Research reportConsequences of ethanol exposure on cued and contextual fear conditioning and extinction differ depending on timing of exposure during adolescence or adulthood
Introduction
The adolescent brain is in a dynamic state of remodeling (see [1], [2] for review), which may make adolescence an especially vulnerable period for exposure to negative environmental agents [3], [4], [5], [6]. This is particularly concerning given that initiation of alcohol consumption is common during adolescence, and alcohol is often consumed by adolescents in a binge-like fashion (i.e., 4–5 drinks within 2 h, achieving BECs ≥ 80 mg/dl) [7]. Indeed, preclinical and clinical evidence is mounting to suggest that exposure to alcohol during adolescence may be particularly detrimental. For instance, human adolescents that met criteria for an alcohol use disorder (AUD) showed impairments in cognitive tasks such as problem solving, verbal and nonverbal retrieval, visuospatial skills and working memory along with significant brain alterations, such as reduced hippocampal volumes and white matter abnormalities relative to their nonabusing agemates (see [8] for review). Causality cannot be concluded from such investigations, but preclinical studies have also found repeated alcohol (ethanol) exposure during adolescence results in greater impairment in memory [9] as well as greater brain damage in certain brain regions [10] when compared with adult exposed animals when all groups were tested shortly after exposure. An important question is whether enhanced neurobehavioral deficits after adolescent ethanol exposure would persist into adulthood.
Although only a limited number of studies to date have focused on long-term consequences of adolescent alcohol use, these studies generally support the hypothesis that adolescent ethanol exposure results in persistent behavioral and neural changes. For instance, Sircar and Sircar [11] found learning deficits in a Morris water maze task 25 days after exposure in animals exposed to ethanol during adolescence, but not adulthood. Pascual et al. [12] reported deficits on both conditional discrimination and novel object recognition tasks 21 days after repeated ethanol exposure in adolescence. Other recent studies found reversal learning deficits using the Barnes maze [13] and Morris water maze [14] tasks 15 and 35 days, respectively, after adolescent ethanol exposure. In conjunction with the behavioral deficits observed in these studies, adolescent ethanol exposure also produced long-term brain changes, with reports of upregulation of TLR4, TLR3 and HMGB1 expression in the PFC [13] and decreased basal forebrain volume and numbers of cholinergic neurons [14]. These data support the hypothesis that ethanol exposure during adolescence, a period of time during which the brain is still undergoing significant maturational changes, results in long-lasting alterations in neurobehavioral development (see [4], [6] for discussion).
Given that adolescent ethanol exposure previously has been found to influence learning in adulthood on various cognitive tasks, the purpose of this series of experiments was to determine if adolescent or adult ethanol exposure would affect later learning and memory during contextual and auditory Pavlovian fear conditioning and extinction. Pavlovian fear conditioning is an ideal model to assess consequences of adolescent ethanol exposure on learning and memory because the neural mechanisms involved have been examined extensively, with key brain regions that include the amygdala, hippocampus, and medial prefrontal cortex (mPFC) playing a critical role in tone conditioning, context conditioning, and extinction of these tasks, respectively (see [15], [16], [17], [18], [19] for reviews). These structures and connectivity among these regions continue to develop during adolescence (see [1], [2] for review), and therefore may be particularly vulnerable to ethanol exposure at that time, perhaps changing their developmental trajectory. Thus, Pavlovian fear conditioning and extinction may provide useful tools to assess potential structure-relevant cognitive alterations following adolescent ethanol exposure.
One study to date has examined long-term consequences of adolescent ethanol exposure on fear conditioning in adulthood. Bergstrom et al. [20] found a deficit in tone fear conditioning in adult Long–Evans rats 30 days after repeated voluntary access to ethanol during adolescence (P22–45), but not adulthood (P80–97). Interpretation of these results, however, is complicated by the fact that adults consumed significantly less ethanol than the adolescents, thereby confounding amount of ethanol exposure across age. Thus, although that study suggests that adolescent ethanol exposure may result in long-term deficits in tone fear memory, it is unclear if comparable exposure in adults would produce similar deficits. Furthermore, no studies to date have examined context conditioning or extinction after either tone or context conditioning in adolescent ethanol-exposed animals. Given that context conditioning is relatively hippocampal-reliant [21], [22], [23] and the medial prefrontal cortex (mPFC) has been implicated in extinction learning (see [18], [19] for review), assessing these particular learning and memory processes after adolescent ethanol exposure may add valuable information in terms of potential structure-specific changes after ethanol exposure.
Two experiments were conducted to assess tone and context fear acquisition, retention and extinction 22 days after repeated intermittent ethanol exposure consisting of 4 g/kg intragastric (i.g.) ethanol or water every 48 hrs for a total of 11 exposures. In the first experiment, exposure started in early adolescence [postnatal day (P) 28–48] or adulthood (P70–90), with tone and context conditioning and extinction assessed 22 days later. In a follow-up to the first experiment, a comparable study was conducted in a second adolescent exposure group where the exposure began in mid-adolescence, continuing through late adolescence (P35–55).
Section snippets
Subjects
Male Sprague–Dawley rats bred and reared in our colony at Binghamton University were used in this experiment. On the day after birth, postnatal day (P) 1, litters were culled to 8–10 pups, with a sex ratio of 6 males and 4 females retained whenever possible. Pups were housed with their mother in a standard clear plastic tub with shavings until pair-housed with same-sexed littermates at the time of weaning (P21). Animals were maintained in a temperature-controlled vivarium on a 12-h:12-h
Subjects and design
A total of 96 male Sprague–Dawley rats were used in this 2 exposure age (adolescent: P28–48; adult: P70–90) × 2 exposure (H2O; EtOH) × 2 conditioning stimulus (tone; context) factorial study, with an n = 12/group.
Animal exclusion
A total of 2 adolescent and 3 adult EtOH-exposed, tone conditioned animals were excluded due to elevated baseline (pre-CS) freezing on the tone test day. Also, 2 adolescent-exposed animals from the H2O-exposure, tone conditioned group were excluded, with one excluded as a statistical outlier
Experiment 1 and 2: exploratory analyses
Given that the effects of repeated ethanol on fear retention and extinction varied with exposure age, potential age differences in the impact of ethanol on measures of body weight and intoxication collected during the exposure periods were explored. As described below, these exploratory analyses revealed that weight deficits emerging during the ethanol exposure period were particularly marked in adults, though also evident to some extent in animals exposed to ethanol in mid-late, but not
Discussion
The purpose of these experiments was to assess potential long-lasting consequences of early-adolescent, mid-adolescent and adult ethanol exposure on tone and context fear conditioning retention and extinction. Early adolescent animals were relatively less sensitive than mid-adolescent and adult animals during the ethanol exposure period in terms of weight deficits and signs of intoxication. These results add to an established literature that adolescents are less sensitive than adults to the
Acknowledgments
The research presented in this paper was supported by NIAAA grants R01AA018026 and U01AA019972-NADIA Project.
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