Central Administration of Cyclosporine A Decreases Ethanol Drinking

Central Administration of Cyclosporine A Decreases Ethanol Drinking Abstract Aims Abstinence among alcohol dependent liver graft recipients is remarkably high. The routine use of anti-immune agents in these patients led to rodent studies showing that immunosuppressants acting through inhibition of calcineurin (CLN) are highly effective in decreasing alcohol consumption. It remained unclear, however, whether the decreased alcohol consumption in rodent models is mediated through peripheral suppression of immune response or centrally through direct inhibition of cyclophilin-CLN in the brain. We tested the hypothesis that direct brain inhibition of CLN with intracerebroventricular (ICV) injections of the immunosuppressant cyclosporine A (CsA) is sufficient to decrease ethanol consumption in a rodent model of binge-like drinking. Methods Male C57BL/6NHsd mice were put through a modified ‘drinking in the dark’ (DID) paradigm. Effects of both peripheral (IP) and central (ICV) injections of CsA on ethanol consumption were assessed. Results Here, as in earlier work, IP CsA administration significantly decreased alcohol consumption. Supporting our hypothesis, central administration of CsA was sufficient to decrease alcohol consumption in a dose-dependent manner. There was no significant effect of CsA on water or sucrose consumption. Conclusions These results clearly implicate a CLN-mediated mechanism in brain in the inhibitory effects of CsA on ethanol consumption and provide novel targets for investigation of treatment for Alcohol Use Disorders (AUD). These results also add to the growing body of literature implicating neuroimmune mechanisms in the etiology, pathophysiology and behaviors driving AUD. Short Summary The unusually high abstinence rate and routine use of immunosuppressants in AUD liver graft recipients led us to rodent studies showing that immunosuppressants acting through inhibition of calcineurin (CLN) are highly effective in decreasing drinking. Here we demonstrate that this effect is mediated by brain rather than peripheral immune mechanisms. INTRODUCTION Liver graft recipients with alcohol use disorder (AUD) show a surprisingly high rate of abstinence (Lucey, 2007; DiMartini et al., 2010). Approximately 90% are abstinent 1 year after transplant and about 75–80% after 3 years. This is remarkable when compared to rates from other treatment studies demonstrating abstinence rates at 1 year closer to 35–45% (Yates et al., 1993; Allen et al., 1995). We initially hypothesized that this high abstinence among liver transplant patients could be explained by pre-transplant AUD case selection and/or post-transplant psychosocial factors known to facilitate abstinence (Lucey et al., 1994). However, these factors could not account for the persistent high abstinence rates among AUD liver graft recipients (Beresford et al., 1992, 1990; Beresford, 1997). One common factor among these patients is the near universal use of immunosuppressants such as cyclosporine A (CsA) post-transplant. We considered the possibility that these immunosuppressants could be playing a role in promoting abstinence. We were the first to report an effect of immunosuppressants on alcohol drinking in an animal model. Mice treated with CsA drank significantly less ethanol in a controlled, continuous-access, two-bottle choice model while having no effect on total liquid intake (Beresford et al., 2005). We sought to determine the mechanism of this effect by comparing three different immunosuppressants acting though different modes of action in a modified ‘drinking in the dark’ (DID) paradigm of excessive drinking (Beresford et al., 2012). Again, immunosuppressants were highly effective in decreasing alcohol consumption, but only those acting through inhibition of the phosphatase calcineurin (CLN) and that crossed the blood-brain barrier. CsA and tacrolimus (FK-506) inhibit CLN activity through different mechanisms but both decreased ethanol consumption. A third immunosuppressant, sirolumus (SRL) that acts through CLN-independent pathways had no effect on ethanol consumption. Though inhibition of CLN was clearly implicated, it was still unclear whether the anti-drinking effects were due to suppression of peripheral immune responses through CLN inhibition or direct inhibition of CLN in brain or via another mechanism in brain. Both CsA and TRL crossed the blood-brain barrier in this study. They also have known inhibitory effects on CLN in brain where immunophilins and calcineurin are abundant and widespread (Dawson et al., 1994). Sirolimus did not effectively penetrate brain suggesting that a general suppression of peripheral immune function was not sufficient to decrease excessive alcohol consumption. We sought to determine in these experiments if this CLN-mediated inhibitory effect on drinking is acting via suppression of peripheral immune response or acting directly in the central nervous system (CNS). We hypothesized that central administration of CsA via intracerebroventricular (ICV) injection would be sufficient to inhibit excessive drinking behavior similar to what we have observed after peripheral administration (IP). The goal of this study was to assess the effects of inhibition of brain calcineurin, separate from any immunosuppressant effect, on ethanol drinking in a mouse model of binge-like drinking. We hypothesized that centrally (ICV) administered Cyclosporine A (CsA) would reduce alcohol consumption in a drinking in the dark model. MATERIALS AND METHODS Animals Adult male C57BL/6NHsd mice (8 weeks; Envigo, Barrier 208 A, Frederick, MD) were initially housed five per group (Microvent Positive Ventilated Housing, Allentown Inc., Allentown, NJ) on a reversed light cycle (off 9:00 AM: on 9:00 PM) in a temperature controlled (23 ± 1°C) AALAC approved animal facility at the Sioux Falls VA. Mice were given ad libidum access to food and water. Ethanol consumption in the DID model has been shown to be significantly altered depending on the variety of standard rodent chow chosen (Marshall et al., 2015). Teklad 2018, 18% Protein Rodent Diet (Envigo) facilitates ethanol consumption. Mice were randomly assigned to five experimental groups (n = 7–8/group): (a) ICV vehicle, (b) ICV Low CsA (15 μg in 2 μl), (c) ICV High CsA (30 μg/2 μl), (d) IP Vehicle, (e) IP CsA (30 mg/kg). Following surgical implantation of ICV guide cannulae, mice were housed individually. All experimental procedures complied with NIH guidelines and were approved by the University of South Dakota IACUC and the Sioux Falls VA Research Service. Materials Ethanol (190 proof, Fisher Scientific) and sucrose (Sigma-Aldrich) were mixed with tap water to 20% (v/v) and 10% (w/v) solutions, respectively. All solutions were made fresh daily. Cyclosporine A (LC Laboratories, Woburn, MA) was dissolved in 85% DMSO (vehicle). Doses of CsA (30 mg/kg IP, 15 and 30 μg ICV) were based on previous work demonstrating that 30 mg/kg IP was the most effective dose in reducing ethanol consumption in similar ethanol drinking model in mice (Beresford et al., 2012) and studies showing a range of behavioral and physiological effects of ICV CsA (Dougherty and Dafny, 1988; Francischi et al., 1997; Almeida-Correa et al., 2015). ICV cannulation Guide cannulae were surgically implanted to allow for ICV injections. Mice were placed in an induction chamber and briefly exposed to isoflurane (5%). Mice were removed and anesthesia was maintained using a nosecone. Ketorolac (5 mg/kg, SC) was administered and their scalps shaved. They were then transferred to a stereotaxic unit (Kopf Model 962 Ultra Precise Small Animal Stereotaxic Instrument, David Kopf Instruments, Tujunga, CA) equipped with a gas anesthesia head holder (Model 923-B). Isoflurane anesthesia was maintained at 1.5–2.5% and the pedal response was continually checked to ensure adequate sedation. The scalp was scrubbed with a chlorhexidine solution followed by sterile saline. An incision was made in the scalp and tissue clamps applied to expose the skull. Fascia was scored and scraped to the sides of the skull with a scalpel and a hydrogen peroxide solution (3%) was applied. Stereotaxic coordinates targeting the lateral ventricle (AP: −0.46; ML: 1.0; DV: 2.3) were determined (Franklin and Paxinos, 2013). With the aid of a surgical stereomicroscope on an articulating arm, a mark was made with surgical ink at these coordinates using a sterile needle attached to an electrode holder (Model 1770). A precision drill (Model 1474 High Speed Stereotaxic Drill; David Kopf Instruments, Tuluma, CA) with a sterile burr bit was used to drill through the skull at this mark, being careful to leave the dura intact. Dura and any remaining bone disc were gently removed using a sterile 25-g needle. Two more holes were drilled anterior and posterior and anchor screws inserted (1.6 diameter × 1.6 mm length; Plastics One). A guide cannula (26-g, C315GA, Plastics One, Roanoke, VA) was inserted into a cannula holder, lowered into the hole and secured in place with a self-curing acrylic resin (OrthoJet Acrylic, Lang Dental, Wheeling, IL). A dummy cannula was inserted and the incision was closed with sutures (simple interrupted, silk, 3.0) and an antibiotic cream applied to the wound. Mice were allowed to recover for 2 weeks before any further experimental procedures were performed. Drinking in the dark Rodent models of ethanol drinking involving voluntary consumption are limited in their ability to generate blood ethanol concentrations (BEC) believed to be pharmacologically meaningful in regard to characteristics of binge-drinking patterns (Thiele and Navarro, 2014). We employed a ‘drinking in the dark’ paradigm (DID) that has been reported to work well inducing BEC of 0.08 (80 mg/100 ml of blood) (Rhodes et al., 2005; Thiele et al., 2014). This model promotes binge-like excessive ethanol consumption during a limited access schedule (2–4 h) during the peak activity period in the dark