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

Abstract

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.

1. Introduction

Adolescence is a developmental period characterized by risk-taking and sensation-seeking behavior relative to other stages of development; unsurprisingly then, adolescence is also the period during which many individuals first drink alcohol (Leung et al., 2019). Data from 2020 indicate that 25.6% of 8^th graders and 61.5% of 12^th graders acknowledged ever drinking alcohol, and 10% of 8^th graders and 33.6% of 12^th graders reported alcohol use in the past month (Johnston et al., 2021). Moreover, 4.5% of 8^th graders and 16.8% of 12^th graders had consumed 5 or more drinks in a single session, qualifying as binge drinkers, and 20–25% of these individuals reported extreme binge drinking, defined as 10+ drinks in a session (Johnston et al., 2021). Binge drinking is a particularly harmful pattern of alcohol consumption, as it is often associated with injurious and risky behavior and blackouts in the short term, and growing evidence indicates that it is also associated with long-term consequences to the brain and behavior.

Both male and female adolescents binge drink alcohol, and although the sex-dependent consequences of problematic alcohol use are less understood, adolescent heavy drinking has negative consequences for both sexes. Several countries have reported recent binge and extreme drinking (Bell et al., 2013; Kang, Min, & Min, 2020; Nazareno et al., 2020; Patrick et al., 2013), particularly in female youth (A. White et al., 2015; A. M. White, 2020), suggesting that the behavior is less male-dominated than observed in earlier decades. In the United States in 2016–2017, similar numbers of male and female 14-year-olds engaged in binge drinking in the past two weeks, with approximately 4% of 14-year-olds consuming 5+ drinks and 1% consuming 10+ drinks in a session (Patrick & Terry-McElrath, 2019). At age 16, similar numbers of males (9%) and females (10%) engaged in binge drinking, but statistically more males (3.8%) than females (2.7%) reported extreme binge drinking. By age 18, both patterns of drinking were more prevalent in males (5+ drinks: 16.4%; 10+ drinks: 7.7%) but were present in females as well (5+ drinks: 11.8%; 10+ drinks: 3.3%). Increased levels of excessive and binge drinking among females underscores the urgency of directing specific attention to understanding the pathological effects of heavy alcohol consumption across sexes.

In light of the incidence of binge drinking in youth, an outstanding question is whether this exposure impacts adolescent brain development in a way that produces long-lasting effects. This is relevant to public health: while many adolescents binge drink, most will “age out” as they reach adulthood. Nevertheless, long-lasting consequences of prior alcohol drinking, especially on those brain functions that continue to develop during adolescence, like executive control and decision making, can negatively impact many aspects of adult life. Preclinical animal studies are useful to address this question as the timing and dose of alcohol exposure can be controlled. Moreover, animal studies avoid the individual genetic, behavioral, or environmental influences that confer risk for or resilience to binge drinking in people. The Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium has investigated the persistent effects of adolescent binge ethanol exposure in rodents and identified behavioral, neurochemical, neurophysiological, and epigenetic consequences that continue into adulthood, long after the original alcohol exposure. However, as with many areas of biomedical research, most of the research conducted in the NADIA Consortium and in the field only recently has included sex as a biological variable in experimental designs (Guizzetti et al., 2016). As a result, the effect of adolescent alcohol exposure in females is less understood, despite the fact that increasing numbers of young women engage in binge drinking behavior.

This review focuses on how biological sex impacts the long-term effects of adolescent ethanol exposure with emphasis on the preclinical literature. Many of the studies described are from the NADIA Consortium and use well-established regimens of adolescent intermittent ethanol (AIE) exposure (Crews et al., 2019; Crews, Vetreno, Broadwater, & Robinson, 2016). Broadly speaking, AIE regimens involve intragastric, inhaled (vapor) or intraperitoneal ethanol administration to adolescent animals, typically producing blood ethanol levels over 100 mg/dl – that is, meeting or exceeding binge levels – followed by abstinence until adulthood, when dependent variables are assessed. This model is well suited to determine whether adolescent alcohol exposure, mimicking the periodic binge drinking many youth engage in, alters neurobiology and behavior in ways that impact adult outcomes. Here, we focus on a subset of behavioral, neurochemical, physiological and epigenetic measures that are now known to be affected by AIE, discuss the contribution of sex in these AIE-induced effects, and identify some gaps in the literature.

2. Adolescent Alcohol and Sex: Ethanol Intake and Sensitivity

Alcohol use commonly begins during early adolescence (Masten, Faden, Zucker, & Spear, 2009; Morean, L’Insalata, Butler, McKee, & Krishnan-Sarin, 2018). While early experimentation with alcohol is common, initiation of drinking early in adolescence is associated with an increased risk for developing alcohol use disorders (AUDs) in adulthood (Dawson, Goldstein, Chou, Ruan, & Grant, 2008; DeWit, Adlaf, Offord, & Ogborne, 2000; Ehlers, Slutske, Gilder, Lau, & Wilhelmsen, 2006). Boys tend to initiate alcohol use at an earlier age than girls (Alvanzo et al., 2011; Keyes, Martins, Blanco, & Hasin, 2010), with this difference in initiation possibly contributing to a higher prevalence rate of AUDs in men than women (Ehlers et al., 2010; Grant et al., 2015). Therefore, understanding the relationship between adolescent drinking, sex, and vulnerability to developing AUDs later in life is pivotal.

Animal models offer an opportunity to understand the extent to which biological factors drive developmental changes in ethanol consumption, and rodents have proved to be an especially powerful and well-characterized model with adolescence occurring over a brief period of time (Spear, 2000). Rodent adolescence is conservatively denoted as postnatal day [P] 28 – P42, with late adolescence/emerging adulthood extending to approximately P55 (Vetter-O’Hagen & Spear, 2012). Important milestones of adolescent behavior that are relevant to alcohol consumption include increased impulsivity, novelty-seeking, and final maturation of executive function, roughly parallel key events during human adolescence (Spear, 2000, 2011; Varlinskaya, Vetter-O’Hagen, & Spear, 2013). In addition, the time line of how rodent puberty overlaps with adolescence is also well defined and primary sex differences, especially the earlier pubertal development of females than males, also parallels human development (Varlinskaya et al., 2013).

