Animal handling, anesthetic procedures, and experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The project was approved by the Institutional Animal Care and Use Committees at the University of Utah. We assessed retinal TRPV1 expression using a knock-in mouse in which Cre was inserted into Exon 15 of Trpv1 (TRPV1Cre; Jackson Laboratory 017769). This line was crossed to B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Ai9; 007909) in which the LoxP-STOP-LoxP TdTomato construct is knocked in at the Gt(ROSA)26Sor locus (Madison et al., 2010; Jo et al., 2017). Trpv4^-/- mice have an excised exon 12-encoding transmembrane pore domains 5 and 6 (Liedtke and Friedman, 2003). C57BL/6J (C57), bacterial artificial chromosome (BAC)-transgenic Tg(TRPV4- EGFP)MT43Gsat mice (referred to as TRPV4^eGFP), TRPV1^-/-, TRPV4^-/-, TRPV1Cre:Ai3, and Trpv1Cre:Ai9 mice were maintained in a pathogen-free facility with a 12-h light/dark cycle and unrestrained access to food and water. Data were gathered from male and female mice with no noted gender differences.

The TRPV4 agonist GSK1016790A (GSK101) and antagonist HC-067047 (HC-06) were purchased from Sigma. The TRPV1 agonist capsaicin (CAP; 8-methyl-N-vanillyl-6-nonenamide) and the TRPV1 antagonist capsazepine (CPZ; N-[2-(4-Chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide) and the endogenous agonist of CB1 receptors 2-arachidonoylglycerol (2-AG) were obtained from Cayman Chemicals. BDNF and CNTF used to culture RGCs were obtained from GenWay Biotech. Other salts and reagents were purchased from Sigma, VWR, Across Organics, or Thermo Fisher. GSK101 (1 mM), HC-06 (10 mM), CAP (10 mM), and CPZ (20 mM) stocks in DMSO were diluted in extracellular saline before use and placed into reservoirs connected to gravity-fed perfusion systems (Warner Instruments).

The retinas were incubated in an enzyme solution containing 16 U/ml papain (Worthington), 0.2 mg/ml L-cysteine (Sigma), and 50 U/ml DNase I recombinant (Roche) for 45 min at 37°C with gentle agitation and triturated with D-PBS solution containing 1.5 mg/ml BSA, 1.5 mg/ml Trypsin inhibitor, pH: 7.4, to yield a single cell suspension that was passed through a 30 μm pre-separation filter and centrifuged. The cell pellet was re-suspended and incubated in 0.5% BSA solution containing CD90.1 MicroBeads (1:10; Miltenyi Biotech) for 15 min at 4°C. After additional washing and centrifugation, cells were separated using MACS LS columns and incubated in serum-free neurobasal medium (Gibco/ThermoFisher) with 1% penicillin/streptomycin (Sciencell), transferrin (0.1 mg/ml), putrescine (16 ng/ml), insulin (5 μg/ml), 3,5,3-triiodothyronine T3 (100 nM), progesterone (20 nM), 2% B27, N-acetyl cysteine (5 ng/ml), sodium pyruvate (1 mM), L-glutamine (2 mM), brain-derived neurotrophic factor (BDNF, 50 ng/ml), ciliary neurotrophic factor (CNTF, 10 ng/ml), and forskolin (5 μM). The growth medium was changed every 2–3 days.

Total RNA was isolated using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) as described (Phuong et al., 2017). One microgram of total RNA was used for reverse transcription. First-strand cDNA synthesis and PCR amplification of cDNA were performed using qScript^TM XLT cDNA SuperMix cDNA synthesis kit (Quanta Biosciences). The PCR products were run on 2% agarose gels and visualized by ethidium bromide staining, along with 100-bp DNA ladder (ThermoFisher). SYBR Green-based real-time PCR was performed using Apex qPCR Master Mix (Genesee Scientific). The results were performed in triplicate of at least four separate experiments. The comparative C_T method (ΔΔC_T) was used to measure relative gene expression where the fold enrichment was calculated as: 2^{-[ΔCT(sample)-ΔCT(calibrator)]} after normalization. To normalize fluorescence signals, GAPDH and β-actin were utilized as endogenous controls. The primer sequences and expected product sizes are given in Table 1.

