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

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