The vast majority of newborn neurons are generated in the mammalian brain during perinatal development (Bayer, 1989). This rapid rate of neurogenesis in humans results from an astounding rate of cell division, with approximately 250,000 nerve cells formed every minute and over 80 billion neurons formed in a newborn human, 70 million in a newborn mouse, and 200 million in a newborn rat brain (Bandeira et al., 2009; von Bartheld et al., 2016). Cortical neurons populate their respective regions first, whereas dentate granule neurons of the hippocampus develop later, beginning in utero in humans during the third trimester and in the rodent at birth (Bayer, 1980a; 1980b). Concurrently, the hippocampus also begins to express nicotinic cholinergic receptors that regulate hippocampal neurogenesis and regional neurodevelopment (Naeff et al., 1992; Zhang et al., 1998; Adams et al., 2002). Thus, the human third trimester and the first 10 days of rodent postnatal development are equivalent, and constitute the developmental timeframe known as the brain’s growth spurt, which is characterized by rapid hippocampal maturation and the beginnings of hippocampal cholinergic innervation (Dobbing and Sands, 1979; Matthews et al., 1974; Nadler et al., 1974) (see Figure 2). In fact, unilateral hippocampal ablation during this early postnatal developmental window reduces cholinergic immunoreactivity in the basal forebrain by 40% in adulthood, highlighting the importance of the reciprocal feedback between the hippocampus and basal forebrain during perinatal neurodevelopment (Plaschke et al., 1997). This reciprocal development between the hippocampus and basal forebrain-to-hippocampus cholinergic projections is dependent upon the production and release of nerve growth factor (NGF), which is selectively produced and released by cells targeted by these cholinergic projections (Higgins et al., 1989). NGF binds to tropomyosin receptor kinase A (TrkA) receptors on cholinergic terminals, providing critical signaling feedback that regulates gene expression necessary for cellular differentiation, influencing somal size, and neurite outgrowth and arborization (as reviewed by (Niewiadomska et al., 2011)), thereby providing continual reciprocal maintenance between cholinergic projection neurons with their target regions during development (Li et al., 1995).

The correlation of human third trimester to the first 10 postnatal days of rodent neurodevelopment (Dobbing and Sands, 1979) has engendered technical complications for the development of FASD rodent models with variations in patterns of ethanol administration (e.g., acute, repeated), developmental period of ethanol exposure (e.g., prenatal, neonatal, or perinatal), and route of ethanol administration (e.g., vapor, intragastric intubation, intraperitoneal injection) (for review see (Patten et al., 2014)). In addition, models of FASD must contend with the complications associated with untangling neurological and behavioral outcomes driven by ethanol itself versus alterations in maternal behavior in ethanol-exposed dams, variation in maternal-pup dynamics in ethanol-exposed pups in neonatal split litter models, and other considerations that arise with cross-fostering and artificial rearing. As such, the vast majority of rodent models of FASD use either prenatal (human first and second trimester equivalent) or neonatal (colloquially termed “third-trimester” models) with few studies utilizing models that encompass the entire human three trimesters of pregnancy due to the aforementioned complications. Thus, for the purpose of this review, rodent models of FASD will be referred to generally as perinatal exposure paradigms unless otherwise stated.

The myriad of developmental and pharmacological variations in rodent FASD models complicates translation of findings to the human literature, particularly when results vary by experimental design. For example, the characteristic facial dysmorphology evidenced in children with FASD (i.e., microcephaly, smooth philtrum, small eyes, short nose with a low nasal bridge, and thin upper vermilion) has been specifically linked to ethanol-induced cell death during early embryonic exposure (mouse embryonic days 7–9) (Sulik, 1984) are not evident in rodent neonatal exposure studies that model the human third trimester. These findings highlight alcohol’s unique pathogenic constellation of teratogenic effects that are often dependent on the developmental window of alcohol exposure. However, some consistencies have emerged in the field that are strengthened by their reliability across models despite these technical nuances. For example, reductions in hippocampal volume and cell number as well as deficits on hippocampal-dependent cognitive-behavioral tasks are consistent across various preclinical models of FASD (West et al., 1986; Tran and Kelly, 2003; Gil-Mohapel et al., 2014) and also corroborate findings from humans studies on FASD (Willoughby et al., 2008). This suggests that the hippocampus and related cognitive behavioral outcomes are especially sensitive to the teratogenic effects of ethanol.

