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. 2024 Jan 18;134(5):e171995.
doi: 10.1172/JCI171995.

Targeting mitochondrial dynamics of morphine-responsive dopaminergic neurons ameliorates opiate withdrawal

Changyou Jiang  1   2 , Han Huang  1   2 , Xiao Yang  1   2 , Qiumin Le  1   2 , Xing Liu  1   2 , Lan Ma  1   2 , Feifei Wang  1   2
Affiliations

Targeting mitochondrial dynamics of morphine-responsive dopaminergic neurons ameliorates opiate withdrawal

Changyou Jiang et al. J Clin Invest. .

Abstract

Converging studies demonstrate the dysfunction of the dopaminergic neurons following chronic opioid administration. However, the therapeutic strategies targeting opioid-responsive dopaminergic ensembles that contribute to the development of opioid withdrawal remain to be elucidated. Here, we used the neuronal activity-dependent Tet-Off system to label dopaminergic ensembles in response to initial morphine exposure (Mor-Ens) in the ventral tegmental area (VTA). Fiber optic photometry recording and transcriptome analysis revealed downregulated spontaneous activity and dysregulated mitochondrial respiratory, ultrastructure, and oxidoreductase signal pathways after chronic morphine administration in these dopaminergic ensembles. Mitochondrial fragmentation and the decreased mitochondrial fusion gene mitofusin 1 (Mfn1) were found in these ensembles after prolonged opioid withdrawal. Restoration of Mfn1 in the dopaminergic Mor-Ens attenuated excessive oxidative stress and the development of opioid withdrawal. Administration of Mdivi-1, a mitochondrial fission inhibitor, ameliorated the mitochondrial fragmentation and maladaptation of the neuronal plasticity in these Mor-Ens, accompanied by attenuated development of opioid withdrawal after chronic morphine administration, without affecting the analgesic effect of morphine. These findings highlighted the plastic architecture of mitochondria as a potential therapeutic target for opioid analgesic-induced substance use disorders.

Keywords: Addiction; Mitochondria; Neurological disorders; Neuroscience; Therapeutics.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Dysregulation of the spontaneous activity in the VTA dopaminergic Mor-Ens mediates withdrawal-induced aversion and anxiety after chronic morphine administration.
(A) Schematic of virus injection and fiber photometry recordings. Viruses combining Cre-loxp and Flpo-FRT systems were used to label TH+ neuronal ensembles with GCaMP7b, and the optic fiber was unilaterally implanted in the VTA of WT mice. (B) Representative images of GCaMP7b expression in the VTA. Dashed white lines outline the VTA and optic fiber tract. Green, GCaMP7b; Blue, DAPI. Scale bar: 200 μm. (C) Representative photometric traces of GCaMP7b signals in Mor-Ens before and after morphine EDA. Marked circles indicate detected events above the threshold. (D) Relative frequency of calcium events in Mor-Ens. Paired t test, n = 14. (E) Experimental scheme of the ensembles labeling and behavioral testing in TH-Cre mice. AAV-RAM-tTA-TRE-hM3Dq-HA was injected into the VTA of TH-Cre mice to label RAM-driven expression of hM3Dq-HA in TH+ neuronal ensembles. (F) Representative images of hM3Dq-HA expression in VTA ensembles. Red, hM3Dq-HA; Blue, DAPI. Scale bar: 100 μm. White dashed line outlines the boundary of VTA. (G and H) The effects of CNO activation of Mor-Ens on CPA and anxiety during morphine withdrawal. CPA score (G), the representative traces in EPM test, and the quantification of time in the open arm (H) are represented. 2-way RM ANOVA, Sal-Ens, n = 14; Mor-Ens, n = 15 in G. Unpaired t test, n = 10 mice per group in H. Data are presented as mean ± SEM; *P < 0.05, ***P < 0.0001.
Figure 2
Figure 2. Chronic morphine administration alters dysregulation of the mitochondria-related signaling pathways in the VTA dopaminergic Mor-Ens.
