Pulsed Infrared Releases Ca from the Endoplasmic Reticulum of Cultured Spiral Ganglion Neurons

Inner ear spiral ganglion neurons were cultured from day 4 postnatal mice and loaded with a fluorescent Ca 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 concentration ([Ca]) of 0.20-1.4 µM, with a simultaneous reduction of [Ca] in regions containing endoplasmic reticulum (ER). IR-induced increases in cytosolic [Ca] continued in medium containing no added Ca (±Ca buffers) and low [Na], indicating that the [Ca] increase was mediated by release from intracellular stores. Consistent with this hypothesis, the IR-induced [Ca] response was prolonged and eventually blocked by inhibition of ER Ca-ATPase with cyclopiazonic acid, and was also inhibited by a high concentration of ryanodine and by inhibitors of inositol (1,4,5)-trisphosphate (IP)-mediated Ca 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] 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] elevations is conferred by TRP channels on ER membranes, which facilitate Ca efflux into the cytosol and thereby contribute to Ca-induced Ca-release via IP and ryanodine receptors. NEW & NOTEWORTHY Infrared radiation-induced photothermal effects release Ca from the endoplasmic reticulum of primary spiral ganglion neurons. This Ca release is mediated by activation of transient receptor potential (TRPV4) channels and involves amplification by Ca-induced Ca-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, studying Ca regulatory mechanisms, and influencing neuronal excitability.