Acute and Chronic Ethanol Effects during Adolescence on Neuroimmune Responses: Consequences and Potential Pharmacologic Interventions.

Heavy ethanol consumption during adolescence has been linked to neuroimmune response dysregulation and cognitive deficits in the developing adolescent brain. During adolescence, the brain is particularly susceptible to the pharmacological effects of ethanol that are induced by acute and chronic bouts of exposure. Numerous preclinical rodent model studies have used different ethanol administration techniques, such as intragastric gavage, self-administration, vapor, intraperitoneal, and free access, and while most models indicated proinflammatory neuroimmune responses in the adolescent brain, there are various factors that appear to influence this observation. This review synthesizes the most recent findings of the effects of adolescent alcohol use on toll-like receptors, cytokines, and chemokines, as well as the activation of astrocytes and microglia with an emphasis on differences associated with the duration of ethanol exposure (acute vs. chronic), the amount of exposure (e.g., dose or blood ethanol concentrations), sex differences, and the timing of the neuroimmune observation (immediate vs. persistent). Finally, this review discusses new therapeutics and interventions that may ameliorate the dysregulation of neuroimmune maladaptations after ethanol exposure.

The Influence of Sex on Hippocampal Neurogenesis and Neurotrophic Responses on the Persistent Effects of Adolescent Intermittent Ethanol Exposure into Adulthood.

In the United States, approximately 90% of alcohol consumed by adolescents is binge drinking. Binge-like ethanol exposure during adolescence promotes dysregulation of neurotrophic responses and neurogenesis in the hippocampus. These effects include changes in proliferation, regulation, differentiation, and maturation of neurons, and there is indication that such effects may be disproportionate between sexes. This study determined whether sex impacts neurotrophic responses and neurogenesis in adulthood after adolescent intermittent ethanol (AIE) exposure. To determine this, adolescent rats underwent AIE with ethanol (5 g/kg). In adulthood, animals were euthanized, and immunohistochemical techniques and ELISAs were utilized to determine AIE effects on sex-specific neurogenesis factors and neurotrophic markers, respectively. AIE exposure led to a significant decrease in neurogenesis in the dentate gyrus of the hippocampal formation indicated by reductions in the numbers of DCX+, SOX2+ and Ki-67+ cells in male and female AIE-exposed rats. Additionally, AIE increased markers for the pro-inflammatory cytokines, TNF-α and IL-1ß, in the hippocampus into adulthood in male AIE-exposed rats only. No significant AIE-induced differences were observed in the anti-inflammatory cytokines, IL-10 and TGF-ß, nor in the neurotrophic factors BDNF and GDNF. Altogether, our findings indicate that although AIE did not have a persistent effect on hippocampal neurotrophic levels, there was still a reduction in neurogenesis. The neurogenic impairment was not sex specific, but the neurogenic deficits in males may be attributed to an increase in pro-inflammatory cytokine expression. A persistent impairment in neurogenesis may have an impact on both behavioral maladaptations and neurodegeneration in adulthood.

Sex-specific effects of adolescent intermittent ethanol exposure-induced dysregulation of hippocampal glial cells in adulthood.

Adolescent alcohol abuse is a significant public health concern, with approximately 4.3 million U.S. adolescents reporting monthly binge drinking. Excessive ethanol consumption during adolescence has been linked to dysregulation of the neuroimmune system, particularly in the hippocampus. Because there are sex differences in both neuroimmune responses and ethanol's pharmacologic actions, this study tested whether there were disparate effects based on sex in glial cells and neurodegeneration in adulthood after the adolescent intermittent ethanol (AIE) model. Male and female adolescent Sprague-Dawley rats underwent AIE. In adulthood, immunohistochemical techniques were utilized to determine the effects of AIE on astrocytes and microglia, and Fluoro-Jade C (FJC) was used to assess neurodegeneration in the hippocampus. AIE exposure significantly increased astrocyte activation in the cornu ammonis 1 (CA1), CA2/3, and dentate gyrus (DG) in both male and female rats with no discernible sex differences in immunoreactivity. Likewise, the number of GFAP + cells was significantly increased by AIE across the hippocampus. In our microglial assessment, AIE only led to increased Iba1 immunoreactivity in the CA1 but not CA2/3 or DG regions. However, the number of Iba1+ cells was increased by AIE in both the CA1 and DG subregions. In the DG, the ethanol effect was observed in both sexes, but in the CA1, AIE-induced increased Iba1 cells were only observed in females. In regard to neurodegeneration, there were no persisting AIE effects on FJC + cells. These findings indicate that AIE alters hippocampal glial cells in adulthood, in the absence of active neurodegeneration. However, while AIE induced long-term elevation of astroglial measures in both males and females, persisting AIE-induced microglial activation was more sparse and sex-dependent. While the majority of these findings suggest that AIE has similar effects on glial morphology and number between males and females, additional work should determine whether there are molecular differences as well as innate sex differences in glial interaction with AIE's influence on glial functions in behavior.

