Alcohol (ethanol) is one of the most used and misused drugs in the world (Ferreira and Willoughby, 2008) resulting in great personal and financial loss (Manthey et al., 2021). Recent data suggest that alcohol use is increasing due to the COVID-19 pandemic in most age groups including adolescents (Pollard et al., 2020) and the increased alcohol use in general is resulting in greater alcohol-related deaths (White et al., 2022). While it is often reported that adolescents consume alcohol at high levels and in a dangerous, binge fashion (Patrick and Terry-McElrath, 2019; U.S. Department of Health and Human Services, 2019), current world health concerns have likely exacerbated adolescent alcohol use and misuse (Dodge et al., 2021). Consequently, understanding the impact of binge alcohol use during adolescence across the lifespan is a critical public health need.

Alcohol consumption during adolescence can have life-long consequences. For example, chronic intermittent ethanol (CIE) during adolescence will block the age-related increase in the hypnotic effects of high dose ethanol (Silvers et al., 2003; Matthews et al., 2008) and reduce ethanol-induced hypothermia (Swartzwelder et al., 1998; White et al., 2002). Furthermore, CIE during adolescence can produce effects that last into young adulthood. For example, CIE during adolescence can produce anxiety-like behaviors that last into adulthood (Coleman et al., 2014; Van Skike et al., 2015; Varlinskaya et al., 2015; Vetreno et al., 2016; Kokare et al., 2017; Lee et al., 2017). However, the vast majority of these studies have only used male subjects and there are a limited number of studies that have included both female and male subjects (see Robinson et al., 2021 for a review). For example, male rats, but not female rats, exposed to chronic ethanol early in adolescence have a social anxiety-like phenotype (Varlinskaya et al., 2015). Furthermore, both male and female subjects exposed to ethanol early in adolescence have increases in anxiety-like behavior in the elevated plus maze, while only males have an anxiety-like phenotype following ethanol exposure later in adolescence (Towner and Varlinskaya, 2020). Finally, a recent study has shown that chronic exposure to ethanol during adolescence can produce a sex-specific effect on the elevated plus maze, but this effect is driven by an increased amount of time on the open arms in females (Healey et al., 2021). Conversely, other studies have found minimal effects of chronic ethanol exposure during adolescence on anxiety-like behaviors (Amodeo et al., 2018; Gamble and Diaz, 2020). Given the non-uniform results on the sex-specific effects of CIE on anxiety-like behavior coupled with the lack of studies investigating the impact of CIE on anxiety-like behaviors late in life, additional research in this area is needed.

Chronic intermittent ethanol exposure (CIEg) during adolescence may also impair cognition (Reitz et al., 2021), specifically behavioral flexibility, in young adulthood (Coleman et al., 2014; Gass et al., 2014; Barker et al., 2017; Fernandez et al., 2017; Contreras et al., 2019; Sey et al., 2019; Macht et al., 2020). Behavioral flexibility, the ability to learn something new in the face of old learning contingencies, has been identified as a critical cognitive measure that correlates with ethanol consumption in abstinent alcoholics and predicts ethanol consumption in non-human primate models (Shnitko et al., 2020). However, while several studies have investigated if CIE during adolescence impairs cognition and behavioral flexibility in young adulthood, only a single study investigated if CIE during adolescence impairs behavioral flexibility beyond young adulthood into older life stages (Matthews et al., 2022). In addition, few studies have investigated sex differences in cognition and behavioral flexibility in late adulthood or in aging following CIE during adolescence.

The average age of the world’s population is increasing, and it has been predicted by the year 2050, the number of adults over the age of 65 will double. Older adults continue to consume alcohol often in a dangerous binge pattern (Keyes, 2023) and it has been suggested that understanding the impact of alcohol use in the older population is a critical public health concern (Matthews and Koob, 2023; Matthews and Rossmann, 2023; White et al., 2023). Unfortunately, while much research has investigated the lasting behavioral effects of CIE during adolescence into young adulthood (see Crews et al., 2019 for review), little is known how CIE during adolescence impacts aged animals.

