Persistent loss of hippocampal neurogenesis and increased cell death following adolescent, but not adult, chronic ethanol exposure

Introduction

Evidence is mounting to suggest that the hippocampus may be particularly susceptible to negative consequences of adolescent alcohol (ethanol) exposure. Previous research assessing age-related differences in sensitivity to ethanol has shown that adolescent rats are more affected by ethanol-induced impairments on a hippocampal-dependent spatial task, the Morris water maze MWM), than adults [1, 2]. In line with these behavioral effects, in vitro studies have shown more potent ethanol-induced enhancement of tonic inhibition [3] and antagonism of NMDA receptor synaptic function [4] in the hippocampus of adolescents relative to adults. Research in human adolescents has also suggested that the hippocampus may be susceptible to the effects of adolescent alcohol use, with decreased hippocampal volumes reported in youth who met criteria for alcohol use disorder (AUD) compared to controls [5]; however, it is unclear if these brain differences were present prior to alcohol use. Together these data suggest that the hippocampus may be particularly vulnerable to alcohol exposure during adolescence, which has led to the hypothesis that adolescent alcohol use may lead to long-lasting hippocampal alterations that persist into adulthood.

Recently our lab investigated long-term effects of adolescent ethanol exposure on learning and memory using a Pavlovian fear conditioning paradigm in male Sprague-Dawley rats [6]. Our results were consistent with the suggestion that the hippocampus is persistently affected by adolescent ethanol exposure, with animals that were exposed to ethanol during adolescence (P28–48) showing less retention of context fear, a task thought to be hippocampally-related [7, 8, 9] when tested in adulthood after an ~4 week ethanol free period. No effect of adolescent ethanol exposure emerged during tone fear conditioning, and comparable exposure to ethanol during adulthood (P70–90) did not produce a deficit in context fear memory [6]. These findings are consistent with other studies that reported poor performance on other hippocampally-related tasks, such as Morris water maze [2] and trace fear conditioning [10] in adult animals with a history of ethanol exposure during adolescence. Together these studies suggest that adolescent alcohol use may lead to persistent disruptions in hippocampal functioning.

The mechanisms underlying this apparent adolescent ethanol-induced hippocampal dysfunction have yet to be elucidated. One potential contributor to performance deficits on hippocampally-related tasks may be a long-lasting ethanol-induced disruption of neurogenesis. Indeed, previous reports suggest that irradiation-induced decreases in adult neurogenesis disrupted retention of context fear in rats [11, 12, 13, 14, 15], but not tone fear retention [12, 14], as well as impairing performance on other tasks thought to be hippocampal-dependent (see [16, 17] for review). Furthermore, adolescents given large doses of ethanol showed decreased neurogenesis [18], and chronic exposure to repeated ethanol vapor during adolescence was reported to significantly decrease neurogenesis in adulthood [19]. The latter study, however, did not explore whether a similar decrease would have been evident after comparable adult exposure.

The purpose of the present experiment was to determine whether doublecortin (DCX), an endogenous marker of immature neurons [20], would be decreased in adulthood after adolescent and adult ethanol exposure regimens used in our previous fear conditioning studies. Also explored was whether changes in neurogenesis would be specific to the hippocampus, or were more widespread, affecting another neurogenic niche, the subventricular zone (SVZ). Levels of Ki67, a marker of cell proliferation (see [21] for review), and cleaved caspase-3, a marker of apoptosis [22], were determined to assess the potential influence of neural progenitor cell proliferation and/or cell death, respectively, on DCX expression in the DG of adolescent ethanol exposed adults.

Materials & Methods

Results

Discussion

This is the first study to show that identical ethanol treatments of adolescent and adult rats results in a persistent loss of neurogenesis only in adolescents and not in adults. Despite adolescents showing decreased sensitivity to ethanol during the exposure period in terms of disruption in weight gain, repeated ethanol exposure during adolescence, but not in adulthood, significantly reduced DCX+IR in the DG when assessed ~4 weeks after the final ethanol exposure. DCX+IR did not differ as a function of prior exposure at either age in the SVZ, suggesting that the effect of adolescent ethanol exposure on DCX expression was specific to the hippocampus. Previous studies have found adolescent hippocampal neurogenesis was more sensitive to acute ethanol inhibition than adult neurogenesis [18], and that high levels of neurogenesis in adolescence decline progressively with aging [24, 25]. Consistent with previous studies, we found a significant age3 related decline in both the hippocampal DG and forebrain SVZ, with significantly less DCX+IR observed in P116 (adult exposure groups) than P74 (adolescent exposure groups) animals, regardless of exposure history.