The cholinergic system rapidly develops during the early neonatal period in rodents (see Figure 2), and is a key regulator of both hippocampal neuroimmune signaling and neurogenesis (Kaneko et al., 2006; Field et al., 2012; Terrando et al., 2014), suggesting that disruptions in maturation of the cholinergic system may have cascading consequences on adult hippocampal neurogenesis in models of FASD. Perinatal ethanol exposure disrupts basal forebrain cholinergic system development, as a single ethanol exposure on postnatal day (P)7 was reported to decrease basal forebrain cholinergic neurons in adulthood (P70) by 34–42% in both male and female rats, relative to age-matched controls (Smiley et al., 2021).

Perinatal ethanol exposure also induces large-scale induction of apoptotic cascades within 24 h after acute ethanol, with variations in regional sensitivity to induction of cell death cascades corresponding with variations in developmental timing of various brain regions (Ikonomidou et al., 2000). For example, peak ethanol-induced induction of the apoptotic marker cleaved caspase-3 in the basal forebrain is most prominent on P7, corresponding with development of the basal forebrain system (Ikonomidou et al., 2000; Olney et al., 2002; Young et al., 2005; Farber et al., 2010). The persistent loss of basal forebrain cholinergic neurons after neonatal ethanol exposure suggests that 1) ethanol-induced decreases in adult basal forebrain cholinergic neurons may result from caspase-3-mediated apoptotic cell death during critical neonatal developmental windows, and 2) reductions in adult populations of basal forebrain cholinergic neurons after neonatal ethanol exposure do not recover despite long periods of abstinence. These findings have been reproduced across a variety of mammalian species, from mice to primates, suggesting a high level of congruency in this literature. Moreover, neonatal ethanol-induced loss of basal forebrain cholinergic neurons is accompanied by diminished evoked acetylcholine efflux in the adult hippocampus as assessed using in vivo microdialysis, indicating that perinatal ethanol exposure persistently disrupts basal forebrain-hippocampal cholinergic neurocircuitry (Perkins et al., 2015). These findings further highlight that discrepancies in the effects of perinatal ethanol exposure on adult hippocampal neurogenesis may vary across FASD models – particularly prenatal versus postnatal exposure models – as prenatal exposure models miss critical neonatal developmental periods of cholinergic systems, which could subsequently shift the ability of acetylcholine to modulate neuroprogenitors and the hippocampal environmental milieu to regulate adult hippocampal neurogenesis.

Children with FASD have several complications due to large-scale cell death induced by prenatal alcohol exposure coupled with broad shifts in epigenetic regulation of gene expression. Epigenetics is the modulation of gene expression in the absence of alterations to DNA, making it a master director of brain development by altering gene accessibility to transcriptional machinery across time (Podobinska et al., 2017). For example, the most frequently investigated epigenetic modification is changes in patterns of methylation across the genome. Increases in DNA methylation on CpG dinucleotides at gene promoter regions restrict transcriptional machinery’s access to specific genes, thereby reducing transcription and translation into protein. In contrast, removal of these methyl groups at CpG-rich promoter islands increases gene access to transcription machinery, resulting in increased gene transcription. Changes in methylation at various gene loci are a critical part of normal neurodevelopment and can be further modified by environmental exposures, including maternal behavior (Weaver et al., 2004) and alcohol exposure (Kobor and Weinberg, 2011), with increased sensitivity across different periods over the lifespan. Epigenetics, including DNA methylation, therefore has the capacity to alter gene expression with tight temporal regulation during critical developmental periods. These neurological and epigenetic changes during critical brain developmental windows shift the brain’s developmental trajectory in children with FASD, resulting in a variety of social, emotional, cognitive, and behavioral deficits that emerge with age (Kodituwakku, 2009). This suggests several considerations in the context of FASD: 1) ethanol-induced alterations during perinatal development persistently impact development by changing gene transcription, 2) some consequences of perinatal ethanol will emerge over time in conjunction with developmental changes in epigenetic regulation of gene expression, and 3) pharmacological interventions which aim to reverse perinatal ethanol-induced developmental effects will be most effective if they also reverse perinatal ethanol-induced epigenetic changes.