(A) Experimental scheme for single-cell RNA–Seq of the VTA dopaminergic ensembles. Red, tdTomato. Scale bar: 200 μm. (B) Signaling network enrichment analysis between dopaminergic Sal-Ens and Mor-Ens. (C) Signaling network enrichment analysis between dopaminergic Mor-Ens treated without or with morphine EDA groups. n = 4 mice per group.
Figure 3
Figure 3. Chronic morphine administration induces increased oxidative stress and impairs Ca2+ transport in dopaminergic Mor-Ens and mitochondrial respiration in the VTA.
(A) Experimental scheme to assess the oxidative stress in the dopaminergic Mor-Ens of VTA from mice with or without morphine EDA. (B) Representative images of nitrotyrosine staining of the brain slices containing VTA. White dashed lines outline Mor-Ens. Red, tdTomato; Green, nitrotyrosine; Blue, DAPI. Scale bar: 20 μm. (C) The normalized expression level of nitrotyrosine in VTA tdTomato+ ensembles in saline and morphine EDA groups. Unpaired t test, n = 4 mice per group. (D) Cumulative frequency distribution of nitrotyrosine intensity in tdTomato+ neurons. 2-sample Kolmogorov-Smirnov test, Saline Ctrl, 229 cells from 4 mice; Morphine EDA, 194 cells from 4 mice. (E) Schematic of fiber photometry setup for detecting mitochondrial Ca2+ signal in Mor-Ens in freely moving mice. (F) Heatmap of relative mito-GCaMP fluorescence intensity in Mor-Ens after intracerebral injection of kaempferol into VTA (1.6 μL, 2 nmol/μL) in mice with or without morphine EDA. (G) Average ΔF/F (%) and (H) the AUC quantification of mito-GCaMP fluorescence. Dashed vertical line indicates kaempferol injection. Paired t test, n = 7. (I) Experimental scheme to assess the mitochondrial respiration of the VTA tissues. G, glutamate; P, pyruvate; M, malate; Scc, succinate; CCCP, mitochondrial oxidative phosphorylation uncoupler; Rot, rotenone; Ant, antimycin; C, complex; ER, endoplasmic reticulum; ETS max, maximal electron transport system capacity; NAD, nicotinamide adenine dinucleotide, oxidized form; NADH, nicotinamide adenine dinucleotide, reduced form. (J) Oxygen consumption rate of mitochondrial respiration in the VTA of mice with or without morphine EDA. 2-way RM ANOVA, n = 4–5 mice per group. Data are presented as mean ± SEM; *P < 0.05, ***P < 0.001, ****P < 0.0001.
Figure 4
Figure 4. Chronic morphine administration increases the VTA neuronal mitophagy and mitochondrial fragmentation in the VTA dopaminergic Mor-Ens.
(A) Experimental scheme for fiber photometry to detect mitophagy in Mor-Ens. Representative images show the expression of mt-keima in the VTA neurons. Dashed white lines outline the VTA and fiber optic tract. Scale bar, left: 200 μm; right: 20 μm. (B) Relative mitochondrial autophagy induction (% normalized to baseline) in the VTA neurons after morphine EDA. Paired t test, n = 9. (C) Experimental scheme to analyze mitochondrial morphology in Mor-Ens of mice in saline ctrl and morphine EDA groups. (D) Representative images of dopaminergic Mor-Ens expressing EYFP and mito-tdTomato. ChR2-EYFP was used to label dendrites and mito-tdTomato was used to label mitochondria. The white solid lines indicate primary dendrites and the dashed lines indicate secondary dendrites in each channel. Scale bar: 20 μm. (E and I) Representative images of primary dendrites (E) and secondary dendrites (I) containing labeled mitochondria from saline-ctrl, and withdrawal (WD) mice 1 day and 21 days after morphine EDA. Red, mito-tdTomato; Green, EYFP. Scale bars: 10 μm in E; 5 μm in I. (F–H) Quantification of mitochondrial aspect ratio (F), length (G), and area (H) in primary dendrites of dopaminergic Mor-Ens in saline-ctrl (38 neurons in 6 mice), morphine-EDA WD 1 day (40 neurons in 8 mice), or WD 21 day (40 neurons in 5 mice) groups. (J–L) Quantification of mitochondrial aspect ratio (J), length (K), and area (L) in dopaminergic Mor-Ens in saline-ctrl (36 neurons in 6 mice), morphine-EDA WD 1 day (35 neurons in 8 mice), or WD 21 day (38 neurons in 5 mice) groups. 1-way ANOVA with Bonferroni’s test and Kolmogorov-Smirnov test (F–H and J–L). Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 5
Figure 5. The expression of Mfn1 is decreased in VTA dopaminergic Mor-Ens after chronic morphine administration.
