Polymodal TRPV1 and TRPV4 Sensors Colocalize but Do Not Functionally Interact in a Subpopulation of Mouse Retinal Ganglion Cells

Retinal ganglion cells (RGCs) are projection neurons that transmit the visual signal from the retina to the brain. Their excitability and survival can be strongly influenced by mechanical stressors, temperature, lipid metabolites, and inflammatory mediators but the transduction mechanisms for these non-synaptic sensory inputs are not well characterized. Here, we investigate the distribution, functional expression, and localization of two polymodal transducers of mechanical, lipid, and inflammatory signals, TRPV1 and TRPV4 cation channels, in mouse RGCs. The most abundant vanilloid mRNA species was Trpv4, followed by Trpv2 and residual expression of Trpv3 and Trpv1. Immunohistochemical and functional analyses showed that TRPV1 and TRPV4 channels are expressed as separate molecular entities, with TRPV1-only (∼10%), TRPV4-only (∼40%), and TRPV1 + TRPV4 (∼10%) expressing RGC subpopulations. The TRPV1 + TRPV4 cohort included SMI-32-immunopositive alpha RGCs, suggesting potential roles for polymodal signal transduction in modulation of fast visual signaling. Arguing against obligatory heteromerization, optical imaging showed that activation and desensitization of TRPV1 and TRPV4 responses evoked by capsaicin and GSK1016790A are independent of each other. Overall, these data predict that RGC subpopulations will be differentially sensitive to mechanical and inflammatory stressors

Emergent temporal signaling in human trabecular meshwork cells: role of TRPV4-TRPM4 interactions

Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca2+ signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated polymodal cation channel. Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4 (Transient Receptor Potential Melastatin 4) channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway

Stratified organization and disorganization of inner plexiform layer revealed by TNAP activity in healthy and diabetic rat retina.

Tissue non-specific alkaline phosphatase (TNAP), an abundant ectophosphatase, is present in various organs including the brain and retina of several vertebrate species. Evidence is emerging that TNAP influences neural functions in multiple ways. In rat, strong TNAP activity has been found in retinal vessels, photoreceptors, and both synaptic layers. In the present study, we identified eleven strata of the inner plexiform layer (IPL) by using TNAP histochemistry alone. The TNAP strata corresponded exactly to the strata seen after combined immunohistochemistry with four canonical IPL markers (TH-ChAT-CR-PKCalpha). Therefore, as described in other mammalian species, our data support the existence of multiple morphologically and functionally discernible IPL strata in rats. Remarkably, the stratification pattern of the IPL was severely disrupted in a diabetic rat model, even before changes in the canonical IPL markers were detectable. These findings indicate that TNAP histochemistry offers a more straightforward, but also more sensitive, method for investigating retinal strata and their diabetes-induced degeneration