All procedures were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and related regulations of the Institutional Animal Care and Use Committee. The animals were 3-7-month-old male and female C57BL/6J mice purchased from Jackson Laboratory (Bar Harbor, ME, USA). Chemicals were purchased primarily from Sigma-Aldrich (St. Louis, MO, USA) and Tocris Bioscience (Bristol, United Kingdom) except otherwise specified.

Animals were dark-adapted for 1–2 h before recording, and all related procedures were performed under infrared illumination. RGCs were recorded from living whole-mount retinas under the loose patch, voltage-clamp, or current clamp mode. The dual-cell patch-clamp recording used MultiClamp 700A or 700 B amplifiers connected to DigiData 1322a interfaces and operated by pClamp software (Axon Instruments, Foster City, CA, USA). Two EPC10 quadruplet amplifiers with digitizers (HEKA instrument, Holliston, MA, USA) provided eight independent channels, which enabled us to record eight cells simultaneously under either voltage-or current-clamp mode. The recording was performed under infrared illumination (>750 nm) or dim red light and monitored by a Nano video camera (Stemmer Imaging AG, Puchheim, Germany). The novel technology has been recently applied to retinal study for the first time by Pang and colleagues (22, 52, 53).

Electrodes were filled with an internal solution containing 0.1–0.5% Lucifer yellow (LY) and/or 2% neurobiotin (NB) with pH adjusted to 7.3. (54–57). Patch pipettes had 5–8 MΩ tip resistance when filled with an internal solution containing 112 mM Cs-methanesulfonate, 12 mM CsCl, 5 mM EGTA, 0.5 mM CaCl₂, 4 mM ATP, 0.3 mM GTP, and 10 mM Tris, adjusted to pH 7.3 with CsOH. For current-clamp and some voltage-clamp recordings, the pipettes were filled with internal solutions containing 112 mM K-gluconate, 10 mM KCl, 10 mM EGTA, 10 mM HEPES, 0.5 mM CaCl₂, 1 mM MgCl₂, 4 mM Na₂-ATP, and 0.3 mM Na₃-GTP, adjusted to pH 7.3 by KOH. The internal solution and the external normal Ringer’s solution yield an E_Cl of −59 mV at room temperature. Recorded cells were visualized by LY and/or NB fluorescence with a confocal microscope (LSM 800, Carl Zeiss, Germany).

A photostimulator delivered light spots of a diameter of 600–1,200 μm and 500 nm wavelength (λ_max = 500 nm, full width-half max 10 nm) at various intensities (−10 to −1 log I) to stimulate the retina via the epi-illuminator of the microscope (55, 56, 58). The intensity of unattenuated [0 in log unit (log I)] 500 nm light from a halogen light source was 4.4 × 10⁵ photons. μm⁻² sec⁻¹. An LED emitting 505 nm light of the unattenuated intensity of 4 × 10³ photons.μm⁻² sec⁻¹ will be used for multi-cell patch-clamp recording, delivering light spots of 700–1,200 μm in diameter.

Double-and triple-immuno-labeling followed the published experimental protocols (15, 55, 59–62). All fixed retinas were blocked with 10% donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) in TBS [D-PBS with 0.5% Triton X-100 (Sigma-Aldrich) and 0.1% NaN3 (Sigma-Aldrich)] for 2 h at room temperature or at 4°C overnight to reduce nonspecific labeling. A vibratome (Pelco 102, 1000 Plus; Ted Pella, Inc., Redding, CA, USA) was used to prepare retinal slices. Whole retinas were imbedded in low gel-point agarose (Sigma-Aldrich) and trimmed into a 10 × 10 × 10 mm³ block. The block was glued onto a specimen chamber mounted on the vibratome and subsequently cut into 40-mm thick vertical sections in PBS solution. Whole retinas and free-floating sections were incubated in primary antibodies in the presence of 3% donkey serum-TBS for 3–5 days at 4°C. Controls were also processed without primary antibodies. Following several rinses, the slices and whole retinas were then transferred into Cy3-, Cy5-, or Alexa Fluor 488-conjugated streptavidin (1:200, Jackson ImmunoResearch), with Cy3-and/or Cy5-conjugated secondary antibodies (1:200, Jackson ImmunoResearch) and/or Alexa Fluor 488-conjugated secondary antibodies (1:200; Molecular Probes, Eugene, OR), in 3% normal donkey serum-TBS solution at 4°C overnight. A nuclear dye, TO-PRO-3 (0.5 mL/mL, Molecular Probes, Eugene, Oregon, USA) was used with the secondary antibody to visualize the nuclei in the retina. After extensive rinsing, retinal preparations were cover-slipped.

