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: 3.18). 07/2012; 36(8):3040-50. DOI: 10.1111/j.1460-9568.2012.08226.x
Source: PubMed


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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Available from: Jason Jacoby, Jul 30, 2014
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    • "In seven cells bathed in lanthanum, the average change in fluorescence of cells stimulated with the kainate puff solution was 0.002 Ϯ 0.002 ⌬F/F (Fig. 5C). All of these agents have also been shown to block the extracellular alkalinization previously detected from horizontal cells by self-referencing H ϩ -selective electrodes (Jacoby et al. 2012; Kreitzer et al. 2012). "
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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.
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    • "In addition, students help to craft manuscripts for submission to research journals and thus become involved in learning how to prepare their work for publication. Thus far, two articles have been published in peer reviewed scientific journals, with 6 undergraduates as co-authors (Jacoby et al., 2012; Kreitzer et al., 2012). RESULTS/DISCUSSION A significant component of our undergraduate program is the weekly collaborative laboratory discussions run through video-conferencing with Skype or Google Hangout. "
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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: Lateral inhibition is one of the most fundamental building blocks of neuronal processing. Its pervasive presence throughout the nervous system is especially important in enabling neural computations that mediate our sensory systems. One of the first descriptions of the importance of lateral inhibition in the central nervous system came from studies examining the processing of signals within the retina (Hartline et al., 1956). Yet despite a significant number of investigations and the passage of over fifty years, the precise molecular mechanism(s) that generates lateral inhibition in the outer retina is still unknown and contentiously debated. In 2007, two separate papers using two different techniques were published that came to completely opposite conclusions while examining the extracellular H+ dynamics of horizontal cells following cell activation. Jouhou et al. (2007) attempted to monitor extracellular changes in H+ from horizontal cells isolated from goldfish and carp using the pH-sensitive dye 5-hexadecanoylamino-fluorescein (HAF). They reported that depolarization of horizontal cells altered the fluorescence of the dye indicating an extracellular acidification response, consistent with the H+ hypothesis of lateral inhibition. Conversely, using H+-selective microelectrodes in a self-referencing format, Kreitzer et al. (2007) reported that the depolarization of isolated horizontal cells of skate and catfish, respectively, induced an alkalinization of the extracellular milieu; the H+ hypothesis of lateral inhibition predicts an acidification instead. The self-referencing and HAF imaging studies mentioned above differed in the species used, the extracellular pH buffer used (HEPES vs. bicarbonate), and the presence or absence of cobalt, a known regulator of Ca2+-dependent signaling. To verify that these experimental differences were not responsible for these conflicting results, I performed both techniques using horizontal cells from the same species (catfish) and in the same experimental conditions (HEPES as pH buffer, no cobalt). Surprisingly, opposite results were still obtained; fluorescent imaging using HAF reported an acidification response when cells were challenged with either glutamate or high extracellular potassium, whereas H+-selective microelectrodes in the self-referencing format consistently reported an extracellular alkalinization to the same stimuli. Using the same HAF staining protocol as performed by Jouhou et al., laser-scanning confocal microscopy revealed that HAF was not exclusively isolated to the extracellular surface of the plasma membrane as previously assumed, but instead was found to be present extensively throughout the intracellular compartment. These findings introduced the possibility that HAF is not reporting extracellular pH changes, but rather pH changes taking place within the cell’s interior. A center optical section of an HAF-stained cell was imaged and its level of fluorescence monitored as a function of time; when the horizontal cell was challenged with glutamate, this center optical section produced a decrease in the fluorescent ratio indicative of an acidification response reported directly from dye within the cell. In a follow-up study, we established a new staining protocol whereby intracellular dye accumulation was minimized and membrane-associated staining was prevalent, creating a functional extracellular pH sensor to monitor changes in extracellular H+. Isolated horizontal cells exhibiting high membrane-associated labeling of the pH-sensitive dye HAF now reported an extracellular alkalinization when cells were activated by glutamatergic agents. Furthermore, by breaking the imaged cell down to a single-pixel matrix to examine the spatial distribution of the changes in extracellular H+, an overall global alkalinization around the cell was observed with no signs of discrete microdomains of acidification. By implementing a modified staining protocol that maximizes extracellular staining over intracellular staining, we were able to reverse the signal from an intracellular acidification to an extracellular alkalinization solely dependent upon the anatomical location of the pH-reporter HAF. Taken together, the research presented here sheds new light onto a recurrently proposed mechanism of lateral inhibition in the outer retina. Our data suggests that protons released from horizontal cells do not appear to be the mediating agent responsible for facilitating inhibitory feedback onto photoreceptor synaptic terminals. Rather, activation of calcium-dependent plasmalemma Ca2+/H+ antiporters (PMCA) on horizontal cells may take up protons from within the synaptic cleft, working to disinhibit voltage-gated calcium channels and thereby enhancing the effects of glutamate onto second order neurons. Discovering how lateral feedback inhibition is carried out in the retina will give us great insight into how this prolific neuronal process is facilitated throughout the nervous system.
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