Agmatine reduces alcohol drinking and produces antinociceptive effects in rodent models of alcohol use disorder

Abstract

Alcohol use disorder (AUD) is a chronic, relapsing disorder characterized by an escalation of drinking and the emergence of negative affective states over time. Within this framework, alcohol may be used in excessive amounts to alleviate withdrawal-related symptoms, such as hyperalgesia. Future effective therapeutics for AUD may need to exhibit the ability to reduce drinking as well as to alleviate co-morbid conditions such as pain, and to take mechanistic sex differences into consideration. Agmatine is an endogenous neuromodulator that has been previously implicated in the regulation of reward and pain processing. In the current set of studies, we examined the ability of agmatine to reduce escalated ethanol drinking in complementary models of AUD where adult male and female mice and rats were made dependent via chronic, intermittent ethanol vapor exposure (CIE). We also examined the ability of agmatine to modify thermal and mechanical sensitivity in alcohol-dependent male and female rats. Agmatine reduced alcohol drinking in a dose-dependent fashion, with somewhat greater selectivity in alcohol-dependent female mice (versus non-dependent female mice), but equivalent efficacy across male mice and both groups of male and female rats. In mice and female rats, this efficacy did not extend to sucrose drinking, indicating some selectivity for ethanol reinforcement. Female rats made dependent on alcohol demonstrated significant hyperalgesia symptoms, and agmatine produced dose-dependent antinociceptive effects across both sexes. While additional mechanistic studies into agmatine are necessary, these findings support the broad-based efficacy of agmatine to treat co-morbid excessive drinking and pain symptoms in the context of AUD.

Keywords: alcohol dependence; analgesia; ethanol; pain; self-administration.

Figure 1:

A) Voluntary ethanol intake (g/kg) for CIE exposed and CTL (air-exposed) male mice during baseline and the first two ethanol drinking test cycles while all mice received vehicle injections before drinking (N=48/group). * Indicates lower intake compared to their own baseline level (p<0.001). # Indicates higher ethanol intake in CIE mice compared to CTL mice and their own baseline level of intake (p<0.001). B) Ethanol intake (g/kg) for male mice that received pretreatment with different doses of agmatine during Test Cycle 3 (N=11–12/group). The ANOVA indicated a significant main effect of CIE vs. CTL condition due to significantly higher ethanol intake in CIE mice. ^ Indicates a main effect of agmatine dose with lower intake in mice that received agmatine compared to vehicle independently of CIE or CTL condition (p<0.05). C) Ethanol intake (g/kg) for male mice that received pretreatment with different doses of agmatine during Test Cycle 4 (second cycle of treatment) (N=11–12/group). The ANOVA indicates a significant main effect of CIE vs. CTL condition due to significantly higher ethanol intake in CIE mice. ^ Indicates lower intake in mice that received agmatine compared to vehicle independently of CIE or CTL condition (p<0.01). D) Ethanol intake (g/kg) for male mice during Test Cycle 5 (washout test) that received pretreatment with different doses of agmatine during Test cycles 3 and 4 (N=11–12/group). The ANOVA indicated a significant main effect of CIE vs. CTL condition due to significantly higher ethanol intake in CIE mice (***p<0.001). Values are mean ±SEM

Figure 2:

A) Voluntary ethanol intake (g/kg) for CIE-exposed and CTL (air-exposed) female mice during baseline and the first three ethanol drinking test cycles (all mice received vehicle injections) (N=46 CTL, 48 CIE). # Indicates higher ethanol intake in CIE mice compared to CTL mice and their own baseline level of intake (p<0.001). B) Ethanol intake (g/kg) for female mice that received different doses of agmatine during Test Cycle 4 (N=11–12/group). # Indicates higher intake in CIE vs. CTL mice that received vehicle injections (p<0.01). * Indicates lower intake in CIE mice that receive agmatine pre-treatment (any dose) and CTL mice that received the highest dose compared to their respective vehicle condition (p<0.01). C) Ethanol intake (g/kg) for female mice that received different doses of agmatine during Test Cycle 5 (second cycle of agmatine treatment) (N=11–12/group). # Indicates higher intake in CIE vs. CTL mice that received vehicle injections (p<0.001). * Indicates lower intake in CIE mice that receive agmatine pre-treatment compared to CIE mice that received vehicle (p<0.001). D) Ethanol intake (g/kg) for female mice during Test Cycle 6 (washout test) that received pretreatment with different doses of agmatine during Test cycles 4 and 5 (N=10–12/group). The ANOVA indicated a significant main effect of CIE vs. CTL condition due to significantly higher ethanol intake in CIE mice (**p<0.01). Values are mean ±SEM

Figure 3:

Sucrose intake (g/kg) in male (left) and female (right) mice during baseline (all mice received vehicle injections) and during the test cycle when subjects received different doses of agmatine (N=9–10/group). The ANOVA failed to indicate any effect of agmatine dose or interaction of dose and phase (baseline vs. test). Values are mean ± SEM

Figure 4:

Operant alcohol (left) and water (right) self-administration in male Wistar rats. Agmatine reduces alcohol self-administration in a dose-dependent manner across both alcohol-dependent and non-dependent male Wistar rats. Responding for water was not significantly altered in either group. N=5–6/group. ###p<0.001 main effect of agmatine dose in male rats. Values are mean ± SEM

Figure 5:

Operant alcohol (left) and water (right) self-administration in female Wistar rats. Agmatine reduces alcohol self-administration in a dose-dependent manner across both alcohol-dependent and non-dependent female Wistar rats. N=8–12/group. ###p<0.001 main effect of agmatine dose in female rats. Values are mean ± SEM

Figure 6:

Operant sucrose self-administration in male (left) and female (right) Wistar rats. Agmatine reduces responding for sucrose in a dose-dependent fashion in male Wistar rats but does not alter operant sucrose self-administration in female Wistar rats. N=15 (males) and N=6 (females). ##p<0.01 main effect of agmatine dose in male rats. **p<0.01 and ***<0.001 difference in agmatine dose from vehicle in male rats. Values are mean ± SEM

Figure 7:

A) Thermal sensitivity in male (left, n=6–7/group) and female (right, n=8–11/group) Wistar rats. Agmatine produces thermal antinociception in both male and female rats. ^†p<0.05 lower paw withdrawal thresholds in alcohol-dependent group. **p<0.01 and ***<0.001 interaction of dose and dependence state in male and female rats (respectively). B) Mechanical sensitivity in male (left) and female (right) Wistar rats. Agmatine produces mechanical antinociception in both male and female rats. N= 6–11/group. ^†p<0.05 lower paw withdrawal thresholds in alcohol-dependent group. *p<0.05 interaction of dose and dependence state in male and female rats. Values are mean ± SEM

Figure 7:

A) Thermal sensitivity in male (left, n=6–7/group) and female (right, n=8–11/group) Wistar rats. Agmatine produces thermal antinociception in both male and female rats. ^†p<0.05 lower paw withdrawal thresholds in alcohol-dependent group. **p<0.01 and ***<0.001 interaction of dose and dependence state in male and female rats (respectively). B) Mechanical sensitivity in male (left) and female (right) Wistar rats. Agmatine produces mechanical antinociception in both male and female rats. N= 6–11/group. ^†p<0.05 lower paw withdrawal thresholds in alcohol-dependent group. *p<0.05 interaction of dose and dependence state in male and female rats. Values are mean ± SEM