Effects of chronic intermittent ethanol exposure during early and late adolescence on anxiety-like behaviors and behavioral flexibility in adulthood

Journal Pre-proof Effects of chronic intermittent ethanol exposure during early and late adolescence on anxiety-like behaviors and behavioral flexibility in adulthood ´ Trevor Towner, David F. Elena I. Varlinskaya, Dominika Hosova, Werner, Linda P. Spear

PII:

S0166-4328(19)30472-3

DOI:

https://doi.org/10.1016/j.bbr.2019.112292

Reference:

BBR 112292

To appear in:

Behavioural Brain Research

Received Date:

26 March 2019

Revised Date:

19 August 2019

Accepted Date:

7 October 2019

Please cite this article as: Varlinskaya EI, Hosova´ D, Towner T, Werner DF, Spear LP, Effects of chronic intermittent ethanol exposure during early and late adolescence on anxiety-like behaviors and behavioral flexibility in adulthood, Behavioural Brain Research (2019), doi: https://doi.org/10.1016/j.bbr.2019.112292

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Effects of chronic intermittent ethanol exposure during early and late adolescence on anxiety-like behaviors and behavioral flexibility in adulthood. Elena I. Varlinskaya, Dominika Hosová, Trevor Towner, David F. Werner, Linda P. Spear Neurobiology of Adolescent Drinking in Adulthood Consortium (NADIA) Center for Development and Behavioral Neuroscience Department of Psychology Binghamton University

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Binghamton, NY 13902-6000

Corresponding Author: David Werner

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Email: [email protected] Phone: 607-777-5782

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Fax: 607-777-4890

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The authors have no conflict of interest to disclose.

The work presented in this manuscript was funded by grants U01 AA019972 (Neurobiology of Adolescent Drinking in Adulthood Consortium - NADIA Project) and T32 AA025606 (

Abstract

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Development and Neuroadaptation in Alcohol and Addictions – DNAA Project)

Although both humans and laboratory rodents demonstrate cognitive and affective alterations

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associated with adolescent alcohol exposure, it is still unknown whether the consequences of early initiation of alcohol use differ from those of later binge drinking within the adolescent developmental period. The present study was designed to assess the effects of early and late AIE on (1) anxiety-like behavior under social (modified social interaction test) and non-social test circumstances (modified light/dark box test, elevated plus maze), and (2) behavioral flexibility, indexed via set shifting in males and females. Early-mid adolescent intermittent exposure (early

AIE) occurred between postnatal days (P) 25 and 45, whereas late adolescent intermittent exposure (late AIE) was conducted between P45 and P65, with behavioral testing initiated not earlier than 25 days after repeated exposure to ethanol (4.0 g/kg intragastrically, every other day for a total of 11 exposures). Anxiety-like behavior on the EPM was evident in males and females following early AIE, whereas only males demonstrated non-social anxiety on the EPM following late AIE. Social anxiety-like alterations and deficits in behavioral flexibility were

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evident only in males following early AIE. Taken together, the results of the present study

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demonstrate a particular vulnerability of young adolescent males to long-lasting detrimental effects of repeated ethanol and an insensitivity of older adolescent females to the intermittent

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ethanol exposure paradigm.

1. Introduction

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Exposure timing; Sex differences

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Keywords: Adolescent ethanol exposure; Anxiety; Behavioral flexibility; Social interaction;

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Alcohol use typically begins during early adolescence [1], with this use potentially contributing to the risk of later alcohol abuse/dependence. Early initiation of use is especially concerning given that adolescents who begin drinking at 14 years of age or earlier are four times more likely to become alcohol-dependent relative to those who started drinking at 20 years of

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age or later [2-4]. According to recent data from the Monitoring the Future survey, annual prevalence rates for alcohol use are 21%, 42% and 58% for 8th, 10th and 12th graders, respectively [5]. Over 20% of high school seniors report consumption of 5 or more drinks per occasion (i.e., binge levels of drinking) within the past 2 weeks, with 10% endorsing consumption of 10 or more drinks, and over 5% reporting consumption of 15 or more drinks over the same period [6]. Binge patterns of alcohol consumption (five or more drinks in males, or

four or more drinks in females within a 2-hour period that result in blood alcohol levels of 80 mg/dL and higher) are thought to be particularly harmful to the developing adolescent brain [7, 8]. Binge patterns of drinking among early adolescents (ages from 10 to 14) are even more alarming, since estimated peak blood alcohol concentrations (BECs) following 5 drinks are around 280 mg/dL for 10-year-olds and about 160 mg/dL for 14-year-old adolescents [see: 9]. Adverse consequences of adolescent alcohol use in humans are not limited to an increased

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vulnerability for alcohol use disorders. Although few studies have investigated the impact of

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adolescent alcohol exposure on other adult psychiatric disorders, the research available reports relatively strong associations between alcohol use during adolescence and adult anxiety and

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depression disorders [10, 11]. Alcohol-related problems are especially common in adolescents

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with social anxiety [12, 13], although it is still not clear whether alcohol use during adolescence can enhance or even elicit social anxiety. Alcohol exposure during adolescence in humans has

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also been associated with poor performance on a variety of cognitive measures, ranging from deficits in attention, memory and visuospatial function to impaired executive functions [14-17].

