Abstract
Progression of neurodegeneration in disease and injury is influenced by the response of individual neurons to stressful stimuli and whether this response includes mechanisms to counter declining function. Transient receptor potential (TRP) cation channels transduce a variety of disease-relevant stimuli and can mediate diverse stress-dependent changes in physiology, both presynaptic and postsynaptic. Recently, we demonstrated that knock-out or pharmacological inhibition of the TRP vanilloid-1 (TRPV1) capsaicin-sensitive subunit accelerates degeneration of retinal ganglion cell neurons and their axons with elevated ocular pressure, the critical stressor in the most common optic neuropathy, glaucoma. Here we probed the mechanism of the influence of TRPV1 on ganglion cell survival in mouse models of glaucoma. We found that induced elevations of ocular pressure increased TRPV1 in ganglion cells and its colocalization at excitatory synapses to their dendrites, whereas chronic elevation progressively increased ganglion cell Trpv1 mRNA. Enhanced TRPV1 expression in ganglion cells was transient and supported a reversal of the effect of TRPV1 on ganglion cells from hyperpolarizing to depolarizing, which was also transient. Short-term enhancement of TRPV1-mediated activity led to a delayed increase in axonal spontaneous excitation that was absent in ganglion cells from Trpv1−/− retina. In isolated ganglion cells, pharmacologically activated TRPV1 mobilized to discrete nodes along ganglion cell dendrites that corresponded to sites of elevated Ca2+. These results suggest that TRPV1 may promote retinal ganglion cell survival through transient enhancement of local excitation and axonal activity in response to ocular stress.
Introduction
Neurodegeneration in disease and injury involves not only pathogenic processes but also prosurvival mechanisms to counter degradation of function in response to stress. Members of the transient receptor potential (TRP) family of cation-selective ion channels respond to a variety of stress-related stimuli (Lin and Corey, 2005; Ho et al., 2012; Vennekens et al., 2012). The capsaicin (CAP)-sensitive TRP vanilloid-1 (TRPV1) channel contributes to tactile sensitivity, diabetic sensory neuropathy, pressure-induced pain, injury monitoring, and visceral distension (Mutai and Heller, 2003; Hwang et al., 2004; Rong et al., 2004; Scotland et al., 2004; Jones et al., 2005; Ma et al., 2005; Liedtke, 2006; Plant et al., 2006; Daly et al., 2007; Pingle et al., 2007). Activation of TRPV1 induces a robust Ca2+ conductance that supports intracellular signaling cascades for both normal physiology and stress-related processes (Agopyan et al., 2004; Aarts and Tymianski, 2005; Reilly et al., 2005; Kim et al., 2006; Miller, 2006). Stimuli that activate TRPV1 can also increase the expression, sensitization, and translocation of the receptor to the plasma membrane (Kanai et al., 2005; Zhang et al., 2005; Facer et al., 2007; Schumacher and Eilers, 2010). In injury, TRPV1 is shuttled to damaged axons, in which it enhances spontaneous excitation (Biggs et al., 2008), and TRPV1-gated Ca2+ can potentiate glutamatergic signaling and contribute to cytoskeletal remodeling (Medvedeva et al., 2008; Jiang et al., 2009; Goswami et al., 2010; Peters et al., 2010). TRPV1 is also implicated in forms of synaptic remodeling, including long-term potentiation and depression (Li et al., 2008; Chávez et al., 2010).
Retinal ganglion cell neurons express TRPV1 and other TRP subunits, which can increase intracellular Ca2+ when activated and influence survival of ganglion cells challenged by stressors such as ischemia and elevated ocular pressure (Nucci et al., 2007; Maione et al., 2009; Sappington et al., 2009; Wang et al., 2010; Ryskamp et al., 2011; Leonelli et al., 2013).The latter is especially relevant for neurodegenerative disease, because pressure is a critical risk factor in glaucoma, the most common optic neuropathy and leading cause of irreversible blindness worldwide (Quigley and Broman, 2006). Recently, we demonstrated that knock-out of TRPV1 in mice (Trpv1−/−) or prolonged pharmacological antagonism of TRPV1 in rats using an inducible model of glaucoma accelerated ganglion cell axonal and somatic degeneration with exposure to elevated intraocular pressure (IOP) for 5 weeks (Ward et al., 2014). Importantly, ganglion cells from Trpv1−/− retina lacked a compensatory increase in spontaneous action potentials with elevated pressure and required greater depolarization to reach firing threshold. Here we demonstrate that exposure to elevated IOP in the same inducible model increased expression and localization of TRPV1 at excitatory synapses to ganglion cell dendrites. This effect was early and transient, as was the capacity of TRPV1 to promote physiological excitation by reversal of its net influence from hyperpolarizing to depolarizing. Finally, activation of TRPV1 in ganglion cells induced translocation to dendritic nodes that gated an increase in intracellular Ca2+. Together, these results suggest that TRPV1 promotes neuronal survival in response to disease-relevant stressors by transient enhancement of local excitation at postsynaptic sites.
Materials and Methods
Animals.
Adult (1–4 months old) C57BL/6 (C57) mice were obtained from Charles River Laboratories, whereas adult DBA/2J (3–12 months old) and the age-matched transgenic control strain D2-Gpnmb+ (D2; Howell et al., 2007a,b) were obtained from The Jackson Laboratory, as were adult (1 month old) male Trpv1−/− (B6.129X1–Trpv1tm1Jul/J) mice; Caterina et al., 2000; Ciura and Bourque, 2006; Treesukosol et al., 2007). Following Ward et al. (2014), these were genotyped before experimentation to confirm the transgene, following protocols provided by The Jackson Laboratory. For primary cultures of purified retinal ganglion cells, eyes from postnatal days 4–7 Sprague Dawley rats were enucleated, and their retinas were removed as described previously (Sappington et al., 2006, 2009). The Vanderbilt University Medical Center Institutional Animal Care and Use Committee approved all experimental procedures. Mice were maintained in a 12 h light/dark cycle with standard rodent chow available ad libitum.
