Initial sensitivity to the intoxicating effects of ethanol is a genetically mediated trait. Using an animal model, these results support this finding and suggest that in addition to genetic susceptibility, environmental factors (e.g., restraint stress, CORT administration) also influence sensitivity to the sedative-hypnotic effects of ethanol, albeit in divergent directions and in a genotype-dependent manner. In ILS mice, the more ethanol sensitive of the genotypes, acute-and repeated-restraint stress decreased sensitivity to the sedative-hypnotic effects of ethanol as measured by LORE duration. The increase in BECRR in the ILS ACUTE group suggests that a decrease in CNS sensitivity underlies the shortened LORE duration in the ACUTE treatment; however, CNS sensitivity does not fully account for the shortened LORE duration in the REPEATED group because BECRR did not change. In the ISS mice (the ethanol-insensitive genotype), repeated-restraint stress caused an increase in sensitivity to ethanol at the 4.1 g/kg dose as indicated by increased LORE duration and decreased BECRR. This effect was contrary to that of the ILS mice. When ISS mice were given a higher dose of ethanol, the results were less clear. At the 6.0 g/kg dose, ISS mice showed a similar increase in duration of LORE after repeated stress as they did at the 4.1 g/kg dose. However, their BECRR was also higher than the NO STRESS and ACUTE STRESS groups, thus suggesting a decrease in sensitivity. As Radcliffe et al. (2005)
have demonstrated, ISS mice display a large drop in body temperature in response to a 6 g/kg dose of ethanol, and it is possible that the high dose of ethanol induced severe hypothermia in the ISS, thus decreasing their rate of metabolism; that is, the ISS became less sensitive, but the metabolic effect caused a longer LORE duration. It is also feasible that at such a high dose, the increased BECRR in the ISS may have been caused by the development of acute functional tolerance. However, little is known of the effect of acute or repeated stress on acute tolerance.
Duration of LORE is due to the effects of both CNS ethanol sensitivity and pharmacokinetic parameters such as ethanol absorption and clearance. A change in either will alter the duration of LORE, although the latter would probably have a much greater effect than the former after i.p. administration. Although LORE and BECRR are generally expected to be correlated, BEC is a more sensitive indicator of the effects of ethanol in the CNS. To determine if changes in metabolism were causing the differences in LORE duration, ethanol absorption and clearance experiments were performed on acutely stressed and unstressed ILS and ISS mice. A significant decrease in ethanol absorption was observed in acutely stressed mice, amounting to about a 10% difference in BEC values. However, a three-way ANOVA found no significant differences in ethanol clearance across treatments, thus suggesting that pharmacokinetic parameters were not responsible for the effects of acute stress on waking blood ethanol values. Although the linear regression showed modest strain differences in slope and BEC at time = 0, there were no differences in ethanol elimination between stress treatments. The slight differences in ethanol metabolism between strains have been reported before (Smolen et al., 1986
) and might marginally contribute to the overall differences in sensitivity; however, acute stress had no significant effect on clearance and is not sufficient to explain the behavioral differences that were observed. In addition, it is possible that repeated stress could alter ethanol absorption or clearance in ILS and ISS mice. Unfortunately, the emergence of breeding problems in the colony at the Institute for Behavioral Genetics has severely limited the availability of these strains of mice, making them unavailable for additional testing. Further study on the effects of repeated stress on ethanol pharmacokinetics would be informative.
Because of the observed differences between ILS and ISS in their LORE and BECRR following restraint stress, the mice were next examined to determine if they differed in their CORT responsiveness to restraint stress. This is the first time the ISS and ILS mice have been examined for differences in CORT responses following psychological stress (previous work has used the outbred SS and LS only). Minnick et al. (1995)
reported that stressed LS mice released significantly more CORT than stressed SS mice; however, our findings do not concur with their observation. Here, ILS and ISS mice did not differ in plasma CORT levels immediately following acute-restraint stress. However, measurement of CORT levels at various time points following restraint would provide important information on how the strains adapt to repeated-restraint stress. The fact that our CORT results in ILS and ISS mice do not follow the same relative patterns as those seen in the progenitor strains does not necessarily imply that these differences in CORT responsiveness are a result of inbreeding; rather, they may be a function of the different stressors used by Minnick et al. (45 min of exposure to the open arm of an Elevated Plus Maze). Additionally, results indicated that the CORT response of the ILS mice began to habituate to repeated-restraint stress, whereas the CORT response of the ISS mice did not (). Further research on the effects of repeated stress and CORT habituation in ISS mice would be informative.
Previous studies in rodents suggest that exposure to stress may modulate sensitivity to ethanol, yet the ways in which this occurs remain unclear. In experiment 5, we sought to determine if enhanced CORT levels alone (in the absence of the psychological stress component) were sufficient to alter sensitivity to the sedative-hypnotic effects of ethanol.
Interestingly, there were no effects of saline or CORT injections on LORE duration in ILS mice compared to their noninjected littermates (). However, ISS mice injected with CORT showed a decrease in duration of LORE compared to both saline and non-injected littermates (). There were no effects of treatment on BECRR in either strain, thus suggesting that some property of ethanol pharmacokinetics was altered in the ISS, rather than a change in CNS sensitivity. Interestingly, the effect of CORT injection on LORE duration is in the opposite direction to the effect of restraint stress in ISS mice, and may be due to a variety of reasons. One explanation for these strain differences may be that the elevated CORT levels caused by the CORT injections dissipated in the ILS mice before having the opportunity to alter LORE duration due to their inherently longer sleep times. However, this possibility is unlikely, because the exogenous CORT injections were matched to mimic the rise in CORT following 30 min of restraint stress (which did have a significant effect on the LORE duration of ILS mice).
Alternatively, it is feasible that other aspects of activated neurocircuitry following psychological stress are primarily responsible for the influence of stress on LORE independent of elevated CORT concentrations. In fact, numerous labs have shown that genetic differences between ILS and ISS in 5HT, DA, norepinephrine, GABA, and NMDA (Hanania et al., 2000
; Hanania & Zahniser, 2002
; Haughey et al., 2005
; Proctor et al., 2004
; Zahniser et al., 1992
) receptors may be responsible for habituation (or lack thereof) of the HPA-axis response to stress and underlie their differing sensitivity to ethanol following stress or glucocorticoid injection. Further research using the ILS and ISS strains to determine how these neural systems are differentially regulated by stress exposure might prove fruitful in further elucidating genes and gene networks involved in stress and ethanol sensitivity.
In conclusion, the present study demonstrated that exposure to restraint stress and exogenous CORT injection modified the sedative-hypnotic effects of ethanol on ILS and ISS mice in different ways and in a genotype-dependent fashion. This suggests that differences in CORT levels alone are not sufficient to explain the influence of stress on sensitivity to the sedative-hypnotic effects of ethanol; and further research on the neuroanatomical, biochemical, and molecular mechanisms underlying this phenomenon are needed. Ongoing studies are being conducted to continue examining the effects of restraint stress on other ethanol sensitivity-related phenotypes (such as the hypothermic, locomotor activating, or anxiolytic effects of ethanol) across inbred, congenic, and mutant strains of mice. These will be useful in further exploring the impact of stress-induced alterations of critical responses to ethanol using a mouse model.