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Much of this work has been cross-sectional and hence cannot be used to draw strong conclusions about causality.

In contrast, substantial animal research has emerged to assess long-lasting, causal, behavioral, cognitive, affective, neural and molecular consequences of adolescent ethanol

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exposure [see: 18 for review]. In such studies, ethanol has been intermittently administered to adolescent rodents through a variety of routes, including intragastric gavage (for advantages and limitations see [19]) for varying lengths of time during the broad adolescent developmental period ranging from postnatal day (P) 25 up to P65 (see [18, 20, 21] for references and review). In rats, a conservative age range during which both males and females exhibit adolescent-typical

neurobehavioral characteristics initially was characterized as P28–42 [22, 23]. More recently, based on physiological and hormonal changes, the age span has been expanded to consider the period between P25 and P45 as the pre- and peri-pubertal period of early-mid adolescence and the interval between P45 and P65 as late adolescence/emerging adulthood [21, 24]. These age spans correspond to approximately 10–18 and 18–25 years of age in humans, respectively [21]. Research conducted by the Neurobiology of Adolescent Drinking in Adulthood

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Consortium (NADIA) [reviewed in: 18, 20, 21] has revealed highly specific cognitive

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impairments as well as anxiety-like behavioral alterations in adulthood following adolescent ethanol exposure in rodents. For instance, although adolescent intermittent ethanol exposure

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(AIE) had little impact on simple spatial learning tasks [25] and more challenging five-choice

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serial reaction time tests [26], AIE-associated deficits have been reported on tasks requiring behavioral flexibility, including impaired set shifting [27, 28], decreased reversal acquisition [29,

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30], and delayed context fear extinction [31]. Enhancements of anxiety-like behavior following AIE have also been reported for non-social (elevated plus maze, light/dark box) as well as social

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tests of anxiety [32-34].

Although both humans and laboratory rodents demonstrate cognitive and affective alterations associated with adolescent alcohol exposure, it is still unknown whether the consequences of alcohol use during early adolescence differ from those of later drinking within

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the adolescent developmental period. Indeed, early drinking onset individuals are often continuing drinking later in adolescence [35], making it almost impossible to separate adverse consequences of early exposure from those associated with heavy drinking later in adolescence. In rats, cognitive and anxiety-like alterations associated with AIE have emerged following

repeated intermittent exposure to ethanol initiated during early adolescence and continuing through the entire adolescent period [27, 28, 32, 36, 37]. Our recent work has shown, however, that social anxiety-like alterations following AIE are sex- and exposure timing-dependent [34]. Specifically, when effects of AIE during early-mid adolescence (P25–45) versus late adolescence/emerging adulthood (P45–65) were assessed, adult male rats exposed to ethanol during early-mid adolescence (early AIE) but not late adolescence

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(late AIE) were found to exhibit long-lasting social anxiety-like alterations (indexed via

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decreases in social investigation and social preference); these effects were not evident in early exposed females [33, 34]. Given these findings, it appears that the cognitive and affective

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with ethanol exposure initiated later in adolescence.

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consequences of ethanol exposure during early adolescence may differ from those associated

The proposed differences in the adverse consequences of early and late AIE may stem

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from differential maturation of brain regions critical for emotional responding and those implicated in cognitive, top-down control. Prefrontal cortical regions implicated in behavioral

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flexibility show delayed maturation [38, 39] relative to more rapidly maturing limbic regions that are critically involved in processing of emotional and rewarding stimuli [40]. Therefore, affective alterations may be associated with early AIE as a result of disrupted limbic maturation, whereas cognitive alterations may be more pronounced following late AIE as a result of

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disrupted maturation of prefrontal cortical regions. The present study was designed to test this hypothesis by assessing the effects of early and late AIE on (1) anxiety-like behavior under social and non-social test circumstances, and (2) behavioral flexibility, indexed via set shifting in males and females.

2. Material and methods

2.1. Subjects Male and female Sprague Dawley rats bred and reared at Binghamton University were used in all experiments. Animals were housed in a temperature controlled (22°C) vivarium on a 12/12-hour light/dark cycle (lights on at 0700) with ad libitum access to food and water prior to the start of testing in adulthood. Litters were culled to 8-10 pups, maintaining a sex ratio of six

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males and four females whenever possible. At P21 animals were weaned and pair-housed either

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with a same-sex littermate (for assessments of anxiety-like behavior) or a same-sex nonlittermate (for assessment of behavioral flexibility). Animal use and maintenance was in

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accordance with the guidelines for animal care established by the National Institutes of Health,

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and all protocols used were approved by the Binghamton University Institutional Animal Care

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and Use Committee.

2.2 Intermittent Ethanol Exposure

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Males and females were exposed to ethanol intragastrically (i.g.) at a dose of 4 g/kg (25% ethanol solution in tap water, v/v) every other day between 1100 and 1500 hours (11 exposures). Control animals were given an isovolumetric amount of tap water i.g. on each exposure day. Early-mid adolescent animals were exposed to water or ethanol from P25 to P45 (early AIE),

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whereas late adolescent animals were given water or ethanol from P45 to P65 (late AIE). Following the final exposure, all animals were left undisturbed for 20-25 days, depending on the specific experiment (see below), thereby allowing animals to mature into adulthood prior to behavioral testing. Intragastric ethanol exposure at this level produces peak BECs well into the binge range at the start (~ 200 mg/dl) and end (~ 130 mg/dl) of the chronic exposure period [41].

