Male-selective effects of oxytocin agonism on alcohol intake: behavioral assessment in socially housed prairie voles and involvement of RAGE

The study addresses the need for effective treatments for alcohol use disorder (AUD), noting that current pharmacotherapies have limited clinical success despite preclinical efficacy. Oxytocin (OXT), known for its roles in social behavior, is a candidate therapy whose effects may leverage social support to improve abstinence. The research aims to improve translational validity by: (1) using intranasal (IN) administration of OXT, the route commonly used in human trials; (2) modeling social context using socially housed prairie voles in a continuous-access two-bottle choice (CA-2BC) paradigm with mixed-cage designs; and (3) probing mechanisms of OXT brain penetration, particularly the role of the Receptor for Advanced Glycation End-products (RAGE). The central questions are whether OXT/OXTR agonism reduces voluntary alcohol intake in socially housed prairie voles in a sex-dependent manner, whether exogenous OXT reaches the brain, and whether RAGE facilitates its brain transport, with implications for individualized AUD treatment.

Prior work has implicated OXT in modulating alcohol-related behaviors preclinically and clinically, including reduced alcohol self-administration and cue-reactivity. Evidence suggests central OXT action is important for reducing alcohol intake, with centrally active OXTR antagonists blocking these effects in rodents. Studies in rodents and non-human primates indicate small but detectable brain penetration of peripherally administered OXT, yet mechanisms of transport remain unclear. RAGE has been identified as a transporter for OXT into the brain in mice. Small-molecule OXTR agonists have been developed; among them, LIT-001 shows high OXTR affinity with much lower affinity for AVPR1a and has efficacy in other models (e.g., social deficits, inflammatory pain). Social factors significantly influence alcohol consumption, underscoring the need for models that incorporate social housing and interactions.

Animals: Adult male and female prairie voles from an in-house colony (OHSU) were used. All procedures were IACUC-approved and followed NIH guidelines. Design for consumption studies: Animals were socially housed in Herdsman-2 (HM2) cages enabling unrestricted social interaction with individualized fluid intake measurement via balances and RFID implants. A continuous-access two-bottle choice (CA-2BC) paradigm provided 6 days of ad libitum access to 5% (v/v) ethanol and water. A mixed-cage design placed treated and control animals together. Baseline intake was recorded; then animals received treatment and intake was measured hourly up to 6 h post-treatment. Treatments and doses: Intranasal OXT acetate at 5 mg/kg or 10 mg/kg in 25 µl saline; vehicle saline. Small-molecule OXTR agonist LIT-001 (synthesized in Dr. Hibert’s lab) at 10 mg/kg intraperitoneal (IP) in 5% DMSO/saline; vehicle control. For transport studies, RAGE antagonist FPS ZM1 at 1 mg/kg IP or saline 30 min prior to exogenous labeled OXT. Brain penetration and RAGE involvement: Confirmed RAGE expression in prairie vole brain by RT-PCR and immunohistochemistry, with immunoreactivity in hypothalamus, choroid plexus, hippocampus. Assessed brain levels of deuterium-labeled oxytocin (d5 OXT; oxytocin-(leucine-5,5,5-d3 glycine-2,2-d2) trifluoroacetate) administered either IN (3 µg/25 µl) or IP (12 µg/0.1 ml). Ten minutes after d5 OXT, animals were anesthetized and perfused, brains snap-frozen, and d5 OXT quantified by LC-MS/MS (Shimadzu Nexera-LCMS-8060). Outcome measures: Alcohol and water intake (g/kg), drink size per event (ml), number of consumptive and non-nutritive visits, computed hourly for 24 h with focus on first 6 h. Brain d5 OXT levels reported as pg/mg tissue. Statistics: Non-normal distributions prompted non-parametric analyses. Group comparisons used Kruskal–Wallis tests with Mann–Whitney U post hoc tests. Visualization in Prism; analyses in IBM SPSS 27. Sample sizes for IN OXT consumption study: female vehicle n=29, female 5 mg/kg OXT n=13, female 10 mg/kg OXT n=14, male vehicle n=27, male 5 mg/kg OXT n=12, male 10 mg/kg OXT n=16. Brain penetration groups ranged n=6–7 per sex/treatment. LIT-001 consumption study: female vehicle n=15, female LIT-001 n=16, male vehicle n=12, male LIT-001 n=11.

