Richard H. Kramer, Ph.D. - US grants

DESCRIPTION (Adapted from applicant's abstract): This application proposes to study the properties of CNG channels in cone synaptic terminals of the lizard. These terminals are large and therefore suitable for localized electrophysiological experiments and calcium imaging. The premise is that these CNG channels are involved in calcium influx and regulation of cone transmitter release. The first specific aim is to characterize the pharmacology of the CNG channel, and in particular to find specific antagonists. Two prospects are a Conus toxin and cyclic nucleotide analogs. The second specific aim is to investigate the role of CNG channels in calcium influx and to compare this influx with that generated by voltage dependent calcium channels in the terminal. The third specific aim is to determine the role of CNG channels in triggering transmitter release from cones. Catfish horizontal cells will be used as a "biosensor" to detect the cone's glutamate release. The final specific aim is to determine the role of CNG channels in mediating the release of cone transmitter in the presence of nitric oxide (NO). Since NO affects both CNG and voltage-gated calcium channels, the relative significance of these two pathways will be determined. Preliminary results indicate that NO produces a sag in light response of horizontal cells in the intact retina. Whether this is due to effects on cones or effects on horizontal cells will be examined.

Insulin-like growth factor l (IGF-l) is a peptide that is structurally homologous and has overlapping functions with insulin. IGF-l is produced in tissues throughout the body including the brain and retina, where it acts as a paracrine signal from pigment epithelial to rod photoreceptors. IGF-l plays important roles in cell differentiation, growth, and survival, and recent studies show that IGF-l also has acute physiological effects on ion channels in the brain. The levels of IGF-1 and IGF-1 receptors are changed dramatically in diabetes, and have been proposed to play an integral part in diabetic neuropathies. The goal of this project is to understand more about the acute effects of IGF-l on the retina, with a specific focus on rod photoreceptors. Our preliminary experiments show that IGF-l alters the sensitivity of rods to light. Moreover, IGF-1 modulates the activity of the ion channels responsible for generating the light response. Thus, IGF-1 alters the sensitivity of cyclic nucleotide-gated (CNG) channels to cyclic GMP, the crucial "internal transmitter" of phototransduction. The effect of IGF-l is mediated by a tyrosine kinase cascade, perhaps resulting in changes in the tyrosine phosphorylation state of the CNG channels. The aims of our project are to understand more about how IGF-l affects CNG channels in rods, and how changes in the activity of the channels might underlie changes in the light response. First, we will investigate the biochemical cascade that couples the activated IGF-1 receptor to modulation of the channels. Second, we will investigate how changes in tyrosine phosphorylation modulate the channels, and we will use site-directed mutagenesis of exogenously expressed CNG channels to locate the crucial phosphorylation site(s) on the CNG channel protein. Finally, we will examine the effect of IGF-l on single human photoreceptors from diabetic and non-diabetic donors, to understand how CNG channel modulation may alter the light response. These studies will lead to a molecular-level understanding of the effects of IGF-l on the retina, and will provide important insights into cellular and molecular processes that may be related to normal retinal physiology and diabetic retinopathy.

The connections between cone photoreceptors and their postsynaptic targets, bipolar and horizontal cells (BCs and HCs), are the first synapses in the visual system. Neurotransmitter release from cones is regulated intrinsically, by light, and extrinsically, by feedback signals from HCs. Our long-term goal is to understand at a molecular level how these signals regulate release. Cone terminals contain a specialized structure called the synaptic ribbon. The ribbon binds synaptic vesicles and is thought to deliver them to the plasma membrane where they undergo Ca2+-dependent exocytosis. Our first specific aim is to understand the mechanism of synaptic vesicle delivery by the ribbon, and to evaluate the role of Ca2+ in regulating this process. We propose three steps in ribbon-mediated vesicle delivery: Vesicle binding to the ribbon, vesicle movement along the ribbon, and vesicle detachment from the ribbon. To address the first step, we will ask whether Rab3a, a vesicle- associated small G-protein, is responsible for the initial binding of synaptic vesicles to the ribbon. To address the second step, we will use fluorescent markers of synaptic vesicles to measure vesicle mobility on the ribbon with Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS). To address the third step, we will use electron microscopy to evaluate whether vesicles vacate the ribbon when Ca2+ is elevated in the cytoplasm. Finally, to better understand how Ca2+ might regulate these events, we will measure the Ca2+ profile along the ribbon with a novel Ribbon-Associated Ca2+ Indicator (RACI). Together, these experiments will help explain the fundamental events that control synaptic vesicle delivery in cones. Our second specific aim is to investigate the mechanisms of HC feedback onto cone terminals. Protons have been proposed to be the signal underlying HC negative feedback. We will measure the local pH at the cone synapse of zebrafish with pH-sensitive GFP (pHluorin). The pHluorin probe will be spliced onto synaptic proteins enabling high spatial resolution pH measurement at the very site of HC feedback. We will evaluate a second ephaptic hypothesis with caged glutamate receptor agonists to locally alter current flow into individual dendrites of HCs. Finally, we will explore a newly-discovered positive feedback system from HCs to cones, investigating the nature of the retrograde signal and determining its mechanism of action. These studies are important for three reasons: 1) they will improve our understanding of the fundamental processes underlying the first steps in seeing, 2) they may provide insights into the mechanisms and consequences of several blinding disorders, including Ushers Syndrome and autosomal dominant cone-rod dystrophy (CORD7), which are associated with disruptions in photoreceptor synapses, and 3) by elucidating normal mechanisms of synaptic information transfer in the retina, they may provide a clearer template for the design and programming of prosthetic devices for restoring vision to blind patients.

