Spontaneous action potentials in retinal horizontal cells of goldfish (Carassius auratus) are dependent upon L-type Ca²⁺ channels and ryanodine receptors

Abstract

Horizontal cells (HCs) are interneurons of the outer retina that undergo graded changes in membrane potential during the light response and provide feedback to photoreceptors. We characterized spontaneous Ca2+-based action potentials (APs) in isolated goldfish ( Carassius auratus) HCs with electrophysiological and intracellular imaging techniques. Transient changes in intracellular Ca2+ concentration ([Ca2+]i) were observed with fura-2 and were abolished by removal of extracellular Ca2+ or by inhibition of Ca2+ channels by 50 µM Cd2+ or 100 µM nifedipine. Inhibition of Ca2+ release from stores with 20 µM ryanodine or 50 µM dantrolene abolished Ca2+ transients and increased baseline [Ca2+]i. This increased baseline was prevented by blocking L-type Ca2+ channels with nifedipine, suggesting that Ca2+-induced Ca2+ release from stores may be needed to inactivate membrane Ca2+ channels. Caffeine (3 mM) increased the frequency of Ca2+ transients, and the store-operated channel antagonist 2-aminoethyldiphenylborinate (100 μM) counteracted this effect. APs were detected with voltage-sensitive dye imaging (FluoVolt) and current-clamp electrophysiology. In current-clamp recordings, regenerative APs were abolished by removal of extracellular Ca2+ or in the presence of 5 mM Co2+ or 100 µM nifedipine, and APs were amplified with 15 mM Ba2+. Collectively, our data suggest that during APs Ca2+ enters through L-type Ca2+ channels and that Ca2+ stores (gated by ryanodine receptors) contribute to the rise in [Ca2+]i. This work may lead to further understanding of the possible role APs have in vision, such as transitioning from light to darkness or modulating feedback from HCs to photoreceptors. NEW & NOTEWORTHY Horizontal cells (HCs) are interneurons of the outer retina that provide inhibitory feedback onto photoreceptors. HCs respond to light via graded changes in membrane potential. We characterized spontaneous action potentials in HCs from goldfish and linked action potential generation to a rise in intracellular Ca2+ via plasma membrane channels and ryanodine receptors. Action potentials may play a role in vision, such as transitioning from light to darkness, or in modulating feedback from HCs to photoreceptors.

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

“…Additionally, GABA-positive cells comprised 41.1% (56/136) of all fixed cells observed, which is in line with reports that H1 is the most common subtype in fish (e.g., Ariel, Lasater, Mangel, & Dowling, 1984;Young & Dowling, 1984). Verification of other HC subtypes within HCs of teleosts (e.g., goldfish, carp, catfish) and skates are known to exhibit Ca 2+ -dependent APs (Country et al, 2019;Johnston & Lam, 1981;Lasater et al, 1984;Murakami & Takahashi, 1987;Tachibana, 1981). In many reports, the APs occurred spontaneously (Country et al, 2019), especially in the presence of Ba 2+ or K + channel blockers (Murakami & Takahashi, 1987;Tachibana, 1981).…”

“…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).…”

“…In darkness, HCs are chronically stimulated with glutamate from photoreceptors, which leads to depolarization and constant Ca 2+ influx through L‐type Ca 2+ channels and Ca 2+ ‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPARs) (Linn & Christensen, 1992; Sun, Jiang, Liu, Gong, & Liang, 2010; Tachibana, 1983, 1985). In fish, HCs produce slow (seconds‐ to minutes‐long) Ca 2+ ‐based action potentials (APs; Country, Campbell, & Jonz, 2019; Johnston & Lam, 1981; Lasater, Dowling, & Ripps, 1984; Murakami & Takahashi, 1987; Tachibana, 1981), which are not well understood. These APs are dependent on Ca 2+ entry through L‐type Ca 2+ channels and ryanodine receptors, and they occur in the absence of glutamate (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).…”

Codonopsis lanceolata Contributes to Ca2+ Homeostasis by Mediating SOCE and PLC/IP3 Pathways in Vascular Endothelial and Smooth Muscle Cells

“…Cells were treated with dantrolene to exclude the effects of RyR, another major Ca 2+ pathway in ER/SR, to identify the mechanism involving the PLC/IP 3 Ca 2+ pathway of CL or LMA. Dantrolene suppression of RyR increased [Ca 2+ ] c. Furthermore, dantrolene treatment of goldfish retinal neuron cells abolished Ca 2+ transients and increased baseline Ca 2+ levels, whereas concurrent treatment with dantrolene and nifedipine suppressed the dantrolene-induced increase in baseline Ca 2+ , suggesting that this increased Ca 2+ was introduced into cells through L-type Ca 2+ channels [34]. The present study showed that simultaneous treatment with dantrolene and CL resulted in a greater increase in [Ca 2+ ] c than treatment with dantrolene alone, suggesting that the CL-or LMA-induced increase in Ca 2+ was introduced through a pathway other than L-type Ca 2+ channels.…”