Two previous studies from our laboratory subjected male rats or male and female rats to CIE during adolescence and then tested them on a variety of behaviors several times over the next 18–22 months (Matthews et al., 2017, 2022). In the first study (Matthews et al., 2017), CIE administration during adolescence produced both long-lasting (over 500 days after CIE during adolescence) tolerance to a high dose ethanol challenge, as measured by sleep time, while also producing sensitization to ethanol-induced spatial memory impairments, an impairment likely driven by altered hippocampal function (see Van Skike et al., 2019 for a review of this literature). In the second study (Matthews et al., 2022), CIE during adolescence impaired behavioral flexibility in male and female rats but the pattern of behavior was different between the two sexes. Specifically, CIE during adolescence interacted with age in male rats to impair behavioral flexibility primarily later in life and only in subjects administered 5.0 g/kg ethanol during the adolescent exposure period while CIE during adolescence impaired behavioral flexibility across the lifespan in female subjects. Given the brain and hippocampus are still developing during adolescence (Bayer et al., 1982), these data suggest that exposure to ethanol in a chronic binge-like fashion during adolescence may produce changes which alter behavior across the entirety of the lifespan. However, these studies left several important issues unanswered. First, there was limited investigation into how CIE during adolescence impacted behavior in females later in life. Second, the studies did not investigate if CIE during adolescence produced a long-lasting anxiety-like effect in either male and/or female rats. Third, it is unknown if cognitive performance to a novel test late in life is differentially impacted by sex of subject and CIE during adolescence. Finally, no corresponding neurobiological measure was assessed to gain information into possible mechanisms mediating the reported behavioral changes.

Microglia are innate glial cells that play a variety of roles in the central nervous system including in neurodevelopment, synaptic pruning and plasticity (see Salter and Stevens, 2017 for review). Microglia also react to insults such as those achieved with binge-like ethanol administration (see Melbourne et al., 2021 for review). For example, postmortem analysis of brains from humans with an AUD have shown reactive microglia (He and Crews, 2008) and this effect may be age dependent (Carlson et al., 2023). We have shown that alcohol exposure during adolescence causes a microglia reaction (McClain et al., 2011; Peng and Nixon, 2021) that persists at least a month if not several months according to our adult models (Obernier et al., 2002; Nixon et al., 2008; see also Melbourne et al., 2021 for review). Therefore, it is possible that exposure to ethanol during adolescence may persistently alter microglia phenotype and state later in life.

The current project is designed to investigate if CIE during adolescence in both female and male rats produce alterations in anxiety-like behavior across the lifespan and impairs cognition, including behavioral flexibility, 19 months following CIE exposure during adolescence. Finally, as microglia effects can be long lasting, we used autoradiography for [³H]PK11195 binding to the translocator protein 18 kDa (TSPO) to measure neuroimmune activation and microglial reactivity (Chen and Guilarte, 2008). The TSPO is expressed on the outer membrane of mitochondria and therefore contributes to various mitochondrial functions such as respiration, opening of the mitochondrial permeability transition pore and putatively, cholesterol transport, for which it is named (Chen and Guilarte, 2008; Rupprecht et al., 2022). Autoradiography for TSPO has long been used to identify neuroimmune reactivity and especially microglia reactivity following insult (Chen and Guilarte, 2008). [³H]PK11195 binding has been a highly sensitive indicator of microglia effects in alcohol models (Marshall et al., 2013; Tyler et al., 2019) and one that persists at least months after alcohol exposure (Obernier et al., 2002). Therefore, we chose this approach to increase our likelihood of detecting long-term effects in brain tissue as microglia reactivity increases with aging, neurodegeneration, and excessive alcohol use (Carlson et al., 2023). Accordingly, [³H]PK11195 was examined in three brain regions important for spatial learning (entorhinal cortex, dentate gyrus, and CA1 region of the hippocampus) from female and male ethanol treated animals.

