Morphine regulates dopaminergic neuron differentiation via miR-133b

Abstract

Morphine is one of the analgesics used most to treat chronic pain, although its long-term administration produces tolerance and dependence through neuronal plasticity. The ability of morphine to regulate neuron differentiation in vivo has been reported. However, the detailed mechanisms have not yet been elucidated because of the inability to separate maternal influences from embryonic events. Using zebrafish embryos as the model, we demonstrate that morphine decreases miR-133b expression, hence increasing the expression of its target, Pitx3, a transcription factor that activates tyrosine hydroxylase and dopamine transporter. Using a specific morpholino to knock down the zebrafish μ-opioid receptor (zfMOR) in the embryos and selective mitogen-activated protein kinase inhibitors, we demonstrate that the morphine-induced miR-133b decrease in zebrafish embryos is mediated by zfMOR activation of extracellular signal-regulated kinase 1/2. A parallel morphine-induced down-regulation of miR-133b was observed in the immature but not in mature rat hippocampal neurons. Our results indicate for the first time that zebrafish embryos express a functional μ-opioid receptor and that zebrafish serves as an excellent model to investigate the roles of microRNA in neuronal development affected by long-term morphine exposure.

Fig. 1.

Effect of morphine on the expression of miRNA-133b, Pitx3, TH and DAT. A, qRT-PCR analyses of miRNA-133b levels in 24-hpf zebrafish embryos at two different morphine concentrations, 10 and 1 nM. B, the Pitx3 levels measured at both concentrations of morphine with qRT-PCR. C, the level of TH transcript measured with qRT-PCR in morphine-exposed embryos. D, the level of DAT transcript measured with qRT-PCR in embryos exposed to morphine. In all cases, the levels of miR-133b and all three transcripts were measured by qRT-PCR in control embryos (no drug exposure), embryos exposed to naloxone only, or embryos exposed to morphine and naloxone. *, P ≤ 0.05; **, P ≤ 0.005 (unpaired Student's t test with Welch correction), n = 3.

Fig. 2.

Effect of the injection of Pitx3 3′UTR on the expression of miRNA-133b, Pitx3, TH, and DAT. Analyses of the level of miR-133b (A), Pitx3 (B), TH (C), and DAT (D) by qRT-PCR in 24-hpf zebrafish embryos injected with 100 pg of Pitx3 3′UTR. *, P ≤ 0.05; **, P ≤ 0.005 (unpaired Student's t test with Welch correction), n = 3.

Fig. 3.

Effect of MOR knockdown on the expression of miRNA-133b, Pitx3, TH, and DAT. The morpholino oligonucleotide technique was used to knock down the μ-opioid receptor from zebrafish as described under Materials and Methods. The consequence of MOR knockdown on the levels of miR-133b (A), Pitx3 (B), TH (C), and DAT (D) in control and 1 or 10 nM morphine-treated embryos were determined by qRT-PCR as described under Materials and Methods. Data were analyzed by two-way analysis of variance with post hoc Bonferroni test for comparisons. Error bars, S.E.M.. *, P ≤ 0.05; **, P ≤ 0.005, n = 3.

Fig. 4.

Effect of the inhibition of ERK 1/2 on the expression of miRNA-133b, Pitx3, TH, and DAT. A, qRT-PCR analyses of miRNA-133b expression levels in 24-hpf zebrafish embryos exposed to the ERK 1/2 inhibitors PD98059 (PD) and U0126 (U) added at 5 or t 16 hpf. B, the Pitx3 gene expression levels are also measured in the same embryos, as well as TH (C) and DAT (D). qRT-PCR analyses were also carried out in 24-hpf zebrafish embryos treated simultaneously with 10 nM morphine and ERK 1/2 inhibitors. Data were analyzed by two-way analysis of variance with post hoc Bonferroni test for comparisons. Error bars, S.E.M. *, P ≤ 0.05; **, P ≤ 0.005, n = 3.

Fig. 5.

Effect of morphine on the expression of miRNA-133b in mammalian neurons. The level of miRNA-133b in 1-week (A) and 3-week (B) hippocampal neuron culture treated with 100 nM morphine, 1 μM naloxone, or morphine and naloxone simultaneously were determined by qRT-PCR. *, P ≤ 0.05; **, P ≤ 0.005 (unpaired Student's t test with Welch correction), n = 4.