Retinal glial cells respond differently to the activation of the mechanosensitive channel TRPV4

Abstract

Purpose : The three glial cell types in the mammalian retina: Müller cells, microglia and astrocytes have been implicated in blinding diseases that involve mechanical stress but it is not clear whether their ability to transduce pressure, swelling and strain is comparable or mediated by similar molecular mechanisms. We used biophysical, electrophysiological and physiological tools to characterize the expression and activation properties of putative mechanosensing ion channels intrinsic to retinal glia and investigated their functional interactions with purinergic signaling and gliotransmitter release.

Methods : We examined retinas from Cxcr3^eGFP and Polr2a^{CAG-GCaMP5,tdTomato} mice, respectively. Paraformaldehyde fixed cryosections and acutely isolated cells were labeled with antibodies against glutamine synthetase (Müller cell), GFAP (astrocyte), P2Y12/Iba1 (microglia) and TRPV4. Explants were imaged with 2-Photon/confocal microscopy or whole cell/cell-attached patch clamp to detect changes in calcium and transmembrane currents in the presence of a selective agonists and inhibitors of TRPV4 and purinergic receptors. Acutely isolated glia were loaded with calcium indicators and studied with whole cell clamp and CCD camera-based imaging.

Results : Antibody labeling and calcium imaging revealed localization and functional expression of TRPV4 in Müller cells and microglia, but not astrocytes. However, in intact retinas TRPV4 activation evoked intracellular calcium ([Ca²⁺]_i) increases in Müller cells, microglia and astrocytes. In the astrocyte processes and Müller cell endfeet large, rapid transient TRPV4-dependent [Ca²⁺]_i elevations were followed by a period of oscillatory activity. The increase in astrocyte [Ca²⁺]_i was inhibited by non-selective P2X and P2Y receptor antagonists which however had no effect on Müller cell responses. Removal of Ca²⁺ from the perfusate reduced ATP-induced [Ca²⁺]_i elevations in astrocytes, suggesting preferential activation of ionotropic P2X receptors.

Conclusions : We demonstrate that retinal glial TRPV4 expression is class-specific, with Muller cells and microglia exhibiting unique electrophysiological and spatial activation patterns of channel activation. Importantly, although astrocytes do not utilize TRPV4 as a polymodal transducer of ambient stimuli they remain TRPV4 activation-sensitive due to purinergic signaling stimulated by gliotransmitter release from Muller cells.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.