Adolescence represents a unique time period where significant brain maturation occurs (Bayer et al., 1982). In addition to changes in the nervous system, adolescence is a time when many individuals begin to experiment with new behaviors including alcohol consumption. Unfortunately, alcohol consumption in this age group is often high and occurs in a dangerous binge pattern (Patrick and Terry-McElrath, 2019). Animal research has been necessary to determine the impact of binge-like ethanol exposure in adolescent rats and has demonstrated significant negative effects, including increases in anxiety-like behaviors (Coleman et al., 2014; Van Skike et al., 2015; Varlinskaya et al., 2015; Vetreno et al., 2016; Kokare et al., 2017; Lee et al., 2017), impairments in cognition (Coleman et al., 2014; Gass et al., 2014; Barker et al., 2017; Fernandez et al., 2017; Contreras et al., 2019; Sey et al., 2019; Macht et al., 2020) and alterations in microglia reactivity (McClain et al., 2011, Grifasi et al., 2021; Melbourne et al., 2021; Silva-Gotay et al., 2021). In addition, previous research has shown that some effects of binge-like ethanol exposure during adolescence can last beyond the immediate time following exposure and into young adulthood (for review see Crews et al., 2019). However, little is known if these effects are transitory or if they last throughout most of the lifespan. Using both female and male rats, we provide evidence that chronic binge-like ethanol exposure during adolescence produces initial anxiety-like effects in male, but not female, rats. However, anxiety-like effects resolved as they were not found in adult or aged subjects of either sex. In addition, we provide data revealing a subtle, yet significant, impairment in reversal learning in aged male rats due to CIEg exposure during adolescence, an effect that remarkably was found several hundred days following the ethanol exposure during adolescence. Furthermore, CIEg during adolescence did not produce persistent microglia reactivity in either the entorhinal cortex, dentate gyrus or CA1 region of the hippocampus in male or female rats. Finally, we report that microglia reactivity was greater in female aged rats compared to male age rats in the entorhinal cortex but not the dentate gyrus or CA1 region of the hippocampus.

Chronic intermittent ethanol during adolescence has been shown to increase anxiety-like behaviors in rodents (Coleman et al., 2014; Van Skike et al., 2015; Varlinskaya et al., 2015; Vetreno et al., 2016; Kokare et al., 2017; Lee et al., 2017; Healey et al., 2021; See Crews et al., 2019 for review). Specifically, CIEg during adolescence in male rats produces a significant anxiety-like effect in the elevated plus maze that can last for several weeks (Pandey et al., 2015; Van Skike et al., 2015; Kokare et al., 2017; Kyzar et al., 2017), however increased in anxiety-like behavior is not always found (see Towner and Varlinskaya, 2020 for a review of this field). While it has been reported that CIE can produce a sex-dependent increase in social anxiety behavior (effect in males significantly greater than in females) when subjects are tested in young adulthood (Varlinskaya et al., 2015) few studies have investigated sex-dependent effects of CIE during adolescence. For example, CIEg during adolescence will produce differential behavioral effects on the elevated plus maze that are sex dependent (Healey et al., 2021). Specifically, ethanol exposure during adolescence results in increased open arm time in female rats, but not male rats at PND 70, suggesting altered anxiety-like effects due perhaps to a blunted or exaggerated response in females. Furthermore, late adolescence ethanol exposure also produces sex-specific effects whereas males, but not females, have an anxiety-like phenotype while early adolescence ethanol exposure produces an anxiety-like effect in both sexes (Towner and Varlinskaya, 2020). However, the sex-specific effect in anxiety-like behavior due to adolescent ethanol exposure has not always been reported (Amodeo et al., 2018; Gamble and Diaz, 2020). The current work helps clarify some of these issues while also producing additional questions that require further research. First, the results from the current study support previous research that demonstrated CIE throughout the majority of adolescence can result in sex-specific effects where male subjects display an anxiety-like effect while females do not display an anxiety-like effect. Second, the results provide important information concerning the temporal limitations of any anxiety-like effect produced by CIEg during adolescence. Specifically, we did not find a significant anxiety-like effect in the open field or elevated plus maze on PND 244 or PND 579. Third, any resultant residual anxiety-like effect that does exist late in life will be very subtle, requiring a large number of animals to have sufficient statistical power. Finally, the natural increase in anxiety-like behavior over the course of normal aging (Imhof et al., 1993) and present results with males) will make teasing apart any long lasting anxiety-like effect late in life by CIEg during adolescence difficult.