phase. It has shown predictive face validity for binge-like ethanol drinking in humans (Kamdar et al., 2007; Thiele et al., 2014). We used a slightly modified 2-day version of the DID procedure (Thiele et al., 2014). Briefly, during Week 1 mice were acclimated to handling and modified sipper tubes and given 4 days of alcohol access without any drug treatment. We created custom sipper tubes both to accurately measure consumption and to reduce any leakage as described by Thiele et al. (2014). Briefly, stainless steel bearing sipper tubes (Ancare Corp.) were inserted into polystyrene 10 ml graduated serological pipets (Fisher Scientific) and sealed with heat shrink tubing. Silicone stoppers (Fisher, European size 10D) were used to seal the opposite end. All tubes were tested for leakage. Binder clips were used to attach the tubes to the cages to prevent leakage as mice will climb on them. On Day 1 homecage water bottles were removed at the start of the dark period and replaced with the custom pipette sipper tubes filled with water. Water bottles were replaced after 8 h. On Days 2–5, homecage water bottles were removed and replaced with a 20% ethanol solution for 2 h, and 3 h into the dark phase. We and others have noted that extra days of ethanol access increase average alcohol consumption Figure 1 (Thiele et al., 2014). The DID procedure was carried out on Days 9 and 10. Again, they were given 2 h access to 20% ethanol 3 h into the dark phase. On Day 10, CsA or Vehicle was administered 30 min prior to ethanol access and consumption over 2 h was measured. Two hours was chosen because it has been shown that during the second week of DID procedures mice ‘front-load’ their intake and consume roughly half of the total volume consumed over 4 h in the first 30 min (Wilcox et al., 2014). We used the same 2-day DID model with 10% sucrose or water in place of EtOH to avoid bias when assessing the effects of CsA on alcohol consumption. All mice were weighed before the dark period on the first day of ethanol access and those weights were used to calculate consumption on the following days. To verify ICV injections, mice were anesthetized and 2.0 μl methylene-blue was injected ICV using identical procedures to those used during the experiment. Brains were removed, frozen on dry ice and sectioned on a cryostat microtome (Cryostar NX70, Thermo Fisher Scientific). Injections were verified by the presence of dye in ventricles. Statistics Data were analyzed using SigmaStat 4.0 and graphed with SigmaPlot 10.0 (Systat Software Inc., San Jose, CA). Analysis of Variance (ANOVA) tests were used for all experiments (α = 0.05). Bonferonni Post-hoc tests were used if a significant interaction or main effect of drug was observed. All data are expressed as mean ± SEM. RESULTS Cyclosporine significantly decreased ethanol consumption whether given peripherally (IP) or centrally in brain (ICV). There was a significant main effect for treatment, F(4,32) = 6.17, P < 0.001. We repeated earlier findings demonstrating that a 30 mg/kg IP dose of CsA decreases ethanol intake in a DID model (P = 0.006). Here we show that central administration of CsA also decreases ethanol intake in a similar fashion (30 μg dose, P = 0.002). Though the mean consumption was slightly less for ICV vehicle compared to IP vehicle groups this difference was not significant (P = 0.467). There were no significant main effects of ICV or IP administration of CsA on consumption of sucrose, F(4,31) = 0.927, P = 0.461, or water, F(4,31) = 1.594, P = 0.201. Access to alcohol for 2 h session for 4 days in the week prior to the DID procedure significantly increased overall ethanol consumption, F(4,32) = 6.299, P = 0.003 (Figs 2–4). Fig. 1. View largeDownload slide Ethanol intake increased over a 4 day access period. Mice were given access to 20% ethanol for 2 h/day for 4 days; *P = 0.011. Fig. 1. View largeDownload slide Ethanol intake increased over a 4 day access period. Mice were given access to 20% ethanol for 2 h/day for 4 days; *P = 0.011. Fig. 2. View largeDownload slide Both central and peripheral administration of cyclosporine A significantly reduced alcohol intake in mice. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg); *P < 0.05. Fig. 2. View largeDownload slide Both central and peripheral administration of cyclosporine A significantly reduced alcohol intake in mice. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg); *P < 0.05. Fig. 3. View largeDownload slide Cyclosporine had no effect on sucrose intake. There were no significant differences in sucrose consumption between groups. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg). Fig. 3. View largeDownload slide Cyclosporine had no effect on sucrose intake. There were no significant differences in sucrose consumption between groups. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg). Fig. 4. View largeDownload slide Cyclosporine had no effect water intake. There were no significant differences in water consumption between groups. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg). Fig. 4. View largeDownload slide Cyclosporine had no effect water intake. There were no significant differences in water consumption between groups. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg). DISCUSSION This experiment demonstrated that central administration of cyclosporine A is sufficient to decrease alcohol intake in mice in a binge-drinking model of ethanol consumption in a dose-dependent fashion. These results combined with our previous research (Beresford et al., 2012) clearly show that the anti-drinking effects of CsA are mediated by direct effects in brain and not by peripheral immunosuppression. We also replicated and extended earlier findings demonstrating that peripheral administration (IP) of CsA significantly reduces alcohol intake (Beresford et al., 2005, 2012) using a slightly different mouse strain and drinking model. Administration of CsA in brain is sufficient to decrease ethanol consumption suggesting that the effects seen after peripheral administration are due to direct actions in brain as well. Additionally, the fact that there were no significant effects of CsA on water or sucrose consumption indicates a specific effect of CsA on ethanol intake. An indiscriminate antidipsic effect cannot explain the results of this study nor can a reduced need for caloric intake or generalized taste aversion mechanism. The specific pathways mediating this effect still need to be elucidated. Suppression of neuroimmune activation is one hypothesis. As an immunosuppressant, CsA acts in brain much like it does in peripheral immune cells. Similar to its role in peripheral immune/inflammatory cells, CLN plays an integral role in processes regulating neuroimmune function. Calcineurin regulates glial cell activation and cytokine expression and is induced during disease, injury and aging (Furman and Norris, 2014). Calcineurin is a major regulator of immune or inflammatory processes in both T-cells and glial cells through activation of the nuclear factor of activated T-cells (NFAT) family of transcription factors. Cyclosporine binds the immunophilin cyclophilin and this complex then inhibits the activity of CLN, a Ca2+ and calmodulin-dependent serine-threonine phosphatase also known as protein phosphatase 3 (PPP3) (Liu et al., 1991). Dephosphorylation of NFAT by CLN causes activation and nuclear translocation where it stimulates transcription of a number of proinflammatory genes including interleukin-2 (Clipstone and Crabtree, 1993; Ho et al., 1996; Musson and Smit, 2011). It is now widely accepted that immune mechanisms, and specifically neuroimmune mechanisms, play important, if poorly understood, roles in the etiology and progression of alcohol abuse (Mayfield and Harris, 2017). The hypothesis that an overactive neuroimmune system promotes alcohol consumption is gaining widespread support among scientists (Mayfield et al., 2013; Cui et al., 2014; Crews et al., 2017). The first mention of a possible connection between immunosuppressants and alcohol consumption was published as a brief clinical report in 1990 (Giles et al., 1990). It was noted that AUD liver graft patients were more abstinent while on immunosuppressants. It has taken many years for the research community to embrace and investigate this possibility. Our 2005 study, stemming from insights gained from liver graft recipients, was the first to test the hypothesis that suppression of immune processes could limit ethanol intake (Beresford et al., 2005). This notion has been extended by a number of other groups in the last few years. A range of compounds or genetic approaches that inhibit neuroimmune function have been shown to limit ethanol intake to varying degrees in a variety of animal models (Mayfield et al., 2013; Cui et al., 2014; Crews et al., 2017). These studies target different specific mechanisms but they all converge on the common outcome of reducing neuroimmune activation. Along with reducing neuroinflammation, CsA also plays key roles in neuronal signaling processes. The effectiveness of CsA in reducing ethanol consumption seen in this and our past studies may be due to its complementary roles in other brain signaling pathways related to brain reward and addiction. During the past few decades, the diverse and important roles CLN plays in neuronal signaling processes have been more and more appreciated. Calcineurin is one of the most abundant phosphatases in brain and is known to play important roles in a wide variety of signal transduction pathways. Not only involved in neuroimmune processes, CLN is also expressed at especially high levels in the brain in neurons. There are particularly high concentrations of CLN in the hippocampus, neocortex, striatum and amygdala (Furman and Norris, 2014) where CLN modulates diverse cellular functions including receptor and ion channel trafficking, ion channel function, apoptosis and gene regulation (Baumgartel and Mansuy, 2012). Calcineurin is also abundantly expressed