Adolescent binge drinking modifies gene expression in a fashion that increases susceptibility to anxiety and alcohol use disorders in adulthood, according to research in rats recently published in eNeuro.
Targeting the microRNAs responsible could be a new route for undoing the damage of alcohol use caused during adolescence.
Previous researchers found that microRNA, small bits of RNA that modify how genes are expressed rather than coding for a protein, are involved in how early-life alcohol exposure changes the brain, but the exact mechanism was unknown.
Kyzar et al. exposed adolescent rats to alcohol and measured the resulting levels of miR-137, one type of microRNA, in the rats’ amygdalae.
The rats exposed to alcohol displayed increased levels of miR-137, resulting in lower expression of proteins necessary for healthy neuron growth and branching.
In adulthood, the rats displayed higher levels of anxiety behaviors and a higher preference for alcohol compared to control rats.
Inhibiting miR-137 in the amygdala reversed the anxiety and alcohol preference in the rats with an adolescent drinking history.
Diagram of epigenetic reprogramming that occurs from adolescent alcohol use. The image is credited to Kyzar et al., eNeuro 2019.
Adolescent binge drinking remains a significant public health issue in the United States. In 2017, more than 4.5 million youth in the United States aged 12 to 20 (11.7%) reported binge drinking (4 + drinks for women, 5 + drinks for men), and almost 1 million (2.7%) reported frequent binge drinking (5 + binge drinking days in the past month) (SAMHSA, 2018). Extreme binge drinking of 10 to 15 or more drinks in a row is reported by many adolescents (Nguyen‐Louie et al., 2016; Patrick and Terry‐McElrath, 2019). It is clear that the short‐term consequences of adolescent binge drinking can be detrimental (Kuntsche and Gmel, 2013; White and Hingson, 2014), including effects such as impaired judgment, intoxicated driving, unintended sex, injury, and death. What is less clear is how binge drinking affects brain development, and whether such neurobiological effects resolve with time or persist into adulthood. To address the causes of excessive adolescent drinking, the National Institutes of Health has funded longitudinal research in youth to track which factors may predispose adolescents to binge drink. The National Consortium on Alcohol and Neurodevelopment in Adolescence (NCANDA) (Brown et al., 2015) and the Adolescent Brain Cognitive Development study (ABCD) (Auchter et al., 2018) are 2 large, multisite projects that aim to pinpoint environmental, social, and other factors that lead to drinking and drug use in adolescents. However, factors that predict alcohol use in humans can confound determination of the consequences of binge drinking, another major aspect of the public health issue and a question that can be systematically examined by modeling binge exposure in rodents that controls alcohol exposure across development.
In 2011, the National Institute on Alcohol Abuse and Alcoholism funded the Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium with the charge to use animal models to define the enduring effects of binge‐like adolescent alcohol exposure and explore the associated neurobiological mechanisms. The Consortium gathered basic neuroscientists with multidisciplinary expertise to explore the impact of adolescent binge alcohol on adult neurobiology and psychopathology using rodent models. Individual laboratories would conduct independent but coordinated experiments, building a body of data that in aggregate could support or refute hypotheses on adolescent alcohol exposure. To integrate the projects and the resulting data, the Consortium agreed to a set of procedural guidelines. First, all studies used rats, and NADIA recommended procedures to control early‐life environments before adolescence, to reduce unintended impacts on adult neurobiology, alcohol drinking, and other phenotypes (Chappell et al., 2013; Yorgason et al., 2016). Second, alcohol exposure targeted the rat adolescent period, involving early adolescence, puberty, and young adulthood (i.e., broadly rat postnatal days [P] 25 to 55). Third, the alcohol exposure procedures were designed to mimic the intermittent, high blood alcohol levels common among binge‐drinking adolescent humans (Kuntsche and Gmel, 2013). Fourth, testing occurred in adulthood, typically P70 or later, to focus on the long‐lasting consequences of adolescent intermittent ethanol (AIE) exposure. Assessments following maturation to stable adult characteristics reduce developmental confounds that muddle multilaboratory replication efforts. The goal of these guidelines was to increase replication and extension of findings across NADIA Consortium components as well as to other alcohol research laboratories.
