Periadolescent ethanol vapor exposure persistently reduces measures of hippocampal neurogenesis that are associated with behavioral outcomes in adulthood
Introduction
Adolescence is a period of cognitive, emotional, and social maturation suggested to encompass the entire second decade of life in humans (10–20 years) (Spear, 2000, Dahl and Spear, 2004). In addition to the onset of puberty, adolescence is also a critical stage of brain development associated with changes in synapse numbers (Huttenlocher, 1984). During adolescence, there are also major changes that occur in brain morphology including the selective removal of 40–50% of the synapses (i.e., synaptic pruning) in cortical and subcortical brain regions (van Eden, 1990, Lidow et al., 1991, Johnston, 1995, Seeman, 1999), continued myelination of cortical regions (Giedd et al., 1996, Sowell et al., 1999b), and dramatic changes in receptor levels and sensitivity (Gould et al., 1991, Lidow et al., 1991). Adolescence is also a time when humans are initially exposed to a number of potentially toxic external stimuli such as ethanol and other drugs of abuse (Johnston, 1995, Clark et al., 2008, Squeglia et al., 2009). Given that the brain continues to develop before and throughout the adolescent period into early adulthood (Markus and Petit, 1987, Pfefferbaum et al., 1994, Sowell et al., 1999a, Sowell et al., 1999b), ethanol and other drug exposures during that time period may have unique deleterious consequences including changes in brain morphology.
In spite of these well-documented changes in brain structure and function, compared with the study of fetal ethanol effects, fewer studies have assessed the long-term effects of chronic drug or ethanol exposure on adolescent brain or behavior during this crucial time of development (Trauth et al., 2000, White et al., 2000, Dahl and Spear, 2004). It is known that the adolescent brain is highly sensitive to excitotoxic insult, particularly in cortical and hippocampal regions (Johnston, 1995). Several MRI (magnetic resonance imaging) studies of adolescent alcohol users have found hippocampal volume decreases associated with their alcohol use (see De Bellis et al., 2000, Nagel et al., 2005, Medina et al., 2007). However, studies in human adolescent studies cannot distinguish whether hippocampal volume decreases represent a consequence of alcohol use or exposure to other adverse environmental conditions (Staff et al., 2012), or are a pre-existing condition associated with risk for alcohol use. However, recently, reductions in the volume of the hippocampus were detected in MRI studies in rats exposed to ethanol vapor during adolescence and subsequently evaluated as adults (Ehlers et al., 2013). Although the mechanisms that result in this reduction in adult hippocampal volume following periadolescent ethanol exposure remain unknown, these findings suggest that adolescent ethanol exposure can impact adult hippocampal structure.
The hippocampus represents a unique brain structure in that it is one of two brain regions where neurogenesis extends into adulthood (see Zhao et al., 2008). Neurogenesis emerges from a population of neural progenitor cells (NPC) that exist in the forebrain subventricular zone (SVZ) and in the hippocampal dentate gyrus (DG) (Altman and Das, 1965, Doetsch et al., 1999). The formation of new neurons progresses over 3–6 weeks through a set of stages that include: NPC proliferation, cell migration, neuronal differentiation and survival of new neurons or neuronal death. It is thought that a number of the components of hippocampal neurogenesis may be impacted by stress (Barha et al., 2011), inflammation (Whitney et al., 2009), and chronic sleep restriction (Novati et al., 2011) that may also potentially result in reduced hippocampal volume. Neural stem cells of the hippocampus have been linked to a number of functions of the hippocampus including, memory, learning, depression and anxiety (see Balu and Lucki, 2009, Hanson et al., 2011). Adolescent rats have been shown to have higher levels of hippocampal neurogenesis than adults, with measures of neurogenesis decreasing during brain maturation from adolescence to adulthood (He and Crews, 2007). Reduced hippocampal volume and lower levels of hippocampal neurogenesis have been suggested to underlie the symptomatology and etiology of emotional and depressive disorders that typically arise during adolescence (see Sapolsky, 2000, Czeh and Lucassen, 2007, Perera et al., 2008, Boldrini et al., 2009). There is also a growing body of data to suggest that neurogenesis during adolescence is potently impacted by alcohol exposure during that time period.
