Ontogeny and adolescent alcohol exposure in Wistar rats: open field conflict, light/dark box and forced swim test
Introduction
Adolescence is a period between childhood and adulthood that is defined both behaviorally and biologically, and has been suggested to encompass almost the entire second decade of life (10–20 yrs) (Spear, 2000). It has been suggested that during this time individuals learn to function more independently, and fluency in emotional and social functioning is acquired (Dahl and Spear, 2004). While adolescence is usually defined by a series of endocrine events resulting in puberty, it actually involves changes in a number of organ systems including the brain that may occur in a separate time frame from endocrine events (Spear and Varlinskaya, 2010). During this developmental period there are changes in neurobiological organization and behavior that appear to be generally conserved during evolution with similarities seen across a number of mammalian species. In the rat it has been suggested, as a conservative estimate, that the adolescent period may span post-natal days 28-42 (Ojeda and Skinner, 2006, Spear and Brake, 1983, Varlinskaya et al., 1999). However depending on gender and the measures used to define adolescence, periadolescence may be seen as early as post-natal day (PD) 22 in females, and it may last until PD55 in males (Spear, 2000).
Adolescence is also a critical stage of brain development when humans initiate a range of exploratory behaviors that can result in exposure to ethanol and other drugs of abuse (Clark et al., 2008, Squeglia et al., 2009a). Since the brain continues to develop throughout the adolescent period into early adulthood (Markus and Petit, 1987, Sowell et al., 1999a, Sowell et al., 1999b), ethanol exposure during this time period may have unique and deleterious consequences including changes in disinhibitory, cognitive, and affectively driven behaviors. Several studies in humans have demonstrated that early alcohol exposure is associated with neurobehavioral deficits as measured by magnetic resonance imaging (MRI) scans and psychological testing (Hanson et al., 2011, McQueeny et al., 2009, Schweinsburg et al., 2011, Squeglia et al., 2009b). However, it is still not entirely clear whether these deficits are caused by alcohol exposure or are a result of pre-existing conditions (Nagel et al., 2005). The use of animal models of adolescent alcohol exposure allows for the testing of the direct effects of alcohol on the developing brain under more controlled conditions.
Exposure to chronic intermittent ethanol (CIE) during adolescence in rats has been demonstrated to cause a number of changes in brain and behavior that persist into adulthood. It has been demonstrated that chronic intermittent exposure to ethanol vapor during adolescence (14 hours exposure/10 hours no exposure, daily, over a 5-week period) can cause increases in voluntary ethanol drinking, reductions in the size of the hippocampus as imaged by diffusion tensor imaging, reductions in measures of hippocampal neurogenesis, increases in the latency of the P300 component of the event-related potential, signs of behavioral disinhibition in the light/dark box and open field conflict test, immobility in the forced swim test, and reductions in cholinergic tone in the basal forebrain (see Criado and Ehlers, 2013, Ehlers et al., 2011, Ehlers et al., 2013a, Ehlers et al., 2013b, Ehlers et al., 2014).
Studies in animal models have also shown that adolescent rats are less sensitive than adult rats to the effects of acute alcohol administration on hypothermia, motor in-coordination, sedation, as well as electrophysiological effects (Pian et al., 2008a, Silveri and Spear, 1998, Silveri and Spear, 2000). Adolescent rats display greater tolerance to the sedative effects of acute ethanol administration than adult rats, and regain consciousness at higher blood alcohol levels (Pian et al., 2008b, Silveri and Spear, 1998). For adult rats exposed to CIE during adolescence, the adolescent phenotype of reduced sensitivity to ethanol has been shown to be “retained” in behavioral measures (Slawecki, 2002, White et al., 2000a, White et al., 2000b), electrophysiological measures in the hippocampus (Fleming et al., 2012, Fleming et al., 2013, Slawecki, 2002), and in the cortex (Ehlers et al., 2013c, Ehlers et al., 2014, Slawecki, 2002). Some authors have suggested that adolescent ethanol exposure may “lock-in” adolescent sensitivity to ethanol and then sustain it into adulthood (Fleming et al., 2012). However, whether adolescent alcohol exposure “locks-in” the adolescent phenotype of other behaviors is not as well understood.
The present study investigated whether adolescent intermittent ethanol (AIE) could “lock-in” the adolescent phenotype in behaviors using three different test apparatus: the open field conflict, the light/dark box and the forced swim test. Data on the normal ontogenic development of behaviors in these tests over the course of adolescence and adulthood is sparse. Therefore we examined whether adolescent Wistar rats at different ages would display more or less “anxiety-like” and “depressive-like” behaviors in these tests as compared to adult rats. We examined three age groups of rats ranging from middle adolescence to early adulthood to determine the normal ontogenic responses in adolescence (group 1: PD34-42; group 2: PD55-63; group 3: PD69-77). We compared these groups to previously published data from control adult rats and adult rats that underwent AIE as adolescents and were tested in the behavioral apparatuses between PD70 and PD126. It was our hypothesis that adult rodents with ethanol exposure during adolescence may exhibit a behavioral profile that is more reminiscent of an adolescent animal rather than an adult.
Section snippets
Animals
The animals used in this study were male Wistar rats that were received and weaned at post-natal day (PD) 21 (n = 144 juveniles, Charles River, USA). On PD22 the adolescent animals were housed three per cage in standard plastic cages until PD63 to PD70, and then they were pair-housed for the rest of the experiment. Animals were kept in a light/dark (12 hours light/12 hours dark, lights on at 6:00 am or 8:00 am) and temperature-controlled environment. Food and water were available ad libitum
Light/dark box
Behavior in the light/dark box was assessed in three groups of animals: group 1: post-natal day (PD) 34; group 2: PD55; group 3: PD69 and the results are presented in Table 1. Two different data analyses were conducted to determine if there was (1) an ontogenic effect between young animals and older adults and (2) an effect due to AIE exposure.
When comparing the three ontogenic adolescent/young adult groups directly with the older adults, younger animals differed from older animals in the
Discussion
This study evaluated whether there were ontogenic differences in behavior in three behavioral testing apparatuses the light/dark, the open field conflict test, and the forced swim test. These data were then compared to previous data collected from adult rats with a history of ethanol exposure during adolescence, and their controls, in order to determine whether alcohol exposure caused retention of the “adolescent phenotype”. In behavioral circumstances involving disinhibition or food motivated
Acknowledgements
This study was supported in part by the National Institutes of Health (NIH), National Institute on Alcoholism and Alcohol Abuse grants, AA006059 and AA019969 awarded to CLE. The authors thank Shirley Sanchez for assistance in editing the manuscript.
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