Distinctive patterns of alterations in proton efflux from goldfish retinal horizontal cells monitored with self-referencing H(+) -selective electrodes.

Department of Biology, Indiana Wesleyan University, 4201 South Washington Street, Marion, IN 46953, USA Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA University of Texas, Austin, TX, USA University of Colorado, Colorado Springs, CO, USA National Eye Institute, Bethesda, MD, USA Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA.
European Journal of Neuroscience (Impact Factor: 3.67). 07/2012; 36(8):3040-50. DOI: 10.1111/j.1460-9568.2012.08226.x
Source: PubMed

ABSTRACT The H(+) hypothesis of lateral feedback inhibition in the outer retina predicts that depolarizing agents should increase H(+) release from horizontal cells. To test this hypothesis, self-referencing H(+) -selective microelectrodes were used to measure extracellular H(+) fluxes from isolated goldfish horizontal cells. We found a more complex pattern of cellular responses than previously observed from horizontal cells of other species examined using this technique. One class of cells had an initial standing signal indicative of high extracellular H(+) adjacent to the cell membrane; challenge with glutamate, kainate or high extracellular potassium induced an extracellular alkalinization. This alkalinization was reduced by the calcium channel blockers nifedipine and cobalt. A second class of cells displayed spontaneous oscillations in extracellular H(+) that were abolished by cobalt, nifedipine and low extracellular calcium. A strong correlation between changes in intracellular calcium and extracellular proton flux was detected in experiments simultaneously monitoring intracellular calcium and extracellular H(+) . A third set of cells was characterized by a standing extracellular alkalinization which was turned into an acidic signal by cobalt. In this last set of cells, addition of glutamate or high extracellular potassium did not significantly alter the proton signal. Taken together, the response characteristics of all three sets of neurons are most parsimoniously explained by activation of a plasma membrane Ca(2+) ATPase pump, with an extracellular alkalinization resulting from exchange of intracellular calcium for extracellular H(+) . These findings argue strongly against the hypothesis that H(+) release from horizontal cells mediates lateral inhibition in the outer retina.

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    ABSTRACT: This report describes a unique undergraduate research and teaching collaboration between investigators at two institutions, one a relatively small, primarily undergraduate institution and the other a large, urban research-intensive university. The program incorporates three major facets. First, undergraduates participate in a weekly collaborative lab meeting involving instructors from both institutions and held via remote video. Student-led discussions and presentations dominate these meetings, and the unique format promotes novel interactions between students and instructors. Second, students carry out investigative studies centered on understanding the role extracellular pH dynamics play in regulating neuronal processing. Students carry out studies on isolated neurons and glia throughout the fall and spring semesters, and primarily use a noninvasive electrophysiological technique, termed self-referencing, for extracellular pH measurements. The technique is relatively simple and readily learned and employed by undergraduates, while still being powerful enough to provide novel and meaningful research results. The research component is expanded for several students each summer who are selected to participate in summer research with both PIs and graduate students at the major research institution. Finally results gathered during the year and over the summer are disseminated at institutional symposia, undergraduate neuroscience symposia, national society meetings, and in submitted journal manuscripts. Preliminary observations and findings over three years support the aim of this research experience; to create a productive environment that facilitates deep-level understanding of neurophysiological concepts at the undergraduate level and promotes intellectual development while cultivating an excitement for scientific inquiry in the present and future.
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    ABSTRACT: Extracellular acidification induced by retinal horizontal cells has been hypothesized to underlie lateral feedback inhibition onto vertebrate photoreceptors. To test this hypothesis, the H(+)-sensitive fluorophore HAF (5-hexadecanoylaminofluorescein) was used to measure changes in H(+) from horizontal cells isolated from the retina of the catfish. HAF staining conditions were modified to minimize intracellular accumulation of HAF and maximize membrane-associated staining, and ratiometric fluorescent imaging of cells displaying primarily membrane-associated HAF fluorescence was conducted. Challenge of such HAF-labeled cells with glutamate or the ionotropic glutamate receptor agonist kainate produced an increase in the fluorescence ratio, consistent with an alkalinization response of +0.12 pH units and +0.23 pH units, respectively. This alkalinization was blocked by the AMPA receptor antagonist CNQX, the L-type calcium channel blocker nifedipine, and lanthanum. The alkalinization reported by HAF was consistent with extracellular alkalinizations detected in previous studies using self-referencing H(+)-selective microelectrodes. The spatial distribution of the kainate-induced changes in extracellular H(+) was also examined. An overall global alkalinization around the cell was observed, with no obvious signs of discrete centers of acidification. Taken together these data argue against the hypothesis that glutamatergic-induced efflux of protons from horizontal cells mediates lateral feedback inhibition in the outer retina.
    Journal of Neurophysiology 12/2013; 111(5). DOI:10.1152/jn.00768.2013 · 3.04 Impact Factor


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Jul 30, 2014