Our studies for the first time tested in parallel four lines of mice with genetic deletions of components of the CRF system in a model of binge alcohol consumption, i.e., the DID paradigm. While CRFR2 KO mice did not show any distinct differences in binge drinking or BECs compared to their WT counterparts, CRFR1 KO mice showed lower alcohol intakes on all four days and lower BECs than the WTs but greater sucrose intakes on days 1 to 3, indicating that the CRF receptor type 1 is involved in alcohol binge drinking. Furthermore, Ucn1 KO mice were found to drink similar amounts of alcohol and showed similar BEC levels as the WTs, whereas CRF KO mice had clearly reduced alcohol intakes and BEC levels when compared to their WT littermates, indicating that CRF acting on CRFR1 promotes binge alcohol consumption. Importantly, the genetic deletion of CRFR1 and CRF prevented these mutants from reaching BECs that are considered intoxicating. These results cannot be attributed to potential differential alcohol metabolism between genotypes, as several studies have consistently shown no difference in blood alcohol elimination rates between these genotypes (Olive et al., 2003
: Pastor et al., 2008
; Sharpe et al., 2005
: Sillaber et al., 2002
Interestingly, for all the genotypes tested, female mice had higher alcohol intakes than male mice, but there was no difference in the BECs between the sexes, suggesting that alcohol clearance was different between females and males. This is in agreement with previous studies showing that female rodents consume greater amounts of alcohol and appear to be more sensitive to the effects of alcohol than males (Crippens et al., 1999
; Lancaster and Spiegel, 1992
; Middaugh et al., 1999
; Peterson et al., 1991
). This difference is most likely attributed to faster alcohol metabolism rates in female than male mice and rats (Crippens et al., 1999
; Peterson et al., 1991
). These findings may explain the discrepancy between alcohol intake and BECs between the sexes that we observed. However, for none of the genotype effects was there a significant interaction with sex, indicating that the contribution of CRF and CRFR1 receptors to binge alcohol consumption occurs in animals of both sexes.
Our results showing that CRFR1 KO mice consume less alcohol and have lower BECs than the corresponding WT mice in a binge drinking model bear out and strengthen recent pharmacological evidence showing that administration of a CRFR1 antagonist results in decreased binge drinking (Lowery et al., 2008
; Sparta et al., 2008
). Antagonists of the CRF receptor type 1 have also been reported to reduce alcohol intakes in dependent rats (Funk et al., 2007
; Gehlert et al., 2007
; Gilpin et al., 2008
; Ji et al., 2008
; Sommer et al., 2008
). Further evidence showing the involvement of the CRFR1 in excessive drinking comes from a study carried out by Treutlein et al. (2006)
where two SNPs in the CRFR1 gene were associated with heavy drinking in adolescents with little previous exposure to alcohol and in alcohol-dependent adults. The recent finding that CRFR1 KO mice had lower intakes of 20% ethanol but not of 3%, 6% and 10% ethanol when compared to WT mice in a 24 hour access two-bottle choice paradigm (Pastor et al. 2011
) also provides suggestive evidence that binge, and not modest, drinking is affected. Taken together, our current genetic studies in KO mice, genetic studies in humans and previous and recent pharmacological studies clearly implicate the CRFR1 in excessive alcohol consumption in both non-dependent and dependent subjects.
Mice deficient in CRFR2 did not show any distinct differences in alcohol intakes or in BECs, except on day 1, which may be due to a delay in acquisition of this behavior. Pharmacological studies in dependent rats have shown that administering a CRFR2 agonist, Ucn 3, into the CeA resulted in decreased drinking (Funk and Koob, 2007
). Lowery et al. (2010)
recently reported that Ucn 3, administered intracerebroventricularly in C57BL/6J mice, protected against binge drinking in a DID procedure similar to ours. Interestingly, Sharpe et al. (2005)
showed that CRFR2 KOs did not behave any differently to WTs after continuous alcohol exposure but showed increased intakes at two out of ten stages of their limited access alcohol drinking procedure. Both these findings can be considered different from our observation of no difference between CRFR2 KO and WT mice in the DID procedure. However, in contrast to Sharpe et al. (2005)
our procedure involves 4 hour access to ethanol and used a higher concentration of ethanol, and it has been argued that this protocol appears to be a better measure of the binge aspect of alcohol intake (Sparta et al., 2008
). While our procedure is similar to the one used by Lowery et al. (2010)
, the mice in their study underwent stereotaxic implantation of cannulae and central administration of the CRFR2 agonist whereas our CRFR2 KO and WT mice were allowed to drink undisturbed. Another possible explanation for the discrepancy could be potential developmental compensations occurring in the CRFR2 KO mice. Importantly, none of the studies observed a decrease in intake in the CRFR2 KO mice, which implies that CRF type 2 receptors do not play a critical role in moderate or excessive binge-like alcohol consumption.