The ability to artificially control neuronal activity is important both experimentally, for understanding neural circuits, and therapeutically, for compensating for damage or degeneration of neural structures. Commonly used electrical or chemical methods of neural control are invasive and can be spatially and temporally inaccurate. Targeted expression of genes encoding K+or C1- channel has been used to "silence" specific neurons, but initiation of channel expression takes hours and is not easily reversible. We are developing a rapid and reversible method for silencing the activity of individual neurons that involves expression of channels that are chemically modified to render them light-sensitive. Because light flashes can be applied rapidly and accurately, this approach allows greater temporal and spatial control . Our light-activated channels consist of a derivative of the small photoisomerizable molecule azobenzene (AZO), and a Shaker K+ channel. The AZO derivative has a cysteine-reactive maleimide (MAL) group on one end, allowing attachment to a specific cysteine in Shaker, and a pore-blocking tetraethylalnmonium (TEA) group on the other end. In its elongated trans form, the MAL-AZO-TEA molecule can reach the pore and block, but upon exposure to 360 nm light, the AZO photoisomerizes to its bent cis form, which is too short. Illumination with 420 nm light accelerates the reverse cis to trans conversion, restoring the blocked state. Hence illumination with different wavelengths extends or retracts the TEA group, blocking and unblocking the channel. To maximize the impact of the channel on neural activity, we will introduce mutations in the Shaker channel that eliminate inactivation and shift its voltage-dependent activation to hyperpolarized potentials, making the channel constitutively active in its unblocked state. We will first express the channel in Xenopus oocytes and characterize light-sensitivity. We will then introduce the gene encoding the channel into cultured mammalian neurons, apply the modified AZO, and use light to hyperpolarize and silence electrical activity. Finally, light-activated channels will be expressed in ganglion cells in intact retina. Appropriate illumination should alter action potential firing, even in retina that are lacking functional rods and cones. Light-activated channels provide an accurate and reversible way to regulate neural activity and open a new opto-bioelectronic interface for influencing the nervous system for experimental and therapeutic purposes.

DESCRIPTION (provided by applicant): The vision science community at UC Berkeley has a long and distinguished history, having contributed many seminal discoveries in the fields of visual system development, physiology, psychophysics, and pathology. UC Berkeley vision scientists come from many academic disciplines, increasing our understanding of vision at many different levels. Our group consists of 20 CORE investigators, ranging in expertise and interest from molecular mechanisms of synaptic function to human visual perception. We request continued support for our CORE grant to ensure further success in vision research through shared resources and services. We seek funding for four modules which will support current faculty and attract new faculty to investigate the visual system. The modules are:: (1) Gene Delivery (Xiaohua Gong & John Flannery, co-directors), designed to provide molecular biology expertise and support in the use of viral vectors for delivering genes into tissues of the visual system and for creating transgenic animal models of ocular disease. (2) Microscopic Imaging (Maria Feller, director), which will apply and develop advanced imaging methods for vision research - facilitating the use of shared-access microscopes on the Berkeley Campus and customizing microscopes in the labs of participating investigators. (3) Software Development (Marty Banks, director), which will provide custom software solutions for shared use by visual system investigators using psychophysical and physiological methods. (4) Translational Research, to promote translation of scientific knowledge into clinical applications by supporting design, data collection, and data analysis of experiments involving human subjects. To build on our current strengths, the UC Berkeley Depts. of Molecular & Cell Biology, Psychology, Optometry, and the Helen Wills Neuroscience Institute have all targeted vision research as a priority area for new faculty hiring. Further demonstrating their commitment, these administrative groups and the University Administration have committed to collectively provide resources equivalent to $800,000 over the next 5 years to support the Vision Science CORE.