(A and B) Experimental scheme to label (A) and purify (B) the ribosome-associated mRNA from the dopaminergic ensembles expressing NBL10-HA. (C and D) Quantification of the relative mRNA levels of dnm1l, fis1, mfn1, mfn2, and opa1 in VTA dopaminergic Mor-Ens at different time points after morphine EDA (normalized to the mice without morphine EDA). 6–7 mice per group, 1-way ANOVA with Bonferroni’s test. (E) Experimental scheme of the time line for VTA tissue collection. (F) Representative smFISH images of Mfn1 mRNA expressing in the VTA Mor-Ens at different time points after morphine EDA. Red, tdTomato; Green, Mfn1; Blue, DAPI. Dashed white lines outline the tdTomato+ cells. Scale bar: 20 μm. (G) Quantification of the Mfn1 mRNA in the tdTomato+ Mor-Ens at different time points after morphine-EDA groups (normalized to the mice without morphine EDA). Cumulative frequency distribution of Mfn1 mRNA intensity in the tdTomato+ Mor-Ens at different time points after morphine-EDA groups. n = 5 mice per group, baseline, 707 cells; wd 1 day, 743 cells; wd 1 week, 594 cells; wd 2 weeks, 669 cells; wd 4 weeks, 253 cells. 1-Way ANOVA with Bonferroni’s test, Kolmogorov-Smirnov test for cumulative frequency distribution. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 6
Figure 6. Overexpression of MFN1 in dopaminergic Mor-Ens alleviates withdrawal symptoms after chronic morphine administration in both male and female mice.
(A) Experimental scheme to assess the effect of MFN1 overexpression in dopaminergic Mor-Ens. (B) Representative smFISH images of Mfn1 mRNA expressed in the VTA Mor-Ens. Red, Mfn1; Green, Egfp; Blue, DAPI. Dashed white lines outline the Egfp+ cells. Scale bars: 20 μm; 5 μm. (C) Quantification of the Mfn1 mRNA in the Egfp+ cells from EGFP and MFN1-EGFP groups. 3–4 mice per group, Egfp, 121 cells; Mfn1, 91 cells. (D) Representative images of nitrotyrosine immunostaining in the VTA Mor-Ens expressing EGFP or MFN1-EGFP. Red, nitrotyrosine; Green, EGFP; Blue, DAPI. Dashed white lines outline the EGFP+ cell. Scale bars: 20 μm; 5 μm. (E) The normalized expression level of nitrotyrosine in the VTA EGFP+ Mor-Ens. 3 mice per group, EGFP, 381 cells; MFN1, 434 cells. Unpaired t test or Kolmogorov-Smirnov test. (F–O) The effect of MFN1 overexpression in the VTA dopaminergic Mor-Ens on naloxone-precipitated withdrawal symptoms in both male and female mice. Weight loss, diarrhea, jumps, wet dog shakes, and body tremors were analyzed in EGFP and MFN1-EGFP groups. Male: 12–13 mice per group; female: 13 mice per group. Unpaired t test or Mann-Whitney test. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 7
Figure 7. Restoration of MFN1 expression in dopaminergic Mor-Ens alleviates withdrawal-induced negative affects in both male and female mice.