We measured IOP bilaterally in anesthetized (2.5% isoflurane) mice using a TonoPen XL rebound tonometer (Medtronic Solan) as described previously (Inman et al., 2006; Sappington et al., 2010). For C57 and male Trpv1−/− mice, we monitored IOP for 2–3 d to give an average baseline IOP value (day 0). We elevated IOP by injecting only once 1.5 μl of 15 μm polystyrene microbeads (Invitrogen) into the anterior chamber of one eye, with the contralateral eye serving as internal control by receiving an equivalent volume of saline, as described previously (Crish et al., 2010; Sappington et al., 2010). We measured IOP in a subset of DBA/2 mice up to 10 months of age using the Tono-Pen XL (Medtronic Solan) as described previously (Inman et al., 2006).
Immunolabeling and confocal imaging.
For immunohistochemistry studies of TRPV1 localization, cohorts of mice (n = 3–5 animals each) were killed at 4 d and 1, 3, 5, and 7 weeks after microbead injection. Immunolabeling of retinal sections was performed as described previously and at identical conditions between sets using a highly specific rabbit anti-TRPV1 (1:100; Neuromics) and mouse anti-postsynaptic density protein 95 (PSD-95; 1:200; Millipore; Sappington et al., 2009; Dapper et al., 2013). Sections were counterstained for cell nuclei using 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; 1:100 in ddH2O).
Confocal imaging was done through the Vanderbilt University Medical Center Cell Imaging Shared Resource using identical microscope settings to acquire images for signal quantification. This was done by a naive observer using custom routines in NIH ImageJ. Fluorescent intensity of immunolabeled proteins was determined within outlined retinal layers and averaged over the selected area of pixels. Using FluoView software (Olympus), colocalization of TRPV1 and PSD-95 in the retina was examined by using stacked micrographs through multiple optical planes of retinal tissue. For each section, we drew multiple random horizontal lines across the region of interest and identified the number of pixels above an intensity threshold of 50 for either the TRPV1 or PSD-95 channel. Of that subset, the fraction of them above threshold for both channels was calculated, and colocalization was expressed as the ratio of pixels above threshold for TRPV1 and PSD-95 to the number of pixels above threshold for either.
Quantitative RT-PCR.
We extracted RNA from retina of naive and saline- or microbead-injected C57 eyes and of DBA2J and D2 eyes as described previously (Hanna and Calkins, 2006; Crish et al., 2013). Quantitative PCR (qPCR) was performed using an ABI Prism 7300 Real-Time PCR System and a FAM dye-labeled gene-specific probe for Trpv1 (Applied Biosystems). Cycling conditions and cycle threshold values were automatically determined by the supplied ABI software (SDS version 1.2). Relative product quantities for the Trpv1 transcript were performed in triplicate and determined using the 2ΔΔCt analysis method (Livak and Schmittgen, 2001), with normalization to 18S rRNA as an endogenous control.
Fluorescent in situ hybridization with immunolabeling.
Following our published protocol (Crish et al., 2013), to generate Trpv1 mRNA probes, total RNA from C57 mouse brain was extracted using RNeasy Mini kit (Qiagen), and first-strand cDNA synthesis was performed using Superscript III reverse transcriptase (Invitrogen). An antisense probe recognizing Trpv1 mRNA was made against a nucleotide sequence present in mouse Trpv1 (nucleotides 226–500 of GenBank accession number NM_001001445). A transcript generated by PCR using primers to Trpv1 (forward, 5′-ATC ACC GTC AGC TCT GTT GTC ACT-3′ and reverse, 5′-TGC AGA TTG AGC ATG GCT TTG AGC-3′) was inserted into pGEM-T Easy Vector (Promega), and orientation was verified by sequencing. Isolated plasmids were linearized and purified, and labeled Trpv1 RNA probes were generated using SP6 and T7 RNA polymerases and Digoxigenin RNA Labeling Mix (Roche Applied Science). Probe concentration and quality (A260/A280 ratio) were determined using a NanoDrop spectrophotometer. Probes were stored at −80°C.
Fluorescent in situ hybridization was performed using flat-mounted retinas as described previously (Crish et al., 2013). Immmunodetection of labeled Trpv1 mRNA was performed using anti-DIG-Fab-POD conjugate (Roche) diluted 1:100 in blocking buffer [1% blocking reagent (Roche) in 0.1 m Tris, pH 7.5, and 0.15 m NaCl], followed by detection using the TSA plus Fluorescein system (PerkinElmer Life and Analytical Sciences). Immunolabeling for retinal ganglion cells in the same tissue was performed with antibodies to phosphorylated heavy-chain neurofilament (1:1000, SMI31; Sternberger Monoclonals). For quantification, five confocal images were captured per retina, and labeled Trpv1 mRNA was quantified in 5–10 randomly selected retinal ganglion cells per image using ImagePro Analyzer 6.3 (Media Cybernetics).
Whole-cell patch-clamp recordings.
Additional C57 mice were killed up to either 2 weeks (4–15 d) or 4 weeks (18–28 d) after microbead injections as described above. These mice yielded 71 and 28 saline-injected and 85 and 26 microbead-injected eyes for the 2 and 4 week groups, respectively. Similarly, Trpv1−/− mice were killed 12–28 d after injections, yielding 14 each of saline and microbead retinas. Following our previous work (Ward et al., 2014), retinas were removed, hemisected, and transferred to oxygenated Ames solution in a temperature-controlled perfusion chamber with the ganglion cell layer up in the dark at room temperature. Ganglion cells (typically from the midperipheral retina) were whole-cell current clamped with borosilicate glass electrodes (5–10 MΩ) containing 130 mm K-gluconate, 10 mm KCl, 10 mm HEPES, 2 mm MgCl2, 1 mm EGTA, 2 mm Na2ATP, 0.3 mm NaGTP, and 1% Lucifer yellow dye (Life Technologies) to visualize dendritic morphology. For cells that did not show a spontaneous action potential firing rate >0.5 Hz, a 1 s depolarizing step current ranging from 25 to 200 pA was delivered at 0.1 Hz until the firing rate exceeded 3 Hz. Ganglion cells for which action potentials did not maintain a constant firing rate for a minimum of 4 min were excluded, as were cells for which membrane resistance changed by >20%. Drugs were bath perfused and included the TRPV1-specific agonists CAP (2 μm) and N-oleoyldopamine (N-OLDA; 10 μm; Medvedeva et al., 2008) and the TRPV1-specific antagonist iodoresiniferatoxin (IRTX; 100 nm). CAP in the 1–10 μm concentration range enhances excitatory transmission in a variety of brain structures (Marinelli et al., 2003; Li et al., 2004; Xing and Li, 2007). A 10 μm dose of N-OLDA has similar effects (Spicarova and Palecek, 2009), whereas IRTX is an effective inhibitor at submicromolar concentrations (Wahl et al., 2001). After recording, retinas were fixed in 4% paraformaldehyde overnight and mounted for confocal microscopy as described above.
