During the two days prior to microinjections, when mice were sham injected, the average ethanol intake was 2.85 ± 0.27 g/kg, with a preference ratio for ethanol of 0.87 ± 0.03. There were no significant differences between the two vehicle injections for intake, preference or any other dependent variable. This indicated that microinjection volume (1.0 μl vs. 2.0 μl) had no significant impact and that there was no significant drift in baseline response to vehicle over the term of the study. Thus, vehicle injection data were averaged to create a single vehicle value for subsequent data analysis.
Injection of Ucn 3 dose-dependently decreased the volume of ethanol consumed when expressed in ml (; F(1,38)=5.1, P<0.03) or normalized to the weight of the mice (g/kg; ; F(1,38)=5.3, P<0.03). Ucn 3 did not significantly affect water intake (; P=0.42) or ethanol preference ratio (; P=0.44). However, Ucn 3 did decrease the size of the largest run of licking for ethanol during the 2-h limited access session (; F(1,38)=6.5, P<0.02). No other variable reflecting pattern of drinking was significantly affected by Ucn 3 (), nor did the amount of food consumed change significantly with dose of Ucn 3 during the 2 hour session or in the 24 hours following the injection ().
Ucn 3 significantly decreased ethanol intake (* P<0.03), but not water intake (P=0.42), when measured as volume consumed during the 2-hr limited access session. Data are presented as means ± SEM.
Figure 2 Ucn 3 significantly decreased g/kg ethanol consumption (panel A; * P<0.03) and the size of the largest run of licking for ethanol (panel B; * P<0.02). A run of licking was defined as a minimum of 25 licks with no separation between licks (more ...)
Ethanol drinking and food consumption data (mean ± SEM) from the 2-hr limited-access session.
The findings presented here suggest that the CRF2
R is involved in regulation of ethanol intake when ethanol is offered during limited periods of time. Taken with our previous demonstration of an increase in limited access ethanol intake in CRF2
R deficient mice (Sharpe et al., 2005
), these data suggest that CRF2
R activation may decrease ethanol intake. These results also confirm, via pharmacology in non-genetically altered mice, that developmental compensation in the CRF2
R null mice was not responsible for our previously published effects (Sharpe et al., 2005
). Analysis of the pattern of ethanol consumption using lickometers showed a decrease in the size of the largest run of ethanol drinking, while other parameters were not significantly changed by Ucn 3 administration. Thus, it is not clear from these results if Ucn 3 specifically affects the satiety, initiation or maintenance of ethanol drinking. Subsequent studies using operant self-administration techniques, combined with appetitive vs. consummatory models (e.g., Samson et al., 1998
; Samson and Czachowski, 2003
), could provide more insight into the role of CRF2
Ethanol and food ingestion were both measured in the present study because both processes are regulated by many of the same neurotransmitters, peptides, and neural circuits. Due to the length of the alcohol limited-access session, food intake was measured at 2 and 24 hours post injection. We observed no significant change in food intake in non-food deprived mice in the 2 hours following Ucn 3 injection. This is consistent with previously published reports suggesting that central administration of Ucn 3 decreases food intake in non-food deprived rats after a minimum 2-hour delay (Ohata and Shibasaki, 2004
; Fekete et al., 2007
). While there was no significant change in food intake during the limited access session, there was a significant decrease in ethanol intake during this same time, suggesting that the effect of CRF2
R on ethanol intake may be differentially regulated from that seen with food. In addition to a decrease in food intake following Ucn 3, Ohata and Shibasaki (2004)
also observed a significant decrease in locomotor activity in mice injected i.c.v. with Ucn 3. Locomotor activity was not measured in the studies presented here, thus we cannot rule out the possibility that a general decrease in locomotor activity contributed to the decrease in ethanol intake. However, there was no significant difference in the rate of licking in the first ethanol drinking run, or in the 2-h food intake following Ucn 3 injection in our study, suggesting that any effect of Ucn 3 on locomotor behavior that could have affected consumptive behavior was minor.
