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 alcohol exposure alters threat avoidance in adulthood.

Adolescent binge-like alcohol exposure impairs cognitive function and decision making in adulthood and may be associated with dysfunction of threat avoidance, a critical mechanism of survival which relies upon executive function. The present study investigated the impact of binge-like alcohol exposure during adolescence on active avoidance in adulthood. Male and female rats were subjected to adolescent intermittent ethanol (AIE) exposure by vapor inhalation and then tested in adulthood using a platform-mediated avoidance task. After training to press a lever to receive a sucrose reward, the rats were conditioned to a tone that co-terminated with a foot-shock. A motivational conflict was introduced by the presence of an escape platform that isolated the rat from the shock, but also prevented access to the sucrose reward while the rat was on the platform. During the task training phase, both male and female rats exhibited progressive increases in active avoidance (platform escape) in response to the conditioned tone, whereas innate fear behavior (freezing) remained relatively constant over training days. A history of AIE exposure did not impact either active avoidance or freezing behavior during task acquisition. On the test day following platform acquisition training, female rats exhibited higher levels of both active avoidance and freezing compared to male rats, while AIE-exposed male but not female rats exhibited significantly greater levels of active avoidance compared to controls. In contrast, neither male nor female AIE-exposed rats exhibited alterations in freezing compared to controls. Following 5 days of extinction training, female rats continued to display higher levels of active avoidance and freezing during tone presentation compared to males. Male AIE-exposed rats also had higher levels of both active avoidance and freezing compared to the male control rats. Together, the results demonstrate that female rats exhibit elevated levels of active avoidance and freezing compared to males and further reveal a sex-specific impact of AIE on threat responding in adulthood.

Adolescent alcohol exposure reduces dopamine 1 receptor modulation of prelimbic neurons projecting to the nucleus accumbens and basolateral amygdala.

Binge drinking during adolescence is highly prevalent despite increasing evidence of its long-term impact on behaviors associated with modulation of behavioral flexibility by the medial prefrontal cortex (mPFC). In the present study, male and female rats underwent adolescent intermittent ethanol (AIE) exposure by vapor inhalation. After aging to adulthood, retrograde bead labelling and viral tagging were used to identify populations of neurons in the prelimbic region (PrL) of the mPFC that project to specific subcortical targets. Electrophysiological recording from bead-labelled neurons in PrL slices revealed that AIE did not alter the intrinsic excitability of PrL neurons that projected to either the NAc or the BLA. Similarly, recordings of spontaneous inhibitory and excitatory post-synaptic currents revealed no AIE-induced changes in synaptic drive onto either population of projection neurons. In contrast, AIE exposure was associated with a loss of dopamine receptor 1 (D1), but no change in dopamine receptor 2 (D2), modulation of evoked firing of both populations of projection neurons. Lastly, confocal imaging of proximal and apical dendritic tufts of viral-labelled PrL neurons that projected to the nucleus accumbens (NAc) revealed AIE did not alter the density of dendritic spines. Together, these observations provide evidence that AIE exposure results in disruption of D1 receptor modulation of PrL inputs to at least two major subcortical target regions that have been implicated in AIE-induced long-term changes in behavioral control.

Adolescent high-fructose corn syrup consumption leads to dysfunction in adult affective behaviors and mesolimbic proteins in male Sprague-Dawley rats.

Adolescence is a critical period of development, during which the brain undergoes rapid maturation. Problematically, adolescents are the top consumers of high fructose corn syrup (HFCS) sweetened beverages and snacks, which may have neurodevelopmental consequences. While HFCS consumption has been linked to an increased likelihood of obesity and other physical health impairments, the link between HFCS and persistent behavioral changes is not yet fully established. The present study aimed to assess whether adolescent HFCS consumption could lead to alterations in adult behaviors and protein expression, following cessation. Adolescent HFCS-exposure contributed to deficits in learning and motivation on an effort-related T-Maze procedure, as well as increased immobility time in the forced swim paradigm during adulthood. Molecularly, protracted decreases in accumbal dopamine D1 and D2 receptors and protein kinase G (PKG), as well as increases in tyrosine hydroxylase and GluA2 receptor subunits, were observed following HFCS-exposure. Taken together, these data suggest that adolescent HFCS-consumption leads to protracted dysfunction in affective behaviors and alterations in accumbal proteins which persist following cessation of HFCS-consumption.