Purified retinal ganglion cell primary cultures and Ca2+ imaging.
Primary cultures of purified ganglion cells were prepared as described previously by immunomagnetic separation using mouse anti-rat Thy1.1/CD90 IgG (5 μg/ml; BD Pharmingen) and metallic microbeads conjugated with anti-mouse IgG (Sappington et al., 2006, 2009). To assess TRPV1-mediated changes in accumulated intracellular Ca2+, we used the BAPTA-based Ca2+ indicator dye Fluo-4 AM (Invitrogen; Kreitzer et al., 2000; Sappington et al., 2009), which exhibits a 40-fold increase in fluorescence intensity with Ca2+ binding (Gee et al., 2000). Primary cultures were loaded with 5 μm Fluo-4 for 30 min, assessed for comparable loading, and then treated for 30 min with either CAP (100 nm or 1 μm) or its vehicle (ETOH) or CAP (1 μm) and 950 μm EGTA (Gibco), which reduced the concentration of available Ca2+ to 100 μm, as determined by Maxchelator (Stanford University, Stanford, CA). Live cultures were coverslipped with physiological saline and imaged using confocal microscopy. For each sample, 15–20 independent fields were acquired, and Fluo-4 intensity was quantified using Image Pro Plus (version 5.1.2; Media Cybernetics). After imaging of accumulated Ca2+, ganglion cell cultures were fixed briefly, immunolabeled for TRPV1 and SMI31, and imaged using confocal microscopy as described above.
Statistical analyses.
Unless otherwise indicated, all data are expressed as mean ± SE; the number of samples used in each experiment is given in the appropriate description in Materials and Methods or figure legend. Statistical comparisons between two independent measurements were made using two-sided t tests, after confirmation of normality for each using the Shapiro–Wilk normality test; samples for which normality failed were compared using the Mann–Whitney rank-sum test (SigmaPlot 11.1; Systat Software). Comparisons of a sample mean to a hypothesized or predicted value were made using a one-sided t test. Comparisons between multiple groups were made using Kruskal–Wallis one-way ANOVA on ranks (SigmaPlot 11.1; Systat Software). Actual pvalues of significance are indicated when appropriate in Results or figure legends. All comparisons for which significance is reported achieved or exceeded a post hoc calculation of power of 0.80.
Results
Elevated ocular pressure increases TRPV1 transiently at retinal synapses
We elevated IOP for various durations in cohorts of C57 and Trpv1−/− mice by microbead occlusion of aqueous fluid in the anterior eye (Sappington et al., 2010; Chen et al., 2011). A single unilateral microbead injection elevated IOP by 31–34% for up to 7 weeks in C57 eyes intended for expression studies (Fig. 1A) and by 32–34% for up to 4 weeks in C57 or Trpv1−/− eyes intended for physiological studies (Fig. 1B). All IOP measurements were similar to those we described recently in C57 and Trpv1−/− eyes for a slightly lesser volume injection (1.0 μl; Ward et al., 2014).
Microbead-induced elevations in mouse IOP. A, Daily average of IOP for C57 eyes used in labeling studies before (day 0) and after (days ≥1) a single unilateral injection of polystyrene microbeads (1.5 μl) or an equivalent volume of saline. Cohorts followed from 4 d (circles) to 7 weeks (diamonds). Microbead eyes exhibited an increase of 31–34% (19.68–19.84 ± 0.15–0.39) that was significant compared with saline eyes (14.69–15.04 ± 0.12–0.25) for all cohorts (p < 0.001). Between cohorts, IOP was similar for saline (p = 0.38) and microbead (p = 0.65) eyes. B, Average daily IOP in eyes used for physiological studies was similarly elevated (32–34%) by microbead injection for up to 2 (circles) or 4 (squares) weeks for C57 eyes (19.56 ± 0.26 and 19.65 ± 0.39, respectively) and for just under 4 weeks in Trpv1−/− eyes (19.73 ± 0.42). Errors indicated are SD. IOP was similar for both saline (p ≥ 0.15) and microbead (p ≥ 0.15) eyes for all cohorts.
Immunolabeling for TRPV1 in retina from saline-injected control eyes was generally modest in all cohorts (Fig. 2A, top row), with clear localization near ganglion cell neurons and in the nerve fiber layer in which their axons extend. This is consistent with previous work in naive retina (Leonelli et al., 2009, 2010; Sappington et al., 2009). Microbead-induced elevated IOP increased TRPV1 transiently by 1 week, with a return to saline-eye levels by later times (Fig. 2A, bottom row). Although TRPV1 label increased throughout the retina, when quantified, the only significant changes were in the ganglion cell layer and inner plexiform layer, in which ganglion cell dendritic arbors ramify and receive synaptic contacts (Fig. 2B). The increase in TRPV1 was transient, diminishing to levels in saline retina by 5 weeks. Quantitative RT-PCR measurements showed that expression of Trpv1 mRNA in the retina increased transiently after 1–2 weeks of elevated IOP, diminishing significantly by 7 weeks (Fig. 2C).