The animals were euthanized by isoflurane inhalation. The retinas were isolated in ice-cold Leibovitz 15 (L15) medium containing 11 mg/ml L15 powder, with (in mM) 20 D-glucose, 10 Na-HEPES, 2 sodium pyruvate, 0.3 sodium ascorbate, and 1 glutathione. Incubation in L15 containing papain (7 U/ml; Worthington) digested the extracellular matrix, and was terminated by rinsing with cold L15 solution; 500 μm pieces of retina were mechanically dissociated and cells were plated onto concanavalin A (0.2–0.5 mg/ml) coated coverslips. Calcium imaging followed established protocols (Ryskamp et al., 2011; Jo et al., 2016; Lakk et al., 2017), with the cells loaded with the Fura-2 AM (5–10 μM, Life Technologies) indicator dye for 45–60 min. Extracellular saline contained: (in mM) 133 NaCl, 10 HEPES hemisodium salt, 10 glucose, 2.5 KCl, 2 CaCl₂, 1.5 MgCl₂, 1.25 NaH₂PO₄, 1 pyruvic acid, 1 lactic acid, and 0.5 glutathione. Epifluorescence images were acquired using an inverted Nikon microscope with a 40x (1.3 NA oil) objective. Subsets of cells were stimulated with agonists and antagonists of TRPV1/4 channels. 340 and 380 nm excitation was delivered from an Xe lamp (Lambda DG-4, Sutter Instruments). Emissions were collected at 510 nm with 14-bit CoolSNAPHQ2 or Delta Evolve cameras and analyzed using NIS-Elements. ΔR/R (peak F340/F380 ratio – baseline/baseline) was used to quantify the amplitude of Ca²⁺ signals.

The immunolabeling protocol for vertical sections followed the procedures described in Molnar et al. (2016) and Jo et al. (2017). The retinas were fixed for 1 h in 4% paraformaldehyde, rinsed with PBS, dehydrated, and embedded in OCT compound mounting medium (Electron Microscopy Sciences); 12 μm thick cryosections were incubated in a blocking buffer (5% FBS and 0.3% Triton X-100 in 1X PBS) for 20 min. Primary antibodies (rabbit anti-TRPV4, 1:1000, LifeSpan Biosciences; rabbit anti-RBPMS, 1:500, PhosphoSolutions; mouse anti-Thy1.1, Sigma, 1:500; mouse SMI-32, 1:100, Covance; mouse anti-GFP, 1:500, Santa Cruz) were diluted in the diluent (2% BSA and 0.2% Triton X-100 in 1X PBS) and applied overnight at 4°C, followed by incubation in fluorophore-conjugated secondary antibodies (1:500; goat anti-mouse AlexaFluor 405, 488, or 647, goat anti-rabbit AlexaFuor 488 or 594, Life Technologies) for 1 h at RT. Images were acquired on an Olympus CV1200 confocal microscope using 20x (NA water) and 40x (0.9 NA water) objectives.

Data are presented as means ± SEM. Statistical comparisons were made with one-way ANOVA test followed by post hoc Tukey’s multiple comparison of means (Origin 8.0, Origin Lab Corporation). A difference of P ≤ 0.05 (^∗), P ≤ 0.01 (^∗∗), P ≤ 0.001 (^∗∗∗), and P ≤ 0.0001 (^∗∗∗∗) were considered statistically significant.

Vanilloid TRP channels are osmo- and thermosensitive non-selective cation channels with critical functions in neuronal neural plasticity, synaptic transmission, synapse formation, neurogenesis, apoptosis, and survival (Sousa-Valente et al., 2014; Ramírez-Barrantes et al., 2016; White et al., 2016). The mouse retina expresses multiple members of the vanilloid subfamily (Gilliam and Wensel, 2011) but their relative expression in RGCs is not known. RNA profiling shows that mouse RGCs express all four thermoTrp transcripts (Trpv1-4) (Figure 1A), with expression dominated by Trpv4, followed by Trpv2, Trpv3, and Trpv1 mRNAs, respectively (Figure 1B). TRPV4^-/- RGCs showed a trend toward Trpv1 upregulation but these changes were not significant (Figure 1C). Examination of mRNAs in TRPV4^-/- RGCs showed little evidence of cross-isoform plasticity apart from a (non-significant) trend toward Trpv1 (Figure 1C). We conclude that mouse RGCs as a group express all non-epithelial vanilloid Trp genes. Lack of compensatory upregulation in KO mice lacking the dominant TRPV4 channel might indicate absence of regulatory interaction at the transcriptional level, or an absence of obligatory heteromerization.

