MEASUREMENT OF MEMBRANE-POTENTIAL AND [CA2- APPLICATION TO THE STUDY OF GLUTAMATE TASTE IN MICE(](I) IN CELL ENSEMBLES )

We have studied the spectral properties of the voltage-sensitive dye, opyl)-4-[beta[2-(di-n-octylamino)-6-naphtyl]vinyl] pyridinium betaine (di-8-ANEPPS), and the Ca2+-sensitive dye, fura-2, in azolectin liposo mes and in isolated taste buds from mouse, We find that the fluorescen ce excitation spectra of di-8-ANEPPS and fura-2 are largely nonoverlap ping, allowing alternate ratio measurements of membrane potential and intracellular calcium ([Ca2+](i)), There is a small spillover of di-8- ANEPPS fluorescence at the excitation wavelengths used for fura-2 (340 and 360 nm), However, voltage-induced changes in the fluorescence of di-8-ANEPPS, excited at the fura-2 wavelengths, are small, In addition , di-8-ANEPPS fluorescence is localized to the membrane, whereas fura- 2 fluorescence is distributed throughout the cytoplasm. Because of thi s, the effect of spillover of di-8-ANEPPS fluorescence in the [Ca2+](i ) estimate is <1%, under the appropriate conditions, We have applied t his method to study of the responses of multiple taste cells within is olated taste buds, We show that membrane potential and [Ca2+](i) can b e measured alternately in isolated taste buds from mouse, Stimulation with glutamate and glutamate analogs indicates that taste cells expres s both metabotropic and ionotropic receptors, The data suggest that th e receptors responding to 2-amino-4-phosphonobutyrate (L-AP4), presuma bly metabotropic L-glutamate receptors, do not mediate excitatory glut amate taste responses.