Pulsed infrared releases Ca²⁺from the endoplasmic reticulum of cultured spiral ganglion neurons

Abstract

Inner ear spiral ganglion neurons were cultured from day 4 postnatal mice and loaded with a fluorescent Ca 2+ indicator (fluo-4, -5F, or -5N). Pulses of infrared radiation (IR; 1,863 nm, 200 µs, 200–250 Hz for 2–5 s, delivered via an optical fiber) produced a rapid, transient temperature increase of 6–12°C (above a baseline of 24–30°C). These IR pulse trains evoked transient increases in both nuclear and cytosolic Ca 2+ concentration ([Ca 2+ ]) of 0.20–1.4 µM, with a simultaneous reduction of [Ca 2+ ] in regions containing endoplasmic reticulum (ER). IR-induced increases in cytosolic [Ca 2+ ] continued in medium containing no added Ca 2+ (±Ca 2+ buffers) and low [Na + ], indicating that the [Ca 2+ ] increase was mediated by release from intracellular stores. Consistent with this hypothesis, the IR-induced [Ca 2+ ] response was prolonged and eventually blocked by inhibition of ER Ca 2+ -ATPase with cyclopiazonic acid, and was also inhibited by a high concentration of ryanodine and by inhibitors of inositol (1,4,5)-trisphosphate (IP 3 )-mediated Ca 2+ release (xestospongin C and 2-aminoethoxydiphenyl borate). The thermal sensitivity of the response suggested involvement of warmth-sensitive transient receptor potential (TRP) channels. The IR-induced [Ca 2+ ] increase was inhibited by TRPV4 inhibitors (HC-067047 and GSK-2193874), and immunostaining of spiral ganglion cultures demonstrated the presence of TRPV4 and TRPM2 that colocalized with ER marker GRP78. These results suggest that the temperature sensitivity of IR-induced [Ca 2+ ] elevations is conferred by TRP channels on ER membranes, which facilitate Ca 2+ efflux into the cytosol and thereby contribute to Ca 2+ -induced Ca 2+ -release via IP 3 and ryanodine receptors. NEW & NOTEWORTHY Infrared radiation-induced photothermal effects release Ca 2+ from the endoplasmic reticulum of primary spiral ganglion neurons. This Ca 2+ release is mediated by activation of transient receptor potential (TRPV4) channels and involves amplification by Ca 2+ -induced Ca 2+ -release. The neurons immunostained for warmth-sensitive channels, TRPV4 and TRPM2, which colocalize with endoplasmic reticulum. Pulsed infrared radiation provides a novel experimental tool for releasing intracellular Ca 2+ , studying Ca 2+ regulatory mechanisms, and influencing neuronal excitability.