Opposing effects of aIC vs. pIC inactivation on cued reinstatement of heroin seeking after extinction in males

In this experiment, the aIC or pIC was inactivated during either a cued or heroin-primed reinstatement test after extinction to determine the effect on active lever pressing (Fig. 1C). Rats underwent either cued or heroin-primed reinstatement tests (but not both) and received BM or vehicle immediately prior to the tests in a counterbalanced manner. The total number of extinction sessions preceding each reinstatement test did not significantly differ between conditions (Table 1). Figures 2A, B show self-administration and extinction data, respectively, for rats that would receive aIC injections before cued reinstatement. Figure 2C shows active lever presses during cued reinstatement for those rats receiving aIC injections. A two-way repeated measures ANOVA of active lever presses revealed a significant main effect of reinstatement (F1,10 = 21.35, p < 0.001), a significant main effect of manipulation (F1,10 = 6.60, p < 0.05), and a significant interaction (F1,10 = 7.84, p < 0.05). Post hoc tests revealed that rats had increased active lever pressing during cued reinstatement in both the vehicle- and BM-treated conditions compared with extinction baselines (p < 0.01; p < 0.0001, respectively). However, rats had significantly more active lever presses during cued reinstatement in the BM-treated condition compared to the vehicle-treated condition (p < 0.05), indicating that aIC inactivation increased cued reinstatement.

Table 1 Total number of extinction sessions before each reinstatement test condition.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.

Figures 2D, E show self-administration and extinction data, respectively, for rats that would receive aIC injections before heroin-primed reinstatement. Figure 2F shows active lever presses during heroin-primed reinstatement for those rats receiving aIC injections. A two-way repeated measures ANOVA of active lever presses revealed a significant main effect of reinstatement (F1,9 = 16.87, p < 0.01), but no main effect of manipulation (F1,9 = 0.12, p > 0.15) or interaction (F1,9 = 0.46, p > 0.15). Post hoc tests revealed that, although rats had increased active lever pressing during heroin-primed reinstatement in both the vehicle- and BM-treated conditions compared to extinction baselines (both p < 0.05), the vehicle- and BM-treated conditions did not differ in terms of reinstatement of active lever pressing (p > 0.15).

Figures 2G, H show self-administration and extinction data, respectively, for rats that would receive pIC injections before cued reinstatement. Figure 2I shows active lever presses during cued reinstatement for those rats receiving pIC injections. A two-way repeated measures ANOVA of active lever presses revealed a significant main effect of reinstatement (F1,10 = 19.62, p < 0.01), a significant main effect of manipulation (F1,10 = 5.67, p < 0.05), and a significant interaction (F1,10 = 6.51, p < 0.05). Post hoc tests revealed that, although rats had increased active lever pressing during cued reinstatement in the vehicle-treated condition compared to extinction baseline (p < 0.01), they did not in the BM-treated condition (p > 0.15) and this condition had significantly fewer active lever presses compared with the vehicle-treated condition (p < 0.05). Thus, pIC inactivation significantly reduced cued reinstatement of active lever pressing.

Figures 2J, K show self-administration and extinction data, respectively, for rats that would receive pIC injections before heroin-primed reinstatement. Figure 2L shows active lever presses during heroin-primed reinstatement for those rats receiving pIC injections. A two-way repeated measures ANOVA of active lever presses revealed a significant main effect of reinstatement (F1,9 = 15.06, p < 0.01), but no main effect of manipulation (F1,9 = 1.18, p > 0.15) or interaction (F1,9 = 1.37, p > 0.15). Post hoc tests revealed that rats had significant reinstatement in both the vehicle- and BM-treated conditions compared to their extinction baselines (p < 0.01; p < 0.05, respectively), but the vehicle- and BM-treated conditions did not differ in terms of reinstatement of active lever pressing (p > 0.15).

