Orbitofrontal Neuroadaptations and Cross-Species Synaptic Biomarkers in Heavy-Drinking Macaques

Abstract

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 techniques 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.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 chronically 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.

Keywords: alcohol; electrophysiology; genetics; orbitofrontal cortex; proteomics.

Figure 1.

Long-term alcohol drinking reduces evoked spiking in area 13L pyramidal neurons. A, B, Alcohol consumption levels (A; mean ± SD) and BACs (B; mean ± SD) during the 6 months of 22 h access to alcohol self-administration in the INIA Cyno 13 cohort of monkeys used for electrophysiological analysis. C, Representative correlations between alcohol intake and BACs. D, Example image of the ventral aspect of a cynomolgus macaque brain showing prominent anatomical markers and areas 10, 12, 13L, 13M, and 14. E, A representative example of a coronal section of the OFC-containing area 13L that was used for functional and biochemical analyses. F, Representative traces showing reduced evoked spiking in a control and a drinking monkey. G, Number of action potentials at current injections ranging from −40 to −240 pA in control and drinking monkeys. H, Rheobase was increased in the drinking monkeys. I, J, Representative traces and average number of evoked firing in the absence and presence of 22 mm alcohol bath application in control (I) and drinking (J) monkeys. Electrophysiological data are expressed as the mean ± SEM.

Figure 2.

Long-term alcohol self-administration enhances the amplitude and frequency of sPSCs in deep-layer area 13L pyramidal neurons. A, B, Representative traces of sPSCs from a control (black trace) and a drinking (gray trace) monkey. C, Average amplitude, frequency, half-width, rise time, and decay in controls and drinkers. Data are expressed as the mean ± SEM. D, E, Frequency histograms for binned and cumulative sPSC amplitude distributions in controls and drinking monkeys.

Figure 3.

Synaptomic profiling of PSD-enriched fractions from control and drinking cynomolgus macaques identifies novel alcohol-sensitive proteins (controls, n = 3; drinkers, n = 5). A, B, Average daily alcohol intake (A) and BACs (B) during the 6 months of self-administration. The monkeys used for MS/MS analysis are shown in the hatched bars. Two of the monkeys (10201 and 10207) reached criteria for heavy drinking (≥3 g/kg on 48% and 38% of the open-access drinking days), and the remaining monkeys fit the criteria for low drinkers achieving ≤3 g/kg intake on <1% of the open access days. Data are the mean ± SD. C, Correlation analysis of average BAC and alcohol intake (g/kg) at 7 h into the daily session for all eight drinking monkeys. D, E, Frequency distribution of the normalized log-transformed median expression ratios of the 976 proteins. The histogram was fit by a normal distribution. Frequency distribution of the ratios is shown in blue bars, and a red line shows the results of the normal fit. F, IPA Core analysis identified highly ranked biological functions of the Triton X-100 insoluble proteins in the area 13L that were differentially expressed by alcohol self-administration. The red line denotes threshold, and the number above each bar shows the number of proteins contained within that annotation. G, Presynaptic and postsynaptic proteins related to glutamatergic signaling that were significantly different between control and drinking monkeys. H, GeneWeaver search results of alcohol-related gene sets (GS) containing GPM6A and PRRT2.

Figure 4.

Western blot characterization and analysis of synaptic GluA1 expression in the OFC of drinking monkeys. A, Characterization of GluA1 Western blot in macaque cortical tissue (protein loading range, 1.25–40 μg). B, Positive correlation between the amount of protein loaded and GluA1 optical density values. C, Representative images and quantitation of an intentional 17% decrease in total protein load. D, Representative GluA1 blot and quantitation of normalized GluA1 expression in controls and drinkers. Data are the mean ± SEM.

Figure 5.

Exploratory analysis of the orbitofrontal cortex synaptome identified proteins that best discriminate between low- and high-drinking cynomolgus monkeys and genes with cross-species links to alcohol- and cocaine-related phenotypes. A, Selectivity ratio plot for all proteins identified in the synaptomics screen. Proteins with selectivity ratios above the 95% confidence limit are identified by gene name. B, GeneWeaver search results of alcohol-related gene sets (GS) containing C2CD2L, DIRAS2, and PYCR2. C, D, Correlations between C2cd2l transcript levels in the PFC and cocaine self-administration and cocaine-conditioned place preference in BXD RI strains.

Figure 6.

Alcohol consumption in BXD RI strains of mice that inherited the B or D allele at the C2cd2l interval. A, Alcohol (20%, v/v) intake across 15 BXD RI strains in a single-bottle, 4 h DID model. The number within each bar for A shows the number of BXD RI mice within each strain. B–D, BXD mice that inherited the D allele at C2cd2l consumed significantly less alcohol in the DID model (B) and two-bottle choice (C) 24 and 2 h access (D) models. The number within each bar shows the number of strains with the B or D alleles for the C2cd2l interval for B–D.