in all of the regions of the brain where CaMKII is found including the dopaminergic reward pathway of the ventral tegmental area (VTA) and the nucleus accumbens (NAc) (Sola et al., 1999). Calcineurin directly regulates dopaminergic and other monoaminergic signaling. Along with direct effects on monoamine synthesis and release it has been shown that CLN activity regulates an important dopamine and addiction-related signal transduction protein—dopamine and cAMP-regulated neuronal phosphoprotein (DARPP-32). This protein regulates both dopaminergic and glutaminergic (NMDA) receptor responses in extended brain reward pathways (Greengard et al., 1999; Svenningsson et al., 2004) and is critically involved in regulating responses to ethanol and other drugs of abuse (Donohue et al., 2005; Svenningsson et al., 2005). DARPP-32 is a direct CLN substrate. Dopamine causes increased phosphorylation and CN acts to maintain low levels of phosphorylated DARPP (Nishi et al., 1999). It has been hypothesized that CsA inhibition of CLN acts as a dopamine mimetic via dopamine-like increases in phosphorylated DARPP-32 (Wera and Neyts, 1994). In this way CLN inhibition could potentially dampen temporally linked dopamine reward cues elicited by alcohol and thereby lessen reinforcement. Monoaminergic activity is integral to processes related to reward and addiction and CLN has been shown to regulate monoaminergic signaling. In rats, CsA alone has an inhibitory effect on monoamine production but, interestingly, when given along with a chronic stressor it has the opposite effect, leading to enhanced tissue levels of serotonin and norepinephrine (Fujisaki et al., 2003). It also had antidepressant-like activity in the unpredictable chronic stress model that paralleled imipramine. Calcineurin regulates several other signaling pathways known to be associated with addiction and stress including corticotropin releasing factor (CRF). While playing a critical role in the regulation of behavior and neuroendocrine responses to stress, CRF has also proven to be a significant contributor to the processes of alcohol consumption and abuse (Heilig and Koob, 2007; Ronan and Summers, 2011; Phillips et al., 2015). Calcineurin regulates CRF gene expression and CRF-mediated behaviors. Transcription of CRF is mediated via cAMP response element binding protein (CREB) phosphorylation. Phosphorylation of CREB is necessary but not sufficient for CRF transcription (Liu et al., 2008). A CLN-regulated coactivator called TORC (tranducer of regulated CREB activity) is also needed for CREB-induced CRF transcription (Liu et al., 2010; Spencer and Weiser, 2010). Dephosphorylation of TORC by CLN facilitates translocation to the nucleus where it interacts with the dimerization and DNA binding domain of CREB and facilitates the recruitment of the preinitiation complex. This, in turn, facilitates transcriptional activation of CREB-dependent genes including CRF. CLN activity is required for depolarization-induced CREB-dependent gene transcription in cortical neurons (Kingsbury et al., 2007). Stress alone induces nuclear translocation of TORC and transcription of CRF in rodents (Liu et al., 2011). This TORC mediated mechanism for enhanced stress-induced CRF expression may underlie the enhanced sensitivity to stressors seen in models of ethanol abuse. Cyclosporine blocks CRF-receptor mediated catecholamine synthesis and release through inhibition of NFAT activation in cell culture (Dermitzaki et al., 2012). CRF has been shown to modulate brain reward function directly through signaling in the ventral tegmental area, nucleus accumbens and the prefrontal cortex, among other structures (Ronan and Summers, 2011; Quadros et al., 2016). One of the strengths of this study is the use of a well-validated and widely used rodent drinking model. The ‘drinking in the dark’ procedure has high face validity as a model of human binge drinking (for review see, Thiele and Navarro, 2014). Many strains of mice have the natural tendency to display excessive bouts of drinking under short term access to ethanol. The fact that they are not forced to drink as in other models of binge drinking (i.e. gavage, vapor, injection) can help us get at the underlying drinking behaviors. It also is not necessary in this model to deprive mice of food or water to study these drinking behaviors. Varying lines of evidence have converged to demonstrate that the high ethanol consumption in this model is not due to caloric need. This model has been successfully used to identify pharmacological targets and genetic mechanisms that promote binge drinking. This study is an example of research going from clinical observations to the bench. The central question answered by this study, whether CsA acts centrally or peripherally to suppress alcohol consumption, could not have been answered in humans. The initial hypotheses gleaned from clinical observations lacked real proof. Empirical data on CsA and other immunosuppressants versus control with respect to suppression of alcohol consumption are not available in human studies. And performing a placebo controlled study in this population is ethically unfeasible. In this study we were able to employ a ‘placebo’-controlled study design to properly assess the effects of CsA. The steps from bench back to clinic: the need for mechanistic clarity While our mouse study data are remarkably encouraging in pursuing this innovative approach to treatment development, exploring this observed effect begins with an assessment of whether or not a similar effect actually occurs in people. The mouse data do not translate directly into human experiments with these agents. For one example, the effective mg/kg doses in mice would be toxic in human beings. For another, their immunosuppressant effects at standard doses given to transplant patients postoperatively cannot be clinically justified as a widespread treatment. In this regard, therefore, pursuing CLN inhibition as a new treatment option for AD first requires careful investigation with respect to specific mechanisms of action. Identification of the specific pathways and substrates involved in this immunosuppressant effect on drinking needs to be studied taking a variety of approaches. One line of inquiry we are pursuing is to identify specific CLN pathways involved in this effect using a genetic approach. We are using conditional knockouts using both Cre-loxP and RNA interference techniques (Scammell et al., 2003; Callahan et al., 2013), employing a transgenic mouse line with a floxed regulatory subunit of calcineurin (a generous gift from Dr. S. Tonegawa, MIT) along with ‘cre-cutter’ mouse lines and viral vectors, to determine the role of calcineurin-mediated pathways in specific brain regions and cell types (e.g. dopaminergic, glia). These studies and others investigating intracellular CLN substrates and downstream pathways will yield information that will allow us to target specific aspects of CLN signaling to better enable us to develop effective treatments with minimal side effects. CONCLUSIONS Our results clearly implicate a central brain mechanism in the inhibitory effects of CsA on alcohol consumption. This provides a rich avenue for the pursuit of therapeutic targets for treatment of AUD in humans and adds to the growing body of evidence that various neuro-immuno-modulatory pathways could provide novel targets for the development of pharmacotherapies for AUD (Coller and Hutchinson, 2012; Cui et al., 2014). Cyclosporine itself is not a viable treatment option given its possible long-term nephrotoxicity and range of adverse side effects. Deciphering the specific proximal mechanisms underlying the anti-drinking effects of CsA holds the promise for the development of effective treatments where few exist. FUNDING This work was supported by the Department of Veterans Affairs (T.B. and P.R.) and an Department of Veterans Affairs, Office of Academic Affiliations, Advanced VA Research Fellowship (P.R.) and the Great Plains Medical Research Foundation (P.R.). It also received support from the University of South Dakota Center for Brain and Behavior Research (P.R., G.P.) and the U. Discover Summer Research Scholars Program (S.S.). CONFLICT OF INTEREST STATEMENT The authors declare no conflicts of interest. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs or the United States government. ACKNOWLEDGEMENTS The authors would like to thank Allison Rollag, Paige Baker, Belissa Fernandez and Jessica Fernandez for their technical assistance with this study. REFERENCES Allen JP, Litten RZ, Fertig JB. ( 1995) NIDA-NIAAA workshop: efficacy of therapies in drug and alcohol addiction. Strategies for treatment of alcohol problems. Psychopharmacol Bull  31: 665– 9. Google Scholar PubMed  Almeida-Correa S, Moulin TC, Carneiro CF, et al.  . ( 2015) Calcineurin inhibition blocks within-, but not between-session fear extinction in mice. Learn Mem  22: 159– 69. Google Scholar CrossRef Search ADS PubMed  Baumgartel K, Mansuy IM. ( 2012) Neural functions of calcineurin in synaptic plasticity and memory. Learn Mem  19: 375– 84. Google Scholar CrossRef Search ADS PubMed  Beresford HF, Deitrich R, Beresford TP. ( 2005) Cyclosporine-A discourages ethanol intake in C57bl/6j mice: a preliminary study. J Stud Alcohol  66: 658– 62. Google Scholar CrossRef Search ADS PubMed  Beresford T, Fay T, Serkova NJ, et al.  . ( 2012) Immunophyllin ligands show differential effects on alcohol self-administration in C57BL mice. J Pharmacol Exp Ther  341: 611– 6. Google Scholar CrossRef Search ADS PubMed  Beresford TP. ( 1997) Predictive factors for alcoholic relapse in the selection of alcohol-dependent persons for hepatic transplant. Liver Transpl Surg  3: 280– 91. Google Scholar CrossRef Search ADS PubMed  Beresford TP, Schwartz J, Wilson D, et al.  . ( 1992) The short-term psychological health of alcoholic and non-alcoholic liver transplant recipients. Alcohol Clin Exp Res  16: 996– 1000. Google Scholar CrossRef Search ADS PubMed  Beresford TP, Turcotte JG, Merion R, et al.  . ( 1990) A rational approach to liver transplantation for the alcoholic patient. Psychosomatics  31: 241– 54. Google Scholar CrossRef Search ADS PubMed  Callahan LB, Tschetter KE, Ronan PJ. ( 2013) Inhibition of corticotropin releasing factor expression in the central nucleus of the amygdala attenuates stress-induced behavioral and endocrine responses. Front Neurosci  7: 195. Google Scholar CrossRef Search ADS PubMed  Clipstone NA, Crabtree GR. ( 1993) Calcineurin is a key signaling enzyme in T lymphocyte activation and the target of the immunosuppressive drugs cyclosporin A and FK506. Ann N Y Acad Sci  696: 20– 30. Google Scholar CrossRef Search ADS PubMed  Coller JK, Hutchinson MR. ( 2012) Implications of central immune signaling caused by drugs of abuse: mechanisms, mediators and new therapeutic approaches for prediction and treatment of drug dependence. Pharmacol Ther  134: 219– 45. Google Scholar CrossRef Search ADS PubMed  Crews FT, Lawrimore CJ, Walter TJ, et al.  . ( 2017) The role of neuroimmune signaling in alcoholism. Neuropharmacology  122: 56– 73. Google Scholar CrossRef Search ADS PubMed  Cui C, Shurtleff D, Harris RA. ( 2014) Neuroimmune mechanisms of alcohol and drug addiction. Int Rev Neurobiol  118: 1– 12. Google Scholar CrossRef Search ADS PubMed  Dawson TM, Steiner JP, Lyons WE, et al.  . ( 1994) The immunophilins, FK506 binding protein and cyclophilin, are discretely localized in the brain: relationship to calcineurin. Neuroscience  62: 569– 80. Google Scholar CrossRef Search ADS PubMed  Dermitzaki E, Tsatsanis C, Gravanis A, et al.  . ( 2012) The calcineurin-nuclear factor of activated T cells signaling pathway mediates the effect of corticotropin releasing factor and urocortins on catecholamine synthesis. J Cell Physiol  227: 1861– 72. Google Scholar CrossRef Search ADS PubMed  DiMartini A, Dew MA, Day N, et al.  . ( 2010) Trajectories of alcohol consumption following liver transplantation. Am J Transplant  10: 2305– 12. Google Scholar CrossRef Search ADS PubMed  Donohue T, Hoffman PL, Tabakoff B. ( 2005) Effect of ethanol on DARPP-32 phosphorylation in transgenic mice that express human type VII adenylyl cyclase in brain. Alcohol Clin Exp Res  29: 310– 6. Google Scholar CrossRef Search ADS PubMed  Dougherty PM, Dafny N. ( 1988) Cyclosporine affects central nervous system opioid activity via direct and indirect means. Brain Behav Immun  2: 242– 53. Google Scholar CrossRef Search ADS PubMed  Francischi JN, Pereira LS, Castro MS. ( 1997) Cyclosporin inhibits hyperalgesia and edema in arthritic rats: role of the central nervous system. Braz J Med Biol Res  30: 101– 11. Google Scholar CrossRef Search ADS PubMed  Franklin KBJ, Paxinos G. ( 2013) Paxinos and Franklin’s The Mouse Brain in Stereotaxic Coordinates . Amsterdam: Academic Press, an imprint of Elsevier. Fujisaki C, Utsuyama M, Kuroda Y, et al.  . ( 2003) An immnosuppressive drug, cyclosporine-A acts like anti-depressant for rats under unpredictable chronic stress. J Med Dent Sci  50: 93– 100. Google Scholar PubMed  Furman JL, Norris CM. ( 2014) Calcineurin and glial signaling: neuroinflammation and beyond. J Neuroinflammation  11: 158. Google Scholar CrossRef Search ADS PubMed  Giles HG, Orrego H, Sandrin S, et al.  . ( 1990) The influence of cyclosporine on abstinence from alcohol in transplant patients. Transplantation  49: 1201– 2. Google Scholar CrossRef Search ADS PubMed  Greengard P, Allen PB, Nairn AC. ( 1999) Beyond the dopamine receptor: the DARPP-32/protein phosphatase-1 cascade. Neuron  23: 435– 47. Google Scholar CrossRef Search ADS PubMed  Heilig M, Koob GF. ( 2007) A key role for corticotropin-releasing factor in alcohol dependence. Trends Neurosci  30: 399– 406. Google Scholar CrossRef Search ADS PubMed  Ho S, Clipstone N, Timmermann L, et al.  . ( 1996) The mechanism of action of cyclosporin A and FK506. Clin Immunol Immunopathol  80: S40– 5. Google Scholar CrossRef Search ADS PubMed  Kamdar NK, Miller SA, Syed YM, et al.  . ( 2007) Acute effects of naltrexone and GBR 12909 on ethanol drinking-in-the-dark in C57BL/6J mice. Psychopharmacology (Berl)  192: 207– 17. Google Scholar CrossRef Search ADS PubMed  Kingsbury TJ, Bambrick LL, Roby CD, et al.  . ( 2007) Calcineurin activity is required for depolarization-induced, CREB-dependent gene transcription in cortical neurons. J Neurochem  103: 761– 70. Google Scholar CrossRef Search ADS PubMed  Liu J, Farmer JD Jr., Lane WS, et al.  . ( 1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell  66: 807– 15. Google Scholar CrossRef Search ADS PubMed  Liu Y, Coello AG, Grinevich V, et al.  . ( 2010) Involvement of transducer of regulated cAMP response element-binding protein activity on corticotropin releasing hormone transcription. Endocrinology  151: 1109– 18. Google Scholar CrossRef Search ADS PubMed  Liu Y, Kamitakahara A, Kim AJ, et al.  . ( 2008) Cyclic adenosine 3’,5’-monophosphate responsive element binding protein phosphorylation is required but not sufficient for activation of corticotropin-releasing hormone transcription. Endocrinology  149: 3512– 20. Google Scholar CrossRef Search ADS PubMed  Liu Y, Knobloch HS, Grinevich V, et al.  . ( 2011) Stress induces parallel changes in corticotrophin-releasing hormone (CRH) Transcription and nuclear translocation of transducer of regulated cAMP response element-binding activity 2 in hypothalamic CRH neurones. J Neuroendocrinol  23: 216– 23. Google Scholar CrossRef Search ADS PubMed  Lucey MR. ( 2007) Liver transplantation for alcoholic liver disease: past, present, and future. Liver Transpl  13: 190– 2. Google Scholar CrossRef Search ADS PubMed  Lucey MR, Merion RM, Beresford TP. ( 1994) Liver Transplantation & the Alcoholic Patient: Medical, Surgical, and Psychosocial Issues. Cambridge, New York: Cambridge University Press. Marshall SA, Rinker JA, Harrison LK, et al.  . ( 2015) Assessment of the Effects of 6 Standard Rodent Diets on Binge-Like and Voluntary Ethanol Consumption in Male C57BL/6J Mice. Alcohol Clin Exp Res  39: 1406– 16. Google Scholar CrossRef Search ADS PubMed  Mayfield J, Ferguson L, Harris RA. ( 2013) Neuroimmune signaling: a key component of alcohol abuse. Curr Opin Neurobiol  23: 513– 20. Google Scholar CrossRef Search ADS PubMed  Mayfield J, Harris RA. ( 2017) The neuroimmune basis of excessive alcohol consumption. Neuropsychopharmacology  42: 376. Google Scholar CrossRef Search ADS PubMed  Musson RE, Smit NP. ( 2011) Regulatory mechanisms of calcineurin phosphatase activity. Curr Med Chem  18: 301– 15. Google Scholar CrossRef Search ADS PubMed  Nishi A, Snyder GL, Nairn AC, et al.  . ( 1999) Role of calcineurin and protein phosphatase-2A in the regulation of DARPP-32 dephosphorylation in neostriatal neurons. J Neurochem  72: 2015– 21. Google Scholar CrossRef Search ADS PubMed  Phillips TJ, Reed C, Pastor R. ( 2015) Preclinical evidence implicating corticotropin-releasing factor signaling in ethanol consumption and neuroadaptation. Genes Brain Behav  14: 98– 135. Google Scholar CrossRef Search ADS PubMed  Quadros IM, Macedo GC, Domingues LP, et al.  . ( 2016) An update on CRF mechanisms underlying alcohol use disorders and dependence. Front Endocrinol (Lausanne)  7: 134. Google Scholar PubMed  Rhodes JS, Best K, Belknap JK, et al.  . ( 2005) Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiol Behav  84: 53– 63. Google Scholar CrossRef Search ADS PubMed  Ronan PJ, Summers CH. ( 2011) Molecular signaling and translational significance of the corticotropin releasing factor system. Prog Mol Biol Transl Sci  98: 235– 92. Google Scholar CrossRef Search ADS PubMed  Scammell TE, Arrigoni E, Thompson MA, et al.  . ( 2003) Focal deletion of the adenosine A1 receptor in adult mice using an adeno-associated viral vector. J Neurosci  23: 5762– 70. Google Scholar PubMed  Sola C, Tusell JM, Serratosa J. ( 1999) Comparative study of the distribution of calmodulin kinase II and calcineurin in the mouse brain. J Neurosci Res  57: 651– 62. Google Scholar CrossRef Search ADS PubMed  Spencer RL, Weiser MJ. ( 2010) TORC: a new twist on corticotropin-releasing hormone gene expression. Endocrinology  151: 855– 8. Google Scholar CrossRef Search ADS PubMed  Svenningsson P, Nairn AC, Greengard P. ( 2005) DARPP-32 mediates the actions of multiple drugs of abuse. AAPS J  7: E353– 60. Google Scholar CrossRef Search ADS PubMed  Svenningsson P, Nishi A, Fisone G, et al.  . ( 2004) DARPP-32: an integrator of neurotransmission. Annu Rev Pharmacol Toxicol  44: 269– 96. Google Scholar CrossRef Search ADS PubMed  Thiele TE, Crabbe JC, Boehm SL II. ( 2014) ‘Drinking in the Dark’ (DID): a simple mouse model of binge-like alcohol intake. Curr Protoc Neurosci  68: 9 49 1– 12. Google Scholar CrossRef Search ADS   Thiele TE, Navarro M. ( 2014) ‘Drinking in the dark’ (DID) procedures: a model of binge-like ethanol drinking in non-dependent mice. Alcohol  48: 235– 41. Google Scholar CrossRef Search ADS PubMed  Wera S, Neyts J. ( 1994) Calcineurin as a possible new target for treatment of Parkinson’s disease. Med Hypotheses  43: 132– 4. Google Scholar CrossRef Search ADS PubMed  Wilcox MV, Cuzon Carlson VC, Sherazee N, et al.  . ( 2014) Repeated binge-like ethanol drinking alters ethanol drinking patterns and depresses striatal GABAergic transmission. Neuropsychopharmacology  39: 579– 94. Google Scholar CrossRef Search ADS PubMed  Yates WR, Booth BM, Reed DA, et al.  . ( 1993) Descriptive and predictive validity of a high-risk alcoholism relapse model. J Stud Alcohol  54: 645– 51. Google Scholar CrossRef Search ADS PubMed  Medical Council on Alcohol and Oxford University Press 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Alcohol and Alcoholism Oxford University Press

Central Administration of Cyclosporine A Decreases Ethanol Drinking

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Oxford University Press
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Medical Council on Alcohol and Oxford University Press 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.