Since results from our first experiments showed that CRFR1 is critical in binge alcohol consumption and since both CRF and Ucn1 bind to CRFR1, it was logical to test Ucn1 and CRF KO and WT mice in the same DID protocol to determine the ligand responsible. A major caveat of pharmacological studies is the inability of pharmacological agents to distinguish between multiple ligands acting at the same receptor. In contrast, this can be investigated using KO mice. Ucn1 KO mice were not different from their WT counterparts in either alcohol intakes or BECs. Even though studies showing higher levels of Ucn1 immunoreactivity in mice and rats selectively bred to prefer alcohol suggest that Ucn1 plays a role in the predisposition to alcohol consumption (Fonareva et al., 2009
; Ryabinin and Weitemier, 2006
; Turek et al., 2005
), the Ucn1
gene appears not to be critical in the regulation of binge alcohol intake. We interpret these results as suggesting that Ucn1 may be critical in regulating alcohol acceptance or moderate drinking, but not in excessive alcohol consumption. In support of this, Bachtell et al. (2004)
showed that electrolytic lesions of the Edinger-Westphal nucleus, which is the main source of Ucn1 in the brain, resulted in reduced alcohol preference and consumption in a two-bottle choice continuous access paradigm when the mice were given access to 3, 6 or 10% ethanol, but no difference was seen in preference or consumption between lesioned and sham mice when access was to 20% ethanol.
In contrast to our results with the Ucn1 KOs, CRF KO mice consumed lower amounts of alcohol than their WT littermates and had lower BECs. Our data contrast with findings reported by Olive et al. (2003)
. In their study, CRF KO mice were found to consume more alcohol than the control mice in continuous and limited access experiments. A key difference between their study and ours is their use of F2 hybrids, obtained by breeding C57BL/6J and 129S1/SvImJ mice, as control mice. It is expected that F2 offspring from crossing high drinking and low drinking mouse strains would show low alcohol intake (Carr et al., 1998
), as was observed in their study. In contrast, our study used WT littermates of the CRF KO mice, which have been largely backcrossed on to the C57BL/6J background and therefore show the characteristic high C57BL/6J-like alcohol intake. In addition, the previous study did not use the DID model, so the investigated phenotypes across these studies are different, and this could serve as another argument that CRF acting on CRFR1 receptors is critical only for binge drinking, but not for moderate alcohol consumption.
Other alcohol-related phenotypes have also been investigated in these lines of knockout mice. Pastor et al. (2008)
showed that that CRFR1 KO and CRFR1+R2 KO, but not CRFR2 KO, mice showed attenuated psychomotor sensitization to ethanol. In the same set of experiments, Pastor et al. (2008)
also showed that Ucn1 KO mice were not different compared to WTs and concluded that CRF and CRFR1 were required for sensitization to ethanol to manifest. These findings on psychomotor sensitization to ethanol compare favorably with our data on binge alcohol consumption. Pastor et al. (2008)
hypothesized that the hypothalamic-pituitary-adrenal (HPA) axis may underlie sensitization to alcohol. CRF, acting at the CRFR1, activates the HPA axis, which results in adrenocorticotropic hormone (ACTH) release by the anterior pituitary and glucocorticoid release by the adrenal glands in response to stress (for reviews see Armario, 2006
; Hauger et al., 2006
; Smith and Vale, 2006
). Alcohol is also reported to activate the HPA axis and this effect is blocked by administering CRF antiserum or antagonists and is absent in CRFR1 knockout mice, indicating that the effects of alcohol are exerted via the CRF system (Lee et al., 2001
; Rivier, 1996
; Rivier et al., 1996
; Rivier et al., 1984
; for review see Armario, 2010
). Furthermore, activation of the HPA axis is also hypothesized to be a key factor in alcohol addiction (Fahlke et al., 2000
; Higley et al., 1991
; Uhart et al., 2006
). Conversely, activating the HPA axis has also been shown to decrease alcohol consumption (Krishnan et al., 1991
) or prevent relapse (Kiefer et al., 2006
). Pastor et al. (2008)
observed attenuated corticosterone levels in CRFR1 and CRFR1+R2 double KOs and concluded that CRFR1-mediated corticosterone release and subsequent activation of glucocorticoid receptors was necessary for sensitization to ethanol to occur.
