Mu-Opioid Receptors Expressed in Glutamatergic Neurons are Essential for Morphine Withdrawal

Abstract

Although opioids still remain the most powerful pain-killers, the chronic use of opioid analgesics is largely limited by their numerous side-effects, including opioid dependence. However, the mechanism underlying this dependence is largely unknown. In this study, we used the withdrawal symptoms precipitated by naloxone to characterize opioid dependence in mice. We determined the functional role of mu-opioid receptors (MORs) expressed in different subpopulations of neurons in the development of morphine withdrawal. We found that conditional deletion of MORs from glutamatergic neurons expressing vesicular glutamate transporter 2 (Vglut2⁺) largely eliminated the naloxone-precipitated withdrawal symptoms. In contrast, conditional deletion of MORs expressed in GABAergic neurons had a limited effect on morphine withdrawal. Consistently, mice with MORs deleted from Vglut2⁺ glutamatergic neurons also showed no morphine-induced locomotor hyperactivity. Furthermore, morphine withdrawal and morphine-induced hyperactivity were not significantly affected by conditional knockout of MORs from dorsal spinal neurons. Taken together, our data indicate that the development of morphine withdrawal is largely mediated by MORs expressed in Vglut2⁺ glutamatergic neurons.

Keywords: Dorsal spinal cord; Locomotor hyperactivity; Morphine; Mu-opioid receptor; Naloxone-precipitated withdrawal.

Fig. 1

Generation and verification of the MOR conditional-knockout mouse line. A Targeting strategy for generating the Oprm1^fl/fl mouse line. B Representative images of Oprm1 expression in Oprm1^fl/fl and Vglut2-Cre/Oprm1^fl/fl mice determined by in situ hybridization (scale bar, 500 μm). C Representative images of MOR expression in Oprm1^fl/fl and Vglut2-Cre/Oprm1^fl/fl mice determined by fluorescent immunostaining (scale bar, 200 μm). Cpu, striatum; NAc, nucleus accumbens; AcbC, accumbens nucleus, core; MHb, medial habenula; PV, paraventricular thalamic nucleus; CM, central medial thalamic nucleus; CeA, central amygdala; BLA, basolateral amygdala; IPN, interpeduncular nucleus; VTA, ventral tegmental area; PBN, parabrachial nucleus; scp, superior cerebellar peduncle; LPB, lateral parabrachial nucleus.

Fig. 2

Deletion of MORs in glutamatergic neurons alleviates naloxone-precipitated withdrawal symptoms. A Experimental timeline for chronic morphine delivery and naloxone-precipitated withdrawal. B, C Time course of scores and total scores for naloxone-precipitated withdrawal in Oprm1^fl/fl and Vglut2-Cre/Oprm1^fl/fl mice (n = 6–8 mice/group). D–K Bar graphs showing bouts and scores for each withdrawal symptom in Oprm1^fl/fl and Vglut2-Cre/Oprm1^fl/fl mice. Jumping, teeth-chattering, wet-dog shakes, headshakes, grooming, piloerection, paw tremor, and twitching behaviors were analyzed from recorded videos. L, M Effects of morphine on locomotor activity of Oprm1^fl/fl and Vglut2-Cre/Oprm1^fl/fl mice. Travel distance and average speed were measured in the open field test before and after morphine administration (n = 9–12 mice/group). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant (one-way ANOVA in C–K; two-way ANOVA between genotypes or morphine treatment in L and M, followed by a post hoc Bonferroni test). Data are presented as the mean ± SEM.

Fig. 3

MOR expression is abolished in multiple brain regions of Vgat-Cre/Oprm1^fl/fl mice. A Representative images of Oprm1 expression in Oprm1^fl/fl and Vgat-Cre/Oprm1^fl/fl mice (scale bar, 500 μm). B Representative images of MOR expression in Oprm1^fl/fl and Vgat-Cre/Oprm1^fl/fl mice determined by fluorescent immunostaining (scale bar, 200 μm). NAc, nucleus accumbens; MHb, medial habenula; CeA, central amygdala; IPN, interpeduncular nucleus; PBN, parabrachial nucleus. Cpu, striatum; NAc, nucleus accumbens; AcbC, accumbens nucleus, core; MHb, medial habenula; PV, paraventricular thalamic nucleus; CM, central medial thalamic nucleus; CeA, central amygdala; BLA, basolateral amygdala; IPN, interpeduncular nucleus; VTA, ventral tegmental area; PBN, parabrachial nucleus; scp, superior cerebellar peduncle; LPB, lateral parabrachial nucleus.

Fig. 4

Deletion of MORs in GABAergic neurons has limited effects on naloxone-precipitated withdrawal symptoms. A Experimental timeline for chronic morphine delivery and naloxone-precipitated withdrawal. B, C Time course of scores and total scores for naloxone-precipitated withdrawal in Oprm1^fl/fl and Vgat-Cre/Oprm1^fl/fl mice (n = 6–8 mice/group). D–K Bar graphs showing bouts and scores for each withdrawal symptom in Oprm1^fl/fl and Vgat-Cre/Oprm1^fl/fl mice. Jumping, teeth-chattering, wet-dog shakes, headshakes, grooming, piloerection, paw tremor, and twitching behaviors were analyzed from recorded videos. L, M Effects of morphine on the locomotor activity of Oprm1^fl/fl and Vgat-Cre/Oprm1^fl/fl mice. Travel distance and average speed were measured in the open field test before and after morphine administration (n = 9–10 mice/group). **P < 0.01, ***P < 0.001, n.s., not significant (one-way ANOVA in C–K; two-way ANOVA between genotypes or morphine treatment in L and M, followed by a post hoc Bonferroni test). Data are presented as the mean ± SEM.

Fig. 5

Deletion of MORs in the dorsal spinal cord has no significant effect on naloxone-precipitated withdrawal symptoms. A Schematic of the protocol for deleting MORs from the dorsal spinal cord. Lbx1-Cre mice were crossed with Oprm1^fl/fl mice, and exons 2 and 3 of Oprm1 were excised. B, C Representative images of Oprm1 expression in the dorsal cord of Oprm1^fl/fl and Lbx1-Cre/Oprm1^fl/fl mice (scale bar, 200 μm). D Experimental timeline for chronic morphine delivery and naloxone-precipitated withdrawal. E, F Time course of scores and total scores for naloxone-precipitated withdrawal in Oprm1^fl/fl and Lbx1-Cre/Oprm1^fl/fl mice (n = 6–7 mice/group). G–N Bar graphs showing bouts and scores for each withdrawal symptom in Oprm1^fl/fl and Lbx1-Cre/Oprm1^fl/fl mice. Jumping, teeth-chattering, wet-dog shakes, headshakes, grooming, piloerection, paw tremor, and twitching behaviors were analyzed from recorded videos. O, P Effect of morphine on locomotor activity of Oprm1^fl/fl and Lbx1-Cre/Oprm1^fl/fl mice. Travel distance and average speed were measured in the open field test before and after morphine administration (n = 6 mice/group). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant (one-way ANOVA in F–N; two-way ANOVA between genotypes or morphine treatment in O and P, followed by a post hoc Bonferroni test). Data are presented as the mean ± SEM.