We first examined the correspondence between the response characteristics of the intrinsically photoreceptive ganglion cells recorded in vitro with those of pupillary responses recorded in vivo under equivalent conditions. shows the response of the macaque pupil to a 10-second pulse of light (493 nm) under photopic conditions. At light ON, the pupil rapidly constricted and the constriction was maintained for the duration of the stimulus. At light OFF, the pupil dilated transiently, but then reconstricted and displayed a sustained, post-stimulus constriction in darkness. shows the response of an intrinsically-photoreceptive retinal ganglion cell recorded from the in vitro macaque retina under similar conditions to those in . Overall, the retinal ganglion cell activity closely matched the observed pupillary responses in time course. At light ON, the ganglion cell depolarized rapidly (latency to first spike ~35 msec) and exhibited sustained firing for the stimulus duration. (Dacey et al., 2005
). At light OFF, the cell hyperpolarized transiently to briefly cancel the sustained intrinsic response; the cell then repolarized to give rise to the sustained late discharge in darkness before slowly returning to the resting potential in darkness (Dacey et al., 2005
). The complex dynamics of the ganglion cell response derives from an interaction between cone input, which provides the short-latency responses at light onset and offset, and the inherent photoresponse, which maintains depolarization and firing for the duration of the stimulus followed by an extremely slow decay at light OFF (Dacey et al., 2005
Figure 1 Pupillary and ganglion cell responses in macaques. (A) Averaged pupillary responses (n=5) showing sustained pupilloconstriction to a 10-second pulse of light (493 nm, 13.3 log quanta/cm2/second). At light OFF, there is a transient pupil dilation followed (more ...)
The hypothesis that combined cone and melanopsin-derived light responses account respectively for transient and sustained pupil behavior can be evaluated by measurement of the spectral sensitivity of these responses. The slow and sustained intrinsic photoresponse of the retinal ganglion cells is characterized by an absorbance template corresponding to a single retinal1
-based pigment with a peak at 482 nm, whereas the fast and relatively transient cone-mediated response is complex and broadband, with significant sensitivity to longer wavelengths derived from L and M cone inputs (Dacey et al., 2005
). Under normal photopic conditions, substantial and prompt pupilloconstriction was elicited by retinal illumination at both 493 nm (activating both cones and the intrinsic photoresponse) and 613 nm (activating predominantly cones) (). However, during pharmacological blockade of rod and cone inputs (confirmed by the substantial reduction in the b-wave of the electroretinogram; see ), the pupillary response was delayed by approximately one second and was more sluggish than normal (); at the same time, substantial pupilloconstriction was elicited only by retinal illumination at 493 nm, consistent with the idea that the intrinsic photoresponse of ganglion cells controls this pupillary response. Similarly, sustained pupilloconstriction after light cessation was present only after retinal illumination at 493 nm ( D,E).
To more completely characterize the spectral sensitivity of these macaque pupillary responses, the irradiance-response relations for pupil constriction were measured at ten wavelengths between 430 nm and 613 nm, and fitted with the Hill equation. As an example, the filled circles in show the normal relation during the light stimulus at 532 nm. During pharmacological blockade (open squares), the response was absent at irradiance levels below ~11 log quanta/cm2/sec, presumably due to the elimination of rod-driven input. At higher irradiance levels, substantial pupilloconstriction occurred, despite the elimination of cone-driven responses.
shows Hill equation fits to the irradiance-response relations of the sustained response during illumination for all ten wavelengths under normal conditions. Pupil response-irradiance functions obtained for stimuli between 452 nm and 552 nm were comparable, with a light-evoked pupil constriction of ~3.2 mm at a retinal irradiance of 14.0 log quanta/cm2/sec. However, at longer wavelengths, pupil constriction was reduced. The collective spectral sensitivity data could not be well fit by a single pigment absorbance template for a retinal1-based pigment (, triangles), indicating the presence of more than one receptoral component. shows Hill equation fits to the irradiance-response relations during pharmacological blockade to isolate the intrinsic photoresponse. The pupillary responses for a given retinal irradiance were largest for wavelengths between 453 and 510 nm, with light-evoked pupil constriction of ~2.1 mm at a retinal irradiance of 14.0 log quanta/cm2/sec. At longer wavelengths, light-evoked pupillary responses were significantly reduced, and were essentially absent at 613 nm (). The collective spectral sensitivity data were well fit by the melanopsin spectrum (λmax 482 nm) (, circles).
Figure 2 Pupillary responses of macaques during a light stimulus under normal conditions and during pharmacological blockade. (A) Retinal irradiance-pupillary response plots under normal conditions. (B) Retinal irradiance-pupillary response plots after pharmacological (more ...)
Corresponding experiments on the sustained pupillary constriction after light OFF showed that this response was driven exclusively by the intrinsic photoresponse. More specifically, a large pupilloconstriction was produced by prior exposure to 10-second light pulses between 432 nm and 510 nm (, blue and green traces), whereas little response was observed after 10-second light pulses at 613 nm, a wavelength that does not readily evoke the intrinsic response (, red trace). At all wavelengths, the magnitude of the sustained pupilloconstriction after light pulses of a given retinal irradiance in the absence of pharmacological blockers was comparable to that of the sustained light-evoked pupillary response evoked by light of the same irradiance in the presence of blockers (cf. and ). Further, a comparable retinal irradiance was required in both cases to produce half-maximal pupilloconstriction at 493 nm. With or without pharmacological blockade, the irradiance-response relation for sustained, post-stimulus pupilloconstriction was the same at all wavelengths (), and had the same spectral sensitivity, being well fit by the melanopsin spectrum (λmax 482 nm) ().
Figure 3 Post-stimulus, sustained pupillary responses of macaques under normal conditions and during pharmacological blockade. (A) Retinal irradiance-pupillary response plots under normal conditions. (B) Retinal irradiance-pupillary response plots after pharmacological (more ...)
A melanopsin-associated photosensitive pathway appears to exist in humans (Dacey et al., 2005
; Rollag et al., 2003
; Hannibal et al., 2004
), but definitive evidence linking it to a functional role is still lacking. Because the sustained, post-stimulus pupil response appears to arise primarily from the melanopsin pathway in macaque, we sought to investigate the same phenomenon in normal human subjects. We found that pupilloconstriction in human subjects persisted after exposure to a 10-second light at 493 nm (, blue trace), but not at 613 nm for the same irradiance (, red trace). The irradiance-response relation at 493 nm () showed a half-maximal retinal irradiance (13.6 log quanta/cm2
/sec) very similar to that in macaque (cf. ). Next, for eight wavelengths between 452 nm – 592 nm, we calculated the retinal irradiance required to produce a given criterion pupil response if mediated by a Vitamin A1
pigment nomogram with a peak sensitivity at 482 nm (pilot data suggested a spectral sensitivity peaking between 480 and 485 nm). Repeated measures were obtained for the sustained pupil response at each wavelength and retinal irradiance. We found that the actual data obtained in this experiment departed only slightly from the predicted values (). Indeed, these data, following correction for minor departures from the predicted values as described in the legend to , were well fit (R2
= 0.99) by a Vitamin-A1
pigment nomogram with a peak sensitivity at 482 nm (), closely matching our results in macaques.
Figure 4 Post-stimulus, sustained pupillary responses in humans. (A) Averaged responses (n=3) of the pupil to 493 nm light of 14.1 log quanta/cm2/second irradiance (blue trace), and 613 nm light of 14.1 log quanta/cm2/second irradiance (red trace). (B) Retinal (more ...)