Because DID procedures involve providing C57BL/6J mice with limited access to ethanol during the time of day in which food intake is at its highest levels, it was possible that the high levels of ethanol drinking that are stimulated by DID procedures result from increased caloric need (that is, mice treat ethanol as another calorie-rich food source). If this caloric need hypothesis is correct, three predictions should be satisfied: 1) Removal of food should further augment ethanol drinking during DID procedures since ethanol becomes the sole source of calories, 2) pretreatment with an orexigenic peptide (i.e., a peptide that stimulates food intake) should further augment ethanol intake during DID procedures, and 3) pretreatment with an anorectic protein (i.e., an agent that reduces feeding) should attenuate ethanol intake during DID procedures. Contrary to the caloric need hypothesis, none of these predictions were confirmed. Thus, varying the amount of food deprivation time did not significantly alter ethanol drinking or blood ethanol levels. Furthermore, i.p. injection of ghrelin, a peptide that stimulates food intake (Chen et al., 2004
; Wang et al., 2002
), failed to increase ethanol drinking in a dose that increased food intake in C57BL/6J mice. Similarly, i.p. injection of leptin, a protein that attenuates feeding (Halaas et al., 1995
; Prpic et al., 2003
), failed to decrease ethanol intake in a dose that reduced feeding in C57BL/6J mice. Taken together, these observations suggest that the high levels of ethanol drinking promoted by DID procedures are unlikely motivated by caloric need, but rather by other factors such as the pharmacological post-ingestive effects of ethanol.
Interestingly, during the period of time that C57BL/6J mice received ethanol with DID procedures, they exhibited their highest levels of food intake when measures were collected from 4-hours before to 4-hours after the period of time used with DID procedure (). At first glance, this observation appears to be consistent with the hypothesis that high levels of ethanol intake might be related to caloric need. However, we also observed that C57BL/6J mice showed high levels of saccharin solution consumption during this same period of time (). Since saccharin is a non-caloric compound with reinforcing properties, increased consumption of saccharin cannot be related to increased caloric need. An alternative explanation is that C57BL/6J mice exhibit an increased motivation to consume reinforcing stimuli (such as food, sweet flavors, and ethanol) during the time of day that DID procedures are performed, and increased motivation to consume these reinforcing stimuli is independent of the reinforcer’s caloric content.
The present observation that ghrelin did not alter ethanol consumption in C57BL/6J mice is consistent with a recent report showing that hypothalamic infusion of ghrelin increased food intake but did not alter ethanol drinking in Sprague-Dawley rats (Schneider et al., 2007
). Interestingly, plasma ghrelin levels were found to be elevated in alcoholics relative to normal individuals (Kraus et al., 2005
) and plasma ghrelin levels were positively correlated with self-reports of craving in alcoholics (Addolorato et al., 2006
). These observations suggest that while ghrelin may modulate neurobiological pathways involved in craving, this peptide may not directly modulate the ingestion of ethanol. Similarly, the present work with leptin is consistent with the observation that repeated daily injections of leptin failed to alter ethanol drinking in rats, although leptin did appear to augment deprivation-induced increases of ethanol drinking (Kiefer et al., 2001
). However, disruption of leptin signaling in mutant mice was associated with reduced ethanol intake (Blednov et al., 2004
). Circulating levels of leptin have also been found to be elevated in alcoholics, and are correlated with subjective reports of craving in alcoholics (Kiefer et al., 2005
; Nicolas et al., 2001
). Thus, as with ghrelin, while leptin may be involved with craving in human alcoholics, its role in modulating ethanol consumption in animal models remains unclear.
In the present report, we observed BECs in C57BL/6J mice that ranged from approximately 100 to 150 mg%. These levels of BECs are consistent with previous reports that have used DID procedures (Kamdar et al., 2007
; Rhodes et al., 2005
; Rhodes et al., 2007
). Because C57BL/6J mice exhibit signs of behavioral intoxication with BECs in this range (Rhodes et al., 2007
), DID procedures appear to provide a valid animal model of drinking to the point of physiological intoxication, and may be useful for identifying targets that may be protective against binge-like ethanol drinking (Kamdar et al., 2007
; Moore et al., 2007
; Sparta et al., 2008
). The present results further strengthen the usefulness of this model by showing that elevated drinking with DID procedures is unlikely related to caloric need.
In conclusion, data obtained in the present study are inconsistent with the hypothesis that C57BL/6J mice consume large amounts of ethanol during DID procedures in order to satisfy a caloric need. Neither food deprivation nor administration of orexigenic or anorectic compounds significantly alter ethanol drinking with DID procedures. A more likely explanation is that increased ethanol drinking is motivated by other factors associated with the pharmacological post-ingestive effects of ethanol.