These BECs are also in line with estimates for early adolescents following 5 standard drinks [9] and are well within the range of BECs obtained from adolescents drinking in a field setting where levels up to 300 mg/dl have been observed [e.g., 42]. Cage-mates were assigned to the same exposure condition, with only one male and one female subject from a given litter assigned to each exposure/timing/testing condition to minimize litter effects [43, 44].

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2.3. Anxiety-like Behavior

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2.3.1. Experimental Design

Effects of AIE on anxiety-like behavior were assessed using three different paradigms:

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light/dark box, modified social interaction test, and elevated plus maze. The design was a 2

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(Exposure: water, ethanol) X 2 (Timing: early adolescence, late adolescence) X 2 (Sex: male, female) factorial (n = 7-9 per condition), with data analyzed separately for males and females

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given known sex differences following early AIE [e.g., 33, 34]. In this study, animals were pairhoused with same-sex littermates, since one animal from each littermate pair was tested in the

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light/dark box while the other animal was assessed in the modified social interaction test. These housing conditions allowed for utilization of all animals in a given litter, with all experimental conditions for a certain exposure timing presented within a litter. Testing occurred on P70 for the early-exposed animals and P90 for animals in the late adolescent exposure group. For testing,

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subjects were first habituated to the light/dark box testing room in their home cage for one hour. One animal was then tested in the light/dark box while the other was immediately placed into the social interaction chamber in an adjacent room, to habituate there. Following testing of both animals, they were returned to the colony room. Two days later, all subjects were tested using the elevated plus maze (P72 or P92).

2.3.2. Modified Light/Dark Box (LDB) Test The apparatus was composed of a larger, brightly lit (30 lux) chamber (40.64 cm wide x 49.5 cm long x 29.85 cm high) with a clear Plexiglas lid, connected to a smaller, dark (0-2 lux) chamber (40.64 cm wide x 40.64 cm long x 29.85 cm high) with an opaque lid; both chambers had Plexiglas flooring which was left bare for testing. Animals were free to cross between the two sides of the apparatus through a circular aperture (7.6 cm diameter). Testing occurred on P70

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or P90 between 1400-1700, lasted five minutes, was conducted in the presence of a white noise

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generator, and recorded by video camera. Measures scored from the recordings included latency to enter the other apparatus side (all four paws), total number of crosses between sides, and time

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spent on each side.

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Unlike many studies using the LDB, each test session was initiated by placing the animal into the dark compartment, rather than the light one. We found recently (unpublished data) that

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initially placing animals onto the light side of the apparatus resulted in a substantial portion of animals freezing and remaining there for the duration of the session with clear signs of

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discomfort (e.g., large amounts of defecation) as has been noted elsewhere [45, 46]. Between animals, the LDB was cleaned with 6% hydrogen peroxide and wiped dry. 2.3.3. Modified Social Interaction (SI) Test The modified social interaction (SI) test was conducted on P70 or P90 between 1400-

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1700 hours using standard procedures in our laboratory [34]. Testing was conducted under dim light (15-20 lux) with white noise, in a Plexiglas chamber (46 x 30.3 x 30.5 cm) containing pine shavings and divided into two equally-sized compartments with an aperture in the partition to allow free movements of the animals between compartments. At the onset of this procedure, each experimental animal was placed alone inside the chamber for a 30-minute period of habituation

to the novel environment. Then, an unfamiliar social partner of the same sex and age was placed on the opposite side of the chamber from the experimental animal for a ten-minute social interaction test. The partner was non-manipulated, drug-naive, non-socially deprived prior to the test and weighed approximately 10-20 g less than the experimental animal. In order to differentiate experimental animals from their social partners during the test, each experimental animal was marked with a line on its back with an indelible marker.

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Test sessions were recorded by a video camera and scored at a later date by a trained

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experimenter without knowledge of the experimental condition of any given animal. Two anxiety-sensitive behavioral measures, namely social investigation and the coefficient of social

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preference/avoidance [47-49], were scored and analyzed. Social investigation frequency was

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defined as the sniffing of any part of the body of the partner whereas social preference/avoidance was indexed via movements of the experimental animal through the aperture toward or away

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from the non-manipulated partner using a social preference/avoidance coefficient calculated as: (crossovers to the partner – crossovers away from the partner) / (total number of crossovers both

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to and away from the partner) x 100 [50]. The total number of crossovers (movements between compartments) demonstrated by each experimental subject was used as an index of general locomotor activity under social test circumstances. The apparatus was cleaned with 6% hydrogen peroxide and bedding replaced between animals.

