Assessment of Rod, Cone, and Intrinsically Photosensitive Retinal Ganglion Cell Contributions to the Canine Chromatic Pupillary Response

Abstract

Purpose: The purpose of this study was to evaluate a chromatic pupillometry protocol for specific functional assessment of rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) in dogs.

Methods: Chromatic pupillometry was tested and compared in 37 dogs in different stages of primary loss of rod, cone, and combined rod/cone and optic nerve function, and in 5 wild-type (WT) dogs. Eyes were stimulated with 1-s flashes of dim (1 cd/m2) and bright (400 cd/m2) blue light (for scotopic conditions) or bright red (400 cd/m2) light with 25-cd/m2 blue background (for photopic conditions). Canine retinal melanopsin/Opn4 was cloned, and its expression was evaluated using real-time quantitative reverse transcription-PCR and immunohistochemistry.

Results: Mean ± SD percentage of pupil constriction amplitudes induced by scotopic dim blue (scDB), scotopic bright blue (scBB), and photopic bright red (phBR) lights in WT dogs were 21.3% ± 10.6%, 50.0% ± 17.5%, and 19.4% ± 7.4%, respectively. Melanopsin-mediated responses to scBB persisted for several minutes (7.7 ± 4.6 min) after stimulus offset. In dogs with inherited retinal degeneration, loss of rod function resulted in absent scDB responses, followed by decreased phBR responses with disease progression and loss of cone function. Primary loss of cone function abolished phBR responses but preserved those responses to blue light (scDB and scBB). Although melanopsin/Opn4 expression was diminished with retinal degeneration, melanopsin-expressing ipRGCs were identified for the first time in both WT and degenerated canine retinas.

Conclusions: Pupil responses elicited by light stimuli of different colors and intensities allowed differential functional assessment of canine rods, cones, and ipRGCs. Chromatic pupillometry offers an effective tool for diagnosing retinal and optic nerve diseases.

Figure 1

(A) Average (top row, bold traces) and individual (bottom row) PLRs in WT dogs. The gray-shaded area represent the 1-s light stimulus presentation. Open circles indicate the light artifact. (B) Correlation between baseline pupil size and constriction amplitude are shown with regression lines for WT dogs. Calibration bars: 1 s (time, horizontal) and 20% (normalized pupil size, vertical).

Figure 2

(A) Effects of mutations in PDE6B, CNGB1, and PDE6A on PLRs compared to average WT PLRs. Representative recordings are shown here. Recordings from all dogs are shown in Supplementary Figure S2. ★ indicates undetectable PLRs from PDE6A- and CNGB1-mutants overshadowed by a light artifact. Shaded area represents 1-s stimulus presentation. (B) Representative full-field ERGs. Compared to WT dogs, rod-mediated response were nonrecordable in young PDE6B- and PDE6A-mutants, whereas cone-mediated function was recordable but severely reduced in amplitude. (A) Calibration bars: 1 s (time, horizontal) and 20% (normalized pupil size, vertical).

Figure 3

(A) PLRs from dogs carrying the CNGB3 mutation with ACHM with the expected abrogation of phBR responses. In contrast, a subset of CNGB3-mutants with incomplete ACHM showed persistent but reduced phBR responses (arrow). Representative recordings are shown here. Recordings from all dogs are shown in Supplementary Figure S3. Shaded area represents the 1-s light stimulus presentation. (B) Comparison of representative full-field ERGs recorded from CNGB3 mutants show normal rod-mediated function but lost cone-mediated function compared to WT dogs. (A) Calibration bars: 1 s (time, horizontal) and 20% (normalized pupil size, vertical).

Figure 4

(A) Representative PLRs of older dogs bearing mutations in PDE6B, RD3, STK38L, and PRCD. Recordings from all dogs with mutations in STK38L, IQCB1, RPGR, RPE65, and PRCD and older dogs carrying PDE6B, RD3, and PDE6A-mutations are shown in Supplementary Figure S4. Shaded area represents the 1-s light stimulus presentation. ★ indicates absent PLR to scBB in one older STK38L-mutant. Arrows indicate small residual rod function and near-normal preserved cone function in an older dog with mutated PRCD. (B) Representative full-field ERGs recorded from older dogs bearing mutations in PDE6B, STK38L, and PRCD. Responses of a WT dog are shown for comparison. For PDE6B- and STK38K-mutant dogs, rod- and cone- mediated function was non-recordable. For the PRCD-mutant dog, cone-mediated function was recordable, but the amplitude was reduced compared with that of the WT dog. (A) Calibration bars: 1 s (time, horizontal) and 20% (normalized pupil size, vertical).

Figure 5

All PLRs were absent in the dog affected by a mutation in RD3 and concurrent severe optic nerve head coloboma due to a mutation in NEHJ1. Shaded area indicates the 1-s light stimulus presentation. Calibration bars: 1 s (time, horizontal) and 20% (normalized pupil size, vertical).

Figure 6

Results of PLR testing in scotopic and photopic light intensity series. Average PLRs for the 2 WT dogs to photopically matched red and blue light at five intensity levels are shown (left and middle columns). Results of photopic light intensity series testing in 2 dogs with CNGB3 mutation (right column). Average PLRs for the affected dogs to the red and blue light at five intensity levels are shown. Arrow indicates light artifact noted at high red light intensity (400 cd/m²) with 25 cd/m² blue background. For all graphs, shaded areas indicate 1-s light stimulus presentation. Calibration bars: 1 s (time, horizontal) and 20% (normalized pupil size, vertical).

Figure 7

Gene and protein expression patterns in dogs with inherited retinal diseases compared to those in WT dogs. (A) Effects of inherited retinal disease in various canine models on retinal melanopsin/Opn4 mRNA expression levels, quantified by qRT-PCR and compared with those in age-matched WT dogs. Melanopsin/Opn4 was variable but not significantly altered in CNGB3-ACHM. Retinas in PRCD-mutants showed a significant decrease (**) compare with those in WT retinas (P < 0.05). Expression levels in individual RPGR- and PDE6B-mutants also appeared decreased. (B) Melanopsin immunostaining of representative retinal sections from WT and PDE6B-mutant dogs confirms the presence of ipRGCs. Other RGCs are shown in green (NeuN). Cell nuclei are shown in blue with DAPI staining. Calibration bar: 20 μm. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer.