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

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.

Retinal horizontal cells of goldfish (Carassius auratus) display subtype‐specific differences in spontaneous action potentials in situ

“…APs in situ involved a sharp rise in [Ca 2+ ] i , a slow decay, and then a precipitous return to baseline [Ca 2+ ] i , similar to APs of isolated cells in this study and in previous reports (Country et al, 2019; Kreitzer et al, 2012). However, APs in situ were less frequent (median of 1 event per 5 min compared to 7 events per 5 min in Country et al, 2019) and lasted longer (120 s vs. 18 s).…”

“…Significant positive correlations were found for AUC (r 2 = .059, p < .01) and duration (r 2 = .043, p < .05); frequency was negatively correlated with r d/s (r 2 = .047, p < .01). No significant correlations were found for amplitude, TTP, or baseline recently, spontaneous, all-or-nothing Ca 2+ transients were found in goldfish in Ca 2+ imaging experiments (Country et al, 2019;Kreitzer et al, 2012). These transients represent APs, and involve Ca 2+ influx into the cytosol via L-type Ca 2+ channels and ryanodine receptors (Country et al, 2019).…”

“…Depolarizing stimuli or K + channel blockers have been shown to trigger APs in a variety of preparations, including the intact retina (Murakami & Takahashi, 1987), cultured cells (Lasater et al, 1984; Tachibana, 1981), and acutely dissociated cells (Shingai & Christensen, 1983). Even without depolarizing stimuli, APs have been shown to occur spontaneously in electrophysiological and intracellular Ca 2+ imaging experiments (Country et al, 2019; Kreitzer et al, 2012; Tachibana, 1981). While a role for APs in HCs has not yet been firmly established, it has been proposed that they may be important in the light‐to‐dark transition or in modulating feedback from HCs to photoreceptors (Country et al, 2019).…”

Activation of retinal glial (Müller) cells by extracellular ATP induces pronounced increases in extracellular H+ flux

“…H+-selective microelectrodes were prepared as previously described and used in a self-referencing format to record differential voltage measurements reflective of extracellular H+ flux [ 13 – 20 ]. To make a self-referencing measurement from isolated cells, the tip of an H+-selective electrode was placed 1–2 μm from the cell membrane and the voltage of the H+-selective electrode recorded; the microelectrode was then moved 30 μm away parallel to the bottom of the dish and a second reading taken; the second reading was subtracted from that obtained close to the cell.…”

Vesicular Release of GABA by Mammalian Horizontal Cells Mediates Inhibitory Output to Photoreceptors

“…In 1970, Baylor et al demonstrated that turtle retinal horizontal cells contribute a negative feedback signal to the cone photoreceptor light response. When our studies began over 30 years later, the proposed cellular mechanisms of horizontal cell neurotransmission were multiple, controversial, and unconventional: voltage-and sodium-dependent, calciumindependent plasmalemmal γ-aminobutyric acid (GABA) transporter (GAT) activity, as characterized in non-mammalian vertebrates (Schwartz, 2002), ephaptic coupling between photoreceptor calcium channel gating and current flow in horizontal cell glutamate receptors and hemichannels shown in fish retina (Byzov and Shura-Bura, 1986;Kamermans et al, 2001), and photoreceptor calcium current regulation by synaptic cleft pH (Hirasawa and Kaneko, 2003;Vessey et al, 2005;Cadetti and Thoreson, 2006;Kreitzer et al, 2012;Wang et al, 2014;Kramer and Davenport, 2015;Tchernookova et al, 2018;Grove et al, 2019). The apparent lack of the cone surround response block by GABAergic pharmacological agents in turtle, goldfish, mouse, and primate retinas (Thoreson and Burkhardt, 1990;Verweij et al, 1996Verweij et al, , 2003Endeman et al, 2012;Kemmler et al, 2014) was used to argue against a direct role for GABA in feedback inhibition.…”