RGCs were identified with a double-retrograde labeling technique previously established by Pang and colleagues (60, 61). Briefly, eyeballs of dark-adapted animals were enucleated under the illumination of dim red light. The nerve stump of the freshly dissected eyeball was dipped into a small drop (3 μL) of 3% Lucifer yellow (Sigma) and/or 8% neurobiotin (NB, Vector Laboratories, CA) in the internal solution (61) for 20 min. Then, the eyeball was thoroughly rinsed with the oxygenated Ames medium (Sigma) to remove the extra dye and dissected under infrared illumination. The dark-adapted eyecup with intact retina and sclera tissue was transferred into fresh oxygenated Ames medium and kept at room temperature for 40 min under a 10 min-dark/10 min-light cycle. Subsequently, the whole retina was isolated from the sclera, fixed in darkness for 30–45 min at room temperature, and visualized with Cy3-, Cy5-, or Alexa Fluor 488-conjugated streptavidin (1,200, Jackson ImmunoResearch). The technique brightly labeled the entire population of RGCs in the mouse retina (60, 61).

Antibodies were summarized in Table 1. Polyclonal rabbit anti-TRPV4 (LS-C135, 1: 200; LS-A8583 1:200 and LS-C94498 1: 100) was purchased from LifeSpan Biosciences, Inc. (Seatle, WA, USA). LS-C94498 was raised against a synthetic peptide from the cytoplasmic domain (aa100-150) of mouse TRPV4 conjugated to an immunogenic carrier protein. LS-A8583 targets a synthetic 20-amino acid peptide from the internal region of human TRPV4, and LS-C135 was raised against rat TRPV4 (Q9ERZ8, aa853-871, peptide immunogen sequence: CDGHQQGYAPKWRAEDAPL). LS-C135 provided the best signal-to-noise ratio in the primate retina (15). The specificity of LS-A8583 (17), LS-C94498 (17), and LS-C135 (15, 23) for labeling retinal TRPV4 were confirmed in TRPV4 transgenic mice.

Purified polyclonal goat-anti TRAAK was purchased from Santa Cruz (sc-11324, goat, 1: 100). It was raised against a peptide mapping at the C-terminus of TRAAK of human origin, which identified a single band at ~47 kDa in IMR-32 and Y79 whole cell lysates, matching TRAAK (63). TRPV2 antibodies included a rabbit anti-rat (1: 200, PC241, Calbiochem, San Diego, CA, USA) and a goat anti-mouse (LSbio, Lynnwood, WA, USA) polyclonal antibody, and the antigens were a synthetic peptide aa 744–761 of rat TRPV2 (44, 64) and a peptide aa743-756 of mouse TRPV2 with the sequence C-SEEDHLPLQVLQSH. Monoclonal mouse anti-BK was purchased from MilliporeSigma (1: 500, MABN70, anti-Slo1, clone L6/60, pore-forming alpha unit), and it primarily identifies a single band ~100 kDa close to MW 100–130 kDa of BK (65, 66). Polyclonal rabbit anti-ENaCα was from Abcam (1, 200, Cambridge, MA, USA) (67–69), which identified a single band at ~75 kDa in human platelet lysates, matching ENaCα.