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0735-0414
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10.1093/alcalc/agx102
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Abstract

Abstract Aims Abstinence among alcohol dependent liver graft recipients is remarkably high. The routine use of anti-immune agents in these patients led to rodent studies showing that immunosuppressants acting through inhibition of calcineurin (CLN) are highly effective in decreasing alcohol consumption. It remained unclear, however, whether the decreased alcohol consumption in rodent models is mediated through peripheral suppression of immune response or centrally through direct inhibition of cyclophilin-CLN in the brain. We tested the hypothesis that direct brain inhibition of CLN with intracerebroventricular (ICV) injections of the immunosuppressant cyclosporine A (CsA) is sufficient to decrease ethanol consumption in a rodent model of binge-like drinking. Methods Male C57BL/6NHsd mice were put through a modified ‘drinking in the dark’ (DID) paradigm. Effects of both peripheral (IP) and central (ICV) injections of CsA on ethanol consumption were assessed. Results Here, as in earlier work, IP CsA administration significantly decreased alcohol consumption. Supporting our hypothesis, central administration of CsA was sufficient to decrease alcohol consumption in a dose-dependent manner. There was no significant effect of CsA on water or sucrose consumption. Conclusions These results clearly implicate a CLN-mediated mechanism in brain in the inhibitory effects of CsA on ethanol consumption and provide novel targets for investigation of treatment for Alcohol Use Disorders (AUD). These results also add to the growing body of literature implicating neuroimmune mechanisms in the etiology, pathophysiology and behaviors driving AUD. Short Summary The unusually high abstinence rate and routine use of immunosuppressants in AUD liver graft recipients led us to rodent studies showing that immunosuppressants acting through inhibition of calcineurin (CLN) are highly effective in decreasing drinking. Here we demonstrate that this effect is mediated by brain rather than peripheral immune mechanisms. INTRODUCTION Liver graft recipients with alcohol use disorder (AUD) show a surprisingly high rate of abstinence (Lucey, 2007; DiMartini et al., 2010). Approximately 90% are abstinent 1 year after transplant and about 75–80% after 3 years. This is remarkable when compared to rates from other treatment studies demonstrating abstinence rates at 1 year closer to 35–45% (Yates et al., 1993; Allen et al., 1995). We initially hypothesized that this high abstinence among liver transplant patients could be explained by pre-transplant AUD case selection and/or post-transplant psychosocial factors known to facilitate abstinence (Lucey et al., 1994). However, these factors could not account for the persistent high abstinence rates among AUD liver graft recipients (Beresford et al., 1992, 1990; Beresford, 1997). One common factor among these patients is the near universal use of immunosuppressants such as cyclosporine A (CsA) post-transplant. We considered the possibility that these immunosuppressants could be playing a role in promoting abstinence. We were the first to report an effect of immunosuppressants on alcohol drinking in an animal model. Mice treated with CsA drank significantly less ethanol in a controlled, continuous-access, two-bottle choice model while having no effect on total liquid intake (Beresford et al., 2005). We sought to determine the mechanism of this effect by comparing three different immunosuppressants acting though different modes of action in a modified ‘drinking in the dark’ (DID) paradigm of excessive drinking (Beresford et al., 2012). Again, immunosuppressants were highly effective in decreasing alcohol consumption, but only those acting through inhibition of the phosphatase calcineurin (CLN) and that crossed the blood-brain barrier. CsA and tacrolimus (FK-506) inhibit CLN activity through different mechanisms but both decreased ethanol consumption. A third immunosuppressant, sirolumus (SRL) that acts through CLN-independent pathways had no effect on ethanol consumption. Though inhibition of CLN was clearly implicated, it was still unclear whether the anti-drinking effects were due to suppression of peripheral immune responses through CLN inhibition or direct inhibition of CLN in brain or via another mechanism in brain. Both CsA and TRL crossed the blood-brain barrier in this study. They also have known inhibitory effects on CLN in brain where immunophilins and calcineurin are abundant and widespread (Dawson et al., 1994). Sirolimus did not effectively penetrate brain suggesting that a general suppression of peripheral immune function was not sufficient to decrease excessive alcohol consumption. We sought to determine in these experiments if this CLN-mediated inhibitory effect on drinking is acting via suppression of peripheral immune response or acting directly in the central nervous system (CNS). We hypothesized that central administration of CsA via intracerebroventricular (ICV) injection would be sufficient to inhibit excessive drinking behavior similar to what we have observed after peripheral administration (IP). The goal of this study was to assess the effects of inhibition of brain calcineurin, separate from any immunosuppressant effect, on ethanol drinking in a mouse model of binge-like drinking. We hypothesized that centrally (ICV) administered Cyclosporine A (CsA) would reduce alcohol consumption in a drinking in the dark model. MATERIALS AND METHODS Animals Adult male C57BL/6NHsd mice (8 weeks; Envigo, Barrier 208 A, Frederick, MD) were initially housed five per group (Microvent Positive Ventilated Housing, Allentown Inc., Allentown, NJ) on a reversed light cycle (off 9:00 AM: on 9:00 PM) in a temperature controlled (23 ± 1°C) AALAC approved animal facility at the Sioux Falls VA. Mice were given ad libidum access to food and water. Ethanol consumption in the DID model has been shown to be significantly altered depending on the variety of standard rodent chow chosen (Marshall et al., 2015). Teklad 2018, 18% Protein Rodent Diet (Envigo) facilitates ethanol consumption. Mice were randomly assigned to five experimental groups (n = 7–8/group): (a) ICV vehicle, (b) ICV Low CsA (15 μg in 2 μl), (c) ICV High CsA (30 μg/2 μl), (d) IP Vehicle, (e) IP CsA (30 mg/kg). Following surgical implantation of ICV guide cannulae, mice were housed individually. All experimental procedures complied with NIH guidelines and were approved by the University of South Dakota IACUC and the Sioux Falls VA Research Service. Materials Ethanol (190 proof, Fisher Scientific) and sucrose (Sigma-Aldrich) were mixed with tap water to 20% (v/v) and 10% (w/v) solutions, respectively. All solutions were made fresh daily. Cyclosporine A (LC Laboratories, Woburn, MA) was dissolved in 85% DMSO (vehicle). Doses of CsA (30 mg/kg IP, 15 and 30 μg ICV) were based on previous work demonstrating that 30 mg/kg IP was the most effective dose in reducing ethanol consumption in similar ethanol drinking model in mice (Beresford et al., 2012) and studies showing a range of behavioral and physiological effects of ICV CsA (Dougherty and Dafny, 1988; Francischi et al., 1997; Almeida-Correa et al., 2015). ICV cannulation Guide cannulae were surgically implanted to allow for ICV injections. Mice were placed in an induction chamber and briefly exposed to isoflurane (5%). Mice were removed and anesthesia was maintained using a nosecone. Ketorolac (5 mg/kg, SC) was administered and their scalps shaved. They were then transferred to a stereotaxic unit (Kopf Model 962 Ultra Precise Small Animal Stereotaxic Instrument, David Kopf Instruments, Tujunga, CA) equipped with a gas anesthesia head holder (Model 923-B). Isoflurane anesthesia was maintained at 1.5–2.5% and the pedal response was continually checked to ensure adequate sedation. The scalp was scrubbed with a chlorhexidine solution followed by sterile saline. An incision was made in the scalp and tissue clamps applied to expose the skull. Fascia was scored and scraped to the sides of the skull with a scalpel and a hydrogen peroxide solution (3%) was applied. Stereotaxic coordinates targeting the lateral ventricle (AP: −0.46; ML: 1.0; DV: 2.3) were determined (Franklin and Paxinos, 2013). With the aid of a surgical stereomicroscope on an articulating arm, a mark was made with surgical ink at these coordinates using a sterile needle attached to an electrode holder (Model 1770). A precision drill (Model 1474 High Speed Stereotaxic Drill; David Kopf Instruments, Tuluma, CA) with a sterile burr bit was used to drill through the skull at this mark, being careful to leave the dura intact. Dura and any remaining bone disc were gently removed using a sterile 25-g needle. Two more holes were drilled anterior and posterior and anchor screws inserted (1.6 diameter × 1.6 mm length; Plastics One). A guide cannula (26-g, C315GA, Plastics One, Roanoke, VA) was inserted into a cannula holder, lowered into the hole and secured in place with a self-curing acrylic resin (OrthoJet Acrylic, Lang Dental, Wheeling, IL). A dummy cannula was inserted and the incision was closed with sutures (simple interrupted, silk, 3.0) and an antibiotic cream applied to the wound. Mice were allowed to recover for 2 weeks before any further experimental procedures were performed. Drinking in the dark Rodent models of ethanol drinking involving voluntary consumption are limited in their ability to generate blood ethanol concentrations (BEC) believed to be pharmacologically meaningful in regard to characteristics of binge-drinking patterns (Thiele and Navarro, 2014). We employed a ‘drinking in the dark’ paradigm (DID) that has been reported to work well inducing BEC of 0.08 (80 mg/100 ml of blood) (Rhodes et al., 2005; Thiele et al., 2014). This model promotes binge-like excessive ethanol