Adolescent intermittent ethanol (AIE) produces lasting, sex-specific changes in rat body fat independent of changes in white blood cell composition.

Early initiation of alcohol use during adolescence, and adolescent binge drinking are risk factors for the development of alcohol use disorder later in life. Adolescence is a time of rapid sex-dependent neural, physiological, and behavioral changes as well as a period of heightened vulnerability to many effects of alcohol. The goal of the present studies was to determine age-related changes in blood (leukocyte populations) and body composition across adolescence and early adulthood, and to investigate whether adolescent intermittent ethanol (AIE) exposure would alter the trajectory of adolescent development on these broad physiological parameters. We observed significant ontogenetic changes in leukocyte populations that were mirrored by an age-related increase in cytokine expression among mixed populations of circulating leukocytes. Despite these developmental changes, AIE did not significantly alter overall leukocyte numbers or cytokine gene expression. However, AIE led to sex-specific changes in body fat mass and fat percentage, with AIE-exposed male rats showing significantly decreased fat levels and female rats showing significantly increased fat levels relative to controls. These changes suggest that while AIE may not alter overall leukocyte levels, more complex phenotypic changes in leukocyte populations could underlie previously reported differences in cytokine expression. Coupled with long-term shifts in adipocyte levels, this could have long-lasting effects on innate immunity and the capacity of individuals to respond to later immunological and physiological threats.

Adolescent intermittent ethanol exposure enhances adult stress effects in male rats.

Binge patterns of alcohol use, prevalent among adolescents, are associated with a higher probability of developing alcohol use disorders (AUD) and other psychiatric disorders, like anxiety and depression. Additionally, adverse life events strongly predict AUD and other psychiatric disorders. As such, the combined fields of stress and AUD have been well established, and animal models indicate that both binge-like alcohol exposure and stress exposure elevate anxiety-like behaviors. However, few have investigated the interaction of adolescent intermittent ethanol (AIE) and adult stressors. We hypothesized that AIE would increase vulnerability to restraint-induced stress (RS), manifested as increased anxiety-like behavior. After AIE exposure, in adulthood, animals were tested on forced swim (FST) and saccharin preference (SP) and then exposed to either RS (90 min/5 days) or home-cage control. Twenty-four hours after the last RS session, animals began testing on the elevated plus maze (EPM), and were re-tested on FST and SP. A separate group of animals were sacrificed in adulthood after AIE and RS, and brains were harvested for immunoblot analysis of dorsal and ventral hippocampus. Consistent with previous reports, AIE had no significant effect on closed arm time in the EPM (anxiety-like behavior). However, in male rats the interaction of AIE and adult RS increased time spent in the closed arms. No effect was observed among female animals. AIE and RS-specific alterations were found in glial and synaptic markers (GLT-1, FMRP and PSD-95) in male animals. These findings indicate AIE has sex-specific effects on both SP and the interaction of AIE and adult RS, which induces a propensity toward anxiety-like behavior in males. Also, AIE produces persistent hippocampal deficits that may interact with adult RS to cause increased anxiety-like behaviors. Understanding the mechanisms behind this AIE-induced increase in stress vulnerability may provide insight into treatment and prevention strategies for alcohol use disorders.

Sex differences in the effects of adolescent intermittent ethanol exposure on exploratory and anxiety-like behavior in adult rats.

Adolescent intermittent ethanol (AIE) exposure in rodents has been shown to alter adult behavior in several domains, including learning and memory, social interaction, affective behavior, and ethanol self-administration. AIE has also been shown to produce non-specific behavioral changes that compromise behavioral efficiency. Many studies of these types rely on measuring behavior in mazes and other enclosures that can be influenced by animals' activity levels and exploratory behavior, and relatively few such studies have assessed sex as a biological variable. To address the effects of AIE and its interaction with sex on these types of behavioral assays, male and female adolescent rats (Sprague Dawley) were exposed to 10 doses of AIE (5 g/kg, intra-gastrically [i.g.]), or control vehicle, over 16 days (postnatal day [PND] 30-46), and then tested for exploratory and anxiety-like behaviors on the novelty-induced hypophagia (NIH) task in an open field, the elevated plus (EPM) maze, and the Figure 8 maze. AIE reduced activity/exploratory behaviors in males on the anxiety-producing NIH and EPM tasks, but reduced activity in both males and females in the Figure 8 maze, a task designed to create a safe environment and reduce anxiety. Independent of AIE, females engaged in more rearing behavior than males during the NIH task but less in the EPM, in which they were also less active than males. AIE also increased EPM open arm time in females but not in males. These findings demonstrate previously unrecognized sex differences in the effects of AIE on activity, exploratory behavior, and anxiety-like behavior; additionally, they underscore the need to design future behavioral studies of AIE using sex as a variable and with rigorous attention to how AIE alters these behaviors.

Effects of adolescent intermittent ethanol on hippocampal expression of glutamate homeostasis and astrocyte-neuronal tethering proteins in male and female rats.

