June 2015
Volume 56, Issue 6
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Visual Neuroscience  |   June 2015
The Post-Illumination Pupil Response (PIPR)
Author Affiliations & Notes
  • Prakash Adhikari
    Medical Retina and Visual Science Laboratories, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia
  • Andrew J. Zele
    Medical Retina and Visual Science Laboratories, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia
  • Beatrix Feigl
    Medical Retina and Visual Science Laboratories, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia
    Queensland Eye Institute, South Brisbane, Queensland, Australia
  • Correspondence: Beatrix Feigl, Institute of Health and Biomedical Innovation, School of Biomedical Sciences, Queensland University of Technology, 60 Musk Avenue, Brisbane, QLD 4059, Australia; b.feigl@qut.edu.au. Andrew J. Zele, Institute of Health and Biomedical Innovation, School of Optometry and Vision Science, Queensland University of Technology, 60 Musk Avenue, Brisbane, QLD 4059, Australia; andrew.zele@qut.edu.au
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3838-3849. doi:10.1167/iovs.14-16233
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      Prakash Adhikari, Andrew J. Zele, Beatrix Feigl; The Post-Illumination Pupil Response (PIPR). Invest. Ophthalmol. Vis. Sci. 2015;56(6):3838-3849. doi: 10.1167/iovs.14-16233.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The post-illumination pupil response (PIPR) has been quantified using four metrics, but the spectral sensitivity of only one is known; here we determine the other three. To optimize the human PIPR measurement, we determine the protocol producing the largest PIPR, the duration of the PIPR, and the metric(s) with the lowest coefficient of variation.

Methods.: The consensual pupil light reflex (PLR) was measured with a Maxwellian view pupillometer. Experiment 1: Spectral sensitivity of four PIPR metrics (plateau, 6 seconds, area under curve early and late recovery) was determined from a criterion PIPR to a 1-second pulse and fitted with vitamin A1 nomogram (λmax = 482 nm). Experiment 2: The PLR was measured as a function of three stimulus durations (1 second, 10 seconds, 30 seconds), five irradiances spanning low to high melanopsin excitation levels (retinal irradiance: 9.8–14.8 log quanta.cm−2.s−1), and two wavelengths, one with high (465 nm) and one with low (637 nm) melanopsin excitation. Intra- and interindividual coefficients of variation (CV) were calculated.

Results.: The melanopsin (opn4) photopigment nomogram adequately describes the spectral sensitivity of all four PIPR metrics. The PIPR amplitude was largest with 1-second short-wavelength pulses (≥12.8 log quanta.cm−2.s−1). The plateau and 6-second PIPR showed the least intra- and interindividual CV (≤0.2). The maximum duration of the sustained PIPR was 83.0 ± 48.0 seconds (mean ± SD) for 1-second pulses and 180.1 ± 106.2 seconds for 30-second pulses (465 nm; 14.8 log quanta.cm−2.s−1).

Conclusions.: All current PIPR metrics provide a direct measure of the intrinsic melanopsin photoresponse. To measure progressive changes in melanopsin function in disease, we recommend that the PIPR be measured using short-duration pulses (e.g., ≤1 second) with high melanopsin excitation and analyzed with plateau and/or 6-second metrics. Our PIPR duration data provide a baseline for the selection of interstimulus intervals between consecutive pupil testing sequences.

