This Research Insight covers a publication from the Kerschensteiner Lab. Here, we highlight how Michael Fitzpatrick, PhD and colleagues uncovered a new pupil response in the mouse, disentangled the neural pathway in the retina that drives it, and discovered that it is also present in humans. These findings shed new light on strategies the visual system uses to achieve precise, high-acuity vision for the things that matter.
In their recent paper published in Neuron, scientists in the lab of Daniel Kerschensteiner, MD, professor of ophthalmology at WashU Medicine, demonstrate a novel pupil response that is present in both mice and humans. Michael Fitzpatrick, PhD—a former graduate student in the Kerschensteiner Lab—and colleagues show that a specific neural circuit in the retina drives this response—termed the pupillary contrast response (PCoR)—and that the PCoR is sufficient to sharpen image clarity and improve visual acuity.
In mice, like humans, changes in brightness—or illuminance—cause the pupil to change its size, becoming smaller and larger under bright and dim conditions, respectively. This pupillary light reflex (PLR) has been extensively studied and, in part, helps to gate the amount of light that must then be processed by the retina. Given that individuals experience light levels ranging from darkness to bright sunlight, the authors hypothesized that changing pupil size may serve a second, less appreciated function to improve image contrast.
Measuring pupil responses in the lab
Pupil constriction in response to bright light (the PLR) occurs when light information is relayed from light-sensing photoreceptors, through bipolar relay cells, to a unique subtype of intrinsically photosensitive retinal ganglion cells (M1 ipRGCs). M1 ipRGCs send illuminance information—integrated from their own intrinsic light detection and inputs from photoreceptors—to the brain to change the size of the pupil.
In the lab, the PLR is typically assessed by measuring the pupil while a constant-intensity light shines in the eye. Rather than shining a constant-intensity light in the mouse’s eye, Fitzpatrick and colleagues chose to flicker the light at varying rates to test whether changes in contrast, in addition to illuminance, would be mirrored by changes in pupil size.
Flickering the light enabled the authors to uncover the novel PCoR, as it resulted in dynamic changes in pupil size. At slower frequencies, the pupil underwent a pulsing constriction that reliably tracked the frequency at which the light was flickered. However, at higher frequencies, the pupil constricted robustly to the stimulus (the PCoR). This constriction was significantly greater than would be expected for the average intensity of the light (i.e., the PLR), suggesting that the PCoR represents a novel pupil response to contrast.
Identifying the neural basis of the PCoR
Fitzpatrick and colleagues endeavored to test whether the neural basis underlying the PCoR was distinct from that of the PLR. The authors therefore used a battery of genetic strategies to interrogate the visual pathways in which the M1 ipRGCs participate to drive the PLR and PCoR.
As expected, removing the M1 ipRGCs from the retina abolished both the PLR and PCoR, confirming that both pupil responses rely on the information sent by ipRGCs to the brain. In contrast, neither the PLR nor PCoR relied solely on the ability of M1 ipRGCs to intrinsically detect light. These results indicated that any difference in the neural basis of the PLR and PCoR would occur at the level of bipolar cell and photoreceptor inputs to the M1 ipRGCs.
Fitzpatrick and colleagues performed patch clamp recordings of M1 ipRGCs, which revealed that B6 bipolar cells carry information from photoreceptors to the M1 ipRGCs. When the authors remove the B6 bipolar cells from the retina, they successfully abolished the PCoR, whereas the PLR persisted at high light levels. This result indicated that the PCoR—but not PLR—is driven exclusively by the presynaptic inputs to M1 ipRGCs. This result was further confirmed by blocking the light-sensing ability of rod photoreceptors, which likewise prevented the PCoR, but not PLR at high light levels.
Together, these results indicate that the PLR and PCoR are driven by distinct neural mechanisms, which converge at the level of the M1 ipRGCs. M1 ipRGCs drive the PLR in part via their intrinsic photosensitivity, whereas they drive the PCoR exclusively using inputs from B6 bipolar cells, which relay photoreceptor information from the primary rod photoreceptor pathway.
Characterizing the functional significance of the PCoR
Given that two independent mechanisms cooperate to measure illuminance, detect contrast, and control pupil size, the authors used pharmacological and genetic strategies to probe the functional significance of pupil size to visual acuity. To do so, Fitzpatrick and colleagues applied pupil-dilating or constricting drugs to the mouse eyes while testing two visual behaviors: the gaze-stabilizing optokinetic reflex and predation.
The optokinetic reflex is a tracking eye movement that is elicited by the movement of a drifting grating with varying size and contrast. This reflex depends on the ability of a mouse to distinguish the varying size and contrast of the moving bars and was impaired in mice that lacked M1 ipRGCs or that had artificially dilated pupils. The authors demonstrated that this impairment was linked specifically to pupil size, as artificially constricting the pupil in mice lacking M1 ipRGCs restored normal function.
Likewise, mice with dilated pupils were slower hunters than mice with normal pupils. These mice were slower to detect their prey and struggled to actively pursue them during the hunt. These deficits were consistent, regardless of how the pupils were dilated – whether by loss of M1 ipRGCs, application of a pupil-dilating drug to the eye, or manipulation of the brain areas that control pupil size.
The important role of pupil size in both the optokinetic reflex and predation suggests that the pupil has a vital role in tuning visual acuity. The conservation of these types of behaviors across species further motivated the authors to test whether the PCoR occurs alongside the PLR in humans, or whether this is a unique phenomenon in mice.
Discovery of the PCoR in humans
Like mice, humans exhibit strong pupil constrictions in response to increases in light level (the PLR). Given the similarities between species in the organization of the retina, and the conservation of specific cell types—including the M1 ipRGC—the PLR likely occurs via similar signaling mechanisms in the two species. The authors therefore recruited human volunteers to determine whether the PCoR—like the PLR—is conserved between mice and humans.
Humans were shown the flickering light while their pupils were monitored. Like mice, their pupils exhibited a dynamic response that incorporated both the PLR and PCoR, suggesting that pupil size also contributes to humans’ remarkable high visual acuity.
Importantly, the flickering frequencies that generated an optimal response in human pupils differed from those that generated an optimal response in mouse pupils. The authors noted that both mice and humans make small, unconscious head and eye movements while surveying their environment. Further they posit that the optimal flickering frequencies for each species match the small changes in visual input that are generated from these saccadic head and eye movements.
The authors therefore concluded that the PCoR has been conserved evolutionarily from mice to humans to cooperate with minute head and eye movements and translate static images into dynamic, high contrast scenes. As a result, dynamic changes in pupils size help to achieve the visual acuity necessary for mice to efficiently hunt and for humans to engage in their everyday lives.
About WashU Medicine
WashU Medicine is a global leader in academic medicine, including biomedical research, patient care and educational programs with 2,900 faculty. Its National Institutes of Health (NIH) research funding portfolio is the second largest among U.S. medical schools and has grown 56% in the last seven years. Together with institutional investment, WashU Medicine commits well over $1 billion annually to basic and clinical research innovation and training. Its faculty practice is consistently within the top five in the country, with more than 1,900 faculty physicians practicing at 130 locations and who are also the medical staffs of Barnes-Jewish and St. Louis Children’s hospitals of BJC HealthCare. WashU Medicine has a storied history in MD/PhD training, recently dedicated $100 million to scholarships and curriculum renewal for its medical students, and is home to top-notch training programs in every medical subspecialty as well as physical therapy, occupational therapy, and audiology and communications sciences.