Distinct mu, delta, and kappa opioid receptor mechanisms underlie low sociability and depressive-like behaviors during heroin abstinence

Abstract

Addiction is a chronic disorder involving recurring intoxication, withdrawal, and craving episodes. Escaping this vicious cycle requires maintenance of abstinence for extended periods of time and is a true challenge for addicted individuals. The emergence of depressive symptoms, including social withdrawal, is considered a main cause for relapse, but underlying mechanisms are poorly understood. Here we establish a mouse model of protracted abstinence to heroin, a major abused opiate, where both emotional and working memory deficits unfold. We show that delta and kappa opioid receptor (DOR and KOR, respectively) knockout mice develop either stronger or reduced emotional disruption during heroin abstinence, establishing DOR and KOR activities as protective and vulnerability factors, respectively, that regulate the severity of abstinence. Further, we found that chronic treatment with the antidepressant drug fluoxetine prevents emergence of low sociability, with no impact on the working memory deficit, implicating serotonergic mechanisms predominantly in emotional aspects of abstinence symptoms. Finally, targeting the main serotonergic brain structure, we show that gene knockout of mu opioid receptors (MORs) in the dorsal raphe nucleus (DRN) before heroin exposure abolishes the development of social withdrawal. This is the first result demonstrating that intermittent chronic MOR activation at the level of DRN represents an essential mechanism contributing to low sociability during protracted heroin abstinence. Altogether, our findings reveal crucial and distinct roles for all three opioid receptors in the development of emotional alterations that follow a history of heroin exposure and open the way towards understanding opioid system-mediated serotonin homeostasis in heroin abuse.

Figure 1

Social, depressive-like, and spatial working memory deficits develop during the 7 weeks of heroin abstinence. (a) Experimental procedure. Independent animal cohorts were used to assess each time point during heroin abstinence. Following repeated opiate injections, mice were maintained drug free for 1, 4, or 7 weeks. Then, emotional-like responses were evaluated using classical paradigms performed every other day, as previously described (Goeldner et al, 2011; Lutz et al, 2013b). Mice body weights were measured as indices of global opiate effects during chronic injections and abstinence (Supplementary Figure S4). In the social interaction test (SI; n=12 pairs of mice/treatment group/abstinence duration), during heroin abstinence, social avoidance progressively emerged, with decreased duration of social behaviors (b) and increased grooming (c). Stimulus mice for social interactions tests were used only once. Increased despair-like behavior was also detected following a 7-week abstinence period in the forced swim test (FS; n=24 mice/treatment group/abstinence duration), with both increased immobility (d) and decreased latency to first immobilization (e). In the sucrose-preference test (n=24 mice/treatment group/abstinence duration), there was no effect of heroin abstinence on hedonic tone (f). Finally, abstinent mice showed decreased working memory performance in the Y-maze (YM; n=24 mice/treatment group/abstinence duration) task (g). Values are mean±SEM.⁺p<0.05, ANOVA main effect of heroin; *p<0.001, post-hoc effects of heroin for each abstinence duration (see text for other post-hoc comparisons and also Supplementary Figure S2).

Figure 2

During heroin abstinence, delta opioid receptor (DOR) activity prevents the emergence of anhedonia-like symptom, and kappa opioid receptor (KOR) activity mediates the development of social withdrawal. Heroin physical dependence (a and c) and emotional consequences of 4-week abstinence (b and d) were studied in DOR knockout (KO) mice and in their wild-type (WT) littermates (a and b), as well as in KOR knockout (KO) mice and in their wild-type (WT) littermates (c and d). Naloxone-precipitated withdrawal, as a measure of physical dependence achieved at the end of opiate exposure, and the analysis of emotional-like behaviors during protracted abstinence, after spontaneous heroin withdrawal had occurred (no naloxone), were performed in separate animal cohorts. In DOR KO mice, (a) acute heroin withdrawal (n=8–10 mice/treatment group/genotype) and (b, left panel) social avoidance following a 4-week abstinence period (n=14–15 mice/treatment group/genotype) are comparable to levels in WT controls. In contrast, (b, right panel) decreased sucrose preference specifically developed in DOR KO but not in WT mice during heroin abstinence (n=14–15 mice/treatment group/genotype). In KOR KO mice, (c) physical dependence to heroin is slightly decreased (n=8–10 mice/treatment group/genotype), and (d, left panel) social avoidance is fully prevented (n=16–18 mice/treatment group/genotype). No modification of sucrose preference (d, right panel) is observed for KOR KO mice under our experimental conditions (n=16–18 mice/treatment group/genotype). Values are mean±SEM.⁺p<0.001, ANOVA main effects of heroin; *p<0.01, post-hoc comparisons between the saline and heroin groups; ^#p<0.05, post-hoc comparisons between the saline and heroin groups within each genotype.

Figure 3

Chronic antidepressant treatment prevents the development of social avoidance in heroin-abstinent mice. Following chronic saline or heroin injections, mice were fed fluoxetine-supplemented pellets (≈10 mg/kg/24 h) for 4 weeks, or nature pellets as controls, as previously described (Goeldner et al, 2011). In the social interaction (SI) test (n=7–8 pairs of mice/treatment group/food type), chronic treatment with fluoxetine prevented protracted heroin effects on (a) social and (b) individual behaviors. In contrast (c), fluoxetine did not prevent the effect of abstinence on working memory (n=15–16 mice/treatment group/food type). Values are mean±SEM.+p<0.001, ANOVA main effect of heroin; *p<0.01, post-hoc effects of heroin in control food mice; ^#p<0.05, post-hoc effects of fluoxetine in the heroin-treated groups.

Figure 4

Virally Cre-mediated recombination efficiently downregulates mu opioid receptors (MOR) in the dorsal raphe nucleus (DRN). (a) Experimental procedure. Independent animal cohorts (n=170 in total) were used for [³⁵S]-GTPγS binding, heroin physical dependence, and heroin abstinence (see Figure 5). (b) Pattern of eGFP expression following stereotactic infusion of AAV–eGFP shows viral diffusion restricted to the DRN. (c) Four weeks following surgery, MOR activity was significantly reduced in the DRN, as shown by lower DAMGO-induced [³⁵S]-GTPγS binding (n=14–16/group). In contrast, MOR signaling was preserved in the periaqueductal gray (d) or median raphe nucleus (e). Values are mean±SEM. ^#p<0.01, post-hoc comparison between the MOR^fl/fl and MOR cKO groups at each agonist concentration (M, molar concentration).

Figure 5

Conditional knockout of the mu opioid receptor (MOR cKO) in the dorsal raphe nucleus (DRN) prevents emergence of social avoidance during heroin abstinence. Naloxone-precipitated withdrawal (a) served as a measure of physical dependence achieved at the end of opiate exposure. Analyses of emotional-like behaviors during abstinence (b–d) were performed in separate animal cohorts, after they experienced spontaneous withdrawal from the chronic heroin treatment. MOR cKO in the DRN does not modify the severity of heroin physical dependence (n=6–7 mice/treatment group/virus type). In contrast, DRN MOR cKO in independent cohorts (n=9–13/treatment group/virus type) prevents the emergence of decreased social interactions (b) and decreases grooming duration (c), following a 4-week abstinence period. Finally, deleterious consequence of heroin abstinence on spatial working memory still manifested despite MOR cKO (d). Values are mean±SEM.⁺p<0.001, ANOVA main effects of heroin; ^‡p<0.001, ANOVA main effect of genotype; *p<0.001, post-hoc comparison between saline- and heroin-treated mice.