Jordan M. Renna - US grants

DESCRIPTION (provided by applicant): Retinal waves comprise zones of spontaneous activation that propagate across the immature retina. These waves are thought to participate in the development of retinotopic maps and the segregation of binocular inputs to the superior colliculus and lateral geniculate nucleus. Retinal waves occur before rod and cone photoreceptors are mature, at a time when the retina has been considered insensitive to light. However, recent findings invalidate this assumption. There is a novel class of photoreceptor, the intrinsically photosensitive retinal ganglion cell (ipRGC), that is fully functional at this stage. Virtually nothing is known about possible interactions between ipRGCs, light exposure, and retinal waves. This proposal explores not only how these photoreceptors are affected by retinal waves, but also how they, in turn, may affect the waves. In the adult retina, ipRGCs not only receive excitatory and inhibitory synaptic input, but also are thought to feed signals back into the inner retina. This raises the possibility of bidirectional interactions between retinal waves and ipRGCs, although the intraretinal synaptic connectivity of ipRGCs at this developmental stage has not been determined. Preliminary data indicate that not only do ipRGCs discharge in association with retinal waves, but also that photic activation of this class of ganglion cells alters retinal wave activity. Therefore, the specific aims of this proposal are: 1. Characterize excitation of ipRGCs during retinal wave activity and identify the mechanism of activation;2. Test the hypothesis that light-induced activation of ipRGCs alters retinal wave activity and assess the mechanism responsible. These studies will shed new light on the mechanism of retinal wave activity and will extend our understanding of ipRGCs, which play an instructive role in circadian rhythm photoentrainment and the pupillary light reflex. PUBLIC HEALTH RELEVANCE: This study will examine the role of a specialized class of retinal cells in the regulation of normal visual system development. Further, it will shed new light on how these cells function early in development. These cells play a central role in the adult body's response to daylight and therefore, these studies are relevant to such public health issues as jet lag, seasonal affective disorder, circadian disruption in the blind, and the negative consequences of shift work including impaired performance, increased risk of injury and even elevated cancer rates.

? DESCRIPTION (provided by applicant): In the developing retina, melanopsin ganglion cells are the first functional photoreceptors and play an important role in the proper wiring of the visual system from retina to brain. In fact, for the first ten days of life, they are the sole soure of light information to the brain because the bipolar cells which link rods and cones to ganglion cells are not yet mature. In other words, the outer retina is functionally disconnected from the inner retina until postnatal day 10 (P10). We have recently documented a surprising new observation in mouse retina that many immature melanopsin ganglion cells extend their dendrites during the first week of life not only to the conventional location in the inner plexifor layer, but also all the way out to the outer plexiform layer, where in the mature retina, rods and cones synapse onto bipolar cells. We have shown that these melanopsin ganglion cell Complex Outer Retinal Dendrites (CORDs) can be found in close association with cone axon terminals and express post-synaptic density marker 95, suggestive of direct synaptic contact. We have shown that some CORDs persist into adulthood. Thus, CORDs may represent a novel anatomical circuit in the mammalian retina connecting the outer retina to inner retina early in development and bypassing the conventional cone-to- bipolar-cell pathway. However, the functional significance of this circuit is unknown. The goal of this study is to determine the anatomical and physiological properties of this unique circuit and to determine its functional significance in the developing mouse retina. This work is novel because it challenges the canonical view of how melanopsin ganglion cells influence visual system development and proposes a new circuit by which cones may influence the brain far earlier than previously recognized.

Project Summary/Abstract Treating retinal degenerative diseases has been hampered by the lack of suitable systems that can evaluate how new treatment strategies affect the function of the human retina. Human stem cell-derived 3- dimensional retinal organoid technologies have been recently developed. Remarkably, human retinal organoids mimic the native tissue's histological organization, cellular composition, and are able to respond to light. These organoids are an ideal model system for investigating novel therapies to treat blinding diseases. However, to fully realize the unprecedented potential of human retinal organoids for the development of treatments for retinal degenerative diseases, we need new technologies that can rapidly measure retinal function under a wide variety of conditions. Current techniques such as patch-clamp electrophysiology, calcium imaging, and multi-electrode array recordings, measure how individual cells function within the circuitry of the retina. However, these techniques are laborious and ineffective at assessing the health, reproducibility, and functional responsivity of the retina as a whole - features that are key to the application of organoid systems to drug screening and validation. The lack of a device and techniques that allow for rapid, non-invasive screening of the functional status of retinal organoids constitutes a major unmet need. In Aim 1 of this proposal, we will develop an electroretinogram (ERG) recording chamber and a recording protocol for real-time assessment of light-evoked retinal organoid physiology by measuring photoreceptor and bipolar cell function. Our findings will be used to establish critical metrics associated with normal organoid light-evoked responsivity. Further, we will evaluate the power of this approach to detect changes in photoreceptor function, to provide evidence of its applicability to the assessment of disease models and therapeutic screening. To take full advantage of this technology for downstream applications it is critical that it be combined with robust organoid models. The variability and low yield of current protocols for retinal organoid generation and the extended time required for functional maturation of organoid photoreceptors hinder their application in drug development and disease evaluation. In Aim 2 of this proposal, we will address this critical gap by developing and evaluating improved protocols for human retinal organoid generation that increase yield and accelerate photoreceptor differentiation. We will then evaluate retinal function in these improved organoids using our ERG platform. Through the combination of these technologies, we will have created the first system for rapid, non- invasive functional screening in human retinal organoids that can be applied to the evaluation of normal, diseased, and drug-treated conditions. Our system has the potential to greatly accelerate the development of novel therapies to reverse vision loss.