Our results show that rod-cone photoreceptors play an important role in driving pupillary constriction during exposure to continuous low-irradiance light, whereas melanopsin is the primary photopigment that drives sustained pupillary constriction in response to high-irradiance light. Rod-cone photoreceptors mediate rapid constriction of the pupils following light stimulus onset, and allow the pupils to track high-frequency intermittent light stimuli. During exposure to continuous light, the relative contribution of cone photoreceptors to pupillary constriction decreases over time. By applying short-duration intermittent dark pulses, however, pupillary responses can be sustained and even enhanced, presumably by allowing cone photoreceptors time to dark-adapt between light pulses.
In a blind individual without rod-cone function, pupillary constriction was preserved during exposure to high-irradiance 480 nm light, but weak or absent at lower irradiance levels. Similar results have been reported in rodless/coneless mice (Lucas et al., 2001
) and dogs with sudden acquired retinal degeneration syndrome (Grozdanic et al., 2007
). Our findings confirm in humans that melanopsin dominates the PLR at high irradiances (Lucas et al., 2003
), whereas visual photoreceptors mediate pupillary constriction during exposure to continuous low-irradiance light. The threshold for pupillary constriction in the blind individual was close to 11 log photons cm−2
, which is consistent with results in rd/rd cl
mice and gnat1−/−
mice that lack rod-cone function (Lucas et al., 2003
; Do et al., 2009
), and monkeys with pharmacologic blockade of rod-cone signaling (Gamlin et al., 2007
). These findings suggest that we were able to isolate melanopsin-driven pupillary responses in the blind patient, and that ipRGC function and sensitivity might be conserved across different mammalian species.
In sighted participants, we observed a short-wavelength shift in sensitivity during exposure to 2 min of light, suggesting a contribution from middle- and/or long-wavelength sensitive cones early in the exposure (Mure et al., 2009
). Consistent with this interpretation, the pupils showed sustained constriction during exposure to 4 min of 620 nm red light (13 log photons cm−2
; ), but we cannot exclude a contribution from rod photoreceptors. By comparison, there was little or no response to long-wavelength red light in the blind participant. Rather, the PLR was most sensitive to blue light, even though less short-wavelength light is transmitted to the retina as the lens ages (Brainard et al., 1997
; Kessel et al., 2010
) and pupillary responses were not corrected for pre-retinal lens absorption. Our findings in the blind patient are consistent with the spectral sensitivity of pupillary responses in an older blind individual (Zaidi et al., 2007
; 87-year old participant), in mice without rod and cone function (Lucas et al., 2001
), and in macaques with synaptic blockade of rod-cone responses (Gamlin et al., 2007
The onset of pupillary light constriction was abnormally slow in the blind patient, suggesting that rods/cones mediate the initial rapid pupillary response to light in sighted individuals. After the light stimulus was extinguished, the rate of pupillary dilation was also unusually slow in the blind patient, even though post-illumination pupillary constriction is thought to be driven primarily by melanopsin (Gamlin et al., 2007
; Kankipati et al., 2010
). Our findings are consistent, however, with recent reports demonstrating that patients with outer retinal degeneration show abnormally slow re-dilation of the pupils following exposure to blue light (Markwell et al., 2010
; Kardon et al., 2011
; Leon et al., 2012
), suggesting a possible role for rod-cone signaling in determining the rate of pupillary re-dilation after light offset.
The blind individual’s pupil was unable to track intermittent light. Instead, pupil diameter decreased in size over the first minute until reaching a stable size, thereafter behaving as if his eyes were being exposed to continuous light. By comparison, in sighted participants, pupillary constriction and dilation responses were closely time-locked to the intermittent light stimulus. Our results are similar to light responses measured from melanopsin cells in rat using a flickering 0.33 Hz stimulus (1 s on, 2 s off) (Wong et al., 2007
). With intact rod-cone signaling, each light pulse elicits a fast depolarization event, whereas during synaptic blockade only the sustained melanopsin-dependent depolarization remains. These findings suggest that visual photoreceptors are required for melanopsin cells to encode fast modulations of light intensity.
