This work illustrates that stress coupled with a single ethanol withdrawal can reduce social interaction in adolescent rats. This reduction in social interaction is a validated measure of increased anxiety-like behavior (File and Seth, 2003
). Importantly, it was demonstrated that the combination of both stress and ethanol withdrawal was critical, as neither stress nor a single ethanol withdrawal alone produced this anxiety-like behavior (Wills et al., 2009
). Similar findings were previously found in adult rats, where stress substituted for early withdrawals to produce anxiety-like behavior (Breese et al., 2004
). Further, the changes in locomotor activity following the stress/withdrawal paradigm in adolescent rats were also similar to data in adults. In both age groups, reduced locomotor activity was found for rats given two stress episodes followed by a single 5-day cycle of ethanol diet (Breese et al., 2004
), although this effect was modest and unlikely to impact on the social interaction of this group. Additional support can be found in the duration experiment, where this same stress/withdrawal treatment (2.5% CY1-Str at 5 hours) elicited a similar reduction in social interaction without a reduction in activity. These results reconfirm the idea that social interaction and locomotor activity are independently manipulatable and not necessarily contingent on one another through changes in ethanol treatment and drug treatments (Breese et al., 2004
; Knapp et al., 2005
; Overstreet et al., 2002
In other experiments, the anxiety-like behavior produced from the stress/withdrawal paradigm was present at 5 hours but had returned to baseline by 24 hours into withdrawal in both adolescent and adult rats. This profile of recovery is similar to that seen with repeated ethanol withdrawals in adult rats (Wills et al., 2009
). However, this duration is very different from that found with repeated ethanol withdrawals in adolescent rats (Wills et al., 2009
). In these previous results, it was illustrated that anxiety-like behavior after repeated ethanol withdrawals could be detected for up to 1 week following the final withdrawal. The reason for differences in the duration of reduced social interaction between these paradigms may be because of a lower sensitivity to the anxiogenic effects of acute restraint stress in adolescent rats. This behavioral divergence between paradigms (repeated ethanol withdrawal vs. stress/withdrawal) suggests that different mechanisms likely contribute to the development of this anxiety-like behavior. Adolescent rats were also shown to display higher baseline levels of activity and social interaction than adult rats. This result was consistent with previous reports of elevated exploration and social behavior in adolescent rats (Adriani et al., 1998
; Primus and Kellogg, 1989
; Vanderschuren et al., 1997
). Corrections for these baseline differences were made (converted to % decrease from baseline) and illustrated that adolescent and adult rats showed equivalent reduction in social interaction (45 to 50%). The next set of experiments provided evidence for a role of CRF in the sensitization of anxiety-like behavior. Adolescent rats given 7.5 µg of CRF in combination with a single ethanol withdrawal exhibited anxiety-like behavior (reduction in social interaction), which was not found with either of these treatments alone. This dose of CRF required to elicit sensitization of withdrawal-induced anxiety in adolescents was higher than doses effective in producing this anxiety in adult studies (5 µg; Overstreet et al., 2004a
). Therefore, it seems that adolescent rats may also be less sensitive to the anxiogenic effects of CRF. There were modest effects on locomotor activity in these experiments caused by an increased in activity from CRF-CD groups (7.5 and 5 µg). It is unlikely that these differences in activity had any effect on social interaction for the reasons described earlier. Additionally, the differences do not affect the primary comparison between 7.5 µg-ED and controls as these groups were not different.
