Modulations of NeuroD activity contribute to the differential effects of morphine and fentanyl on dendritic spine stability - PubMed (original) (raw)

Modulations of NeuroD activity contribute to the differential effects of morphine and fentanyl on dendritic spine stability

Hui Zheng et al. J Neurosci. 2010.

Abstract

The cellular level of neurogenic differentiation 1 (NeuroD) is modulated differentially by mu-opioid receptor agonists; fentanyl increases NeuroD level by reducing the amount of microRNA-190 (miR-190), an inhibitor of NeuroD expression, whereas morphine does not alter NeuroD level. In the current study, NeuroD activity was demonstrated to be also under agonist-dependent regulation. After 3 d of treatment, morphine and fentanyl decreased the activity of the Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIalpha), which phosphorylates and activates NeuroD. Because NeuroD activity is determined by both the CaMKIIalpha activity and the cellular NeuroD level, the overall NeuroD activity was reduced by morphine, but maintained during fentanyl treatment. The differential effects of agonists on NeuroD activity were further confirmed by measuring the mRNA levels of four NeuroD downstream targets: doublecortin, Notch1, neurogenic differentiation 4, and Roundabout 1. Decreased dendritic spine stability and mu-opioid receptor signaling capability were also observed when NeuroD activity was attenuated by miR-190 overexpression or treatment with KN93, a CaMKIIalpha inhibitor. The decrease could be rescued by NeuroD overexpression, which restored NeuroD activity to the basal level. Furthermore, elevating NeuroD activity attenuated the morphine-induced decrease in dendritic spine stability. Therefore, by regulating NeuroD activity, mu-opioid receptor agonists modulate the stability of dendritic spines.

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Figures

Figure 1.

Figure 1.

Morphine, but not fentanyl, decreases NeuroD activity. Primary rat hippocampal cultures were treated with PBS (control), 1 μ

m

morphine, or 10 n

m

fentanyl for 3 d. A, The amount of phosphorylated serine was determined in immunoprecipitated NeuroD to indicate NeuroD phosphorylation (pND). Total NeuroD (ND) was measured in the nuclear extracts. The total CaMKIIα (CaM), phosphorylated CaMKIIα (pCaM), total ERK, and β-actin were detected in whole-cell lysis. Total ERK and β-actin served as internal controls. IP, Immunoprecipitation; IB, immunoblotting. B, The levels of miR-190 and NeuroD mRNA were measured by real-time PCR. C, mRNA levels of DCX, ND4, Notch1, and Robo1 were determined by real-time PCR. A–C, Results were normalized against those in control samples. Error bars indicate SD. *p < 0.05.

Figure 2.

Figure 2.

NeuroD level correlates with its activity. Primary cultures were treated with PBS (control), con-vir, 190-vir, 190-vir plus nd-vir, and nd-vir for 3 d. A, The amounts of phosphorylated NeuroD (pND), total NeuroD (ND), total CaMKIIα (CaM), phosphorylated CaMKIIα (pCaM), total ERK, and β-actin were determined as in Figure 1_A_. B, The levels of miR-190 and NeuroD mRNA were measured by real-time PCR. C, The mRNA levels of DCX, ND4, Notch1, and Robo1 were determined with real-time PCR. A–C, Results were normalized against those in control samples. IP, Immunoprecipitation; IB, immunoblotting. Error bars indicate SD. *p < 0.05.

Figure 3.

Figure 3.

NeuroD activity correlates with its phosphorylation by CaMKIIα. Primary cultures were treated with PBS (control), KN92 (2 μ

m

), KN93 (2 μ

m

), or KN93 (2 μ

m

) plus nd-vir for 3 d. A, The amount of phosphorylated NeuroD (pND), total NeuroD (ND), total CaMKIIα (CaM), phosphorylated CaMKIIα (pCaM), total ERK, and β-actin were determined as in Figure 1_A_. B, The levels of miR-190 and NeuroD mRNA were measured by real-time PCR. C, The mRNA levels of DCX, ND4, Notch1, and Robo1 were determined with real-time PCR. A–C, Results were normalized against those in control samples. IP, Immunoprecipitation; IB, immunoblotting. Error bars indicate SD. *p < 0.05.

Figure 4.

Figure 4.

Spine collapse and new spine formation during control PBS treatment. Primary cultures from rat hippocampal neurons were treated with PBS (control) for 3 d. A, B, Live-cell images on day 0 (A) and day 3 (B) of a representative neuron were presented. Spine densities on day 0 and day 3, the numbers of newly formed spines, and the number of spines that collapsed, shrank, or enlarged during 3 d are also summarized in C. Error bars indicate SD.

Figure 5.

Figure 5.

NeuroD activity is required to maintain spine stability. Primary cultures were treated with PBS (control), con-vir, 190-vir, 190-vir plus nd-vir, nd-vir, KN92 (2 μ

m

), KN93 (2 μ

m

), or KN93 (2 μ

m

) plus nd-vir for 3 d. A, Live-cell images of a portion of a single dendrite from a representative neuron on day 0 and day 3. EGFP and ECFP were used to indicate successful infection of con-vir/190-vir and nd-vir, respectively. Stars indicate the spines' decrease in size. B, Spine densities on day 0 and day 3 were quantified. C, Spine volumes were calculated by the overall DsRed fluorescence and the fluorescence intensity on day 3 was normalized against that on day 0. D, he apoptosis rates were also determined, with neurons treated with 1 μ

m

staurosporin for 24 h serving as positive control. Error bars indicate SD. *p < 0.05.

Figure 6.

Figure 6.

Overexpressing NeuroD counteracts morphine-induced decrease in NeuroD activity. Primary cultures were treated with PBS, morphine (1 μ

m

), morphine (1 μ

m

) plus nd-vir, fentanyl (10 n

m

), fentanyl (10 n

m

) plus 190-vir, or fentanyl (10 n

m

) plus KN93 for 3 d. A, The amount of phosphorylated NeuroD (pNeuroD), total NeuroD, total CaMKIIα (CaM), phosphorylated CaMKIIα (pCaM), total ERK, and β-actin were determined as in Figure 1_A_. B, The levels of miR-190 and NeuroD (ND) mRNA were measured by real-time PCR. IP, Immunoprecipitation; IB, immunoblotting. C, The mRNA levels of DCX, ND4, Notch1, and Robo1 were determined by real-time PCR. A–C, Results were normalized against those in control samples. Error bars indicate SD. *p < 0.05.

Figure 7.

Figure 7.

Elevating NeuroD activity attenuates morphine-induced decrease in spine stability. Primary hippocampal neuron cultures were treated with PBS (control), DMSO (0.1%), morphine (1 μ

m

), morphine (1 μ

m

) plus nd-vir, fentanyl (10 n

m

), fentanyl (10 n

m

) plus 190-vir, or fentanyl (10 n

m

) plus KN93 for 3 d. A, Live-cell images of a portion of a dendrite from a representative neuron on day 0 and day 3. Stars indicate the spines' decrease in size. B, Spine densities on day 0 and day 3. C, Spine volumes were measured by the overall DsRed fluorescence, and the fluorescence intensity on day 3 was normalized against that on day 0. D, Apoptosis rates were measured as in Figure 5_D_. Error bars indicate SD. *p < 0.05.

Figure 8.

Figure 8.

Schematic summary of agonist-dependent regulation on NeuroD activity.

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