Central serotonergic neurons are differentially required for opioid analgesia but not for morphine tolerance or morphine reward

Abstract

Opioids remain the most effective analgesics despite their potential adverse effects such as tolerance and addiction. Mechanisms underlying these opiate-mediated processes remain the subject of much debate. Here we describe opioid-induced behaviors of Lmx1b conditional knockout mice (Lmx1bf/f/p), which lack central serotonergic neurons, and we report that opioid analgesia is differentially dependent on the central serotonergic system. Analgesia induced by a kappa opioid receptor agonist administered at the supraspinal level was abolished in Lmx1bf/f/p mice compared with their wild-type littermates. Furthermore, compared with their wild-type littermates Lmx1bf/f/p mice exhibited significantly reduced analgesic effects of mu and delta opioid receptor agonists at both spinal and supraspinal sites. In contrast to the attenuation in opioid analgesia, Lmx1bf/f/p mice developed tolerance to morphine analgesia and displayed normal morphine reward behavior as measured by conditioned place preference. Our results provide genetic evidence supporting the view that the central serotonergic system is a key component of supraspinal pain modulatory circuitry mediating opioid analgesia. Furthermore, our data suggest that the mechanisms of morphine tolerance and morphine reward are independent of the central serotonergic system.

Fig. 1.

Morphine-induced analgesia in Lmx1b^f/f/p and wild-type mice. (A–C) Saline and escalating doses of morphine (3, 6, 10, and 30 mg/kg, multiple s.c. injections, n = 10–12 per genotype) or multiple doses of morphine (0.1, 0.2, and 0.3 nmol i.t. or 0.3 and 0.6 nmol i.c.v., n = 8–12 every dose per genotype) were administered to wild-type and Lmx1b^f/f/p mice, and morphine analgesia was measured with the tail-flick assay. Immediately after the withdrawal latency measurement after 30 mg/kg s.c., 0.3 nmol i.t., or 0.6 nmol i.c.v. morphine injections, mice were injected with naloxone (1 mg/kg i.p.), and antinociception was assessed again after 15 min. The analgesic effect was presented as the percentage of maximum possible effect [%MPE = (drug latency − baseline latency) × 100/(cutoff latency − baseline latency)]. (D–F) Time course of morphine-induced analgesia of wild-type and Lmx1b^f/f/p mice in the tail-flick test after a single morphine dose of 6 mg/kg s.c. (D), 0.2 nmol i.t. (E), or 0.3 nmol i.c.v. (F). All data are presented as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (repeated-measures ANOVA compared with wild type). Nal, naloxone.

Fig. 2.

Analgesia evoked by KOR or DOR agonists in Lmx1b^f/f/p and wild-type mice. (A) Saline and escalating doses of the KOR agonist U50,488H (1, 3, 6, 10, and 30 mg/kg s.c.) were administered to wild-type and Lmx1b^f/f/p mice, and the analgesic effect was measured with the tail-flick assay. (B and C) The analgesic effect of the i.t. injection of U50,488H (60 μg) in Lmx1b^f/f/p mice was comparable to wild-type mice (B), whereas effect of the i.c.v. injection of U50,488H (60 μg) was almost absent in Lmx1b^f/f/p mice compared with that in wild type (C). (D and E) The analgesic effect of i.t. (D) and i.c.v. (E) injections of the DOR agonist [D-Pen2, D-Pen5]enkephalin (5 μg) in wild-type and Lmx1b^f/f/p mice. All data are mean ± SEM. *, P < 0.05; **, P < 0.01 (repeated-measures ANOVA compared with wild type).

Fig. 3.

Morphine tolerance developed in parallel in Lmx1b^f/f/p and wild-type mice. (A) Wild-type and Lmx1b^f/f/p mice were treated with morphine (15 mg/kg s.c.) daily for 7 days. The analgesic effect was assessed 30 min after the injection by water-immersion tail-flick assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (repeated-measures ANOVA followed by Newman–Keuls test compared with the first-day data of the same genotype). ###, P < 0.001 (repeated-measures ANOVA compared with wild type). (B) The rate of development of morphine tolerance denoted by the slope of the %MPE across 7 days (P > 0.05, two-tailed t test, genotype effect). All data are presented as means ± SEM.

Fig. 4.

Morphine-induced CPP and locomotor activity in Lmx1b^f/f/p and wild-type mice. (A and B) Dose–response study of morphine as measured by percentage of time spent (A) or travel distance ratio (B) comparing time or travel distance in the morphine-paired chamber before (pretest) and after (posttest) conditioning. (C–E) Extinction of morphine-induced CPP in Lmx1b^f/f/p and wild-type mice at doses of 5 (C), 10 (D), and 20 (E) mg/kg. Data are described as the percentage of mice meeting the extinction criterion [see supporting information (SI) Methods] (P > 0.05, Fisher's exact test, genotype effect). (F) Days of extinction at doses of 5, 10, and 20 mg/kg morphine. Data are shown as mean ± SEM (P > 0.05, unpaired t test, genotype effect). n = 6–7 in each genotype per dose in C–F. (G) Reinstatement of CPP at different doses of morphine. (H) Locomotor activity reflected by travel distance during the 30-min period after injection of saline or different doses of morphine. In all experiments morphine was administered i.p. n = 9–14 in each genotype per dose in A, B, and H, and n = 6–7 in G. Except in C–E, all data are means ± SEM (P > 0.05, two-tailed paired or unpaired t test comparing genotypes within treatment in A, B, G, and H). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed paired or unpaired t test, compared with pretest within the same genotype and dose in A, B, and G, or ANOVA followed by Newman–Keuls tests compared with different doses in H).

Fig. 5.

MOR expression in Lmx1b^f/f/p and wild-type mice assessed by in situ hybridization and Western blotting assays. (A–L) MOR expression detected in various brain areas of wild-type and Lmx1b^f/f/p animals by in situ hybridization. Except the raphe system including dorsal raphe nucleus (DRN) (A and B) and caudal raphe nuclei (CRN) (C and D) where 5-HT neurons are clustered along the midline, no major differences of MOR expression was detected (E–L) in various brain regions between wild-type and Lmx1b^f/f/p mice. Asterisks in A, B, G, and H indicate the cerebral aqueduct. Arrows in A–D and I–L indicate the typical MOR expression areas. (Scale bar: 100 μm.) (M and N) Western blot analysis showing no significant difference in MOR protein levels found in four brain areas of wild-type (left sample in M and open bars in N) and Lmx1b^f/f/p (right sample in M and black bars in N) mice (n = 4 per genotype). Data are expressed as the ratio of MOR band densities to band densities of the corresponding β-actin on the same film. Error bars represent mean ± SEM (P > 0.05, one-way ANOVA followed by Newman–Keuls post hoc test; the difference in the same areas between genotypes were compared). HP, hippocampus; PAG, periaqueductal gray; PB, parabrachial nucleus; LC, locus ceruleus; BS, brain stem; CPu, caudate putamen; SC, spinal cord.