CFTR Involves In Ultrasound Induced Neural Modulation In Cultured Cortical Neurons

Background:Ultrasound(US)is widely used in diagnostic imaging in clinic.As ultrasound can penetrate long distance with minimal energy lost,and can precisely focus on local tissue,so ultrasound can be more widely applied.Focused ultrasound can be used to treat tumor and lithiasis.In central nervous system,high intensity ultrasound can treat some diseases such as Parkinson's disease and serious depression.Moderate intensity ultrasound can regulate the activity of neurons without killing neurons.Recent years,the use of ultrasound in noninvasive regulation of neuronal activity has become a new research direction in the field of neurology.Ultrasound can aim and affect the brain at high precision,enhance or inhibit neuronal activity and has great potential in the treatment of movement disorders,depression,anxiety and some refractory neuropsychiatric disorders.In vivo experiments in animals showed that ultrasound can act on motor cortex and directly induce muscle contraction and cause limb movement,act on sensory cortex and induce sense,which makes it possible for ultrasound to carry out noninvasive brain stimulation.There are several function mechanisms of ultrasound on the body.At present,it is more accepted to be a kind of mechanical force.Ultrasound could act on mechanical sensitive channel on the cell membrane.It has been reported that ultrasound can open the double pore potassium channel heterologous expressed on Xenopus oocytes.However,the research about effect of ultrasound on the neurons related to mechanical sensitive channel is rare.Cystic fibrosis transduction regulator(CFTR)as an anion channel mediates chloride ion through membrane,it has been shown to be mechanosensitive,which can feel the deformation of cell membrane and then cause current.But we don't know if CFTR can be activated by ultrasound,and if CFTR channel involves in the effect of ultrasound on neurons.The purpose of this study is to investigate the role of CFTR in the ultrasound neuronal effect and to reveal the cell and channel mechanism of the neuronal regulatory role of ultrasound.Methods:We designed four parts of experiments to measure the role of CFTR in ultrasound activation of cortical neurons:the activation effect of ultrasound on wild type neurons;the role of CFTR in ultrasound activation of wild type neurons;the effect of ultrasound on CFTR knockout neurons;and the effect of ultrasound on CFTR transfected HEK293 cells.Firstly,C57 fetal mice cortical neurons were primary cultured to 7-12 days.Electric physiological experiment was performed by both voltage clamp and current clamp to test the effect of ultrasound on cortical neuron,spikes/action potential and current was recorded with or without ultrasound.Anti-cFos immunofluorescent staining was performed to test the effect of ultrasound to the whole coverslip and fluorescent intensity was compared between none US stimulation and US stimulation.Second,confirm CFTR expression on neurons by immunofluorescent staining and Western blot.Thirdly,CFTR gene knockout fetal mice CFTR-/-cortical neurons were also primary cultured to 7-12 days.Voltage clamp was used to record the current stimulated by ultrasound,and the current frequency and amplitude was measured and compared with wild type mice.The whole neuron crawling coverslip was stained with c-Fos antibody after ultrasonic stimulation,and c-F os expression was measured and compared with the wild type mice.Finally,CFTR plasmid was transfected to HEK293 cells,and the current induced by US stimulation was recorded using whole cell voltage clamp.Results:1.US increased firing rate(both intracellular and extracellular recording)(p<0.001),triggered inward current(current frequency compared with PreUS p<0.05),increased c-Fos expression(fluorescent intensity compared with PreUS p<0.01)on primary cultured mice neurons.2.CFTR expression on mice cortical neurons was confirmed by immunofluorescent staining,RT PCR and Western Blot.The increased firing rate and inward current induced by US was inhibited by CFTR inhibitor GlyH 101(compared with pre GlyH101 application p<0.001 and p<0.01,respectively).Increased c-Fos expression induced by US was also inhibited by GlyH 101(compared with pre GlyH 101 application,p<0.05).3.Inward currents were also triggered by ultrasound in CFTR-/-cortical neurons but with less frequency compared with wild type neurons(p<0.05).c-Fos expression was also increased but extent was less than wild type neurons(p<0.05).4.Inward currents were triggered by US stimulation in CFTR tansfescted HEK293 cells but not none tansfescted HEK293 cells,and CFTR inhibitor GlyH101 could inhibit the current(compared with pre GlyH101 application p<0.01).Conclusion:The research showed that US can activate mice cultured cortical neuron.CFTR expressed on cortical neurons and CFTR involved in US activation of mice cortical neurons.CFTR knock out neurons showed reduced reaction to US stimulation;CFTR transfected HEK293 cells gained the reaction to US stimulation.To summary,CFTR involved in US activation of mice cultured cortical neurons.