The pupil light reflex (PLR) is a fundamental diagnostic tool for objective and noninvasive measurement of retinal and optic nerve function in neuro-ophthalmic disorders.1 The pupil control pathway receives retinal input from intrinsically photosensitive retinal ganglion cells (ipRGCs), which also project to the suprachiasmatic nucleus (SCN) for photoentrainment,27 and there is circadian modulation of the post-illumination pupil response (PIPR).8,9 Given that outer retinal extrinsic rod, cone, and inner retinal intrinsic melanopsin photoresponses influence the human PLR,2,3,8,1016 there has been interest in developing PLR protocols that quantify outer and inner retinal input.14,15,1725 An established marker of direct, intrinsic melanopsin activity is the PIPR, the sustained pupilloconstriction after light offset.11,26 With ipRGCs affected in optic nerve and retinal disease such as glaucoma,21,24,27 retinitis pigmentosa,14,17,20 diabetes,22 age-related macular degeneration,28 Leber's congenital amaurosis,17 as well as in circadian disorders,10 the PLR techniques may complement other clinical measures of retinal function in the healthy and diseased eye, such as the electroretinography (ERG) and perimetry. Depending on the measurement paradigms, ERGs measure the summed and local photoreceptor, bipolar, and ganglion cell responses. Visual field testing with standard automated perimetry (SAP) and other modes, including frequency-doubling technology (FDT), short-wavelength automated perimetry (SWAP), and flicker perimetry, measure the integrity of visual pathways. In contrast, the PLR can be used to simultaneously differentiate inner retinal function (mediated via ipRGCs) and outer retinal function (mediated via rods and cones) to provide a clinical tool for diagnosis and monitoring progression of ocular disorders, with the PIPR being a specific measure of ipRGCs. The PIPR has been reported in response to a range of stimulus durations, irradiances, and wavelengths8,12,14,18,2124,29 and quantified using five metrics, namely the plateau PIPR,12,14 redilation velocity,8,21 6-second PIPR,17 area under curve (AUC) early and late recovery18 (metrics are defined in the Methods). 
There are outstanding questions before the PIPR can be translated to clinical practice. First, the plateau PIPR metric in response to 10-second light pulses is the only metric shown to match the spectral sensitivity of opn4 melanopsin photopigment12,14,28; there are no reported measurements of the PIPR spectral sensitivity for the other metrics. Second, there has been no direct comparison of these different stimuli and metrics under the same conditions and hence no consensus on which metric(s) should be used to quantify the PIPR for clinical application. For application in a clinical setting, the intra- and interindividual variability of the metrics for the different stimulus conditions must be determined in a single cohort to determine the optimum test conditions. 
This study addresses these two questions. First, we determine the spectral sensitivity of the PIPR for each of the metrics. Second, we present measurements of the human PLR as a function of stimulus duration (1, 10, and 30 seconds), wavelength (465 and 637 nm), and irradiance (9.8–14.8 log quanta.cm−2.s−1) to define the stimulus parameters that produce the largest melanopsin response and the PIPR metrics with the lowest intra- and interindividual coefficient of variation (CV) in the same cohort. Given that in vitro recordings in rat ipRGCs show up to a 10-hour response to continuous (480 nm) light stimulation26 at 12.8 log quanta.cm−2.s−1 and the PIPR has been only measured up to 60 seconds in humans,12,14 we measured the duration of the PIPR and demonstrate that the return to baseline pupil diameter after melanopsin excitation can be as long as 3 minutes post illumination. 
Methods
Participants
A total of seven healthy participants with no ocular pathology were enrolled. None of the participants were taking any prescription medication. All participants had a visual acuity (≥6/6), normal contrast sensitivity (Pelli-Robson chart), normal color vision (Lanthony desaturated D-15 test), an intraocular pressure of <21 mm Hg (tonometer; iCare Finland Oy, Helsinki, Finland), a normal central visual field (MP-1 microperimeter; Nidek Co., Ltd., Padova, Italy), and normal retinal nerve fiber layer thickness (RS-3000 OCT RetinaScan Advance; Nidek Co., Ltd., Tokyo, Japan). Anterior and posterior eye examination using slit lamp biomicroscopy revealed no pathology. The PIPR spectral sensitivity is reported for two participants (32-year-old female, 31-year-old male). The PLR and PIPR measurements are reported for five participants (four male, one female; mean age = 32.6 ± 5.4 years SD; range, 29–42 years). The research followed the tenets of the Declaration of Helsinki, and informed consent was obtained from the participants after explanation of the nature of the study. All experiments were conducted in accordance with Queensland University of Technology Human Research Ethics Approval (080000546). Participants were tested between 10 AM and 5 PM to minimize circadian variation on ipRGC contribution to the PIPR.8,9 Each participant was tested for up to 1.5 hours per day to minimize fatigue, and each participated for approximately 30 hours in total. 
Pupillometer
The PLR was measured using a custom-built, extended Maxwellian view pupillometer.23,28,30 The calibrated optical system comprised narrowband light-emitting diode (LED) light sources (see Pupillometry Protocol for stimulus wavelengths) imaged in the pupil plane of the right eye via two Fresnel lenses (100-mm diameter, 127-mm and 70-mm focal lengths; Edmund Optics, Singapore) and a 5° light-shaping diffuser (Physical Optics Corp., Torrance, CA, USA) to provide a 35.6° diameter light stimulus (retinal image diameter: 15.4 mm). The consensual image of the left eye was recorded under infrared LED illumination (λmax = 851 nm) with a Pixelink camera (IEEE-1394, PL-B741 Fire Wire, 640 × 480 pixels, 60 frames/second; PIXELINK, Ottawa, ON, Canada) through a telecentric lens (2/3-inch, 55 mm, with 2X extender C-Mount; Computar, Singapore, Malaysia). The stimulus presentation, pupil recording, and analysis were controlled by custom Matlab software (version 7.12.0; Mathworks, Nitick, MA, USA). The blink artefacts were identified and extracted by a customized algorithm during software analysis of pupil recordings using linear interpolation. The spectral outputs of the LED stimuli were measured with a spectroradiometer (StellarNet, Tampa, FL, USA), and light output was calibrated with a radiometer (ILT1700 Research Radiometer; International Light Technologies, Inc., Peabody, MA, USA). Details of the recording procedure can be found elsewhere.8 
Pupillometry Protocol
Spectral sensitivity of the PIPR was measured with the Maxwellian view optical system in response to a 1-second rectangular pulse at 5 wavelengths (409, 464, 508, 531, and 592 nm). The participant's left eye was dilated (1% tropicamide), and the criterion consensual PIPR of the fellow eye was measured in response to a 1-second light pulse ranging between 13.0 and 15.7 log quanta.cm−2.s−1. The 409-nm LED had a maximum irradiance of 0.00015 W.cm−2 at 14.6 log quanta.cm−2.s−1. This is equivalent to a stimulus luminance of 9.64 cd.m−2 (the retinal illuminance in trolands [td] for an 8.0-mm pupil is 484.56 td). The maximum output of the LED is therefore below the upper exposure limits (0.003 W.cm−2) to prevent any phototoxicity from UV radiation.31 The wavelength of successive test stimuli was always greater than 100 nm to control for melanopsin bistability.5 The criterion PIPR amplitude was defined as 8% for the plateau PIPR, 10% for the 6-second PIPR, 4 log units for the AUC early and AUC late. The retinal irradiances required at each wavelength to produce the criterion PIPR were normalized and fitted with a vitamin A1 pigment nomogram.32 
The PLR was measured with the Maxwellian view optical system at two wavelengths (short wavelength: λmax = 465 nm [bluish]; long wavelength: λmax = 637 nm [reddish]) over a 5-log unit range of retinal irradiances to span low to high melanopsin excitation levels (9.8–14.8 log quanta.cm−2.s−1 [−2.0 to 2.8 log cd.m−2 luminance] for the 465-nm light; 9.9–14.9 log quanta.cm−2.s−1 [−2.3 to 2.8 log cd.m−2 luminance] for the 637-nm light). Figure 1 shows the temporal sequence of the pupillometry protocols. Three stimulus durations (1, 10, and 30 seconds) were chosen to reflect the durations commonly adopted in published protocols. The 1-second duration pulse was chosen because the 6-second and net 6-second PIPR amplitudes are largest with 1-second pulses.17 The 10-second pulse has been widely used in clinical studies of the PIPR, but only three different parameters have been quantified (redilation velocity, plateau, and 6-second PIPR).8,12,13,17,2124 The 30-second pulse was studied because ipRGCs dominate the steady-state pupil response during light presentation compared to rod and cone inputs when stimulus durations are >10 seconds.13 All irradiances were above rod threshold.33 Retinal irradiances are photopic when >11.8 log quanta.cm−2.s−1.11 The prestimulus duration was 10 seconds for all conditions. The poststimulus recording period ranged from 40 to 600 seconds to ensure that the sustained pupilloconstriction returned to baseline before remeasurement. Pilot studies determined the interstimulus interval (ISI) for return to baseline to be between 100 and 660 seconds; the ISI increased with increasing retinal irradiance and stimulus duration. To consider the effect of dilation of the stimulated eye on the PIPR of the fellow eye, a subset of two participants underwent pilot testing with their right eye dilated with 1% tropicamide (Minims; Chauvin Pharmaceuticals Ltd., Romford, UK). There was <4% CV between the metrics for the undilated and dilated conditions within the acceptable range of CV (see Statistical Analysis for details on CV). Since there is evidence of unequal consensual and direct PLR in some normal persons,34 we compared the metrics between the consensual and direct PLR in these two participants and found <7% CV for our test protocols. 
Figure 1
 
