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Exposure to ethanol in utero can result in Fetal Alcohol Spectrum Disorders, which may cause long-lasting cognitive and behavioral abnormalities. Preclinical studies indicate that choline ameliorates the behavioral effects of developmental alcohol exposure in rodents, and clinical studies on the effectiveness of choline, administered early in pregnancy, showed that the adverse effects of heavy prenatal alcohol exposure on postnatal growth, and cognition in human infants were mitigated. However, little is known on the mechanisms behind the effects of choline. We have previously reported that astrocyte pre-treatment with 75 mM ethanol, in vitro, reduces neurite outgrowth in hippocampal neurons co-cultured with the pre-treated astrocytes. Our in vitro system allows us to study the effects of chemicals on astrocyte functions, able to modulate neuronal development. The main objective was to test the hypothesis that choline can ameliorate the astrocyte-mediated effects of ethanol on neurite growth. In this study, we exposed primary rat cortical astrocytes to ethanol, choline, ethanol plus choline, or control conditions for 24 h. Culture media was then removed, replaced with fresh media containing no ethanol or choline treatments and primary rat hippocampal neurons were plated on top of the astrocyte monolayer and cultured for 16 h. Neurons were then stained for β-III Tubulin and neurite outgrowth was measured. Astrocyte exposure to ethanol (25, 50, and 75 mM) decreases neurite outgrowth in co-cultured hippocampal pyramidal neurons, while astrocyte treatment with choline had no effect. Astrocyte treatment with ethanol and choline in combination, however, prevented the effect of ethanol, leading to levels of neurite outgrowth similar the control condition. Choline prevents the inhibitory effect of ethanol-treated astrocytes on neurite outgrowth while not altering normal neuronal development. These results suggest a new, astrocyte-mediated mechanism by which choline ameliorates the effects of developmental alcohol exposure.

Binge drinking is a dangerous pattern of behavior. We tested whether chronically manipulating nucleus accumbens (NAc) activity (via clozapine-N-oxide (CNO) and Designer Receptors Exclusively Activated by Designer Drugs (DREADD)) could produce lasting effects on ethanol binge-like drinking in mice selectively bred to drink to intoxication. We found chronically increasing NAc activity (4 weeks, via CNO and the excitatory DREADD, hM3Dq) decreased binge-like drinking, but did not observe CNO-induced changes in drinking with the inhibitory DREADD, hM4Di. The CNO/hM3Dq-induced reduction in ethanol drinking persisted for at least one week, suggesting adaptive neuroplasticity via transcriptional and epigenetic mechanisms. Therefore, we defined this plasticity at the morphological and transcriptomic levels. We found that chronic binge drinking (6 weeks) altered neuronal morphology in the NAc, an effect that was ameliorated with CNO/hM3Dq. Moreover, we detected significant changes in expression of several plasticity-related genes with binge drinking that were ameliorated with CNO treatment (e.g., Hdac4). Lastly, we found that LMK235, an HDAC4/5 inhibitor, reduced binge-like drinking. Thus, we were able to target specific molecular pathways using pharmacology to mimic the behavioral effects of DREADDs.

In utero alcohol exposure can cause fetal alcohol spectrum disorders (FASD), characterized by structural brain abnormalities and long-lasting behavioral and cognitive dysfunction. Neuronal plasticity is affected by in utero alcohol exposure and can be modulated by extracellular proteolysis. Plasmin is a major extracellular serine-protease whose activation is tightly regulated by the plasminogen activator (PA) system. In the present study we explored the effect of ethanol on the expression of the main components of the brain PA system in sex-specific cortical astrocyte primary cultures in vitro and in the cortex and hippocampus of post-natal day (PD) 9 male and female rats. We find that ethanol alters the PA system in astrocytes and in the developing brain. In particular, the expression of tissue-type PA (tPA), encoded by the gene Plat, is consistently upregulated by ethanol in astrocytes in vitro and in the cortex and hippocampus in vivo. Astrocytes exhibit endogenous plasmin activity that is increased by ethanol and recombinant tPA and inhibited by tPA silencing. We also find that tPA is expressed by astrocytes of the developing cortex and hippocampus in vivo. All components of the PA system investigated, with the exception of Neuroserpin/Serpini1, are expressed at higher levels in astrocyte cultures than in the developing brain, suggesting that astrocytes are major producers of these proteins in the brain. In conclusion, astrocyte PA system may play a major role in the modulation of neuronal plasticity; ethanol-induced upregulation of tPA levels and plasmin activity may be responsible for altered neuronal plasticity in FASD.

Some of the neurobehavioral deficits identified in children with Fetal Alcohol Spectrum Disorders have been recapitulated in a binge model of gestational third trimester-equivalent ethanol exposure, in which Sprague-Dawley rats are intragastrically intubated between post-natal day (PD) 4 and PD9 with high doses of ethanol. In this model, the ameliorating effects of choline administration on hippocampal-dependent behaviors altered by ethanol have also been extensively documented. In the present study, we investigated the effects of ethanol (5g/kg/day) and/or choline (100mg/kg/day) on morphometric parameters of CA1 pyramidal neurons by Golgi-Cox staining followed by Neurolucida tracing and analysis. We found that ethanol increased apical dendrite complexity in male and female pups neonatally exposed to ethanol. Ethanol did not significantly affect basal dendrite parameters in female and male rats. Interestingly, choline treatments decreased basal dendrites’ length, number, and maximal terminal distance in male pups. When pups were co-treated with ethanol and choline, choline did not rescue the effect of ethanol. In conclusion, ethanol increases while choline decreases dendritic length and arborization of hippocampal CA1 neurons in PD9 rats. We hypothesize that developmental ethanol exposure induces a premature maturation of neurons, leading to early restriction of neuronal plasticity while choline treatments delay the normal program of neuronal maturation and therefore prolong the window of maximal plasticity. Choline does not prevent the effects of developmental alcohol exposure on hippocampal pyramidal neurons’ morphology characterized in the present study, although whether prolonged choline administration after developmental ethanol exposure rectifies ethanol damage remains to be assessed.