[unreadable] DESCRIPTION (provided by applicant): Neurons possess ion channels that are directly activated by voltage, ligands, temperature, and mechanical forces, but not by light. Our goal in this project is to use a combination of organic chemistry and molecular biology to engineer new types of ion channels that can be directly regulated by light. We will then use these channels to answer questions focused on activity dependent synaptic plasticity in the retina that are unapproachable with presently available methods. Specifically, this project will make several major contributions. 1) It will add to the growing toolbox of light-activated channels that are of great utility for remote control of different aspects of neuronal activity. 2) It will elucidate whether retinal ganglion cells, like hippocampal neurons, exhibit homeostatic synaptic plasticity, revealing whether synapses in the retina are hard-wired or can change with use. 3) It will elucidate whether synaptic homeostasis operates locally within a portion of a dendritic tree, or only globally, across an entire neuron, providing clues as to the functional importance and mechanism of homeostatic plasticity in the hippocampus and retina. 4) It will elucidate the changes in the physiology of the retinal circuit that occur as a consequence of photoreceptor degeneration in mouse and rat models of retinitis pigmentosa. By elucidating the functional effects and time course of retinal remodeling, this study will provide information of key importance for evaluating and designing new therapeutic strategies that rely on intact synaptic signaling through the retina, including gene therapy for restoring photoreceptor function and stem cell therapy for regenerating rod or cones. PUBLIC HEALTH RELEVANCE: In this project we will engineer new molecules that will allow nerve cells to be turned on and off with light. We will use these tools to answer important questions about how synaptic connections between nerve cells change with activity and how signaling through the retina changes after rods and cones are lost during degenerative blinding diseases such as retinitis pigmentosa. This will provide fundamental information for understanding how the retina functions and adapts to different light conditions and will be useful for designing and evaluating future therapeutic strategies for restoring vision. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): One of the most exciting technical developments in biology in recent years is the emergence of photochemical methods for controlling electrical activity with light. Our goal in this project is to develop a broadly applicable method for controlling other aspects of cellular function by generating tools for conferring light sensitivity on a broad range of cell surface receptors and ion channels. To meet this challenge we will use a modular approach, utilizing a single Universal Photoswitch as the key light-sensing component. The photoswitch contains at its core the small isomerizable azobenzene moiety, which shortens and lengthens in response to 380 and 500 nm light, respectively. We will use this photoswitch to indirectly regulate receptor and channel activity, through an adapter peptide, which contains a capture domain, which recognizes the short, but not the long configuration of the photoswitch, and a ligand domain, which contains a peptide activator or inhibitor of the targeted cell surface receptor or ion channel. The capture domain is kept constant among all adapter peptides, allowing control by a single Universal Photoswitch, but the nature of ligand domain is tailored to regulate a specific receptor. Several strategies will be used to translate light-dependent capture of the adapter peptide into receptor activation or inhibition. These include dimerizing the adapter peptide with a dimeric photoswitch to activate growth factor receptors, and delivering the adapter peptide, including the ligand, to a G-protein coupled receptor via an antibody-tethered photoswitch. Other strategies may be developed to activate different types of receptors and ion channels. We will test the effectiveness of the Universal Photoswitch approach on two example receptors: the TrkB receptor for brain-derived growth factor (BDNF), a member of the neurotrophin family of receptor tyrosine kinases, and the receptor for neuropeptide Y, which is a GPCR-type receptor. Generating a method for light-sensitive regulation of these receptors will allow examination of their roles in development and neural function in intact tissue with unprecedented precision, but more importantly, it will demonstrate the emergence of a powerful new technique for receptor regulation that can be applied to any cell surface protein for which a known peptide ligand exists.

The primary purpose of the Translational Research module is to promote and facilitate inter-disciplinary collaborations among vision scientists, clinician scientists, and clinicians who do not have the means or expertise to conduct innovative, translational, patient-based clinical research. The UC Berkeley Clinical Research Center (CRC) housed in the School of Optometry was established in 2004. The CRC was created with the aim of supporting vision researchers, academic institutional partners, and private sector companies to explore new models and strategies for the prevention, diagnosis, and treatment of ocular anomalies. To accomplish this, the CRC established an infrastructure with a professional staff to conduct basic-science and translational research as well as patient-based clinical studies and trials. To date, the CRCs collaborations with basic science researchers have been successful but on a small scale, due primarily to funding constraints. Examples of these research activities include collaborations with principal investigators (PIs) within UC Berkeley campus on various vision-related research studies, including accommodation (Co-PI: Cliff Schor/Optometry) innovative non-thermal and non-chemical sterilization technique of contact lenses (Co-PI: Boris Rubinsky/Bioengineering); myopia (Co-PI: Chris Wildoset/Optometry); genetic influence on contact lens-induced adverse events (Co-PI: Lisa Barcellos/Public Health); environmental stress on tear film biophysical and biochemical properties (Co-PI: Randy Maddalena/LBNL); a novel OCT technique to examine retinal layers (Co-PI: Brandon Lujan/Optometry); Amblyopia (Co-PI: Dennis Levi/Optometry), and rheology of tear film (Co-Pl: Tatyana Svitova and Clayton Radke/Chemical Engineering).^* These projects are in progress with manuscripts in preparation. A Core Translational Research module would enable expansion of the CRC to support significantly larger scale (e.g., longitudinal clinical trials) multi-disciplinary, interdepartmental collaborations. This support would include clinical research study design, recruitment of research subjects and compliance with institutional review boards (IRB) overseeing human subject research, conduct of clinical studies and trials, statistical analysis, and collaborative reporting of study results. The resources ofthe CRC, under the direction ofthe module co-directors, would be available to basic science researchers in the greater vision science community (with priority given to CORE grant P.l.s), including professional and support staff.