Chronic intermittent ethanol and lipopolysaccharide exposure differentially alter Iba1-derived microglia morphology in the prelimbic cortex and nucleus accumbens core of male Long-Evans rats.

Accumulating evidence has linked pathological changes associated with chronic alcohol exposure to neuroimmune signaling mediated by microglia. Prior characterization of the microglial structure-function relationship demonstrates that alterations in activity states occur concomitantly with reorganization of cellular architecture. Accordingly, gaining a better understanding of microglial morphological changes associated with ethanol exposure will provide valuable insight into how neuroimmune signaling may contribute to ethanol-induced reshaping of neuronal function. Here we have used Iba1-staining combined with high-resolution confocal imaging and 3D reconstruction to examine microglial structure in the prelimbic (PL) cortex and nucleus accumbens (NAc) in male Long-Evans rats. Rats were either sacrificed at peak withdrawal following 15 days of exposure to chronic intermittent ethanol (CIE) or 24 hr after two consecutive injections of the immune activator lipopolysaccharide (LPS), each separated by 24 hr. LPS exposure resulted in dramatic structural reorganization of microglia in the PL cortex, including increased soma volume, overall cellular volume, and branching complexity. In comparison, CIE exposure was associated with a subtle increase in somatic volume and differential effects on microglia processes, which were largely absent in the NAc. These data reveal that microglial activation following a neuroimmune challenge with LPS or exposure to chronic alcohol exhibits distinct morphometric profiles and brain region-dependent specificity.

General Anesthetic Exposure During Early Adolescence Persistently Alters Ethanol Responses.

BACKGROUND: Adolescent alcohol abuse can lead to behavioral dysfunction and chronic, relapsing alcohol use disorder (AUD) in adulthood. However, not all adolescents that consume alcohol will develop an AUD; therefore, it is critical to identify neural and environmental risk factors that contribute to increases in susceptibility to AUDs following adolescent alcohol (ethanol [EtOH]) exposure. We previously found that adolescent anesthetic exposure led to strikingly similar behavioral and neural effects as adolescent alcohol exposure. Therefore, we tested the hypothesis that general anesthetic exposure during early adolescence would alter EtOH responses consistent with an exacerbation of the adolescent alcohol phenotype. METHODS: To test this hypothesis, early-adolescent male Sprague-Dawley rats were exposed for a short duration to the general anesthetic isoflurane and tested on multiple EtOH-induced behaviors in mid-late adolescence or adulthood. RESULTS: Adolescent rats exposed to isoflurane exhibited decreases in sensitivity to negative properties of EtOH such as its aversive, hypnotic, and socially suppressive effects, as well as increases in voluntary EtOH intake and cognitive impairment. Select behaviors were noted to persist into adulthood following adolescent isoflurane exposure. Similar exposure in adults had no effects on EtOH sensitivity. CONCLUSIONS: This study demonstrates for the first time that early-adolescent isoflurane exposure alters EtOH sensitivity in a manner consistent with an exacerbation of adolescent-typical alcohol responding. These findings suggest that general anesthetic exposure during adolescence may be an environmental risk factor contributing to an enhanced susceptibility to developing AUDs in an already vulnerable population.

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.

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.

Inhibitors of Calcium-Activated Anion Channels Modulate Hypnotic Ethanol Responses in Adult Sprague Dawley Rats.

BACKGROUND: Ethanol is widely known for its depressant effects; however, the underlying neurobiological mechanisms are not clear. Calcium-activated anion channels (CAACs) contribute to extracellular chloride levels and thus may be involved in regulating inhibitory mechanisms within the central nervous system. Therefore, we hypothesized that CAACs influence ethanol behavioral sensitivity by altering CAAC expression. METHODS: We assessed the role of CAACs in ethanol-induced loss of righting reflex (LORR) and locomotor activity using intracerebroventricular infusions of several nonselective CAAC blockers. CAAC expression was determined after ethanol exposure. RESULTS: Ethanol-induced LORR (4.0 g/kg, intraperitoneally [i.p.]) was significantly attenuated by all 4 CAAC blockers. Blocking CAACs did not impact ethanol's low-dose (1.5 g/kg, i.p.) locomotor-impairing effects. Biochemical analysis of CAAC protein expression revealed that cortical Bestrophin1 (Best1) and Tweety1 levels were reduced as early as 30 minutes following a single ethanol injection (3.5 g/kg, intraperitoneally [i.p.]) and remained decreased 24 hours later in P2 fractions. Cortical Best1 levels were also reduced following 1.5 g/kg. However, CAAC expression was unaltered in the striatum following a single ethanol exposure. Ethanol did not affect Tweety2 levels in either brain region. CONCLUSIONS: These results suggest that CAACs are a major target of ethanol in vivo, and the regulation of these channels contributes to select behavioral actions of ethanol.