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2.3.4. Elevated Plus Maze (EPM)

EPM testing occurred on P72 or P92 between 1100 – 1300 hours. The maze consisted of

two open arms and two closed arms, with each arm being 17.5 cm wide and 48.3 cm in length. The open arms had small plastic edges (1.3 cm high) located along each side and end of the open arms to prevent the animals from slipping off the edge. The closed arms were surrounded by

walls 29.2 cm tall, with the entire maze elevated 50.0 cm above the floor. Prior to testing, animals were brought from the vivarium in their home cages into a room adjacent to the testing room, with dim overhead lighting (15-20 lux) and white noise. At that time, cage-mates were separated by a mesh divider in the home cage where they remained for one hour to decrease initial anxiety levels [47]. EPM testing was conducted in an adjacent testing room, with lighting of 15-20 lux in the open arms, 5-10 lux in the closed arms, and a white noise generator to help

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mask extraneous noise. Each animal was allowed to explore the maze for 5 minutes while being

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video recorded. Cage-mates were tested sequentially, with the apparatus cleaned with 6% hydrogen peroxide between animals. Measures scored from the video recordings include time

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spent in the open arms, open arm entries, and closed arm entries. Percent open arm time and

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percent open arm entries were analyzed as the most common measures of anxiety-like behavior on the EPM, with closed arm entries used to index general locomotor activity [51]. Since prior SI

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testing or testing in the LDB had no effects on the EPM measures, means from each pair of littermates tested on the EPM were calculated and used for statistical analyses to avoid including

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more than one animal of each sex from a given litter in a given test condition.

2.4. Behavioral Flexibility (BF) 2.4.1. Experimental Design

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The consequences of AIE and timing of exposure on behavioral flexibility were assessed

using a 2 (Exposure: water, ethanol) X 2 (Timing: early adolescence, late adolescence) X 2 (Sex: male, female) factorial experimental design (n=12 per each condition), with data analyzed separately for each sex, given our prior findings of sex differences in the consequences of AIE. In contrast to measures of anxiety, animals that underwent adolescent exposure and behavioral

flexibility testing in adulthood were pair-housed with non-littermates at the time of weaning (P21). Whereas multiple tests of anxiety were used, only a single measure of behavioral flexibility (set-shifting) was administered and rehousing with non-littermates allowed for both animals in a cage to undergo the same adolescent exposure and adulthood operant procedure.

2.4.2. Procedure

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Behavioral flexibility was assessed using an operant set-shifting task as described

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elsewhere [52]. Five days prior to the initiation of the operant procedure (P65 or P85), animals were gradually food restricted to 85% of free-feeding weights. Animals were exposed to the

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reinforcer (banana-flavored sucrose pellets, Bio-Serv) used in the operant procedure for three

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days prior to the initiation of operant training by placing roughly 20 pellets per animal in their home cages. The operant procedure was initiated on P70 or P90. In brief, the set-shifting task

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requires animals to first associate a light cue with a rewarded lever (set) and, upon meeting criterion, switch to a location-based rule. A criterion of 10 consecutive correct responses was

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used for both the cue and location rules [52, 53]. The number of trials required for acquiring the cue rule (cue acquisition) was used as an indication of baseline differences in cognitive ability. The number of trials required to acquire the location rule (set shift) was used to index behavioral flexibility. Errors made during the shift were broken into three categories: perseverative

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(responding on the cue rule), regressive (responding on the cue rule following a number of correct responses on set shift), and never reinforced (responding on lever that was not associated with the cue or location rules) [52]. Omissions (defined as trials for which no response on either lever was recorded) were excluded from consideration. Animals that failed to meet criterion at any point during the set-shifting task (training, acquisition, or shift) and those that scored two

standard deviations beyond the mean on trials to completion during cue acquisition were characterized as outliers and removed from the study (resulting in the elimination of 1 early water-exposed male, 1 early ethanol-exposed male, 2 late ethanol-exposed males, 2 late waterexposed females, 1 early ethanol-exposed female, and 1 late ethanol-exposed female).

2.5. Statistical analyses

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All anxiety and behavioral flexibility measures were initially assessed using separate 2

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(Exposure: ethanol, water) X 2 (Timing: early adolescence, late adolescence) analyses of variance (ANOVAs) within each sex. Males and females were analyzed separately due to

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previous research showing AIE-induces social anxiety-like alterations in males, but not females

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(Varlinskaya et al. 2014, 2017). Results of these analyses are presented in Table 1. When main effects of exposure or timing were evident or approached significance, Student’s one-tailed t-

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tests or Mann-Whitney U tests (in the cases of a non-normal distribution) were subsequently used to assess whether these main effects were driven by one age only. This approach allowed us

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to assess the alterations resulting from the specific timing of adolescent exposure rather than exposure per se and to decrease concerns regarding false negatives. Significant results in all figures are direct comparisons between ethanol- and water-exposed groups within each

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timing/sex condition made with Student’s one-tailed t-tests or Mann-Whitney U tests.

3. Results

3.2 Light/Dark Box (LDB)

When littermates of animals assessed socially were tested in the LDB, an overwhelming majority (86.3%) of subjects remained in the dark compartment for the entire test session (see Table 2). Due to floor effects, these data were not analyzed further.