The NADIA Consortium designed dependent measures to probe a wide variety of behavioral, physiological, cellular, and molecular endpoints. Behavioral outcomes in adulthood, such as cognition, affect, and alcohol drinking, were essential to provide face validity for the AIE models. Next, the Consortium focused attention on mechanistic studies that cannot be performed in humans, using sophisticated neurobiological measures to characterize neural systems altered by AIE exposure and evaluating potential approaches for prevention or reversal of effects. After a brief summary of the behavioral alterations observed in the NADIA studies after AIE, this review focuses on NADIA Consortium findings that detail enduring AIE‐induced neurobiological alterations at the molecular, cellular, and physiological levels that likely underlie these behavioral changes. The use of distinct but overlapping measures, coupled with broad standard procedures, has allowed the NADIA laboratories to generate converging data, provide cross‐validation, and extend findings across a variety of alcohol exposures. The adolescent brain appears to be particularly vulnerable to the effects of binge alcohol exposure as compared to the adult brain, according to comparisons of animals exposed to ethanol (EtOH) during adolescence versus adulthood (Broadwater et al., 2014; Centanni et al., 2014; Fleming et al., 2013; Li et al., 2013; McClory and Spear, 2014; Risher et al., 2013; Vetreno et al., 2014; White et al., 2000, 2002a). While these comparisons provided the foundation for the formation of the NADIA Consortium, the current mandate is to focus on persistent AIE effects on brain and behavior and their underlying mechanisms. Thus, while the specificity of AIE effects as compared to adult exposure provided the foundation for the NADIA Consortium, it is not a focus of this review. This review describes NADIA discoveries with particular emphasis on emerging mechanistic findings in specific brain circuitry underlying AIE‐induced adult psychopathology.
Persistent AIE‐Induced Effects on Behavior
AIE exposure affects a variety of behavioral measures, as summarized below and more thoroughly documented in recent focused NADIA reviews (Crews et al., 2016; Pandey et al., 2017; Spear, 2015, 2016a,2016b, 2018; Spear and Swartzwelder, 2014; Varlinskaya and Spear, 2015).
Alcohol consumption in rodents is known to vary by species, strain, method, and environment (Crabbe et al., 1999; Fritz and Boehm, 2016). Nevertheless, the majority of rodent studies, including those from the NADIA Consortium, report that AIE often, although not always (Moaddab et al., 2017; Nentwig et al., 2019; Toalston et al., 2015; Varlinskaya et al., 2017), promotes adult alcohol drinking in multiple rat strains (Alaux‐Cantin et al., 2013; Broadwater et al., 2013; Gass et al., 2014; Lee et al., 2017; Pandey et al., 2015; Pascual et al., 2009; Rodd‐Henricks et al., 2002; Toalston et al., 2015; Wille‐Bille et al., 2017). These studies use a variety of AIE exposures as well as alcohol consumption paradigms.
For example, adult EtOH drinking assessed by 2‐bottle choice in home cage increased following AIE exposure via self‐administration of sweetened alcohol (Broadwater et al., 2013), via i.p. injection (Pandey et al., 2015), or via a combination of self‐administration and vapor exposure (Criado and Ehlers, 2013).
Similarly, a combined vapor/self‐administration AIE exposure (intermittent pattern of vapor exposure via voluntary drinking 20% unsweetened alcohol in male and female Wistar rats P22 to 62) increased adult EtOH consumption in both sexes, with females drinking more than males (Amodeo et al., 2018).
Further, individual rats that consumed more EtOH during adolescence (high responders) showed the largest AIE‐induced increases in alcohol drinking in adulthood (Amodeo et al., 2017).
AIE drinking or vapor exposure also increases operant responding for EtOH and reduces extinction of EtOH self‐administration (Amodeo et al., 2017; Gass et al., 2014). Thus, across rat strains, laboratories, and routes of administration, AIE often increases adult alcohol drinking.
