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.