Ethanol inhibition of neurogenesis has been primarily studied in adult rats, with previous studies showing that acute and chronic ethanol reduces hippocampal progenitor proliferation and neurogenesis [26, 27,28,29]. However, ethanol inhibition of neurogenesis in adults returns to age appropriate control levels after about a month of abstinence due to bursts of proliferating progenitors that after a few days mature to microglia [27] and then after about a week of abstinence begin to mature to new dentate gyrus neurons that over time return to a control level of neurogenesis [28]. Although adult neurogenesis rebounds during abstinence following chronic ethanol to age-appropriate levels, treatment of rats with an 8 week ethanol chronic relapsing model of alcoholism did lead to a persistent loss of SVZ neurogenesis [29]. Adolescent intermittent ethanol treatment by oral gavage in this study or by vapor ethanol treatment in a previous study [13] resulted in decreases in neurogenesis that last into adulthood. These intermittent treatments model adolescent weekend drinking and are different from continuous alcoholic-like models that induce a marked physical dependence to alcohol. Studies using a 4 day multiple dose binge treatment in P35 Sprague-Dawley rats that induces physical dependence, find alcohol inhibits neurogenesis, but during withdrawal a rapid rebound increase in proliferation of progenitors restores DG neurogenesis and induces ectopic DCX expression [30] McClain et.al. [30] found abstinence induced neurogenesis correlated with blood alcohol levels over 300 mg/dl. Thus, our finding of a persistent loss of adult neurogenesis after AIE may be specific to intermittent adolescent treatments that do not induce withdrawal seizures or other withdrawal symptoms that can increase neurogenesis.

Adult brain neurogenesis is unique to specific areas that maintain a unique “neurogenic niche”, i.e. a local cellular environment that contains and supports stem cell-like progenitors that can differentiate into multiple brain cell types. DCX has been used as an endogenous marker of neurogenesis, since it is a protein that is transiently expressed in immature neurons from the time neuroblasts are formed until expression of the mature neuronal marker, NeuN [20, 31]. The functional role of DCX is unknown, but may be associated with migration [32, 33] and neurite development [33]. Using DCX as an index of neurogenesis, these data suggest that adolescents may be more susceptible to long-lasting disruptions of hippocampal neurogenesis than adults under the circumstances of the current study. Increased cell death, as indexed by cleaved caspase-3 within the DG, likely contributed to the reduction in DCX+IR in the DG of adults exposed to ethanol during adolescence. These results are consistent with a previous report using an intermittent ethanol vapor exposure regimen during adolescence and also found that decreased survival of progenitors (indexed via cleaved caspase-3) contributed to the loss of neurogenesis [19]. Importantly, the present study extends those findings by providing evidence that persistent attenuations of DCX in adulthood after adolescent ethanol exposure are evident with both oral and vapor routes of ethanol administration (ie., after i.g. administration used in the present study and vapor used in [19]) and not evident when comparable exposure is conducted in adulthood. Therefore, reminiscent of research with prenatal alcohol exposure (see [35] for review), these data suggest that adolescence is another vulnerable period of brain development during which alcohol exposure can result in persistent disruptions of hippocampal neurogenesis.

