Oxytocin as an adolescent treatment for methamphetamine addiction after early life stress in male and female rats

Abstract

Early life stress (ELS) is associated with perturbed neural development and augmented vulnerability to mental health disorders, including addiction. How ELS changes the brain to increase addiction risk is poorly understood, and there are no therapies which target this ELS-induced vulnerability. ELS disrupts the oxytocin system, which can modulate addiction susceptibility, suggesting that targeting the oxytocin system may be therapeutic in this ELS-addiction comorbidity. Therefore, we determined whether adolescent oxytocin treatment after ELS could: (1) reduce vulnerability to anxiety, social deficits, and methamphetamine-taking and reinstatement; and (2) restore hypothalamic oxytocin and corticotropin-releasing factor expressing neurons and peripheral oxytocin and corticosterone levels. Long Evans pups underwent maternal separation (MS) for either 15 min or 360 min on postnatal days (PND) 1-21. During adolescence (PNDs 28-42), rats received a daily injection of either oxytocin or saline. In Experiment 1, adult rats were assessed using the elevated plus-maze, social interaction procedure, and methamphetamine self-administration procedure, including extinction, and cue-, methamphetamine- and yohimbine-induced reinstatement. In Experiment 2, plasma for enzyme immunoassays and brain tissue for immunofluorescence were collected from adult rats after acute stress exposure. Adolescent oxytocin treatment ameliorated ELS-induced anxiety and reduced methamphetamine- and yohimbine-induced reinstatement in both sexes, and suppressed methamphetamine intake and facilitated extinction in males only. Additionally, adolescent oxytocin treatment after ELS restored oxytocin-immunoreactive cells and stress-induced oxytocin levels in males, and attenuated stress-induced corticosterone levels in both sexes. Adolescent oxytocin treatment reverses some of the ELS effects on later-life psychopathology and vulnerability to addiction.

Fig. 1. Effect of early life stress and adolescent oxytocin treatment on anxiety-like behaviour and social aggression.

a Experimental timeline depicting behavioural testing in male and female Long Evans rats after ELS and adolescent oxytocin injections. This figure was created using BioRender (academic subscription). ELS = early life stress, METH = methamphetamine, VEH = vehicle, OXY = oxytocin. b Maternal separation (MS360) increased anxiety-like behaviour relative to separation controls (MS15), which was prevented by adolescent oxytocin treatment in males and females. Percentage (± SEM) of time spent in the closed arms of the EPM by condition (n = 12–15/condition/sex). c Maternal separation (MS360) increased aggressive behaviour towards a novel conspecific relative to separation controls (MS15). Mean (± SEM) duration spent engaging in aggressive behaviour by condition (n = 6–12 pairs/condition/sex). @p < 0.05 significant MS effect. *p < 0.05 significant interaction effect.

Fig. 2. Methamphetamine intake across the 22-day IVSA period.

a males and b females had access to a low METH dose (0.03 mg/kg/infusion) for the first 10 days, which was increased to the high dose (0.1 mg/kg/infusion) for the remaining 12 days. The inset graphs show total METH intake across MS and treatment conditions for each METH dose and total METH intake is depicted in c for males and d for females. Lever pressing during the daily extinction sessions for e male and f female rats. Inset graphs depict the number of extinction sessions required until rats reached the extinction criterion. Data are shown as mean ± SEM. *p < 0.05 significant interaction effect. OXY = Oxytocin, VEH = Vehicle. Sample size (N = 112) was reduced by the removal of 22 rats due to loss of catheter patency (n = 4 males), for not discriminating between the active and inactive lever (did not have a ratio of active to inactive lever presses of 2:1 per session for the last three acquisition sessions), or for not acquiring METH IVSA (did not take greater than 10 infusions per session over the last three acquisition sessions; male: n = 9, MS15 OXY = 4, MS360 VEH = 3, MS360 OXY = 2; female: n = 9, MS15 OXY = 5, MS360 VEH = 1, MS360 OXY = 3). This resulted in a sample size of 90 rats (males = 45, females = 45) with 10–13/condition/sex.

Fig. 3. The effect of early life stress and adolescent oxytocin treatment on cue-, METH-, and yohimbine-induced reinstatement of drug-seeking.

In males, early life stress increased cue- and METH (1 mg/kg) -induced reinstatement, which was reduced by oxytocin treatment in maternally separated rats. For females, early life stress increased METH (1 mg/kg)- and stress (yohimbine hydrochloride (yh) 0.625 mg/kg dose)-induced reinstatement, which was reduced by adolescent oxytocin treatment (n = 10–13/condition/sex). Mean (±SEM) active lever presses on cue-induced reinstatement (a) and (b), METH-primed reinstatement (c) and (d), and yohimbine-induced reinstatement (e) and (f) for males and females. VEH = Vehicle, OXY = Oxytocin. *p < 0.05 significant interaction effect, ^$p < 0.05 significant adolescent treatment effect, @p < 0.05 significant MS effect ^p < 0.05 vs respective extinction or vehicle session.

Fig. 4. Effect of early life stress and adolescent oxytocin treatment on hypothalamic oxytocin and CRF neuronal expression.

a experimental timeline depicting blood and brain analysis in malae and female Long Evan rats after ELS and adolescent oxytocin injections. Figure created in Biorender (academic subscription). Oxytocin and CRF neuronal staining in the PVN of the hypothalamus (−1.8 mm from Bregma) from a representative male from each MS and adolescent oxytocin treatment condition. b Images were taken at 20x magnification and have been adjusted for presentation purposes. Mean number (±SEM) in each condition of (c) oxytocin (n = 5–6/condition/sex) and (d) CRF positive neurons (n = 5–6/condition/sex) and (e) circulating oxytocin (n = 6–8/condition/sex) and (f) corticosterone plasma levels (n = 5–6/condition/sex). CRF corticotropin releasing factor, PVN paraventricular nucleus of the hypothalamus, VEH Vehicle, OXY Oxytocin *p < 0.05 significant interaction effect, ^#p < 0.05 significant sex effect, ^@p < 0.05 significant MS effect.