Jonathan A. Coles - US grants

Physiological investigations of glia-neurone interactions will be continued using the retina of the honeybee drone as a model preparation. This preparation has useful features that make possible experiments that have not yet been performed on mammalian preparations. These features include: (1) The retina is composed of essentially one type of neurone, the photoreceptor cells, which can be stimulated by light, and one type of glial cell. (2) Activities of intracellular ions, particularly K+ and Na+, can be measured with ion-sensitive microelectrodes, in both photo-receptors and glial cells. (3) Ionic and metabolic interactions between glial cells and photoreceptors have been demonstrated. (4) The kinetics of oxygen consumption by the photoreceptors, after a singe flash of light, can be measured. Investigation of the mechanisms by which K+ enters the glial cells when the photoreceptors are stimulated will be continued. Measurements will be made with intracellular Cl- sensitive microelectrodes, and a quantitative synthesis will be attempted of the measured changes in K+, Na+ and Cl- in the photoreceptors, glial cells and extracellular space. The possibility that some of the intracellular Na+, K+ and Cl- is 'bound' and not detected by ion-sensitive electrodes will be investigated by measuring total concentrations of these elements with an electron beam microprobe. To measure the volume of the extra-cellular space a new method will be used in which the diffusion of extra-cellular probe ions through a slice of retina will be measured with ionsensitive electrodes outside the tissue. This method will also permit an investigation of possible interactions between fixed extracellular charges and mobile ions. Control of mitochondrial respiration in functioning tissue will be investigated by looking for an increase in ATP concentration after a brief stimulus.

Investigation will continue of glial-neurone interactions and related questions for which the retina of the honeybee drone is a uniquely advantageous preparation. This tissue can be considered as composed of only three compartments: photoreceptor cells, extracellular space and a syncytium of glial cells. Measurements with ion-selective microelectrodes have shown that a physiological stimulus, light, causes changes in the free concentrations of K, Na and C1 in the three compartments. These measured changes do not maintain electroneutrality. A search for fluxes of additional, unidentified, ions will be made by investigating further the transmembrane fluxes of C1: coupled transport will be investigated with specific inhibitors and by use of pH microelectrodes. The emphasis will be on quantitative estimates of ion fluxes, and on the differences between the neurones and the glia. (Health relatedness: pathologies involving massive redistribution of ions such as epilepsy, ischemia.) Metabolic compartmentation in the drone retina is very marked: nearly all the mitochondria are in the photoreceptors and all detectable glycogen is in the glia. Stimulation with a single flash of light causes a transient threefold increase in 02 consumption and an increase in (ATP) in the photoreceptors has been predicted. This will be investigated by measuring light emission from luciferin/lucificerase injected into the photoreceptors. Light stimulation of the phtoreceptors modifies metabolism in the glia: changes in glial (ATP) and pH will be looked for. Glial Nai in superfused retinas is high and variable: studies will be made with electron microprobe X-ray analysis on tissue rapidly frozen on the living animal to see if the glia are used as a reservoir of Na in vivo. Starting from the desirability of more reliable estimates of the volume of the extracellular space, measurements will be made with ion-selective micro-electrodes of the diffusion of various ions through thin slices of retina. Comparison of different ions (monovalent, polyvalent, cations, anions) will provide information about the biophysical properties of the extracellular space.