The emergence of ethanol’s cascading consequences on neurodevelopment over time complicates interventions aimed at reversal of neurological and behavioral deficits in models of FASD. In fact, the majority of current pharmacological intervention strategies approved for children with FASD target other neuropsychiatric comorbidities, such as the use of stimulants for the highly co-morbid diagnosis of attention deficit hyperactivity disorder in FASD children, with few treatment strategies aimed to prevent or reverse FASD pathophysiology itself (Martinez and Egea, 2007; Wozniak et al., 2019). These challenges in specifically treating FASD are highlighted in preclinical findings surrounding the cholinergic system. For example, using a third-trimester model (P4-P9), Milbocker and Klintsova (Milbocker and Klintsova, 2021) reported that ethanol reduced cholinergic neuron populations (identified by choline acetyltransferase (ChAT)) in the young adult basal forebrain (P72). This deficit was not recovered with either voluntary exercise, complex environmental housing, or a combination of the two (P30-72). Interestingly, choline supplementation has yielded some success with preclinical studies, suggesting that prenatal choline supplements given in conjunction with ethanol exposure across gestation can mitigate loss of brain weight in models of FASD (Thomas et al., 2009) as well as prevent adult working memory deficits in the Morris water maze (Thomas et al., 2010). Choline supplementation also attenuates deficits in cholinergic muscarinic receptor binding (Monk et al., 2012) and causes broad changes in brain gene regulation including cholinergic content as well as receptor expression and function (Otero et al., 2012). However, while one mechanism of choline supplementation is through enhanced acetylcholine synthesis, choline supplementation also reverses perinatal ethanol’s increases in DNA methylation in the hippocampus (Otero et al., 2012). The ability of choline supplementation to reverse alternations in hippocampal DNA methylation after perinatal ethanol is an exciting finding, but these interactions are complex as choline supplementation exhibited opposing effects in the absence of ethanol, highlighting that the role of choline supplementation in DNA methylation must be taken in context with expression of methyltransferase activity and other epigenetic regulators. To further complicate the mechanism of choline supplementation as a therapeutic in models of FASD, choline also affects lipid metabolism and enhances liver oxidation of fatty acids. This suggests that choline supplementation may affect hippocampal gene transcription indirectly through broader impacts on the body in general. These results highlight the multifaceted mechanisms by which choline supplementation may help mitigate developmental consequences of ethanol, as choline is a precursor for the synthesis of acetylcholine, a methyl donor, influencing epigenetic regulation of gene transcription (Zeisel, 2006), and a regulator of lipid metabolism. Collectively, this suggests that ethanol and choline supplementation effect gene transcription broadly, and hippocampal gene transcription specifically.

The positive preclinical effects of choline supplementation on FASD behavioral and neurological outcomes have given it a spotlight in clinical trials, several of which are ongoing (Wozniak et al., 2020). However, clinical trials have further highlighted that choline supplementation efficacy relies heavily on a preventative and/or early intervention strategy and has not yielded clinical success when implemented later in life (e.g., in school-age children) (Nguyen et al., 2016), suggesting the effective therapeutic window for choline supplementation is narrow. The narrow therapeutic window in FASD remains an ongoing clinical challenge (Idrus and Thomas, 2011), particularly for intervention strategies aimed at mitigating deficits in females with FASD, as they are often diagnosed later in life than males (DiPietro and Voegtline, 2017; Osborne et al., 2018). Thus, sex differences in FASD presentation and biological alterations are important to consider in the search for novel treatments for FASD in older populations.

Adolescence is a period of rapid brain maturation with extensive myelination, synaptic pruning, and neurogenesis, highlighting that remodeling of molecular circuitry is a critical component of this developmental period that parallels rapid behavioral and cognitive growth (Arain et al., 2013). Adolescence is also a period when alcohol experimentation and use is typically initiated across both sexes (Petit et al., 2013). As adolescents are less sensitive than adults to the soporific effects of alcohol, they tend to achieve higher BECs than adults in a single drinking session. This means that adolescents are far more likely than adults to consume alcohol in a binge drinking pattern (Kuntsche and Gmel, 2013), which is defined by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) as consuming 4+/5+ drinks in a 2-h period for women and men, respectively (Alcohol Policy Information System, 2020; Drinking Levels Defined, 2021). In fact, the vast majority of alcohol consumption in adolescents is characterized by intake during weekend binge drinking sessions (Chung et al., 2018), where periods of high levels of alcohol intake are intermittently dispersed with short periods of abstinence. The percentage of individuals engaged in binge drinking sessions escalates across adolescence and into college age, with approximately 4% of eighth grade, 9% of 10th grade, 14% of 12th grade, and 44% of college students reporting recent binge drinking episodes (Wechsler et al., 1995; O’Malley et al., 1998; Johnston et al., 2019). Moreover, adolescent-specific neuromaturation persists in humans through age 25 (Crews et al., 2019), well beyond the legal drinking age, suggesting that these persistent neurodevelopmental disruptions which are continuing to be elucidated in clinical and preclinical studies potentially impact a broad spectrum of the legal drinking population.