consumption during a limited access schedule (2–4 h) during the peak activity period in the dark phase. It has shown predictive face validity for binge-like ethanol drinking in humans (Kamdar et al., 2007; Thiele et al., 2014). We used a slightly modified 2-day version of the DID procedure (Thiele et al., 2014). Briefly, during Week 1 mice were acclimated to handling and modified sipper tubes and given 4 days of alcohol access without any drug treatment. We created custom sipper tubes both to accurately measure consumption and to reduce any leakage as described by Thiele et al. (2014). Briefly, stainless steel bearing sipper tubes (Ancare Corp.) were inserted into polystyrene 10 ml graduated serological pipets (Fisher Scientific) and sealed with heat shrink tubing. Silicone stoppers (Fisher, European size 10D) were used to seal the opposite end. All tubes were tested for leakage. Binder clips were used to attach the tubes to the cages to prevent leakage as mice will climb on them. On Day 1 homecage water bottles were removed at the start of the dark period and replaced with the custom pipette sipper tubes filled with water. Water bottles were replaced after 8 h. On Days 2–5, homecage water bottles were removed and replaced with a 20% ethanol solution for 2 h, and 3 h into the dark phase. We and others have noted that extra days of ethanol access increase average alcohol consumption Figure 1 (Thiele et al., 2014). The DID procedure was carried out on Days 9 and 10. Again, they were given 2 h access to 20% ethanol 3 h into the dark phase. On Day 10, CsA or Vehicle was administered 30 min prior to ethanol access and consumption over 2 h was measured. Two hours was chosen because it has been shown that during the second week of DID procedures mice ‘front-load’ their intake and consume roughly half of the total volume consumed over 4 h in the first 30 min (Wilcox et al., 2014). We used the same 2-day DID model with 10% sucrose or water in place of EtOH to avoid bias when assessing the effects of CsA on alcohol consumption. All mice were weighed before the dark period on the first day of ethanol access and those weights were used to calculate consumption on the following days. To verify ICV injections, mice were anesthetized and 2.0 μl methylene-blue was injected ICV using identical procedures to those used during the experiment. Brains were removed, frozen on dry ice and sectioned on a cryostat microtome (Cryostar NX70, Thermo Fisher Scientific). Injections were verified by the presence of dye in ventricles. Statistics Data were analyzed using SigmaStat 4.0 and graphed with SigmaPlot 10.0 (Systat Software Inc., San Jose, CA). Analysis of Variance (ANOVA) tests were used for all experiments (α = 0.05). Bonferonni Post-hoc tests were used if a significant interaction or main effect of drug was observed. All data are expressed as mean ± SEM. RESULTS Cyclosporine significantly decreased ethanol consumption whether given peripherally (IP) or centrally in brain (ICV). There was a significant main effect for treatment, F(4,32) = 6.17, P < 0.001. We repeated earlier findings demonstrating that a 30 mg/kg IP dose of CsA decreases ethanol intake in a DID model (P = 0.006). Here we show that central administration of CsA also decreases ethanol intake in a similar fashion (30 μg dose, P = 0.002). Though the mean consumption was slightly less for ICV vehicle compared to IP vehicle groups this difference was not significant (P = 0.467). There were no significant main effects of ICV or IP administration of CsA on consumption of sucrose, F(4,31) = 0.927, P = 0.461, or water, F(4,31) = 1.594, P = 0.201. Access to alcohol for 2 h session for 4 days in the week prior to the DID procedure significantly increased overall ethanol consumption, F(4,32) = 6.299, P = 0.003 (Figs 2–4). Fig. 1. View largeDownload slide Ethanol intake increased over a 4 day access period. Mice were given access to 20% ethanol for 2 h/day for 4 days; *P = 0.011. Fig. 1. View largeDownload slide Ethanol intake increased over a 4 day access period. Mice were given access to 20% ethanol for 2 h/day for 4 days; *P = 0.011. Fig. 2. View largeDownload slide Both central and peripheral administration of cyclosporine A significantly reduced alcohol intake in mice. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg); *P < 0.05. Fig. 2. View largeDownload slide Both central and peripheral administration of cyclosporine A significantly reduced alcohol intake in mice. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg); *P < 0.05. Fig. 3. View largeDownload slide Cyclosporine had no effect on sucrose intake. There were no significant differences in sucrose consumption between groups. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg). Fig. 3. View largeDownload slide Cyclosporine had no effect on sucrose intake. There were no significant differences in sucrose consumption between groups. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg). Fig. 4. View largeDownload slide Cyclosporine had no effect water intake. There were no significant differences in water consumption between groups. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg). Fig. 4. View largeDownload slide Cyclosporine had no effect water intake. There were no significant differences in water consumption between groups. Experimental groups: ICV vehicle, ICV CsA Low (15 μg), ICV CsA High (30 μg), IP Vehicle, IP CsA (30 mg/kg). DISCUSSION This experiment demonstrated that central administration of cyclosporine A is sufficient to decrease alcohol intake in mice in a binge-drinking model of ethanol consumption in a dose-dependent fashion. These results combined with our previous research (Beresford et al., 2012) clearly show that the anti-drinking effects of CsA are mediated by direct effects in brain and not by peripheral immunosuppression. We also replicated and extended earlier findings demonstrating that peripheral administration (IP) of CsA significantly reduces alcohol intake (Beresford et al., 2005, 2012) using a slightly different mouse strain and drinking model. Administration of CsA in brain is sufficient to decrease ethanol consumption suggesting that the effects seen after peripheral administration are due to direct actions in brain as well. Additionally, the fact that there were no significant effects of CsA on water or sucrose consumption indicates a specific effect of CsA on ethanol intake. An indiscriminate antidipsic effect cannot explain the results of this study nor can a reduced need for caloric intake or generalized taste aversion mechanism. The specific pathways mediating this effect still need to be elucidated. Suppression of neuroimmune activation is one hypothesis. As an immunosuppressant, CsA acts in brain much like it does in peripheral immune cells. Similar to its role in peripheral immune/inflammatory cells, CLN plays an integral role in processes regulating neuroimmune function. Calcineurin regulates glial cell activation and cytokine expression and is induced during disease, injury and aging (Furman and Norris, 2014). Calcineurin is a major regulator of immune or inflammatory processes in both T-cells and glial cells through activation of the nuclear factor of activated T-cells (NFAT) family of transcription factors. Cyclosporine binds the immunophilin cyclophilin and this complex then inhibits the activity of CLN, a Ca2+ and calmodulin-dependent serine-threonine phosphatase also known as protein phosphatase 3 (PPP3) (Liu et al., 1991). Dephosphorylation of NFAT by CLN causes activation and nuclear translocation where it stimulates transcription of a number of proinflammatory genes including interleukin-2 (Clipstone and Crabtree, 1993; Ho et al., 1996; Musson and Smit, 2011). It is now widely accepted that immune mechanisms, and specifically neuroimmune mechanisms, play important, if poorly understood, roles in the etiology and progression of alcohol abuse (Mayfield and Harris, 2017). The hypothesis that an overactive neuroimmune system promotes alcohol consumption is gaining widespread support among scientists (Mayfield et al., 2013; Cui et al., 2014; Crews et al., 2017). The first mention of a possible connection between immunosuppressants and alcohol consumption was published as a brief clinical report in 1990 (Giles et al., 1990). It was noted that AUD liver graft patients were more abstinent while on immunosuppressants. It has taken many years for the research community to embrace and investigate this possibility. Our 2005 study, stemming from insights gained from liver graft recipients, was the first to test the hypothesis that suppression of immune processes could limit ethanol intake (Beresford et al., 2005). This notion has been extended by a number of other groups in the last few years. A range of compounds or genetic approaches that inhibit neuroimmune function have been shown to limit ethanol intake to varying degrees in a variety of animal models (Mayfield et al., 2013; Cui et al., 2014; Crews et al., 2017). These studies target different specific mechanisms but they all converge on the common outcome of reducing neuroimmune activation. Along with reducing neuroinflammation, CsA also plays key roles in neuronal signaling processes. The effectiveness of CsA in reducing ethanol consumption seen in this and our past studies may be due to its complementary roles in other brain signaling pathways related to brain reward and addiction. During the past few decades, the diverse and important roles CLN plays in neuronal signaling processes have been more and more appreciated. Calcineurin is one of the most abundant phosphatases in brain and is known to play important roles in a wide variety of signal transduction pathways. Not only involved in neuroimmune processes, CLN is also expressed at especially high levels in the brain in neurons. There are particularly high concentrations of CLN in the hippocampus, neocortex, striatum and amygdala (Furman and Norris, 2014) where CLN modulates diverse cellular functions