3646 • The Journal of Neuroscience, March 29, 2017 • 37(13):3646–3660 Neurobiology of Disease Orbitofrontal Neuroadaptations and Cross-Species Synaptic Biomarkers in Heavy-Drinking Macaques X Sudarat Nimitvilai,1* Joachim D. Uys,2* John J. Woodward,1,3 Patrick K. Randall,3 Lauren E. Ball,2 X Robert W. Williams,4 XByron C. Jones,4 X Lu Lu,4 X Kathleen A. Grant,5 and Patrick J. Mulholland1,3 Departments of 1Neuroscience, 2Cell and Molecular Pharmacology, and 3Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, 4Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, Tennessee 38120, and 5Department of Behavioral Neuroscience, Oregon Health and Science University, Portland, Oregon 97239 Cognitive impairments, uncontrolled drinking, and neuropathological cortical changes characterize alcohol use disorder. Dysfunction of the orbitofrontal cortex (OFC), a critical cortical subregion that controls learning, decision-making, and prediction of reward outcomes, contributes to executive cognitive function deficits in alcoholic individuals. Electrophysiological and quantitative synaptomics tech- niques were used to test the hypothesis that heavy drinking produces neuroadaptations in the macaque OFC. Integrative bioinformatics and reverse genetic approaches were used to identify and validate synaptic proteins with novel links to heavy drinking in BXD mice. In drinking monkeys, evoked firing of OFC pyramidal neurons was reduced, whereas the amplitude and frequency of postsynaptic currents were enhanced compared with controls. Bath application of alcohol reduced evoked firing in neurons from control monkeys, but not drinking monkeys. Profiling of the OFC synaptome identified alcohol-sensitive proteins that control glutamate release (e.g., SV2A, synaptogyrin-1) and postsynaptic signaling (e.g., GluA1, PRRT2) with no changes in synaptic GABAergic proteins. Western blot analysis confirmed the increase in GluA1 expression in drinking monkeys. An exploratory analysis of the OFC synaptome found cross-species genetic links to alcohol intake in discrete proteins (e.g., C2CD2L, DIRAS2) that discriminated between low- and heavy-drinking monkeys. Validation studies revealed that BXD mouse strains with the D allele at the C2cd2l interval drank less alcohol than B allele strains. Thus, by profiling of the OFC synaptome, we identified changes in proteins controlling glutamate release and postsynaptic signaling and discovered several proteins related to heavy drinking that have potential as novel targets for treating alcohol use disorder. Key words: alcohol; electrophysiology; genetics; orbitofrontal cortex; proteomics Significance Statement Clinical research identified cognitive deficits in alcoholic individuals as a risk factor for relapse, and alcoholic individuals display deficits on cognitive tasks that are dependent upon the orbitofrontal cortex (OFC). To identify neurobiological mechanisms that underpin OFC dysfunction, this study used electrophysiology and integrative synaptomics in a translational nonhuman primate model of heavy alcohol consumption. We found adaptations in synaptic proteins that control glutamatergic signaling in chroni- cally drinking monkeys. Our functional genomic exploratory analyses identified proteins with genetic links to alcohol and cocaine intake across mice, monkeys, and humans. Future work is necessary to determine whether targeting these novel targets reduces excessive and harmful levels of alcohol drinking. Introduction (Whiteford et al., 2013). Accordingly, alcohol ranks higher than Alcohol abuse disorder (AUD) is a detrimental worldwide public 20 abused substances, including heroin, cocaine, and metham- health problem with increased prevalence over the past 20 years Spectrometer was acquired through NIH/National Center for Research Resources Grant S10-OD-010731. We thank Received Jan. 16, 2017; revised Feb. 17, 2017; accepted Feb. 28, 2017. Susana Comte-Walters. We also thank Dr. James B. Daunais for the macaque frontal cortex images. Author contributions: S.N., J.D.U., J.J.W., B.C.J., L.L., K.A.G., and P.J.M. designed research; S.N., J.D.U., R.W.W., *S.N. and J.D.U. contributed equally to this work. B.C.J.,L.L.,andP.J.M.performedresearch;S.N.,J.J.W.,P.K.R.,R.W.W.,K.A.G.,andP.J.M.analyzeddata;S.N.,J.D.U., The authors declare no competing financial interests. J.J.W., L.E.B., R.W.W., B.C.J., L.L., K.A.G., and P.J.M. wrote the paper. Correspondence should be addressed to Dr. Patrick J. Mulholland, Associate Professor, Medical University of This work was supported by National Institutes of Health (NIH) Grants AA-020930, AA-023288, RR-024485, South Carolina, Charleston Alcohol Research Center, Department of Neuroscience and Psychiatry and Behavioral