The effects of adolescent ethanol exposure on neurogenesis in the rat hippocampus have only been studied by a few investigators using primarily short-term (1–4 days) ethanol exposure paradigms. These studies have found that adolescents are more sensitive than adults to ethanol-induced inhibition of brain neurogenesis (Crews et al., 2006). Acute oral administration (by gavage) of doses of ethanol (1.0, 2.5, and 5.0 g/kg), that mimic “binge drinking”, often seen during adolescence, have been demonstrated to dose dependently inhibit NPC proliferation in both forebrain and hippocampus (Crews et al., 2006). Multi-day exposure to “binge-like” levels of blood alcohol during adolescence has additionally been shown to result in both reduced cell proliferation and impaired survival as well as increases in several cell death markers in the dentate gyrus (Morris et al., 2010). Adolescent binge alcohol may also accelerate progression through the cell cycle (McClain et al., 2011). It appears that most of the cells that do survive this treatment become neurons and that newborn cells differentiate into neurons in a slighter higher rate in adolescents than adults (He et al., 2005, Morris et al., 2010). Hippocampal neurogenesis has also been evaluated in primate brain following “heavy drinking” over an 11-month period during adolescence. In that study alcohol associated reductions in the division and migration of hippocampal preneuronal progenitors were found (see Taffe et al., 2010). Although these studies demonstrate clear evidence of alcohol-induced interference with adolescent neurogenesis, it is not known whether these findings are associated with behavioral pathology in the alcohol-exposed animals nor is it known how long lasting the effects are.
The present investigation was designed to extend previous studies in adolescent rats in order to study the effects of moderate levels of chronic ethanol vapor exposure during adolescence and early adulthood on behaviors and measures of neurogenesis in adulthood at 2 and 8 weeks following withdrawal from ethanol vapor exposure. The measures of neurogenesis used in the study were: Ki-67 labeling, an endogenous cell cycle protein expressed in actively dividing cells from G1-phase through M-phase (Scholzen and Gerdes, 2000), and Doublecortin (DCX) a marker of immature neurons at both time points (71/72 (collectively referred to as 72) and 113/114 (collectively referred to as 114) days, 2 and 8 weeks following withdrawal), as well as Bromo-deoxy-Uridine (BrdU), a thymidine analog that is incorporated into cells in place of a thymine base pair as the cell undergoes DNA replication during the S phase of the mitotic cell cycle, and as such is a measure of cell proliferation at the second time point (114 days, 8 weeks following withdrawal). Cleaved caspase-3 + IR (immunoreactivity), a marker of cell death, was also measured at 72 and 114 days. The behaviors in both groups (72 and 114 days) included: locomotion, behavior in the modified open field, and immobility and defecation in the forced swim test (FST). The results of the body weights, blood ethanol concentrations (BECs), behaviors, diffusion tensor imaging (DTI) and choline acetyl transferase IR in a larger group of animals that included some of those in the present study, who were sacrificed at 72 days, have been reported previously (Ehlers et al., 2011, Ehlers et al., 2013).
Section snippets
Subjects
Male Wistar rats and their dams who were received at postnatal day (PD) 21 (n = 42), Charles River, USA) were used in this study. The adolescent animals (PD 21) were housed three per cage respectively, in standard cages, until PD70 (for study 2 only) when they were housed two per cage for the duration of the experiment. Animals were kept in a light/dark (12-h light/12-h dark) and temperature-controlled environment. Food and water were available ad libitum throughout the experiment. All
Body weight and BECs
Fig. 1 represents a time line of the ethanol exposure and withdrawal as well as the behavioral tests and the time of sacrifice for both experimental groups. As seen in Fig. 2, all rats gained weight over the course of the experiment. The 19 rats in the 72-day sacrifice group, who had neurogenesis data, grew from about 50 g at PD22 to about 360 g being (365 ± 27) and (356 ± 37) g for control and ethanol at 7 weeks respectively. No significant overall differences in body weight were seen between the
Discussion
In the present study, rats were exposed to ethanol vapors during the periadolescent period in order to examine ethanol’s effects on neurogenesis and its relationship to behavior in adulthood. BECs over the 5-week exposure period were maintained at 170 mg/dL, these blood ethanol levels are consistent with this protocol being a model of blood alcohol levels seen during adolescent binge drinking (Donovan, 2009). Two groups of animals were evaluated, group one was sacrificed at 72 days of age, 2 weeks
Conclusions
Thus our data support the hypothesis that adolescent ethanol exposure can have significant effects on brain and behavior in an animal model where control of ethanol exposure can help delineate environmental effects from genetic background. In the present study, rats were exposed to ethanol vapors during the periadolescent period in order to examine ethanol’s effects on neurogenesis and its relationship to behavior in adulthood. Ethanol vapor-exposed rats, as compared to controls, displayed
Acknowledgements
This study was supported in part by the NADIA Initiative Project of the NIH National Institute on Alcoholism and Alcohol Abuse grants AA019969 (CLE) and AA020022, AA020023 and AA020024 (FTC) and the UNC Bowles Center for Alcohol Studies. The authors thank Jose Criado and Greta Berg for their assistance in data collection and Shirley Sanchez for editing the manuscript.
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