Pupil diameter was previously thought to reach a steady-state size within a few minutes of exposure to continuous light (Mure et al., 2009
). By using longer-duration exposures, we show that pupil diameter increases monotonically in sighted participants for at least 30 min. As the rate of pupillary escape is log-linear over time, i.e. higher near the beginning of the light stimulus, pupillary responses could appear to reach a steady-state when viewed on shorter time scales. Since we provided full-field illumination of both retinae, it is possible that part of the decrease in pupillary constriction early in the light exposure was due to reduction of retinal illumination caused by the PLR itself. The time-course of pupillary escape that we observed during the first 5 min of exposure to light was similar, however, to that described in a study that examined the consensual light reflex, in which the pupil of the stimulated eye was dilated using a mydriatic agent (Mure et al., 2009
). Although we chose a stimulus that would not be expected to drive the intrinsic melanopsin cell response (543 nm, 12 log photons cm−2
), we cannot exclude the possibility that melanopsin contributed to the weak sustained response to green light in sighted individuals. Similarly, these experiments do not distinguish between contributions from rods versus cones, as rod photoreceptors can signal non-visual light responses at photopic intensities (Altimus et al., 2010
With intermittent light in the 0.1–4 Hz range, we prevented pupillary escape and enhanced pupillary constriction for at least 30 min, similar to findings for exposure to shorter-duration sinusoidal light (Varju, 1964
; Troelstra, 1968
; Clarke et al., 2003
). Our findings are analogous to the Brucke-Bartley effect (i.e., brightness enhancement) for vision, in which a flickering light stimulus appears brighter than the same light presented continuously, but only within a specific frequency range (Bartley, 1939
). The spectral sensitivity of brightness enhancement matches the photopic luminosity function (Walters and Harwerth, 1978
), suggesting involvement of cone photoreceptors. We hypothesize that short intermittent dark pulses allow cone photoreceptors the opportunity to dark-adapt prior to each light pulse, thus preventing pupillary escape.
Similar to our findings for the PLR, exposure to intermittent red light enhances circadian phase shift responses in Opn1mwR
mice that express human long-wavelength sensitive opsin (Lall et al., 2010
). Taken together, these results raise the possibility that non-visual light responses in humans can be driven, and perhaps enhanced, by intermittent light therapy that targets activation of cone photoreceptors. Since circadian and pupillary light responses are driven by different populations of melanopsin cells in mice (Chen et al., 2011
), additional studies are required to determine whether our findings for the PLR are generalizable to other non-visual light responses in humans.
Although we did not study age-matched controls, pupillary light responses in the blind individual (aged 58 years) were many times slower than predicted for a normally-sighted individual in the same age group (Feinberg and Podolak, 1965
; Alexandridis and Manner, 1977
; Pfeifer et al., 1983
; Straub et al., 1992
; Bitsios et al., 1996
). To account for age-dependent differences, we normalized PLR responses to dark-adapted pupil size, i.e. we determined percentage pupillary constriction, which is stable across age groups (Birren et al., 1950
; Daneault et al., 2012
). In doing so, we found that the sensitivity of pupillary responses in the blind individual was similar to mice without rod-cone function (Lucas et al., 2003
; Do et al., 2009
) and macaques with pharmacologic blockade of synaptic input to ipRGCs (Gamlin et al., 2007
); hence we consider it unlikely that age-related decline in sympathetic or retinal function contributed substantially to differences we observed in pupillary responses between sighted individuals and the older blind patient.
Our findings have potential implications for how rod/cone and melanopsin dysfunction is assessed in patient populations (Kankipati et al., 2010
; Kawasaki et al., 2010
; Kardon et al., 2011
; Kankipati et al., 2011
). Here, we characterized PLR responses in a blind patient who was previously described as having pupils that were unresponsive to light based on a standard penlight examination performed by an ophthalmologist (Zaidi et al, 2007
). Based on our findings, clinical testing of pupillary light responses in patients with visual loss should be performed in darkness, using longer-duration light stimuli in addition to short light pulses. Our results may explain why, in a previous study, totally visually blind individuals with intact circadian responses failed to show a PLR during a routine ophthalmologic exam (Czeisler et al., 1995
). Pupillary light responses could potentially be used to screen for blind patients with intact circadian photoreception who should continue to expose themselves to light-dark cues in order to entrain to the 24-h solar day. This test might be particularly important in visually-impaired patients considering enucleation, to avoid removing a light-sensitive eye. Hence, in future studies it will be important to examine the relationship between pupillary light constriction and other non-visual light responses in patients with visual dysfunction.