There is now considerable evidence that three types of photoreceptors (rods, cones and

intrinsically photosensitive retinal ganglion cells [ipRGCs]) contribute photic information to the

neural circuitry that mediates the pupillary light reflex (see Markwell et al., 2010 for review). In

light of this evidence, one potential reason for why current pupil testing strategies are relatively

insensitive to early retinal damage is that there is likely considerable redundancy in the neural

circuitry that mediates the pupil response. For example, genetically modified mice that lack all

rod and cone function still exhibit ipRGC-driven pupillary reflexes, and the magnitude of the

pupil constriction evoked by bright white light in these mice is not significantly different from

control mice (Hattar et al., 2003).

Thus, there has been considerable interest in developing clinical protocols that isolate

the separate components of the pupillary light reflex that are mediated by the different

photoreceptors (Kankipati et al., 2011, Park et al., 2011, Tsujimura and Tokuda, 2011, Zele et

al., 2011). By utilizing stimuli that unmask the contribution of the different photoreceptor types

to the pupillary reflex, the goal is to develop novel pupil testing strategies that are more sensitive

to early retina and optic nerve disease.

With previous funding from OLERF, my lab has been developing a rodent pupil testing

strategy that assesses the sensitivity of the pupil response to flickering light. The light

responses of ipRGCs are strikingly different from those in rod and cone photoreceptors.

Notably, ipRGCs slowly depolarize in response to light, while rods and cones exhibit faster

hyperpolarizing responses. Our guiding hypothesis is that one consequence of the

sluggishness of ipRGC light responsiveness is that these photoreceptors will have poor

temporal resolution as compared to rod and cone photoreceptors. In other words, I predict that

ipRGCs exhibit similar responses when stimulated with either continuous light or light that is

flickering at a relatively slow rate (0.1 Hz).

continuous and flickering light, we have been characterizing the pupil responses of rats exposed

to flickering light of different intensities and frequencies (see Figure 1 for an example). We

have shown that the rat pupil exhibits sustained constriction following light offset, and the rate of

re-dilation is dependent on the intensity of the stimulus. As a consequence, there is a decrease

in the magnitude of pupil flicker that occurs in response to flashing light stimuli of increasing

intensities. We propose that the relative insensitivity of the rat pupil to bright flickering light is

due to the increased contribution of ipRGCs at high light intensities. This work has been

presented in abstract form (Doerning et al., 2011 ARVO E-abstract #3461) and is currently

In this proposed project, we will specifically ablate ipRGCs in adult rats using intravitreal

injections of saporin immunotoxin conjugated to melanopsin antibodies. We will test the guiding

hypothesis that the sensitivity of the rat pupillary reflex to flickering light (i.e. the ability of the

pupil response to discriminate the individual light flashes) will increase after ipRGC ablation.

Specific Aim. We will characterize the magnitude of fluctuation in the pupil response to a

slowly flickering (0.1 Hz) at different light intensities in rat eyes in which ipRGCs are ablated. By

varying the concentration of the saporin conjugate used for the intravitreal injections, we will

correlate the flicker sensitivity of the pupil response to varying degrees of ipRGC loss

We have previously found that amplitude of pupil fluctuation decreases as the intensity

of the light flashes increases (Doerning et al. 2011 ARVO E-abstract #3461). The pupil

responses for a representative control rat, exposed to 0.1 Hz flickering light at intensities

Figure 1A. As evident by the summary data (Figure 1B), the amplitude of the pupil fluctuations

pupil exhibited negligible fluctuation in response to the flashing light.

I expect that the saporin/antibody conjugate intravitreal injections will result in specific

ablation of the ipRGC population in the rat retinas. Thus, we expect that the counts of ipRGCs

will be lower in the retinas of the saporin-injected retinas. We have tested the antibodies in

immunohistochemistry experiments on rat retinal flatmounts and confirmed that these antibodies

stain neurons that have a morphology consistent with that known for ipRGCs. The specificity of

the primary antibodies indicates that other retinal cells should not be affected by the saporin

injection, as shown in work by other researchers (Goz et al., 2008, Ingham et al., 2009). To

verify this, a sample of the retinas will be stained with a general nuclear stain (DAPI) and

histological counts of the nuclei in the inner and outer retinal nuclear layers will be performed.

We expect these counts to be unaltered in the saporin-injected eyes versus controls.

Figure 1. A) Example trace of pupil size from a rat exposed to 0.1 Hz flickering blue light of

amplitude of pupil flicker at each intensity (n = 5).

The preliminary results shown in Figure 2 are from a rat that was injected with 400 ng of

saporin/antibody conjugate in the right eye and saline in the left eye. The pupil response to

flickering blue light (same protocol as in Figure 1A) is shown for the saline-injected eye (Figure

2A) and for the saporin-injected eye (Figure 2B). The results represent the average for 2 trials

performed on each eye two months after the saporin injection. Note the increased fluctuation in

the pupil constriction in the saporin-injected eye. These retinas are currently being processed

for immunohistochemistry to determine the overall ipRGC numbers in these two retinas.

Figure 2. Example traces of pupil size from both eyes of a rat exposed to 0.1 Hz flickering blue

was obtained from the right eye that was injected with 400 ng of saporin/antibody conjugate.

These results in Figure 2 are consistent with our hypothesis that pupil size will exhibit

more fluctuation in response to the flickering blue light after ipRGC ablation. We predict that the

different concentrations of injected saporin will cause ipRGC death that ranges from mild (i.e.

10% ipRGC loss) to severe (i.e. >70% ipRGC loss), and that the flicker sensitivity of the pupil

response will correlate to ipRGC death. These results will verify whether our proposed flickering

light pupil test can serve as a sensitive marker of early ipRGC death. The ultimate goal of this

work is to develop pupil tests that will better enable the earlier detection of optic nerve diseases

such as glaucoma.

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photosensitive retinal ganglion cells with a saporin conjugate alters the effects of light on

mouse circadian rhythms. PLoS ONE 3:e3153.2008).

Hattar S, Lucas RJ, Mrosovsky N, Thompson S, Douglas RH, Hankins MW, Lem J, Biel M,

Hofmann F, Foster RG, Yau KW (Melanopsin and rod-cone photoreceptive systems

account for all major accessory visual functions in mice. Nature 424:76-81.2003).

Ingham ES, Gunhan E, Fuller PM, Fuller CA (Immunotoxin-induced ablation of melanopsin

retinal ganglion cells in a non-murine mammalian model. J Comp Neurol 516:125-

140.2009).

Kankipati L, Girkin CA, Gamlin PD (The post-illumination pupil response is reduced in glaucoma

patients. Invest Ophthalmol Vis Sci 52:2287-2292.2011).

Markwell EL, Feigl B, Zele AJ (Intrinsically photosensitive melanopsin retinal ganglion cell

contributions to the pupillary light reflex and circadian rhythm. Clin Exp Optom 93:137-

149.2010).

Park JC, Moura AL, Raza AS, Rhee DW, Kardon RH, Hood DC (Toward a Clinical Protocol for

Assessing Rod, Cone and Melanopsin Contributions to the Human Pupil Response.

Invest Ophthalmol Vis Sci.2011).

Tsujimura SI, Tokuda Y (Delayed response of human melanopsin retinal ganglion cells on the

pupillary light reflex. Ophthalmic Physiol Opt.2011).

Zele AJ, Feigl B, Smith SS, Markwell EL (The circadian response of intrinsically photosensitive

retinal ganglion cells. PLoS ONE 6:e17860.2011).