodes) and measured using Imaris™ imaging software (Bitplane). Myelin ultrastructure was evaluated by calculating the G‐ratio (axonal diameter/myelinated fiber diameter) on images acquired using transmission electron microscopy.ResultsConsistent with our previous study, high resolution DTI at P50 showed lower FA in the CC of alcohol‐exposed mice (p = 0.0014). Here, we show that while AD (diffusion parallel to CC axons) was similar between treatment groups (p = 0.30), RD (diffusion perpendicular to CC axons) in alcohol‐exposed subjects was significantly higher than in controls (p = 0.0087). In the posterior CC, where we identified the highest degree of abnormal diffusion, node of Ranvier length did not differ between treatment groups (p = 0.41); however, the G‐ratio of myelinated axons was significantly higher in alcohol‐exposed animals than controls (p = 0.023).ConclusionsHigh resolution DTI revealed higher RD at P50 in the CC of alcohol‐exposed animals, suggesting less myelination of axons, particularly in the posterior regions. In agreement with these findings, ultrastructural analysis of myelinated axons in the posterior CC showed reduced myelin thickness in alcohol‐exposed animals, evidenced by a higher G‐ratio.

Prenatal alcohol exposure (PAE) is associated with long-term neurodevelopmental deficits resulting in impaired executive functioning and motor control. Intriguingly, PAE has been linked with an increased risk of transient systemic hypoxia–ischemia (TSHI), which alone results in suboptimal fetal growth and neurodevelopmental consequences. Here, using two translationally relevant preclinical models, we investigated the short-term and lasting effects of PAE and TSHI on the morphology of the medial prefrontal cortex (mPFC), a region important in executive function, and tested whether PAE interacts with TSHI to produce a distinct pattern of injury relative to either condition alone. The four experimental groups included sham (saccharin water, no TSHI), PAE (5% alcohol, no TSHI), TSHI (saccharin water, TSHI), and PAE+TSHI (5% alcohol, TSHI). Brains were extracted for Golgi–Cox staining at Postnatal Day 35 (P35) or P100 and processed for 3D Sholl analysis. The analysis of the mPFC at P35 showed no significant differences in the number of branches or dendritic length overall, although the impact of TSHI compared to alcohol was significant for both. There were no significant differences in the number of Sholl intersections overall at P35, although a sex difference was noted in PAE offspring. At P100, analysis of filament dendritic length and branching number was also significantly impacted by TSHI compared to alcohol. Interestingly, sex was also a significant factor when assessing the impact of alcohol. PAE and TSHI both had an insignificantly increased number of Sholl intersections at P100 compared to the control. The observed changes to dendritic complexity at P100 demonstrate altered neuronal morphology in the mPFC that endure into adulthood. Given the importance of the mPFC in executive functioning, these pilot data provide insight into morphological changes that may contribute to the neurobehavioral deficits observed following exposure to PAE and TSHI and highlight the need for additional investigations into this area.

AbstractAlcohol exposure during central nervous system (CNS) development can lead to fetal alcohol spectrum disorder (FASD). Human imaging studies have revealed significant white matter (WM) abnormalities linked to cognitive impairment in children with FASD; however, the underlying mechanisms remain unknown. Here, we evaluated both the acute and long‐term impacts of alcohol exposure on oligodendrocyte number and WM integrity in a third trimester‐equivalent mouse model of FASD, in which mouse pups were exposed to alcohol during the first 2 weeks of postnatal development. Our results demonstrate a 58% decrease in the number of mature oligodendrocytes (OLs) and a 75% decrease in the number of proliferating oligodendrocyte progenitor cells (OPCs) within the corpus callosum of alcohol‐exposed mice at postnatal day 16 (P16). Interestingly, neither mature OLs nor OPCs derived from the postnatal subventricular zone (SVZ) were numerically affected by alcohol exposure, indicating heterogeneity in susceptibility based on OL ontogenetic origin. Although mature OL and proliferating OPC numbers recovered by postnatal day 50 (P50), abnormalities in myelin protein expression and microstructure within the corpus callosum of alcohol‐exposed subjects persisted, as assessed by western immunoblotting of myelin basic protein (MBP; decreased expression) and MRI diffusion tensor imaging (DTI; decreased fractional anisotropy). These results indicate that third trimester‐equivalent alcohol exposure leads to an acute, albeit recoverable, decrease in OL lineage cell numbers, accompanied by enduring WM injury. Additionally, our finding of heterogeneity in alcohol susceptibility based on the developmental origin of OLs may have therapeutic implications in FASD and other disorders of WM development.