While there was no significant change in the amount of food consumed during the limited access session, the pattern of food consumption was not monitored. Thus, it is not possible to determine if the pattern of food consumption changed or if the interactions between feeding and drinking changed following Ucn 3 injection. Recently, a drinking-implicit method of meal patterning has been used by Zorrilla and colleagues (2005)
that includes both eating and drinking events in determining the definition of a meal, since eating and drinking behaviors are often intertwined. A drinking-inclusive analysis of ingestive behavior for food, ethanol and water, during the 2 hr ethanol limited-access procedure may have yielded more detailed information on the effect of Ucn 3 on both ethanol consumption and feeding. A visual analysis of the cumulative records suggests a possible interaction of feeding and drinking after Ucn 3 injection, specifically a trend towards longer times between drinking runs after Ucn 3 injection (data not shown). This increase in inter-run interval after Ucn 3 could reflect a decrease in feeding between runs, resulting in longer pauses between runs of drinking. Since only drinking (but not eating) behavior was monitored on a constant basis during the limited access session in this study, the effect of Ucn 3 on ethanol drinking pattern is difficult due to the normal interplay between eating and drinking in meals.
These results extend, to non-dependent mice, the previous finding that Ucn 3 decreases ethanol self-administration in dependent and withdrawing rats (Valdez et al., 2004
). Dependent, withdrawing mice were not tested in the current study, thus no comparisons between rodent species can be made. In contrast to the current results, Valdez et al. (2004)
did not see a significant effect of Ucn 3 in non-dependent and non-withdrawing rats. This may be explained by the considerable differences in the procedures used for the two studies. The design of the Valdez study, with regard specifically to the non-dependent control group, included a break, during which ethanol self-administration was not available and rats were not placed into the operant chambers. Rats were initially trained to self-administer ethanol in daily sessions, followed by 21 days without exposure to ethanol, during which time they consumed a palatable liquid diet containing sucrose. It is possible that use of this procedure with prolonged periods without ethanol or operant chamber exposure may have unintentionally affected the self-administration behavior of these animals. For example, there could have been loss of training to the operant task. In fact, examination of ethanol versus water responding shows that there was little or no preference for ethanol in this control group, following the abstinent period. In addition, ethanol self-administration may have been altered due to the daily exposure to a highly-palatable, sucrose-containing, liquid diet available in the home cage. In the present study, mice self-administered ethanol in continuous, uninterrupted daily sessions prior to all Ucn 3 treatments. An advantage of this procedure (with no breaks in self-administration) is that the consistent treatment produced subjects with very stable day-to-day self-administration and maintained a high ethanol-preference (see , ethanol preference ratio) despite the Ucn 3-induced decrease in ethanol intake. This stable ethanol-drinking baseline was sensitive to changes in intake, which may not have been possible with a more complicated, variable experimental design. Finally, the differences between the results of the two studies could also be explained by the use of operant self-administration by Valdez et al. (2004)
, as opposed to the non-operant, home cage self-administration presented here. Due to the significant differences in procedure between the two studies, it is not possible to conclude if there is a differential effect of Ucn 3 on ethanol drinking between mice and rats.
The results of this study establish a role for CRF2R in a non-dependent mouse model of ethanol self-administration. Although much of the focus in the alcohol field has been on CRF and activity at CRF1R, results from this study and others suggest that CRF2R may also regulate ethanol self-administration in both dependent and non-dependent animals. This effect may be mediated by the hypothalamus, which is involved in regulation of ingestive processes, or limbic areas such as the amygdala or bed nucleus of the stria terminalis that are hypothesized to regulate drug reward and use. Future work using microinjections could elucidate the specific region or regions of the brain where CRF2R regulate ethanol intake, and perhaps determine if chronic drug use has an effect on endogenous levels of Ucn 3 or CRF2R.