Decreased cued heroin seeking after prolonged withdrawal with pIC, but not aIC, inactivation in males, but not females

In this experiment, the aIC or pIC was inactivated during a cued heroin-seeking test after 14 days of withdrawal (Fig. 1D). Figure 3A shows self-administration data for the aIC groups, with males and females showing similar levels of lever pressing and heroin infusions during late self-administration days. Two-way ANOVA of inactive lever presses, active lever presses, and heroin infusions during the final 10 d of self-administration revealed no differences between groups that would be treated with BM vs vehicle during the seeking test. (Table 2). Figure 3B shows active lever presses during the day 1 and day 14 cued seeking tests for those rats receiving aIC injections, with injections of vehicle or BM given on day 14 in a between-subjects manner. Analysis of active lever presses during the first 30 min of each seeking test revealed a significant main effect of day (F1,23 = 51.31, p < 0.0001), but no main effect of manipulation (F1,23 = 0.14, p > 0.15) or interaction (F1,23 = 0.48, p > 0.15). Post hoc tests revealed that both vehicle- and BM-treated groups had increased active lever pressing on day 14 compared to day 1 (p < 0.0001; p < 0.001, respectively). However, there was no effect of manipulation on day 14 lever pressing (p > 0.15), indicating that aIC inactivation did not alter the incubation of heroin craving.

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.

Table 2 Statistics for self-administration measures from incubation of craving experiments.

Figure 3C shows self-administration data for the pIC groups. Because sex differences were observed in incubation of craving, both sexes were fully powered, and analyses were performed for each sex separately. Two-way ANOVA of inactive lever presses, active lever presses, and heroin infusions during the final 10 days of self-administration revealed no differences between groups subsequently treated with BM vs vehicle during the seeking test (Table 2). Figure 3D shows active lever presses, separated by sex, during the day 1 and day 14 tests for those rats receiving pIC injections, with injections of vehicle or BM given on day 14 in a between-subjects manner. For the males (left panel), a two-way ANOVA of active lever presses during the first 30 min of each seeking test revealed a significant main effect of day (F1,15 = 38.38, p < 0.0001), a trend toward a main effect of manipulation (F1,15 = 3.39, p = 0.0857), and a significant interaction (F1,15 = 6.26, p < 0.05). Post hoc tests revealed that both vehicle- and BM-treated groups had increased active lever pressing on day 14 compared to day 1 (p < 0.0001; p < 0.05, respectively). However, the BM group had significantly fewer active lever presses compared to the vehicle group (p < 0.05), indicating that pIC inactivation decreased the incubation of heroin craving in males. For the females (right panel), a two-way ANOVA of active lever presses during the first 30 min of each seeking test revealed a significant main effect of day (F1,16 = 7.24, p < 0.05), but no main effect of manipulation (F1,16 = 0.12, p > 0.15) or interaction (F1,16 = 0.08, p > 0.15). Post hoc tests revealed that both vehicle- and BM-treated groups had increased active lever pressing on Day 14 compared to Day 1 that, when separately analyzed, produced non-significant trends (p = 0.1070; p = 0.1019, respectively). However, there was no effect of manipulation on day 14 lever pressing (p > 0.15), indicating that pIC inactivation did not alter the incubation of craving in females.

No sex differences in 6 h heroin self-administration

We also conducted ancillary analyses of self-administration data, collapsed across the aIC and pIC incubation-of-craving experiments, to determine whether there were any sex differences in 6 h heroin self-administration. Figure 4A–C show, respectively, the total daily active lever presses, total daily heroin infusions, and total daily mg/kg heroin intake across the final 10 days of self-administration for males and females. A two-way ANOVA of active lever presses revealed a significant main effect of day (F3.06,177.30 = 2.73, p < 0.05), but no main effect of sex (F1,58 = 0.76, p > 0.15) and no interaction (F9,522 = 1.53, p = 0.1355). Analysis of heroin infusions revealed a significant main effect of day (F3.38,195.80 = 4.62, p < 0.01), no effect of sex (F1,58 = 2.13, p = 0.1496), and no interaction (F9,522 = 1.03, p > 0.15). When adjusted for body weight, the amount of heroin self-administered did not differ between the sexes, as analysis of mg/kg heroin intake revealed a significant main effect of day (F3.00,174.00 = 4.49, p < 0.01), but no main effect of sex (F1,58 = 0.19, p > 0.15) or interaction (F9,522 = 0.83, p > 0.15).

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.

No effect of pIC and aIC inactivation on locomotor activity

Figure 5A, B show, respectively, the total distance traveled in an open field test following aIC and pIC inactivation. Paired t-tests of total distance travelled revealed no effect of aIC (t(9) = 0.94, p > 0.15) or pIC (t(8) = 0.06, p > 0.15) inactivation on locomotor activity in males and females.

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.