EFFECTS OF GLUTAMATE ON EXTRACELLULAR H+ FLUXES OF HORIZONTAL CELLS ISOLATED FROM THE RETINA OF THE SHARK (MUSTELUS CANIS)
Travis Bautista1, Robert Paul Malchow2.
1University of South Florida, Tampa, FL, 2University of Illinois at Chicago, Chicago, IL.
There is considerable current evidence to suggest that alterations in the concentration of extracellular protons (H+) may mediate feedback inhibition from retinal horizontal cells onto vertebrate photoreceptors, creating the classic surround portion of the receptive fields of retinal bipolar cells. If this hypothesis is correct, then depolarization of horizontal cells should induce an extracellular acidification of the environment directly adjacent to the cell membrane. We sought to test this hypothesis by examining proton fluxes from horizontal cells isolated from the retina of the smooth dogfish (Mustelus canis). Ultra-sensitive, self-referencing H+-selective microelectrodes were used to monitor extracellular H+ fluxes from horizontal cells isolated using a papain dissociation protocol. We found that unstimulated cells typically displayed a standing proton flux indicating a higher standing concentration of protons adjacent to the membrane as compared to the point 30 μm distant in the surrounding solution. Stimulation of horizontal cells with 1 mM glutamate induced a decrease in proton flux, lowering the concentration of free hydrogen ions around the cell. These data suggest that shark horizontal cells respond to the presumed photoreceptor neurotransmitter glutamate by reducing the overall level of protons at the external face of the cells, precisely opposite to the prediction of the H+ hypothesis for lateral inhibition. These data are also consistent with data from several other species (catfish, goldfish, and skate), for which horizontal cells also show an extracellular alkalinization upon stimulation with glutamate.
(This work was supported by NSF grants IOS-0924372 and DBI-1005378 “REU Site: Biological Discovery in Woods Hole”.)