Temporal sequence of the stimulus protocol for the pupillometry experiments. Retinal irradiance is specified on the left ordinate and poststimulus time on the abscissa. Stimulus (three durations, 30 seconds: upper, 10 seconds and 1 second: lower). PRE, prestimulus period.
Figure 1
 
Temporal sequence of the stimulus protocol for the pupillometry experiments. Retinal irradiance is specified on the left ordinate and poststimulus time on the abscissa. Stimulus (three durations, 30 seconds: upper, 10 seconds and 1 second: lower). PRE, prestimulus period.
All measurements were preceded by 10-minute dark adaptation in a darkened (<1 lux) laboratory. For the PLR measurements, short- and long-wavelength stimulus lights were alternated in all sessions to control for the effect of melanopsin bistability.5 Every measurement for each stimulus wavelength, irradiance, and duration combination was repeated at least three times, with the time interval equal to the corresponding ISI. Table 1 specifies the individual photoreceptor excitations for the stimuli35; the L cones have higher sensitivity to the 637-nm light, whereas melanopsin, rods, M cones, and S cones have higher excitation to the 465-nm light compared to L cones. It should be noted that narrowband lights do not provide photoreceptor isolation and that the high (or low) photoreceptor excitations specified in Table 1 do not imply that a photoreceptor does (or does not) contribute to the PLR; these factors depend on the relative contributions of these photoreceptors' inputs to the pupil pathway and their variation with the stimulus properties (e.g., spatial, temporal, and wavelength), for which many of these factors are unknown. 
Table 1
 
Individual Photoreceptor Excitation (in Log10 Units) With 465 nm and 637 nm Light Stimuli at Different Retinal Irradiances (Based on Lucas et al.35)
Table 1
 
Individual Photoreceptor Excitation (in Log10 Units) With 465 nm and 637 nm Light Stimuli at Different Retinal Irradiances (Based on Lucas et al.35)
Because the shape of the pupil image is elliptical when measured during off-axis fixation,36 we determined that estimated pupil diameter measured in our Maxwellian view optical system would be underestimated by 0.113 ± 0.024 mm when the fixation eccentricity was up to 8.13° off axis. For all pupil recordings used in the analysis, the eye movements were within 5° of central fixation axis of the optical system and infrared camera plane, introducing an error of ≤0.07 mm in estimated pupil diameter. 
Analysis of the PLR and PIPR
The PLR and PIPR were described by the 12 metrics outlined in Table 2 and Figure 2. The metrics were derived from the best fit of the linear and exponential model to the data.8,14,21,22 For the peak constriction amplitude, 6-second PIPR, and plateau PIPR, a smaller value indicates a larger pupil response. Larger PIPR amplitudes are defined by smaller values of the redilation velocity, 6-second PIPR, plateau PIPR, and larger values of the AUC early and late and PIPR duration. Though the models yield negative values for pupil dynamics, absolute values are used in Figures 5 and 6
Table 2
 
Description and Definition of the PLR Metrics During Light Stimulation and PIPR Metrics After Light Offset
Table 2
 
Description and Definition of the PLR Metrics During Light Stimulation and PIPR Metrics After Light Offset
Figure 2
 
An exemplar of the PLR and PIPR in response to a short-wavelength (465 nm), 30-second light pulse. The metrics used to quantify the pupil light response during and after light stimulation are indicated on the pupil trace and defined in Table 2. The blue trace indicates the PLR and PIPR; the gray trace shows the model.
Figure 2
 
An exemplar of the PLR and PIPR in response to a short-wavelength (465 nm), 30-second light pulse. The metrics used to quantify the pupil light response during and after light stimulation are indicated on the pupil trace and defined in Table 2. The blue trace indicates the PLR and PIPR; the gray trace shows the model.
Statistical Analysis
Statistical data analysis was conducted using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). Means ± SD were used to describe data. Shapiro-Wilk tests indicated that all data were normally distributed. One-way repeated measures ANOVA (95% confidence interval, P < 0.05, Tukey's test for pairwise multiple comparisons, Geisser-Greenhouse correction) was applied to compute the differences in the pupil responses between different stimulus durations. To determine variability of the PIPR and net PIPR metrics, the intra- and interindividual CV was calculated (SD/mean). The CV provides a more precise measurement of variability than SD because it is dimensionless and is not affected by changes in measurement units.37 A CV of ≤0.2 was considered acceptable based on the target acceptance criteria for immunoassay applications38,39; we are unaware of a literature reference for a CV for human behavioral studies. 
Results
The spectral sensitivity of the PIPR metrics is shown in Figure 3 for the two observers (circle and square symbols). The data for all metrics (plateau, 6-second, early and late AUC) are well described by a vitamin A1 nomogram with a peak spectral sensitivity at 482 nm. There were no differences in spectral sensitivity derived from the modeled data (shown) and the raw unmodeled data (not shown). 
Figure 3
 