Protein Kinase-C alpha (PKCα) is a classic marker for rod BCs (55, 70, 71). The anti-PKCα antibody from Sigma (P4334, 1: 1000, rabbit, polyclonal) was tested in immunoblotting with rat brain extract, and it recognized a heavy band at ~76 kDa and a very weak band at 40 kDa, while the predicted molecular weight of the PKCα was 76–93 kDa. The staining was specifically inhibited by PKCα immunizing peptide (659–672). The monoclonal anti-PKCα antibody from BD transduction [610107, Clone 3/PKCα (RUO), 1: 200, mouse] identified a single band at 82 kDa from a rat cerebrum lysate. Two calbindin D-28 k antibodies were used to identify horizontal cells (72), one of which was a rabbit polyclonal antibody raised against the recombinant rat calbindin D-28 K (CB) protein purchased from Swant (Marly 1, Switzerland) (CB38, 1: 1000) (73, 74), and the other was a mouse monoclonal antibody purchased from Sigma and produced with the bovine kidney calbindin-D (C9848, clone CB955, 1: 200) (73, 74). Monoclonal mouse anti-glutamine synthetase (GS) (1: 1000, clone 6, BD Transduction Laboratories, Palo Alto, CA) was used to identify Mȕller cells (60). The antibody was raised against the human glutamine synthetase aa 1–373 and recognized a band at ~45 kDa, consistent with the predicted molecular weight of GS. Other primary antibodies included in this study have also been used in previous reports, including polyclonal guinea pig anti-GABA (1:1000, AB175; Chemicon, Temecula, CA, USA) (75) and rat anti-glycine antiserum (1:1000, a generous gift from Dr. David Pow, University of Queensland, Brisbane, QLD, Australia) (76). The specificity of these primary antibodies has been demonstrated in previous studies, and their staining patterns in our results were similar to the reports. Controls were also processed with blocking peptides or without primary antibodies. All controls did not show positive results.

We used confocal microscopes (510 and LSM 800, Carl Zeiss, Germany) for morphological observation. Pixel intensities of MSC immunoreactivities were analyzed in different retinal layers with the confocal (Zen Blue, Zeiss), Sigma plot (v12 and 15), and Excel software and fitted to an exponential function (15).

where a is the pixel count at the distribution peak, I is the pixel intensity, and I₀ is I at the distribution peak, and b is a slope/width factor. The ATP consumed by RGCs for maintaining resting membrane potential (ATP_RP, molecules/s) was calculated by the previously established equation (95)

where V_Na (+50 mV) and V_K (−100 mV) are Nernst potentials for Na⁺ and K⁺, N_A/F = 6.2 × 10¹⁸, and N_A and F are the Avogadro and Faraday constant, respectively. Data were further analyzed by Sigmaplot (v12 and v15, Systat, Point Richmond, CA, USA), Clampfit (v10.3 and v9.2, Axon Instruments, Foster City, CA, USA), Matlab, and Microsoft Excel for statistical significance. The α level to reject the null hypothesis is 0.05.

BK was the most heavily expressed in the outer segment layer (OSL) (Figures 1C,D), and weaker immunoreactivity (IR) was also present in the OPL, IPL, some somas in the INL and GCL (Figure 1A), and large globules of axon terminals of rod bipolar cells (RBCs) (Figure 1B and the inset of Figure 1A). Horizontal cells identified by calbindin D-28 K antibody were nearly negative for BK (Figure 1E). The peak intensity of the fitting curve of the pixel histogram (Equation 1) showed that the relative intensity of BK-IR was in the order of OSL > IPL≈RBC axons > OPL≈GCLINL. BK-IR identified some GABAergic somas of ACs and dendrites of putative A17 ACs that contacted the axon terminal of PKCα-positive RBCs (Figure 1A).

In triple-labeled retinal slices, TRAAK (Figure 2A₁) was heavily expressed in Müller cells, including the somas in the middle of the INL, end feet in the GCL, and descending processes passing the INL and IPL. The OSL and OPL were weakly labeled. The relative intensity of TRAAK-IR was in the order of Müller cells > OSL≈ OPL. In Figures 2A,B, RGCs were retrogradely identified, and RGC somas were nearly negative for TRAAK.

In labeled retinal slices, TRPV4-IR was more intensive in retrograde-labeled RGC somas in the GCL (Figures 2A₂,B). It was weaker in other layers and nearly absent in the inner segment layer (ISL) of photoreceptors. TRPV4-IR was mostly diminished in homozygotes with TRPV4 expression suppressed (TRPV4^−/− mice), demonstrating the specificity of the antibody (Figure 2D). TRPV4-IR was also present in PKCα-identified RBCs (Figure 2C, asterisk), some PKCα-negative cone bipolar cells (Figure 2C, triangle), and GS-identified Mȕllar cells (Figure 2B). Some somas of putative cone bipolar cells were negative for TRPV4 (Figure 2B, inset b). The relative intensity of TRPV4-IR was in the order of GCL > OSL≈ OPL≈ IPL≈ INL.

