Persistent myelin abnormalities in a third trimester-equivalent mouse model of fetal alcohol spectrum disorder.

BACKGROUND: Abnormal 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. METHODS: Mouse 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 Nav 1.6 (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. RESULTS: Consistent 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). CONCLUSIONS: High 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.

Resistance of Postnatal Hippocampal Neurogenesis to Alcohol Toxicity in a Third Trimester-Equivalent Mouse Model of Gestational Alcohol Exposure.

BACKGROUND: The 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. METHODS: All 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. RESULTS: Early 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. CONCLUSIONS: Both 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.

Acute oligodendrocyte loss with persistent white matter injury in a third trimester equivalent mouse model of fetal alcohol spectrum disorder.

Alcohol 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.

Ethanol exposure during the third trimester equivalent does not affect GABAA or AMPA receptor-mediated spontaneous synaptic transmission in rat CA3 pyramidal neurons.

BACKGROUND: Ethanol exposure during the rodent equivalent to the 3(rd) trimester of human pregnancy (i.e., first 1-2 weeks of neonatal life) has been shown to produce structural and functional alterations in the CA3 hippocampal sub-region, which is involved in associative memory. Synaptic plasticity mechanisms dependent on retrograde release of brain-derived neurotrophic factor (BDNF) driven by activation of L-type voltage-gated Ca(2+) channels (L-VGCCs) are thought to play a role in stabilization of both GABAergic and glutamatergic synapses in CA3 pyramidal neurons. We previously showed that ethanol exposure during the first week of life blocks BDNF/L-VGCC-dependent long-term potentiation of GABAA receptor-mediated synaptic transmission in these neurons. Here, we tested whether this effect is associated with lasting alterations in GABAergic and glutamatergic transmission. METHODS: Rats were exposed to air or ethanol for 3 h/day between postnatal days three and five in vapor inhalation chambers, a paradigm that produces peak serum ethanol levels near 0.3 g/dl. Whole-cell patch-clamp electrophysiological recordings of spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs, respectively) were obtained from CA3 pyramidal neurons in coronal brain slices prepared at postnatal days 13-17. RESULTS: Ethanol exposure did not significantly affect the frequency, amplitude, rise-time and half-width of either sIPSCs or sEPSCs. CONCLUSIONS: We show that an ethanol exposure paradigm known to inhibit synaptic plasticity mechanisms that may participate in the stabilization of GABAergic and glutamatergic synapses in CA3 pyramidal neurons does not produce lasting functional alterations in these synapses, suggesting that compensatory mechanisms restored the balance of excitatory and inhibitory synaptic transmission.

Moderate Alcohol Exposure during the Rat Equivalent to the Third Trimester of Human Pregnancy Alters Regulation of GABAA Receptor-Mediated Synaptic Transmission by Dopamine in the Basolateral Amygdala.

Fetal ethanol (EtOH) exposure leads to a range of neurobehavioral alterations, including deficits in emotional processing. The basolateral amygdala (BLA) plays a critical role in modulating emotional processing, in part, via dopamine (DA) regulation of GABA transmission. This BLA modulatory system is acquired during the first 2 weeks of postnatal life in rodents (equivalent to the third trimester of human pregnancy) and we hypothesized that it could be altered by EtOH exposure during this period. We found that exposure of rats to moderate levels of EtOH vapor during the third trimester-equivalent [postnatal days (P) 2-12] alters DA modulation of GABAergic transmission in BLA pyramidal neurons during periadolescence. Specifically, D1R-mediated potentiation of spontaneous inhibitory postsynaptic currents (IPSCs) was significantly attenuated in EtOH-exposed animals. However, this was associated with a compensatory decrease in D3R-mediated suppression of miniature IPSCs. Western blot analysis revealed that these effects were not a result of altered D1R or D3R levels. BLA samples from EtOH-exposed animals also had significantly lower levels of the DA precursor (L-3,4-dihydroxyphenylalanine) but DA levels were not affected. This is likely a consequence of reduced catabolism of DA, as indicated by reduced levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid in the BLA samples. Anxiety-like behavior was not altered in EtOH-exposed animals. This is the first study to demonstrate that the modulatory actions of DA in the BLA are altered by developmental EtOH exposure. Although compensatory adaptations were engaged in our moderate EtOH exposure paradigm, it is possible that these are not able to restore homeostasis and correct anxiety-like behaviors under conditions of heavier EtOH exposure. Therefore, future studies should investigate the potential role of alterations in the modulatory actions of DA in the pathophysiology of fetal alcohol spectrum disorders.

AMPAR-mediated synaptic transmission in the CA1 hippocampal region of neonatal rats: unexpected resistance to repeated ethanol exposure.

Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate glutamatergic receptors (AMPAR) mediate most of the fast excitatory synaptic transmission in mature neurons. In contrast, a number of developing synapses do not express AMPARs; these are gradually acquired in an activity-driven manner during the first week of life in rats, which is equivalent to the third trimester of human pregnancy. Neuronal stimulation has been shown to drive high conductance Ca(2+)-permeable AMPARs into the synapse, strengthening glutamatergic synaptic transmission. Alterations in this process could induce premature stabilization or inappropriate elimination of newly formed synapses and contribute to the hippocampal abnormalities associated with fetal alcohol spectrum disorder. Previous studies from our laboratory performed with hippocampal slices from neonatal rats showed that acute ethanol exposure exerts potent stimulant effects on CA1 and CA3 neuronal networks. However, the impact of these in vitro actions of acute ethanol exposure is unknown. Here, we tested the hypothesis that repeated in vivo exposure to ethanol strengthens AMPAR-mediated neurotransmission in the CA1 region by means of an increase in synaptic expression of Ca(2+)-permeable AMPARs. We exposed rats to ethanol vapor (serum ethanol concentration approximately 40 mM) or air for 4h/day from postnatal day (P) 2-6. In brain slices prepared at P4-6, we found no significant effect of ethanol exposure on input-output curves for AMPAR-mediated field excitatory postsynaptic potentials (fEPSPs), the contribution of Ca(2+)-permeable AMPARs to these fEPSPs, or the acute effect of ethanol on fEPSP amplitude. These results suggest that homeostatic plasticity mechanisms act to maintain glutamatergic synaptic strength and ethanol sensitivity in response to repeated developmental ethanol exposure.

Prenatal ethanol exposure persistently impairs NMDA receptor-dependent activation of extracellular signal-regulated kinase in the mouse dentate gyrus.

The dentate gyrus (DG) is the central input region to the hippocampus and is known to play an important role in learning and memory. Previous studies have shown that prenatal alcohol is associated with hippocampal-dependent learning deficits and a decreased ability to elicit long-term potentiation (LTP) in the DG in adult animals. Given that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascade by NMDA receptors is required for various forms of learning and memory, as well as LTP, in hippocampal regions, including the DG, we hypothesized that fetal alcohol-exposed adult animals would have deficits in hippocampal NMDA receptor-dependent ERK1/2 activation. We used immunoblotting and immunohistochemistry techniques to detect NMDA-stimulated ERK1/2 activation in acute hippocampal slices prepared from adult fetal alcohol-exposed mice. We present the first evidence linking prenatal alcohol exposure to deficits in NMDA receptor-dependent ERK1/2 activation specifically in the DG of adult offspring. This deficit may account for the LTP deficits previously observed in the DG, as well as the life-long cognitive deficits, associated with prenatal alcohol exposure.