Negative and positive allosteric modulators of the α7 nicotinic acetylcholine receptor regulates the ability of adolescent binge alcohol exposure to enhance adult alcohol consumption.

Rationale and Objectives: Ethanol acts directly on the α7 Nicotinic acetylcholine receptor (α7). Adolescent-binge alcohol exposure (ABAE) produces deleterious consequences during adulthood, and data indicate that the α7 receptor regulates these damaging events. Administration of an α7 Negative Allosteric Modulator (NAM) or the cholinesterase inhibitor galantamine can prophylactically prevent adult consequences of ABAE. The goals of the experiments were to determine the effects of co-administration of ethanol and a α7 agonist in the mesolimbic dopamine system and to determine if administration of an α7 NAM or positive allosteric modulator (PAM) modulates the enhancement of adult alcohol drinking produced by ABAE. Methods: In adult rats, ethanol and the α7 agonist AR-R17779 (AR) were microinjected into the posterior ventral tegmental area (VTA), and dopamine levels were measured in the nucleus accumbens shell (AcbSh). In adolescence, rats were treated with the α7 NAM SB-277011-A (SB) or PNU-120596 (PAM) 2 h before administration of EtOH (ABAE). Ethanol consumption (acquisition, maintenance, and relapse) during adulthood was characterized. Results: Ethanol and AR co-administered into the posterior VTA stimulated dopamine release in the AcbSh in a synergistic manner. The increase in alcohol consumption during the acquisition and relapse drinking during adulthood following ABAE was prevented by administration of SB, or enhanced by administration of PNU, prior to EtOH exposure during adolescence. Discussion: Ethanol acts on the α7 receptor, and the α7 receptor regulates the critical effects of ethanol in the brain. The data replicate the findings that cholinergic agents (α7 NAMs) can act prophylactically to reduce the alterations in adult alcohol consumption following ABAE.

The role of sex in the persistent effects of adolescent alcohol exposure on behavior and neurobiology in rodents.

Alcohol drinking is often initiated during adolescence, and this frequently escalates to binge drinking. As adolescence is also a period of dynamic neurodevelopment, preclinical evidence has highlighted that some of the consequences of binge drinking can be long lasting with deficits persisting into adulthood in a variety of cognitive-behavioral tasks. However, while the majority of preclinical work to date has been performed in male rodents, the rapid increase in binge drinking in adolescent female humans has re-emphasized the importance of addressing alcohol effects in the context of sex as a biological variable. Here we review several of the consequences of adolescent ethanol exposure in light of sex as a critical biological variable. While some alcohol-induced outcomes, such as non-social approach/avoidance behavior and sleep disruption, are generally consistent across sex, others are variable across sex, such as alcohol drinking, sensitivity to ethanol, social anxiety-like behavior, and induction of proinflammatory markers.

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.

Strategies for Referring Cancer Patients in a Smoking Cessation Program.

Most people who smoke and develop cancer are unable to quit smoking. To address this, many cancer centers have now opened smoking cessation programs specifically designed to help cancer patients to quit. An important question has now emerged-what is the most effective approach for engaging smokers within a cancer center in these smoking cessation programs? We report outcomes from a retrospective observational study comparing three referral methods-traditional referral, best practice advisory (BPA), and direct outreach-on utilization of the Duke Cancer Center Smoking Cessation Program. We found that program utilization rate was higher for direct outreach (5.4%) than traditional referral (0.8%), p < 0.001, and BPA (0.2%); p < 0.001. Program utilization was 6.4% for all methods combined. Inferring a causal relationship between referral method and program utilization was not possible because the study did not use a randomized design. Innovation is needed to generate higher utilization rates for cancer center smoking cessation programs.

