Monitoring calcium concentration in dendritic spines of cultured hippocampal neurons with cameleons

摘要

Transient and substantial elevation of postsynaptic calcium was important for hippocampal long-term potentiation (LTP),so detection of calcium changes in spine was necessary to understand the mechanisms underlying synaptic plasticity.Unfortunately most recent calcium fluorescence indicators severely perturbed calcium transients, and traditionalcameleons’ poor dynamic ranges prevented detection of changes of calcium. We presented a new method to monitorquantificationally free calcium concentration in dendritic spines with a new yellow cameleon (YC3.60) basing on cultureof hippocampal neurons and calcium phosphate transfection technique and confocal microscopy with 458nm laser. Intransiently transfected hippocampal neurons, the ratio of YFP to CFP was detected as FRET level. In our study, we gotthe parameters of YC3.60 excited with 458nm laser. Under control conditions, FRET levels in different dendritic spinesof cultured hippocampal neurons were diverse but showed robust increases upon treatment with potassium chloride.FRET levels in different parts of hippocampal neurons were also different, the calcium concentration decreased with thedistance from soma. These results suggested that the FRET methodology with YC3.60 could monitor calciumconcentration in spines and it might be useful in analyzing mechanisms underlying synaptic plasticity.