Effects of adolescent intermittent ethanol exposure on cortical perineuronal net and parvalbumin expression in adulthood mediate behavioral inflexibility.

BACKGROUND: Alcohol is commonly consumed by adolescents in a binge-like pattern, which can lead to long-lasting cognitive deficits, including reduced behavioral flexibility. We and others have determined that adolescent intermittent ethanol (AIE) exposure leads to increased number of perineuronal net (PNN) numbers in brain regions that are important for behavioral flexibility. However, whether altered neurochemistry stemming from AIE exposure plays a significant role in reduced behavioral flexibility is unknown. METHODS: We measured the number and size of parvalbumin expressing (PV+) interneurons and associated PNNs within the orbitofrontal cortex (OFC), prelimbic cortex (PrL), infralimbic cortex (IL), and anterior insular cortex (AIC) of female and male rats following AIE or control exposure and subsequent training on an attentional set-shift task (ASST). We then ran analyses to determine whether AIE-induced changes in PV and PNN measures statistically mediated the AIE-induced behavioral deficit in reversal learning. RESULTS: We demonstrate that AIE exposure impaired behavioral flexibility on reversal two of the ASST (i.e., recalling the initial learned associations), and led to smaller PV+ cells and increased PNN numbers in the AIC. Interestingly, PNN size and number were not altered in the PrL or IL following AIE exposure, in contrast to prior reports. Mediation analyses suggest that AIE alters behavioral flexibility, at least in part through changes in PV and PNN fluorescent measures in the AIC. CONCLUSIONS: This study reveals a significant link between AIE exposure, neural alterations, and diminished behavioral flexibility in rats, and highlights a potential novel mechanism comprising changes in PV and PNN measures within the AIC. Future studies should explore the impact of PNN degradation within the AIC on behavioral flexibility.

Impact of adolescent intermittent ethanol exposure on interneurons and their surrounding perineuronal nets in adulthood.

BACKGROUND: Binge alcohol exposure during adolescence results in long-lasting alterations in the brain and behavior. For example, adolescent intermittent ethanol (AIE) exposure in rodents results in long-term loss of functional connectivity among prefrontal cortex (PFC) and striatal regions as well as a variety of neurochemical, molecular, and epigenetic alterations. Interneurons in the PFC and striatum play critical roles in behavioral flexibility and functional connectivity. For example, parvalbumin (PV) interneurons are known to contribute to neural synchrony and cholinergic interneurons contribute to strategy selection. Furthermore, extracellular perineuronal nets (PNNs) that surround some interneurons, particularly PV+ interneurons, further regulate cellular plasticity. The effect of AIE exposure on the expression of these markers within the PFC is not well understood. METHODS: The present study tested the hypothesis that AIE exposure reduces the expression of PV+ and choline acetyltransferase (ChAT)+ interneurons in the adult PFC and striatum and increases the related expression of PNNs (marked by binding of Wisteria floribunda agglutinin lectin) in adulthood. Male rats were exposed to AIE (5 g/kg/day, 2-days-on/2-days-off, i.e., P25 to P54) or water (CON), and brain tissue was harvested in adulthood (>P80). Immunohistochemistry and co-immunofluorescence were used to assess the expression of ChAT, PV, and PNNs within the adult PFC and striatum following AIE exposure. RESULTS: ChAT and PV interneuron densities in the striatum and PFC were unchanged after AIE exposure. However, PNN density in the PFC of AIE-exposed rats was greater than in CON rats. Moreover, significantly more PV neurons were surrounded by PNNs in AIE-exposed subjects than controls in both PFC subregions assessed: orbitofrontal cortex (CON = 34%; AIE = 40%) and medial PFC (CON = 10%; AIE = 14%). CONCLUSIONS: These findings indicate that, following AIE exposure, PV interneuron expression in the adult PFC and striatum is unaltered, while PNNs surrounding these neurons are increased. This increase in PNNs may restrict the plasticity of the ensheathed neurons, thereby contributing to impaired microcircuitry in frontostriatal connectivity and related behavioral impairments.

Altered Cortico-Subcortical Network After Adolescent Alcohol Exposure Mediates Behavioral Deficits in Flexible Decision-Making.

Behavioral flexibility, the ability to modify behavior according to changing conditions, is essential to optimize decision-making. Deficits in behavioral flexibility that persist into adulthood are one consequence of adolescent alcohol exposure, and another is decreased functional connectivity in brain structures involved in decision-making; however, a link between these two outcomes has not been established. We assessed effects of adolescent alcohol and sex on both Pavlovian and instrumental behaviors and resting-state functional connectivity MRI in adult animals to determine associations between behavioral flexibility and resting-state functional connectivity. Alcohol exposure impaired attentional set reversals and decreased functional connectivity among cortical and subcortical regions-of-interest that underlie flexible behavior. Moreover, mediation analyses indicated that adolescent alcohol-induced reductions in functional connectivity within a subnetwork of affected brain regions statistically mediated errors committed during reversal learning. These results provide a novel link between persistent reductions in brain functional connectivity and deficits in behavioral flexibility resulting from adolescent alcohol exposure.