The long-term consequences of alcohol drinking in adolescence have primarily been examined through two collaborative consortiums: 1) National Consortium on Alcohol and Neurodevelopment in Adolescence (NCANDA), which evaluates the progressive disruption of neurodevelopment and behavioral alterations with alcohol exposure across adolescence in humans, and 2) the Neurobiology of Adolescent Drinking in Adulthood (NADIA) consortium, which uses rodent models of adolescent intermittent binge ethanol exposure to examine mechanisms underlying ethanol’s long-term molecular and cognitive-behavioral changes in adulthood (Crews et al., 2019). As adolescents who binge drink often continue drinking in adulthood, discerning the discrete effects of adolescent versus adult alcohol exposure can be complex in human studies. Thus, the NADIA consortium has modeled patterns of human adolescent binge drinking in rodents using a paradigm of adolescent intermittent ethanol (AIE) exposure in rats. In this model, ethanol exposure occurs specifically across periadolescent development (P25-P55) via an intermittent dosing regimen that results in the high BECs (>150 mg/dl) that are achieved in human adolescents (Lamminpää et al., 1993; Kraus et al., 2005; White et al., 2006; Donovan, 2009; Patrick et al., 2013; Orio et al., 2018), followed by a period of abstinence to determine the long-lasting, persistent effects of adolescent ethanol exposure on the brain and behavior in adulthood (for a review of the model, see (Crews et al., 2019)). Findings from the NADIA consortium and other rodent models of adolescent ethanol exposure have indicated that the effects of ethanol on the brain and behavior during adolescence are distinct from perinatal or even adult exposure, instead being molded from the landscape of adolescent development where neurocircuitry is being remodeled and refined versus formed. The result is a plethora of cognitive-behavioral and neurobiological changes that persist into adulthood despite abstinence (as reviewed by (Crews et al., 2019)). This section will highlight some of these persistent effects, particularly in relation to adult hippocampal neurogenesis, the cholinergic system, and neuroinflammation.

Neurogenesis during adolescent development is on average four-fold greater than during early adulthood (He and Crews, 2007; Kozareva et al., 2019), and this developmental finding is consistent across mammalian species (Snyder, 2019). High levels of adolescent neurogenesis are thought to reflect a heightened need for neuroplasticity during this period, which corresponds with adolescent-related behavioral changes (Kozareva et al., 2019). Generally, adolescence is characterized by increased exploration, risk-taking behaviors, inhibition of juvenile behavioral patterns, and acquisition of new behaviors essential for transition from parental care to independence as an adult (Spear L. P., 2000). As neurogenesis is an index of neuroplasticity and facilitates pattern separation, spatial learning and memory, and cognitive flexibility, it follows that heightened neurogenesis during adolescence contributes to adaptive behavioral development during this period. However, the increased need for hippocampal neuroplasticity during normal adolescent neurodevelopment also confers a period of neurogenic vulnerability with ensuing consequences on behaviors that rely upon this mechanism of cellular plasticity.

Adolescent binge ethanol exposure causes a loss of basal forebrain cholinergic neurons immediately following the conclusion of ethanol treatment (i.e., P55) that persists well into adulthood (i.e., P220 [165 days post-ethanol]), paralleling AIE-induced lasting reductions in hippocampal neurogenesis and proinflammatory induction. The observed 20–30% reduction in basal forebrain cholinergic neuron number following adolescent binge ethanol exposure is consistent across both mouse and rat studies (Coleman et al., 2011; Vetreno and Crews, 2018; Macht et al., 2020a; Vetreno et al., 2020; Crews et al., 2021) in both sexes (Vetreno and Crews, 2018). However, unlike in models of FASD, the reduction in cholinergic neuronal markers after adolescent binge ethanol exposure is preventable and reversible with exercise (Vetreno and Crews, 2018) as well as with repeated treatment with the cholinesterase inhibitor galantamine (Crews et al., 2021) and the non-steroidal anti-inflammatory compound indomethacin (Vetreno and Crews, 2018). Restoration of basal forebrain cholinergic neurons after AIE also restores hippocampal neurogenesis, highlighting the reciprocal dynamic between these systems (Macht et al., 2021). These findings suggest not only that the persistent loss of cholinergic anti-inflammatory feedback after AIE is a central mechanism in the long-term induction of hippocampal proinflammatory signaling cascades, but also that proinflammatory signaling plays a role in AIE-induced cholinergic pathology more locally within the basal forebrain, evidenced by AIE increased phosphorylation of NF-κB p65 within cholinergic neurons (Vetreno et al., 2020).