AA-024426,AA-019431,AA-013541,AA-016662,AA-013499,AA-010761,AA-009986,AA-021951,andOD-11092. Sciences, 67 President Street, MSC 861/IOP 462N, Charleston, SC 29425-8610. E-mail: [email protected]. TheMedicalUniversityofSouthCarolinaMassSpectrometryFacilityreceivessupportfromtheSCCOBREinOxidants, DOI:10.1523/JNEUROSCI.0133-17.2017 Redox Balance, and Stress Signaling (Grant P20 GM103542) and the Office of the Provost. The Orbitrap Elite Mass Copyright © 2017 the authors 0270-6474/17/373646-15$15.00/0 Nimitvilai, Uys et al. • Alcohol and the OFC Synaptome J. Neurosci., March 29, 2017 • 37(13):3646–3660 • 3647 phetamine, as the most harmful drug (Nutt et al., 2010). AUD is and 11 monkeys (3 controls and 8 drinkers; MATRR.org INIA Cohort 9) a chronic relapsing disease characterized by functional and met- were used for quantitative proteomics and Western blot analyses. Be- abolic tolerance, a distinctive withdrawal syndrome, recurrent cause of the individual differences in average daily intake in this volun- and heavier alcohol use, and cognitive impairments. Impor- tary alcohol-drinking procedure (Baker et al., 2014, 2017) that may tantly, compelling evidence from studies that have examined the preclude identifying functional or proteomic neuroadaptations, alcohol- drinking monkeys were oversampled in these studies. neurobiology of cognitive deficits in alcoholic individuals has Preparation of brain slices. Brain slices containing area 13L were pre- demonstrated that poor cognitive function is a risk factor for pared for whole-cell patch-clamp electrophysiology experiments with increased vulnerability to relapse (Charlet et al., 2014), suggesting the experimenter blind to the treatment conditions. Following the nec- that improving cognitive performance during abstinence may ropsy procedure and rapid removal of the brain, the tissue was blocked prevent relapse. However, current pharmacotherapies for treat- coronally for the frontal cortex and mounted in a Vibroslicer (Leica) ing AUD do not target alcohol-induced cognitive impairments. containing ice-cold oxygenated (95% O2,5%CO2) sucrose containing Subregions of the prefrontal cortex (PFC) are key regulators of buffer, and coronal sections (300 ␮m) were cut. Slices containing area executive functions that are often critically impaired following 13L were immediately placed in a holding chamber containing oxygen- heavy alcohol use. Among these, the orbitofrontal cortex (OFC) ated artificial CSF (aCSF) at 34°C for 45 min and kept at room temper- ature for at least 30 min before transferring to the recording chamber. contributes to outcome-based decision-making related to the ex- ␮ pected rewards and the pleasurable properties of rewarding stim- The glutamate NMDA receptor blocker AP5 (50 M) was added to the aCSF during incubation to prolong the viability of neurons, and slices uli (Stalnaker et al., 2015). Imaging and behavioral studies show were washed with regular oxygenated aCSF for at least 20 min before the that patients with AUD have greater activation of the OFC and whole-cell recordings were performed. Although AP5 is reported to wash reduced performance when completing OFC-dependent tasks out of acute slice preparations (Dozmorov et al., 2004; Herman et al., compared with healthy control subjects, demonstrating that pro- 2011), application of AP5 for Ն12 h enhances evoked firing (Ishikawa et longed drinking can impair OFC function (Fortier et al., 2008, al., 2009; Lee and Chung, 2014), suggesting that there may be a compen- 2009). Consistent with deficits reported in individuals with AUD, satory increase in intrinsic excitability under these recording conditions. rodent models of alcohol dependence induce OFC-dependent However, all groups were treated identically, so this is unlikely to explain behavioral deficits and adaptations in synaptic physiology and a difference in firing between treatment groups. intrinsic excitability in OFC neurons (Crews and Boettiger, 2009; The composition of the sucrose-containing cutting solution used was as follows (in mM): 194 sucrose, 30 NaCl, 4.5 KCl, 1.2 NaH PO , 1 MgCl , Badanich et al., 2011; McGuier et al., 2015; Nimitvilai et al., 2 4 2 10 glucose, and 26 NaHCO , adjusted to 305–315 mOsm. The composi- 2016). Although rodent studies on the effects of alcohol on cor- 3 tion of the aCSF was as follows (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, tical function are valuable, they are limited by the reduced com- 1.3 MgCl , 2.0 CaCl , 0.4 ascorbate, 10 glucose, and 25 NaHCO , adjusted plexity of the rodent cortex present in primates (Carmichael and 2 2 3 to 290–310 mOsm. Both solutions were saturated with 95% O2/5% CO2,pH Price, 1994; Ongu¨r and Price, 2000). To circumvent these limita-