Insular cortex subregions have distinct roles in cued heroin seeking after extinction learning and prolonged withdrawal in rats

Abstract

Evidence indicates that the anterior (aIC), but not posterior (pIC), insular cortex promotes cued reinstatement of cocaine seeking after extinction in rats. It is unknown whether these subregions also regulate heroin seeking and whether such involvement depends on prior extinction learning. To address these questions, we used baclofen and muscimol (BM) to inactivate the aIC or pIC bilaterally during a seeking test after extinction or prolonged withdrawal from heroin. Male Sprague-Dawley rats in the extinction groups underwent 10+ days of heroin self-administration, followed by 6+ days of extinction sessions, and subsequent cued or heroin-primed reinstatement. Results indicate that aIC inactivation increased cued reinstatement of heroin seeking after extinction, whereas pIC inactivation prevented cued reinstatement. To determine whether these effects were extinction-dependent, we conducted a subsequent study using both sexes with prolonged withdrawal. Male and female rats in the withdrawal groups underwent 10+ days of heroin self-administration, followed by cued seeking tests after 1 and 14 days of homecage withdrawal to measure incubation of heroin craving. In this case, the findings indicate that aIC inactivation had no effect on incubation of heroin craving after withdrawal in either sex, whereas pIC inactivation decreased heroin craving only in males. These findings suggest that the aIC and pIC have opposing roles in suppressing vs promoting cued heroin seeking after extinction and that these roles are distinct from those in cocaine seeking. Moreover, the incubation of craving results suggest that new contingency learning is necessary to recruit the aIC in cued heroin seeking.

Fig. 1. Histology and procedures.

A, B Schematic (left) and representative images (right) of microinjector termination in the aIC and pIC, respectively. C Timeline of extinction-reinstatement procedures. D Timeline of incubation of craving procedures.

Fig. 2. Opposing effects of aIC vs pIC inactivation on cued reinstatement of heroin seeking in males.

A Lever presses and infusions during the final 10 d of heroin self-administration for rats that would receive aIC injections before cued reinstatement. B Lever presses during the first 6 d of extinction for rats that would receive aIC injections before cued reinstatement. C Active lever presses during cued reinstatements and extinction baselines (left) and within-subjects comparison of reinstatement conditions (right). Intra-aIC baclofen/muscimol infusions increased lever pressing during cued reinstatement compared to vehicle controls. D Lever presses and infusions during the final 10 d of heroin self-administration for rats that would receive aIC injections before heroin-primed reinstatement. E Lever presses during the first 6 d of extinction for rats that would receive aIC injections before heroin-primed reinstatement. F Active lever presses during heroin-primed reinstatements and extinction baselines (left) and within-subjects comparison of reinstatement conditions (right). Intra-aIC baclofen/muscimol infusions had no effect on heroin-primed reinstatement compared to vehicle controls. G Lever presses and infusions during the final 10 d of heroin self-administration for rats that would receive pIC injections before cued reinstatement. H Lever presses during the first 6 d of extinction for rats that would receive pIC injections before cued reinstatement. I Active lever presses during cued reinstatements and extinction baselines (left) and within-subjects comparison of individual animals (right). Intra-pIC baclofen/muscimol infusions decreased lever pressing during cued reinstatement compared to vehicle controls. J Lever presses and infusions during the final 10 d of heroin self-administration for rats that would receive pIC injections before heroin-primed reinstatement. K Lever presses during the first 6 days of extinction for rats that would receive pIC injections before heroin-primed reinstatement. L Active lever presses during heroin-primed reinstatements and extinction baselines (left) and within-subjects comparison of individual animals (right). Intra-pIC baclofen/muscimol infusions had no effect on heroin-primed reinstatement compared to vehicle controls. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3. Attenuated incubation of heroin craving with pIC, but not aIC, inactivation in males.

A Lever presses and infusions during the final 10 d of heroin self-administration for both sexes (left), males and females (right) in the aIC groups. B Intra-aIC baclofen/muscimol infusions had no effect on incubation of heroin craving, as measured by active lever presses during the first 30 min of a day 14 incubation test, compared to vehicle controls in males or females. C Lever presses and infusions during the final 10 days of heroin self-administration for males (left) and females (right) in the pIC groups. D Intra-pIC baclofen/muscimol infusions decreased incubation of heroin craving, as measured by active lever presses during the first 30 min of a day 14 incubation test, compared to vehicle controls in males (left) but not females (right). ^#p < 0.11, *p < 0.05, ****p < 0.0001.

Fig. 4. No sex differences in 6-h heroin self-administration.

To analyze for potential sex differences in 6 h heroin self-administration, rats were collapsed across the aIC and pIC experiments and the baclofen/muscimol and vehicle groups, and data were analyzed for the final 10 days of self-administration. A Daily active lever presses. B Daily heroin infusions. C Daily heroin intake (mg/kg). There were no significant differences between males and females across the three measures.

Fig. 5. No effect of aIC or pIC inactivation on locomotor activity in either sex.

A Intra-aIC baclofen/muscimol infusions had no effect on distance traveled in an open field compared to vehicle controls in males or females. B Intra-pIC baclofen/muscimol infusions had no effect on distance traveled compared to vehicle controls in males or females.