A Fluorescence Technique to Measure Intracellular pH of Single Neurons in Brainstem Slices

摘要: We have developed a technique to measure the pHi of single neurons in brainstem slices using fluorescence imaging system. Slices were loaded with pH-sensitive fluorescent dye BCECF and was visualized by exciting alternately at 500 440 nm. The emitted 530 nm directed through an MTI GenIISys image intensifier CCD72 camera. images processed Image-1/FL software. ratio excitation wavelengths measured converted pH constructing calibration curve high K+/nigericin solutions values ranging from 5.8 8.6. BCECF-loaded showed distinct spheres intense diffuse background fluorescence. labeled neuron-specific antibody, enolase, staining that correlated cells. glial-specific glial fibrillary acidic protein, diffuse, staining. Neurons retrograde-labeled rhodamine beads fluoresced as large exactly Further, had membrane potentials about −60 mV generated action potentials. These findings indicate are neurons. these (neurons) dorsal ventral medullary chemosensitive regions, 7.32 ± 0.02 (n = 110) 7.38 85), respectively.