TRPV1 and TRPV4 channels have been implicated in optic nerve degeneration (Križaj et al., 2014; Sappington et al., 2015) and axonal neuropathies (Echaniz-Laguna et al., 2014), and shown to be sensitive to mechanical stressors (such as changes in cell volume and strain; Sudbury et al., 2010; Ryskamp et al., 2016). To categorize the relative fractions of TRPV1- and TRPV4-expressing RGCs, we used microfluorimetry. Dissociated cells were identified by the size and morphology of the somata, and responsiveness to glutamate (100 μM) and high K⁺ (35 mM; Ryskamp et al., 2011). After loading with the calcium indicator Fura-2-AM, cells were stimulated with pharmacological activators and inhibitors of TRPV1 and TRPV4 channels. The largest RGC cohort (35.08%) showed intracellular Ca²⁺ ([Ca²⁺]_i) increases in response to the TRPV4 agonist GSK101 (25 nM) and lack of sensitivity to the TRPV1 agonist CAP (10 μM; Figures 2A,B). On average, in these cells, GSK101 evoked an increase in the 340/380 ratio of 0.51 ± 0.05 (n = 87; P < 0.001). As shown previously (Ryskamp et al., 2011, 2014a), the responses to the TRPV4 agonist were characterized by a transient [Ca²⁺]_i peak that inactivated in the continued presence of GSK101. Subsequent co-applications of the drug evinced lower-amplitude or missing Ca²⁺ responses due to tachyphylaxis (continued channel desensitization). Pretreatment with CAP did not affect the amplitude of GSK101-evoked [Ca²⁺]_i responses in this cohort, suggesting that tachyphylaxis is isoform-specific.

Another population, encompassing 11.69% of RGCs, responded to CAP administration with increased [Ca²⁺]_i with an average CAP-evoked ratio increase of 0.45 ± 0.07 (n = 29; P < 0.001; Figures 2C,D). These cells were unresponsive to GSK101, and co-application of GSK101 and CAP yielded a ratio increase of 0.39 ± 0.08 that was not significantly different from the exposure to CAP alone. The third functional type (10.89%) responded to both TRPV1 and TRPV4 agonists (0.43 ± 0.09 and 0.48 ± 0.06 ratio increases, respectively; n = 27; Figures 2E,F). A representative example of a cell classified into the third cohort is shown in Figure 3, with vertical lines in Figure 3A, corresponding to fluorescence images of free [Ca²⁺]_i elevations in the RGC cytosol shown in Figure 3B. As expected (Ryskamp et al., 2011; Jo et al., 2017), the response to both agonists desensitized in the continued presence of the agonist (Figures 3Biv,viii). The dataset in Figure 2G shows that ∼46% of total glutamate responder RGCs express TRPV4, and ∼20% express TRPV1. Another way of parsing the data shows that ∼48% of TRPV1 expressing RGCs (22.6% of total glutamate-responding cells) functionally express TRPV4 channels whereas ∼24% of TRPV4-expressing cells express TRPV1 as well.

We next investigated whether the results from functional studies (Figures 2, 3) can be mirrored by proof-of-principle histochemical evidence. TRPV4 channels can be studied with validated antibodies (Ryskamp et al., 2011; Jo et al., 2016) but the specificity of commercial TRPV1 antibodies is questionable (Gilliam and Wensel, 2011; Molnar et al., 2012). We therefore studied TRPV1Cre:Ai9 and TRPV1Cre:Ai3 retinas in which channel expression manifests in the fluorescence patterns of tdTomato and GFP reporters, respectively (Mishra and Hoon, 2010; Jo et al., 2017). Cells were evaluated in vertical sections from the central- to mid-peripheral retina in order to increase the likelihood of hitting on TRPV1-expressing RGCs (e.g., Jo et al., 2017).

We found that TRPV4 is localized to a substantial population of putative RGCs (identified by Thy1 or RBPMS-ir). TRPV1⁺ cells, identified by tdT⁺ and GFP⁺ fluorescence (Figure 4), typically colocalized with TRPV4-ir, with rare examples (arrow in Figure 4A) that were immunonegative for TRPV4. Two examples of TRPV1^GFP cell that colocalized the RGC marker Thy1 (CD90) with TRPV4 are shown in Figure 4B (arrowheads). Although Thy1 labels a subset of displaced cholinergic syntaxin⁺ cells in the RGC layer (Raymond et al., 2008), the presence of TRPV4 (which is absent from amacrines; Ryskamp et al., 2011) indicates that the labeled cell is a RGC.