A number of animal models, including those that use voluntary consumption paradigms (two-bottle choice, drinking in the dark, scheduled high-alcohol consumption) or forced ethanol administration (intragastric gavage, intraperitoneal injections, ethanol vapor inhalation, forced drinking), have been used to determine whether adolescent ethanol exposure affects ethanol intake in adulthood. Although preclinical studies have shown that ethanol exposure during adolescence can, in some models, increase ethanol intake in adulthood (c.f., Towner & Varlinskaya, 2020), relatively few studies included both sexes, making it difficult to assess sex differences in long-lasting effects of adolescent ethanol exposure on ethanol intake. Furthermore, results of the studies that included both sexes are mixed (see Table 1), with some papers reporting no effects of AIE on ethanol intake (Garcia-Burgos, Gonzalez, Manrique, & Gallo, 2009; Varlinskaya, Kim, & Spear, 2017), others reporting similar effects in males and females (Amodeo et al., 2018; Broadwater, Varlinskaya, & Spear, 2013; Moore, Mariani, Linsenbardt, Melon, & Boehm, 2010; Younis et al., 2019), and still others reporting sex-dependent AIE-associated changes in ethanol consumption (Gamble & Diaz, 2020; Maldonado-Devincci, Alipour, Michael, & Kirstein, 2010; Maldonado-Devincci et al., 2021; Siciliano & Smith, 2001; Strong et al., 2010). A number of factors may contribute to these inconsistent findings, including exposure regimens, duration, route of ethanol administration, blood ethanol concentrations attained, mode of testing, and animal strain. For instance, studies in C57BL/6J mice that underwent a drinking-in-the-dark procedure during adolescence demonstrated an increase in adult intake, regardless of sex (Moore et al., 2010; Younis et al., 2019), while adult female C57BL/6J mice exhibited higher intake than males after exposure to an intermittent schedule, high-alcohol drinking procedure in adolescence that produced blood ethanol concentrations that exceed typical binge drinking levels (Strong et al., 2010). In contrast, using an inhaled (vapor) exposure, AIE-exposed male mice, but not females, exhibited increased alcohol intake in a two-bottle choice procedure (Maldonado-Devincci et al., 2021). Using an AIE exposure model that combined intermittent ethanol vapor and voluntary drinking, Amodeo and colleagues demonstrated increased adult ethanol consumption in both male and female Wistar rats (Amodeo et al., 2018). Adolescent exposure to vapor alone was not found to increase voluntary ethanol consumption in one study (Slawecki & Betancourt, 2002), but seemed to increase ethanol intake in male, but not female, Sprague Dawley rats in another (Gamble & Diaz, 2020). AIE exposure via the intragastric route of ethanol administration in Sprague Dawley rats either enhanced ethanol intake later in life, with males showing more enhanced ethanol intake in a two-bottle choice protocol than females (Maldonado-Devincci et al., 2010), or had no effect on social drinking in both sexes (Varlinskaya et al., 2017). More pronounced sex differences were evident in Long-Evans rats following forced ethanol consumption in adolescence, with males increasing and females decreasing ethanol intake later in life (Siciliano & Smith, 2001). Furthermore, in rats, males but not females demonstrated enhanced ethanol intake following AIE, with opposite AIE effects on ethanol drinking evident in mice (Strong et al., 2010). Overall, the studies to date suggest that sex is a significant factor to consider in determining the impact of adolescent ethanol exposure on drinking behavior later in life, although the direction and degree of sex effects are likely dependent on specific experimental parameters.

Table 1.

Adolescent Ethanol Exposure and Ethanol Intake Later in Life.

Strain	Adolescent Exposure			Test	Results	Reference
	Route and Ethanol Dose	Pattern	Timing
Sprague Dawley rats	1 bottle 10% EtOH in SS, 30 min	Intermittent	P24–33 P69–78	1 bottle limited access	Increased intake of a familiar solution regardless of sex	Broadwater et al., 2013
	VI	Intermittent	P30–40	30-min access to 10% sweet EtOH	Increased intake in males, but not females	Gamble & Diaz, 2020
	IG 1.5, 3.0, or 5.0 g/kg	Intermittent	P28–45	2BC	Intake increase more pronounced in males than females	Maldonado-Devincci et al., 2010
	IG 3.5 g/kg	Intermittent	P25–45	Social drinking	No change in intake	Varlinskaya et al., 2017
Wistar rats	2BC 20% EtOH + VI	Variable	P22–67	2BC	Increased intake regardless of sex	Amodeo et al. 2018
	4BC 5, 10, and 20% EtOH	Continuous	P19–28 P28–37 P90–99	4BC	No change in intake	Garcia-Burgos et al., 2009
Long-Evans rats	1 bottle 5% or 10% EtOH	Continuous	P21–70	2BC	Increased intake in males, decreased intake in females	Siciliano & Smith, 2001
C57BL/6J DBA/2J mice	DID 20% EtOH	Consecutive limited 2-hr access	P28–42	DID	Increased intake in C57BL/6J regardless of sex	Moore et al., 2010
C57BL/6J mice	SHAC 5% EtOH	Limited intermittent	P26–47 P58–79	DID 2BC	Increased intake following adolescent exposure predominantly in females	Strong et al., 2010
	Modified DID 1 bottle 20% EtOH	Consecutive limited 2-hr access	P28–36 P72–80	DID 2BC	Increased intake following adolescent, but not adult, exposure regardless of sex	Younis et al., 2019
	VI	Intermittent	P28–42	2BC	Increased intake following adolescent exposure in males but not females	Maldonado-Devincci et al., 2021

EtOH - Ethanol, 2BC - Two-bottle choice, 3BC - Three-bottle choice, 4BC - Four-bottle choice, IP - Intraperitoneal, IG - Intragastric Gavage, VI - Vapor Inhalation, SS – Supersac (sucrose + saccharin), DID - Drinking in the Dark, SHAC - Scheduled High-Alcohol Consumption

In addition to alcohol intake, researchers have studied the potential impact of AIE on more broad behavioral responses to alcohol, including sensitivity to locomotor effects that range from activation to sedation. In general, AIE-associated changes in responsiveness to ethanol in adults resemble adolescent-typical patterns of response to alcohol. Adolescent-typical responsiveness to ethanol (Spear, 2014) is characterized by enhanced sensitivity to some ethanol effects that may foster high intake levels (socially facilitating, stimulatory, rewarding) and reduced sensitivity to other effects of ethanol that could limit intake (sedative, motor-impairing, socially impairing, aversive). One NADIA study analyzed a large data set generated over an 8-year period to provide a population-level assessment of variables influencing level of intoxication of adolescent and adult rats in response to ethanol vapor exposure (Glover et al, 2021). The results indicated important differences with respect to strain, sex, and age during ethanol exposure in the relationship between blood ethanol concentration and behavioral signs of intoxication.