(A–J) The effect of MFN1 overexpression in Mor-Ens on negative affect during the spontaneous and prolonged morphine withdrawal in both male and female mice. Morphine withdrawal-induced CPA (A and F), locomotor activity (B and G), immobility time in TST test (C and H), time in the open arm in EPM test (D and I), and social novelty scores (E and J) were analyzed in EGFP and MFN1-EGFP groups. Male: 11–22 mice per group; female: 12–14 mice per group. Unpaired t test or Mann-Whitney test, 2-way RM ANOVA with Bonferroni’s test in CPA. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 8
Figure 8. Mitochondrial division inhibitor Mdivi-1 restores the mitochondrial respiration of the VTA and ameliorates mitochondrial fragmentation in VTA dopaminergic Mor-Ens.
(A) Experimental scheme to assess the mitochondrial respiration of VTA of mice in vehicle and Mdivi-1 groups. (B) Oxygen consumption rate of mitochondria in the VTA following morphine EDA in vehicle and Mdivi-1 groups. 5 mice per group, 2-way RM ANOVA by Bonferroni’s test. (C) Experimental scheme for tracing mitochondrial morphology in dopaminergic Mor-Ens after morphine EDA in mice of Mdivi-1 or vehicle groups. (D) Representative images of primary dendrites (green) containing mitochondria (red) after morphine EDA in Mdivi-1 or vehicle groups. Red, mito-tdTomato; Green, EYFP. Scale bar: 10 μm. (E–G) Quantification of mitochondrial aspect ratio (E), length (F), and area (G) in primary dendrites of dopaminergic Mor-Ens in vehicle (36 neurons in 6 mice) and Mdivi-1 groups (30 neurons in 8 mice). Unpaired t test and Kolmogorov-Smirnov test. Data are presented as mean ± SEM; n.s. not significant; *P < 0.05, **P < 0.01, ****P < 0.0001.
Figure 9
Figure 9. Mdivi-1 restores the maladaptation of neuronal plasticity in VTA dopaminergic Mor-Ens.
(A) Experimental scheme of electrophysiological recording in dopaminergic Mor-Ens from saline and morphine EDA groups treated with vehicle or Mdivi-1. (B) Quantification of the rheobase and threshold of the action potentials in VTA tdTomato+ neurons. 4–5 mice per group; rheobase: 16–22 neurons per group, threshold: 17–24 neurons per group. (C) Representative AP traces and (D) quantification of the induced spike numbers of tdTomato+ neurons in the VTA. 13–18 neurons from 3–4 mice per group. (E) Representative traces and quantification of the spontaneous firing rate of tdTomato+ neurons in the VTA. 15–21 neurons from 3–4 mice per group. (F) Representative traces and quantification of the EPSC/IPSC ratio of tdTomato+ neurons in the VTA. 12–19 neurons from 3–4 mice per group. 2-way ANOVA by Bonferroni’s test in B, E,and F, 2-way RM ANOVA in D. Sal, saline; Mor, morphine. Data are presented as mean ± SEM; n.s. not significant; *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 10
Figure 10. Mdivi-1 alleviates withdrawal symptoms and negative affects after chronic morphine administration in both male and female mice.
(A) Experimental scheme to measure the naloxone-precipitated withdrawal symptoms in both male and female mice. (B–F) The effects of Mdivi-1 on weight loss (B), diarrhea (C), wet dog shakes (D), body tremor (E), and backward locomotion (F) were analyzed in mice from vehicle and Mdivi-1 groups. Male, 10-12 mice per group; female, 15 mice per group. Unpaired t test or Mann-Whitney test. (G–O) The effect of Mdivi-1 on negative affects during spontaneous and chronic morphine withdrawal in both male and female mice. (G) Experimental scheme of the behavioral tests. Immobility time (H and I), morphine withdrawal-induced CPA (J and M), time in the open arm (K and N), and social preference (L and O) were analyzed in vehicle and Mdivi-1 pretreated groups. Male, 11–12 mice per group; female, 14–15 mice per group. Unpaired t test or Mann-Whitney test for 2 groups, 2-way RM ANOVA with Bonferroni’s test in CPA. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 11
Figure 11. Mdivi-1 alleviates the development of morphine-induced reinforcement and drug seeking after prolonged withdrawal.