Adolescent intermittent ethanol exposure does not alter responsiveness to ifenprodil or expression of vesicular GABA and glutamate transporters.

Adolescent intermittent ethanol (AIE) exposure in the rat results in a retention of adolescent-like responsiveness to ethanol into adulthood characterized by enhanced sensitivity to socially facilitating and decreased sensitivity to socially suppressing and aversive effects. Similar pattern of responsiveness to social and aversive effects of the selective glutamate NMDA NR2B receptor antagonist ifenprodil is evident in adolescent rats, suggesting that AIE would also retain this pattern of ifenprodil sensitivity into adulthood. Social (Experiment 1) and aversive (measured via conditioned taste aversion; Experiment 2) effects of ifenprodil were assessed in adult male and female rats following AIE exposure. Sensitivity to the social and aversive effects of ifenprodil was not affected by AIE exposure. Experiment 3 assessed protein expression of vesicular transporters of GABA (vGAT) and glutamate (vGlut2) within the prelimbic cortex and nucleus accumbens in adolescents versus adults and in AIE adults versus controls. vGlut2 expression was higher in adolescents relative to adults within the PrL, but lower in the NAc. AIE adults did not retain these adolescent-typical ratios. These findings suggest that AIE is not associated with the retention of adolescent-typical sensitivity to NR2B receptor antagonism, along with no AIE-induced shift in vGlut2 to vGAT ratios.

Excitatory/inhibitory balance across ontogeny contributes to age-specific behavioral outcomes of ethanol-like challenge in conditioned taste aversion.

Adolescent-typical sensitivities to ethanol (EtOH) are characterized in part by reduced sensitivity to EtOH's aversive effects. Rodent studies have shown that adolescents are less sensitive than adults to aversive properties of EtOH in a conditioned taste aversion (CTA) paradigm. To the extent that EtOH exerts antagonist-like actions upon glutamate receptors and/or agonist-like actions upon γ-aminobutyric acid (GABA) receptors, age differences in excitatory/inhibitory balance may regulate age-specific EtOH sensitivities, such as attenuated sensitivity of adolescents to EtOH aversion. In our experiments, adolescent and adult Sprague-Dawley rats were tested for CTA following challenge with one of the following pharmacological agents: glutamatergic AMPA1 receptor antagonist NBQX, glutamatergic N-methyl-d-aspartate NR2B receptor antagonist ifenprodil, and extrasynaptic GABAA receptor agonist THIP to determine whether these induced age-specific aversive sensitivities like those seen with EtOH. NBQX administration did not induce CTA. The highest dose of extrasynaptic GABAA agonist THIP induced CTA in adolescents but not adults, an opposite ontogenetic profile as seen following EtOH. Ifenprodil exerted an age-specific pattern of CTA similar to that seen with EtOH in males, with adolescents being insensitive to ifenprodil's aversive effects relative to adults. Thus, only antagonism of NR2B receptors in male rats mimicked age-specific sensitivities to the aversive effects of EtOH.

Adolescent intermittent ethanol exposure: Effects on pubertal development, novelty seeking, and social interaction in adulthood.

Alcohol use initiated early in adolescence is a major predictor for the development of alcohol use disorders. This risk may be increased when drinking is initiated around the time of puberty, given evidence of bidirectional relationships between alcohol and gonadal hormones. The current study examined the effects of adolescent intermittent ethanol exposure (AIE) on pubertal timing and expression of novelty-seeking and peer-directed behaviors as well as neural correlates of these behaviors. AIE did not affect pubertal timing or the later expression of novelty-seeking and peer-directed behaviors. AIE increased corticosterone (CORT) levels in females not tested behaviorally in adulthood or tested in the novel-object exploration paradigm, whereas social interaction blunted CORT levels in AIE females. Delays in pubertal timing and decreases in CORT levels were correlated, however, with increased novelty seeking in adult males - a phenotype associated with increased addiction vulnerability. In females, social testing elevated oxytocin receptor (OXTR) mRNA expression in the central amygdala (CeA), with this social testing-associated elevation evident in the lateral septum (LS), regardless of sex. Vasopressin receptor 1a (AVP-1aR) mRNA expression in the CeA was enhanced by social testing in females, but not males, with expression of this gene suppressed by social testing in the LS in males, but not females. Together, these data demonstrate that behavioral and neural alterations that may serve as risk factors in later drug vulnerabilities are likely not the result of a single insult, but may reflect interactions among several variables including sex, pubertal timing, stress reactivity, and test circumstances.

Oxytocin and vasopressin modulation of social anxiety following adolescent intermittent ethanol exposure.

