Increase In Intracellular Sodium Evoked by Glutamate Transporter Activation In Cortical Astrocytes: A Fluorescence Microscopy Analysis

Extract

Primary cultures of mouse cortical astrocytes were used to investigate the changes in intracellular Na⁺ concentration ([Na⁺]_i) following exposure to the excitatory neurotransmitter glutamate. The fluorescent probe sodium-binding benzofuran isophthalate (SBFI) was used to measure [Na⁺]_i by epifluorescence microscopy imaging. Detection of the low light level fluorescent signal was accomplished using a Gen III+ intensified CCD camera. Fluorescence excitation ratio images of dye-loaded cells were obtained after sequential illumination at 340nnm and 380nm, with an emission observed at >520nm. Ratio images were proportional in intensity to [Na⁺]_i. In situ calibration of the fluorescent signals was obtained for each experiment and each cell under study by equilibrating [Na⁺]_i with external Na⁺ after treatment with the cation ionophores monensin and gramicidin, and with ouabain, a specific inhibitor of the Na+/K+ ATPase. Cells on glass coverslips were perfused at 35°C in a closed microscope chamber using solutions buffered with 25 mM bicarbonate/5%CO₂.