Pulsed Infrared Releases Ca2+ 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-2+ indicator (fluo-4, -5F, or -5N). Pulses of infrared radiation (IR; 1,863 nm, 200 mu s, 200-250 Hz for 2-5 s, delivered via an optical fiber) produced a rapid, transient temperature increase of 6-12 degrees C (above a baseline of 24-30 degrees C). These IR pulse trains evoked transient increases in both nuclear and cytosolic Ca2+ concentration ([Ca2+]) of 0.20-1.4 mu M, with a simultaneous reduction of [Ca2+] in regions containing endoplasmic reticulum (ER). IR-induced increases in cytosolic [Ca2+] continued in medium containing no added Ca2+ (+/- Ca(2+ )buffer) b and low [Na+], indicating that the [Ca2+] increase was mediated by release from intracellular stores. Consistent with this hypothesis, the IR-induced [Ca2+] response was prolonged and eventually blocked by inhibition of HR Ca2+-ATPase with cyclopiazonic acid, and was also inhibited by a high concentration of ryanodine and by inhibitors of inositol (1,4,5)-trisphosphate (IP3)-mediated Ca2+ 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 [Ca2+] increase was inhibited by TRPV4 inhibitors (HC-067047 and GSK2193874), and immunostaining of spiral ganglion cultures demonstrated the presence of TRPV4 and TRPM2 that colocalized with HR marker GRP78. These results suggest that the temperature sensitivity of IR-induced [Ca2+] elevations is conferred by TRP channels on ER membranes, which facilitate Ca2+ efflux into the cytosol and thereby contribute to Ca2+ -induced Ca2+ -release via IP3 and ryanodine receptors.NEW & NOTEWORTHY Infrared radiation-induced photothermal effects release Ca2+ from the endoplasmic reticulum of primary spiral ganglion neurons. This Ca2+ release is mediated by activation of transient receptor potential (TRPV4) channels and involves amplification by Ca2+ -induced Ca2+ -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 Ca2+ studying Ca2+ regulatory mechanisms, and influencing neuronal excitability.