RATIONALE: Adolescent intermittent ethanol exposure (AIE) produces lasting, sex-specific social anxiety-like alterations in male, but not female rats. Oxytocin (OXT) and vasopressin (AVP) brain systems play opposite roles in regulating social preference/avoidance, with OXT increasing approach to, and AVP increasing avoidance of social stimuli. OBJECTIVES: To test the hypothesis that social anxiety-like alterations seen in adult males after AIE are associated with a shift in the balance between OXT and AVP toward AVP, effectiveness of pharmacological activation of the OXT system and blockade of endogenous activity at AVP receptors for reversing AIE-induced social anxiety-like alterations was assessed, along with examination of the effects of AIE on OXT, vasopressin V1a, and V1b receptor (OXT-R, V1a-R, and V1b-R) surface expression in the hypothalamus. METHODS: Sprague-Dawley male and female rats were given 4 g/kg ethanol (AIE) or water intragastrically every 48 h for a total of 11 exposures during postnatal days (P) 25-45. On P70-72, animals were given a social interaction test following administration of a selective OXT-R agonist WAY-267464, selective V1a-R antagonist SR-49059, or V1b-R antagonist SSR-149415, and hypothalamic tissue was collected. RESULTS: Social anxiety-like behavior was induced by AIE in males but not females, and was selectively reversed by the selective OXT-R agonist and V1b-R antagonist, but not V1a-R antagonist. AIE was also found to decrease OXT-R, but increase V1b-R neuronal surface expression relative to water-exposed controls in the hypothalamus of males, but not females. CONCLUSIONS: These findings demonstrate that AIE induces changes in OXT-R and AVP-R surface expression in the hypothalamus along with social anxiety-like alterations in male rats. These social anxiety-like alterations can be reversed either by activation of the OXT system or by suppression of the AVP system, data that support the hypothesis that social anxiety-like alterations induced by adolescent alcohol exposure in male rats are associated at least in part with an OXT/AVP imbalance.

Effects of AMPA receptor antagonist, NBQX, and extrasynaptic GABAA agonist, THIP, on social behavior of adolescent and adult rats.

Adolescence is characterized by high significance of social interactions, along with a propensity to exhibit social facilitating effects of ethanol while being less sensitive than adults to the inhibition of social behavior that emerges at higher doses of ethanol. Among the neural characteristics of adolescence are generally enhanced levels of glutamatergic (especially NMDA receptor) activity relative to adults, whereas the GABA system is still developmentally immature. Activation of NMDA receptors likely plays a role in modulation of social behavior in adolescent animals as well as in socially facilitating and suppressing effects of ethanol. For instance, adolescent and adult rats differ in their sensitivities to the effects of NMDA antagonists and ethanol on social behavior, with adolescents but not adults demonstrating social facilitation at lower doses of both drugs and adults being more sensitive to the socially suppressing effects evident at higher doses of each. The roles of AMPA and extrasynaptic GABAA receptors in modulation of social behavior during adolescence and in adulthood are still unknown. The present study was designed to assess whether pharmacological blockade of AMPA receptors and/or activation of extrasynaptic GABAA receptors results in age-dependent alterations of social behavior. Adolescent and adult male and female Sprague-Dawley rats were injected with an assigned dose of either a selective AMPA antagonist, NBQX (Experiment 1) or extrasynaptic GABAA agonist, THIP (Experiment 2) and placed into a modified social interaction chamber for a 30-min habituation period prior to a 10-min social interaction test with a novel age- and sex-matched partner. Behaviors such as social investigation, contact behavior and play behavior were scored from video recordings of the interaction tests. In Experiment 1, NBQX produced similar social inhibition at higher doses in both age groups. In Experiment 2, THIP induced inhibition in adolescents, but not adults. No social facilitation was evident following low doses of either drug. Therefore, AMPA and extrasynaptic GABAA receptors appear to play little role if any in modulation of peer-directed social behavior in adolescence and adulthood and not likely to contribute to previously observed age differences in the social effects of acute ethanol.

Age differences in conditioned place preferences and taste aversions to nicotine.

Adolescents and adults differ in their behavioral sensitivities to drugs of abuse, including nicotine. Studies have shown that both rewarding and aversive properties of drugs of abuse can exist concomitantly. The present study investigated the ontogeny of these opposing qualities across a range of doses using a combined conditioned taste aversion and place preference paradigm in pair-housed rats that were not deprived of food or water. Results indicated that adolescents were more sensitive to the rewarding properties of nicotine than adults. In contrast, although all doses produced a taste aversion at both ages in the same rats, the aversion was weaker at lower than high doses in adolescents whereas adults showed strong aversion at all doses, suggesting modest attenuation in nicotine's aversive properties among adolescents relative to adults. Thus, attenuated aversive and accented appetitive sensitivities of adolescents to nicotine can be experienced simultaneously in the same animals. © 2016 Wiley Periodicals, Inc. Dev Psychobiol 58: 660-666, 2016.