3.2. Social Interaction (SI) Test When separate 2-way ANOVAs for each behavioral measure -- social investigation,

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social preference, and total number of crossovers – were conducted within each sex, a significant

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effect of exposure emerged in males, but not females for social investigation, with ethanolexposed males, in general, demonstrating less social investigation than water-exposed males

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(172.7 ± 11.2 for water-exposed males; 139.7 ± 8.6 for ethanol-exposed males). Although no

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significant main effect or interactions were evident for the social preference/avoidance coefficient in males (see Table 1), the main effect of exposure approached significance (p =

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0.07), reflecting a tendency for ethanol-exposed males to show lower social preference than water-exposed males (34.2 ± 5.1 for water-exposed males; 19.3 ± 6.1 for ethanol-exposed Further analyses that were restricted to comparisons between water- and ethanol-

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males).

exposed animals separately for each timing period of adolescent exposure clearly demonstrated that exposure effects were driven by the early adolescent exposure in males only, with early AIE significantly reducing the frequency of social investigation (t15 = 2.343, p = 0.033) as well as the

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preference/avoidance coefficient (t 15 = 2.685, p = 0.017), with no significant differences between water- and ethanol-exposed males evident for these measures (social investigation: t 13 = 0.618, p = 0.547; social preference: t13 = 0.888, p = 0.391) following late AIE (see Figure 1a,b,c,). In females, social investigation, social preference, and total number of crossovers did not differ as a function of exposure or exposure timing (see Table 1, Figure 1a,b,c).

3.3. Elevated Plus Maze (EPM) Two-way ANOVAs for percent open arm time and percent open arm entries revealed significant main effects of exposure for both measures in males (see Table 1), with ethanolexposed males demonstrating lower percent of open arm time (5.1 ± 1.2 % for water-exposed males; 0.5 ± 0.2% for ethanol-exposed males) and open arm entries (1.5% for water-exposed; 2.6 ± 0.7% for ethanol-exposed males) than their water-exposed counterparts. Further analyses

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assessing the effects of AIE within each adolescent timing period in males revealed that late AIE

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significantly decreased percent open arm entries (t 15 = 2.70, p = 0.016; see Figure 1d) and percent open arm time on the EPM (U = 16.00, p = 0.028; Figure 1e). Males exposed to ethanol

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during early adolescence also demonstrated a significant decrease in percent open arm entries (U

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= 19.00, p = 0.05, Figure 1d, e), with a decrease in percent arm time being non-significant (U = 20.00, p = 0.06). Although no main effects for the EPM anxiety measures were evident in

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females, (see Table 1), Student t- test showed that early AIE significantly reduced percent open arm time (t 16 = 2.284, p = 0.018, see Figure 1d) and percent open arm entries (t 16 = 2.199, p =

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0.022, see Fig 1e), whereas late AIE did not affect either of these anxiety-related measures (percent open arm time: t 16 = 0.498, p = 0.312; percent open arm entries: t 16 = 0.123, p = 0.452). Closed arm entries were not affected by early- and late-AIE in either males or females (Table 1, Figure 1f). Overall, these data suggest that unlike social anxiety, early AIE enhances non-social

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anxiety in both sexes, with males continuing to be vulnerable to AIE-associated non-social anxiety into late adolescence. 3.4. Behavioral Flexibility (BF) Two-way ANOVAs of the acquisition data (i.e, number of trials required for acquiring the cue rule) did not reveal significant main effects or interactions in either males or females (see

Table 1, Figure 2a). Similarly, no main effects or interactions were evident in either sex when the number of trials required to acquire the location rule (set shift) as well as the number of total errors made during set shift were analyzed (Table 1, Figure 2b). When the three error types during the set shift were analyzed, a significant main effect of exposure emerged for regressive errors in males (6.83 ± 0.92 for water-exposed, 11.00 ± 1.31 for ethanol-exposed males). In females, significant main effects of timing were evident for both

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regressive (7.30 ± 1.12 for early exposure, 11.71 ± 1.67 for late exposure) and never reinforced

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(2.17 ± 0.43 for early exposure, 3.91 ± 0.41 for late exposure) errors (see Table 1), with older females, in general, demonstrating more errors than their less mature counterparts. Further

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comparisons within each timing/sex condition demonstrated that the number of regressive errors

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was significantly increased in males following early AIE (t 20 = 2.235, p = 0.019, see Figure 2e), whereas late AIE in males (t20 =1.430, p = 0.084) as well as early (t21 =1.230, p = 0.116) or late

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4. Discussion

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(t19 = 0.104, p = 0.459) AIE in females had no effects on these measures.