Regulation of the deleterious effects of binge-like exposure to alcohol during adolescence by α7 nicotinic acetylcholine receptor agents: prevention by pretreatment with a α7 negative allosteric modulator and emulation by a α7 agonist in alcohol-preferring (P) male and female rats.

RATIONALE AND OBJECTIVES: Binge-like alcohol consumption during adolescence associates with several deleterious consequences during adulthood including an increased risk for developing alcohol use disorder (AUD) and other addictions. Replicated preclinical data has indicated that adolescent exposure to binge-like levels of alcohol results in a reduction of choline acetyltransferase (ChAT) and an upregulation in the α7 nicotinic receptor (α7). From this information, we hypothesized that the α7 plays a critical role in mediating the effects of adolescent alcohol exposure. METHODS: Male and female P rats were injected with the α7 agonist AR-R17779 (AR) once during 6 time points between post-natal days (PND) 29-37. Separate groups were injected with the α7 negative allosteric modulator (NAM) dehydronorketamine (DHNK) 2 h before administration of 4 g/kg EtOH (14 total exposures) during PND 28-48. On PND 75, all rats were given access to water and ethanol (15 and 30%) for 6 consecutive weeks (acquisition). All rats were then deprived of EtOH for 2 weeks and then, alcohol was returned (relapse). RESULTS: Administration of AR during adolescence significantly increased acquisition of alcohol consumption during adulthood and prolonged relapse drinking in P rats. In contrast, administration of DHNK prior to binge-like EtOH exposure during adolescence prevented the increase in alcohol consumption observed during acquisition of alcohol consumption and the enhancement of relapse drinking observed during adulthood. DISCUSSION: The data indicate that α7 mediates the effects of alcohol during adolescence. The data also indicate that α7 NAMs are potential prophylactic agents to reduce the deleterious effects of adolescent alcohol abuse.

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.

Alcohol use and consequences in matriculating US college students by prescription stimulant/opioid nonmedical misuse status.

BACKGROUND: US college students have elevated prescription opioid and stimulant misuse rates, with frequent alcohol use and alcohol-related consequences (ARCs). To date, though, no research has examined relationships between opioid and/or stimulant misuse and alcohol quantity/frequency or ARC variables in college students. METHODS: The 2016-17 AlcoholEDU for College™, a web-based alcohol prevention program, provided data (n = 491,849). Participants were grouped into past 14-day: (1) no misuse; (2) opioid misuse only; (3) stimulant misuse only; and (4) combined misuse. Using multilevel logistic regressions, groups were compared on 14-day alcohol use odds, and among those with use, odds of any ARCs and specific ARCs (e.g., hangover). Multilevel negative binomial regressions compared group members with alcohol use on 14-day total drinks, maximum 24-h drinks and drinking days. RESULTS: Alcohol use and any ARCs odds were highest in the stimulant (odds ratios [OR] = 3.47 and 2.97, respectively) or opioid misuse only groups (ORs = 3.31 and 2.43, respectively), with the combined misuse group intermediate (ORs = 1.63 and 1.29; reference: no misuse). Mean 14-day drinks decreased from those with combined misuse, to those with stimulant misuse only, opioid misuse only and no misuse (8.22, 7.1, 6.67, and 4.71, respectively). CONCLUSIONS: College students engaged in 14-day stimulant and/or opioid misuse had higher odds of 14-day alcohol use, higher levels of alcohol use, and a greater likelihood of ARCs, versus students without misuse. These findings suggest that college students with any prescription misuse need alcohol screening, although those with poly-prescription misuse may not need more intensive alcohol interventions.

Mechanisms of Persistent Neurobiological Changes Following Adolescent Alcohol Exposure: NADIA Consortium Findings.

The Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium has focused on the impact of adolescent binge drinking on brain development, particularly on effects that persist into adulthood. Adolescent binge drinking is common, and while many factors contribute to human brain development and alcohol use during adolescence, animal models are critical for understanding the specific consequences of alcohol exposure during this developmental period and the underlying mechanisms. Using adolescent intermittent ethanol (AIE) exposure models, NADIA investigators identified long-lasting AIE-induced changes in adult behavior that are consistent with observations in humans, such as increased alcohol drinking, increased anxiety (particularly social anxiety), increased impulsivity, reduced behavioral flexibility, impaired memory, disrupted sleep, and altered responses to alcohol. These behavioral changes are associated with multiple molecular, cellular, and physiological alterations in the brain that persist long after AIE exposure. At the molecular level, AIE results in long-lasting changes in neuroimmune/trophic factor balance and epigenetic-microRNA (miRNA) signaling across glia and neurons. At the cellular level, AIE history is associated in adulthood with reduced expression of cholinergic, serotonergic, and dopaminergic neuron markers, attenuated cortical thickness, decreased neurogenesis, and altered dendritic spine and glial morphology. This constellation of molecular and cellular adaptations to AIE likely contributes to observed alterations in neurophysiology, measured by synaptic physiology, EEG patterns, and functional connectivity. Many of these AIE-induced brain changes replicate findings seen in postmortem brains of humans with alcohol use disorder (AUD). NADIA researchers are now elucidating mechanisms of these adaptations. Emerging data demonstrate that exercise, antiinflammatory drugs, anticholinesterases, histone deacetylase inhibitors, and other pharmacological compounds are able to prevent (administered during AIE) and/or reverse (given after AIE) AIE-induced pathology in adulthood. These studies support hypotheses that adolescent binge drinking increases risk of adult hazardous drinking and influences brain development, and may provide insight into novel therapeutic targets for AIE-induced neuropathology and AUDs.

General anesthetic exposure in adolescent rats causes persistent maladaptations in cognitive and affective behaviors and neuroplasticity.

Accumulating evidence indicates that exposure to general anesthetics during infancy and childhood can cause persistent cognitive impairment, alterations in synaptic plasticity, and, to a lesser extent, increased incidence of behavioral disorders. Unfortunately, the developmental parameters of susceptibility to general anesthetics are not well understood. Adolescence is a critical developmental period wherein multiple late developing brain regions may also be vulnerable to enduring general anesthetic effects. Given the breadth of the adolescent age span, this group potentially represents millions more individuals than those exposed during early childhood. In this study, isoflurane exposure within a well-characterized adolescent period in Sprague-Dawley rats elicited immediate and persistent anxiety- and impulsive-like responding, as well as delayed cognitive impairment into adulthood. These behavioral abnormalities were paralleled by atypical dendritic spine morphology in the prefrontal cortex (PFC) and hippocampus (HPC), suggesting delayed anatomical maturation, and shifts in inhibitory function that suggest hypermaturation of extrasynaptic GABAA receptor inhibition. Preventing this hypermaturation of extrasynaptic GABAA receptor-mediated function in the PFC selectively reversed enhanced impulsivity resulting from adolescent isoflurane exposure. Taken together, these data demonstrate that the developmental window for susceptibility to enduring untoward effects of general anesthetics may be much longer than previously appreciated, and those effects may include affective behaviors in addition to cognition.

Region-Specific Differences in Morphometric Features and Synaptic Colocalization of Astrocytes During Development.

It is well established that astrocytes play pivotal roles in neuronal synapse formation and maturation as well as in the modulation of synaptic transmission. Despite their general importance for brain function, relatively little is known about the maturation of astrocytes during normal postnatal development, especially during adolescence, and how that maturation may influence astroglial-synaptic contact. The medial prefrontal cortex (mPFC) and dorsal hippocampus (dHipp) are critical for executive function, memory, and their effective integration. Further, both regions undergo significant functional changes during adolescence and early adulthood that are believed to mediate these functions. However, it is unclear the extent to which astrocytes change during these late developmental periods, nor is it clear whether their association with functional synapses shifts as adolescent and young adult maturation proceeds. Here we utilize an astrocyte-specific viral labeling approach paired with high-resolution single-cell astrocyte imaging and three-dimensional reconstruction to determine whether mPFC and dHipp astrocytes have temporally distinct maturation trajectories. mPFC astrocytes, in particular, continue to mature well into emerging adulthood (postnatal day 70). Moreover, this ongoing maturation is accompanied by a substantial increase in colocalization of astrocytes with the postsynaptic neuronal marker, PSD-95. Taken together, these data provide novel insight into region-specific astrocyte-synapse interactions in late CNS development and into adulthood, thus raising implications for the mechanism of post-adolescent development of the mPFC.