A number of studies that used only male rodents reported decreased sensitivity to ethanol-induced sedation (Jury et al., 2017; Matthews et al., 2008, Matthews et al., 2017) and ethanol-induced conditioned taste aversion (Alaux-Cantin et al., 2013; Diaz-Granados & Graham, 2007; Saalfield & Spear, 2015) in adult animals with prior history of AIE. However, these findings may differ by sex. For instance, Wolstenholme and colleagues have reported increased sensitivity to ethanol-induced sedation in adult DBA/2J mice exposed to AIE, with this effect driven by females (Wolstenholme, Mahmood, Harris, Abbas, & Miles, 2017). In the case of conditioned taste aversion, AIE-exposed male Long-Evans rats, but not their female counterparts, showed an attenuated aversion in adulthood (Sherrill et al., 2011). Adult male, but not female, Sprague Dawley rats demonstrated ethanol-induced increases in peer-directed social behavior (i.e., social facilitation) following AIE (Varlinskaya et al., 2017; Varlinskaya, Truxell, & Spear, 2014). Furthermore, adult male, but not female, C57BL/6J mice with prior history of AIE exposure were insensitive to ethanol-induced chrono-disruption (Ruby et al., 2018), retaining adolescent-typical responding to ethanol-associated suppression of photic phase-resetting (Ruby et al., 2017). Other aspects of ethanol sensitivity do not appear to differ by sex. Adolescent-typical enhanced sensitivity to the locomotor stimulant effects of ethanol was evident in both male and female adult Swiss mice following AIE exposure (Quoilin et al., 2014). Similarly, enhanced sensitivity to ethanol’s reinforcing effects following exposure to ethanol during adolescence was evident in adult male and female Wistar rats (Hauser et al., 2019). However, in composite, these data suggest that males are more likely to experience long-term changes in ethanol sensitivity that facilitate ethanol consumption.

The findings reviewed here demonstrate that AIE can increase adult ethanol intake and alter ethanol responsiveness, depending on the strain and route of administration. Adult ethanol consumption increased in eight of ten studies reviewed, and ethanol consumption increased more in males than in females in all but one study that included both sexes. Thus, male rodents may be more vulnerable to enhanced drinking after adolescent ethanol exposure than females, which may be associated with sex differences in sensitivity to ethanol. These findings are somewhat congruent with the epidemiologic literature in humans (Flores-Bonilla & Richardson, 2020; A. M. White, 2020), suggesting that animal models may provide useful insight into the potential risks for humans of significant ethanol exposure during adolescence.

3. Adolescent Alcohol and Sex: Social and Non-social Affect

Anxiety and mood disorders, which are common during adolescence, can lead to and also be a consequence of alcohol use and abuse. Adolescents often drink in social settings, and social anxiety is further linked to problematic drinking during this developmental period (Terlecki, Ecker, & Buckner, 2014), with reductions in interpersonal interactions being predictive of AUDs in older adolescents (Chou, Mackenzie, Liang, & Sareen, 2011). Consequentially, human research has also revealed that alcohol use in adolescence is associated with anxiety and depression (Marmorstein, 2009). The NIAAA-funded National Consortium on Alcohol and Neurodevelopment in Adolescence and others have made progress in determining the contributing factors of human adolescent alcohol use, including sex differences both in sociability and brain activity related to affective responding. For example, while social drinking predicted more frequent alcohol use for both sexes, peer socialization was evident earlier for females than males, and social situations influenced drinking later in adolescence in males but not females (Boyd, Sceeles, Tapert, Brown, & Nagel, 2018). While researchers continue to investigate sex differences during human adolescence associated with alcohol use, more preclinical studies are needed to determine whether social and non-social affective alterations associated with adolescent drinking differ between males and females.

In male rats and mice, long-lasting changes in negative affect associated with AIE exposure have been reported by a number of researchers (c.f., Crews et al., 2019; Towner & Varlinskaya, 2020). However, very few preclinical studies have included both sexes while assessing AIE-induced changes in later social and non-social anxiety-like behavior, and the results are inconsistent. Adult male, but not female, Sprague Dawley rats exposed to AIE during early to mid-adolescence (between P25-P45, 3.5 g/kg per day, intragastrically) demonstrated social anxiety-like alterations indexed via decreases in social investigation and social preference in a modified social interaction test (Varlinskaya et al., 2014). The timing of AIE was important, as neither males nor females demonstrated changes in social behavior following late AIE exposure (P45-P65). Intriguingly from a potential therapeutic perspective, AIE-associated social anxiety-like alterations in adult males were reversed by oxytocin or vasopressin pharmacological manipulations (Dannenhoffer et al., 2018). In contrast, when adult male and female Sprague Dawley rats were assessed in a modified social interaction test following relatively moderate ethanol vapor exposure during early adolescence (12 hours every other day, P30-P40), males demonstrated no changes in social behavior and social preference, while females showed higher levels of play behavior than their air-exposed counterparts (Gamble & Diaz, 2020). Sex differences in the social consequences of AIE were also evident in a recent study that used a tube dominance test for assessment of dominant and submissive behavior. AIE-exposed, Long-Evans, adult male rats (5 g/kg per day, 2-days on/2-days off, intragastrically, P24–54) were initially more submissive, but then became dominant in subsequent test rounds, while AIE-exposed females progressively became more submissive with repeated testing (Macht, Elchert, & Crews, 2020). Together, these findings suggest that AIE-associated social alterations are specific to sex, time of AIE exposure, and social behavior examined.

Preclinical studies have also demonstrated that AIE exposure results in non-social anxiety-like behavior. These tests are generally measurements of locomotor approach and avoidance, and the inhibition of exploratory behavior is interpreted as an anxiety-like phenotype. Enhanced anxiety-like behavior associated with AIE has been reported in the open field, elevated plus maze (EPM), marble burying, and light/dark box (c.f., Towner & Varlinskaya, 2020). The very limited number of studies including both males and females reported mixed results. In the EPM, AIE effects appeared to be dependent on the timing of exposure. Early AIE exposure (4 g/kg, intragastrically, P25-P45) enhanced anxiety-like behavior in both adult male and female rodents on the EPM, in contrast to social anxiety that was male-specific. However, later AIE exposure (P45-P65) enhanced anxiety-like behavior on the EPM only in males (Varlinskaya, Hosova, Towner, Werner, & Spear, 2020). However, other studies did not find anxiogenic effects of AIE exposure in either adult male or female rodents (e.g., Amodeo et al., 2018; Gamble & Diaz, 2020). Interestingly, female mice with a history of AIE vapor exposure showed more enhanced anxiety-like responding in a novelty-induced hypophagia test than their male counterparts, but only if they also experienced prior restraint stress (Kasten et al., 2020), an effect that was reversed by blocking type 5 metabotropic glutamate receptors.