The results (Figures 1, 2) together revealed that the OSL expressed BK, TRAAK, and TRPV4; RGCs possessed the highest level of TRPV4 and a low level of BK and lacked TRAAK; INL, IPL, and OPL weakly co-expressed TRPV4 and BK. The higher level of depolarizing TRPV4 in RGCs is consistent with their higher pressure-vulnerability observed in glaucoma (1, 2) and traumatic retinal injury (3–6) compared to other neurons.

TRPV2 was expressed primarily in the OPL, IPL, and retrograde-identified RGCs, and TRPV2-IR was weak in the OSL and INL and nearly absent in the ISL. The relative intensity of TRPV2-IR was in the order of IPL≈ OPL > GCL > OSL≈ INL for wild-type mice. TRPV2-IR was heavier in the GCL and ACL in the congenital glaucoma model DBA/2J (D2) mice, and the relative intensity was in the order of GCL≈ ACL > OPL≈OSL (Figure 3).

ENaCα was intensively expressed in photoreceptors, OPL, IPL, and RGC somas and dendrites, and the INL was weakly labeled. The relative ENaCα-IR was in the order of photoreceptors≈ GCL≈ OPL≈ IPL > INL (Figure 4). The ENaCβ antibody stained blood vessels and weakly labeled the OSL, OPL, and IPL (Figure 4C). The distribution of these depolarizing MSCs was nearly even in the outer and inner retina but favored neurons over Müller cells. In Figures 3, 4, all RGCs were retrogradely identified.

In summary, the relative expression level was in the order of Müller cells > OSL≈ OPL for TRAAK, OSL > IPL≈ RBC axons > OPL≈ GCL≈ INL for BK, GCL > OSL≈ OPL≈ IPL≈ INL for TRPV4, GCL≈ ACL > OPL≈ OSL for TRPV2 (in D2 mice), and GCL≈ photoreceptors≈ OPL≈ IPL > INL for ENaCα. TRAAK was expressed mainly in Müller cells. Photoreceptors expressed BK and ENaCα intensively and TRAAK, TRPV2, and TRPV4 weakly. RGC somas and axons retrograde-identified clearly expressed ENaCα, TRPV4, and TRPV2 but lacked TRAAK and BK. BCs weakly expressed BK, ENaCα, and TRPV4. Some amacrine cells were positive for BK, ENaCα, and TRPV4. These results indicate a higher ratio of K-MSCs to N-MSCs (R_K/N) in photoreceptors and interneurons than RGCs, which is consistent with the high pressure-vulnerability of RGCs in glaucoma.

We have previously characterized mouse RGCs by the resting potential, synaptic currents, and firing features (57, 77). Since TRPV4 mediates excitatory cation current, we further study the role of TRPV4 in RGCs with TRPV4 agonists 4α-Phorbol 12,13-didecanoate (4αPDD) and GSK101 (GSK, GSK1016790A). The results showed that TRPV4 agonists enhanced the firing rate of spontaneous action potentials (Figures 5A,B) recorded under the loose-patch mode and the frequency and amplitude of the spontaneous and light-evoked excitatory postsynaptic currents (sEPSCs and ΔI_C, respectively) (Figures 5D–G) under the voltage-clamp mode in all RGCs. In Figures 5A,B, the ONαRGC generated light-evoked action potentials (APs) with little visual noise, while 4αPDD induced many spontaneous APs, i.e., visual noises, which were unrelated to the light. Similarly, Figures 5F,G showed that in another ONαRGC, 4αPDD enhanced the amplitude and frequency of sEPSCs unrelated to the light. We further measured the frequency of light-evoked APs (Signal, S) and light-unrelated APs (Noise, N) in the same RGCs before and after application of drugs. In the presence of the drugs, the visual signal reliability (VSR), calculated by (VSR = 1-N/S) (Figure 5C), was reduced by ~50% (p < 0.001) in ON RGCs. Since the versicle release from presynaptic terminals primarily determines the frequency of sEPCSs and the postsynaptic mechanism dominates the amplitude of sEPSCs, these data indicate that the TRPV4-induced excitation in RGCs involves TRPV4 in both BCs and RGCs.