BackgroundThe adult hippocampal dentate is comprised of both developmentally generated dentate granule cells (dDGCs) and adult‐generated dentate granule cells (aDGCs), which play distinct roles in hippocampal information processing and network function. EtOH exposure throughout gestation in mouse impairs the neurogenic response to enriched environment (EE) in adulthood, although the basal rate of adult neurogenesis under standard housing (SH) is unaffected. Here, we tested whether the production and/or survival of either dDGCs or aDGCs are selectively impaired following exposure of mice to EtOH vapors during early postnatal development (human third trimester‐equivalent), and whether this exposure paradigm leads to impairment of EE‐mediated dentate neurogenesis in adulthood.MethodsAll experiments were performed using NestinCreERT2:tdTomato bitransgenic mice, which harbor a tamoxifen‐inducible tdTomato (tdTom) reporter for indelible labeling of newborn hippocampal DGCs. We exposed all mice to EtOH vapor or room air (Control) for 4 h/d from postnatal day (PND) 3 through PND 15. This paradigm resulted in a mean daily postexposure blood EtOH concentration of ~160 mg/dl. One cohort of neonatal mice received a single injection of tamoxifen at PND 2 and was sacrificed at either PND 16 or PND 50 to assess the impact of EtOH exposure on the production and long‐term survival of dDGCs born during the early postnatal period. A second cohort of mice received daily injections of tamoxifen at PND 35 to 39 to label aDGCs and was exposed to SH or EE for 6 weeks prior to sacrifice.ResultsEarly postnatal EtOH exposure had no statistically significant effect on the production or survival of tdTom+ dDGCs, as assessed at PND 16 or PND 50. Early postnatal EtOH exposure also had no effect on the number of tdTom+ aDGCs under SH conditions. Furthermore, early postnatal EtOH exposure had no significant impact on the adult neurogenic response to EE.ConclusionsBoth early postnatal dentate neurogenesis and adult dentate neurogenesis, as well as the adult neurogenic response to EE, are surprisingly resistant to early postnatal EtOH vapor exposure in mice.

BackgroundThe goal of this study was to evaluate the expression and serine 9 phosphorylation of glycogen synthase kinase (GSK‐3β) within the adult hippocampal dentate gyrus (DG) in a preclinical mouse model of fetal alcohol spectrum disorders. GSK‐3β is a multifunctional kinase that modulates many hippocampal processes affected by gestational alcohol, including synaptic plasticity and adult neurogenesis. GSK‐3β is a constitutively active kinase that is negatively regulated by phosphorylation at the serine 9 residue.MethodsWe utilized a well‐characterized limited access “drinking‐in‐the‐dark” paradigm of prenatal alcohol exposure (PAE) and measured p(Ser9)GSK‐3β and total GSK‐3β within adult DG by Western blot analysis. In addition, we evaluated the expression pattern of both p(Ser9)GSK‐3β and total GSK‐3β within the adult hippocampal dentate of PAE and control mice using high‐resolution confocal microscopy.ResultsOur findings demonstrate a marked 2.0‐fold elevation of p(Ser9)GSK‐3β in PAE mice, concomitant with a more moderate 36% increase in total GSK‐3β. This resulted in an approximate 63% increase in the p(Ser9)GSK‐3β/GSK‐3β ratio. Immunostaining revealed robust GSK‐3β expression within Cornu Ammonis (CA) pyramidal neurons, hilar mossy cells, and a subset of GABAergic interneurons, with low levels of expression within hippocampal progenitors and dentate granule cells.ConclusionsThese findings suggest that PAE may lead to a long‐term disruption of GSK‐3β signaling within the DG, and implicate mossy cells, GABAergic interneurons, and CA primary neurons as major targets of this dysregulation.