Sex Differences in Photic Entrainment and Sensitivity to Ethanol-Induced Chronodisruption in Adult Mice After Adolescent Intermittent Ethanol Exposure.

BACKGROUND: Evidence supports a role for the circadian system in alcohol use disorders, but the impact of adolescent alcohol exposure on circadian timing later in life is unknown. Acute ethanol (EtOH) attenuates circadian photic phase-resetting in adult, but not adolescent, rodents. However, nearly all studies have focused on males and it is unknown whether this adolescent-typical insensitivity to EtOH persists into adulthood after adolescent drinking. METHODS: Circadian activity was monitored in C57BL/6J mice receiving adolescent intermittent EtOH (AIE) exposure (15% EtOH and water every other day throughout adolescence) or water alone followed by 24 days wherein EtOH was not available (washout). Mice then received a challenge dose of EtOH (1.5 g/kg, intraperitoneal) or saline 15 minutes prior to a 30-minute phase-delaying light pulse and then were released into constant darkness (DD). To control for possible phase-shifting by EtOH challenge alone, a separate group of mice underwent AIE exposure (or water-only) and washout and then received an EtOH or saline injection, but did not receive a light pulse prior to DD. RESULTS: Striking sex differences in nearly all measures of circadian photic entrainment were observed during adolescence but AIE effects were subtle and few. Only EtOH-naïve adult male mice showed attenuated photic phase-shifts with EtOH challenge, while all other groups showed normal phase-resetting responses to light. AIE-exposed females showed a persistent delay in activity offset. CONCLUSIONS: Adult male AIE-exposed mice retained adolescent-like insensitivity to EtOH-induced suppression of photic phase-resetting, suggesting AIE-induced "lock-in" of an adolescent behavioral phenotype. Adult AIE-exposed females showed delayed initiation of the rest phase. Our results also indicate that intermittent EtOH drinking has subtle effects on circadian activity in mice during adolescence that differ from previously reported effects on adult males. The observed sex differences in circadian activity, EtOH consumption and preference, and responses to EtOH challenge merit future mechanistic study.

Donepezil Reverses Dendritic Spine Morphology Adaptations and Fmr1 Epigenetic Modifications in Hippocampus of Adult Rats After Adolescent Alcohol Exposure.

BACKGROUND: Adolescent intermittent ethanol (AIE) exposure produces persistent impairments in cholinergic and epigenetic signaling and alters markers of synapses in the hippocampal formation, effects that are thought to drive hippocampal dysfunction in adult rodents. Donepezil (Aricept), a cholinesterase inhibitor, is used clinically to ameliorate memory-related cognitive deficits. Given that donepezil also prevents morphological impairment in preclinical models of neuropsychiatric disorders, we investigated the ability of donepezil to reverse morphological and epigenetic adaptations in the hippocampus of adult rats exposed to AIE. Because of the known relationship between dendritic spine density and morphology with the fragile X mental retardation 1 (Fmr1) gene, we also assessed Fmr1 expression and its epigenetic regulation in hippocampus after AIE and donepezil pretreatment. METHODS: Adolescent rats were administered intermittent ethanol for 16 days starting on postnatal day 30. Rats were treated with donepezil (2.5 mg/kg) once a day for 4 days starting 20 days after the completion of AIE exposure. Brains were dissected out after the fourth donepezil dose, and spine analysis was completed in dentate gyrus granule neurons. A separate cohort of rats, treated identically, was used for molecular studies. RESULTS: AIE exposure significantly reduced dendritic spine density and altered morphological characteristics of subclasses of dendritic spines. AIE exposure also increased mRNA levels and H3-K27 acetylation occupancy of the Fmr1 gene in hippocampus. Treatment of AIE-exposed adult rats with donepezil reversed both the dendritic spine adaptations and epigenetic modifications and expression of Fmr1. CONCLUSIONS: 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.