The results of the present study demonstrate that long-lasting detrimental effects of repeated ethanol exposure were sex- and timing of exposure-dependent. In males, but not females, early adolescent ethanol exposure resulted in the emergence of social anxiety-like

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alterations and some deficits in behavioral flexibility. In contrast, males and females exposed to ethanol during early-mid adolescence, as well as males exposed to ethanol during late adolescence, demonstrated anxiety-like behavior on the EPM. These findings suggest that, unlike social alterations, early AIE induces non-social anxiety regardless of sex, with males also showing non-social anxiety when AIE exposure was delayed until late adolescence-emerging

adulthood. Similar anxiety-like alterations on the EPM have been reported in Sprague Dawley males following intraperitoneal administration of ethanol (2 g/kg) during early-mid adolescence [32, 36, 37]. In contrast, no behavioral changes on the EPM were reported in male Wistar rats following intragastric ethanol exposure (3 g/kg, 6 exposures between P37 and P44; Torcaso et al., 2017), while other studies have shown decreased anxiety-like behavior on the EPM following early AIE [27, 54]. For instance, intermittent exposure to ethanol by vapor inhalation of Long-

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Evans adolescent male rats (P28-P42) was found to increase percent open arm time and open

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arm entries, suggesting an early AIE-associated reduction in non-social anxiety [27]. Similarly, ethanol self-administration early in adolescence (P28-P42) was reported to decrease anxiety-like

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behavior on the EPM in male Wistar rats tested in adulthood [54]. These discrepancies may be

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associated with procedural differences between the studies that include, but are not limited to, different strains, different routes of ethanol administration, breeding in-house versus shipping of

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young animals, different levels of ethanol exposure, pre-test manipulations, testing during light versus dark part of the light/dark cycle, and handling duration [55, 56]. In the two studies

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reporting significant decreases in anxiety-like behavior on the EPM, animals were tested during the dark part of the light/dark cycle under extremely low light and hence extremely low anxietyprovoking test conditions [27, 54]. It seems likely that the observed increases in open arm exploration under these non-anxiety-provoking test circumstances may reflect AIE-associated

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disinhibition rather than decreased anxiety-like behavior, since the behavioral expression of anxiety and disinhibition can compete depending on the characteristics of the test situation [57]. The social consequences of AIE differed as a function of exposure timing and sex.

Similar to our earlier findings [33, 34, 58], late AIE did not affect social behavior, whereas socially anxiogenic effects of early AIE were evident only in males. The confirmed differences

in the social consequences of early versus late AIE suggest that neural systems affected by repeated ethanol exposure differ in early-mid and late adolescent males. Social anxiety-like alterations may be associated with ethanol-induced adaptations in neural systems implicated in social behavior and anxiety. For instance, recent studies have shown involvement of frontal cortical regions, the amygdala, and the hippocampus in peer-directed social interactions in juvenile and adolescent animals [59-62], with these regions also implicated in the modulation of

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non-social anxiety-like behavior [63-66]. Substantial remodeling of these brain areas during

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adolescence [22, 67-70] suggests high vulnerability of these regions to disruption by repeated ethanol exposure at the time of early AIE [18].

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Brain oxytocin (OXT) and vasopressin (AVP) systems are among the neural systems that

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are involved in social alterations evident in adult males following early AIE. These neuropeptide systems play a substantial role in the regulation of social behaviors, are sexually dimorphic, and

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are differentially involved in the modulation of anxiety-like behavior, especially anxiety evident under social circumstances [71, 72].

Whereas the brain OXT system is critical for social

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approach and social preference [73], activation of the AVP system typically exerts anxiogenic effects [see: 72 for references and review]. Both V1a [74, 75] and V1b [76] types of AVP receptors are involved in the modulation of anxiety-related behaviors, with V1a receptors playing a more substantial role in the modulation of non-social anxiety [74] and V1b receptors

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contributing to social anxiety [76]. Given the sex differences in OXT and AVP brain systems and the separable roles of these peptides in modulating anxiety-like alterations in social contexts, our recent study tested the hypothesis [see: 72] that social anxiety evident in adult males following early AIE is associated with alterations in OXT and AVP systems [58]. This hypothesis was supported by our confirmed, with the data showing early AIE-induced changes in OXT and AVP

receptor surface expression in the hypothalamus only in males, characterized by significant decreases in hypothalamic neuronal surface exposure of OXT receptors along with increases in V1b receptor expression [58]. Importantly, these social anxiety-like alterations seen in adult males following early AIE were separately reversed either by pharmacological stimulation of OXT or by blockade of V1b receptors; V1a receptor blockade was ineffective [58]. These findings support an involvement of OXT and V1b receptors (but not V1a) receptors) in social

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sex-specific consequences of early AIE. In contrast, given evidence for a role of V1a receptors in

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the modulation of non-social anxiety [74], it is possible that these receptors might play a role in the non-social anxiety evident in the EPM in both males and females following early AIE and in

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males after late AIE. This possibility has yet to be tested.

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Relatively mild alterations in behavioral flexibility were evident in males exposed to ethanol during early adolescence, with no effects observed in females. That is, when using a set-

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shifting operant task as an index of behavioral flexibility, the early AIE males demonstrated a significant increase in the number of regressive during the set-shifting task (i.e., responding on

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the original cue rule following a number of correct responses on the set shift) than their waterexposed counterparts, suggesting certain difficulties in switching from the cue to the location rule. These difficulties suggest alterations in functioning of prefrontal cortical regions associated with early AIE, given that inactivation of the prelimbic region of the medial prefrontal cortex

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(mPFC) impairs the shift to the new rule [77]. In contrast to males, females showed no deficits in behavioral flexibility following early AIE. The observed vulnerability of males, but not females, during early-mid adolescence to ethanol-associated alterations in behavioral flexibility may be related to later maturation of the mPFC in males than in females [see: 78 for references and review]. For instance, in rats, dendritic remodeling of the mPFC during adolescence occurs later

in males than in females, with no sex differences evident in the basolateral amygdala [69]. Since the brain is especially vulnerable to drugs during its development, with periods of pronounced developmental changes in brain regions sensitive to a given drug defining critical periods for induction of lasting drug-induced changes in those and associated regions [79], it is not surprising that males are more vulnerable than females to ethanol-associated cognitive alterations when exposure to ethanol occurs during early adolescence.