The impact of CIE on cognition late in life has not been extensively investigated. While previous work has shown that CIE can impair cognition in young adulthood (for review see Crews et al., 2019) or into middle age (Reitz et al., 2021), we are aware of only two studies that investigated the effect of CIE during adolescence in aged rats (Matthews et al., 2017). In the first study, male rats were exposed to ethanol or to saline during adolescence and then tested over a 500+ day longitudinal study. CIE during adolescence coupled with an ethanol challenge at certain time points during the lifespan resulted in delayed spatial learning, as demonstrated by swim latency to the platform. However, the effect of CIE in female subjects was not determined nor was the impact of CIE on other cognitive measures, such as behavioral flexibility (Matthews et al., 2017). In a more recent project (Matthews et al., 2022), CIEg during adolescence in both female and male subjects resulted in altered behavioral flexibility, but the pattern of the impairment was sex dependent. Specifically female subjects administered 5.0 g/kg ethanol had impaired behavioral flexibility throughout their life (a main effect of ethanol exposure during adolescence) while males administered 5.0 g/kg ethanol primarily had impaired behavioral flexibility only later in life (a significant interaction of ethanol dose and aging).

In the current project, CIEg during adolescence in males produced alterations in spatial learning and impairments in behavioral flexibility late in life, but did not alter spatial learning nor behavioral flexibility in females late in life. Previous work has demonstrated that CIE during adolescence can produce impairments in the swimming version of the radial arm maze task (tested at ∼115 PND) and novel object recognition (tested at ∼210 PND) in both male and female rats (Galaj et al., 2019) and in male rats during middle age (Reitz et al., 2021). The current work supports the recently reported impairment in behavioral flexibility found in male rats (Matthews et al., 2022). However, the lack of an impairment in behavioral flexibility in the female subjects in the current work was surprising. Several possibilities exist to explain the differential results. First examination of Figure 5C and D from Matthews et al. (2022) shows that female subjects had similar performance on the behavioral flexibility test late in life regardless of the ethanol dose (or lack thereof in the control subjects). This suggests that CIEg during adolescence in female subjects produces impairments in female subjects early in life but the normal aging process occludes the effect late in life. Second, animals in the current project were only tested in the water maze once late in life instead of multiple times throughout life as previously done (Matthews et al., 2022). Future research is needed to better understand the impact of CIEg during adolescence on cognitive performance in female subjects. In addition, research is needed to understand the effect of CIEg during adolescence on episodic type tasks in the water maze [i.e., spatial working memory tasks (Foster, 2023)].

Alcohol-induced increases in microglia reactivity as determined by [³H]PK11195 autoradiography for TSPO expression, was not detectable 20-months following CIEg administration in either male or female subjects. Alcohol exposure produces microglia reactions in adolescents (McClain et al., 2011; Vetreno and Crews, 2012; Barton et al., 2017; see also Melbourne et al., 2021 for review), but assessment of TSPO has only been reported in abstract form (Van Doorn et al., in preparation). However, we did find increased microglia reactivity in female rats compared to male rats although the effect was selective to brain region in that [³H]PK11195 binding was greater in the entorhinal cortex compared to either the dentate gyrus or CA1 region of the hippocampus. This finding may reflect that females have greater amounts of microglia compared to males in some limbic regions including greater amounts in the dentate gyrus and CA1 region of the hippocampus of mice though female rats tend to have fewer microglia than males, an effect which does vary across limbic regions and with aging (Perkins et al., 2018; Ince et al., submitted). A heightened state of reactivity has been found in females, but there is significant variability in the literature based on what was measured (Bollinger et al., 2015; reviewed in Cortez et al., 2020; Lynch, 2022; Ugidos et al., 2022). While microglia have greater reactions to alcohol in adult female Sprague Dawley rats (Barton et al., 2017) and in F344 adult rats that consume ethanol for several months, the opposite was true for Wistar strain rats where Toll-like receptor-4 gene expression was higher in the males after alcohol drinking (Silva-Gotay et al., 2021). Intriguingly, sex differences in neuroimmune signatures are found at the level of the brain transcriptome, even when animals are phenotypically similar for high alcohol drinking (Hitzemann et al., 2022). In aging, the opposite may be true: males appear to have amplified inflammatory priming of microglia which may underlie their age-associated cognitive deficits (Ince et al., submitted). Further, CIE during adolescence can produce sex-specific effects in adulthood on hippocampal glial cells suggesting adolescence is a vulnerable period when ethanol exposure can alter glial expression and function. Thus, future research should investigate if differential microglia reactivity or priming exists between CIEg treated adolescent male and female animals and CIEg treated aged male and female animals.