Decabromodiphenyl ether (decaBDE) is an applied brominated flame retardant that is widely-used in electronic equipment. After decades of use, decaBDE and other members of its polybrominated diphenyl ether class have become globally-distributed environmental contaminants that can be measured in the atmosphere, water bodies, wildlife, food staples and human breastmilk. Although it has been banned in Europe and voluntarily withdrawn from the U.S. market, it is still used in Asian countries. Evidence from epidemiological and animal studies indicate that decaBDE exposure targets brain development and produces behavioral impairments. The current study examined an array of motor and learning behaviors in a C57BL6/J mouse model to determine the breadth of the developmental neurotoxicity produced by decaBDE. Mouse pups were given a single daily oral dose of 0 or 20 mg/kg decaBDE from postnatal day 1 to 21 and were tested in adulthood. Exposed male mice had impaired forelimb grip strength, altered motor output in a circadian wheel-running procedure, increased response errors during an operant differential reinforcement of low rates (DRL) procedure and a blunted response to an acute methylphenidate challenge administered before DRL testing. With the exception of altered wheel-running output, exposed females were not affected. Neither sex had altered somatic growth, motor coordination impairments on the Rotarod, gross learning deficits during operant lever-press acquisition, or impaired food motivation. The overall pattern of effects suggests that males are more sensitive to developmental decaBDE exposure, especially when performing behaviors that require effortful motor output or when learning tasks that require sufficient response inhibition for their successful completion.

During the preparation of the paper the authors neglected to list all funders, the following funders were not included in the original version: Department of Veterans Affairs Merit Review Awards #I01BX001819 (MG) and Career Development Award (CDA-2) #IK2BX001650 (DG). The complete Acknowledgement section should read. This study was supported in part by grant AA021876 from the National Institute of Alcoholism and Alcohol Abuse, Department of Veterans Affairs Merit Review Awards #I01BX001819 (MG) and Career Development Award (CDA-2) #IK2BX001650 (DG). The authors are extremely grateful to Mr. Jeff Frkonja for the graphic design of Figures 1, 3, and 4. The authors would like to apologize for any potential inconvenience caused.

During the last 20 years, new and exciting roles for glial cells in brain development have been described. Moreover, several recent studies implicated glial cells in the pathogenesis of neurodevelopmental disorders including Down syndrome, Fragile X syndrome, Rett Syndrome, Autism Spectrum Disorders, and Fetal Alcohol Spectrum Disorders (FASD). Abnormalities in glial cell development and proliferation and increased glial cell apoptosis contribute to the adverse effects of ethanol on the developing brain and it is becoming apparent that the effects of fetal alcohol are due, at least in part, to effects on glial cells affecting their ability to modulate neuronal development and function. The three major classes of glial cells, astrocytes, oligodendrocytes, and microglia as well as their precursors are affected by ethanol during brain development. Alterations in glial cell functions by ethanol dramatically affect neuronal development, survival, and function and ultimately impair the development of the proper brain architecture and connectivity. For instance, ethanol inhibits astrocyte-mediated neuritogenesis and oligodendrocyte development, survival and myelination; furthermore, ethanol induces microglia activation and oxidative stress leading to the exacerbation of ethanol-induced neuronal cell death. This review article describes the most significant recent findings pertaining the effects of ethanol on glial cells and their significance in the pathophysiology of FASD and other neurodevelopmental disorders.

Hexabromocyclododecane (HBCD) is a brominated flame retardant that is widely-used in foam building materials and to a lesser extent, furniture and electronic equipment. After decades of use, HBCD and its metabolites have become globally-distributed environmental contaminants that can be measured in the atmosphere, water bodies, wildlife, food staples and human breastmilk. Emerging evidence suggests that HBCD can affect early brain development and produce behavioral consequences for exposed organisms. The current study examined some of the developmental and lifelong neurobehavioral effects of prenatal HBCD exposure in a rat model. Pregnant rats were gavaged with 0, 3, 10, or 30 mg/kg HBCD from gestation day 1 to parturition. A functional observation battery was used to assess sensorimotor behaviors in neonates. Locomotor and operant responding under random ratio and Go/no-go schedules of food reinforcement were examined in cohorts of young adult and aged rats. HBCD exposure was associated with increased reactivity to a tailpinch in neonates, decreased forelimb grip strength in juveniles, and impaired sustained attention indicated by Go/no-go responding in aged rats. In addition, HBCD exposure was associated with a significant increase in morbidity in the aged cohort. One health complication, a progressive loss of hindleg function, was observed only in the aged, 3 mg/kg HBCD animals. These effects suggest that HBCD is a developmental neurotoxicant that can produce long-term behavioral impairments that emerge at different points in the lifespan following prenatal exposure.