Figure 6 showed that activating MSCs with low osmolarity (Osm) and TRPV4 agonists 4αPDD and GSK101 significantly depolarized membrane potential in all RGCs (Figures 6A,B). 4αPDD elicited spontaneous firing of APs (Figure 6A) was reversibly blocked by a general TRPV antagonist ruthenium red (RR). TRPV4 induced membrane depolarization and spontaneous firing would need to consume extra ATPs. Thus, we plotted the ATP consumption for maintaining normal resting potential based on Equation 2 (Figure 6D, blue curve). The data showed that TRPV4-induced depolarization raised the ATP consumption to the peak, predicting ATP depletion. We also recorded Na⁺ currents (I_Na) in RGCs mediated by voltage-gated Na⁺ channels (NaV) under voltage-clamp conditions by depolarizing RGCs from −110 or − 70 mV with a step of 8–20 mV. The evoked responses could not be significantly affected by BC and AC synapses. I_Na was an inward current at the holding potentials of ≤40 mV and identifiable from potassium, chlorides, and calcium currents by the duration of ~2 ms and the polarity of the spike. I_Na was found to activated at ~ − 50 mV (n = 5 cells) and sensitive to TTX (Figure 6C), consistent with typical NaVs well documented in previous studies (78, 79). The peak of the ATP consumption curve just overlapped with the membrane potential (~ − 37 mV) where half NaVs underwent slow inactivation (purple dot, Figure 6D) (80), and TRPV4 agonists also raised the ATP consumption to the peak level (green dot, Figure 6D), indicating that ATP depletion and RGC malfunction may follow intensive TRPV4 activation.

The results together demonstrated that TRPV4 activation directly induced depolarization and dysfunction of RGCs, and the effect is associated with TRPV4 expressed in BCs and RGCs, expedited inactivation of NaVs in RGCs, and ATP overconsumption.

The retina expresses multiple MSCs (11, 21, 46), while studies often focus on individual types of MSCs. It has been unclear whether individual retinal neurons co-express multiple MSCs and the significance of such design. The elevation in intra-and extra-ocular pressure closely involves the pathogenesis of glaucoma and other retinal conditions (1–7), and the question of whether retinal neurons were directly responsive to the mechanical signals had not been addressed until recent years when mechanical responses were revealed from RGCs (14, 17), BCs, and photoreceptors (15, 22). MSCs generate electric currents in retinal neurons upon activation. Since many MSCs, including TRPV4, TRPV2, BK, TRAAK, and ENaCa, are not classic molecules that serve light pathways in the retina, their physiological and pathophysiological role remains to be defined (11, 46, 81–84).

We discovered the co-expression of several MSCs in retinal layers and individual neurons, including N-MSCs that mediate inward cation currents and K-MSCs that carry outward K+ current at membrane potential levels between −80 and 0 mV (46). The morphological data, consistent with functional results obtained from photoreceptors and BCs in the salamander (22) and mammalian retina (15, 23), demonstrates that outer retinal neurons possess several types of MSCs. We postulate that a cell with more MSCs would theoretically possess better mechanical adaptability, and because of the opposite polarity of the electric currents at K-and N-MSCs, a balanced ratio of K-and N-MSCs would better reduce the pressure-induced instantaneous electric disturbance on neuronal signals. Supporting this idea, our data revealed a distinct R_K/N ratio in retinal neurons, and the more balanced one in the outer retinal neurons well aligns with their lower pressure vulnerability in glaucoma and traumatic retinal injury (1–7). It indicates that the pressure responsiveness of a cell is to be determined by both the level and ratio of K-and N-MSCs, establishing a novel mechanism of cellular mechanical homeostasis in sensory neurons that may provide not only mechano-protection to cellular physical integrity but also electro-protection to neuronal signals by reducing mechanically induced electric noises.