Together, the findings of preclinical studies reviewed here demonstrate that AIE produces social deficits and enhances non-social anxiety-like behavior. AIE-induced effects on social interactions are sex-specific and behavior-dependent: only males are sensitive to the adverse effects of AIE on social investigation and social preference, whereas AIE-exposed females, but not males, demonstrate social submission that is often viewed as an index of depression-like behavior (Malatynska & Knapp, 2005). In contrast, non-social anxiety-like behavior on the EPM is evident in both male and female rodents following early AIE exposure, with only males affected by later AIE exposure. Furthermore, some sex differences in the anxiety-provoking effects of AIE become apparent only following stress challenges. Future studies should focus on exact mechanisms underlying the reviewed timing- and sex-specific or sex-independent effects of AIE.

4. Adolescent Alcohol and Sex: Dopamine and Reward

One of the biggest concerns about long-term consequences of adolescent alcohol exposure is the possibility that it will enhance the development of AUDs in adulthood. The mesocorticolimbic dopamine system, which plays a key role in reinforcement and reward for alcohol and other drugs of abuse (Becker & Chartoff, 2019; Di Chiara et al., 2004; Koob et al., 2014), is a likely target of adolescent alcohol exposure and is sexually dimorphic. While the literature on sex differences in drug-induced dopamine activity in humans is still quite scant (Petersen & London, 2018), there is a large literature on sex differences in rodent models. Behaviorally, female rats and mice consume more ethanol in many, but not all, consumption models (Flores-Bonilla & Richardson, 2020; Priddy et al., 2017), a difference that can emerge during adolescence (Lancaster, Brown, Coker, Elliott, & Wren, 1996; Lancaster & Spiegel, 1992). Dopamine signaling in the nucleus accumbens has been a major focus in explaining mechanisms that mediate these sex differences. The most consistent neurochemical finding is that strong stimuli like electrical stimulation cause greater evoked dopamine release in female rodents than males (Becker & Chartoff, 2019; Rivera-Garcia, McCane, Chowdhury, Wallin-Miller, & Moghaddam, 2020; Walker, Rooney, Wightman, & Kuhn, 2000; Zachry et al., 2021). Multiple anatomical and biochemical mechanisms in females contribute to this sex effect, including more or larger dopamine cell bodies in midbrain nuclei, decreased autoreceptor inhibition of release, greater expression and regulation of dopamine transporter function, and enhanced vesicular uptake to recapture and maintain dopamine stores (Zachry et al., 2021). In addition, sex differences in dopamine receptors are reported but are difficult to interpret, because typical endocrine manipulations like estradiol treatment can affect receptor transcription or function directly or indirectly, which can modulate activity through altered release of dopamine (Becker & Chartoff, 2019; Di Paolo, 1994; Yoest, Quigley, & Becker, 2018). Finally, sex differences in afferent regulation of dopamine via GABA and glutamate may contribute to the relatively enhanced dopamine release in females relative to males (Becker & Chartoff, 2019; Zachry et al., 2021). Estradiol action on estradiol alpha and beta receptors mediates many of these responses (Becker & Chartoff, 2019; Di Paolo, 1994), supporting not only sex differences, but sometimes differences across the estrous cycle.

While the well-described sex differences in dopamine transmission support the expectation of sex differences in AIE effects on mesolimbic dopamine and reward processing, very few sex comparisons have been made. Multiple studies reported that preference for risky choice was increased in adult males after voluntary or experimenter-administered AIE compared to controls (Boutros, Semenova, Liu, Crews, & Markou, 2014; Nasrallah et al., 2011; Schindler, Tsutsui, & Clark, 2014), a behavior tightly linked to mesolimbic dopamine signaling. Other studies assessed sign tracking during Pavlovian conditioned approach (Flagel, Watson, Robinson, & Akil, 2007), another dopamine-dependent phenotype, and observed that a history of AIE shifted conditioned approach from goal-tracking toward sign-tracking behaviors (Kruse, Schindler, Williams, Weber, & Clark, 2017; Madayag, Stringfield, Reissner, Boettiger, & Robinson, 2017; McClory & Spear, 2014; Spoelder, Tsutsui, Lesscher, Vanderschuren, & Clark, 2015) and enhanced cue-associated dopamine release (Spoelder et al., 2015). The one study that compared sexes found that both female sex and AIE exposure promoted sign-tracking behavior (Madayag et al., 2017), with no statistical interaction between the two, indicating an additive rather than synergistic effect. While several studies have documented persistent alterations in dopamine release in the nucleus accumbens after AIE exposure, these studies have exclusively tested males. For example, Schindler et al. (Schindler, Soden, Zweifel, & Clark, 2016) found that tonic extracellular levels of dopamine in the nucleus accumbens were reduced in AIE-exposed males compared to controls. Moreover, while phasic dopamine release evoked by direct stimulation of the ventral tegmental area (VTA) or medial forebrain bundle was unchanged by AIE exposure (Schindler et al., 2016; Shnitko, Spear, & Robinson, 2016), indirect stimulation of dopamine release by electrical stimulation of the pedunculopontine tegmentum, which releases acetylcholine in the VTA, was enhanced (Schindler et al., 2016). These findings suggest the involvement of cholinergic pathways in the AIE effect. Finally, while tonic increases in mesolimbic dopamine induced by an ethanol challenge were unaffected by AIE history (Pascual, Boix, Felipo, & Guerri, 2009), the characteristic ethanol-induced reduction in VTA-evoked dopamine release was blunted in AIE-exposed males (Shnitko et al., 2016). Overall, AIE appears to alter dopamine dynamics, likely through multiple interactions of neurocircuitry, but potential sex differences in these alterations remain unknown.

In the prefrontal cortex, tyrosine hydroxylase immunoreactivity (a marker of dopaminergic and noradrenergic afferents) was reduced in AIE-exposed male rats compared to controls, indicating less catecholaminergic input after AIE (Boutros et al., 2014; Trantham-Davidson et al., 2017). Membrane-bound catechol-O-methyltransferase (COMT) was also reduced in AIE-exposed adult males (Trantham-Davidson et al., 2017), indicating altered catecholamine clearance. One study to date has compared AIE effects on prefrontal dopamine receptor expression in male and female rodents (Marco et al., 2017). Wistar rats consumed alcohol or water in a drinking-in-the-dark procedure throughout adolescence. In young adulthood, expressions of several neurotransmitter markers were measured via Western blots. In females, but not males, AIE exposure reduced expression of D1-type dopamine receptors in the frontal cortex and the hippocampus, while D2-type receptors were not affected (Marco et al., 2017). This is consistent with electrophysiological recordings in prefrontal cortex (PFC) slices from male rats, in which vapor AIE exposure resulted in reduced D1 receptor modulation of excitation in pyramidal cells, although females were not tested in this study (Trantham-Davidson et al., 2017). Considering that vapor AIE produces much higher blood ethanol concentrations (BECs; ~290 mg/dl) than self-administration AIE (~24 mg/dl), one possibility is that AIE alters cortical D1-associated signaling in both males and females, but females are more sensitive to lower levels of BECs induced by AIE. In sum, sex differences in the persistent effects of adolescent alcohol on dopamine transmission is greatly understudied.