Spectral sensitivity of the plateau PIPR, 6-second PIPR, and AUC early and late recovery metrics. The circles and squares indicate the data (average ± SD) from two participants. The data of 32/F observer are horizontally offset from 31/M observer by 3.5 nm. The solid blue lines indicate the vitamin A1 nomogram (λmax = 482 nm), and the insets show the corresponding metrics. The legends in the first panel are common to all.
Figure 3
 
Spectral sensitivity of the plateau PIPR, 6-second PIPR, and AUC early and late recovery metrics. The circles and squares indicate the data (average ± SD) from two participants. The data of 32/F observer are horizontally offset from 31/M observer by 3.5 nm. The solid blue lines indicate the vitamin A1 nomogram (λmax = 482 nm), and the insets show the corresponding metrics. The legends in the first panel are common to all.
The PLR during light stimulation and the PIPR after light offset were analyzed using 12 metrics (Table 2) as described in the following sections for the group data. Figure 4 shows the complete PLR data for one representative participant. While the PLR response is not the primary outcome of this study, it is presented before the PIPR results to follow the natural time sequence during and after light stimulation. 
Figure 4
 
Average pupil response of a representative participant (30-year-old female) to short-(465 nm) and long-wavelength (637 nm) stimuli of retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1, increasing in 1-log unit steps and three durations: 1 second (A), 10 seconds (B), 30 seconds (C). The retinal irradiance is defined in log quanta.cm−2.s−1 (with log trolands given in parentheses) next to the corresponding pupil trace in the upper panels. Stimulus duration is indicated by the colored rectangular bar on the abscissa. Insets show the 30-second PIPR with the dotted vertical lines indicating the 6-second PIPR amplitude and gray lines indicating the models. All data are offset successively by 5% along the ordinates from the 9.8 log quanta.cm−2.s−1 trace. The same color coding is followed throughout.
Figure 4
 
Average pupil response of a representative participant (30-year-old female) to short-(465 nm) and long-wavelength (637 nm) stimuli of retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1, increasing in 1-log unit steps and three durations: 1 second (A), 10 seconds (B), 30 seconds (C). The retinal irradiance is defined in log quanta.cm−2.s−1 (with log trolands given in parentheses) next to the corresponding pupil trace in the upper panels. Stimulus duration is indicated by the colored rectangular bar on the abscissa. Insets show the 30-second PIPR with the dotted vertical lines indicating the 6-second PIPR amplitude and gray lines indicating the models. All data are offset successively by 5% along the ordinates from the 9.8 log quanta.cm−2.s−1 trace. The same color coding is followed throughout.
Effect of Stimulus Irradiance, Wavelength, and Duration on the PLR
Figure 5 reports the mean group data across all stimulus irradiances and shows that with increasing irradiance, the transient PLR increased and the PLR latency shortened with a plateau beyond 12.8 log quanta.cm−2.s−1. The constriction velocity and peak constriction amplitude increased, whereas the time to peak constriction and pupil escape did not change as a function of irradiance. The effect of stimulus duration on the PLR was wavelength and irradiance dependent. The transient PLR was independent of stimulus duration (465 nm: F2,7 = 1.378, P = 0.298; 637 nm: F2,10 = 0.52, P = 0.607) and so was PLR latency (465 nm: F2,8 = 3.89, P = 0.069; 637 nm: F2,7 = 2.15, P = 0.187). However, the transient PLR amplitude was always larger, and the PLR latency was shorter for short wavelengths than for long wavelengths due to higher rod sensitivity; this difference tapered with increasing irradiance showing saturation of the response. When the data were normalized to peak pupil constriction, the PLR latency still showed a trend of shortening with increasing irradiance, indicating that this process is driven by stimulus irradiance. The constriction velocity was dependent on stimulus duration at short wavelengths (465 nm: F1,7 = 26.24, P = 0.001) and was faster for 30-second stimuli than 1- and 10-second stimuli, but independent of duration at long wavelengths (637 nm: F2,8 = 0.17, P = 0.805). The peak constriction amplitude increased with increasing stimulus duration (465 nm: F1,6 = 26.88, P = 0.002; 637 nm: F1,6 = 7.97, P = 0.025). The time to peak constriction was longer for 30- and 10-second pulses than for 1-second pulses (465 nm: F2,7 = 26.66, P = 0.001; 637 nm: F2,10 = 7.73, P = 0.010); for 1-second pulses, the time to peak constriction was longer for 465 nm (1.4–1.9 seconds) than 637 nm (1.2–1.4 seconds) above 11.8 log quanta.cm−2.s−1, indicating a slower temporal response to the short-wavelength stimuli. The pupil escape velocity was independent of stimulus irradiance, but was dependent on stimulus duration (465 nm: F1,5 = 20.33, P = 0.006; 637 nm: F1,5 = 7.97, P = 0.017), with a slower escape with 30-second pulses than 10-second pulses (note that escape velocity is not applicable to 1-second pulses). 
Figure 5
 
Average (±SD) (n = 5 participants) transient PLR (%), PLR latency (ms), constriction velocity (mm.s−1), peak constriction amplitude (% baseline), time to peak constriction (s), and pupil escape (mm.s−1) of the PLR to stimuli of wavelength 465 nm (blue) and 637 nm (red), retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1 increasing in 1-log unit steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles). The numbers in blue and red in the upper left and right indicate the luminance (log cd.m−2) of the short- and long-wavelength stimuli, respectively.
Figure 5
 
