Adolescent morphine exposure affects long-term microglial function and later-life relapse liability in a model of addiction

Abstract

Adolescence in humans represents a unique developmental time point associated with increased risk-taking behavior and experimentation with drugs of abuse. We hypothesized that exposure to drugs of abuse during adolescence may increase the risk of addiction in adulthood. To test this, rats were treated with a subchronic regimen of morphine or saline in adolescence, and their preference for morphine was examined using conditioned place preference (CPP) and drug-induced reinstatement in adulthood. The initial preference for morphine did not differ between groups; however, rats treated with morphine during adolescence showed robust reinstatement of morphine CPP after drug re-exposure in adulthood. This effect was not seen in rats pretreated with a subchronic regimen of morphine as adults, suggesting that exposure to morphine specifically during adolescence increases the risk of relapse to drug-seeking behavior in adulthood. We have previously established a role for microglia, the immune cells of the brain, and immune molecules in the risk of drug-induced reinstatement of morphine CPP. Thus, we examined the role of microglia within the nucleus accumbens of these rats and determined that rats exposed to morphine during adolescence had a significant increase in Toll-like receptor 4 (TLR4) mRNA and protein expression specifically on microglia. Morphine binds to TLR4 directly, and this increase in TLR4 was associated with exaggerated morphine-induced TLR4 signaling and microglial activation in rats previously exposed to morphine during adolescence. These data suggest that long-term changes in microglial function, caused by adolescent morphine exposure, alter the risk of drug-induced reinstatement in adulthood.

Figure 1.

Timeline of Experiments 1, 2, and 3. A, Rats were treated in adolescence [beginning approximately postnatal day (P) 37–42] with a subchronic regimen of either saline or morphine. On P60, rats began CPP for morphine followed by extinction, saline-induced reinstatement, and subsequently drug-induced reinstatement of morphine CPP. Within 48 h after the completion of behavior, the NAcc was collected for the analysis of CD11b and TLR4 gene expression. B, Rats were treated in adulthood (beginning approximately P54–P58) with a subchronic regimen of either saline or morphine. On P90, rats began CPP for morphine followed by extinction, saline-induced reinstatement, and subsequently drug-induced reinstatement of morphine CPP. Within 48 h after the completion of behavior, the NAcc was collected for the analysis of CD11b and TLR4 gene expression. C, Rats were treated during adolescence (beginning approximately P37–P42) with a subchronic regimen of either saline or morphine and the glial modulator, ibudilast, or its vehicle. On P60, rats began CPP for morphine followed by extinction, saline-induced reinstatement, and subsequently drug-induced reinstatement of morphine CPP. Within 48 h after the completion of behavior, the NAcc was collected for the analysis of TLR4 gene expression.

Figure 2.

Adolescent morphine pre-exposure increases the long-term risk of drug-induced reinstatement of morphine CPP. Adolescent rats were treated with a subchronic regimen of morphine or saline (n = 12/group). A, In adulthood, rats from both treatment groups showed similar levels of morphine CPP; and 3 weeks later, rats showed no preference for the morphine-paired chamber (Extinguished CPP). B, Injection of the same rats with saline (Saline Reinstatement) did not cause the reinstatement of morphine CPP; however, re-exposure to morphine (Drug-Induced Reinstatement) caused the reinstatement of morphine CPP only in rats treated with morphine as adolescents. *p < 0.05, compared with saline-treated rats. C, Pretreatment of both groups with a microglial inhibitor, minocycline (25 mg/kg), before morphine re-exposure did not prevent the drug-induced reinstatement of morphine CPP in rats that received morphine as adolescents.

Figure 3.

Adult morphine pre-exposure does not increase the long-term risk of drug-induced reinstatement of morphine CPP. Adult rats, postnatal day 60, were treated with a subchronic regimen of morphine or saline (n = 12/group). A, Thirty days later, rats from both treatment groups showed similar levels of morphine CPP; and 4 weeks later, rats showed no preference for the morphine-paired chamber (Extinguished CPP). B, Injection of the same rats with saline (Saline Reinstatement) did not cause the reinstatement of morphine CPP. Re-exposure to morphine (Drug-Induced Reinstatement) also did not lead to reinstatement of morphine CPP.

Figure 4.