TRPV1 increases early with elevated IOP. A, Vertical sections through C57 retina immunolabeled against TRPV1 and counterstained with DAPI after microbead or saline injection (IOPs in Fig. 1A). TRPV1 localization increases after 1 week of microbead-induced IOP elevation, especially for the ganglion cell layer (GCL) and inner plexiform layer (IPL; bracketed). PRL, Photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; NFL, nerve fiber layer. Scale bar, 20 μm. B, Quantification of TRPV1 for specific retinal layers indicated as ratio of microbead/saline eye. Increased ratio in the ganglion cell and inner plexiform layers at 1 week is significant compared with the ratio at 4 d (*p ≤ 0.026) and with a hypothetical ratio of one (dashed line; †p ≤ 0.046). C, qPCR measurements of TRPV1 mRNA expressed as ratio of microbead/saline retina after normalization to 18S rRNA for pairs of eyes 1–2 and 6–7 weeks after injection. Ratio of TRPV1 mRNA at 1–2 weeks (1.12 ± 0.04; n = 8) exceeds the predicted ratio of one (†p = 0.022), whereas the ratio at 6–7 weeks (0.79 ± 0.08; n = 5) is significantly lower than 1–2 weeks (*p = 0.002). IOP for saline and microbead eyes was similar to that in Figure 1. Average TRPV1 mRNA level for naive retina shown for comparison (1.04 ± 0.16; n = 12).
In naive mouse retina, TRPV1 localizes strongly to both ganglion cell bodies identified by a cell-specific marker and in the inner plexiform layer to neuronal processes expressing PSD-95 (Fig. 3A,B), a marker for excitatory synapses in the retina (Cho and So, 1992; Koulen et al., 1998; Jakobs et al., 2008). Microbead-induced elevations in IOP also increased PSD-95 localization transiently in the inner plexiform layer, reaching a peak by 1 week that subsided by 5 weeks (Fig. 3C). Quantification on a pixel-by-pixel basis indicated a significant threefold increase in pixels colabeled with TRPV1 and PSD-95 expressed as the ratio of microbead/saline retina at 1 week (3.8 ratio) compared with 4 d (1.3 ratio; p = 0.023).
Colocalization of TRPV1 and PSD-95 increases transiently. A, Thy1-labeled ganglion cells in naive C57 retina (arrows) expressing TRPV1. B, Single confocal plane through the border of the inner plexiform and ganglion cell layers of C57 retina shows TRPV1 localizing at discrete positions along neuronal processes (arrows) and around cell bodies (circles) outlined by PSD-95 localization. C, High-magnification single confocal planes through inner plexiform layer after microbead or saline injection (IOPs in Fig. 1). Increased colocalization of PSD-95 and TRPV1 after 1 week of elevated IOP indicated (arrowheads) in orthogonal slices at specific locations in image plane (lines). Scale bars: A, B, 20 μm; C, 5 μm. GCL, Ganglion cell layer; IPL, inner plexiform layer; bracketed; PRL, photoreceptor layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; NFL, nerve fiber layer.
TRPV1 localization to ganglion cells increases with elevated IOP and age in the DBA2J mouse model of hereditary glaucoma (Sappington et al., 2009). This inbred strain presents age-dependent elevations in IOP induced by iris atrophy and pigment dispersion caused by mutations in the tyrp1 and gpnmb genes, respectively (Howell et al., 2007a,b). Here, quantitative RT-PCR of retinal mRNA showed that Trpv1 expression in young (3–5 months old) DBA2J retina is ∼50% of that from retina from the age-matched D2 control strain (Fig. 4A). Elevated IOP increased Trpv1 approximately threefold for both young and aged (8–10 months old) DBA2J retina (Fig. 4A). Even for eyes with a narrow range of low IOPs (<15 mmHg), Trpv1 expression scaled with increasing pressure (Fig. 4A, inset). We then combined fluorescent in situ hybridization for Trpv1 antisense mRNA with immunolabeling for phosphorylated neurofilaments, which selectively marks ganglion cells (Howell et al., 2007b; Soto et al., 2008; Crish et al., 2010). Compared with antisense label against ganglion cell-specific Thy1 mRNA as a positive control (Fig. 4B), Trpv1 mRNA in C57 retina distributed lightly in ∼70% of ganglion cells (Fig. 4C). We observed a similar pattern in 3-month-old DBA2J ganglion cells (Fig. 4D). By 8 months, DBA2J ganglion cells demonstrated a clear increase in Trpv1 expression (Fig. 4E, left) that dissipated by 12 months (Fig. 4E, right). When quantified, signal forTrpv1 antisense increased significantly by 6–8 months compared with 3 months; by 12 months, Trpv1 expression returned to 3 month levels (Fig. 4F). In no case did the control sense sequence elicit detectable label (Fig. 4B–E). Interestingly, DBA2J ganglion cells with accumulation of phosphorylated neurofilaments in their dendritic arbors, which signifies pathogenic progression (Howell et al., 2007b; Soto et al., 2008), had 50% less Trpv1 antisense signal on a cell-by-cell basis (Fig. 4G,H). These results indicate that, like microbead-induced elevated IOP, chronic stress also transiently increases Trpv1 mRNA with changes detectable in single ganglion cells.
