Alapakkam P. Sampath - US grants

[unreadable] DESCRIPTION (provided by applicant): The experiments described in this proposal are aimed understanding the photoreceptor to On-bipolar cell synapse in the vertebrate retina. The On-bipolar cell uses a G-protein-coupled signaling mechanism to relay information it collects from the photoreceptors to the inner retina for processing. While the receptor and G-protein have been identified, the mechanisms linking these to a non-specific cation channel are unknown. I will approach the study of this cascade with the following strategy. While blocking the G-protein's action, I will determine what internal factors are necessary in On-bipolar cells to open transduction channels. To accomplish this I will make whole-cell patch clamp recordings from salamander On-bipolar cells. In particular I will test whether the native state of the transduction channel is open or closed, and whether gating mechanisms directly or indirectly involve cGMP. If cGMP is not involved, the metabolic requirements for channel opening will be identified. In parallel experiments I will determine what internal factors are necessary for maintaining the light response, or what factors are necessary for channel closure. I will also test inhibitors of other known signaling mechanisms that utilize non-specific cation channels. These two types of experiments will be corroborated by two-photon imaging of calcium in the On-bipolar dendrites, to compare the localization of "run-open" channels with those opened by the transduction mechanism. Finally, I will study how low light level information is transferred between rod photoreceptors and rod bipolar cells in the mouse retina. The signal-to-noise ratio at this synapse is maximized by a thresholding non-linearity. I will study the mechanism producing this non-linearity. First I will determine whether the mechanism is pre- or postsynaptic, and if it arises from saturation at the receptors or downstream in the transduction mechanism. Lastly, I will determine whether the non-linearity is dynamic, or whether its position can be influenced by very dim background light. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal is aimed at understanding the properties of the rod circuitry in the mammalian retina. Our rod vision encompasses lights as dim as a single photon absorption in as many as 10,000 rod photoreceptors, up to 1000 or more photons per rod. This wide range of light sensitivity is accomplished by biophysical mechanisms that have been studied for more than a half century, but several outstanding issues of fundamental importance remain. The experiments described in this proposal are designed to address two issues: (1) Which aspects of the rod photoresponse are relevant for downstream processing?, and (2) What are the threshold and dynamic range of light intensities for each retinal pathway that relays the rod photoresponse to ganglion cells? To answer these questions we will record light-evoked responses from rod photoreceptors, bipolar cells and ganglion cells from several transgenic mouse lines lacking cone light responses (cone trasducin -/-) with: (1) knockouts in the rod phototransduction pathway that change the shape of the rod photoresponse, and (2) knockouts in the retinal circuitry that will allow the rod retinal pathways (Rod-Bipolar, Rod-Cone, and Rod-Off pathways) to be studied in isolation. The deletion of the phototransduction proteins (GCAP, Rhodopsin Kinase, Rhodopsin, and Arrestin), or the gap junction protein Connexin 36 in specific cells, will alter the transmission of light responses and allow us to infer how the properties of the photoresponse and circuitry influence rod vision. In parallel with these experiments, we will evaluate the behavioral threshold for rod vision in each mouse studied. Thus alterations in the physiology of rod signaling in the retina can be connected to a constrained and quantifiable visual behavior. Such correlations will provide insights into how the processing of the rod photoresponse influences perception, and will have consequences for understanding the mechanistic basis for deficits in rod vision (i.e. Stationary Night Blindness). Night vision in mammals is mediated by several pathways in the retina that relay information about few photon absorptions to the brain. This proposal is aimed at understanding how this remarkable sensitivity is achieved at the cellular level by establishing the properties of the rod photoreceptor response and the retinal circuitry that are important for visual processing. In order to devise therapies for conditions that influence our night vision, like Stationary Night Blindness, it is necessary to first understand the physiological mechanisms that relay information from our eyes to our brain at low light levels.