- Intranasal OXT reduced alcohol intake selectively in males under social housing. Group differences in cumulative alcohol intake were significant at 2 h (p=0.02), 3 h (p=0.02), and 5 h (p=0.03) post-treatment, with trends at 1 h (p=0.08) and 4 h (p=0.054). At 2 h, alcohol intake decreased while 10 mg/kg OXT increased water intake in males (water p=0.04); by 3 h, effects were alcohol-specific (water p=0.11). - Drink size mirrored intake effects: significant group differences at 2 h for alcohol (p=0.02) and water (p=0.03); alcohol-specific at 3 h (p=0.04). No significant differences in number of consumptive or non-nutritive visits at any time point (ps > 0.94). - Post hoc at 3 h: male 5 mg/kg and 10 mg/kg IN OXT had lower alcohol intake (p=0.01; p=0.02) and smaller drink sizes (p=0.01; p=0.02) vs male controls. Male 5 mg/kg IN OXT consumed less alcohol than female OXT groups (vs female 5 mg/kg p=0.04; vs female 10 mg/kg p=0.01) and had smaller drink sizes than female 10 mg/kg (p=0.01). Male 10 mg/kg OXT also had lower intake than female 10 mg/kg (p=0.01). - RAGE expression was confirmed widely in prairie vole brain, including hypothalamus and regions near ventricles. LC-MS/MS detected d5 OXT in brains after both IN and IP administration. Significant group differences in brain d5 OXT (p=0.01). RAGE antagonist (FPS ZM1) pretreatment significantly reduced brain d5 OXT after IN administration in males vs vehicle controls (p=0.02). With antagonist pretreatment, brain d5 OXT was lower with IN vs IP in both sexes (females p=0.04; males p=0.001). No significant differences between control and antagonist groups when d5 OXT was given IP (ps > 0.21); no route differences in controls (ps > 0.45). This supports a RAGE-dependent component for IN, but not IP, OXT brain entry. - LIT-001 (10 mg/kg IP) decreased cumulative alcohol intake at 4 h post-treatment (overall Kruskal–Wallis p < 0.05) without affecting water intake (p=0.46). No significant effects on drink size or visit counts (ps > 0.14). At 4 h, LIT-001-treated males did not differ significantly from male controls but consumed less than female controls and LIT-001-treated females, indicating a male-selective reduction. - Overall, OXTR agonism decreased alcohol intake in male prairie voles under social housing, with effects primarily via reduced drink size. IN OXT’s brain penetration is at least partly RAGE-mediated.

Findings demonstrate that targeting the OXT/OXTR system reduces alcohol consumption in a sex-dependent manner under socially relevant conditions. IN OXT and IP LIT-001 both selectively reduced alcohol intake in males, suggesting that OXTR agonism can modulate drinking behavior, likely through central mechanisms. The lack of changes in visit counts alongside reduced drink size indicates modulation of consummatory magnitude rather than approach frequency. The confirmation of RAGE expression in prairie vole brain and the reduction of brain d5 OXT after RAGE antagonism with IN, but not IP, administration link RAGE to OXT brain transport in a route-dependent manner. This mechanistic insight enhances translational relevance for IN OXT use in humans and suggests that small-molecule OXTR agonists (e.g., LIT-001) may provide practical alternatives that are less dependent on peptide transport mechanisms. The sex-specific effects may relate to known differences in OXT/OXTR distribution and hormonal modulation; although brain penetration did not differ by sex, circuit- or receptor-level differences could drive male selectivity. These results support individualized AUD treatment strategies focusing on OXTR agonism, with consideration of sex differences and social context.

This study shows that intranasal OXT and the small-molecule OXTR agonist LIT-001 reduce voluntary alcohol intake specifically in male prairie voles under social housing, with effects driven by reduced drink size. It establishes RAGE expression in prairie vole brain and implicates RAGE in OXT brain penetration after intranasal, but not intraperitoneal, administration. Together, these findings advance the translational case for OXTR-targeted therapies for AUD and underscore the importance of administration route and social context. Future work should: (1) assess longer-term and broader AUD-relevant outcomes (e.g., craving, relapse, withdrawal); (2) directly measure social interactions and salience to test the social salience hypothesis; (3) probe neural circuitry and receptor distributions underlying sex-specific effects; (4) optimize dosing and pharmacokinetics of small-molecule OXTR agonists and evaluate alternative delivery routes; and (5) further delineate the role of RAGE and other transport pathways in OXT brain access.

- Short observation window focused on acute (up to 6 h) post-treatment effects; long-term impacts on alcohol consumption, relapse, or withdrawal were not assessed. - Social behavior and anxiety/withdrawal-like measures were not directly measured, limiting conclusions about mechanisms such as social salience or anxiolysis. - Use of a single species (prairie voles) and specific housing paradigm may limit generalizability across models and to humans. - OXT was tested at two IN doses; broader dose–response and pharmacokinetic profiling were not performed. LIT-001 was tested at one IP dose. - While RAGE involvement was supported for IN OXT brain penetration, detailed regional brain distribution and temporal kinetics beyond 10 min were not assessed. - Potential off-target effects of peptide OXT (e.g., AVPR1a) cannot be entirely excluded in this design, though LIT-001 has higher OXTR selectivity.