Adolescent alcohol drinking is widely recognized as a significant public health problem, and evidence is accumulating that sufficient levels of consumption during this critical period of brain development have an enduring impact on neural and behavioral function. Recent studies have indicated that adolescent intermittent ethanol (AIE) exposure alters astrocyte function, astrocyte-neuronal interactions, and related synaptic regulation and activity. However, few of those studies have included female animals, and a broader assessment of AIE effects on the proteins mediating astrocyte-mediated glutamate dynamics and synaptic function is needed. We measured synaptic membrane expression of several such proteins in the dorsal and ventral regions of the hippocampal formation (DH, VH) from male and female rats exposed to AIE or adolescent intermittent water. In the DH, AIE caused elevated expression of glutamate transporter 1 (GLT-1) in both males and females, elevated postsynaptic density 95 expression in females only, and diminished NMDA receptor subunit 2A expression in males only. AIE and sex interactively altered ephrin receptor A4 (EphA4) expression in the DH. In the VH, AIE elevated expression of the cystine/glutamate antiporter and the glutamate aspartate transporter 1 (GLAST) in males only. Compared to males, female animals expressed lower levels of GLT-1 in the DH and greater levels of ephrin receptor B6 (EphB6) in the VH, in the absence of AIE effects. These results support the growing literature indicating that adolescent alcohol exposure produces long-lasting effects on astrocyte function and astrocyte-neuronal interactions. The sex and subregion specificity of these effects have mechanistic implications for our understanding of AIE effects generally.

Enduring alterations in hippocampal astrocytesynaptic proximity following adolescent alcohol exposure: reversal by gabapentin.

Adolescent alcohol abuse is a substantive public health problem that has been the subject of intensive study in recent years. Despite reports of a wide range of effects of adolescent intermittent ethanol (AIE) exposure on brain and behavior, little is known about the mechanisms that may underlie those effects, and even less about treatments that might reverse them. Recent studies from our laboratory have indicated that AIE produced enduring changes in astrocyte function and synaptic activity in the hippocampal formation, suggesting the possibility of an alteration in astrocyte-neuronal connectivity and function. We utilized astrocyte-specific, membrane restricted viral labeling paired with immunohistochemistry to perform confocal single cell astrocyte imaging, three-dimensional reconstruction, and quantification of astrocyte morphology in hippocampal area CA1 from adult rats after AIE. Additionally, we assessed the colocalization of astrocyte plasma membrane labeling with immunoreactivity for AMPA-(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate receptor 1, an AMPA receptor subunit and established neuronal marker of excitatory synapses, as a metric of astrocyte-synapse proximity. AIE significantly reduced the colocalization of the astrocyte plasma membrane with synaptic marker puncta in adulthood. This is striking in that it suggests not only an alteration of the physical association of astrocytes with synapses by AIE, but one that lasts into adulthood - well after the termination of alcohol exposure. Perhaps even more notable, the AIE-induced reduction of astrocyte-synapse interaction was reversed by sub-chronic treatment with the clinically used agent, gabapentin (Neurontin), in adulthood. This suggests that a medication in common clinical use may have the potential to reverse some of the enduring effects of adolescent alcohol exposure on brain function. All animal experiments conducted were approved by the Duke University Institutional Animal Care and Use Committee (Protocol Registry Number A159-18-07) on July 27, 2018.

Effects of ethanol on plasma ghrelin levels in the rat during early and late adolescence.

Ghrelin is an appetite-regulating peptide that is primarily secreted by endocrine cells in the stomach and is implicated in regulation of alcohol consumption and alcohol-reinforced behaviors. In the present study, adolescent Sprague-Dawley rats received intermittent ethanol (AIE) exposure by intragastric intubation (5 g/kg) or vapor inhalation, manipulations conducted between postnatal days (PD) 28-43. On the first and last day of AIE exposure, the level of intoxication was examined 1 h after ethanol gavage or upon removal from the vapor chamber. This was immediately followed by a blood draw for determination of the blood ethanol concentration (BEC) and plasma levels of acylated ghrelin (acyl-ghrelin; active). On PD29, plasma levels of acyl-ghrelin were significantly elevated in male (but not female) rats in response to acute ethanol exposure by both gastric gavage and vapor inhalation. Importantly, assessment of plasma acyl-ghrelin in response to repeated ethanol exposure revealed a complex interaction of both sex and method of AIE exposure. On PD43, vapor inhalation increased plasma acyl-ghrelin in both males and females compared to their air-control counterparts, whereas there was no change in plasma levels of acyl-ghrelin in either male or female rats in response to exposure by intragastric gavage. Assessment of plasma acyl-ghrelin following a 30-day ethanol-free period revealed AIE exposure did not produce a change in basal levels. In addition, an acute ethanol challenge in adult rats of 5 g/kg via gastric gavage had no effect on plasma ghrelin levels when assessed 1 h after initiation of exposure. Collectively, these observations suggest that acyl-ghrelin, a primary gut-brain signaling hormone, is elevated by ethanol during early adolescence independent of administration route, and in gender-dependent fashion.