Currently, it is unknown what are potential neurobiological mechanisms producing impaired cognition late in life following CIEg during adolescence. However, studies have provided potential brain mechanisms that are altered by exposure during adolescence and last into adulthood. For example, exposure to ethanol during adolescence produces a significant reduction in cholinergic interneurons in the nucleus accumbens of male and female rats (Galaj et al., 2019) and altered cholinergic activity in the orbital frontal cortex (Kipp et al., 2021). Further, CIE during adolescence can reduce efflux of acetylcholine 12–14 months later and the reduction is selective to male rats (Reitz et al., 2021). Given it has long been proposed that reductions in cholinergic function may underlie a portion of the cognitive decline observed in aging (Schliebs and Arendt, 2011 for review) it is interesting to speculate that CIEg during adolescence may result in increased cholinergic disfunction thereby leading to larger cognitive deficits late in life. Future studies are needed to test this hypothesis.

Previously, we have reported that CIEg during adolescence in male rats but not female rats results in a significant decrease in survivability over a ∼22 month experimental period compared to control animals (Matthews et al., 2022). In the current project, we did not find a significant effect in survivability based on sex or ethanol exposure during adolescence. However, the point in time at which male subjects in our previous work (Matthews et al., 2022) began to be removed from the study due to either A. death or B. a significant health concern began at approximately 21 months of age. Future studies should systematically investigate the time point between 19 and 22 months of life following CIEg during adolescence. This time period may represent a critical window that results in differential sex-specific health issues in subjects exposed to ethanol during adolescence.

The current results demonstrate not only sex and age-dependent effects but also a significant dose effect, particularly in male subjects. For example, 5.0 g/kg ethanol, but not 3.0 g/kg ethanol, produced an initial anxiety-like effect in male rats. In a similar vein, 5.0 g/kg ethanol altered initial spatial learning and impaired behavioral flexibility only in male rats while the 3.0 g/kg ethanol dose did not alter cognition. These data suggest that male rats may be more sensitive than female rats to the long-term cognitive impairing effects of ethanol. However, the current work has several limitations. First, the studies are underpowered statistically. Specifically, it was found that 5.0 g/kg CIEg in male rats produced a very strong trend toward an impairment in spatial learning based on swim pathlength to the platform in that the significance value for the overall ANOVA was a 0.058. This likely was due to reduced animal numbers because of removal of some animals due to health reasons or death. Given the exploratory post-hoc tests all confirm that the 5.0 g/kg ethanol exposed animals performed worse than control animals and mirrors the significant effects found with swim latency to the platform, it is likely a valid and reliable spatial learning impairment. Second, anxiety-like behavior in animals increases with age (Imhof et al., 1993; current results in male rats) resulting in a floor effect for common dependent variables in the elevated plus maze such as percent open arm time or open arm entries. This floor effect makes it more difficult to determine if CIEg during adolescence results in an increased anxiety-like effect in aged animals. It is likely the development of new experimental designs and/or apparatus will be needed to determine the impact of CIEg during adolescence on anxiety-like behavior in aged animals. Third, few people drink in a binge-pattern during adolescence and then do not consume ethanol again throughout their lifespan. As such the research strategy employed here does not model the typical drinking history of older adults. We employed this strategy to capitalize on the extensive research already conducted that investigates the impact of adolescent ethanol exposure during adolescence and then testing in adulthood (see Crews et al., 2019 for a review of this work). The current work greatly extends these findings into the aging portion of the lifespan. However, it is critical to model alcohol use across the lifespan. These studies are being initiated in our laboratory.

In conclusion, CIEg during adolescence produced anxiety-like behavior in male rats, but not female rats, on the elevated plus maze the day following completion of the ethanol exposure regime. However, the anxiety-like behavioral effect was not seen on PND 224 or PND 579 suggesting the effects are transitory as it relates to the lifespan. Further, when tested on PND 582, male rats, but not female rats, administered 5.0 g/kg ethanol CIEg during adolescence had subtle, yet significant, impairments in spatial learning and behavioral flexibility. Finally, CIEg during adolescence did not alter microglia expression in aging. Further work is needed to determine what brain mechanisms underlie these effects and what is the impact of ethanol exposure across the lifespan.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the University of Wisconsin–Eau Claire IACUC.

SS, ST, AS, GR, BI, QP, and AK collected and edited the data. DM designed the study, analyzed the data, and wrote the manuscript. JP collected and edited the data. KN designed the study, and wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.