Therefore, one could theorize that the effects of CRF and CRFR1 deletion on binge alcohol intake observed here could also be attributed to effects of these deletions on the HPA axis. To our knowledge, no studies on alcohol drinking in mice deficient in the glucocorticoid receptors have been reported. However, pharmacological experiments suggest that in contrast to locomotor sensitization, the HPA axis is not critical for binge alcohol consumption. Specifically, while Lowery and colleagues (2010)
observed that administering a CRFR1 antagonist decreased binge drinking in agreement with the KO studies described here, they also assessed the contribution of the HPA axis by administering metyrapone, a corticosterone synthesis inhibitor; mifepristone, a glucocorticoid receptor antagonist and by investigating binge drinking after administering a CRFR1 antagonist in adrenalectomized (ADX) mice. They reported that metyrapone reduced both alcohol and sucrose consumption and was not selective, that mifepristone had no effect on alcohol drinking, and that plasma corticosterone levels after a DID procedure did not correlate with binge drinking. Also in agreement was the finding that the CRFR1 antagonist was effective in reducing binge drinking in ADX mice. Therefore, the HPA axis does not appear to play a critical role in binge alcohol consumption. It is likely therefore, that the contribution of CRFR1 to excessive alcohol intake is via actions of CRF on its central targets.
One caveat that has to be kept in mind when using genetic mouse models is the potential for developmental compensations to occur. Several compensations have been documented in the mice that we utilized. For example, in CRFR1 KO mice, CRF is over-expressed in the paraventricular nucleus (PVN), amygdala, hippocampus and cerebral cortex (Smith et al., 1998
; Timpl et al., 1998
). CRFR2 KO mice show no significant change in CRF mRNA levels in the PVN but increased levels in the central nucleus of the amygdala and increased levels of Ucn1 in the Edinger-Westphal nucleus (now known as the centrally projecting neurons of the Edinger-Westphal, EWcp; see Kozicz et al., 2011
) (Bale et al., 2000
; Coste et al., 2000
). Ucn 1 mRNA is increased in the EWcp of CRF KO mice (Weninger et al., 1999
). In contrast, there appears to be no change in CRF or CRFR1 in Ucn1 KO mice and a slight decrease in CRFR2 gene expression in the lateral septum (Bale and Vale, 2004
; Vetter et al., 2002
; Wang et al., 2002
). While it is theoretically possible that the increased Ucn1 expression in the CRF KO mice and increased CRF in the CRFR1 KOs contributed to our observations, the fact that both these peptides have also been reported to be increased in CRFR2 KO mice, which show no change in alcohol consumption, contradict this idea. Furthermore, pharmacological and genetic evidence discussed above suggests that these compensations would have potentially counteracted rather than contributed to a decrease in drinking. Therefore, our genetic data suggests that developmental compensations are insufficient to prevent the critical role of CRF, via actions on CRFR1, in binge alcohol consumption.
In conclusion, the studies detailed here and previous pharmacological studies show effects in the same direction and clearly implicate CRFR1 in binge drinking. Therefore, CRFR1 is a promising target for the pharmacotherapy of alcohol use disorders. Moreover, our studies with KO mice with their advantage over pharmacological studies, were able to differentiate between multiple ligands acting at the same receptor type, and identified CRF as the ligand responsible for the involvement of CRFR1 in promoting excessive alcohol drinking.