Another well‐documented effect of AIE is heightened social anxiety in adulthood (Varlinskaya and Spear, 2015), measured via the social interaction test (File and Seth, 2003). This finding is specific to males (Dannenhoffer et al., 2018; Varlinskaya et al., 2014, 2017) and to EtOH exposure during early adolescence (P25 to 45) as compared to late adolescence (P45 to 60) (Varlinskaya et al., 2014).
Enhanced anxiety‐like behavior in adulthood after AIE exposure (via i.p., vapor, i.g., and self‐administration) has also been reported in the elevated plus maze (Kokare et al., 2017; Kyzar et al., 2017; Pandey et al., 2015; Sakharkar et al., 2016), the light–dark box (Lee et al., 2017; Pandey et al., 2015; Sakharkar et al., 2016; Slawecki et al., 2004; Vetreno et al., 2016), the marble‐burying test (Lee et al., 2017), and the open‐field test (Coleman et al., 2014; Vetreno et al., 2014). However, these findings are not universal, perhaps due in part to the induction of disinhibition, which has been reported in adult animals after vapor and self‐administered AIE in several studies (Desikan et al., 2014; Ehlers et al., 2019, 2013a; Gass et al., 2014; Gilpin et al., 2012). Specifically, it is well known that the behavioral expression of anxiety and disinhibition can compete depending on the characteristics of the test situation (Ennaceur, 2014). Thus, evidence supports both AIE‐induced anxiety and disinhibition.
Learning and Behavioral Inflexibility
Investigations of the potential effects of AIE on learning and decision making in adulthood have revealed highly specific cognitive effects. While initial instrumental learning is generally unaffected (Boutros et al., 2016; Gass et al., 2014; Mejia‐Toiber et al., 2014; Risher et al., 2013; Semenova, 2012), operant behavioral inefficiency has been observed after AIE (Miller et al., 2017), and performance is impaired on more complex operant tasks that involve a rule change such as extinction or set‐shifting (Gass et al., 2014) and on nuanced memory tasks such as spatial–temporal object recognition (Swartzwelder et al., 2015).
Similar findings have been reported in spatially based tasks such as the Morris water maze or the Barnes maze: Initial learning is intact, but changing the goal location reveals AIE‐induced deficiencies (Acheson et al., 2013; Coleman et al., 2011, 2014; Vetreno and Crews, 2012; Vetreno et al., 2019).
These findings are interesting and can be interpreted as a loss of executive function and behavioral efficiency (Crews et al., 2016). AIE also has been shown to increase risky choices (Boutros et al., 2014; Schindler et al., 2014, 2016), measured by instrumental responding for a preferred reward, even under conditions where the choice is suboptimal. Both self‐administered and intragastric AIE exposure can enhance conditioning to reward predictive cues in adulthood (Kruse et al., 2017; Madayag et al., 2017; McClory and Spear, 2014; Spoelder et al., 2015), as indicated by approach to the cue (sign‐tracking). Finally, vapor AIE exposure in female rats promoted habit‐based alcohol seeking, indicating a loss of behavioral flexibility (Barker et al., 2017). Together, these findings have led to the hypothesis that AIE promotes behavioral inflexibility: Behavioral choices are biased toward rewards and incentive cues, and behaviors perseverate despite changing circumstances.
“Lock‐in” of an Adolescent Phenotype
Multiple NADIA laboratories have observed that for some dependent measures, AIE induces a persistence of adolescent‐typical behavior or brain function into adulthood. This has been described as a “lock‐in” of adolescent characteristics, as if the adolescent alcohol exposure interrupted or altered neurobehavioral developmental processes, resulting in the expression of adolescent characteristics in adulthood (see Spear and Swartzwelder, 2014, for a review). E
xamples of adolescent characteristics that have been shown to persist into adulthood after AIE include the induction of memory‐related synaptic plasticity (Risher et al., 2015a), local circuit inhibitory processes (Fleming et al., 2012, 2013), increased impulsivity/disinhibition (Desikan et al., 2014; Ehlers et al., 2013a), and the dynamics of fear conditioning (Broadwater and Spear, 2014).