One could assume that loss of expression of ChAT immunoreactivity after AIE – as with findings in fetal alcohol models – suggests cell death of this neuronal population. However, this conclusion from the perinatal literature is fueled by 1) concurrent induction of caspase activity of these neurons during critical neurodevelopmental windows during the brain’s growth spurt, and 2) failure to restore ChAT immunoreactivity with pharmacological or environmental interventions (Ikonomidou et al., 2000; Olney et al., 2002; Young et al., 2005; Farber et al., 2010; Smiley et al., 2021). Findings from AIE do not parallel the fetal alcohol field in this regard. For example, there is no loss of total neuronal number in the basal forebrain after AIE, and expression of cholinergic cells can be recovered with exercise or pharmacological interventions (Vetreno and Crews, 2018; Vetreno et al., 2020; Crews et al., 2021). While neurogenesis could restore cell loss due to cell death, this process is highly confounded by development, and shortly after P10 in rodents, is solely restricted to the subventricular zone and the dentate gyrus of the hippocampus. This suggests that AIE-induced loss and restoration of cholinergic neurons in the basal forebrain is not due to either cell death or spontaneous adult neurogenesis in this region. Rather, the ability to restore ethanol-induced loss of expression of basal forebrain cholinergic neurons in adulthood after adolescent but not perinatal ethanol exposure suggests different mechanisms underlying these long-term changes in cholinergic cell expression. The groundbreaking discovery that cholinergic neurons can be recovered after adolescent but not perinatal ethanol exposure reveals a unique mechanism of neuroplasticity that is centered on epigenetics (Vetreno et al., 2020).

AIE persistent induction of neuroimmune genes as well as reduction in hippocampal neurogenesis corresponds with impairments in a variety of learning and memory tasks, including discrimination learning (Pascual et al., 2007) and reversal learning in males (Crews et al., 2016; Vetreno et al., 2018; Crews et al., 2019). In fact, not only do these neurogenic deficits persist into middle age despite abstinence (Vetreno et al., 2014; Crews et al., 2016), but AIE similarly impairs performance in cognitive flexibility-related tasks in male and female rats long into middle age (Macht et al., 2020b) suggesting that neither AIE-induced deficits in neurogenesis nor AIE-related cognitive flexibility deficits recover with abstinence alone. However, several intervention strategies have emerged that can prevent and/or restore both adult neurogenesis and behavioral flexibility deficits in adulthood in rats exposed to AIE. For example, pre-treatment with the non-steroidal anti-inflammatory drug indomethacin blocks AIE induction of hippocampal inflammatory cascades, restores expression of basal forebrain cholinergic neurons, restores hippocampal neurogenesis (as evidenced by increases in DCX+ immunoreactivity), and restores behavioral deficits (Vetreno and Crews, 2018; Vetreno et al., 2018). The cholinesterase inhibitor galantamine similarly blocks and reverses AIE-induced neuroimmune gene induction as well as blocks and reverses adolescent ethanol-induced loss of neurogenesis (Macht et al., 2021). These findings suggest that AIE-induced reductions in the cholinergic system are a central mechanistic player in both hippocampal neuroinflammatory increases as well as the loss of adult hippocampal neurogenesis and persistence of behavioral flexibility deficits (see Figure 5). Adolescent development therefore presents a unique period of vulnerability to the detrimental effects of binge ethanol on adult behavioral and cellular plasticity deficits in the neurogenic niche.

During the adolescent developmental period, changes are governed by alterations in epigenetic regulation of cholinergic and neuroimmune genes, driving an environmental imbalance in the hippocampal environmental milieu which is unfavorable to the successful adult birth, maturation, and integration of newborn hippocampal neurons. This suggests that in the absence of intervention, both adolescent ethanol-induced loss of neurogenesis (Vetreno and Crews, 2015), basal forebrain cholinergic neuron expression, and cognitive-behavioral deficits (Macht et al., 2020b) persist long into adulthood, suggesting a possible permanence of these neurodevelopmental alterations. This highlights the necessity for active interventions following adolescent ethanol exposure to restore both hippocampal neurogenesis and cognitive function. Surprisingly, as the loss of basal forebrain cholinergic neurons is mediated by epigenetic mechanisms, restoration of cholinergic function after adolescent ethanol has the potential to be restored by reversing epigenetic suppression of the cholinergic phenotype. Collectively, these results suggest enhanced sensitivity of neuroprogenitors to ethanol during adolescence. The persistence of these effects suggest that this period confers a unique developmental susceptibility to ethanol with potential long-lasting consequences on hippocampal neurocircuitry and behavior. However, the reversibility of these factors through voluntary exercise gives hope for interventions aimed at restoring these deficits in adulthood.