Average (±SD) (n = 5 participants) transient PLR (%), PLR latency (ms), constriction velocity (mm.s−1), peak constriction amplitude (% baseline), time to peak constriction (s), and pupil escape (mm.s−1) of the PLR to stimuli of wavelength 465 nm (blue) and 637 nm (red), retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1 increasing in 1-log unit steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles). The numbers in blue and red in the upper left and right indicate the luminance (log cd.m−2) of the short- and long-wavelength stimuli, respectively.
Effect of Stimulus Irradiance, Wavelength, and Duration on the PIPR
Figure 6 displays the effect of stimulus irradiance, wavelength, and duration on the six PIPR metrics. The PIPR redilation velocity decreased with increasing irradiance for 1-second pulses, but was independent of irradiance for 10- and 30-second pulses. At 465 nm, a second redilation phase (Fig. 4) was observed at around 40, 50, and 70 seconds post stimulus for 1-, 10-, and 30-second pulses at 14.8 log quanta.cm−2.s−1, which has, to our knowledge, not been previously reported. The 6-second PIPR, plateau PIPR, AUC early, AUC late, and PIPR duration increased with increasing stimulus irradiance. At the highest measured retinal irradiance (14.8 log quanta.cm−2.s−1), all PIPR metrics (except PIPR duration) for 1-second pulses were larger or equal to those for 10- and 30-second pulses. 
Figure 6
 
Average (±SD) (n = 5) redilation velocity (mm.s−1), 6-second PIPR amplitude (% baseline), plateau PIPR (% baseline), AUC early and late recovery (linear and log units), and PIPR duration (s) of the pupil light response to stimuli of wavelength 465 nm (blue) and 637 nm (red), retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1 increasing in 1-log steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles). The numbers in blue and red in the upper left and right indicate the luminance (log cd.m−2) of the short- and long-wavelength stimuli, respectively.
Figure 6
 
Average (±SD) (n = 5) redilation velocity (mm.s−1), 6-second PIPR amplitude (% baseline), plateau PIPR (% baseline), AUC early and late recovery (linear and log units), and PIPR duration (s) of the pupil light response to stimuli of wavelength 465 nm (blue) and 637 nm (red), retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1 increasing in 1-log steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles). The numbers in blue and red in the upper left and right indicate the luminance (log cd.m−2) of the short- and long-wavelength stimuli, respectively.
Redilation velocity was dependent on stimulus duration at long wavelengths, with higher redilation velocity for 1-second pulses than for 10- or 30-second pulses (637 nm: F1,7 = 37.82, P = 0.0003), but no effect at short wavelengths (465 nm: F1,6 = 1.48, P = 0.278). Stimulus duration had no significant effect on the 6-second PIPR amplitude (465 nm: F1,5 = 1.63, P = 0.258; 637 nm: F1,6 = 5.34, P = 0.052), plateau PIPR amplitude (465 nm: F1,5 = 2.81, P = 0.752; 637 nm: F2,7 = 0.38, P = 0.633), AUC early (465 nm: F2,10 = 3.06, P = 0.094; 637 nm: F2,7 = 8.05, P = 0.019), AUC late (465 nm: F1,7 = 1.25, P = 0.323; 637 nm: F2,10 = 0.79, P = 0.479), and PIPR duration (465 nm: F1,6 = 2.04, P = 0.210; 637 nm: F1,6 = 5.35, P = 0.062). However, at 14.8 log quanta.cm−2.s−1, the PIPR duration increased with increasing stimulus duration. 
Figure 7 shows, as expected, that the net PIPR for irradiances below the melanopsin threshold was not significant for the three stimulus durations. Beyond 11.8 log quanta.cm−2.s−1, which is known to be within the melanopsin range,11 all net PIPR metrics except the net redilation velocity for 10- and 30-second pulses increased with increasing irradiance. There was no significant effect of stimulus duration on the net 6-second PIPR (F1,6 = 4.72, P = 0.068), net plateau PIPR (F1,6 = 2.41, P = 0.174), net AUC late (F1,6 = 3.98, P = 0.094), and net PIPR duration (F1,6 = 0.29, P = 0.635). The net redilation velocity (F1,6 = 11.57, P = 0.016) and net AUC early (F1,6 = 7.93, P = 0.028) were dependent on stimulus duration, with net velocity and net AUC larger for 1-second pulses than for 10- and 30-second pulses. 
Figure 7
 
Average (±SD) (n = 5) net redilation velocity (A), net 6-second PIPR (B), net plateau PIPR (C), net AUC early (D) and late (E) recovery, and net PIPR duration (F) of the pupil light response to stimuli of wavelength 465 nm and 637 nm, retinal irradiance from 9.8 to 14.8 log quanta.cm−2.s−1 increasing in 1-log steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles).
Figure 7
 
Average (±SD) (n = 5) net redilation velocity (A), net 6-second PIPR (B), net plateau PIPR (C), net AUC early (D) and late (E) recovery, and net PIPR duration (F) of the pupil light response to stimuli of wavelength 465 nm and 637 nm, retinal irradiance from 9.8 to 14.8 log quanta.cm−2.s−1 increasing in 1-log steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles).
Intra- and Interindividual CV
To quantify the level of dispersion in the PIPR metrics, we calculated the CV (Fig. 8) and applied a criterion of ≤0.2.38,39 The intraindividual CV for the plateau PIPR and 6-second PIPR was ≤0.2, with the others >0.2 at all measured irradiances. The interindividual CV of the PIPR in the melanopsin range was ≤0.2 for the plateau PIPR, 6-second PIPR, and AUC early and late recovery, whereas the CV was >0.2 for all other PIPR metrics. 
Figure 8
 