Brief prenatal ethanol exposure alters behavioral sensitivity to the kappa opioid receptor agonist (U62,066E) and antagonist (Nor-BNI) and reduces kappa opioid receptor expression.

BACKGROUND: Approximately 10 to 15% of women consume alcohol (ethanol [EtOH]) during pregnancy in the United States. Even low amounts of EtOH consumption during pregnancy can elicit long-term consequences. Prenatal experience with as few as 3 drinks has been associated with increase problem drinking in adulthood. Such effects are corroborated in rodents; however, the underlying neural adaptations contributing to this effect are not clear. In the current set of experiments, we investigated whether changes in EtOH responding following prenatal EtOH exposure involved kappa opioid receptor activation and expression. METHODS: Sprague-Dawley rats were prenatally exposed to low levels of alcohol (1.0 g/kg) during late gestation (gestational days 17 to 20 [GD17-20]) via intragastric intubation of pregnant dams. Following birth, EtOH intake, kappa- and mu-opioid-induced place conditioning, and kappa opioid receptor expression in mesolimbic brain regions were assessed in infant rats (postnatal days 14 to 15 [PD14-15]) that were offspring of dams given EtOH, vehicle, or untreated, during pregnancy. RESULTS: Animals exposed to prenatal alcohol drank more alcohol later in life and exhibited significant changes in the kappa opioid system. While control subjects found kappa opioid activation aversive, animals exposed to EtOH prenatally exhibited either no aversion or appetitive responding. Further analysis revealed that synaptosomal kappa opioid receptor expression was significantly decreased in brain areas implicated in responding to EtOH. CONCLUSIONS: Overall, these data suggest that prenatal EtOH affects kappa opioid function and expression and that these changes may be involved in increased drinking later in life.

Molecular and behavioral characterization of adolescent protein kinase C following high dose ethanol exposure.

RATIONALE: Ethanol is commonly used and abused during adolescence. Although adolescents display differential behavioral responses to ethanol, the mechanisms by which this occurs are not known. The protein kinase C (PKC) pathway has been implicated in mediating many ethanol-related effects in adults, as well as gamma-aminobutyric acid (GABA(A)) receptor regulation. OBJECTIVES: The present study was designed to characterize cortical PKC isoform and GABA(A) receptor subunit expression during adolescence relative to adults as well as assess PKC involvement in ethanol action. RESULTS: Novel PKC isoforms were elevated, while PKCγ was lower during mid-adolescence relative to adults. Whole-cell lysate and synaptosomal preparations correlated for all isoforms except PKCδ. In parallel, synaptosomal GABAA receptor subunit expression was also developmentally regulated, with GABA(A)R δ and α4 being lower while α1 and γ2 were higher or similar, respectively, in adolescents compared to adults. Following acute ethanol exposure, synaptosomal novel and atypical PKC isoform expression was decreased only in adolescents. Behaviorally, inhibiting PKC with calphostin C, significantly increased ethanol-induced loss of righting reflex (LORR) in adolescents but not adults, whereas activating PKC with phorbol dibutyrate was ineffective in adolescents but decreased LORR duration in adults. Further investigation revealed that inhibiting the cytosolic phospholipase A2/arachidonic acid (cPLA2/AA) pathway increased LORR duration in adolescents, but was ineffective in adults. CONCLUSIONS: These data indicate that PKC isoforms are variably regulated during adolescence and may contribute to adolescent ethanol-related behavior. Furthermore, age-related differences in the cPLA2/AA pathway may contribute to ethanol's age-related effects on novel and atypical PKC isoform expression and behavior.