The glutamate receptor-associated protein Homer2 regulates alcohol-induced neuroplasticity within the nucleus accumbens (NAC), but the precise intracellular signaling cascades involved are not known. This study examined the role for NAC metabotropic glutamate receptor (mGluR)-Homer2-phosphatidylinositol 3-kinase (PI3K) signaling in regulating excessive alcohol consumption within the context of the scheduled high alcohol consumption (SHAC) model of binge alcohol drinking. Repeated bouts of binge drinking (ϳ1.5 g/kg per 30 min) elevated NAC Homer2a/b expression and increased PI3K activity in this region. Virus-mediated knockdown of NAC Homer2b expression attenuated alcohol intake, as did an intra-NAC infusion of the mGluR5 antagonist MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride] (0.1-1 g/side) and the PI3K antagonist wortmannin (50 ng/side), supporting necessary roles for mGluR5/Homer2/PI3K in binge alcohol drinking. Moreover, when compared with wild-type littermates, transgenic mice with an F1128R point mutation in mGluR5 that markedly reduces Homer binding exhibited a 50% reduction in binge alcohol drinking, which was related to reduced NAC basal PI3K activity. Consistent with the hypothesis that mGluR5-Homer-PI3K signaling may be a mechanism governing excessive alcohol intake, the "anti-binge" effects of MPEP and wortmannin were not additive, nor were they observed in the mGluR5 F1128R transgenic mice. Finally, mice genetically selected for a high versus low SHAC phenotype differed in NAC mGluR, Homer2, and PI3K activity, consistent with the hypothesis that augmented NAC mGluR5-Homer2-PI3K signaling predisposes a high binge alcohol-drinking phenotype. Together, these data point to an important role for NAC mGluR5-Homer2-PI3K signaling in regulating binge-like alcohol consumption that has relevance for our understanding of the neurobiology of alcoholism and its pharmacotherapy.

Background: Studies in rodents have determined that intermittent exposure to alcohol vapor can increase subsequent ethanol self-administration, measured with operant and 2-bottle choice procedures. Two key procedural factors in demonstrating increased alcohol intake are the establishment of stable alcohol self-administration before alcohol vapor exposure and the number of bouts of intermittent vapor exposure. The present studies provide additional behavioral validation and initial pharmacological validation of this withdrawal-associated drinking procedure.Methods: Studies at 2 different sites (Portland and Scripps) examined the effect of intermittent ethanol vapor exposure (3 cycles of 16 hours of ethanol vapor18 hours air) on 2-hour limited access ethanol preference drinking in male C57BL/6 mice. Separate studies tested 10 or 15% (v/v) ethanol concentrations, and measured intake during the circadian dark. In one study, before measuring ethanol intake after the second bout of intermittent vapor exposure, mice were tested for handlinginduced convulsions (HICs) indicative of physical dependence on ethanol. In a second study, the effect of bilateral infusions of the corticotropin-releasing factor (CRF) receptor antagonist D-Phe-CRF(12-41) (0.25 mg/0.5 mL) into the central nucleus of the amygdala (CeA) on ethanol intake was compared in vapor-exposed animals and air controls.Results: Intermittent ethanol vapor exposure significantly increased ethanol intake by 30 to 40%, and the mice had higher blood ethanol concentrations than controls. Intra-amygdala infusions of D-Phe-CRF(12-41) significantly decreased the withdrawal-associated increase in ethanol intake without altering ethanol consumption in controls. Following the second bout of intermittent vapor exposure, mice exhibited an increase in HICs, when compared with their own baseline scores or the air controls.Conclusions: Intermittent alcohol vapor exposure significantly increased alcohol intake and produced signs of physical dependence. Initial pharmacological studies suggest that manipulation of the CRF system in the CeA can block this increased alcohol intake.

The results obtained clearly demonstrate that the cEA has a role in the regulation of ethanol consumption in the limited-access procedure. However, neither lesions of the CeA nor BNSTLP prevented the intermittent ethanol vapor-induced increase in consumption. These data do not preclude some role of the cEA in the increased ethanol consumption following intermittent ethanol vapor exposure, but would suggest that other brain regions also must have a significant influence.