Intraindividual (upper two rows) and interindividual (lower two rows) CV of the PIPR metrics for short-wavelength stimuli. The CVs for long-wavelength stimuli (not shown) were similar. The traces joined by squares, triangles, and circles represent the data for 1-second, 10-second, and 30-second pulses in all parts. The data points with a CV > 1.0 are not shown.
Figure 8
 
Intraindividual (upper two rows) and interindividual (lower two rows) CV of the PIPR metrics for short-wavelength stimuli. The CVs for long-wavelength stimuli (not shown) were similar. The traces joined by squares, triangles, and circles represent the data for 1-second, 10-second, and 30-second pulses in all parts. The data points with a CV > 1.0 are not shown.
Discussion
This study shows that a nomogram at the peak sensitivity of the melanopsin (opn4) photopigment (λmax = 482 nm) adequately describes the spectral sensitivity derived from all current PIPR metrics, and thus any of these metrics can be used to quantify the PIPR to obtain a measure of the intrinsic melanopsin photoresponse. The PIPR amplitude and intra- and interindividual variability is stimulus dependent. The largest PIPR amplitude was obtained with a 1-second short-wavelength pulse (retinal irradiance ≥ 12.8 log quanta.cm−2.s−1) and the intra- and interindividual variability was lowest for the 6-second and plateau PIPR metrics. Of the test stimuli and six PIPR metrics evaluated, we propose that 1-second stimuli and the plateau and/or 6-second PIPR metrics will be most applicable for clinical studies of ipRGC function. We further observed that the maximum duration of the sustained PIPR was 83 seconds for 1-second pulses and 180 seconds for 30-second pulses (465 nm, 14.8 log quanta.cm−2.s−1), but there is large intra- and interindividual variation. 
Post-illumination Pupil Response
The PIPR amplitude was larger with 1-second than with 10-second pulses, which is larger than with 30-second pulses for retinal irradiances ≥ 12.8 log quanta.cm−2.s−1, as evident in the comparison between 1- and 30-second pulses9 and 1- and 10-second pulses.17 This duration-dependent response amplitude may be due to the peak ipRGC firing, with stimuli longer than 1 second, occurring 2 to 3 seconds after stimulus onset and then gradually decaying11,26,40 with light adaptation.41 Taken together, this may lead to the lower PIPR amplitude observed with longer stimulus durations. However, with 14.8 log quanta.cm−2.s−1, 465-nm pulses, the PIPR duration increases with increasing stimulus duration from 1 to 30 seconds, in agreement with a study in mouse eyes42 that showed the duration of the PIPR increased with stimulus duration from 50 ms to 1 second, possibly due to increased light adaptation of melanopsin signaling over time. 
One study18 reported only the test–retest repeatability of the AUC early (intraclass correlation coefficient [ICC] = 0.6) and late recovery (ICC = 0.8), and another study43 reported variation of the plateau PIPR metric (CV = 0.16, ICC = 0.95, 30° central stimulus) but no other metrics. We report the intra- and interindividual variability of all current PIPR metrics. Another study reported a lower interindividual coefficient of variation for the 6-second PIPR than the plateau PIPR,44 whereas our study showed a low CV (≤0.2) for both 6-second and plateau PIPR metrics compared to all other metrics. However, that study used a larger stimulus (60° × 90°) and defined the plateau PIPR as the average PIPR from 10 to 30 seconds post stimulus; hence, it may not be comparable to our results. In our study with a smaller central stimulus field (35.6°), the PIPR variability increased with increasing irradiance, indicating that at higher irradiances a larger PIPR can be produced, but with larger variability. Lei et al.43,44 showed a lower variation in PIPR at higher irradiances with large stimuli (full field and 60° × 90°), probably because the mass response from ipRGCs at high irradiances with large-field stimulation reduces the interindividual variability. It is known that the pupil constriction amplitudes to large stimuli are greater than to smaller stimuli of equal irradiance.1 For a constant corneal flux density, the pupil constriction amplitude is independent of stimulus size.45,46 With regard to the effect of stimulus size on the PIPR, full-field stimuli presented in Newtonian view produce a larger sustained PIPR with less variability than smaller central-field (60° × 90° and 30°) and hemi-field (half of 30° central field) stimuli.43,44 Larger stimuli, however, will be less sensitive to early local retinal deficits (see Ref. 28 for review). Studies in mouse models have indicated that ipRGCs are robust to axonal injury47,48 and induced chronic ocular hypertension.49 Studies in mouse models of retinal degeneration suggest that ipRGC axons/dendrites remain unaffected in early stages and ipRGC density conserves until the advanced stages of retinal degeneration.50,51 Further work is required to understand the role of redundancy and robustness of ipRGCs during disease in humans to define the complex relationships between ipRGC dysfunction and PIPR amplitude, dynamics, and variability of the response. 
We determined that the PIPR duration is longer (>83.4 ± 48.0 seconds) than previously reported,8,12,14,1719,2123 and subsequently longer than the ISI employed in many studies. The ISI should vary with stimulus irradiance because the PIPR duration increases with increasing irradiance and the in vitro intrinsic response also scales with irradiance in melanopsin excitation range.52 Based on our measurements, we propose that for 1-second short-wavelength pulses ≥ 14.8 log quanta.cm−2.s−1, the ISI should be at least 83 seconds (95% CI; upper: 159.8 seconds), so that the sustained PIPR does not interfere with subsequent recordings. The PIPR durations were longer for 1-second than 10- and 30-second pulses at 12.8 and 13.8 log quanta.cm−2.s−1, possibly indicating different adaptation responses to the stimulus durations. Finally, by measuring the PIPR at high irradiances, we observed that the poststimulus pupil redilation shows two phases (Fig. 4; first phase just after light offset and second phase at approximately 40, 50, and 70 seconds post stimulus for 1-, 10-, and 30-second pulses), with the latter phase for short-wavelength pulses at 14.8 log quanta.cm−2.s−1 not well described by a single exponential function. While the origin of this biphasic redilation is not clear, it may reflect different adaptation processes or the contribution of different ipRGC subtypes.5355 
Pupil Light Reflex During Light Stimulation
Analysis of the PLR metrics during light stimulation indicates that the time to peak constriction is longer for 465 nm than 637 nm with 1-second pulses in melanopsin range, whereas this difference was not present below melanopsin threshold. The time to peak constriction did not differ between 465 nm and 637 nm with 10- and 30-second pulses, in agreement with Tsujimura and Tokuda.56 Pupil escape has been considered previously in detail by Loewenfeld,1 Kardon et al.,19 and McDougal and Gamlin.13 In an extension to their observations, we found that pupil escape with 30-second pulses (≥12.0 log quanta.cm−2.s−1) was slower than with 10-second pulses, which we infer is due to larger relative ipRGC contributions to the steady-state pupil constriction,13,57 a decay in rod–cone response with stimuli longer than 10 seconds,13 and ipRGC adaptation to steady light stimulation.41 Taken together, these markers indicate signature contributions of melanopsin to the pupil constriction amplitude and escape. 
In general, the metrics quantifying the human PLR during light stimulation are in accordance with previous studies using different test stimulus protocols with broadband light stimuli. We found that the pupil constriction velocity is wavelength dependent; with long wavelengths, the velocity is independent of stimulus duration, as per the early findings of Lowenstein and Loewenfeld,58 who used broadband lights, whereas the constriction velocity to the short-wavelength light was duration dependent, with the fastest velocity with 30-second pulses. The wavelength-dependent effect on constriction velocity may be related to the differential rod and cone sensitivity to the wavelength and mediated extrinsically via ipRGCs.13 Consistent with previous studies,43,44 pupil constriction velocity increased with increasing stimulus irradiance.58,59 Our findings confirm for narrowband lights that, with increasing retinal irradiance, the magnitude of pupil constriction increases5861 and the transient PLR increases and the PLR latency shortens.19,59,62 The pupil attains the minimum latent period1 at approximately 12.8 log quanta.cm−2.s−1, indicating that the additional time delay of the PLR originating in the photoreceptors and neural reflex circuit and dependent on stimulus intensity1 is absent at 12.8 log quanta.cm−2.s−1; thus, the minimum latent period cannot be eliminated by further increases in stimulus intensity because the time delay is then limited by iris sphincter muscle strength.1 
In a pilot experiment (n = 2), the PLR to a 1-second pulse (14.8 log quanta.cm−2.s−1) of undilated and dilated eyes was compared using the same metrics for describing the response as in the main experiment; we found <4% CV between two conditions. This is not surprising as we used a Maxwellian view pupillometer to provide an open-loop feedback.1,63 Further studies are needed to show whether a full-field system using Newtonian stimulation64 (closed-loop feedback) detects a difference between stimulated eyes with dilated and undilated pupils. We conclude that for a Maxwellian system, dilation of stimulated eye is not essential unless it is required to minimize accommodative fluctuations on the pupil or for persons whose natural pupil diameter is small. 
In conclusion, we propose that the PIPR produced by short-duration pulses (e.g., ≤1 second) with an irradiance above melanopsin threshold and described with the plateau and/or 6-second PIPR metrics may be the optimum protocol for monitoring disease progression in clinical studies of ipRGCs because short-duration stimuli produce larger PIPR amplitudes, and these two metrics show the least intraindividual CV. 
Acknowledgments
We thank Daniel S. Joyce for contributions to data collection. 
Supported by Australian Research Council Discovery Projects (ARC-DP140100333) to BF and AJZ. 
Disclosure: P. Adhikari, None; A.J. Zele, None; B. Feigl, None 
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Figure 1
 
