Abstract
Purpose:
To characterize the molecular mechanism of temperature and mechanosensation and calcium homeostasis in retinal ganglion cells (RGCs) and place it within the context of RGC output signaling using a combination of biophysical, electrophysiological, genetic and physiological approaches.
Methods:
RGCs were loaded with Ca indicator dyes (Fura-2, OGB-1, Fluo-4) and exposed to rapid increases water temperature (mediated by 1865 nm lasers) or slow (30-60 sec) elevations in bath temperature, or uniaxial/biaxial substrate stretch. Transmembrane currents were recorded directly with the whole cell patch and/or high-speed pressure clamp technique. Calcium signals were recorded with the confocal microscope, two photon micorscope or CCD cameras.
Results:
Short 7 msec infrared pulses at 13.93-22.28 J/cm2, increased local bath temperature by 9-14 deg C and evoked robust and reproducible Ca elevations in wild type HEK293 cells. These responses were observed in the absence of external Ca2+ and were susceptible to the blockers of Ca2+ sequestration/release from the endoplasmic reticulum. An additional Ca2+ component, characterized by rapid temperature-dependent [Ca2+]i kinetics was observed in cells overexpressing TRPV4 channels and in RGCs. Heat-evoked Ca2+ elevations in RGCs were suppressed by Ruthenium Red (non-selective TRP channel blocker) and HC-067047 (selective TRPV4 antagonist) whereas heat -induced responses from stores comprised a minor component of the neuronal thermo-response. Moderate heat, membrane stretch and TRPV4 agonists facilitated NMDA receptor-mediated responses of RGCs. Consistent with this, an increase in bath temperature to 37°C increased the firing rate of RGCs, an effect that was inhibited by the TRPV4 blocker.
Conclusions:
We conclude that force- and temperature-sensitive calcium influx through TRPV4 provides a depolarizing drive that modulates RGC excitability and thus retinal output to the visual brain. Our data further suggests that channels intrinsic to RGCs play a potentially important function by integrating the effects of non-visual inputs such as mechanical stimuli, temperature and vanilloid/cannabinoid agonists with light inputs derived through rod and cone circuits.