Alcohol use disorder (AUD) is highly prevalent in Western society, with estimates of up to 29% of adults suffering from AUD at some point in their lifetime (Grant et al., 2015). This disorder is characterized by patterns of high levels of alcohol intake despite adverse social, occupational, economic, and health consequences. While AUD has historically been more prevalent in adult males, prevalence of AUD in adult females has been rapidly rising over the last decades (Roberta et al., 2017). The issue of AUD has become increasingly prevalent in the face of the COVID-19 pandemic as studies estimate that ∼24% of individuals reported increases in alcohol intake irrespective of prior diagnosis of AUD, and 17% of previously abstinent individuals with AUD relapsed during pandemic lockdowns (Kim et al., 2020). These increases in adult binge drinking escalate over time, with 1.19-fold greater odds of individuals binge drinking for every week at home (Weerakoon et al., 2021). Adult binge alcohol consumption is concerning as it results in a host of neurological changes and damage, including increases in neuroinflammatory factors as well as decreases in hippocampal neurogenesis and some subtle changes in the central cholinergic system. Therefore, understanding the long-term neurobiological consequences to binge drinking in adulthood is critical to intervention strategies, and of escalating importance in today’s society.

Although neurogenesis was originally thought to be isolated to the neonate, Altman and Das (Altman and Das, 1965) revolutionized this view with their 1965 seminal findings. It is now estimated that up to 6% of granular cell neurons are replaced every month in adulthood in rodents as a result of hippocampal neurogenesis (Cameron and Mckay, 2001). Although the prevalence and function of adult neurogenesis in humans is an ongoing scientific debate centered on technical discrepancies (for review see (Kempermann et al., 2018)), rodents have routinely demonstrated quantifiable neurogenesis in adulthood with emerging work centered instead on elucidating the functional roles of neurogenesis in relation to hippocampal circuitry and behavior (van Praag et al., 2002; Clelland et al., 2009; Garthe and Kempermann, 2013). Collectively, results seem to indicate that these new neurons play critical roles in hippocampal physiology and neuronal plasticity, and as with other developmental periods, adult newborn neurons are sensitive to the effects of ethanol.

Preclinical studies indicate that adult chronic binge ethanol exposure decreases hippocampal neuroprogenitor cell proliferation and neurogenesis (Nixon and Crews, 2002) even in models of moderate alcohol blood ethanol concentrations (Anderson et al., 2012), although the severity of effects increases with prolonged dependence (Richardson et al., 2009). However, these results become more complex when comparing these models of adult AUD to adolescent intermittent exposure paradigms as there are important distinctions in the models used, particularly in relation to ethanol dependence and withdrawal symptoms. For example, 4-day binge and liquid diet paradigms which model human AUD result in physical dependence to ethanol in conjunction with pronounced withdrawal symptoms (Morris et al., 2010). Models of AIE do not result in dependence or severe withdrawal symptoms. In fact, when ethanol is administered intermittently across adulthood (P70-90), there are no reductions in hippocampal neurogenesis, unlike in adolescent intermittent ethanol models (Broadwater et al., 2014). This suggests that intermittent ethanol exposure in adulthood is not sufficiently severe to induce long-term neurogenic deficits, and instead models of AUD, which result in physical dependence and/or severe withdrawal systems, are necessary for inducing deficits in adult hippocampal neurogenesis after adult ethanol exposure (Table 1).

Adult ethanol-induced suppression of hippocampal cell proliferation is further complicated by withdrawal periods, as there is some evidence for a brief burst in cell proliferation 1 week into abstinence (Nixon and Crews, 2004). However, this effect appears to be insufficient to recover neurogenesis as 2 weeks of abstinence is insufficient to recover ethanol-induced loss of DCX immunoreactivity after 1 month of ethanol self-administration (Stevenson et al., 2009). Moreover, there is some evidence that loss of neuroprogenitor proliferation after binge ethanol is more dramatic in females than males (Maynard et al., 2018), and that this decrease in female progenitors is similarly due to greater sensitivity to ethanol’s impairment in the availability of trophic support. Unlike perinatal or adolescent binge alcohol exposure, adult binge alcohol exposure does not increase cellular apoptosis in the adult dentate gyrus (Obernier et al., 2002), suggesting a divergence of mechanisms between developmental and adult alcohol impairments in hippocampal neurogenesis. However, adult binge alcohol does induce necrotic cell death in the hippocampus with females again being particularly sensitive to this effect, resulting in greater loss in hippocampal cell number after binge alcohol specifically in females (Maynard et al., 2018). This suggests that although adult binge ethanol exposure reduces neurogenesis in males and females, this effect could be particularly devastating in females due to an inability to repopulate the granular cell layer over time. In support of this, some recent findings suggest that recovery of neurogenesis after recurrent binge adult alcohol exposure by short-term voluntary wheel running (3 days) is insufficient to recover granular layer cell loss in females (West et al., 2019). Therefore, alcohol-induced granular cell layer loss in females may require more prolonged or intensive therapeutic intervention strategies (for summary, see Figure 6).

