The results of these experiments provide strong evidence for the involvement of the CRF/urocortin system in the regulation of alcohol consumption. Importantly, while the majority of studies in this field focus on the importance of CRF, our studies clearly show that Ucn1 could play an equal, if not a greater role in the regulation of alcohol intake. Overall, intra-lateral septum injection of Ucn1 significantly decreased alcohol consumption at picomolar doses in contrast to water, which was not affected by Ucn1 at these doses. Ethanol consumption appeared differentially sensitive to Ucn1 because CRF decreased ethanol consumption only at the highest dose (60 pmol), and both ethanol and water consumption were affected by CRF microinjections. Ucn1 was capable of suppressing ethanol intake both when it was administered prior to the third ethanol drinking session, presumably influencing the expression of ethanol intake, and when it was administered during the first three ethanol drinking sessions, presumably affecting the acquisition of ethanol self-administration. Moreover, while injections of this peptide did not have significant prolonged effects on fluid consumption beyond the 2-hour ethanol drinking session, a history of repeated Ucn1 injections led to decreased ethanol intake in the absence of Ucn1, which may represent a conditioned response. Taken together, these data suggest that Ucn1 affects the regulatory processes governing limited access ethanol consumption.
Since Ucn1 is a peptide related to CRF, much attention has focused on its potential involvement in the regulation of anxiety and the stress response (Bale and Vale, 2004
, Gysling et al., 2004
, Kozicz, 2007
). While the data on its involvement in the regulation of these responses remain not without contradictions, with different results depending on the species and stress paradigm (Weninger et al., 2000
, Vetter et al., 2002
, Wang et al., 2002
, Gaszner et al., 2004
, Kozicz et al., 2004
, Turek and Ryabinin, 2005
), it is important to consider whether anxiety or stress could contribute to the results observed in the present studies. In fact, several studies have shown that intra-lateral septum injection of CRF or related peptides could induce anxiety-related responses (Radulovic et al., 1999
, Henry et al., 2006
), while intra-lateral septum injection of CRF2 receptor antagonists could attenuate them (Radulovic et al., 1999
, Bakshi et al., 2002
). However, the threshold doses of Ucn1 that were capable of decreasing ethanol consumption in our procedure (6 pmol) are substantially lower than doses of CRF (100 but not 20 pmol) or doses of Ucn2 (240 but not 48 pmol) that have been reported to increase anxiety-like behavior in mice (Radulovic et al., 1999
, Henry et al., 2006
It has been suggested that exposure to immobilization stress can increase the sensitivity of anxiety responses in mice to intra-lateral septum Ucn2 injections. Although it is unlikely that mice in our procedure experience levels of anxiety similar to those produced by immobilization stress, it is important to note that our 6 pmol dose was lower than the dose of Ucn2 necessary to produce an increase in anxiety in mice stressed by immobilization (48, but not 4.8 pmol, (Henry et al., 2006
)). Theoretically it is possible that Ucn1 used in our study would be more potent than CRF or Ucn2 in producing anxiety, by acting on both CRF1 and CRF2 receptors. However, previous studies have shown that the effects of stress-induced anxiety are mediated by CRF2, and not CRF1 receptors (Henry et al., 2006
, Todorovic et al., 2007
). Therefore, it is unlikely that activation of CRF1 and CRF2 receptors by Ucn1 would have additive effects exceeding its action at CRF2 receptors.
Taking into account the above mentioned doses of peptides required for anxiogenic effects, it is possible that the non-specific effects of the highest doses of CRF on ethanol and water consumption observed in our experiments were mediated by increased anxiety following these injections. In contrast, Ucn1 did not exert a non-specific effect on fluid consumption, exhibiting a selective effect on ethanol intake. Therefore, it is much more likely that the effects of Ucn1 on ethanol intake were not mediated by concomitant effects on anxiety. This conclusion concurs with early studies showing that intracerebroventricular administration of Ucn1 was more effective than CRF at reducing food intake, but less effective at increasing anxiety (Spina et al., 1996
Since the selectivity of the effects of Ucn1 on ethanol consumption is suggested by lack of effects on water intake in Experiment 3, the possibility of test-order effects should be acknowledged. Theoretically, one can envision that a single exposure to picomolar doses of Ucn1 (as low as 6 and 20 pmol) could lead to insensitivity of lateral septum to subsequent administrations of this peptide. However, such desensitization did not occur in the Experiment 4, even with a higher (40 pmol) dose of Ucn1. Therefore, we believe it is unlikely that test-order effects contributed to the selectivity of Ucn1’s effect on ethanol consumption (and lack of effect on water consumption with subsequent microinjection) in Experiment 3.
Interestingly, it has been shown that intra-lateral septum injections of CRF-like peptides can modulate learning independently of their effects of anxiety (Radulovic et al., 1999
). Importantly, these effects were mediated by lower doses of CRF and Ucn1 (20 pmol). Therefore, we cannot exclude the possibility that Ucn1’s effect on alcohol consumption during acquisition of alcohol drinking (Experiment 4) could be influenced by Ucn1’s effect on learning. While an effect of repeated administration on learning might contribute to the putative conditioned suppression of ethanol intake following the sham injection in Experiment 4, the potential effect on learning cannot account for the Ucn1-mediated suppression of ethanol intake following a single administration of the peptide (e.g., Day 3 of Experiments 1 and 3 or Day 1 of Experiment 4). Therefore, it seems more likely that Ucn1 influenced ethanol intake by interfering with the regulatory processes governing alcohol consumption.