Project Summary Rod photoreceptor death is a significant cause of human blindness, and much research effort has been expended towards their rescue or replacement using gene or stem cell therapy. However, rod death is followed by secondary changes in the inner retina such as dendritic remodeling, cell migration, and rewiring. The extent to which this reorganization obstructs the potential for recovery of vision following photoreceptor rescue is not known. The objective of this proposal is to examine signal processing in the affected retina following the genetic rescue of rods. To this end, we have created a mouse model of retinal degeneration caused by loss of expression of the ?-subunit of the cyclic nucleotide-gated (CNG?1) channel, a model for autosomal recessive retinitis pigmentosa in humans. The novelty of this mouse model is that CNG?1 can be expressed from the endogenous locus in all affected CNGB1-/- rods upon tamoxifen(TX)-inducible Cre- mediated recombination, leading to rod rescue. This proposal utilizes this mouse line to determine the impact of rescuing rod function on retinal signal processing. In Aim 1 we will examine how the structure and function of rod photoreceptors recovers following the restoration of CNG?1 expression. In particular we will examine the extent of functional recovery when TM is administered in mice with increasing severity of retinal degeneration, as this may identify a critical window for the efficacy of rod recovery and halting further degeneration. In Aim 2 we will examine how the synapse between rods and their primary postsynaptic partner, rod bipolar cells, is reformed following rod rescue. Synapses between rods and rod bipolar cells form retinal circuits that regulate our night vision. Finally, in Aim 3 we will evaluate how rod rescue impacts the function of retinal ganglion cells, which are the sole conduit for signals from the retina to reach higher brain areas. The central hypothesis is that while light sensitivity will recover substantially with rod rescue, some deficits in retinal signaling will persist and worsen at late rescue ages due to secondary changes in retinal circuits. These studies will define the window of opportunity for therapeutic intervention and provide a foundation for future studies aimed at reversing the negative effects of neural remodeling.

PROJECT SUMMARY Mammalian rod and cone photoreceptors are indispensible for vision. They convert light into electrical response, which is then propagated across the retina circuit and into the brain. Transmission of the electrical signal generated by the photoreceptors requires their synaptic connectivity with the downstream interneurons, the bipolar cells. Deficits in synaptic communication between photoreceptors and bipolar cells are known to cause congenital stationary blindness in humans, a condition characterized by poor light sensitivity and frequent co-morbidity with many other ocular conditions. Our long-term goal is to elucidate molecular and cellular mechanisms by which photoreceptors establish synapses and transmit their signals with the hope to better understand blinding conditions and devising strategies for their treatment. Two types of the photoreceptors, rods and cones, form distinct connections with different types of the bipolar cells. This synaptic specificity segregates visual inputs and plays an essential role in setting up the fundamental properties of our vision, including a wide dynamic range of light sensitivity and contrast discrimination. However, the molecular mechanisms responsible for selective connectivity between photoreceptors and their downstream bipolar neurons are unknown. We have identified a new cell adhesion- like molecule ELFN1 that specifically present at the photoreceptors synapses. We found that ELFN1 forms a trans-synaptic interaction with the principal neurotransmitter receptor in bipolar cells, mGluR6. Disruption of ELFN1 results in selective loss of rod synapses. We hypothesize that ELFN1-mGluR6 interaction play key roles in mediating selective synaptic connectivity of rod photoreceptors and direct the propagation of light signal across retina circuit. This hypothesis will be tested by pursuing three complementary Specific Aims that will (i) use knockout mouse models, and genetic rescue experiments to determine cellular mechanisms of ELFN1 function in the formation of synapse between rod photoreceptors and ON-RBC, (ii) investigate the role of ELFN1 in directing the propagation light signal across retina circuitry, and (iii) examine molecular mechanisms by which ELFN1 enables its synaptogenic effects. The strategy proposed to address these aims will entail a synergistic combination of biochemical, molecular biological, electrophysiological, and physiological approaches, each exploiting the existence of a powerful array of reagents and animal models.