Increased TRPV1 mRNA in ganglion cells of the DBA2J mouse retina. A, Bar graphs, qPCR measurements of Trpv1 mRNA in DBA2J retina relative to levels in age-matched D2 control retina; both calculated relative to 18S rRNA. For the 3–5 month group, retinas from eyes with higher IOP (17.00 ± 0.52 mmHg) demonstrated threefold higher Trpv1 than those from lower IOP eyes (11.11 ± 0.46 mmHg), as did higher IOP retinas from the 8–10 month group (17.62 ± 0.86 mmHg) compared with low IOP counterparts (14.11 ± 0.84 mmHg; *p ≤ 0.017). The 8–10 month low- and high-IOP samples also had greater Trpv1 than their 3–5 month counterparts (†p ≤ 0.05). Sample size: n = 6 for each. Inset, Trpv1 mRNA in DBA2J relative to D2 retinas from eyes within the low IOP groups (<15 mmHg only) with best-fitting regression line (r2 = 0.53; p = 0.007). B, Ganglion cells in flat-mounted naive C57 retina colabeled with cell-specific Thy1 antisense (AS) probe and phosphorylated neurofilament antibodies (SMI31) as a positive control. Corresponding sense sequence (S) shown as negative control. C, SMI31 immunolabeled ganglion cells in C57 retina stained for Trpv1 antisense sequence. D, SMI31-labeled ganglion cells in DBA2J retina (3 months) colabeled with Trpv1 antisense probe (left) and control sense sequence (right). E, An 8 month DBA2J retina (left) expresses higher levels of Trpv1 mRNA compared with 12 months (right). F, Quantification of Trpv1 antisense signal in individual DBA2J ganglion cells shows progressively increased expression from 3 to 8 months (*p < 0.001), with a subsequent decrease to 3 month levels by 12 months (p = 0.65). Sample size: n = 20–25 cells per group. G, Ganglion cells in flat-mounted 12 month DB2J retina immunolabeled for SMI31 (left) and Trpv1 antisense probe (middle). Ganglion cells with accumulation of SMI31-labeled phosphorylated neurofilaments (arrows) show lower levels of Trpv1 expression (right). H, Ganglion cells with SMI31 accumulation (W/ AC) show significantly lower fraction of cell body area containing Trpv1 signal compared with cells without accumulation (W/O AC; *p < 0.001). Scale bars: B–E, 20 μm; G, 30 μm.
Elevated pressure transiently enhances TRPV1-mediated excitation
Next we probed whether increased TRPV1 at excitatory synapses with elevated IOP in the C57 retina could influence ganglion cell physiology using patch-clamp voltage recording from microbead retinas (IOPs in Fig. 1B). For each ganglion cell targeted for recording in whole-mounted retina, we confirmed an intact axon and classified the cell as ON, OFF, or ON–OFF based on dendritic stratification after intracellular filling with Lucifer yellow to illuminate morphology (Fig. 5A). For an ON–OFF cell from a saline-injected eye (Fig. 5B), bath application of the TRPV1-specific agonist CAP (2 μm) induced a modest (11%) increase in rate of action potential generation from the spontaneous rate of 4.6 Hz (Fig. 5C). For an ON–OFF ganglion cell after 13 d of elevated IOP, CAP induced a 20% increase in firing rate (Fig. 5D). Application of a different TRPV1-specific agonist (N-OLDA, 10 μm) elicited a 49% increase in an ON–OFF ganglion cell after 14 d of elevated IOP (Fig. 5E). In these examples, TRPV1 contributes an excitatory effect on ganglion cell firing rate, with elevated IOP enhancing the effect.
Elevated IOP increases TRPV1-mediated excitation. A, C57 ganglion cell filled with 1% Lucifer yellow during whole-cell patch-clamp recording shows ramified dendrites and axon (arrow). B, Reconstruction of ON–OFF cell with axon (arrow) from saline eye 5 d after the fellow eye received microbead injection. C, Action potential recordings under current clamp from the cell shown in B before (left) and after (right) 3 min bath application of 2 μm CAP, which increases firing 11% from the baseline rate of 4.6 Hz. D, Reconstruction of ON–OFF ganglion cell with axon (arrow) from microbead retina 13 d after injection. Scale bar, 100 μm. Application of 2 μm CAP increases firing rate by 20%. E, Firing rate in ON–OFF cell from microbead retina 14 d after injection increases 49% after application of 10 μm N-OLDA. Scale bar applies to D also. Scale bars: A, B, 50 μm.
Overall, however, TRPV1 had two very different effects on ganglion cell responses. We compared the responses of ganglion cells from microbead retina with those in corresponding saline retina for bath application of CAP or N-OLDA after brief (2 weeks) and more extensive (4 weeks) periods of elevated IOP as shown in Figure 1B. Interestingly, for the 2 week group, ganglion cells from saline retina on average responded to 3 min bath application of CAP or N-OLDA with a net reduction in firing rate relative to the spontaneous rate before application (Fig. 6A). This trend increased over the course of a recording. In contrast, ganglion cells from microbead retina in the 2 week cohort responded with a net increase in firing rate, consistent with the examples in Figure 5. However, by 4 weeks of elevated IOP, this excitatory enhancement had disappeared, with ganglion cells from microbead retina demonstrating the same net reduction in excitation as cells in saline retina (Fig. 6B). Saline eye responses were similar between the 2 and 4 week groups.
Increased TRPV1 excitation is transient. A, Average firing rate over time as a percentage of baseline rate (−3 to 0 min) after application (solid line, 0–3 min) of TRPV1 agonists CAP (2 μm) or N-OLDA (10 μm). Agonists cause a net reduction in rate for ganglion cells from saline eyes (n = 19) but a net increase in cells from microbead eyes (n = 31) after 2 weeks of IOP elevation (p = 0.001). B, After 4 weeks of IOP elevation, response to TRPV1 agonists is similar between microbead (n = 14) and saline (n = 15) cells (p = 0.78). Dashed line indicates no change from baseline rate (100%). C, Mean firing rate in the period after application of TRPV1 agonists (3–10 min) for individual ON, OFF, and ON–OFF ganglion cells from saline versus microbead retinas as a percentage of baseline. IOP elevation for 2 weeks increases the rate by 6% on average (106.3 ± 2.7%), which differs significantly from the rate for saline cells (94.6 ± 2.0%; *p = 0.007). Both means (squares) differ from a prediction of no change in rate (or 100%; †p ≤ 0.026). The response after 4 weeks is similar for ganglion cells from saline versus microbead retinas (p = 0.41), and neither group differs from a prediction of no change (100%; p ≥ 0.10). Saline eye responses did not differ for the 2 and 4 week groups (p = 0.98). D, Top trace, Average response to bath application of the TRPV1-specific antagonist IRTX over time is minimal and similar for saline (n = 20) and microbead (n = 30) ganglion cells 2 weeks after injection (p = 0.52). Bottom trace, Pretreatment (10 min) with IRTX inhibits agonist-induced differences in response between saline (n = 13) and microbead (n = 16) ganglion cells (p = 0.26).
