This report provides novel evidence that NPY signaling selectively modulates binge-like ethanol drinking in C57BL/6J mice. Briefly, we found that i.c.v. infusion of NPY blunted ethanol drinking associated with DID procedures. In addition, a Y1R agonist and a Y2R antagonist blunted binge-like ethanol drinking, implicating the Y1R and Y2R as key receptors in modulating this behavior. We then examined the influence of a history of binge-like ethanol drinking on NPY signaling in the CeA using immunohistochemistry and electrophysiology approaches. In support of the behavioral findings, we found that binge-like ethanol drinking reduced NPY and Y1R IR in the CeA, and removal of ethanol for 24
h following three cycles of binge-like ethanol drinking promoted increases of Y1R and Y2R IR. Electrophysiological recordings of slice preparations from the CeA showed that binge-like ethanol drinking augmented the ability of NPY to inhibit GABAergic transmission. Taken together, the present observations (and previous data with CRF1R antagonist; Lowery et al, 2010
; Sparta et al, 2008
) suggest that signaling in critical NPY pathways is weakened (and CRF signaling is enhanced) when sufficient amounts of ethanol have been consumed during a binge-like drinking episode, at which point administration of exogenous NPY is effective in blunting binge-like ethanol intake (either by restoring NPY signaling and/or by countering the effects of CRF). Our previous finding that a range of centrally administered NPY doses did not alter non-binge-like ethanol drinking in C57BL/6J mice (Thiele et al, 2003
) suggests that NPY selectively modulates excessive ethanol intake, which is also consistent with previous work on dependence-like ethanol intake (Gilpin et al, 2008
). Data from immunohistochemistry and electrophysiology studies point to the CeA as a critical brain region in which NPY modulates binge-like drinking, and future studies that directly manipulate NPY signaling in the CeA will help address this question. Importantly, to the best of our knowledge, these are the first observations of alterations of NPY and NPY receptor signaling in animals voluntarily drinking excessive amounts of ethanol, as previous work has relied on forced ethanol exposure via ethanol vapor (Criado et al, 2011
; Ehlers et al, 1998
; Gilpin et al, 2011
; Slawecki et al, 2005
; Walker et al, 2010
), ethanol-containing diet (Roy and Pandey, 2002
; Zhang et al, 2010
), or ethanol injection (Pandey et al, 2008
; Sakharkar et al, in press
The effects of NPY on binge-like ethanol drinking appear to be reciprocally modulated by Y1R and Y2R. Central infusion of a selective Y1R agonist decreased, whereas a selective Y1R antagonist increased, binge-like ethanol intake. In contrast, central infusion of a selective Y2R agonist did not significantly alter binge-like ethanol drinking whereas central infusion of a selective Y2R antagonist significantly decreased binge-like consumption. Importantly, effective doses of selective NPY compounds (and NPY) that altered binge-like ethanol drinking did not influence 10% sucrose consumption in the same direction, suggesting that the effects of these compounds were specific to ethanol intake. Previous work has implicated roles for the Y1R and Y2R in the modulation of ethanol consumption (Thiele et al, 2002
; Thorsell et al, 2002
). The ability of the Y1R antagonist to significantly increase binge-like ethanol drinking suggests that Y1R signaling is engaged during binge-like drinking episodes (perhaps early on in the drinking episode); however, Y1R signaling in response to endogenous NPY is not sufficient to protect against binge-like drinking, likely because of blunted NPY signaling that develops over the course of the binge-like drinking episode (see below). The ability of the Y2R antagonist to significantly reduce binge-like ethanol drinking suggests that the Y2R is also engaged during binge-like drinking. The Y2R may be acting as an autoreceptor that reduces NPY release (Chen et al, 1997
; King et al, 1999
), and thus blocking Y2R would theoretically increase NPY signaling, upregulating the protective effects of NPY.
We found that binge-like ethanol drinking lead to selective alterations to the NPY systems in the CeA, as sucrose drinking did not alter NPY, Y1R, or Y2R IR. A lack of influence of binge-like drinking on NPY or Y1R IR in the BLA and MeA suggests that alterations of IR were specific to the CeA. Although caution is necessary when inferring the direction of changes in signaling based on IR data (Navarro et al, 2008
), when viewed together, the pharmacological and IR data strongly suggest that over the course of a binge-like drinking episode, endogenous NPY signaling is blunted, which may motivate continued excessive binge-like drinking. Low endogenous NPY signaling can be overcome by administration of exogenous NPY or a selective Y1R agonist, or a selective Y2R antagonist (which as a presynaptic autoreceptor (Chen et al, 1997
; King et al, 1999
) may increase endogenous NPY signaling). Interestingly, chronic exposure to ethanol and withdrawal have been reported to cause decreased levels of NPY in the amygdala (Roy and Pandey, 2002; Zhang and Pandey, 2003
), revealing similar observations between paradigms promoting binge-like drinking and dependence-like states in rodents.