5. Adolescent Alcohol and Sex: Spatial Learning and Hippocampal Physiology

The hippocampus is particularly vulnerable to the persistent effects of AIE, exhibiting alterations in multiple neurotransmitter systems and hippocampal-dependent spatial learning and memory. A large body of work has found alterations of hippocampal neurons and gene expression after AIE which have been linked to changes in cognition, synapses, and synaptic physiology (i.e., plasticity; Mulholland et al., 2018; Risher, Fleming, et al., 2015; Risher, Sexton, et al., 2015; Swartzwelder et al., 2014; Vetreno et al., 2020). Likewise, AIE induces sustained glutamatergic hippocampal modifications such as a reduced threshold for long term potentiation (LTP) induction (Risher, Fleming, et al., 2015), increased NMDA receptor-mediated synaptic currents (Swartzwelder, Park, & Acheson, 2017), impaired NMDA receptor-induced long term depression (LTD) (Contreras et al., 2019), and altered glutamatergic epigenetic (Contreras et al., 2019) and synaptic expression (Healey, Kibble, Hodges, et al., 2020; Swartzwelder et al., 2016). These appear to be largely NDMA receptor-mediated alterations, as there have been no reported alterations in AMPA receptor expression (Healey, Kibble, Bell, Hodges, & Swartzwelder, 2020) or AMPA receptor function (Risher, Fleming, et al., 2015) after AIE. AIE also alters inhibitory processes in hippocampal formation by reducing the density and altering the activation kinetics of the depolarization-induced K⁺ current, I_A, in CA1 GABAergic interneurons, thus increasing their firing rates (Li et al., 2013). Importantly, these AIE effects on I_A density and activation were not observed after chronic intermittent ethanol exposure during adulthood (Li et al., 2013). The majority of the above studies were conducted in male rodents only, underscoring the need for additional studies on the consequences of AIE with female animals. For example, AIE significantly increased the expression of glial glutamate transporters in the hippocampus of both males and females, but other proteins associated with astrocytic-neuronal interactions were changed after AIE in a sex-specific manner (Healey, Kibble, Bell, et al., 2020), supporting likely sex differences in the functional impact of AIE on glutamatergic physiology. Supporting this hypothesis, it was recently reported that LTD was blunted in adolescent (P35–45) female rats with high circulating estrogen 24 hours after an ethanol binge (3 g/kg ethanol; 20% v/v, i.p., 2 doses 9 hrs apart), but not in pre-pubertal females (P21–25), adolescent females with low circulating estrogen, or adolescent male rats (Rabiant, Antol, Naassila, & Pierrefiche, 2021). Further, in adolescent males the addition of estrogen (17β-estradiol, 180 μg/kg), and a higher dose of ethanol (3.75 g/kg, i.p.) to allow for similar blood ethanol concentration, similarly reduced LTD, indicating that estrogen may regulate this sensitivity to ethanol. Estrogen likely contributes to the ethanol-impairing effects via the glutamatergic system, specifically GluN2B receptors, as administration of tamoxifen prevents the effects of estrogen (Rabiant et al., 2021). Serotonergic systems are also differentially affected by AIE in males and females. For example, directly after AIE exposure, male rats exhibited reduced hippocampal serotonin transporter binding while female rats did not (Abreu-Villaça et al., 2019). One week later, this effect was ameliorated in males, but females then exhibited a reduction in serotonin transporter. In adulthood, there were no sustained effects on serotonin transporter binding, suggesting that these effects were not persistent (Abreu-Villaça et al., 2019).

Given the physiological changes that occur in the hippocampus after AIE exposure, it is not surprising that there are also hippocampal-related behavioral changes after adolescent ethanol exposure, as described in reviews (Crews et al., 2019; Crews et al., 2016; Spear, 2018). However, few of these studies have compared males and females. In a recent study, AIE-exposed female rats took longer to strategically minimize errors in the radial arm water maze than male counterparts, and AIE-exposed females exhibited impaired novel object recognition compared to both the female control and male AIE cohorts (Macht et al., 2020). These effects are not universal to all hippocampal-dependent behaviors, as both AIE-exposed males and females displayed deficits in social recognition and reversal of a probabilistic rule compared to controls (Galaj, Kipp, Floresco, & Savage, 2019), the latter involving prefrontal cortical function. One approach that has been used to distinguish the role of sex in AIE-associated behavioral changes is to examine the specific effect of gonadal hormones on memory deficits. For example, a recent study using a single dose of ethanol (2g/kg) in late adolescent rats revealed that females showed greater impairment than males in contextual fear conditioning to the alcohol challenge, and that the impairment was directly tied to estrogen levels (Sircar, 2019). This sex difference in the acute effect of alcohol provides support for further investigations into the effects of repeated alcohol exposure during adolescence, as well as whether these effects persist into adulthood.

6. Adolescent Alcohol and Sex: Behavioral Flexibility and the Prefrontal Cortex

Behavioral flexibility, or cognitive flexibility, is a complex process of adjusting behavior in response to changes in environmental demands and individual factors (Diamond, 2013; Luna, 2009). It builds on other cognitive processes like inhibitory control and working memory (Diamond, 2013) that are dependent on prefrontal cortical activity (Nowrangi, Lyketsos, Rao, & Munro, 2014). Changing perspectives or modifying a particular behavior typically involves inhibiting an ongoing action, activating and retaining a new mindset, and maintaining the new behavior based on feedback. Thus, behavioral flexibility impacts how animals and humans make decisions and the consequences derived from those decisions.