When averaged over the entire postdrug recording period (7 min), the responses of individual cells from microbead retina in the 2 week cohort to the agonists were predominantly excitatory, with 19 of 31 cells (61%) demonstrating an increase in firing rate that averaged 6% in amplitude (Fig. 6C). In contrast, 79% of ganglion cells from saline eyes in the same cohort demonstrated a reduction in firing rate that averaged 5%. Both differed significantly from a prediction of no change from their predrug firing rate. By 4 weeks of elevated IOP, the response of ganglion cells from microbead and saline retinas were the same, and neither differed from their predrug firing rate (Fig. 6C). For each cohort, we did not detect any systematic differences between ON, OFF, or ON–OFF ganglion cells, although the sample size for each type was limited. Finally, preapplication of the synthetic TRPV1-specific antagonist IRTX nearly eradicated any change in firing rate attributable to CAP or N-OLDA for ganglion cells from either saline or microbead retinas (Fig. 6D). Thus, in the absence of stress induced by elevated IOP, TRPV1 activation by agonists primarily reduces ganglion cell excitation in intact retina. This effect was not an artifact attributable to recording, because neither membrane voltage nor membrane resistance changed significantly during the duration of our recordings and were the same for both saline and microbead ganglion cells (p = 0.69 and 0.35, respectively; data not shown).
Recently, we found that 4 weeks of elevated IOP significantly increased spontaneous firing of action potentials for C57 ganglion cells; Trpv1−/− cells lacked this compensatory increase entirely for the same IOP elevation (Ward et al., 2014). Given the transient increase of TRPV1 in expression (Figs. 2, 3) and capacity to induce excitation (Fig. 6), we extended our previous result by comparing firing rates between the 2 versus 4 week cohorts for all ganglion cells that, in the absence of additional stimulation, spontaneously generated action potentials (>0.5 Hz). Ganglion cells in the 4 week C57 cohort demonstrated a 61% increase in spontaneous activity with elevated IOP compared with ganglion cells from corresponding saline retinas (Fig. 7A). This is consistent with our published finding (Ward et al., 2014). Even at 2 weeks, ganglion cells from microbead retinas had a 25% higher spontaneous firing rate compared with cells from saline retina (Fig. 7A), but this difference did not reach significance. In contrast, Trpv1−/− ganglion cells had no systematic difference in spontaneous rate for saline or microbead retinas for the entire range of times we examined (12–28 d; p = 0.49). When pooled, Trpv1−/− ganglion cells from microbead retinas demonstrated a spontaneous rate that was 37 and 42% less than rates for ganglion cells from the 2 and 4 week C57 microbead cohorts, respectively (Fig. 7A).
Trpv1−/− ganglion cells lack compensatory excitation. A, Spontaneous firing rate increases significantly for ganglion cells in C57 4 week microbead retinas (11.41 ± 1.48 Hz, n = 37 cells) compared with those in saline retinas (7.17 ± 1.35 Hz, n = 31; *p = 0.025). The apparent increase for microbead cells in the 2 week group (10.37 ± 0.95 Hz, n = 105 cells) compared with saline (8.31 ± 0.84 Hz, n = 68) was not significant (p = 0.28). Cells in Trpv1−/− microbead retina demonstrated significantly less spontaneous activity compared with their C57 counterparts in either cohort (6.64 ± 1.04 Hz, n = 34 cells; †p ≤ 0.048) and were similar to Trpv1−/− saline ganglion cells (6.52 ± 0.81, n = 33 cells; p = 0.67). Ganglion cell responses in saline retinas did not differ between any group (p = 0.55). B, Minimum depolarizing current required to induce action potentials in ganglion cells with low spontaneous rates (<0.5 Hz) was greater for Trpv1−/− microbead retinas (64.0. ± 8.05, n = 25 cells) compared with Trpv1−/− saline (39.8 ± 5.6, n = 22; *p = 0.013) and when compared with 4 week C57 microbead ganglion cells (42.5 ± 7.7, n = 20; †p = 0.020). Threshold current did not differ between any saline retina cohort (p = 0.55).
For ganglion cells in our sample with very low spontaneous firing rates (<0.5 Hz), we induced a rate of 3 Hz or higher by injecting steps of depolarizing currents (Fig. 7B). Ganglion cells of this class from C57 retinas required the same degree of depolarization (40–50 pA) for both 2 and 4 week cohorts and for saline and microbead eyes (p = 0.38). Although Trpv1−/− ganglion cells from saline retinas required the same threshold current as C57, cells from Trpv1−/− microbead retina required ∼63% more depolarization to reach threshold. This threshold was the same for allTrpv1−/− ganglion cells from microbead retinas, regardless of length of IOP elevation (p = 0.60). Together, our results suggest that ganglion cells from Trpv1−/− retina challenged by elevated IOP consistently demonstrate less spontaneous activity and require greater excitation to generate action potentials regardless of the duration of stress. Furthermore, the effects of increased TRPV1 expression appear to have a delayed influence on spontaneous ganglion cell activity, because only a longer exposure to elevated IOP (4 weeks) elicited a significant increase in firing rate (Fig. 7A).
Activation induces local translocation of TRPV1 and increased Ca2+
Activation of TRPV1 by ligands or physical stimuli can induce translocation of the channel to the plasma membrane with increased sensitization of the receptor (Zhang et al., 2005; Schumacher and Eilers, 2010). We found that, for isolated ganglion cells from naive retina, activation of TRPV1 with a low concentration of CAP (100 nm) caused little change in overall levels of Fluo-4-conjugated Ca2+ but did concentrate signals within local nodes along dendritic processes (Fig. 8A,B). A 10-fold increase in CAP concentration (1 μm) amplified this effect, along with overall levels of Ca2+ (Fig. 8C). Coapplication of CAP with EGTA (950 μm) to chelate extracellular Ca2+prevented the Ca2+increase but not the formation of local nodes (Fig. 8D).