including receptor and ion channel trafficking, ion channel function, apoptosis and gene regulation (Baumgartel and Mansuy, 2012). Calcineurin is also abundantly expressed in all of the regions of the brain where CaMKII is found including the dopaminergic reward pathway of the ventral tegmental area (VTA) and the nucleus accumbens (NAc) (Sola et al., 1999). Calcineurin directly regulates dopaminergic and other monoaminergic signaling. Along with direct effects on monoamine synthesis and release it has been shown that CLN activity regulates an important dopamine and addiction-related signal transduction protein—dopamine and cAMP-regulated neuronal phosphoprotein (DARPP-32). This protein regulates both dopaminergic and glutaminergic (NMDA) receptor responses in extended brain reward pathways (Greengard et al., 1999; Svenningsson et al., 2004) and is critically involved in regulating responses to ethanol and other drugs of abuse (Donohue et al., 2005; Svenningsson et al., 2005). DARPP-32 is a direct CLN substrate. Dopamine causes increased phosphorylation and CN acts to maintain low levels of phosphorylated DARPP (Nishi et al., 1999). It has been hypothesized that CsA inhibition of CLN acts as a dopamine mimetic via dopamine-like increases in phosphorylated DARPP-32 (Wera and Neyts, 1994). In this way CLN inhibition could potentially dampen temporally linked dopamine reward cues elicited by alcohol and thereby lessen reinforcement. Monoaminergic activity is integral to processes related to reward and addiction and CLN has been shown to regulate monoaminergic signaling. In rats, CsA alone has an inhibitory effect on monoamine production but, interestingly, when given along with a chronic stressor it has the opposite effect, leading to enhanced tissue levels of serotonin and norepinephrine (Fujisaki et al., 2003). It also had antidepressant-like activity in the unpredictable chronic stress model that paralleled imipramine. Calcineurin regulates several other signaling pathways known to be associated with addiction and stress including corticotropin releasing factor (CRF). While playing a critical role in the regulation of behavior and neuroendocrine responses to stress, CRF has also proven to be a significant contributor to the processes of alcohol consumption and abuse (Heilig and Koob, 2007; Ronan and Summers, 2011; Phillips et al., 2015). Calcineurin regulates CRF gene expression and CRF-mediated behaviors. Transcription of CRF is mediated via cAMP response element binding protein (CREB) phosphorylation. Phosphorylation of CREB is necessary but not sufficient for CRF transcription (Liu et al., 2008). A CLN-regulated coactivator called TORC (tranducer of regulated CREB activity) is also needed for CREB-induced CRF transcription (Liu et al., 2010; Spencer and Weiser, 2010). Dephosphorylation of TORC by CLN facilitates translocation to the nucleus where it interacts with the dimerization and DNA binding domain of CREB and facilitates the recruitment of the preinitiation complex. This, in turn, facilitates transcriptional activation of CREB-dependent genes including CRF. CLN activity is required for depolarization-induced CREB-dependent gene transcription in cortical neurons (Kingsbury et al., 2007). Stress alone induces nuclear translocation of TORC and transcription of CRF in rodents (Liu et al., 2011). This TORC mediated mechanism for enhanced stress-induced CRF expression may underlie the enhanced sensitivity to stressors seen in models of ethanol abuse. Cyclosporine blocks CRF-receptor mediated catecholamine synthesis and release through inhibition of NFAT activation in cell culture (Dermitzaki et al., 2012). CRF has been shown to modulate brain reward function directly through signaling in the ventral tegmental area, nucleus accumbens and the prefrontal cortex, among other structures (Ronan and Summers, 2011; Quadros et al., 2016). One of the strengths of this study is the use of a well-validated and widely used rodent drinking model. The ‘drinking in the dark’ procedure has high face validity as a model of human binge drinking (for review see, Thiele and Navarro, 2014). Many strains of mice have the natural tendency to display excessive bouts of drinking under short term access to ethanol. The fact that they are not forced to drink as in other models of binge drinking (i.e. gavage, vapor, injection) can help us get at the underlying drinking behaviors. It also is not necessary in this model to deprive mice of food or water to study these drinking behaviors. Varying lines of evidence have converged to demonstrate that the high ethanol consumption in this model is not due to caloric need. This model has been successfully used to identify pharmacological targets and genetic mechanisms that promote binge drinking. This study is an example of research going from clinical observations to the bench. The central question answered by this study, whether CsA acts centrally or peripherally to suppress alcohol consumption, could not have been answered in humans. The initial hypotheses gleaned from clinical observations lacked real proof. Empirical data on CsA and other immunosuppressants versus control with respect to suppression of alcohol consumption are not available in human studies. And performing a placebo controlled study in this population is ethically unfeasible. In this study we were able to employ a ‘placebo’-controlled study design to properly assess the effects of CsA. The steps from bench back to clinic: the need for mechanistic clarity While our mouse study data are remarkably encouraging in pursuing this innovative approach to treatment development, exploring this observed effect begins with an assessment of whether or not a similar effect actually occurs in people. The mouse data do not translate directly into human experiments with these agents. For one example, the effective mg/kg doses in mice would be toxic in human beings. For another, their immunosuppressant effects at standard doses given to transplant patients postoperatively cannot be clinically justified as a widespread treatment. In this regard, therefore, pursuing CLN inhibition as a new treatment option for AD first requires careful investigation with respect to specific mechanisms of action. Identification of the specific pathways and substrates involved in this immunosuppressant effect on drinking needs to be studied taking a variety of approaches. One line of inquiry we are pursuing is to identify specific CLN pathways involved in this effect using a genetic approach. We are using conditional knockouts using both Cre-loxP and RNA interference techniques (Scammell et al., 2003; Callahan et al., 2013), employing a transgenic mouse line with a floxed regulatory subunit of calcineurin (a generous gift from Dr. S. Tonegawa, MIT) along with ‘cre-cutter’ mouse lines and viral vectors, to determine the role of calcineurin-mediated pathways in specific brain regions and cell types (e.g. dopaminergic, glia). These studies and others investigating intracellular CLN substrates and downstream pathways will yield information that will allow us to target specific aspects of CLN signaling to better enable us to develop effective treatments with minimal side effects. CONCLUSIONS Our results clearly implicate a central brain mechanism in the inhibitory effects of CsA on alcohol consumption. This provides a rich avenue for the pursuit of therapeutic targets for treatment of AUD in humans and adds to the growing body of evidence that various neuro-immuno-modulatory pathways could provide novel targets for the development of pharmacotherapies for AUD (Coller and Hutchinson, 2012; Cui et al., 2014). Cyclosporine itself is not a viable treatment option given its possible long-term nephrotoxicity and range of adverse side effects. Deciphering the specific proximal mechanisms underlying the anti-drinking effects of CsA holds the promise for the development of effective treatments where few exist. FUNDING This work was supported by the Department of Veterans Affairs (T.B. and P.R.) and an Department of Veterans Affairs, Office of Academic Affiliations, Advanced VA Research Fellowship (P.R.) and the Great Plains Medical Research Foundation (P.R.). It also received support from the University of South Dakota Center for Brain and Behavior Research (P.R., G.P.) and the U. Discover Summer Research Scholars Program (S.S.). CONFLICT OF INTEREST STATEMENT The authors declare no conflicts of interest. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs or the United States government. ACKNOWLEDGEMENTS The authors would like to thank Allison Rollag, Paige Baker, Belissa Fernandez and Jessica Fernandez for their technical assistance with this study. REFERENCES Allen JP, Litten RZ, Fertig JB. ( 1995) NIDA-NIAAA workshop: efficacy of therapies in drug and alcohol addiction. Strategies for treatment of alcohol problems. Psychopharmacol Bull  31: 665– 9. Google Scholar PubMed  Almeida-Correa S, Moulin TC, Carneiro CF, et al.  . ( 2015) Calcineurin inhibition blocks within-, but not between-session fear extinction in mice. Learn Mem  22: 159– 69. Google Scholar CrossRef Search ADS PubMed  Baumgartel K, Mansuy IM. ( 2012) Neural functions of calcineurin in synaptic plasticity and memory. Learn Mem  19: 375– 84. Google Scholar CrossRef Search ADS PubMed  Beresford HF, Deitrich R, Beresford TP. ( 2005) Cyclosporine-A discourages ethanol intake in C57bl/6j mice: a preliminary study. J Stud Alcohol  66: 658– 62. Google Scholar CrossRef Search ADS PubMed  Beresford T, Fay T, Serkova NJ, et al.  . ( 2012) Immunophyllin ligands show differential effects on alcohol self-administration in C57BL mice. J Pharmacol Exp Ther  341: 611– 6. Google Scholar CrossRef Search ADS PubMed  Beresford TP. ( 1997) Predictive factors for alcoholic relapse in the selection of alcohol-dependent persons for hepatic transplant. Liver Transpl Surg  3: 280– 91. Google Scholar CrossRef Search ADS PubMed  Beresford TP, Schwartz J, Wilson D, et al.  . ( 1992) The short-term psychological health of alcoholic and non-alcoholic liver transplant recipients. Alcohol Clin Exp Res  16: 996– 1000. Google Scholar CrossRef Search ADS PubMed  Beresford TP, Turcotte JG, Merion R, et al.  . ( 1990) A rational approach to liver transplantation for the alcoholic patient. Psychosomatics  31: 241– 54. Google Scholar CrossRef Search ADS PubMed  Callahan LB, Tschetter KE, Ronan PJ. ( 2013) Inhibition of corticotropin releasing factor expression in the central nucleus of the amygdala attenuates stress-induced behavioral and endocrine responses. Front Neurosci  7: 195. Google Scholar CrossRef Search ADS PubMed  Clipstone NA, Crabtree GR. ( 1993) Calcineurin is a key signaling enzyme in T lymphocyte activation and the target of the immunosuppressive drugs cyclosporin A and FK506. Ann N Y Acad Sci  696: 20– 30. Google Scholar CrossRef Search ADS PubMed  Coller JK, Hutchinson MR. ( 2012) Implications of central immune signaling caused by drugs of abuse: mechanisms, mediators and new therapeutic approaches for prediction and treatment of drug dependence. Pharmacol Ther  134: 219– 45. Google Scholar CrossRef Search ADS PubMed  Crews FT, Lawrimore CJ, Walter TJ, et al.  . ( 2017) The role of neuroimmune signaling in alcoholism. Neuropharmacology  122: 56– 73. Google Scholar CrossRef Search ADS PubMed  Cui C, Shurtleff D, Harris RA. ( 2014) Neuroimmune mechanisms of alcohol and drug addiction. Int Rev Neurobiol  118: 1– 12. Google Scholar CrossRef Search ADS PubMed  Dawson TM, Steiner JP, Lyons WE, et al.  . ( 1994) The immunophilins, FK506 binding protein and cyclophilin, are discretely localized in the brain: relationship to calcineurin. Neuroscience  62: 569– 80. Google Scholar CrossRef Search ADS PubMed  Dermitzaki E, Tsatsanis C, Gravanis A, et al.  . ( 2012) The calcineurin-nuclear factor of activated T cells signaling pathway mediates the effect of corticotropin releasing factor and urocortins on catecholamine synthesis. J Cell Physiol  227: 1861– 72. Google Scholar CrossRef Search ADS PubMed  DiMartini A, Dew MA, Day N, et al.  . ( 2010) Trajectories of alcohol consumption following liver transplantation. Am J Transplant  10: 2305– 12. Google Scholar CrossRef Search ADS PubMed  Donohue T, Hoffman PL, Tabakoff B. ( 2005) Effect of ethanol on DARPP-32 phosphorylation in transgenic mice that express human type VII adenylyl cyclase in brain. Alcohol Clin Exp Res  29: 310– 6. Google Scholar CrossRef Search ADS PubMed  Dougherty PM, Dafny N. ( 1988) Cyclosporine affects central nervous system opioid activity via direct and indirect means. Brain Behav Immun  2: 242– 53. Google Scholar CrossRef Search ADS PubMed  Francischi JN, Pereira LS, Castro MS. ( 1997) Cyclosporin inhibits hyperalgesia and edema in arthritic rats: role of the central nervous system. Braz J Med Biol Res  30: 101– 11. Google Scholar CrossRef Search ADS PubMed  Franklin KBJ, Paxinos G. ( 2013) Paxinos and Franklin’s The Mouse Brain in Stereotaxic Coordinates . Amsterdam: Academic Press, an imprint of Elsevier. Fujisaki C, Utsuyama M, Kuroda Y, et al.  . ( 2003) An immnosuppressive drug, cyclosporine-A acts like anti-depressant for rats under unpredictable chronic stress. J Med Dent Sci  50: 93– 100. Google Scholar PubMed  Furman JL, Norris CM. ( 2014) Calcineurin and glial signaling: neuroinflammation and beyond. J Neuroinflammation  11: 158. Google Scholar CrossRef Search ADS PubMed  Giles HG, Orrego H, Sandrin S, et al.  . ( 1990) The influence of cyclosporine on abstinence from alcohol in transplant patients. Transplantation  49: 1201– 2. Google Scholar CrossRef Search ADS PubMed  Greengard P, Allen PB, Nairn AC. ( 1999) Beyond the dopamine receptor: the DARPP-32/protein phosphatase-1 cascade. Neuron  23: 435– 47. Google Scholar CrossRef Search ADS PubMed  Heilig M, Koob GF. ( 2007) A key role for corticotropin-releasing factor in alcohol dependence. Trends Neurosci  30: 399– 406. Google Scholar CrossRef Search ADS PubMed  Ho S, Clipstone N, Timmermann L, et al.  . ( 1996) The mechanism of action of cyclosporin A and FK506. Clin Immunol Immunopathol  80: S40– 5. Google Scholar CrossRef Search ADS PubMed  Kamdar NK, Miller SA, Syed YM, et al.  . ( 2007) Acute effects of naltrexone and GBR 12909 on ethanol drinking-in-the-dark in C57BL/6J mice. Psychopharmacology (Berl)  192: 207– 17. Google Scholar CrossRef Search ADS PubMed  Kingsbury TJ, Bambrick LL, Roby CD, et al.  . ( 2007) Calcineurin activity is required for depolarization-induced, CREB-dependent gene transcription in cortical neurons. J Neurochem  103: 761– 70. Google Scholar CrossRef Search ADS PubMed  Liu J, Farmer JD Jr., Lane WS, et al.  . ( 1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell  66: 807– 15. Google Scholar CrossRef Search ADS PubMed  Liu Y, Coello AG, Grinevich V, et al.  . ( 2010) Involvement of transducer of regulated cAMP response element-binding protein activity on corticotropin releasing hormone transcription. Endocrinology  151: 1109– 18. Google Scholar CrossRef Search ADS PubMed  Liu Y, Kamitakahara A, Kim AJ, et al.  . ( 2008) Cyclic adenosine 3’,5’-monophosphate responsive element binding protein phosphorylation is required but not sufficient for activation of corticotropin-releasing hormone transcription. Endocrinology  149: 3512– 20. Google Scholar CrossRef Search ADS PubMed  Liu Y, Knobloch HS, Grinevich V, et al.  . ( 2011) Stress induces parallel changes in corticotrophin-releasing hormone (CRH) Transcription and nuclear translocation of transducer of regulated cAMP response element-binding activity 2 in hypothalamic CRH neurones. J Neuroendocrinol  23: 216– 23. Google Scholar CrossRef Search ADS PubMed  Lucey MR. ( 2007) Liver transplantation for alcoholic liver disease: past, present, and future. Liver Transpl  13: 190– 2. Google Scholar CrossRef Search ADS PubMed  Lucey MR, Merion RM, Beresford TP. ( 1994) Liver Transplantation & the Alcoholic Patient: Medical, Surgical, and Psychosocial Issues. Cambridge, New York: Cambridge University Press. Marshall SA, Rinker JA, Harrison LK, et al.  . ( 2015) Assessment of the Effects of 6 Standard Rodent Diets on Binge-Like and Voluntary Ethanol Consumption in Male C57BL/6J Mice. Alcohol Clin Exp Res  39: 1406– 16. Google Scholar CrossRef Search ADS PubMed  Mayfield J, Ferguson L, Harris RA. ( 2013) Neuroimmune signaling: a key component of alcohol abuse. Curr Opin Neurobiol  23: 513– 20. Google Scholar CrossRef Search ADS PubMed  Mayfield J, Harris RA. ( 2017) The neuroimmune basis of excessive alcohol consumption. Neuropsychopharmacology  42: 376. Google Scholar CrossRef Search ADS PubMed  Musson RE, Smit NP. ( 2011) Regulatory mechanisms of calcineurin phosphatase activity. Curr Med Chem  18: 301– 15. Google Scholar CrossRef Search ADS PubMed  Nishi A, Snyder GL, Nairn AC, et al.  . ( 1999) Role of calcineurin and protein phosphatase-2A in the regulation of DARPP-32 dephosphorylation in neostriatal neurons. J Neurochem  72: 2015– 21. Google Scholar CrossRef Search ADS PubMed  Phillips TJ, Reed C, Pastor R. ( 2015) Preclinical evidence implicating corticotropin-releasing factor signaling in ethanol consumption and neuroadaptation. Genes Brain Behav  14: 98– 135. Google Scholar CrossRef Search ADS PubMed  Quadros IM, Macedo GC, Domingues LP, et al.  . ( 2016) An update on CRF mechanisms underlying alcohol use disorders and dependence. Front Endocrinol (Lausanne)  7: 134. Google Scholar PubMed  Rhodes JS, Best K, Belknap JK, et al.  . ( 2005) Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiol Behav  84: 53– 63. Google Scholar CrossRef Search ADS PubMed  Ronan PJ, Summers CH. ( 2011) Molecular signaling and translational significance of the corticotropin releasing factor system. Prog Mol Biol Transl Sci  98: 235– 92. Google Scholar CrossRef Search ADS PubMed  Scammell TE, Arrigoni E, Thompson MA, et al.  . ( 2003) Focal deletion of the adenosine A1 receptor in adult mice using an adeno-associated viral vector. J Neurosci  23: 5762– 70. Google Scholar PubMed  Sola C, Tusell JM, Serratosa J. ( 1999) Comparative study of the distribution of calmodulin kinase II and calcineurin in the mouse brain. J Neurosci Res  57: 651– 62. Google Scholar CrossRef Search ADS PubMed  Spencer RL, Weiser MJ. ( 2010) TORC: a new twist on corticotropin-releasing hormone gene expression. Endocrinology  151: 855– 8. Google Scholar CrossRef Search ADS PubMed  Svenningsson P, Nairn AC, Greengard P. ( 2005) DARPP-32 mediates the actions of multiple drugs of abuse. AAPS J  7: E353– 60. Google Scholar CrossRef Search ADS PubMed  Svenningsson P, Nishi A, Fisone G, et al.  . ( 2004) DARPP-32: an integrator of neurotransmission. Annu Rev Pharmacol Toxicol  44: 269– 96. Google Scholar CrossRef Search ADS PubMed  Thiele TE, Crabbe JC, Boehm SL II. ( 2014) ‘Drinking in the Dark’ (DID): a simple mouse model of binge-like alcohol intake. Curr Protoc Neurosci  68: 9 49 1– 12. Google Scholar CrossRef Search ADS   Thiele TE, Navarro M. ( 2014) ‘Drinking in the dark’ (DID) procedures: a model of binge-like ethanol drinking in non-dependent mice. Alcohol  48: 235– 41. Google Scholar CrossRef Search ADS PubMed  Wera S, Neyts J. ( 1994) Calcineurin as a possible new target for treatment of Parkinson’s disease. Med Hypotheses  43: 132– 4. Google Scholar CrossRef Search ADS PubMed  Wilcox MV, Cuzon Carlson VC, Sherazee N, et al.  . ( 2014) Repeated binge-like ethanol drinking alters ethanol drinking patterns and depresses striatal GABAergic transmission. Neuropsychopharmacology  39: 579– 94. Google Scholar CrossRef Search ADS PubMed  Yates WR, Booth BM, Reed DA, et al.  . ( 1993) Descriptive and predictive validity of a high-risk alcoholism relapse model. J Stud Alcohol  54: 645– 51. Google Scholar CrossRef Search ADS PubMed  Medical Council on Alcohol and Oxford University Press 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Alcohol and AlcoholismOxford University Press

Published: Mar 1, 2018

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