(A) Morphine-induced hyperlocomotion in mice treated with Mdivi-1 (50 mg/kg, i.p.) or vehicle 45 minutes before the morphine injection. 10 mice per group, 2-way RM ANOVA. (B) Quantification of the morphine CPP scores in the mice injected with Mdivi-1 or vehicle 45 minutes before each conditioning session. 10 mice per group, 2-way RM ANOVA with Bonferroni’s test. (C) Experimental scheme to assess the effect of Mdivi-1 on drug seeking in a morphine SA paradigm. Mdivi-1 or vehicle was administrated during the 11–16 training sessions. (D) Numbers of morphine infusions in mice during the training sessions. (E and F) Plots of active nose pokes at 1 and 14 days after morphine withdrawal in mice from Mdivi-1 or vehicle groups. 7 to 8 mice per group, 2-way RM ANOVA, unpaired t test or Mann Whitney test. Data are presented as mean ± SEM; n.s. not significant; *P < 0.05.
Figure 12
Figure 12. Mdivi-1 alleviates the development of analgesic tolerance of morphine.
(A) The effect of Mdivi-1 on acute analgesia of morphine (10 mg/kg, i.p.) in hotplate assay. The latency of withdrawal to noxious stimulus is shown as the percentage of the maximum possible effect (% MPE). 12–13 mice per group. (B) The effect of Mdivi-1 on analgesic tolerance of chronic morphine (10 mg/kg, once daily) administration for 6 days. Analgesic tolerance was mirrored by the decreased % MPE. 12–15 mice per group, 2-way RM ANOVA with Bonferroni’s test for Morphine (Mor) + vehicle versus Morphine + Mdivi-1 (12.5, 25, 50, 100 mg/kg). (C) The effect of Mdivi-1 on acute analgesia of morphine (10 mg/kg, i.p.) in the tail flick assay. (D) Quantification of the analgesic tolerance of chronic morphine (10 mg/kg, once daily) in mice from Mdivi-1 (50 mg/kg) or vehicle groups. 12 mice/group, 2-way RM ANOVA with Bonferroni’s test in C and D. (E) Respiratory inhibition of morphine assessed by whole-body plethysmography in mice. Respiratory frequency is decreased 15 minutes after morphine injection (10 mg/kg, i.p). 2-way RM ANOVA by Bonferroni’s test, saline versus morphine, 4 mice/group; Morphine + vehicle versus Morphine + Mdivi-1, 8 mice per group. (F) Constipation effects of morphine assessed by accumulated faecal boli in Mdivi-1 or vehicle groups. 10–12 mice per group, 2-way RM ANOVA by Bonferroni’s test for saline + vehicle versus morphine + vehicle or morphine + vehicle versus Morphine + Mdivi-1. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 13
Figure 13. Strategy of targeting mitochondrial dynamics in morphine-responsive VTA dopaminergic ensembles to alleviate opiate withdrawal.
Schematic diagram illustrating that chronic morphine administration decreases mitochondrial Ca2+ uptake and induces excessive fragmentation and oxidative stress in morphine-responsive VTA dopaminergic neurons, leading to the dysregulated mitochondrial respiration and maladaptation of the neuronal plasticity in the VTA. Genetic and pharmacological targeting the mitochondrial dynamics corrects mitochondrial fragmentation and neuronal plasticity in morphine-responsive VTA dopaminergic neurons and alleviates morphine withdrawal symptoms and negative affects.
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