Activation of TRPV1 induces translocation. A, Fluo-4-conjugated Ca2+ in isolated retinal ganglion cells distributes evenly in dendritic processes with vehicle (Veh) treatment. B, CAP at 100 nm concentrates Ca2+ in local nodes along dendritic processes (arrowheads). C, Increased CAP (1 μm) raises overall Ca2+ including at nodes (arrowheads). D, Cotreatment with EGTA (950 μm) to chelate extracellular Ca2+ reduced levels but not CAP-induced nodes. E, Immunolabeling for TRPV1 and phosphorylated neurofilaments (SMI31) on the same ganglion cells used for Fluo-4 imaging shows concentrations of TRPV1 corresponding to nodes of increased intracellular Ca2+ with CAP activation (arrowheads). F, Quantification of Fluo-4 signal indicates a more than threefold increase with 1 μm CAP compared with vehicle (*p < 0.001), which was prevented by cotreatment with EGTA (†p = 0.004). G, Treatment with 100 nm CAP increases the density of TRPV1-concentrated nodes threefold along stretches of ganglion cell dendrites, as well as their maximum diameter compared with vehicle (*p < 0.001). Scale bars: A–D, 20 μm; E, 40 μm.
Immunolabeling of the same ganglion cells after Fluo-4 imaging indicated that focal concentrations of TRPV1 corresponded to local nodes of concentrated intracellular Ca2+ induced by CAP treatment (Fig. 8E). When quantified across preparations, compared with vehicle treatment, 1 μm CAP increased ganglion cell Ca2+ by threefold, which was quenched entirely by cotreatment with EGTA (Fig. 8F). Whereas 100 nm CAP did not increase overall Ca2+ compared with vehicle (Fig. 8F), it did increase by threefold the density of TRPV1 nodes along stretches of ganglion cell dendrites, as well as node size (Fig. 8G). Thus, activation of TRPV1 by CAP induces translocation of the channel to local dendritic sites that gate focal increases in influx of extracellular Ca2+.
Discussion
Recently, we found that both Trpv1−/− and pharmacological inhibition of the receptor in vivo accelerated degeneration of retinal ganglion cell axons and bodies in response to modest periods (4–5 weeks) of elevated IOP in the microbead occlusion model of induced glaucoma (Ward et al., 2014). Retinal ganglion cells from eyes with elevated IOP had significantly enhanced spontaneous firing rates, a response that was completely absent in Trpv1−/− ganglion cells. We proposed that TRPV1 intrinsically counters functional degradation of stressed ganglion cells and their axons through a neuroprotective mechanism, as in models of ischemic brain injury (Pegorini et al., 2005; Muzzi et al., 2012). Similar studies have implicated TRPV1 and TRP Canonical 6 as protective mediators of ganglion cell survival in ischemic-reperfusion injury (Nucci et al., 2007).
Because of the high Ca2+ conductance of TRPV1, whether activation is protective or toxic depends greatly on expression levels, the mode of activation, and in some cases interactions with other receptor classes, including cannabinoid receptors (Kim et al., 2007). TRPV1 expression is known to rise with exposure to noxious or stressful stimuli, many of which are activators of the channel (Facer et al., 2007; Schumacher and Eilers, 2010). We found that a single week of microbead-induced elevated IOP increases TRPV1 in ganglion cell bodies and in the inner plexiform layer, in which ganglion cells receive synaptic contacts (Fig. 2). Total retinal Trpv1 mRNA also increased slightly (Fig. 2C). Elevations in TRPV1 were transient, returning to control levels by 5 weeks, and correlated with greater colocalization with excitatory postsynaptic densities (Fig. 3). TRPV1 localization also increases in retinal ganglion cells of the DBA2J mouse model of chronic glaucoma (Sappington et al., 2009), which demonstrates progressive elevations in IOP with age (Libby et al., 2005; Inman et al., 2006). Here, as Trpv1 mRNA rose in the DBA2J retina with elevated IOP and age (Fig. 4A), in situ hybridization against Trpv1 combined with a cell-specific marker (SMI31) showed progressively increased expression in ganglion cells from 6 to 8 months of age (Fig. 4D–F), when neurodegeneration in the DBA2J is early (Whitmore et al., 2005; Inman et al., 2006; Crish et al., 2010; Howell et al., 2012; for review, see Calkins, 2012). Supporting a protective role, Trpv1 antisense signal was lower in ganglion cells with overt accumulation of phosphorylated neurofilaments (Fig. 4G,H), an indicator of advanced progression in the DBA2J retina (Howell et al., 2007b; Soto et al., 2008).
Our key physiological finding is that, as TRPV1 localization rises transiently in retinal ganglion cells, the capacity of the channels to increase excitability changes as well, as it does in other axonal injury models (Biggs et al., 2007, 2008). In retinas from saline-injected control eyes, pharmacological activation of TRPV1 had a net hyperpolarizing influence on ganglion cell firing rates with most cells demonstrating a decreased rate with drug application (Fig. 6A–C). However, after 2 weeks of elevated IOP, the net response became depolarizing, with the majority of ganglion cells demonstrating increased excitation with TRPV1 agonism (Fig. 6A,C). This effect was transient. After 4 weeks of elevated IOP, ganglion cells from microbead and saline retinas had the same, mostly hyperpolarizing response to TRPV1 agonists (Fig. 6B,C). The influence of TRPV1 was highly specific, because the synthetic antagonist IRTX nearly completely blunted the effects of the agonists for all ganglion cells (Fig. 6D). Interestingly, although the excitatory effects of TRPV1-specific agonists were early and transient, commensurate with early changes in expression (Figs. 2, 3), the net influence on spontaneous firing rate was delayed. Although ganglion cells from saline retina had the same spontaneous rate at all times, the rate for ganglion cells from microbead retina gradually increased from 2 to 4 weeks of elevated IOP (Fig. 7A). Ganglion cells from Trpv1−/− retina completely lacked this increase, with spontaneous rates that did not differ from saline-retina ganglion cells in the C57 cohorts (Fig. 7A). Trpv1−/− ganglion cells from microbead eyes that lacked appreciable spontaneous rates (<0.5 Hz) also required ∼60% more depolarizing current to fire, again regardless of length of IOP elevation (Fig. 7B). This could indicate that Trpv1−/− ganglion cells lack not only the compensatory response but the capacity altogether to sense IOP-related stress. This idea is supported by our results from isolated ganglion cells showing that TRPV1 antagonism blunts pressure-induced increases in intracellular Ca2+ (Sappington et al., 2009).