The role of the lateral septum in the reinforcing properties of food or drugs of abuse has been studied less than that of the more traditionally investigated “reward” circuits. Therefore, it is important to consider whether the observed effects of intra-lateral septum injections were mediated by leakage of Ucn1 into other brain regions or into the ventricles. Intracerebroventricular effects of peptides in our studies are unlikely because substantially higher doses of Ucn1 are needed to produce significant effects on behavioral responses (Swiergiel and Dunn, 1999
, Bradbury et al., 2000
, Sinnayah et al., 2003
). The effects are also not likely to be mediated by Ucn1 leakage to other brain regions because the majority of other forebrain regions contain higher levels of CRFR1 than CRFR2 receptors, and one would expect stronger or equal effects of CRF than Ucn1 in these brain regions (Potter et al., 1994
, Chalmers et al., 1995
, Perrin et al., 1995
, Turnbull and Rivier, 1997
, Steckler and Holsboer, 1999
, Fekete and Zorrilla, 2006
). In light of these facts, the role of the lateral septum in consumption of ethanol needs to be given strong consideration.
In agreement with this role, lesions of the lateral septum have been shown to increase water consumption (Iovino and Steardo, 1985
, Taghzouti et al., 1985
, Vasudev et al., 1985
) and food consumption in rats (King and Nance, 1986
, Oliveira et al., 1990
). This suggests that the lateral septum has suppressive effects on consummatory behaviors. Since lateral septal neurons are preferentially GABAergic, lesions of this region would presumably disinhibit the hypothalamus, a long-proposed integrator of consummatory responses (Leibowitz, 1992
, Elmquist et al., 1998
, Inui, 1999
). However, interpretation of these data are complicated by the fact that the lateral septum is involved with other functions (including anxiety, as described above), which would also contribute to the regulation of consummatory behaviors. In this regard, it is important to note that one study to date investigated the effects of Ucn1 and CRF microinjection into the lateral septum on food consumption. The results demonstrated that relatively low doses of Ucn1 injected into the lateral septum (3-30 pmol) suppressed food consumption in rats, and that this suppressive effect was longer lasting than that of equimolar CRF (Wang and Kotz, 2002
). No study prior to the present one has investigated the effects of intra-septal injections of any substances on the reinforcing properties of addictive drugs. A study using brain slices has shown that Ucn1 suppresses glutamatergic activation of neurons in the lateral septum, and that repeated cocaine administration in rats reverses this effect (Liu et al., 2004
, Liu et al., 2005
). The involvement of the lateral septum in the reinforcing effects of drugs of abuse clearly deserves further investigation.
Given that Ucn1 exerted stronger and more specific effects on ethanol consumption than CRF, it seems more likely that these effects are mediated through CRF2 and not CRF1 receptors. Since the CRF2 receptor has high affinity to Ucn1, Ucn2 and Ucn3, it is important to note that not only Ucn1, but also Ucn3, innervate this brain structure (Li et al., 2002
). Therefore, we cannot exclude the possibility that the consumption of alcohol could be regulated by either Ucn1 or Ucn3. However, several studies that mapped expression of the immediate early gene product c-Fos have shown that self-administration of alcohol leads to consistent activation of the pIIIu, the major source of Ucn1 in the brain, but not brain areas containing Ucn3 (Topple et al., 1988
, Bachtell et al., 1999
, Ryabinin et al., 2003a
, Sharpe et al., 2005
). Therefore, it seems likely that Ucn1 from the pIIIu contributes to the regulation of alcohol consumption.
An important consideration is that previous studies using genetic and lesion approaches correlated higher number of Ucn1-containing neurons in the pIIIu with higher ethanol intake (Ryabinin and Weitemier, 2006
). In contrast, our present study shows that application of Ucn1 suppresses
ethanol intake. One potential explanation for this apparent discrepancy is that if Ucn1 contributes to a reward signal associated with ethanol, exogenous application of Ucn1 could signal that the reward already has been achieved, resulting in no further effort to obtain it (by alcohol consumption). However, in this case one would expect low doses of Ucn1 to increase ethanol intake during acquisition of this behavior. Since our study included very low doses of Ucn1, this reasoning seems unlikely. As an alternative explanation, since pIIIu neurons contain other peptides besides Ucn1 as potential regulators of the rewarding properties of drugs of abuse [including CART, CCK and neuropeptide B (Maciewicz et al., 1984
, Innis and Aghajanian, 1986
, Couceyro et al., 1997
, Kozicz, 2003
, Tanaka et al., 2003
)], it is possible that synergistic actions of these peptides are needed to increase the rewarding properties of alcohol. Lesions of the pIIIu or a study correlating the number of cells in the pIIIu with alcohol drinking cannot directly test this possibility, but can only point to an importance of the brain region in this behavior.
Taken together, our experiments show that the neuropeptide Ucn1 acts to suppress alcohol consumption through the lateral septum. Further studies will be required to decipher the exact role of the complex neurocircuits expressing Ucn1 in the regulation of the actions of alcohol and other drugs of abuse.