Box plot representation of drug-induced reinstatement scores from rats pre-exposed to morphine or saline in either adolescence or adulthood. Drug-induced reinstatement scores from the rats in Experiment 1 and the rats in Experiment 2 were plotted together to show the median reinstatement scores as well as the upper and lower quartiles for these reinstatement scores. Overall, rats treated with morphine during adolescence showed a significant increase in the reinstatement of morphine CPP compared with their saline controls: p < 0.05. In contrast, the reinstatement scores from rats pre-exposed to morphine or saline in adulthood show greater variability, with approximately half of the rats in each group showing reinstatement (CPP score >100) and half of the rats not showing reinstatement to morphine CPP, resulting in no significant difference between the treatment groups.

Figure 5.

Attenuating glial activation with ibudilast during adolescence prevents the increased risk of drug-induced reinstatement in adulthood and the long-term increase in TLR4 expression within the NAcc. Adolescent rats were treated subchronically with either ibudilast (7.5 mg/kg) or its vehicle along with morphine or saline (n = 12/group). A, In adulthood, rats from all treatment groups showed similar levels of morphine CPP; and 4 weeks later, rats showed no preference for the morphine-paired chamber (Extinguished CPP). B, Injection of the same rats with saline (Saline Reinstatement) did not cause the reinstatement of morphine CPP. Re-exposure to morphine (Drug-Induced Reinstatement) resulted in the selective reinstatement of morphine CPP in rats treated with morphine as adolescents: *p < 0.05, compared with all other groups. This effect was prevented by treatment with ibudilast at the time of adolescent morphine exposure.

Figure 6.

Adolescent morphine pre-exposure, but not adult morphine pre-exposure, results in increased TLR4 mRNA expression within the NAcc in adulthood. A, In the same rats from Experiment 1 that were pre-exposed to morphine or saline in adolescence, we analyzed the baseline mRNA expression of CD11b and TLR4 within the NAcc 48 h after the completion of all behavioral tasks. Analysis revealed no significant effect of adolescent morphine pre-exposure on CD11b but a significant increase in TLR4 mRNA in rats pre-exposed to morphine during adolescence compared with their saline-treated controls: *p < 0.05. B, In the same rats from Experiment 2 that were pre-exposed to morphine or saline in adulthood, we analyzed the mRNA expression of CD11b and TLR4 within the NAcc 48 h after the completion of all behavioral tasks and found no significant differences in either gene.

Figure 7.

Attenuating glial activation with ibudilast during adolescence prevents the long-term increase in TLR4 expression within the NAcc. In the same rats from Experiment 3 collected within 48 h after the completion of all behavioral tasks, expression of TLR4 mRNA was significantly elevated within the NAcc of rats treated with morphine as adolescents: *p < 0.05, compared with all other groups. This effect was prevented by treatment with ibudilast at the time of adolescence.

Figure 8.

Adolescent morphine exposure results in increased TLR4 protein expression specifically on microglia from the NAcc. A, Representative dot plots from dissociated neural tissue of the NAcc represent the gates used to isolate singlets (top) and subsequently cells (bottom). In this particular sample, 46% of all events were identified as singlets; and from that population, 74% were living cells. B, Dot plots of staining controls (unstained control, CD11b-allophycocyanin alone, TLR4-phycoerythrin alone, and phycoerythrin secondary alone) following flow cytometry and analysis. Boxes represent gates for CD11b⁺ cells (top right) and TLR4⁺ cells (bottom left). C, The bar graph represents the MFI of TLR4 staining from distinct cell populations of NAcc tissue collected in adulthood from rats previously exposed to morphine or saline in adolescence. TLR4 staining was significantly elevated in CD11b⁺ cells from the NAcc of rats previously exposed to morphine in adolescence (p ≤ 0.05) but was not significantly different in CD11b⁻ cells (n = 5/group). D, TLR4 staining was not significantly affected by adolescent morphine treatment in hippocampus of either CD11b⁺ or CD11b⁻ cells from the same rats (n = 5/group).

Figure 9.

Adolescent morphine pre-exposure increases the activation of the TLR4 signaling pathway and microglia within the NAcc after re-exposure to morphine in adulthood. Adolescent rats were pre-exposed to a subchronic regimen of morphine or saline and then treated with a single dose of either morphine (4 mg/kg) or saline in adulthood. Analysis of TLR4 mRNA within the NAcc revealed a significant main effect of adolescent morphine treatment: *p < 0.05, compared with adolescent saline treatment. Analysis of CD14, Myd88, NFκb2, and CD11b revealed a significant interaction of adolescent morphine treatment and adult morphine treatment, such that only rats treated with morphine at both time points showed significantly elevated levels of each gene within NAcc: *p < 0.05, compared with all other treatment groups. In contrast, analysis of Map4k4 mRNA revealed a significant decrease in rats treated with morphine at both time points compared with all other treatment groups.