Alcohol-Related Blackouts, Negative Alcohol-Related Consequences, and Motivations for Drinking Reported by Newly Matriculating Transgender College Students.

BACKGROUND: Many transgender college students struggle with identity formation and other emotional, social, and developmental challenges associated with emerging adulthood. A potential maladaptive coping strategy employed by such students is heavy drinking. Prior literature has suggested greater consumption and negative alcohol-related consequences (ARCs) in transgender students compared with their cisgender peers, but little is known about their differing experiences with alcohol-related blackouts (ARBs). We examined the level of alcohol consumption, the frequency of ARBs and other ARCs, and motivations for drinking reported by the largest sample of transgender college students to date. METHODS: A Web survey from an alcohol-prevention program, AlcoholEdu for College™, assessed student demographics and drinking-related behaviors, experiences, and motivations of newly matriculating first-year college students. A self-reported drinking calendar was used to examine each of the following measures over the previous 14 days: number of drinking days, total number of drinks, and maximum number of drinks on any single day. A 7-point Likert scale was used to measure ARCs, ARBs, and drinking motivations. Transgender students of both sexes were compared with their cisgender peers. RESULTS: A total of 989 of 422,906 students (0.2%) identified as transgender. Over a 14-day period, transgender compared with cisgender students were more likely to consume alcohol over more days, more total drinks, and a greater number of maximum drinks on a single day. Transgender students (36%) were more likely to report an ARB than cisgender students (25%) as well as more negative academic, confrontation-related, social, and sexual ARCs. Transgender respondents more often cited stress reduction, social anxiety, self-esteem issues, and the inherent properties of alcohol as motivations for drinking. For nearly all measures, higher values were yielded by male-to-female than female-to-male transgender students. CONCLUSIONS: Transgender compared with cisgender first-year students engage in higher-risk drinking patterns and experience more ARBs and other negative ARCs. Broad institutional efforts are required to address the unique circumstances of transgender men and women and to reduce negative ARCs in college students, regardless of their sex or gender identity.

Differential Sensitivity to Ethanol-Induced Circadian Rhythm Disruption in Adolescent and Adult Mice.

BACKGROUND: Growing evidence supports a central role for the circadian system in alcohol use disorders, but few studies have examined this relationship during adolescence. In mammals, circadian rhythms are regulated by the suprachiasmatic nucleus, a biological clock whose timing is synchronized (reset) to the environment primarily by light (photic) input. Alcohol (ethanol [EtOH]) disrupts circadian timing in part by attenuating photic phase-resetting responses in adult rodents. However, circadian rhythms change throughout life and it is not yet known whether EtOH has similar effects on circadian regulation during adolescence. METHODS: General circadian locomotor activity was monitored in male C57BL6/J mice beginning in adolescence (P27) or adulthood (P61) in a 12-hour light, 12-hour dark photocycle for ~2 weeks to establish baseline circadian activity measures. On the day of the experiment, mice received an acute injection of EtOH (1.5 g/kg, i.p.) or equal volume saline 15 minutes prior to a 30-minute light pulse at Zeitgeber Time 14 (2 hours into the dark phase) and then were released into constant darkness (DD) for ~2 weeks to assess phase-resetting responses. Control mice of each age-group received injections but no light pulse prior to DD. RESULTS: While adults showed the expected decrease in photic phase-delays induced by acute EtOH, this effect was absent in adolescent mice. Adolescents also showed baseline differences in circadian rhythmicity compared to adults, including advanced photocycle entrainment, larger photic phase-delays, a shorter free-running (endogenous) circadian period, and greater circadian rhythm amplitude. CONCLUSIONS: Collectively, our results indicate that adolescent mice are less sensitive to the effect of EtOH on circadian photic phase-resetting and that their daily activity rhythms are markedly different than those of adults.

Changes in the Adult GluN2B Associated Proteome following Adolescent Intermittent Ethanol Exposure.

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.

Adolescent Intermittent Alcohol Exposure: Deficits in Object Recognition Memory and Forebrain Cholinergic Markers.

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.