Direct involvement of orexinergic systems in the activation of the mesolimbic dopamine pathway and related behaviors induced by morphine

Abstract

In this study, we investigated the role of orexinergic systems in dopamine-related behaviors induced by the mu-opioid receptor agonist morphine in rodents. Extensive coexpression of tyrosine hydroxylase with orexin receptors was observed in the mouse ventral tegmental area (VTA). The levels of dopamine and its major metabolites in the nucleus accumbens were markedly increased by the microinjection of orexin A and orexin B into the VTA. The subcutaneous morphine-induced place preference and hyperlocomotion observed in wild-type mice were abolished in mice that lacked the prepro-orexin gene. An intra-VTA injection of a selective orexin receptor antagonist SB334867A [1-(2-methylbenzoxazol-6-yl)-3-[1.5]naphthyridin-4-yl urea] significantly suppressed the morphine-induced place preference in rats. Furthermore, the increased level of dialysate dopamine produced by morphine in the mouse brain was significantly decreased by deletion of the prepro-orexin gene. These findings provide new evidence that orexin-containing neurons in the VTA are directly implicated in the rewarding effect and hyperlocomotion induced by morphine through activation of the mesolimbic dopamine pathway in rodents.

Figure 1.

Expression of orexin 1 or orexin 2 receptors in dopamine neurons of the VTA. Immunostaining for TH (A, D) is visible in many VTA neurons that show immunoreactivity for orexin 1 receptors (B) or orexin 2 receptors (E). Colocalization is shown by yellow neurons in the merged images of TH/orexin 1 receptor (C) or TH/orexin 2 receptor (F). Scale bars, 50 μm.

Figure 2.

Involvement of central orexinergic system in activation of the mesolimbic dopamine neuron. Orexin A (A) and orexin B (B) each induced hyperlocomotion in mice. Pretreatment with haloperidol (0.1 mg/kg, s.c.) or 6-OHDA (150 nmol/mouse, i.c.v.) in combination with desipramine (25 mg/kg, s.c.) blocked the effects of orexins. C, Total locomotor activity induced by saline pretreatment with haloperidol or 6-OHDA in combination with desipramine. Each column represents the mean to tal activity for 120 min with SEM of 8–17 mice. **p < 0.01, ***p < 0.001 versus saline-treated mice. ^##p < 0.01, ^###p < 0.001 versus orexin A- or orexin B-treated mice.

Figure 3.

Effects of orexin administration into the VTA on the dialysate dopamine level in the NAcc. After the collection of baseline fractions, rats were administered with saline, orexin A, or orexin B (1 nmol) into the VTA at time 0 to evoke the release of dopamine. Data are expressed as percentages of the corresponding baseline levels with SEM for three to five rats. Dopamine, saline versus orexin A, F_(1,6) = 37.1, p < 0.001; saline versus orexin B, F_(1,5) = 282.7, p < 0.001. DOPAC, saline versus orexin A, F_(1,6) = 11.6, p < 0.05; saline versus orexin B, F_(1,5) = 10.9, p < 0.05. HVA, saline versus orexin A, F_(1,6) = 14.9, p < 0.01; saline versus orexin B, F_(1,5) = 10.2, p < 0.05.

Figure 4.

Morphine (MRP)-induced rewarding effect in wild-type (WT) and prepro-orexin knock-out (OXKO) mice. A, Loss of prepro-orexin mRNA levels in the mouse whole brain obtained from prepro-orexin knock-out mice. Representative RT-PCR for prepro-orexin mRNA in the whole brain obtained from OXKO and WT mice is shown. B, Morphine-induced place preference in WT and OXKO mice using the conditioned place preference assay. Ordinate, Preference for drug-paired place, as defined by the postconditioning test score minus preconditioning test score in the drug treatment side. Each point represents the mean conditioning score with SEM of 5–10 mice. *p < 0.05 and **p < 0.01 versus wild-type mice and saline-conditioned group. ^#p < 0.05 and ^###p < 0.001 versus wild-type mice and morphine-conditioned group. ^†p < 0.05 versus prepro-orexin knock-out mice and saline-conditioned group. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. PPOX, prepro-orexin.

Figure 5.

Morphine (MRP)-induced hyperlocomotion in wild-type (WT) and prepro-orexin knock-out (OXKO) mice. A, Time course changes in the locomotor-enhancing effect of morphine in WT and OXKO mice. Each point represents the mean activity counts for 10 min with SEM of 9–14 mice. F_{(1, 20)} = 18.7, p < 0.001 for WT mice treated with 5 mg/kg morphine versus OXKO mice treated with 5 mg/kg morphine. F_{(1, 24)} = 14.3, p < 0.001 for WT mice treated with 10 mg/kg morphine versus OXKO mice treated with 10 mg/kg morphine. B, Total locomotor activity induced by morphine in WT and OXKO mice. Each column represents the mean total activity for 180 min with SEM of 9–14 mice. ***p < 0.001 versus saline-treated WT mice; ^###p < 0.001 versus morphine-treated WT mice.

Figure 6.

Suppression of the rewarding effect of morphine by intra-VTA injection of the selective orexin receptor antagonist. A, Localization of microinjection sites in the rat VTA and SN. Stippled lines show the region in the rat brain in which a cannula was inserted. B, Effect of the microinjection of the selective orexin receptor antagonist SB334867A into the VTA or SN on the morphine-induced place preference. Ordinate, Preference for drug-paired place, as defined by the postconditioning test score minus preconditioning test score in the drug treatment side. Rats were microinjected with SB334867A 10 min before the start of conditioning with morphine. The data represent the mean ± SEM of five to eight rats. ***p < 0.001 versus vehicle-pretreated and saline-conditioned rats; ^##p < 0.01 versus vehicle-pretreated and morphine-conditioned rats.

Figure 7.

Change in the increased dialysate dopamine level induced by morphine in preproorexin knock-out mice. A, Localization of microdialysis probe sites in the mouse NAcc. Stippled lines represent regions in which microdialysis probes were inserted in the mouse brain. B, Effects of treatment with morphine (MRP) on the dialysate dopamine level in the NAcc in wild-type (WT) and prepro-orexin knock-out (OXKO) mice. Morphine (10 mg/kg, s.c.) or saline (SAL) was injected at time 0. The data are expressed as percentages of the corresponding baseline levels with SEM of four to seven mice. F_{(1, 11)} = 5.25, p < 0.05 wild-type mice treated with morphine versus prepro-orexin knock-out mice treated with morphine.