No effect of adolescent exposure history was evident when assessing Ki67, a marker for neural progenitor cell (NPC) proliferation in the current study. Often a decrease in neurogenesis (DCX+IR) is associated with a decrease in progenitor proliferation, e.g. a decrease inKi67+IR [20, 31]. Previous studies of intermittent adolescent ethanol vapor treatment found a loss of Ki67+IR two weeks after ethanol treatment, but not eight weeks after treatment [19]. Other studies using a 4 day multiple dose binge ethanol dependence model in adolescence find decreased neurogenesis, without a change in Ki67+IR [36, 37], although there were changes in progenitor cell cycle kinetics [37]. We assessed Ki67 about 4 weeks after adolescent treatment and did not find any differences in Ki67+IR consistent with the majority of studies in adolescents. Thus, adolescent ethanol exposure appears to reduce immature neurons not via alterations in proliferation per se, as indexed via Ki67, but via increasing their demise, as indexed using cleaved capase-3 as a marker of cell death. Together, these findings suggest hippocampal progenitors continue to proliferate following adolescent ethanol exposure, but cell death is increased such that progenitors die before maturing to dentate granule cell neurons. Therefore, the persistent decrease in adult hippocampal neurogenesis following adolescent intermittent ethanol treatment suggests long term changes that result in increased cell death in the neurogenic niche [25].

Adult brain neurogenesis occurs due to unique properties of glia and other cells within the hippocampal dentate gyrus and forebrain subventricular zone that maintain proliferating stem cells which can form new neurons. The mechanisms for the persistent increase in dentate gyrus caspase-3+IR after adolescent ethanol exposure in the current study is likely related to alterations in the neurogenic niche. The age-related decline in neurogenesis is thought to result from multiple factors, including loss of proliferating progenitors, loss of trophic support for progenitors and increased microglial activation and innate immune gene induction [25]. Adolescent intermittent ethanol treatment has been found to increase innate immune gene expression in frontal cortex [38] as well as to reduce trophic factor support of the neurogenic niche [39]. These findings are consistent with a preserved stem cell population in dentate gyrus following adolescent alcohol exposure, as suggested by continuous progenitor proliferation (Ki-67+IR), but an altered neurogenic niche that increases cell death as indicated by the loss of DCX+IR e.g. neurogenesis and increase in cell death (caspase-3+IR). Loss of trophic factors that support neurogenesis (see [39] for review and [40]), and/or increase immune gene induction [25] may both contribute to the persistent loss of neurogenesis following adolescent ethanol exposure.

Given that neurogenesis is implicated in context fear retention [11, 12, 13, 14, 15], it is possible that alterations in neurogenesis in the DG as a result of adolescent ethanol exposure may have contributed to deficits in context fear retention previously observed in a separate group of animals [6]. Alternatively, since increased survival of newly generated neurons has been reported after trace conditioning [41, 42], a possibility arises that tone conditioning and extinction sessions experienced by animals in the current study may have somehow influenced the subsequent levels of neurogenesis. This interpretation is not likely, however, given lack of evidence that tone conditioning or extinction influences subsequent levels of neurogenesis in the hippocampus, nor were there detectable differences in acquisition of conditioning or extinction observed between adolescent ethanol- and water-treated groups (see [6] for behavioral data).

One caveat for the interpretation that reduced neurogenesis after adolescent intermittent ethanol exposure plays a role in deficits context fear retention is that disruptions in context fear conditioning were not evident in P116 control animals relative to their young adult (P74) counterparts [6], despite the marked age-related declines in DCX levels in DG seen between P74 to P116. These data suggest that levels of neurogenesis (indexed via DCX) may be more important for context fear memory in younger adults (P74) than older adult animals (P116). It is also possible that levels of neurogenesis need to be significantly lower than levels seen in the current study to affect context fear memory in older adults, such as the notable declines reached in studies utilizing irradiation-induced decreases in adult neurogenesis (e.g., [11, 12, 13, 14, 15]). Alternatively, these data may suggest that the disruption of context fear memory seen in Broadwater & Spear [6] may not be dependent on production of new neurons, at least as indexed by number of DCX+ cells.

To the extent that these data extend to humans, alcohol consumption during adolescence may lead to long-lasting negative consequences that involve deficits in hippocampal-dependent memory and alterations in survival of neurons in the DG, even after a period of abstinence. Chronic alcoholism is associated with decreases in hippocampal volumes that might be related to a persistent loss of neurogenesis beginning in adolescence [43]. Therefore, these data provide more evidence that adolescence is a particular sensitive period of development to persisting consequences of alcohol exposure, with the hippocampus being a region of vulnerability to long lasting effects.