The basal forebrain cholinergic system is profoundly affected by ethanol during early development, with both perinatal and AIE exposure reducing basal forebrain cholinergic neuron expression in addition to decreasing cholinergic regulation of the hippocampus. However, adult alcohol exposure does not produce as rapid or robust an effect on the basal forebrain cholinergic system. Just as intermittent ethanol exposure across adolescence but not adulthood produces deficits in adult hippocampal neurogenesis (Vetreno et al., 2018), adolescent but not adult intermittent ethanol exposure reduces basal forebrain ChAT immunoreactivity (Vetreno et al., 2014). Even more dramatically, 20 days of repeated adult binge ethanol exposure in adulthood is still insufficient to reduce basal forebrain cholinergic neuron numbers (Vetreno et al., 2014). Only studies that have employed a forced sole-source ethanol drinking paradigm of 12+ weeks have shown ethanol-reduced basal forebrain cholinergic neuron expression in adults (Arendt et al., 1988b), indicating that these cells are far less susceptible to ethanol-induced damage in adulthood. However, chronic adult ethanol consumption does impact axonal projections, with evidence of decreased cholinergic innervation of the hippocampus (Cadete-Leite et al., 1995). This suggests that even in the absence of reductions in cholinergic cell numbers, chronic adult ethanol exposure can reduce cholinergic projections resulting in impaired function at target regions, including the hippocampus. In support of this, in in vivo microdialysis studies in adults after long-term 3-month ethanol exposure in drinking water, acetylcholine efflux in the hippocampus was significantly reduced by 57%, relative to controls (Casamenti et al., 1993). This effect was reversible with abstinence, suggesting a greater degree of plasticity in cholinergic systems in adulthood, and increased recoverability following ethanol exposure.

Chronic ethanol exposure in adulthood can also disrupt cholinergic receptors, but this finding is complex and largely depends on subtype. For example, 10 weeks of ethanol consumption decreases hippocampal nicotinic receptor expression independently from altering binding affinity (Robles and Sabriá, 2008). In contrast, rodent models using a 28-week liquid diet of ethanol have not reported alterations in number of muscarinic receptor subtypes in the hippocampus (Rothberg et al., 1993, Rothberg et al., 1996). Despite evidence suggesting that there is no impact of ethanol on binding affinity or number of muscarinic receptor subtype, models of chronic ethanol exposure in adulthood do impair acetylcholine-mediated evoked electrophysiological post-synaptic potential responses in pyramidal cells of the CA1 region of the hippocampus, suggesting impaired cholinergic function in this region (Rothberg and Hunter, 1991). Cholinergic activation of pyramidal cells is thought to be mediated through muscarinic receptors, as these effects can be blocked by the muscarinic atropine but not nicotinic agonists (Cole and Nicoll, 1984; Valentino and Dingledine, 2021). This reduced hippocampal cellular responsivity to acetylcholine is postulated to be due to impaired intracellular transduction mechanisms following cholinergic muscarinic receptor activation, suggesting that chronic ethanol may alter muscarinic receptor function in the absence of overarching loss of receptor number. As proliferation of neuroprogenitor pools is dependent on cholinergic activation of muscarinic receptors and subsequent mobilization of intracellular signaling cascades (Ma et al., 2000), these findings suggest that alterations in responsivity to acetylcholine in the hippocampus could underlie some of the deficits in neuroprogenitor proliferation evidenced after chronic ethanol exposure in adulthood.

The loss of hippocampal nicotinic receptor expression could underlie sensitivity to neuroinflammatory induction after chronic ethanol in adulthood, as microglial nicotinic α7 receptors play critical negative feedback role for proinflammatory cascades. Thus, loss of activation at nicotinic α7 receptors is suggestive of a loss of anti-inflammatory feedback and chronic induction of proinflammatory signaling cascades in the brain of individuals with AUD. In support of this concept, an increase in neuroinflammation in post-mortem human brains with AUD is one of the most prevalent clinical findings (Coleman and Crews, 2018), and preclinical models further suggest that neuroinflammation is tightly coupled to continuation of drinking behaviors in nonhuman primates (Beattie et al., 2018). Preclinical models have replicated these results: 6 months of ethanol treatment followed by 2 months of withdrawal in male rats resulted in long-term induction on the activated microglial marker CD11b and the cytokine IL-15 (Cruz et al., 2017). More recent evidence suggests that females may be even more sensitive to this effect as a 4-day binge is sufficient to increase microglial number and frequency of activated phenotypes, indicated by increases in microglia expressing major histocompatibility complex II (MHC II) in the hippocampus of female but not male rats (Barton et al., 2017). These findings support a growing body of clinical and preclinical findings indicating that females may be more sensitive to ethanol-induced damage in adulthood (Alfonso-Loeches et al., 2013).