The progesterone derivative allopregnanolone (ALLO) rapidly potentiates γ-aminobutyric acid A (GABA A ) receptor mediated inhibition. The present studies determined whether specific manipulation of neurosteroid levels in the hippocampus would alter seizure susceptibility in an animal model genetically susceptible to severe ethanol (EtOH) withdrawal, Withdrawal SeizureProne (WSP) mice. Male WSP mice were surgically implanted with bilateral guide cannulae aimed at the CA1 region of the hippocampus one week prior to measuring seizure susceptibility to the convulsant pentylenetetrazol (PTZ), given via timed tail vein infusion. Bilateral intra-hippocampal infusion of ALLO (0.1 g/side) was anticonvulsant, increasing the threshold dose of PTZ for onset to myoclonic twitch and face and forelimb clonus by 2-3 fold. In contrast, infusion of the 5α-reductase inhibitor finasteride (FIN; 2 g/side), which decreases endogenous ALLO levels, exhibited a proconvulsant effect. During withdrawal from chronic EtOH exposure, WSP mice were tolerant to the anticonvulsant effect of intra-hippocampal ALLO infusion, consistent with published results following systemic injection. Finally, administration of intra-hippocampal FIN given only during the development of physical dependence significantly increased EtOH withdrawal severity, measured by handling-induced convulsions. These findings are the first demonstration that bidirectional manipulation of hippocampal ALLO levels produces opposite behavioral consequences that are consistent with alterations in GABAergic inhibitory tone in drug naïve mice. Importantly, EtOH withdrawal rendered WSP mice less sensitive to ALLO's anticonvulsant effect and more sensitive to FIN's proconvulsant effect, suggesting an alteration in the sensitivity of hippocampal GABA A receptors in response to fluctuations in GABAergic neurosteroids during ethanol withdrawal.

Rationale The rapid membrane actions of neuroactive steroids, particularly via an enhancement of γ-aminobutyric acidA receptors (GABAARs), participate in the regulation of central nervous system excitability. Prior evidence suggests an inverse relationship between endogenous GABAergic neuroactive steroid levels and behavioral changes in excitability during ethanol withdrawal. Objectives Previously, we found that ethanol withdrawal significantly decreased plasma allopregnanolone (ALLO) levels, a potent GABAergic neuroactive steroid, and decreased GABAAR sensitivity to ALLO in Withdrawal Seizure–Prone (WSP) but not in Withdrawal Seizure–Resistant (WSR) mice. However, the effect of ethanol withdrawal on levels of other endogenous GABAAR-active steroids is not known. Methods After validation of a gas chromatography-mass spectrometry method for the simultaneous quantification of 10 neuroactive steroids, we analyzed plasma from control male WSP-1 and WSR-1 mice and during ethanol withdrawal. Results We quantified levels of 9 neuroactive steroids in WSP-1 and WSR-1 plasma; levels of pregnanolone were not detectable. Basal levels of 5 neuroactive steroids were higher in WSR-1 versus WSP-1 mice. Ethanol withdrawal significantly suppressed 5 neuroactive steroids in WSP-1 and WSR-1 mice, including ALLO. Conclusions Due to lower basal levels of some GABAAR-active steroids in WSP-1 mice, a withdrawal-induced decrease in WSP-1 mice may have a greater physiological consequence than a similar decrease in WSR-1 mice. Because WSP-1 mice also exhibit a reduction in GABAAR sensitivity to neuroactive steroids during withdrawal, it is possible that the combined decrease in neuroactive steroids and GABAAR sensitivity during ethanol withdrawal in WSP-1 mice represents a neurochemical substrate for severe ethanol withdrawal.

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Background Allopregnanolone (ALLO) is a potent positive modulator of γ-aminobutyric acidA receptors (GABAARs) that affects ethanol withdrawal. Finasteride (FIN), a 5α-reductase inhibitor that blocks the formation of ALLO and other GABAergic neurosteroids, alters ethanol sensitivity. Recently, we found that Withdrawal Seizure-Prone mice from the first genetic replicate (WSP-1) exhibited behavioral tolerance to the anticonvulsant effect of intra-hippocampal ALLO during ethanol withdrawal and that intra-hippocampal FIN significantly increased ethanol withdrawal severity. The purpose of the present study was to determine whether neurosteroid manipulations in the substantia nigra reticulata (SNR) and ventral tegmental area (VTA) produced effects during ethanol withdrawal comparable to those seen with intra-hippocampal ALLO and FIN. Methods Male WSP-1 mice were surgically implanted with bilateral guide cannulae aimed at the SNR or VTA at two weeks prior to ethanol vapor or air exposure for 72 hrs. Initial studies examined the anticonvulsant effect of a single ALLO infusion (0, 100 or 400 ng/side) at a time corresponding to peak withdrawal in the air- and ethanol-exposed mice. Separate studies examined the effect of four FIN infusions (0 or 10 µg/side/day) during the development of physical dependence on the expression of ethanol withdrawal. Results ALLO infusion exerted a potent anticonvulsant effect in ethanol-naïve mice, but a diminished anticonvulsant effect during ethanol withdrawal. Administration of FIN into the SNR exerted a delayed proconvulsant effect in ethanol-naïve mice, whereas infusion into the VTA increased ethanol withdrawal duration. Conclusions Activation of local GABAARs in the SNR and VTA via ALLO infusion is sufficient to exert an anticonvulsant effect in naïve mice and to produce behavioral tolerance to the anticonvulsant effect of ALLO infusion during ethanol withdrawal. Thus, ethanol withdrawal reduced sensitivity of GABAARs to GABAergic neurosteroids in two neuroanatomical substrates within the basal ganglia in WSP-1 male mice.

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