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The results of the present study suggest that early adolescence, particularly in males, is a

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period of enhanced vulnerability to ethanol-associated long-term affective alterations. To a certain extent, these experimental findings are in agreement with human data. For instance,

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recent human research suggests that adolescents, especially males, who reported a shorter

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interval between their first experience with alcohol and the onset of binging as well as firstoccasion binge users are more likely to have alcohol-related problems [80, 81]. Furthermore,

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BECs achieved with intragastric administration of the relatively high dose of ethanol in the rat model of adolescence are in line with estimates for early adolescents following 5 standard drinks

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[9] and are also well in the range of those obtained from breathalyzer data in a local field studies of responsive alcohol drinkers in a college bar setting [42]. In humans, affective disorders, especially social anxiety, are comorbid with alcohol use disorders [82, 83], and our experimental findings are in agreement with the human research. It is still not clear, however, whether alcohol

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use during adolescence can enhance social anxiety or whether pre-existing social anxiety put adolescents at risk for of alcohol use disorder [84], with alcohol perhaps being more appealing for these socially anxious adolescents due to its anxiolytic properties. The results of the present study as well as our previous data [33, 34, 58] support the suggestion that initiation and rapid progression to binge drinking during early adolescence may be a risk factor for the emergence of

anxiety, especially under social circumstances. The extent to which these findings are applicable to human adolescent binge drinking and its consequences later in life is limited by the forced method of ethanol exposure. Forced ethanol exposure, giving the precise control of exposure level and timing, can produce neural adaptations different from those associated with voluntary ethanol consumptions. Nevertheless, the results of the present study demonstrate that affective and cognitive alterations associated with AIE are

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sex- and exposure-timing-dependent. In general, males were more sensitive to ethanol-associated

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alterations than females, with early exposure being more harmful than late exposure. Young adolescent males were particularly vulnerable to long-lasting detrimental effects of chronic

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ethanol exposure, whereas older adolescent females were insensitive to intermittent ethanol

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exposure. However, exact mechanisms underlying timing- and sex-specific effects of AIE are not well understood. Therefore, more studies are needed to focus on the mechanisms of enhanced

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vulnerability of young adolescent males and resilience of older adolescent females to detrimental

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long-lasting effects of repeated ethanol exposure.

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Figure 1. The impact of early and late adolescent intermittent ethanol exposure on (a) social investigation, (b) social preference, (c) locomotor activity during a 10-min social interaction test

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and on (d) percent open arm time, (e) percent open arm entries, and (f) number of closed arm entries during a 5-min elevated plus maze test in adult male (left panels) and female (right

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panels) rats. A significant difference (p≤ 0.05) assessed with Student’s one-tailed t-tests or Mann-Whitney U tests between ethanol- and water-exposed animals within each timing/sex

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condition is indicated with (*).

Elevated Plus Maze

Modified Social Interaction Test

d. Open Arm Time

a. Social Investigation

female

Percent

Percent

*

female

male

Frequency / 10 min

Frequency / 10 min

male

*

* Adolescent Exposure

Adolescent Exposure

b. Social Preference

male

Adolescent Exposure

Percent

Percent

*

female

* Adolescent Exposure

* f. Closed Arm Entries

female

male

Adolescent Exposure

Adolescent Exposure

-p

Adolescent Exposure

Number

Number

Crossovers

Crossovers

female

Adolescent Exposure

Adolescent Exposure

Adolescent Exposure

c. Locomotor Activity

male

*

of

female Coefficient (%)

Coefficient (%)

male

Adolescent Exposure

e. Open Arm Entries

ro

Adolescent Exposure

Figure 1.

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Figure 2. The impact of early and late adolescent intermittent ethanol exposure on (a) number of trials during acquisition, (b) number of trials during set-shifting, (c) total shift errors, (d)

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perseverative, (e) regressive, and (f) never reinforced errors during set-shifting in adult male (left panels) and female (right panels) rats. A significant difference (p≤ 0.05) assessed with Student’s

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one-tailed t-tests or Mann-Whitney U tests between ethanol- and water-exposed animals within

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each timing/sex condition is indicated with (*).

Behavioral Flexibility d. Perseverative Errors

a. Acquisition female

e. Regressive Errors

male

f. Never Reinforced Errors

female

male

ro

Adolescent Exposure

Adolescent Exposure

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Figure 2.