Interestingly, several adolescent‐typical responses to EtOH are observed in AIE‐exposed adults, including elevated EtOH consumption (e.g., Alaux‐Cantin et al., 2013), insensitivity to EtOH on event‐related potential EEG responses (Ehlers et al., 2014), insensitivity to EtOH‐induced motor impairment (White et al., 2002b), increased sensitivity to EtOH‐enhanced social behavior (Varlinskaya et al., 2014), and increased sensitivity to EtOH‐induced impairment of working memory (White et al., 2000).
Importantly, those changes do not appear to be related to tolerance, but rather to enduring physiological, and possibly molecular, alterations.
Collectively, these behavioral and cognitive studies have shown long‐lasting alterations induced by AIE. A similar conclusion has been reached from a recent review of studies in humans and rodents examining predisposing factors that predict adolescent alcohol consumption and the cognitive, behavioral, and neurobiological consequences of adolescent alcohol exposure (Spear, 2018). The animal data, in particular, support the hypothesis that binge‐like alcohol exposure has long‐lasting effects on behavior that may interact with, but are not dependent on, factors that predispose to adolescent drinking.
Conclusion: AIE exposure is sufficient to produce many behavioral characteristics (anxiety, behavioral inflexibility, increased drinking, and altered response to alcohol) observed in humans with alcohol use disorder (AUD). Additional studies are needed to clearly define the contribution of adolescent alcohol abuse to AUD.
Persistent Changes in Adult Molecular Neurobiology Following Aie
NADIA studies have found that AIE persistently changes neuroimmune, neurotrophic, and epigenetic gene regulation and that these are key mechanisms underlying the AIE effects on adult physiology and behavior (for reviews, see Crews et al., 2017a; Crews and Vetreno, 2016; Crews et al., 2016; Kyzar et al., 2016; Pandey et al., 2017).
These mechanisms involving epigenetic regulation of gene expression and noncoding RNA, particularly microRNA, involve signaling across neurons, astrocytes, and microglia that shift transcription, with increases in transcription of proinflammatory genes and reduced transcription of trophic factors.
Several studies report that AIE increases adult neuroimmune signaling through HMGB1, Toll‐like receptors (TLRs), and proinflammatory chemokines and cytokines known to signal in the innate immune system through NFκB transcription mechanisms (Fig. 1).
Moreover, similar increases in gene expression were observed in postmortem brains of humans with AUD (Crews et al., 2017b). While the persistent proinflammatory gene induction by AIE was initially surprising, neuroimmune genes and microglia are known to contribute to brain development at multiple levels (Brenhouse and Schwarz, 2016; Cowan and Petri, 2018; Lenz and Nelson, 2018).
Comparisons of adolescent and adult neuroimmune responses to EtOH and endotoxin (i.e., lipopolysaccharide) suggest that adolescents may have a blunted neuroimmune response (Doremus‐Fitzwater et al., 2015).
Low initial innate immune responses, like those in adolescence, are associated with modest but persistent increases in proinflammatory cytokines that sensitize to repeated exposure, a potential mechanism of the allostatic changes associated with repeated alcohol exposure (Coleman and Crews, 2018; Crews et al., 2017b).
In contrast, large proinflammatory responses, similar to adult responses, trigger initial proinflammatory gene increases that in time shift to increases in other cytokines often associated with wound healing, loss of proinflammatory signals, and desensitized responses (López‐Collazo and del Fresno, 2013; Morris et al., 2014).
Neuroimmune signaling among microglia, astrocytes, and neurons regulates epigenetic enzymes that open or silence chromatin architecture leading to altered gene expression.
Further, neuroimmune signals contribute to brain development through processes such as axon guidance and activity‐dependent synapse development (Boulanger, 2009) that are mediated in part through changes in DNA methylation and histone methylation/acetylation that regulate neurotrophic and neuroimmune gene expression during adolescent brain development (Kyzar et al., 2016).
For example, AIE exposure, but not identical adult EtOH exposure, causes a persistent loss of adult hippocampal dentate gyrus neurogenesis that is associated with increased neuroimmune gene expression (Broadwater et al., 2014). AIE decreases histone acetylation and decreases adult hippocampal brain‐derived neurotropic factor (BDNF), a key trophic factor regulating neurogenesis (Sakharkar et al., 2016; Vetreno and Crews, 2018).