Temporal sequence of the stimulus protocol for the pupillometry experiments. Retinal irradiance is specified on the left ordinate and poststimulus time on the abscissa. Stimulus (three durations, 30 seconds: upper, 10 seconds and 1 second: lower). PRE, prestimulus period.
Figure 1
 
Temporal sequence of the stimulus protocol for the pupillometry experiments. Retinal irradiance is specified on the left ordinate and poststimulus time on the abscissa. Stimulus (three durations, 30 seconds: upper, 10 seconds and 1 second: lower). PRE, prestimulus period.
Figure 2
 
An exemplar of the PLR and PIPR in response to a short-wavelength (465 nm), 30-second light pulse. The metrics used to quantify the pupil light response during and after light stimulation are indicated on the pupil trace and defined in Table 2. The blue trace indicates the PLR and PIPR; the gray trace shows the model.
Figure 2
 
An exemplar of the PLR and PIPR in response to a short-wavelength (465 nm), 30-second light pulse. The metrics used to quantify the pupil light response during and after light stimulation are indicated on the pupil trace and defined in Table 2. The blue trace indicates the PLR and PIPR; the gray trace shows the model.
Figure 3
 
Spectral sensitivity of the plateau PIPR, 6-second PIPR, and AUC early and late recovery metrics. The circles and squares indicate the data (average ± SD) from two participants. The data of 32/F observer are horizontally offset from 31/M observer by 3.5 nm. The solid blue lines indicate the vitamin A1 nomogram (λmax = 482 nm), and the insets show the corresponding metrics. The legends in the first panel are common to all.
Figure 3
 
Spectral sensitivity of the plateau PIPR, 6-second PIPR, and AUC early and late recovery metrics. The circles and squares indicate the data (average ± SD) from two participants. The data of 32/F observer are horizontally offset from 31/M observer by 3.5 nm. The solid blue lines indicate the vitamin A1 nomogram (λmax = 482 nm), and the insets show the corresponding metrics. The legends in the first panel are common to all.
Figure 4
 