The development of behavioral flexibility starts during early childhood and gains complexity through adolescence along with the maturation of the PFC (Best & Miller, 2010; Diamond, 2013; Luna, 2009; Nowrangi et al., 2014; Spear, 2013), which suggests a functional connection between PFC development and executive function (Nowrangi et al., 2014). Thus, it is reasonable to predict that conditions that impact PFC development, such as adolescent exposure to alcohol, might also affect executive function in general and behavioral flexibility in particular. For instance, Coleman and colleagues reported deficits in behavioral flexibility in a spatial learning task and enlarged volume of the orbitofrontal cortex after AIE in male mice, concomitant with increased expression of several extracellular matrix proteins in the same region (Coleman, Liu, Oguz, Styner, & Crews, 2014). Other studies showed AIE-induced deficits in behavioral flexibility as well as changes in levels of brain-derived neurotrophic factor (BDNF; Fernandez, Lew, Vedder, & Savage, 2017) and neuroimmune signaling within the PFC (Vetreno & Crews, 2012). The deleterious effects of AIE on behavioral flexibility have been demonstrated by using diverse behavioral approaches, like operant (Barker, Bryant, Osborne, & Chandler, 2017; Galaj et al., 2019; Gass et al., 2014; Towner & Spear, 2021; Varlinskaya et al., 2020), spatial (Coleman, He, Lee, Styner, & Crews, 2011; Macht et al., 2020; Vetreno et al., 2020), or discriminative tasks (Sey, Gomez, Madayag, Boettiger, & Robinson, 2019). Interestingly, in all of these reports, the observed AIE-induced deficits were most associated with changes to the original rule rather than initial learning of each task. Thus, impairments in behavioral flexibility appear to be a core consequence of adolescent ethanol exposure, at least in preclinical studies.

While all of the above cited studies included males, less than half of them included females, and none used only females. Of particular interest for the focus of this review, AIE effects on behavioral flexibility appear to occur in both sexes, with some specific differences between male and female individuals. For example, Barker et al. (2017) exposed animals to AIE from P28 to P48. After reaching adulthood, they were trained to self-administer 10% sucrose via an operant paradigm. AIE exposure promoted a habit-like strategy (typically considered a form of behavioral inflexibility) in adult female, but not male, rats. In contrast, the opposite sex effect was observed in animals exposed to alcohol during adulthood (Barker et al., 2017). In a separate study, Varlinskaya et al. (2020) used an operant set-shifting task, and observed deficits in behavioral flexibility in males exposed to alcohol during early adolescence (P25-P45), but not during late adolescence (P45-P65), whereas behavioral flexibility in females was unaffected by AIE at either timepoint (Varlinskaya et al., 2020). Remarkably, Towner & Spear (2021), using a sensory-specific satiation procedure after instrumental training, discovered that AIE-exposed male and female rats were resistant to reward devaluation, indicating that AIE promoted a habitual behavioral strategy. Interestingly, that effect was observed in animals exposed to alcohol during late (P45–65) but not during early (P25–45) adolescence (Towner & Spear, 2021). These studies illustrate that ethanol effects on flexible behavior depend upon both sex of the animal and the age of exposure. Not surprisingly, these observations also suggest that not all aspects of adult behavioral flexibility are equally impacted by AIE. Finally, using an operant probabilistic learning task, Galaj and colleagues reported that male and female rats exhibited impaired behavioral flexibility as evidenced by a higher number of trials needed to complete three reversals and longer latencies to select the correct lever (Galaj et al., 2019). These investigators also observed that AIE-exposed male rats committed more errors than control rats after reversal, similar to the effects observed by Varliskaya and colleagues (2020). Additional studies describing AIE-induced deficits in behavioral flexibility that included male and female rats either found no effect of sex (Macht et al., 2020) or were underpowered to detect sex differences (Sey et al., 2019).

Taken together, the above observations suggest that AIE induces behavioral flexibility deficits that are independent of the experimental behavioral approach, and different manipulations, used in the studies. This strongly implicates deficits in behavioral flexibility as a core persistent consequence of AIE exposure. However, it is important to point out that even though most experiments are now including both male and female subjects, substantial work remains to fully define sex-specific effects of AIE on cognitive control.

7. Adolescent Alcohol and Sex: Sleep Regulation and the Basal Forebrain

Adolescent binge drinking has been associated with reduced sleep quality in young adulthood (Ehlers, Wills, & Gilder, 2018; Ogeil et al., 2019). There is considerable evidence suggesting that sleep and adolescent alcohol abuse have a bidirectional relationship, with prolonged adolescent alcohol use not only increasing the likelihood of alcohol-induced insomnia and sleep disruptions in the short term (Thakkar, Sharma, & Sahota, 2015), but sleep disturbances also increasing risk for later substance use (Brower, 2015; Brower & Perron, 2010; Clark et al., 1998; Conroy et al., 2006; Stein & Friedmann, 2005). While alcohol-induced sleep disturbances have been found in both women and men, women describe more problems related to waking intermittently at night, having more bad dreams, and going to bed habitually later compared to men (Arnedt et al., 2011; Ehlers, Wills, et al., 2018). Preclinical studies have also demonstrated that adolescent ethanol exposure can result in compromised sleep later in adulthood. Specifically, a 5‐week AIE vapor exposure of male Wistar rats produced a decrease in the mean duration of slow wave sleep (SWS), an increase in the number of episodes, and an increase in total time in SWS in adulthood, well after the end of alcohol exposure (Criado, Wills, Walker, & Ehlers, 2008; Ehlers, Benedict, Wills, & Sanchez-Alavez, 2020). Similarly, female rats exposed to the same AIE vapor paradigm also exhibited persistent reductions in SWS duration together with an increase in the number of SWS episodes (Amodeo, Wills, Sanchez-Alavez, & Ehlers, 2020). Thus, AIE exposure results in adult “fragmented” sleep in both male and female rats, characterized by more frequent SWS episodes that are shorter in duration (Amodeo, Wills, et al., 2020; Ehlers et al., 2020). In humans, this pattern of fragmented sleep has been associated with less “restful” or “restorative” sleep (Buysse, 2013). Moreover, AIE vapor exposure in both male and female rats reduces sleep-associated spectra power measured with electroencephalogram (EEG) in adulthood, with decreases in delta and theta during SWS (Amodeo, Wills, et al., 2020; Ehlers, Sanchez-Alavez, & Wills, 2018). Reduced delta and theta power during sleep has also been repeatedly found in clinical populations with AUD (Colrain, Turlington, & Baker, 2009; Irwin, Miller, Gillin, Demodena, & Ehlers, 2000). While rodent estrous phase has been shown to impact sleep states and spectra power (Swift et al., 2020), estrous cycle seems to only minimally influence sleep patterns and has no effect on sleep EEG spectra in AIE-exposed Wistar rats (Amodeo, Wills, et al., 2020). Although additional studies are needed to fully understand the interaction of estrous cycles with alcohol and AIE-induced sleep disruption, studies have established long-lasting changes in sleep for those with an AUD and following AIE in both male and female rats.