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Adolescent Exposure

Number

Number

Number

Number

female

Adolescent Exposure

Adolescent Exposure

Adolescent Exposure

c. Total Shift Errors

of

Adolescent Exposure

female

*

Number

Number of Trials Adolescent Exposure

male

Adolescent Exposure

Adolescent Exposure

female

Number of Trials

male

Number

Number Adolescent Exposure

b. Set-shift

Number

Adolescent Exposure

female

male

Number of Trials

Number of Trials

male

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Table 1. ANOVA Results for Social Interaction, Elevated Plus Maze, and Behavioral Flexibility Measures.

Test/Behavioral Factors Measure Exposure SI test:

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Social Investigation

Chamber Crosses

Timing Exposure X Timing Exposure Timing Exposure X Timing Exposure Timing

EPM:

Exposure X Timing Exposure

SI test:

Preference/ Avoidance

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SI test:

% Open Arm Time

EPM:

Timing Exposure X Timing Exposure

% Open Arm Timing

Male

F(1,28) =5.06 =0.03* F(1,28) = 2.08 F(1,28) = 1.74 F(1,29) = 3.63 F(1,29) = 1.38 F(1,29) = 0.61 F(1,29) = 0.32 F(1,29) = 4.15 0.051 F(1,29) = 0.07 F(1,30) = 7.08 <0.01* F(1,30) = 0.15 F(1,30) = 0.03 F(1,30) = 10.36 0.01* F(1,30) = 0.13

Female F(1,29) = 0.01

p = 0.93

F(1,29) = 0.03 F(1,29) = 1.56 F(1,30) = 0.38 F(1,30) = 0.07 F(1,30) = 0.27 F(1,29) = 0.07 F(1,29) = 0.05

p = 0.87 p = 0.22 p = 0.54 p = 0.79 p = 0.61 p = 0.80 p = 0.83

p = 0.80 F(1,29) = 0.00 F(1,32) = 3.05 p

p = 0.99 p = 0.09

p = 0.70 F(1,32) = 0.33 p = 0.87 F(1,32) = 0.89 F(1,32) = 2.01 p<

p = 0.57 p = 0.35 p = 0.17

p = 0.73 F(1,32) = 0.47

p = 0.50

p p = 0.16 p = 0.19 p = 0.07 p = 0.25 p = 0.44 p = 0.57 p=

Exposure X Timing Exposure Timing Exposure X Timing Exposure Timing Exposure X Timing

F(1,30) = 0.16 p = 0.69 F(1,32) = 1.49 p = 0.23 F(1,30) = 0.01 p = 0.98 F(1,32) = 0.00 p = 0.96 EPM: F(1,30) = 0.10 p = 0.64 F(1,32) = 0.00 p = 0.96 Closed Arm F(1,30) = 0.22 p = 0.76 F(1,32) = 0.11 p = 0.75 Entries F(1,40) = 0.91 p = 0.35 F(1,40) = 3.82 p = 0.06 BF: F(1,40) = 2.95 p = 0.09 F(1,40) = 0.36 p = 0.55 Cue Acquisition F(1,40) = 0.00 p= F(1,40) = 0.20 p = 0.66 0.99 Exposure F(1,40) = 0.58 p = 0.45 F(1,40) = 1.26 p = 0.27 BF: Timing F(1,40) = 0.02 p = 0.90 F(1,40) = 2.00 p = 0.17 Set Shift Exposure X Timing F(1,40) = 2.34 p = 0.13 F(1,40) = 1.35 p = 0.25 Exposure F(1,40) = 1.85 p = 0.18 F(1,40) = 0.84 p = 0.37 BF: Timing F(1,40) = 0.00 p = 0.96 F(1,40) = 1.21 p = 0.28 Total Errors Exposure X Timing F(1,40) = 0.24 p = 0.63 F(1,40) = 0.65 p = 0.42 Exposure F(1,40) = 0.77 p = 0.39 F(1,40) = 2.79 p = 0.10 BF: Timing F(1,40) = 0.43 p = 0.52 F(1,40) = 1.93 p = 0.17 Perseverative Exposure X Timing F(1,40) = 0.02 p = 0.87 F(1,40) = 0.00 p = 0.96 Errors Exposure F(1,40) = 0.35 p = 0.56 F(1,40) = 6.66 p < BF: 0.05* Regressive Errors Timing F(1,40) = 1.37 p = 0.25 F(1,40) = 4.73 p < 0.05^ Exposure X Timing F(1,40) = 0.30 p = 0.61 F(1,40) = 0.59 p = 0.45 Exposure F(1,40) = 1.70 p = 0.20 F(1,40) = 0.00 p = 0.98 BF: Never F(1,40) = 0.04 p = 0.84 F(1,40) = 8.13 p < Reinforced Errors Timing 0.01^ Exposure X Timing F(1,40) = 0.50 p = 0.48 F(1,40) = 0.82 p = 0.37 Note: bold text denotes a significant main (p ≤ 0.05) effect of Exposure (*) or Timing (^).

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Entries

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Table 2. Light/Dark Box. Percent of animals remained in the dark chamber for the entire test session. Timing of Adolescent Staying in Dark Adolescent Sex Exposure Chamber (%) Exposure Water 87.5 Male Ethanol 85.7 Early Water 100.0 Female Ethanol 77.8 Water 88.9 Male Ethanol 88.9 Late Water 85.7 Female Ethanol 75.0