Recent work has also identified the interaction between TRPs and BK in smooth muscles and between TRPV4 and another calcium-activated potassium channel KCa2.3 in the endothelial cells of blood vessels (85–87). BK mediates muscle relaxation, and TRPV4 mediates contraction after the relaxation (86), supporting the notion of the counterbalance between these channels.

RGCs showed higher pressure vulnerability in glaucoma and traumatic retinal injury than other retinal neurons (1–7). Depolarizing N-MSCs such as TRPV4 and TRPV1 can mediate RGC depolarization, spontaneous firing, and cell death (14, 15, 17, 88), and our patch-clamp data was in line with previous reports. More importantly, we used VSR to demonstrate TRPV4-mediated RGC dysfunction, and we further calculated TRPV4-related ATP depletion and the likelihood of inactivating voltage-gated sodium channels (NaVs). Meanwhile, our immunological data confirmed a much higher expression level of depolarizing N-MSCs relative to K-MSCs in RGCs but similar levels of N-and K-MSCs in outer retinal neurons. These results together support the idea that a low R_K/N or a high R_N/K plays a pro-neurodegenerative role in neurons.

The excitotoxic effect of TRPVs previously reported in RGCs has suggested that blockage or removal of TRPVs may be beneficial (14, 15, 17, 18, 88), but whether it would compromise the pressure adaptability of the neurons is still uncertain. ENaC and TRPVs carry cation currents to reverse at ≥ ~0 mV, while K-MSCs like BK and TRAAK mediate background leak currents to reverse at ~ −80 mV (46). Thus, at the membrane potential (RP) level of 0 > RP > −80 mV, K-MSCs would counterbalance N-MSC-induced membrane depolarization. Based on current results, raising R_K/N appears to be a safer strategy to reduce pressure-induced excitotoxicity than solely reducing N-MSCs.

Membrane potential alters the driving force of MSCs and is another factor that modulates currents at MSCs besides R_K/N, while RGCs maintain RPs less depolarized than outer retinal neurons (22, 57, 89–93). Hence, we postulate that RGCs are less protected by K-MSCs and more easily activated by N-MSCs compared to outer retinal neurons, and this mechanism could also contribute to the high vulnerability of RGCs. Our data discovered a low R_K/N in RGCs, suggesting R_K/N as a novel determinant of pressure-induced excitotoxicity and improving R_K/N as a new therapeutic strategy.

RGCs express TRPV4 in the mouse (16, 17, 94), porcine (18), and primate retina (15), and TRPV4 agonists excite RGCs (15, 17). Our data are consistent with previous findings. Besides membrane depolarization, we revealed that TRPV4 agonists caused more robust excitatory postsynaptic current (EPSC_TRPV4) and dramatic reduction of VSR, predicting ATP depletion in RGCs. EPSC_TRPV4 may involve TRPV4-IR in both BCs and RGCs.

Using VSR (1-N/S, %), we measured the reliability of the light-evoked action potentials under the disturbance of TRPV4 opening, creating an accurate measure for the dysfunction of individual RGCs. Neurons consume most energy to maintain the normal membrane potential (i.e., the normal electrochemical gradient of ions), as the latter is frequently disrupted by the opening of ion channels on the plasma membrane (95). Cation fluxes via opened ion channels follow and, thus, consume their electrochemical gradients across the membrane. The reduced gradients need ATPase and energy to recover (95). We first explored the relationship between the effect of TRPV4 activation, RP, activities of NaVs, and ATP consumption in RGCs. Our results showed that TRPV4 activation depolarized RGCs to the RP level that would inactivate NaVs (78–80, 96) and maximize ATP consumption. The results further suggest that improving ATP levels should be considered in treating pressure-related visual diseases.

Although currents at K-and N-MSCs are generally mutually compensating because of the opposite polarity, TRPs are known to mediate transient currents (15, 43, 84, 97), and K-MSCs carry leak currents (22, 28, 29). The difference in their kinetics does not allow complete cancelation of the pressure-induced current noise even with a balanced R_K/N. Our data indicates that opening TRPVs in retinal neurons can reduce VSR and increase ATP consumption in RGCs, predicting ATP crisis and pathologies if the pressure stress is persistent. Further studies on MSCs in retinal neurons will likely substantially facilitate the early diagnosis and treatment of pressure-related retinal disorders.