There is limited literature focused on the mechanisms underlying alcohol-induced sleep pathology, with even less examining sex as a variable in that pathology. Adenosine and wake‐promoting cholinergic neurons in the basal forebrain may play a pivotal role in alcohol-induced insomnia (Sharma, Engemann, Sahota, & Thakkar, 2010; Sharma, Sahota, & Thakkar, 2017). Specifically, lesions of the basal forebrain cholinergic neurons in male rats attenuated the effects of acute alcohol on release of adenosine and latency to sleep (Sharma et al., 2017). A well-described consequence of AIE is a reduction in cholinergic neurons within the basal forebrain, as indicated by cholinergic phenotypic markers such as choline acetyltransferase (ChAT+) (Crews et al., 2019; Crews et al., 2016); thus, reduced acetylcholine from basal forebrain projection neurons may underlie AIE-induced sleep disruptions. As no sex differences were observed in AIE-induced loss of ChAT+ in basal forebrain cholinergic neurons of Wistar rats (Vetreno & Crews, 2018), similar acetylcholine/sleep interactions may be expected in AIE-exposed males and females. Another mechanism by which AIE exposure can disrupt sleep in adulthood is via the neuropeptide orexin (also called hypocretin; Hct/OX). Several studies demonstrated that chronic alcohol exposure can increase orexin signaling (Amodeo, Liu, et al., 2020; Lawrence, Cowen, Yang, Chen, & Oldfield, 2006), and antagonism of this system might serve as a potential target for alcohol-induced insomnia. Using multiple AIE exposure models (vapor and intragastric gavage), binge levels of ethanol intoxication during adolescence significantly increased orexin-A immunoreactivity in the anterior hypothalamus (Amodeo, Liu, et al., 2020). Additionally, Hct/OX receptor antagonists can hasten the onset of SWS in both male and female AIE vapor-exposed rats, similar to control rats (Amodeo, Wills, et al., 2020; Ehlers et al., 2020). Understanding the intersecting relationship between homeostatic mechanisms, sleep, and adolescent alcohol use is important for designing future therapeutics for sleep pathologies in AUD. While AIE effects on sleep appear generally consistent across sexes, it is critical that future research continues to include male and female individuals. Although studies find sleep improvements with recovery from AUD, and that lack of improvement in sleep predicts relapse, the value of sleep therapeutics on the long-lasting effects of AIE and possible efficacy for AUD therapy is poorly understood and needs further study.

8. Adolescent Alcohol and Sex: Neuroinflammation and Epigenetic Alterations

One of the primary mechanisms by which alcohol alters neural function is through its induction of proinflammatory cascades across the brain including the prefrontal cortex and hippocampus (Alfonso-Loeches, Pascual-Lucas, Blanco, Sanchez-Vera, & Guerri, 2010; Guerri & Pascual, 2019; Oliveira et al., 2015; Sanchez-Alavez et al., 2019; Valles, Blanco, Pascual, & Guerri, 2004; Vetreno & Crews, 2012; Vetreno, Qin, & Crews, 2013). When repeated alcohol exposure occurs during adolescence, these neuroinflammatory effects persist into adulthood, long after alcohol exposure ends. Specifically, AIE exposure leads to (1) chronic upregulation of innate immune factors, including damage associated molecular pattern (DAMP) molecules, cytokines, chemokines, and nitric oxide, which are paralleled by (2) a persistent loss of cholinergic neurons and the associated anti-inflammatory effects of acetylcholine (Coleman et al., 2011; Coleman et al., 2014; Crews et al., 2019; Evrard et al., 2006; Pascual, Blanco, Cauli, Minarro, & Guerri, 2007; Vetreno et al., 2020; Vetreno & Crews, 2012; Vetreno et al., 2013). Moreover, reducing AIE effects on innate immune activation with anti-inflammatory drugs, exercise, cholinergic drugs or with transgenic mice both prevents and restores many adult behavioral deficits observed after AIE, as determined by anti-inflammatory pharmacological interventions and genetic models, such as toll-like receptor-4 (TLR4) knockout mice (Montesinos et al., 2015; Pascual et al., 2007; Vetreno et al., 2020). This suggests that alcohol effects on neuroinflammation may be a mechanistic cornerstone of the enduring impact of AIE on the brain and cognitive function, although few studies have included female subjects.

Human studies have demonstrated that both 20-year-old males and females adults with a history of binge drinking exhibit an acute induction of a variety of proinflammatory markers relative to non-drinking controls (Orio et al., 2018). Moreover, the magnitude of the induction of these pro-inflammatory cascades was negatively correlated with performance in executive function and memory tests specifically in women, highlighting the importance of sex as a biological variable in the investigation of immune mechanisms of alcohol-induced impairment in cognitive function (Orio et al., 2018). Preclinical studies have also found AIE increases cytokine and other neuroimmune genes in adult male rats. Although fewer studies have investigated the effects of AIE in females, one study by Pascual and colleagues reported similar findings in female rats, showing that acute adolescent alcohol exposure increased plasma serum levels of the chemokines IL-10, IL1β, IL-4, fractalkine, and monocyte-chemoattractant protein-1 (MCP-1; Pascual et al., 2017), findings consistent with other studies done in males. In contrast, adolescent alcohol administration blunted blood cell mRNA cytokine (i.e., IL-6, IL-1, and IĸBα) responses to an adult lipopolysaccharide (LPS) challenge in males but not females, suggesting that females exhibit less habituation to chronic adolescent alcohol-associated immune activation in the periphery (Vore, Doremus-Fitzwater, Gano, & Deak, 2017). However, this could reflect global sex differences in innate peripheral immune reactivity, as in the same study, males exhibited greater cytokine responses to LPS. Important sex differences in the innate immune system after AIE have also been observed in the central nervous system. The molecular profile of the two primary regulators of innate immune function – astrocytes (Healey, Kibble, Hodges, et al., 2020) and microglia (Barton, Baker, & Leasure, 2017) – are differentially impacted by the effect of sex-of-subject following AIE exposure. With astrocytes, AIE exposure elevated expression of the cystine/glutamate antiporter and glutamate aspartate transporter 1 (GLAST) in adult males only, while the glutamate transporter-1 (GLT-1) was elevated in both sexes (Healey, Kibble, Hodges, et al., 2020). With microglia, surface presentation of major histocompatibility complex II (MHCII+) was increased in females but not males after AIE, indicating increased microglial activation in females (Barton et al., 2017). These findings could indicate that ethanol induction of inflammatory responses may activate and/or disrupt glial populations in a sex-specific manner. However, other studies have found similar increases in other proinflammatory microglial markers, such as CD11b, in adult males after AIE (Vetreno, Patel, Patel, Walter, & Crews, 2017), suggesting that a more comprehensive examination of sex differences in microglia and astrocyte activation across the brain is an important future direction for the field.