Colocalization of TRPV1 with PSD-95 on ganglion cell dendrites suggests that the receptor could modulate postsynaptic activity directly (Fig. 3), as does the localization of TRPV1 to dendritic nodes of heightened Ca2+ accumulation in isolated ganglion cells (Figs. 8). Upregulation and activation of TRPV1 are often concurrent with translocation from intracellular binding sites either to different neuronal compartments or the plasma membrane (Zhang et al., 2005; Biggs et al., 2008; Goswami et al., 2010; Schumacher and Eilers, 2010). TRPV1-gated Ca2+ promotes spontaneous excitation and can potentiate responses to glutamate (Marinelli et al., 2003; Xing and Li, 2007; Medvedeva et al., 2008; Jiang et al., 2009; Peters et al., 2010). Evidence for potentiation points to a presynaptic role for TRPV1, through facilitation of glutamate release (Marinelli et al., 2003; Medvedeva et al., 2008). This potentiation supports increased axonal spontaneous firing (Xing and Li, 2007). However, in studies of long-term depression in dentate gyrus, TRPV1 exerts its influence postsynaptically by suppressing excitatory transmission (Chávez et al., 2010). In this case, EPSCs are reduced by CAP treatment, resembling the reduction in activity we observed for ganglion cells from control retinas (Fig. 6). Typically, synaptic actions of TRPV1—whether presynaptic or postsynaptic—involve complex interactions with glutamatergic signaling machinery and/or cannabinoid pathways (Marinelli et al., 2005; Kim et al., 2007; Chávez et al., 2010). In the retina, multiple cell types besides ganglion cells express TRPV1 at different levels, including both excitatory and inhibitory neurons (for review, see Ryskamp et al., 2014). It is possible that increased TRPV1 expression with elevated IOP reflects also a change in the relative influence of TRPV1-expressing neurons presynaptic to ganglion cells.
Degeneration of retinal ganglion cells in glaucoma is likely initiated by IOP-related stress directed to the highly vulnerable unmyelinated segment of the axon as it passes through the optic nerve head (Burgoyne, 2011; Calkins, 2012). Thus, early pathogenesis is axogenic, involving physiological challenges to the optic projection before outright degeneration (Whitmore et al., 2005; Calkins, 2012; Howell et al., 2012). One of the earliest characteristics is degradation of active transport from the retina to the superior colliculus, the most distal projection for ganglion cell axons (Crish et al., 2010; Lambert et al., 2011; Dapper et al., 2013). Our earlier study showed that both depletion of functional transport to the colliculus and axonal degeneration in the optic nerve proceeded approximately twice as rapidly in Trpv1−/− than in age-matched C57 mice for the same 5 week period of IOP elevation; pharmacological antagonism also accelerated degeneration (Ward et al., 2014).
Together, our results suggest an intriguing possibility. The transient TRPV1-mediated enhancement of ganglion cell excitation induced by elevated IOP appears to support, in unknown ways, a longer-lasting higher spontaneous firing rate that is critical to ganglion cell axon survival, because its absence in Trpv1−/− retina accelerates degeneration. In the microbead model, even as axon transport is challenged before outright axon loss (Crish et al., 2010; Chen et al., 2011; Dapper et al., 2013), glutamatergic synapses from ganglion cell axons to interneurons in the colliculus persist, defining a window of opportunity for therapeutic intervention (Crish et al., 2010, 2013). Although we have focused on ganglion cells as postsynaptic neurons in the retina, their central purpose is in fact presynaptic, in conveying visual information to brain targets via glutamatergic signaling. Transient activation by CAP or N-OLDA of TRPV1 in axonal boutons of dorsal root ganglion neurons elevates presynaptic Ca2+ to enhance glutamate release to postsynaptic spinal cord neurons (Medvedeva et al., 2008). In a similar way, retinal TRPV1 acting on ganglion cell dendrites could have a distal influence conveyed via enhanced spontaneous activity to conserve glutamatergic signaling at central brain targets. Enhanced TRPV1 excitation in the ganglion cell circuit could serve an early and critical role in boosting spontaneous axonal activity to maintain functional connectivity with brain targets in response to ocular stress.
Footnotes
This work was supported by National Institutes of Health (NIH) Grants EY017427 (D.J.C.), 5T32EY007135-18 (N.J.W.), and T32GM007628-32 (K.W.H.), Senior Scientific Investigator and Departmental Unrestricted Awards from Research to Prevent Blindness (D.J.C.), the BrightFocus Foundation (D.J.C.), and the Glaucoma Research Foundation (D.J.C.). Imaging was supported through the Vanderbilt University Medical Center Cell Imaging Shared Resource core facility (Clinical and Translational Science Award Grant UL1 RR024975 from National Center for Research Resources/NIH) and the Vanderbilt Vision Research Center (NIH Grant P30EY008126). We acknowledge Brian J. Carlson and Kelsey Karas for their assistance with the project.
The authors declare no competing financial interests.
- Correspondence should be addressed to David J. Calkins, Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, Vanderbilt University Medical Center, 11425 Medical Research Building IV, Nashville, TN 37232-0654. david.j.calkins{at}vanderbilt.edu
