This proposal aims to develop and exploit new optical methods for probing the intracellular ionic signals resulting from neuronal activation and signaling. The initial focus is on Ca2+ and Na+, which are of enormous importance in neuronal electrophysiology, exocytosis, nutritive homeostasis, growth, and plasticity. Molecular designs and synthetic routes are proposed for 1) fluorescent Ca2+ indicator dyes with visible excitation wavelengths and a shift in emission wavelengths upon binding Ca2+ for use in scanning confocal microscopy of living neuronal assemblies; 2) dyes that are triggered by a light flash to memorize the instantaneous Ca2t concentration in the form of an irreversible photochemical isomerization whose extent can be assessed after tissue sectioning; 3) Ca2+-specific, photolabile chelators in which light increases or decreases their affinity by a larger factor than presently available; 4) reversible photolabile Ca2+ chelators which can be repeatedly cycled between low and high affinity states; and 5) Na+ indicators with better optical responsiveness and K+ rejection than currently available. Both presently existing probes and their projected siblings would be exploited in a wide variety of neuronal preparations. For example, with an emission-shifting Ca2+ indicator and a ratiometric, video-rate-scanning confocal microscope, it should be possible to quantify the diffusion gradients of Ca2+ spreading from active membranes of isolated neurons and to visualize dynamically the receptive fields and signal propagation in sensory ganglia and tissue slices of significant thickness. Flash-triggered Ca2+-memorizing dyes would give even higher spatial resolution, ultimately down to the electron-microscopic level using immunocytochemistry, at the expense of continuous recording over time. Photochemical release or uptake of Ca2+ would permit calibration of the Ca2+ sensitivity of channels, exocytotic mechanisms, synaptic modulation, and neurite growth, or in general the extent to which Ca2+ is necessary or sufficient for cell function such as transmitter release or long-term potentiation. A photo-reversible chelator would be even more versatile, permitting generation of prolonged artificial trains or spatial gradients of Ca2+ by alternating two wavelengths in time or shining them on adjacent spatial regions. Na+ indicators should be used to examine how well neurons regulate cytosolic Na+ after bursts of action potentials, how effective Na+Ca+ exchange is, and what role Na+ accumulation may play in mediating post-tetanic potentiation and longer-term trophic effects.
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