We investigated whether TRPV1 and 4 channels might be expressed in SMI-32 cells, which label αRGCs, large-diameter monostratified cells that arborize in ON or OFF sublaminae of the inner plexiform layer (Coombs et al., 2006), play a role in contrast sensitivity and are sensitive to ocular hypertension (Della Santina et al., 2013; Schmidt et al., 2014; Ou et al., 2016). All TRPV1-expressing RGCL cells were immunopositive for SMI-32 and TRPV4. Figures 5A,B show a TRPV1-expressing (RBPMS⁺) RGC that strongly expresses SMI-32. Another example, shown in Figures 5C,D, showcases a TRPV1-expressing αRGC that also expresses TRPV4 (arrowhead in Figure 5D), whereas a TRPV1-expressing putative amacrine cell (arrow in Figure 5C) was TRPV4 immunonegative. We conclude that most RBPMS⁺ TRPV1⁺ cells are SMI-32⁺ and can thus be classified as αRGCs. Of SMI-32⁺ cells, 35.9% were TRPV1⁺. TRPV1 expression was detected in some Müller cells that were immunopositive for TRPV4 (asterisks in Figure 4A). These data suggest that RGCs sense their ambient environment through different combinations of sensory transducers, with TRPV4 channels as dominant non-epithelial vanilloid transducers. Moreover, Müller glia appear to express both vanilloid isoforms, which is consistent with their function as regulators in the retinal microenvironment (Ryskamp et al., 2015).

Vanilloid TRP channels preferentially assemble into homomeric channels (Hellwig et al., 2005); however, formation of macromolecular complexes between TRPV4 and TRPC1 (Ma et al., 2015), TRPP2 (Stewart et al., 2010), and TRPV1 (Sappington et al., 2015) has been reported for endothelial, kidney, and ganglion cells, respectively. We recently reported that pharmacological blockade of TRPV4 has no effect on TRPV1-mediated calcium signals (Jo et al., 2017). Here, we took advantage of transgenic mice to test whether heteromerization with TRPV1 is obligatory for TRPV4 functionality. To test TRPV1–V4 interactions, we assessed the responsiveness to CAP in RGCs expressing a fluorescent reporter (eGFP) under the control of the TRPV4 promoter (Gu et al., 2016). Recordings from CAP responding TRPV4^eGFP+ RGCs (Figures 6Ai,ii) showed an absence of effect of the TRPV1 antagonist CPZ on baseline [Ca²⁺]_i. The amplitude of GSK101-induced [Ca²⁺]_i elevations in the presence of CPZ was 0.66 ± 0.12 (n = 12, P < 0.001; Figures 6A,B), not significantly different from GSK101 responses in WT cells (0.51 ± 0.05; Figure 2D). These data indicate that TRPV1 in mouse RGCs is not required for TRPV4 function and vice versa, TRPV4-mediated responses are largely unaffected by TRPV1 knockdown.

Expression of cannabinoid receptors in most, if not all, mammalian retinal cells (Ryskamp et al., 2014b; Jo et al., 2017) suggests that tonic and/or activity dependent release of endocannabinoids might be important for processing of visual signals. Endocannabinoids regulate TRPV1 directly and through CB1R-dependent intracellular messengers (Zygmunt et al., 1999) but were also suggested to influence TRPV4 activation (Watanabe et al., 2003; Ho et al., 2015). Exposure to the endogenous agonist of CB1 receptor 2-AG suppresses TRPV1 channels in mouse RGC (Jo et al., 2017). To establish whether 2-AG has comparable effects on RGC TRPV4 activation we recorded calcium signals from TRPV4^eGFP (Figures 7A,B), as well as wild type (black traces, Figures 7C–E) and TRPV1^-/- RGCs (orange traces, Figures 7C–E) in the presence or absence of 2-AG. Figures 7B,C show that 2-AG (1 μM) does not affect the [Ca²⁺]_RGC baseline and GSK101-evoked [Ca²⁺]_i signals in both TRPV4⁺ (0.63 ± 0.12; n = 10) and TRPV1-TRPV4⁺ (0.56 ± 0.12; n = 10) RGCs. Moreover, in TRPV1^-/- RGCs (Figures 7C–E), GSK101-evoked [Ca²⁺]_i signals recorded in cells preincubated with 2-AG (0.62 ± 0.05; n = 24) were also indistinguishable from control responses (0.59 ± 0.05; n = 24). Consistent with the pharmacological experiments (Figure 6), we found that ablation of TRPV1 has no effect on GSK101-evoked signals (n = 8; Figure 7A). These data suggest that, unlike TRPV1 signals, TRPV4 signaling in the mouse retina will resist endocannabinoid modulation.