Description:

This study seeks to establish the metabolic cost to an excitable cell associated with neurotransmitter-induced depolarization. Measurements of oxygen consumption from single cells were made using a modulation electrochemical technique. The cell chosen for these studies was the retinal horizontal cell, an important player in the processing of visual signals in the outer retina. Enzymatically isolated cells have a resting potential of approximately -70mV and depolarize in response to the excitatory neurotransmitter glutamate and its analogue, kainate. The depolarization induced by these agents mimics the tonic depolarization of horizontal cells normally observed in the intact retina under dark-adapted conditions. Our technique allows a direct comparison between the metabolic state measured at rest and following tonic depolarization. We found that the average resting O2 consumption for horizontal cells was 0.024 µmol.cm-2.sec-1. Application of FCCP (10µM), an uncoupler of oxidative phosphorylation, elevated oxygen consumption to 0.035 µmol.cm2.sec-1; this elevation was followed by a sharp decline to below basal levels after 10min. 100µM kainate elevated oxygen consumption from a basal average of 0.03 µmol.cm-2.sec-1 to 0.07µmol.cm-2.sec-1. Further, we were able to follow changes in O2 consumption in response to voltage clamp at 0 and -70mV.
This data is one of the few examples where the metabolic cost of information processing has been measured from a single identifiable cell. The development of this approach has great promise for furthering our understanding of neuronal metabolism, degenerative diseases and aging.

Progress: Horizontal cells modulate the receptive field and signal transmission between photoreceptors and bipolar cells. Retinal tissue has a uniquely high demand for energy; moreover, it has been reported that oxygen (O2) is its limiting metabolite. Horizontal cells are located in the avascular zone of the retina and are immediately distal to the metabolically-active photoreceptors, therefore they have a need to conserve O2 during respiration. We are attempting to characterize the metabolic pathways of skate horizontal cells. The large size of these cells and homogeneity of retinal cell types in this animal model facilitates the ease of study. O2 flux was measured over a 20µm gradient with a self-referencing polarized O2 microsensor and current output was calibrated in air-saturated and nitrogen-saturated saline. Electrophysiology was performed with a 30MO microelectrode filled with 250mM KCl. Successful voltage clamp was verified by the presence of the A-type K+ current. Horizontal cells had a basal O2 flux of 2.1+0.4 pmol.cm-2.s-1. FCCP (1µM) increased this flux, confirming the O2 specificity of the microsensor. Kainate (20µM) was used to simulate input from dark-adapted rod photoreceptors, and increased the flux to 2.4+0.5 pmol.cm-2.s-1 (p<0.05). Cyclopiazonic acid (CPA; 50µM) increased the flux by 50+4 % possibly by stimulating intracellular Ca2+ release and thus stimulating the plasma membrane Ca2+ ATPase. Interestingly, CPA appears to block the effect of kainate suggesting a role for the ER Ca2+ pump. Blockade of the Na+/K+ ATPase with ouabain (100µM) did not affect basal or kainate-stimulated O2 fluxes. Horizontal cells had a mean resting potential of -63+2 mV. Kainate depolarized the cell to approximately 0mV. Voltage clamping from -70mV to 0mV replicated the effects of kainate on the O2 flux (-70mV: 0.9+0.2 pmol.cm-2.s-1; 0mV: 1.4+0.3 pmol.cm-2.s-1; p<0.05), signifying kainate acts via voltage-activated ion channels. These data suggest that depolarization of horizontal cells, during dark-adapted conditions, increases metabolic demand via enhanced Ca2+ influx and thus utilization of the intracellular Ca2+ pump.