N-oleoyl glycine and N-oleoyl alanine attenuate alcohol self-administration and preference in mice

Abstract

The endocannabinoid system (ECS) plays a key modulatory role during synaptic plasticity and homeostatic processes in the brain and has an important role in the neurobiological processes underlying drug addiction. We have previously shown that an elevated ECS response to psychostimulant (cocaine) is involved in regulating the development and expression of cocaine-conditioned reward and sensitization. We therefore hypothesized that drug-induced elevation in endocannabinoids (eCBs) and/or eCB-like molecules (eCB-Ls) may represent a protective mechanism against drug insult, and boosting their levels exogenously may strengthen their neuroprotective effects. Here, we determine the involvement of ECS in alcohol addiction. We first measured the eCBs and eCB-Ls levels in different brain reward system regions following chronic alcohol self-administration using LC-MS. We have found that following chronic intermittent alcohol consumption, N-oleoyl glycine (OlGly) levels were significantly elevated in the prefrontal cortex (PFC), and N-oleoyl alanine (OlAla) was significantly elevated in the PFC, nucleus accumbens (NAc) and ventral tegmental area (VTA) in a region-specific manner. We next tested whether exogenous administration of OlGly or OlAla would attenuate alcohol consumption and preference. We found that systemic administration of OlGly or OlAla (60 mg/kg, intraperitoneal) during intermittent alcohol consumption significantly reduced alcohol intake and preference without affecting the hedonic state. These findings suggest that the ECS negatively regulates alcohol consumption and boosting selective eCBs exogenously has beneficial effects against alcohol consumption and potentially in preventing relapse.

Fig. 1. Intermittent access to EtOH induces an escalation of EtOH intake and preference in females and males.

A Alcohol intake in female (red) and male (blue) mice exposed to intermittent access to 20% EtOH. Female mice exhibited significantly increased EtOH consumption relative to males mice [N = 13–15 mice/group; Mixed-effects analysis, followed by Sidak`s multiple comparisons test: F (1,27) = 5.509, sex effect P = 0.0265; F (6.055, 160.1) = 20.6, session P < 0.0001; F (16, 423) = 3.763, sex × session interaction P < 0.0001]. Sidak’s post-hoc *P < 0.05, **P < 0.01. B Cumulative intake of EtOH (per day) for female or male mice. Inset, absolute EtOH intake for the two groups (N = 13–15, P = 0.0108, Student’s t-test). *P < 0.05. C There were no differences in EtOH preference between female and male mice. [N = 13-15; Mixed-effects analysis: F (1,27) = 0.7908, sex effect P = 0.3817; F (6.117, 162.5) = 11.98, session P < 0.0001; F (16, 425) = 2.673, sex × session interaction P = 0.0005].

Fig. 2. Lipidomic analysis of eCBs and eCB-Ls following chronic EtOH self-administration in female and male mice.

A–F One day following the last EtOH self-administration session, animals were sacrificed and the PFC, NAc, VTA, HIP, AMY, and CER were dissected and analyzed for the expression levels of eCBs using LC–MS. The levels of OlGly, OlAla, 2-AG, AEA, PEA, and OEA are expressed as ng/mg of tissue weight. Two-way ANOVA followed by Sidak’s multiple comparisons revealed that chronic EtOH self-administration significantly elevated OlGly level in the PFC of female mice [red, N = 4–5 mice/group; F (1,14) = 14.19, P = 0.0021; Ctrl vs. EtOH, P = 0.0230] and OlAla levels were elevated in PFC and NAc of male (blue) and PFC of female and male mice. *P < 0.05, **P < 0.01, ***P < 0.001. Detailed statistical analysis for all the panels are presented in Supplementary Table S3. NS not significant, PFC prefrontal cortex, NAc nucleus accumbens, VTA ventral tegmental area, HIP hippocampus, AMY amygdala, CER cerebellum, eCBs endocannabinoids, OlGly N-oleoyl glycine, OlAla N-oleoyl alanine, 2-AG 2-arachidonoyl glycerol, AEA N-arachidonoyl ethanolamine, OEA N-oleoyl ethanolamide, PEA N-palmitoyl ethanolamide.

Fig. 3. OlGly attenuates EtOH self-administration and preference but not water consumption in male mice.

A Systemic administration of OlGly (60 mg/kg) before each EtOH self-administration session significantly attenuated EtOH intake [N = 9–10; Mixed-effects analysis: F (1,17) = 5.349, treatment effect P = 0.0335; F (5.896, 97.65) = 3.212, session P = 0.0067; F (16, 265) = 1.791, sex × session interaction P = 0.0323]. Student’s t-test: *P < 0.05, **P < 0.01. B Cumulative EtOH intake (per day) of animals injected with either OlGly (orange, 60 mg/kg) or vehicle (blue). Inset, absolute EtOH intake for the two groups (N = 9–10 mice/group, Student’s t-test, P = 0.0238,). *P < 0.05. C OlGly attenuates EtOH preference. Inset, EtOH preference at the last session for the two groups was compared (N = 9–10, Student’s t-test, P = 0.0483), *P < 0.05. D Mixed-effects analysis followed by Sidak’s multiple comparisons test revealed no difference in the water consumption between the two groups [F (1,9) = 0.1972, P = 0.6675]. E Mixed-effects analysis, followed by Sidak’s multiple comparisons test revealed no difference in the total fluid intake between the two groups. [F (1,9) = 1.322, P = 0.2799]. F Mixed-effects analysis revealed no significant differences in sucrose (1% W/V) preference between OlGly and vehicle-treated mice [F (1,18) = 0.6480, P = 0.4313].

Fig. 4. OlAla attenuates EtOH self-administration and preference but not water consumption in male mice.

A Systemic administration of OlAla (gray, 60 mg/kg) or vehicle (blue) before each EtOH self-administration session significantly attenuated EtOH intake [N = 10 mice/group; Mixed-effects analysis: F (1,18) = 4.989, treatment effect P = 0.0384; F (6.118, 108.3) = 4.175, session P = 0.0008; F (10, 177) = 0.5726, sex × session interaction P = 0.8349]. Student’s t-test: *P < 0.05. B Cumulative EtOH intake (per day) of animals injected with either OlAla or vehicle. Inset, absolute EtOH intake for the two groups (N = 10, Student’s t-test, P = 0.014). *P < 0.05. C OlAla attenuates EtOH preference. Inset, EtOH preference was compared at the last session for the two groups (N = 10, Student’s t-test, P = 0.0014). **P < 0.01. D Mixed-effects analysis, followed by Sidak’s multiple comparisons test revealed no difference in the water consumption between the two groups. [F (1, 9) = 0.3549, P = 0.5660]. E Mixed-effects analysis, followed by Sidak’s multiple comparisons test revealed no difference in the fluid intake between the two groups. [F (1, 9) = 0.02550, P = 0.8767].