Alcohol operant self-administration paradigms are critical tools for studying the neural circuits implicated in both alcohol-seeking and consummatory behaviors and for understanding the neural basis underlying alcohol-use disorders. In this study, we investigate the predictive value of two operant models of oral alcohol self-administration in mice, one in which alcohol is delivered into a cup following nose-poke responses with no accurate measurement of consumed alcohol solution, and another paradigm that provides access to alcohol via a sipper tube following lever presses and where lick rate and consumed alcohol volume can be measured. The goal was to identify a paradigm where operant behaviors such as lever presses and nose pokes, as well as other tracked behavior such as licks and head entries, can be used to reliably predict blood alcohol concentration (BAC). All mice were first exposed to alcohol in the home cage using the "drinking in the dark" (DID) procedure for 3 weeks and then were trained in alcohol self-administration using either of the operant paradigms for several weeks. Even without sucrose fading or food pre-training, mice acquired alcohol self-administration with both paradigms. However, neither lever press nor nose-poke rates were good predictors of alcohol intake or BAC. Only the lick rate and consumed alcohol were consistently and significantly correlated with BAC. Using this paradigm that accurately measures alcohol intake, unsupervised cluster analysis revealed three groups of mice: high-drinking (43%), low-drinking (37%), and non-drinking mice (20%). High-drinking mice showed faster acquisition of operant responding and achieved higher BACs than low-drinking mice. Lick rate and volume consumed varied with the alcohol concentration made available only for high- and low-drinking mice, but not for non-drinking mice. In addition, high- and low-drinking mice showed similar patterns during extinction and significant cue-induced reinstatement of seeking. Only high-drinking mice showed insensitivity to quinine adulteration, indicating a willingness to drink alcohol despite pairing with aversive stimuli. Thus, this study shows that relying on active presses is not an accurate determination of drinking behavior in mice. Only paradigms that allow for accurate measurements of consumed alcohol and/or lick rate are valid models of operant alcohol self-administration, where compulsive-like drinking could be accurately determined based on changes in alcohol intake when paired with bitter-tasting stimuli.

The mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in dendritic translation and in learning and memory. We previously showed that heavy alcohol use activates mTORC1 in the orbitofrontal cortex (OFC) of rodents (Laguesse et al., 2017a). Here, we set out to determine the consequences of alcohol-dependent mTORC1 activation in the OFC. We found that inhibition of mTORC1 activity in the OFC attenuates alcohol seeking and restores sensitivity to outcome devaluation in rats that habitually seek alcohol. In contrast, habitual responding for sucrose was unaltered by mTORC1 inhibition, suggesting that mTORC1's role in habitual behavior is specific to alcohol. We further show that inhibition of GluN2B in the OFC attenuates alcohol-dependent mTORC1 activation, alcohol seeking and habitual responding for alcohol. Together, these data suggest that the GluN2B/mTORC1 axis in the OFC drives alcohol seeking and habit.

Fyn kinase in the dorsomedial striatum (DMS) of rodents plays a central role in mechanisms underlying excessive alcohol intake. The DMS is comprised of medium spiny neurons (MSNs) that project directly (dMSNs) or indirectly (iMSNs) to the substantia nigra. Here, we examined the cell-type specificity of Fyn's actions in alcohol use. First, we knocked down Fyn selectively in DMS dMSNs or iMSNs of mice and measured the level of alcohol consumption. We found that downregulation of Fyn in dMSNs, but not in iMSNs, reduces excessive alcohol but not saccharin intake. D1Rs are coupled to Gαs/olf, which activate cAMP signaling. To examine whether Fyn's actions are mediated through cAMP signaling, DMS dMSNs were infected with GαsDREADD, and the activation of Fyn signaling was measured following CNO treatment. We found that remote stimulation of cAMP signaling in DMS dMSNs activates Fyn and promotes the phosphorylation of the Fyn substrate, GluN2B. In contract, remote activation of GαsDREADD in DLS dMSNs did not alter Fyn signaling. We then tested whether activation of GαsDREADD in DMS dMSNs or iMSNs alters alcohol intake and observed that CNO-dependent activation of GαsDREADD in DMS dMSNs but not iMSNs increases alcohol but not saccharin intake. Finally, we examined the contribution of Fyn to GαsDREADD-dependent increase in alcohol intake, and found that systemic administration of the Fyn inhibitor, AZD0503 blocks GαsDREADD-dependent increase in alcohol consumption. Our results suggest that the cAMP-Fyn axis in the DMS dMSNs is a molecular transducer of mechanisms underlying the development of excessive alcohol consumption.

The mammalian target of rapamycin complex 1 (mTORC1), a transducer of local dendritic translation, participates in learning and memory processes as well as in mechanisms underlying alcohol-drinking behaviors. Using an unbiased RNA-seq approach, we identified Prosapip1 as a novel downstream target of mTORC1 whose translation and consequent synaptic protein expression are increased in the nucleus accumbens (NAc) of mice excessively consuming alcohol. We demonstrate that alcohol-dependent increases in Prosapip1 levels promote the formation of actin filaments, leading to changes in dendritic spine morphology of NAc medium spiny neurons (MSNs). We further demonstrate that Prosapip1 is required for alcohol-dependent synaptic localization of GluA2 lacking AMPA receptors in NAc shell MSNs. Finally, we present data implicating Prosapip1 in mechanisms underlying alcohol self-administration and reward. Together, these data suggest that Prosapip1 in the NAc is a molecular transducer of structural and synaptic alterations that drive and/or maintain excessive alcohol use.