Continued support is requested for the application of extrinsic potential-sensitive molecular probes to charge separation in energy-transducing membranes, primarily mitochondria and submitochondrial particles and for the utilization of these probes in the detection of electrical activity changes that accompany normoxic/anoxic transitions and spreading depression episodes in the exposed cerebral cortex of the gerbil. In membrane preparations, investigations include the mechanism(s) by which the probe energy-dependent optical signals occur, the time course of their development which bears on the kinetic competence of the probe to respond to charge separation, the effect of the probe on intrinsic energy-dependent processes due to the permeation of the membrane by the indicator, and the nature of the charge separation process to which the indicators are sensitive. Mechanistic studies include equilibrium dye-membrane binding work, probe fluorescence lifetime measurements, and the effect of dye structure on the probe energy-dependent signals. Utilization of fast responding charge shift probes such as di-4-ANEPPS is proposed. Investigations designed to address the nature of the charge gradient to which molecular probes are sensitive include the effect of varying the medium ionic strength on the probe energy-dependent optical signals, and the determination of the location of the probe in the membrane bilayer by monitoring the effect of such probes on P31, C13, O17 NMR spectra of phospholipid vesicles and submitochondrial particles and conversely the effect of membrane association on the NMR resonances of F19- and O17-labeled and deuterated dyes. In the cerebral cortex application, emphasis will be on improving the spatial resolution in surface fluorimetry measurements by detecting the development of anoxia in local cortex regions induced by the occlusion of selected blood vessels and the measurment of the oxygen gradient between normoxic and anoxic tissue regions. Certain controls for artifacts due to blood volume changes will be continued. The increase in the number of probes utilized in spreading depression work and the employment of convulsion-inducing drugs to initiate such episodes are also proposed.