7. Project Summary/Abstract: This is a competing renewal for an NRSA Research Training Grant (T32- EY007026). The Vision Science Training Program (VSTP) has its home in the Jules Stein Eye Institute (JSEI), University of California, Los Angeles (UCLA) School of Medicine, and has been continuously active for more than 40 years. During this period the VSTP has trained nearly 140 scientists, a large portion who have gone on to distinguished careers in vision science. The Program covers the training of both predoctoral and postdoctoral fellows, and takes place in the laboratories of 14 faculty mentors. These vision scientists possess expertise in a wide range of disciplines and hold academic appointments in 13 departments at UCLA. Applicants for predoctoral fellowships first enter UCLA through one of several graduate programs, and they complete the curriculum associated with that program. After a year of rotations they join the laboratory of a VSTP mentor. Applicants for postdoctoral fellowships apply directly to the VSTP lab of interest. All trainees that are members of VSTP labs are invited to participate in the curricular offerings of this program. These offerings are collectively designed to provide experimental and conceptual training in vision science, as many of the trainees have no prior experience in vision science. All fellows are required to take Fundamentals of Vision Research, a quarter-long course organized and taught by VSTP members. Additionally fellows are integral members of the scientific environment at JSEI through attendance and participation in a number of events, including the weekly Vision Science Seminar Series, the Vision Science Journal Club, and the Annual Vision Science Retreat held in Lake Arrowhead. A particular focus of the VSTP for this grant period is the stronger integration of basic and clinical science. Several offerings will help bridge the gap between these two research focuses by facilitating interactions between the scientists and the clinicians, including the Annual UCLA Stein Eye Clinical and Research Seminar, Translational Grand Rounds, and a Basic-Clinical Science Exchange program. Such experiences will help trainees to place their research into the context of visual health and disease. A major addition to the VSTP will be a dedicated program focused on the postdoctoral trainees, to support their transition to independent investigators. An NRSA F32 Training Course will be offered to all postdoctoral fellows in VSTP labs to provide formal training in how to craft and submit an effective NIH research proposal. Given the interdisciplinary nature of vision research, the overall goal of the VSTP is to bring together faculty with a wide range of expertise (including anatomy, biochemistry, biophysics, chemistry, molecular biology, physiology, pharmacology, cell biology, and developmental biology) to provide interdisciplinary experimental and conceptual training to predoctoral and postdoctoral candidates seeking to develop careers in vision science. Continued funding of the VSTP will permit the JSEI to carry on its critical mission of training the next generation of first-rate basic and clinical vision scientists.

Project Summary Our sense of vision begins when single rod and cone photoreceptors absorb light and produce an electrical signal, which higher centers in the brain then analyze to alter our behavior. We learn even as children that rods are the photoreceptors we use to see dim light and cones to see bright light and color. This view is supported by behavioral measurements and electrical recording, which all seem to show that rods are primarily used to detect dim light and become essentially non-functional as the ambient illumination increases during daylight. Recent experiments have however challenged this notion and demonstrated that rods can continue to respond even in light so strong that a large fraction of the rod photopigment is bleached. These observations challenge our understanding of rod function in bright light. The purpose of this study is to thoroughly reexamine rod current and voltage responses to persistent bright illumination over extended durations of time. Our preliminary evidence shows surprisingly that the responsiveness of rods can recover over the course of hours during persistent bright illumination. Here we are seeking to investigate the molecular and mechanistic basis of this rod recovery and its dependence on time and light intensity in mice. In particular, we will leverage several lines of transgenic mice having targeted mutations in components of the phototransduction cascade. We also are interested in how photoresponse recovery in rods can be made faster and more robust, as observed in cones. We we will explore these phenomena by genetically transferring certain molecular features of cone phototransduction into the rods by leveraging mice with targeted mutations to reduce the sensitivity of rods and increase the rate of photoresponse and photopigment decay. We hope to show which factors are responsible for the differential responsiveness of the two photoreceptors in bright light. These phenomena are not only important to our understanding of the physiology of photoreceptors, they are also essential for photoreceptor survival because rods die when outer- segment channels remain closed for too long a time. In addition, understanding how to make rod photoreceptors more like cones may have therapeutic value, as deficiencies in cone vision may be mitigated by shifting the responsiveness of rods to brighter background light intensities. Because of the importance of these phenomena to photoreceptor function in health and disease, the Retinal Disease Program of the NEI has as one of its program objectives to ?analyze the mechanisms underlying light adaptation and recovery following phototransduction?.