These sexually divergent innate immune responses to AIE are not surprising considering that several genes involved in innate immunity are X chromosome-linked, including TLR intracellular signaling pathways (Jaillon, Berthenet, & Garlanda, 2019). In addition, mid-adolescence reflects a critical period of pubertal maturation characterized by dramatic sex-specific shifts in hormone production that could further complicate the impact of adolescent alcohol on innate immune function, as both estrogen and testosterone hormones exhibit differential and complex interactions with innate immune function (c.f., Kovats, 2015). Collectively, these results suggest that females and males may exhibit sex-specific changes related to increased innate immune gene expression after AIE, potentially through X chromosome-linked distinctions in TLR sensitivity, reflecting shifts in positive drivers of chronic neuroinflammation after AIE.

In conjunction with neuroimmune gene activation, AIE also induces a partial loss of cholinergic neurons and acetylcholine efflux that persists into adulthood (Fernandez & Savage, 2017) and is reversible by the anti-inflammatory compounds indomethacin and the anti-cholinesterase galantamine (Crews, Fisher, Deason, & Vetreno, 2021; Vetreno & Crews, 2018). As acetylcholine is known to be anti-inflammatory, one may speculate that loss of cholinergic regulation may contribute to neuroimmune gene induction after AIE. However, AIE-associated loss of cholinergic anti-inflammatory feedback may not be differentially impacted by sex, as cholinergic markers in interneurons and projection neurons are similarly decreased in males and females after AIE (Galaj et al., 2019; Vetreno et al., 2020; Vetreno & Crews, 2018). This contrasts with reported sex differences in cholinergic neurons, such as a greater effect in female, compared to male, rodents’ acute responses of cholinergic neurons to stress and drugs (c.f., Rhodes & Rubin, 1999). It is possible that females are differentially sensitive to changes in cholinergic-innate immune signaling induced by adolescent ethanol exposure, but any sex differences present are no longer overtly evident in adulthood. Moreover, as acetylcholine plays a critical role in many behaviors, understanding sex differences in the sensitivity of cholinergic responses during behavior is important but understudied. For example, AIE altered adult social anxiety and caused deficits in behavioral flexibility in adult male, but not female, rodents (Varlinskaya et al., 2020), whereas AIE sensitized adult females to increased corticosterone levels associated with novel object exploration and social interaction, the latter effect not found in males (Kim, Varlinskaya, Dannenhoffer, & Spear, 2019). Thus, AIE induces sex-specific changes in behavior that have yet to be linked to cholinergic deficits or neuroimmune signaling mechanisms.

A final layer of consideration regarding the mechanisms of AIE-induced innate immune gene induction is alcohol-induced shifts in epigenetic programming. Epigenetic programming has emerged as an important mechanism regulating gene expression across development, especially adolescence (c.f., Mychasiuk & Metz, 2016). Indeed, AIE exposure produces long-lasting changes in gene expression through epigenetic programming, although the existing literature has focused on studies with males (c.f., Crews et al., 2019; Pandey, Kyzar, & Zhang, 2017). For example, AIE decreases adult histone acetylation that appears to contribute to suppressed trophic factor expression (Pandey, Sakharkar, Tang, & Zhang, 2015), increased phosphorylation of master regulators of innate immune gene transcription (e.g., Nf-ĸB, AP-1), and increased methylation silencing of cholinergic genes (Vetreno et al., 2020). However, it is unknown whether these findings extend to females. In fact, some studies have suggested that adolescent females exhibit higher levels of histone deacetylase activity and methylation in the hippocampus than males (Elsner, Cechinel, de Meireles, Bertoldi, & Siqueira, 2018), which may be associated with neuroimmune responses that involve as well as initiate epigenetic programming of persistent changes in adult brain gene expression. In sum, sex-specific sensitivities to neuroinflammation and epigenetic programming of the same require additional research due to their likely contribution to downstream AIE effects on brain function and behavior.

9. Conclusion

Biological sex is emerging as an important variable in our understanding of how alcohol impacts the neurodevelopmental landscape. Awareness of the nuances in this impact can influence both our understanding of the mechanisms underlying sex differences in alcohol-induced behavioral deficits, as well as potential sex differences in the efficacy of future therapeutics. AIE exposure clearly affects females and males differently in some outcomes. For example, sex differences exist in the impact of AIE on sensitivity to ethanol, social anxiety-like behavior, and habit formation in adulthood. In other domains, sex has little apparent influence on AIE-induced consequences, including non-social anxiety-like behavior and sleep disruption. Often, the effect of AIE exposure is generally consistent across sexes, but the degree of impact may differ, such as with alcohol drinking, Pavlovian approach, some hippocampal-dependent behaviors, and induction of proinflammatory markers. Finally, some areas of research are woefully lacking in studies on females, such that while sex differences may be hypothesized in AIE effects, they are yet unknown. This research includes the effect of AIE exposure on neurotransmitter systems, neuropeptide systems, and epigenetic programming. A major caveat for these conclusions is that the number of studies that included males and females is small, and many studies were underpowered to detect sex differences. In addition, few studies of AIE exposure included direct comparisons to comparable duration of exposure to ethanol in adulthood, and so the results cannot be automatically attributed to the consequence of exposure during a defined developmental window.

In closing, it is important to reiterate the benefits and limitations of animal models. A limitation is that animal models cannot encompass the range of social, environmental, behavioral, cognitive, and genetic influences that interact to lead individuals to binge drink and sometimes develop a future AUD. However, a major benefit is that animal studies can identify the biological consequences of ethanol exposure that also contribute to the human experience. Moreover, the precise control of AIE variables in animal studies – time of exposure, dose, route, etc. – is both a benefit, as it allows one to test mechanistic hypotheses of AIE effects, and a limitation, as apparently conflicting results between studies may be due to methodological differences. Although preclinical studies have identified some sex differences in the consequences of AIE, the neurobiological bases of those differences are not well understood, prompting a call for more research comparing males to females. This information can help untangle biological consequences of adolescent ethanol exposure from the many factors unique to humans that influence consumption including availability, sex and gender roles, and the influence of religion, education, socioeconomic status, peers, and family.

List of Abbreviations

AIE

adolescent intermittent ethanol

AUDs

alcohol use disorders

BDNF

brain-derived neurotrophic factor

BECs

blood ethanol concentrations

ChAT

choline acetyltransferase

COMT

catechol-O-methyltransferase

DAMP

damage associated molecular pattern

EEG

electroencephalogram

EPM

elevated plus maze

GLAST

glutamate aspartate transporter 1

GLT-1

glutamate transporter-1

Hct/OX

hypocretin/orexin

LPS

lipopolysaccharide

LTD

long term depression

LTP

long term potentiation

MCP-1

monocyte-chemoattractant protein-1

MHCII

major histocompatibility complex II

NADIA

Neurobiology of Adolescent Drinking in Adulthood

P

postnatal day

PFC

prefrontal cortex

SWS

slow wave sleep

TLR

toll-like receptor

VTA

ventral tegmental area