Average pupil response of a representative participant (30-year-old female) to short-(465 nm) and long-wavelength (637 nm) stimuli of retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1, increasing in 1-log unit steps and three durations: 1 second (A), 10 seconds (B), 30 seconds (C). The retinal irradiance is defined in log quanta.cm−2.s−1 (with log trolands given in parentheses) next to the corresponding pupil trace in the upper panels. Stimulus duration is indicated by the colored rectangular bar on the abscissa. Insets show the 30-second PIPR with the dotted vertical lines indicating the 6-second PIPR amplitude and gray lines indicating the models. All data are offset successively by 5% along the ordinates from the 9.8 log quanta.cm−2.s−1 trace. The same color coding is followed throughout.
Figure 4
 
Average pupil response of a representative participant (30-year-old female) to short-(465 nm) and long-wavelength (637 nm) stimuli of retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1, increasing in 1-log unit steps and three durations: 1 second (A), 10 seconds (B), 30 seconds (C). The retinal irradiance is defined in log quanta.cm−2.s−1 (with log trolands given in parentheses) next to the corresponding pupil trace in the upper panels. Stimulus duration is indicated by the colored rectangular bar on the abscissa. Insets show the 30-second PIPR with the dotted vertical lines indicating the 6-second PIPR amplitude and gray lines indicating the models. All data are offset successively by 5% along the ordinates from the 9.8 log quanta.cm−2.s−1 trace. The same color coding is followed throughout.
Figure 5
 
Average (±SD) (n = 5 participants) transient PLR (%), PLR latency (ms), constriction velocity (mm.s−1), peak constriction amplitude (% baseline), time to peak constriction (s), and pupil escape (mm.s−1) of the PLR to stimuli of wavelength 465 nm (blue) and 637 nm (red), retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1 increasing in 1-log unit steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles). The numbers in blue and red in the upper left and right indicate the luminance (log cd.m−2) of the short- and long-wavelength stimuli, respectively.
Figure 5
 
Average (±SD) (n = 5 participants) transient PLR (%), PLR latency (ms), constriction velocity (mm.s−1), peak constriction amplitude (% baseline), time to peak constriction (s), and pupil escape (mm.s−1) of the PLR to stimuli of wavelength 465 nm (blue) and 637 nm (red), retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1 increasing in 1-log unit steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles). The numbers in blue and red in the upper left and right indicate the luminance (log cd.m−2) of the short- and long-wavelength stimuli, respectively.
Figure 6
 
Average (±SD) (n = 5) redilation velocity (mm.s−1), 6-second PIPR amplitude (% baseline), plateau PIPR (% baseline), AUC early and late recovery (linear and log units), and PIPR duration (s) of the pupil light response to stimuli of wavelength 465 nm (blue) and 637 nm (red), retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1 increasing in 1-log steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles). The numbers in blue and red in the upper left and right indicate the luminance (log cd.m−2) of the short- and long-wavelength stimuli, respectively.
Figure 6
 
Average (±SD) (n = 5) redilation velocity (mm.s−1), 6-second PIPR amplitude (% baseline), plateau PIPR (% baseline), AUC early and late recovery (linear and log units), and PIPR duration (s) of the pupil light response to stimuli of wavelength 465 nm (blue) and 637 nm (red), retinal irradiance between 9.8 and 14.8 log quanta.cm−2.s−1 increasing in 1-log steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles). The numbers in blue and red in the upper left and right indicate the luminance (log cd.m−2) of the short- and long-wavelength stimuli, respectively.
Figure 7
 
Average (±SD) (n = 5) net redilation velocity (A), net 6-second PIPR (B), net plateau PIPR (C), net AUC early (D) and late (E) recovery, and net PIPR duration (F) of the pupil light response to stimuli of wavelength 465 nm and 637 nm, retinal irradiance from 9.8 to 14.8 log quanta.cm−2.s−1 increasing in 1-log steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles).
Figure 7
 
Average (±SD) (n = 5) net redilation velocity (A), net 6-second PIPR (B), net plateau PIPR (C), net AUC early (D) and late (E) recovery, and net PIPR duration (F) of the pupil light response to stimuli of wavelength 465 nm and 637 nm, retinal irradiance from 9.8 to 14.8 log quanta.cm−2.s−1 increasing in 1-log steps, and three durations: 1 second (squares), 10 seconds (triangles), and 30 seconds (circles).
Figure 8
 
Intraindividual (upper two rows) and interindividual (lower two rows) CV of the PIPR metrics for short-wavelength stimuli. The CVs for long-wavelength stimuli (not shown) were similar. The traces joined by squares, triangles, and circles represent the data for 1-second, 10-second, and 30-second pulses in all parts. The data points with a CV > 1.0 are not shown.
Figure 8
 
Intraindividual (upper two rows) and interindividual (lower two rows) CV of the PIPR metrics for short-wavelength stimuli. The CVs for long-wavelength stimuli (not shown) were similar. The traces joined by squares, triangles, and circles represent the data for 1-second, 10-second, and 30-second pulses in all parts. The data points with a CV > 1.0 are not shown.
Table 1
 
Individual Photoreceptor Excitation (in Log10 Units) With 465 nm and 637 nm Light Stimuli at Different Retinal Irradiances (Based on Lucas et al.35)
Table 1
 
Individual Photoreceptor Excitation (in Log10 Units) With 465 nm and 637 nm Light Stimuli at Different Retinal Irradiances (Based on Lucas et al.35)
Table 2
 
Description and Definition of the PLR Metrics During Light Stimulation and PIPR Metrics After Light Offset
Table 2
 
Description and Definition of the PLR Metrics During Light Stimulation and PIPR Metrics After Light Offset
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