The Role Of Caveolin-1 In Morphine-induced Structural Plasticity Of Primary Cultured Murine Cortical Neurons

Background As one of the opioid analgesics,morphine is widely used for chronic pain,but its long-term usage may result in addiction.Morphine-induced neuronal plasticity is reported as one of the main molecular mechanisms underlying drug-dependence.Caveolin-1(Cav-1),is a scaffolding protein of membrane/lipid rafts(MLRs)widespread throughout the CNS,organizing neuronal receptors and cytoskeletal dynamics modulators and participates in the processes of signal transduction,neurite growth and synapse formation/stabilization.It is reported that Cav-1 is a key modulator of neuronal plasticity.However,the role of Cav-1 in morphine-induced neuronal structural plasticity was not fully under understood yet.In this research,the effect of Cav-1 on the expression of Growth association protein-43(GAP-43)and Microtube-associated protein2(MAP-2)in the process of morphine-induced neuroplasticity were studied in primary cultured cortical neurons of mice.Methods Primary cultured neurons were isolated from the cortex of C57/BL6 mice at postnatal 24 h.First of all,the morphine exposure model of neuron was built.the neurons were divided into 4 groups and treated with phosphate buffered solution(PBS)or different concentration of morphine(1.0?10.0?100.0 ?mol/L)at cultured day 7 for another 48 h,respectively.Then they were subjected to MTT assay and immunoblot to find out a proper concentration of morphine,which could have significant impact on Cav-1 expression without doing harm to neuronal viability.With that concentration of morphine,q RT-PCR was preformed to figure out the possible mechanism of morphine-induced Cav-1 expression.The level of GAP-43 and MAP-2 labeled neurite outgrowth was detected by immunoblot and immunofluorescence confocal microscopy.Secondly,for further investigate the role of Cav-1 in morphine-induced structural plasticity,neurons were transfected with lenti virus-based Cav-1 si RNA to inhibit the expression level of Cav-1 or scramble si RNA as negative control at cultured day 4 for 72 h,then treated with PBS or morphine at final concentration of 10.0 ?mol/L at day 7 for 48 h,respectively.GAP-43 levels were determined using immunoblot analysis,and MAP-2 possitive neurite using immunofluorescence confocal microscopy and high content analysis system.Results 1)The MTT assay demonstrated that 10.0 ?mol/L morphine exposure had no adverse impact on neuron viability and could cause an up-regulation of Cav-1,assessed by immunoblot analysis.2)The q RT-PCR results revealed a significant increase in m RNA level of Cav-1 in neurons of morphine groups when compared with that of control.Increase of GAP-43 and MAP-2 labeled primary neurite outgrowth and branching following 10.0 ?mol/L morphine treatment was found by immunoblot and immunofluorescence confocal microscopy,indicating an enhanced neuronal plasticity.3)Both morphine-induced up-regulation of GAP-43 and neurite outgrowth labeled by MAP-2 were inhibited by Lentish RNACav1 mediated Cav-1 knockdown.As assessed by western blot analysis,immunofluorescence confocal microscopy and high content analysis(HCA),morphine-induced up-regulation of GAP-43 as well as dendritic growth and arborization of neurons labeled by MAP-2 was inhibited significantly by the Cav-1 si RNA-mediated Cav-1 knockdown,suggesting the key role of Cav-1 in morphine-induced neuronal plasticity.Conclusions Morphine exposure enhanced structural plasticity in primary cultured cortical neurons.Cav-1 was a key modulator in that process.The inhibition of Cav-1 expression reduced the increase of neurite growth markers GAP-43 and MAP-2 by morphine treatment,indicating that the suppression neurite outgrowth.Cav-1 might be a potential target of the treatment of morphine addiction.