| [Objective]The cerebellum is an important structure for motor balance and coordination.The external tactile information is transmitted to the only efferent neuron in the cerebellum,Purkinje cell(PC),through mossy fiber(MF)-granule cell(GrC)-parallel fiber(PF)pathway,to induce PF-PC and PF-molecular layer interneurons(MLIs),However,The mechanism by which tactile stimulation induces synaptic transmission between MLI and PC,as well as the relationship between neuronal electrical signals and Ca2+transient characteristics in this process,are still unclear.Therefore,in this study,we used in vivo laser-beam two-photon Ca2+ imaging technology,electrophysiological and pharmacological methods to study the Ca2+transient characteristics of mouse cerebellar Purkinje cell(PC)and MLIs caused by facial hair blowing stimulation,and to explore the relationship between Ca2+imaging and electrical signals of PC.[Methods]In this study,we use real-time two-photon imaging technology,inject adenovirus with fluorescent dyes in the cerebellar Crus Ⅱ area to make targeted cells express Ca2+signals,and the PC specifically expressed transgenic mice L7-Cre and C57BL/6J mice were injected with red fluorescent protein Ca2+ indicator carried AAV1-CAG-DIORCaMP(5.0×1012 gc/ml)and AAV1-Syn-NES-RGECO(5.73×1012 gc/ml),which labeled PC and MLI.The optical and electrical signals of the targeted cells were observed by combining whole-cell and cell-attached electrophysiological recording techniques,at the same time Gabazine was applied pharmacologically to block GABAA receptors to observe the functions of MLIs on facial air-puff stimuli-evoked Ca2+transients of PCs.All two-photon imaging images and data were collected by ThorImage.LS 3.0 and analyzed and processed by ImageJ.Clampfit 10.4 software was used to analyze the electrophysiological data.The data of each group were expressed as Mean±SEM,and the paired t-test in SPSS software or one-way ANOVA(electrophysiological results and two-photon imaging results)were used for statistical analysis.P<0.05 was considered statistically significant.[Results]1.Air-puff stimulation obviously evoked Ca2+transients on PC dendrites,the fluorescent intensity was 39.6±2.2%,which significantly increased in comparing with pre-stimuli(1.1±1.4%,P<0.05).2.Air-puff stimulation also evoked Ca2+transients on PC somas(Control:0.1±1.2%;Stimuli:2.4 ± 0.8%.P<0.05),but not so obvious as dendrites.3.Setting PC soma as the datum mark,35um as a unit,we marked four locations(same areas of ROIs)on PC dendrites to observe the Ca2+transients by different distances,showing that the variation of Ca2+transients on somas is the least,the middle of dendrites is the biggest.(From soma to the end of dendrites:2.8 ± 1.2%;20.2±1.2%;52.6 ±1.5%;43.8 ± 1.3%.The dendrites compared to somas:P<0.05).4.Blocking GABAA receptors by Gabazine we find that the Ca2+transients on dendrites are decreased(Control:39.6 ± 2.2%;Gabazine:24.2 ±1.4%.P<0.05).5.Combining with the electrophysiology results,the air-puff stimulation-evoked IPSC on PC dendrites are conversed into EPSC by blocking GABAA receptors,meanwhile the Ca2+transients decreased slightly.Nevertheless the incident that the evoked IPSC transform into evoked action potential is not particularly relevant to the variation of Ca2+ transients on PC somas.6.Air-puff stimulation evoked Ca2+ transients on the somas and dendrites of MLIs(Dendrites control:0.2±0.9%;Dendrites stimuli:4.3±2.0%;Somas control:0.2±1.9%;Somas stimuli:4.0±1.5%.P<0.05),which is weaker than that of PCs.[Conclusion]Tactile stimulation increases PC and MLI Ca2+transients through PF-PC/PF-MLI synapses;Blocking MLI inhibitory afferent attenuates PC Ca2+transients induced by tactile stimulation. |
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