The effect of ethanol on hippocampal neuroinflammation can be magnified under conditions which challenge the immune system, such as a LPS or polyI:C innate immune challenge. Ten days of intragastric administration of ethanol in mice potentiates proinflammatory responses in brain to a lipopolysaccharide-TLR4 innate immune challenge which included CCL2, COX-2, gp91^phox NADPH oxidase subunit, TNFα, and IL-1β as well as greater morphological changes in microglia (Qin et al., 2008). Similarly, a polyI:C-TLR3 challenge after 10 days of ethanol administration also results in an exaggerated increase in the microglial marker ionized calcium binding adaptor molecule 1 (Iba1) in the dentate gyrus of the hippocampus (Qin and Crews, 2012). These neuroinflammatory inductions after ethanol were also associated with reductions in the newborn neuronal marker DCX and greater induction of necrotic and apoptotic cell death in the hippocampus, further highlighting that induction of neuroinflammation, sensitivity to activation of cell death cascades, and loss of neurogenesis are tightly coupled. In fact, in studies of rat hippocampal brain slice cultures, blocking IL-1β with either an antagonist or neutralizing antibody blocked adult ethanol inhibition of hippocampal neurogenesis (Zou and Crews, 2012). Neuroimmune signaling involves feed-forward processes that increase expression of proinflammatory agonists and receptors, particularly TLR receptors. Inhibition of proinflammatory signaling at multiple components of the amplification process are effective. These findings highlight that neuroinflammation is a particularly crucial mediator of neurogenic deficits in adulthood.

Therapeutics under investigation for AUD often exhibit anti-inflammatory components. Given the critical role of the cholinergic system in regulating anti-inflammatory feedback, it is unsurprising that many of these restorative interventions center around the cholinergic systems. Rodent and human studies suggest successful therapeutic targets at both cholinergic muscarinic M4 receptors (Walker et al., 2020) and nicotinic α7 and α7β3 receptors through varenicline (Witkiewitz et al., 2019), and since the late 1980s, rat models of AUD have even been treated with cholinergic-rich brain transplants (Arendt et al., 1988a). While the majority of therapies for AUD focus on reducing relapse behaviors and overall ethanol intake, the ability to reverse molecular cascades involved in cognitive deficits after long-term ethanol use is another important consideration. The mechanisms underlying the efficacy of cholinergic-based therapeutics continue to be investigated for use in alcohol-related disorders, but an emerging theme is their regulation of ethanol-induced neuroinflammation. Hippocampal neuroinflammation is a critical long-term consequence of adult ethanol intake, partially through its mediation of caspase-activated apoptotic cascades and reductive consequences on hippocampal neurogenesis (Liu et al., 2021). In fact, targeting cholinergic systems in adulthood after ethanol use may be effective not through direct increases in activation of cholinergic circuits, but rather indirectly through their mediating factors on both neuroinflammation and neurogenesis. To emphasize this point, targeting ethanol’s induction of IL-1β is sufficient for restoration of hippocampal neurogenesis in adulthood in rodents (Zou and Crews, 2012). This suggests that ethanol induction of neuroinflammatory markers and hypersensitivity of inflammatory systems in adulthood is a central mediator to the loss of hippocampal neurogenesis.

These mechanisms linking neuroinflammation and neurogenesis are particularly important when examining sex differences as human females are more likely than men to experience organ damage following long-term alcohol use, including greater brain damage in general and more specifically loss of hippocampal volume (Agartz et al., 1999; Hommer et al., 2001; Mann et al., 2005; Sharrett-Field et al., 2013), and are more likely to need active interventions beyond abstinence than males. For example, preclinical models indicate that abstinence is insufficient for granule cell layer recovery in females in the absence of an intervention such as voluntary exercise (Maynard and Leasure, 2013). Voluntary exercise after adult ethanol exposure restores the proinflammatory-trophic balance in the hippocampal environmental milieu – a balance which exhibits greater ethanol-induced disruptions in females than males (Maynard et al., 2018). These alarming emerging findings suggest that examining the mechanisms for reversing ethanol-related brain damage in females is becoming an increasingly critical area of investigation as females close the gender gap in AUDs.