May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Testing the Proton Hypothesis of Lateral Inhibition: Measurements of H+ Flux From Isolated Horizontal Cells of the Catfish, Skate and Goldfish
Author Affiliations & Notes
  • R. P. Malchow
    Biological Sciences, University Illinois Chicago, Chicago, Illinois
  • M. A. Kreitzer
    Biological Sciences, Indiana Wesleyan University, Indiana, Indiana
  • A. J. A. Molina
    Pharmacology & Experimental Therapeutics, Tufts University, Boston, Massachusetts
  • L. P. Collis
    BioCurrents Research Center, Marine Biological Laboratory, Woods Hole, Massachusetts
  • P. J. S. Smith
    BioCurrents Research Center, Marine Biological Laboratory, Woods Hole, Massachusetts
  • Footnotes
    Commercial Relationships  R.P. Malchow, None; M.A. Kreitzer, None; A.J.A. Molina, None; L.P. Collis, None; P.J.S. Smith, CIR Biomedical Engineering & Technology, C.
  • Footnotes
    Support  NSF 009-1281, NCRR P41 RR001395, & an MBL Summer Research Fellowship
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 2428. doi:https://doi.org/
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      R. P. Malchow, M. A. Kreitzer, A. J. A. Molina, L. P. Collis, P. J. S. Smith; Testing the Proton Hypothesis of Lateral Inhibition: Measurements of H+ Flux From Isolated Horizontal Cells of the Catfish, Skate and Goldfish. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2428. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Hydrogen ions (H+) have been hypothesized to mediate lateral inhibitory feedback by horizontal cells onto photoreceptors in the outer plexiform layer (OPL) of the retina. According to this hypothesis, extracellular levels of H+ should increase upon depolarization of horizontal cells. To test this hypothesis, we have measured and compared the extracellular flux of H+ from horizontal cells isolated from the retina of catfish, skate and goldfish using self-referencing H+- selective electrodes.

Methods: : Primary cultures of isolated retinal neurons were prepared using a papain-based dissociation protocol. Extracellular proton fluxes were measured using H +-selective microelectrodes in a self-referencing format, which increases the useful sensitivity of such electrodes by up to a thousand times.

Results: : At concentrations of 100 µM, glutamate and the ionotropic glutamate analogues kainate and AMPA all promoted an alkalinization of the extracellular surface of the rod-driven external horizontal cells of the skate, rod and cone-driven horizontal cells of the catfish, and the cone-driven H1 horizontal cells of the goldfish. For all species, the AMPA and kainate induced effects could be blocked by the antagonist, CNQX. The glutamate-induced alkalinization occurred regardless of whether the extracellular Ringer’s solution was buffered with HEPES, phosphate or bicarbonate. The effects of glutamate were abolished when calcium was removed from the extracellular solution. The L-type calcium channel blocker nifedipine and the plasmalemma calcium pump antagonist carboxyeosin were also effective in reducing glutamate-evoked changes in H+ flux.

Conclusions: : The results of this comparative study argue strongly against the H+-hypothesis of lateral inhibition. Rather, this study supports an alternate hypothesis, where extracellular alkalinization of the OPL by horizontal cells may relieve suppression of photoreceptor neurotransmitter release that results from exocytosed protons released from photoreceptor synaptic terminals. The striking conservation of response in both rod-and cone-driven horizontal cells and across a wide diversity of species suggests that this glutamate-induced extracellular alkalinization may be a common feature in signal processing within the retina.

Keywords: horizontal cells • pH regulation/protons • calcium 
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