Analyses were also made of CRF levels in adolescent and adult rats that received repeated withdrawals, stress/withdrawals, or control diet. Results illustrated that CRF immunoreactivity was not significantly changed by any of the adult treatments in any of the regions that were evaluated (CeA or PVN). Even higher (7%) ethanol concentrations in one experiment failed to modify CRF levels in adults. However, in adolescent rats that were given repeated ethanol withdrawal, there was a difference in CRF immunoreactivity in the CeA, but not PVN. This difference was caused by a decrease in density of CRF in the CeA without a change in CRF-immunopositive cell number. We hypothesize that this decrease in CRF density levels from repeated ethanol withdrawal relates to an increase in CRF release during withdrawal. An increase in CRF release in this group would presumably deplete the supply of CRF contained within the cell body and would lead to the decreased immuno-density that was found. This idea can be further supported by microdialysis studies which showed that CRF levels increased and peaked 11 to 12 hours into withdrawal from chronic ethanol exposure (Merlo Pich et al., 1995
). Similar results have been found under more chronic ethanol conditions. For example, Zorrilla et al. (2001)
illustrated that during the first day of withdrawal there was a decrease in CRF immunoreactivity in the amygdala. Later studies demonstrated this reduction in CRF immunoreactivity specifically in the CeA during withdrawal of dependent rats (Funk et al., 2006
). In agreement with our results, this study also found no change in CRF-positive cell number (Funk et al., 2006
). Both authors also speculate that this reduction in immunoreactivity was an indication of increased CRF release during this period. This reduction in CRF immunoreactivity in the CeA was only found in adolescent rats that experienced repeated ethanol withdrawals, not following stress/withdrawal procedure or following either treatment in adult rats. It is important to note that both of these treatments in adolescent and adults produced anxiety-like behavior; therefore, this change in CRF is most likely not universally related to this acute (5 hours into ethanol withdrawal) anxiety-like behavior. Further, previous work has demonstrated that CRF1
antagonists are able to prevent the sensitization of anxiety-like behavior in the stress/withdrawal and repeated withdrawal paradigm in adult rats (Breese et al., 2004
; Knapp et al., 2004
). This work illustrates a role of CRF in the development of anxiety-like behavior under these paradigms in adult rats. Thus, the reduction in CRF immunoreactivity seen with repeated withdrawals in adolescent rats is likely related to the greater sensitivity illustrated by an extended anxiety-like behavior (up to 1 week into withdrawal) found only in this group. These results could also suggest that different mechanisms underlie repeated ethanol withdrawal and stress/withdrawal in adolescent rats.
The immunohistochemical experiments also indicated that adolescent rats have higher basal immunoreactivity of CRF (increased density and cell number) in the PVN and CeA compared to adult rats. This result provides unique evidence of increased CRF protein levels and cell number within the PVN and CeA in adolescent rats. An earlier study measuring mRNA levels of CRF found no difference in the PVN and an increase in the CeA from 30- to 60-day-old male rats (Viau et al., 2005
). This study differs from the current results presented here in that only changes in CRF mRNA levels were quantified and somewhat younger animals were assayed (their ages 30 and 60; our ages P45 and P73). Therefore, our results indicate that at least at P45 there are increased levels of CRF protein within the PVN and CeA under our control conditions (liquid diet and isolate housing). The implications of this observation would benefit from further examinations of CRF levels across age and challenge conditions. Additionally, these baseline differences in CRF between adolescent and adult rats in the current experiments might have been responsible for reduced behavioral sensitivity to the stress and CRF withdrawal paradigms.
It is also important to note that these studies contain numerous variables that can be considered stressful aside from the experimentally administered restraint stress. These variables include liquid diet exposure, isolate housing, and ethanol exposure, which could all interact in an age-specific manner to effect social interaction behavior in the current studies. While this current work does not address how these paradigms may be impacted by other control conditions (chow-fed and group-housed), the controls employed facilitate appropriate comparisons that support our conclusions.
In summary, these experiments have illustrated that both stress and CRF substitute for early withdrawals and sensitize anxiety-like behavior. Further, there were higher levels of basal CRF levels with the PVN and CeA in adolescent rats. Finally, it was demonstrated that repeated ethanol withdrawals in adolescent rats caused a decrease in CRF density but not cell number within the CeA. Future studies will extend this work to evaluate the role of increased basal CRF in adolescents and how changes in CRF following repeated withdrawals might be tied to the extended anxiety-like behavior found in this group.