Background Human adolescence is a crucial stage of neurological development during which ethanol (EtOH) consumption is often at its highest. Alcohol abuse during adolescence may render individuals at heightened risk for subsequent alcohol abuse disorders, cognitive dysfunction, or other neurological impairments by irreversibly altering long-term brain function. To test this possibility, we modeled adolescent alcohol abuse (i.e., intermittent EtOH exposure during adolescence [AIE]) in rats to determine whether adolescent exposure to alcohol leads to long-term structural and functional changes that are manifested in adult neuronal circuitry. Methods We specifically focused on hippocampal area CA1, a brain region associated with learning and memory. Using electrophysiological, immunohistochemical, and neuroanatomical approaches, we measured post-AIE changes in synaptic plasticity, dendritic spine morphology, and synaptic structure in adulthood. Results We found that AIE-pretreated adult rats manifest robust long-term potentiation, induced at stimulus intensities lower than those required in controls, suggesting a state of enhanced synaptic plasticity. Moreover, AIE resulted in an increased number of dendritic spines with characteristics typical of immaturity. Immunohistochemistry-based analysis of synaptic structures indicated a significant decrease in the number of co-localized pre- and postsynaptic puncta. This decrease is driven by an overall decrease in 2 postsynaptic density proteins, PSD-95 and SAP102. Conclusions Taken together, these findings reveal that repeated alcohol exposure during adolescence results in enduring structural and functional abnormalities in the hippocampus. These synaptic changes in the hippocampal circuits may help to explain learning-related behavioral changes in adult animals preexposed to AIE.

These findings indicate that AIE produces long-lasting decreases in dendritic spine density and changes in Fmr1 gene expression in the hippocampal formation, suggesting morphological and epigenetic mechanisms underlying previously reported behavioral deficits after AIE. The reversal of these effects by subchronic, post-AIE donepezil treatment indicates that these AIE effects can be reversed by up-regulating cholinergic function.

The long-term effects of intermittent ethanol exposure during adolescence (AIE) are of intensive interest and investigation. The effects of AIE on learning and memory and the neural functions that drive them are of particular interest as clinical findings suggest enduring deficits in those cognitive domains in humans after ethanol abuse during adolescence. Although studies of such deficits after AIE hold much promise for identifying mechanisms and therapeutic interventions, the findings are sparse and inconclusive. The present results identify a specific deficit in memory function after AIE and establish a possible neural mechanism of that deficit that may be of translational significance. Male rats (starting at PND-30) received exposure to AIE (5g/kg, i.g.) or vehicle and were allowed to mature into adulthood. At PND-71, one group of animals was assessed using the spatial-temporal object recognition (stOR) test to evaluate memory function. A separate group of animals was used to assess the density of cholinergic neurons in forebrain areas Ch1-4 using immunohistochemistry. AIE exposed animals manifested deficits in the temporal component of the stOR task relative to controls, and a significant decrease in the number of ChAT labeled neurons in forebrain areas Ch1-4. These findings add to the growing literature indicating long-lasting neural and behavioral effects of AIE that persist into adulthood and indicate that memory-related deficits after AIE depend upon the tasks employed, and possibly their degree of complexity. Finally, the parallel finding of diminished cholinergic neuron density suggests a possible mechanism underlying the effects of AIE on memory and hippocampal function as well as possible therapeutic or preventive strategies for AIE.

Adolescent intermittent ethanol (AIE) exposure diminishes neurogenesis and dendritic spine density in the dentate gyrus. The cholinesterase inhibitor, donepezil (Aricept), reverses AIE effects on dendritic spines, possibly by interacting with inflammatory and/or epigenetic mediators after AIE exposure. This study tests the hypothesis that donepezil reverses AIE-induced neuroimmune, and epigenetic changes in the adult dentate gyrus. Adolescent Sprague-Dawley male rats (PD30-43) were given 10 intermittent, intragastric doses of ethanol (5.0 g/kg) or isovolumetric water (AIW). Twenty-one days later half of the animals from each group were treated with either donepezil or isovolumetric water (i.g.) once daily for four days. Two hours after the last donepezil or water dose animals were sacrificed and brains prepared for immunohistochemical analyses. AIE reduced immunoreactivity for doublecortin (DCX) and increased immunoreactivity for activated caspase-3 and death receptor-3 in adulthood, suggesting an enduring attenuation of neurogenesis and an increase in progenitor death. These effects were reversed by donepezil treatment in adulthood. AIE also increased immunoreactivity for the inflammatory signaling molecules HMGB1 and RAGE, as well as the activated phosphorylated transcription factor pNFκB p65, and the gene silencing marker dimethylated histone H3K9. All of these AIE effects were also reversed by donepezil, with the exception of HMGB1.

Adolescent alcohol use is the strongest predictor for alcohol use disorders. In rodents, adolescents have distinct responses to acute ethanol, and prolonged alcohol exposure during adolescence can maintain these phenotypes into adulthood. One brain region that is particularly sensitive to the effects of both acute and chronic ethanol exposure is the hippocampus. Adolescent intermittent ethanol exposure (AIE) produces long lasting changes in hippocampal synaptic plasticity and dendritic morphology, as well as in the susceptibility to acute ethanol-induced spatial memory impairment. Given the pattern of changes in hippocampal structure and function, one potential target for these effects is the ethanol sensitive GluN2B subunit of the NMDA receptor, which is known to be involved in synaptic plasticity and dendritic morphology. Thus we sought to determine if there were persistent changes in hippocampal GluN2B signaling cascades following AIE. We employed a previously validated GluN2B-targeted proteomic strategy that was used to identify novel signaling mechanisms altered by chronic ethanol exposure in the adult hippocampus. We collected adult hippocampal tissue (P70) from rats that had been given 2 weeks of AIE from P30-45. Tissue extracts were fractionated into synaptic and non-synaptic pools, immuno-precipitated for GluN2B, and then analyzed using proteomic methods. We detected a large number of proteins associated with GluN2B. AIE produced significant changes in the association of many proteins with GluN2B in both synaptic and non-synaptic fractions. Intriguingly the number of proteins changed in the non-synaptic fraction was double that found in the synaptic fraction. Some of these proteins include those involved in glutamate signaling cytoskeleton rearrangement, calcium signaling, and plasticity. Disruptions in these pathways may contribute to the persistent cellular and behavioral changes found in the adult hippocampus following AIE. Further, the robust change in non-synaptic proteins suggests that AIE may prime this signaling pathway for future ethanol exposures in adulthood.

Adolescence is a period of development in neural circuits that are critical for adult functioning. There is a relationship between alcohol exposure and risky decision-making, though the enduring effects of adolescent ethanol exposure on risky decision-making in adulthood have not been fully explored. Studies using positive reinforcement have shown that adolescent intermittent ethanol (AIE) exposure results in higher levels of risky decision-making in adulthood, but the effects of AIE on punishment-mediated decision-making have not been explored. Adolescent rats were exposed to AIE or saline vehicle across a 16-day period, and then allowed to mature into adulthood. They were then trained to lever press for food reward and were assessed for risky decision-making by pairing increased levels of food reward with the probability of footshock punishment. AIE did not alter punishment-mediated risky decision-making. However, it did result in a significant increase in the delay to lever pressing. This finding is consistent with previous reports, using other behavioral tasks, which show decreased behavioral efficiency in adulthood after AIE. These findings indicate that AIE increases behavioral inefficiency, but not punishment-mediated risk-taking, in adulthood. Thus they contribute to a more nuanced understanding of the long-term effects of AIE on adult behavior.

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