Moreover, AIE‐induced decreases in histone acetylation (H3K9/14) of the BDNF gene promotor and reductions in neurogenesis markers in the hippocampus were normalized by treatment with HDAC inhibitors in adulthood. AIE‐induced histone modifications have been extensively studied in amygdala, where AIE increases HDAC2, a deacetylase that reduces histone acetylation and decreases expression of BDNF (particularly Exon IV), activity‐regulated cytoskeleton‐associated (Arc) protein, and dendritic spine density (Pandey et al., 2015). Interestingly, reductions in BDNF expression in the amygdala are also regulated by increases in global and BDNF gene‐specific H3K9me2 by decreases in lysine demethylases (LSD1) and the neuron‐specific LSD1 + 8a splice variant in adult rat amygdala after AIE (Kyzar et al., 2017). Increased melanocortin and decreased neuropeptide Y activity due to altered histone acetylation in the amygdala may also be important in AIE‐induced anxiety phenotypes in adulthood (Kokare et al., 2017).
HDACs and histone acetyltransferases (HATs) interact and regulate histone acetylation mechanisms (Krishnan et al., 2014), and CREB‐binding protein (CBP) and p300 serve as HATs and regulate histone acetylation. AIE decreases CREB and CBP/p300 levels in the rat adult amygdala via decreases in histone acetylation levels at their promoters (Zhang et al., 2018).
Thus, the persistent AIE‐induced decrease in adult neurogenesis is linked to epigenetic histone repression of BDNF as well as to persistent increases in neuroimmune gene signaling, and AIE‐induced condensed chromatin architecture in the amygdala may be related to increases in HDAC2, decreases in HATs and LSD1, and regulation of the expression of BDNF, Arc, and neuropeptide Y. AIE epigenetic modifications in amygdala gene expression are related to AIE‐increased alcohol drinking and anxiety (Kokare et al., 2017; Kyzar et al., 2017, 2019a; Pandey et al., 2015; Zhang et al., 2018), whereas hippocampal and cholinergic neuron AIE alterations in gene expression have been linked to changes in cognition, synapses, and synaptic physiology (Mulholland et al., 2018; Risher et al., 2015a,2015b; Swartzwelder et al., 2015; Vetreno et al., 2019).
Small miRNA are released in vesicles and signal across cells regulating synaptic plasticity (Cohen et al., 2011). In addition to miRNA involvement in gene expression by targeting mRNA stability, miRNA and other noncoding RNA participate in neuroimmune signaling through TLRs (Coleman et al., 2017; Crews et al., 2017b). Studies performed in adult amygdala reveal that miRNA‐494 interacts with CREB transcription factors and CBP/p300 to regulate anxiety‐like behaviors (Teppen et al., 2016).
Moreover, an antagomir (miRNA blocker) of miRNA‐494 injected into central amygdala increases CBP/p300 and histone acetylation and provokes anxiolytic effects similar to acute EtOH exposure in rats (Kyzar et al., 2019b; Teppen et al., 2016). Another miRNA‐mediated mechanism is the recently discovered activation of TLR7 by miRNA let7. Levels of TLR7 are increased by alcohol exposure contributing to neuroimmune activation (Coleman et al., 2017) and are persistently elevated in adults following AIE (Crews et al., 2017b). Thus, AIE‐induced changes in gene expression appear to involve multiple complex epigenetic mechanisms involving alterations in proinflammatory and trophic factors, as well as genes involved in remodeling synapses and neurocircuitry.
Conclusion: AIE exposure induces long‐lasting, persistent increases in proinflammatory neuroimmune genes as well as epigenetic histone‐ and DNA‐modifying enzymes and miRNA. These alterations in turn may contribute to decreased trophic factor gene expression that impacts the adult brain synaptic transcriptome.
Calli McMurray – SfN
The image is credited to Kyzar et al., eNeuro 2019.
Original Research: Closed access
“MicroRNA-137 Drives Epigenetic Reprogramming in the Adult Amygdala and Behavioral Changes After Adolescent Alcohol Exposure”. Evan J. Kyzar, John Peyton Bohnsack, Huaibo Zhang and Subhash C. Pandey.