Article Figures & Data
Figures
- Figure 1.
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.
- Figure 2.
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).
- Figure 3.
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.
- Figure 4.
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.
- Figure 5.
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.
- Figure 6.
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).
- Figure 7.
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).
- Figure 8.
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.
