A FLUORESCENCE TECHNIQUE TO MEASURE INTRACELLULAR PH OF SINGLE NEURONS IN BRAIN-STEM SLICES

We have developed a technique to measure the pH(i) of single neurons i n brainstem slices using a fluorescence imaging system. Slices were lo aded with the pH-sensitive fluorescent dye BCECF and fluorescence was visualized by exciting the slices alternately at 500 and 440 nm. The e mitted fluorescence at 530 nm was directed through an MTI GenIISys ima ge intensifier and MTI CCD72 camera. The images were processed by Imag e-1/FL software. The ratio of emitted fluorescence at excitation wavel engths of 500 and 440 nm was measured and converted to pH by construct ing 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 la beled 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, gli al fibrillary acidic protein, showed a diffuse, background staining. N eurons that were retrograde-labeled with rhodamine beads fluoresced as large spheres that exactly correlated with the fluorescence from BCEC F-loaded cells. Further, large fluorescent spheres had membrane potent ials of about -60 mV and generated action potentials. These findings i ndicate that the large fluorescent spheres are neurons. pH(i) was meas ured in these large spheres (neurons) in the dorsal and ventral medull ary chemosensitive regions, and was 7.32+/-0.02 (n=110) and 7.38+/-0.0 2 (n=85), respectively.