Toward a clinical protocol for assessing rod, cone, and melanopsin contributions to the human pupil response

Abstract

PURPOSE. To better understand the relative contributions of rod, cone, and melanopsin to the human pupillary light reflex (PLR) and to determine the optimal conditions for assessing the health of the rod, cone, and melanopsin pathways with a relatively brief clinical protocol. METHODS. PLR was measured with an eye tracker, and stimuli were controlled with a Ganzfeld system. In experiment 1, 2.5 log cd/m(2) red (640 ± 10 nm) and blue (467 ± 17 nm) stimuli of various durations were presented after dark adaptation. In experiments 2 and 3, 1-second red and blue stimuli were presented at different intensity levels in the dark (experiment 2) or on a 0.78 log cd/m(2) blue background (experiment 3). Based on the results of experiments 1 to 3, a clinical protocol was designed and tested on healthy control subjects and patients with retinitis pigmentosa and Leber's congenital amaurosis. RESULTS. The duration for producing the optimal melanopsin-driven sustained pupil response after termination of an intense blue stimulus was 1 second. PLR rod- and melanopsin-driven components are best studied with low- and high-intensity flashes, respectively, presented in the dark (experiment 2). A blue background suppressed rod and melanopsin responses, making it easy to assess the cone contribution with a red flash (experiment 3). With the clinical protocol, robust melanopsin responses could be seen in patients with few or no contributions from the rods and cones. CONCLUSIONS. It is possible to assess the rod, cone, and melanopsin contributions to the PLR with blue flashes at two or three intensity levels in the dark and one red flash on a blue background.

Figures

Figure 1.

Average PLRs from five normal subjects. (a) PLRs to red stimuli of eight different durations, coded by color. The pupil size at 6 seconds after stimulus offset is marked as filled dots, connected by solid line. (b) Same as (a) for blue stimuli. (c) Pupil size at 6 seconds as a function of stimulus duration with ±1 SE bars. (d) PLRs to 1-second (saturated red and blue tracings) compared to 10-second (desaturated red and blue tracings) stimuli. The filled circles are marked at the same points as in (a) and (b), with dots for the pupil tracings elicited by 1-second stimuli and with stars for the pupil tracings elicited by 10-second stimuli. The stimulus intensity used in this experiment was 2.5 log cd/m2.

Figure 2.

Average PLRs from seven normal subjects to stimuli of different intensities in the dark. (a) PLRs to red stimuli at 14 intensity levels. (b) PLRs to blue stimuli at 14 intensity levels. (c–h) Pairs of PLRs, from (a) and (b), to photopically matched red and blue stimuli. Black bars: 1-second stimulus presentation.

Figure 3.

(a) Peak normalized pupil size versus stimulus intensity is shown for seven normal subjects (small symbols) and their mean values (large symbols connected by the thin lines). The blue thick line is the best fitting linear regression line for the data between −4 to −1 log cd/m2, and the red thick line is this same line horizontally shifted for best fit to the red data. The insets show the PLRs to photopically matched red and blue stimuli at the intensity levels indicated. Note that y-axes are reversed so that larger responses are at the top. Dashed lines: pairs of stimuli used in the lower panels, with the corresponding panels indicated in the boxes. (b–e) Pairs of PLRs to red and blue stimuli separated by 1.94 log units, showing that they are nearly equal in contraction amplitude.

Figure 4.

RvI functions of pupil size at 6 seconds after stimulus offset in the dark. The data are presented as in Figure 3a. Note that the sustained pupil response to stimulus offset starts to occur after 1-second blue light offset of 0.5 log cd/m2 but did not significantly occur after red light offset.

Figure 5.

Average PLRs from five normal subjects to stimuli with various intensities on a blue background. The data are presented as in Figure 2. Unlike the dark-adapted data, the responses to the red stimuli are equal to, or larger than, the responses to the photopically matched blue flashes. There is a suggestion of a small sustained melanopsin contribution only to the brightest, 2.6-log cd/m2 blue stimulus offset (h, arrow)

Figure 6.

(a) Peak normalized pupil size versus stimulus intensity on a blue background is shown for five normal subjects (small symbols) and their mean values (large symbols connected by the thin lines). (b) RvI functions of pupil size at 6 seconds after stimulus offset on a blue background. Note that under blue background conditions, there is no significant sustained pupil response after light offset in control subjects, in contrast to the no background condition. Data are presented as in Figures 3a and 4.

Figure 7.

PLRs for eight control subjects (thin lines) for the three conditions of the clinical protocol. The bold solid and dashed lines show the mean ±2 SD for the eight control subjects. (a, b) Rod condition: −3 and −2 log cd/m2 blue light stimuli in the dark. (c) Melanopsin condition: 2.6 log cd/m2 blue light stimuli in the dark. (d) Cone condition: 2.6 log cd/m2 red light stimuli on a blue background. Though not strictly necessary to assess the contributions of the rods, cones, and melanopsin, photopically equated red stimuli were also included for the rod and melanopsin conditions, and a photopically equated blue stimulus was included for the cone condition. The gaps seen in some of the records are due to the removal of extreme eye blinks.

Figure 8.

PLRs from five patients with RP (columns RP1–5) for the clinical protocol. Solid lines: patients' PLRs; dashed lines: average across eight controls subjects. Rows (a) to (d) correspond to the conditions in Figure 7. (a, b) Rod condition. (c) Melanopsin condition. (d) Cone condition. The extreme eye blinks were removed as in Figure 7.

Figure 9.

PLRs from three patients with LCA (columns) for the clinical protocol. Data are presented as in Figure 8.