Because data from the IR studies indicated an important role for the CeA in the modulation of binge-like ethanol drinking, we used in vitro
slice electrophysiological procedures to study the effects of binge-like ethanol drinking on basal and NPY-induced alterations of GABAergic transmission. We found no significant differences between binge-like ethanol drinking and water control groups in terms of PPR or spontaneous GABAergic transmission, suggesting that a history of binge-like drinking did not alter basal GABAergic function. This contrasts with previous evidence indicating that baseline GABAergic transmission is upregulated in the CeA of rats previously exposed to ethanol vapor (Roberto et al, 2004
), suggesting that on this measure, the effects of a history of binge-like drinking are different than the effects generated by models used to study dependence-like ethanol drinking.
In tissue from mice that had a history of three cycles of binge-like ethanol drinking and 24
h without ethanol access, NPY-induced inhibition of GABA transmission was enhanced, likely through activation of Y2R given the established role of the Y2R in modulating the effects of NPY on GABAergic transmission in the CeA (Gilpin et al, 2011
). This finding was consistent with the IR results from mice in which ethanol was removed for 24
h after three cycles of binge-like ethanol drinking that also exhibited an upregulation of Y2R (and Y1R) in the CeA relative to the water drinking control group. When one considers the potential autoreceptor function of the Y2R, these changes, particularly the increase in Y2R IR, could be maladaptive, potentially leading to reduced levels of both NPY release and Y1R activation. Presynaptic Y2R also functions as a heteroreceptor that inhibits GABA release (Qian et al, 1997
), which is the likely mechanism by which NPY inhibits GABAergic release here and in previous reports (Gilpin et al, 2011
; Kash and Winder, 2006). When the heteroreceptor function is considered, it is possible that NPY-mediated inhibition of GABA release leads to increased excitability in downstream populations of neurons. The functional ramifications of increased downstream excitability are difficult to assess, as there are multiple populations of neurons in the lateral CeA (Haubensak et al, 2010
). The present electrophysiology results complement the pharmacology and IR data, which together strongly suggest that NPY signaling in the CeA is compromised as a result of binge-like ethanol drinking in C57BL/6J mice.
It was recently reported that application of NPY to slice preparations from the CeA decreased baseline GABAergic transmission and inhibited ethanol-induced stimulation of GABAergic activity (Gilpin et al, 2011
). Interestingly, although we observed increased NPY-induced inhibition of GABAergic activity in the CeA of mice with a history of binge-like ethanol drinking, Gilpin et al (2011
) found no such differences between vapor-exposed and naive rats, which is additional evidence that the mechanisms that drive excessive ethanol intake in models of binge-like drinking and dependence-like drinking are not identical. Although we cannot rule out species differences as the cause of discrepant results, one striking dissimilarity between our study and that of Gilpin et al (2011
) is that we studied excessive ethanol intake in rodents that voluntarily drank ethanol, whereas Gilpin et al (2011
) studied excessive ethanol intake in animals that had prior forced ethanol exposure via vapor inhalation.
Importantly, Gilpin et al (2011)
also found that prophylactic application of NPY during ethanol vapor exposure protected against the development of vapor-induced ethanol drinking. These observations, in tandem with data indicating that the direct application of NPY into the CeA protects against vapor-induced dependence-like drinking (Gilpin et al, 2008
), further reinforce the hypothesis that the dysregulation of NPY signaling in the CeA contributes to uncontrolled and excessive ethanol intake (Koob, 2003
; Koob and Le Moal, 2001
). We propose that a similar (although not identical) dysregulation of NPY signaling occurs within the CeA during the course of a binge-like drinking episode. We speculate that blunted NPY signaling that unfolds during the course of a binge can motivate binge-like drinking in a manner similar to the role of blunted NPY signaling in motivating vapor-induced dependence-like drinking, and that dysregulation of NPY becomes rigid with repeated binge episodes, contributing to the transition to dependence. Viewed this way, in addition to being potential pharmaceutical targets for treating excessive ethanol intake resulting from dependence, the current data suggest that NPY compounds (Y1R agonists and Y2R antagonists) may also be useful for curbing and/or preventing binge drinking, ultimately protecting vulnerable individuals from progressing to the point of ethanol dependence. Because neuroplastic changes that develop in the brain with the development of dependence are thought to be longlasting and the underlying cause of uncontrolled excessive ethanol intake characteristic of dependent individuals (Koob, 2003
; Koob and Le Moal, 2001
), treating at-risk individuals suffering from alcohol abuse disorders before they have become dependent may be a more effective approach than treatments that are aimed at individuals who have already become dependent.