Purpose: Software Development Module This module develops shared software tools for the CORE. It has 4 focus areas: eye tracking, displays, neuroimaging, and psychophysics/physiology. The areas were chosen to ensure that the tools will be useful to multiple CORE members and that the projects can be accomplished within the award period.

Purpose: Microscopic Imaging Module Vision scientists have used optical imaging to assay function at a variety of length scales - from subcellular processes to collections of cell organized into networks. With the advent of new fluorescent probes and imaging technologies, the possibilities of both measuring and manipulating cells and circuit function are more powerful than ever. Two notable breakthroughs in the past few years have revolutionized the ability of vision scientists to manipulate and probe excitable cells in intact tissue. First, the development of two-photon microscopy allows for the stimulation of fluorescent probes deep in tissue with a minimum amount of phototoxicity. Second, ion channels have been engineered to open and close in response to light, allowing for spatially localized stimulation and/or silencing of individual cells with millisecond time resolution. The vision science community at UC Berkeley is unique in that several of its members have been at the forefront of developing and utilizing these and other new optical technologies that are revolutionizing vision research. UC Berkeley is also fortunate in having several advanced microscopy systems on campus. In particular, the Molecular Imaging Center (MIC) in the LSA building has five confocal/2-photon imaging systems, a spinning disk confocal system with a spatial-light-modulator used for optical excitation, and soon-to-be installed PALM microscope for super-resolution imaging, all designated for shared use

Purpose: Gene Delivery Module Research aimed at understanding the development, physiology, and pathology ofthe visual system has been aided enormously by the advent of technologies for exogenously expressing foreign genes and inhibiting expression of endogenous genes. Over the past 5 years, the Gene Delivery Module (GDM) has played an important role in helping Berkeley Vision Science researchers employ these technologies, and has fostered collaborations among visual system labs. The Module two facilities in Barker and Minor Hall that have designed, prepared, and purified gene constructs specifically for somatic cell gene transfer to ocular tissues. It has also helped investigators create germline animal models of basic ocular processes and ocular disease.

Administrative Component The PI, Professor Richard H. Kramer, has a primary appointment in the Department of Molecular and Cell Biology. Professor Kramer has been an independent investigator since 1993, first at the University of Miami School of Medicine, and since 2000 at UC Berkeley. He has had continuous NIH funding since 1993 and continuous ROI funding from the NEI since 1998. As a member of the MCB Department who has also been active member of the UCBSO-based Vision Science Graduate Program since 2000, he is ideal for bridging the diverse fields of interest held by members ofthe Vision Science CORE. Dr. Kramer has served in several leadership positions, including Co-Director ofthe UC Berkeley Nanomedicine Development Center, Chair of the Admissions Committee for the UC Berkeley Biophysics Graduate Program, Chair of Faculty Search Committees, and Chair of the Scientific Advisory Board of the Max-Planck institute in Bonn, Germany. Professor Kramer is dedicated to directing the Vision Science CORE to provide modern resources and services to faculty participants in order to better serve their research needs. Dr. Kramer's research is focused on two Vision Science areas. First, his lab has pioneered functional imaging of light-elicited activity in the retina (with fluorescence indicator dyes or proteins) to better understand molecular mechanisms of synaptic transmission and to provide insights into synaptic information processing important for visual perception. His second research area is focused on the design and use of chemical photoswitch molecules that enable optical control of neuronal firing. In recent studies the Kramer lab has developed photoswitch molecules that can restore at least some visual function to blind mice lacking rods and cones. These responses can be measured optically (with functional imaging), electrophysiologically (with patch clamp or multi-electrode array recordings), and behaviorally (though in vivo tests on blind mice injected intravitreally with these compounds). Dr. Kramer's lab collaborates with the Feller, Werblin, Flannery, and Roorda labs, and all are extensive users of the Microscopic Imaging and Gene Delivery Modules. Dr. Kramer's role as CORE Director will be to oversee the Modules, participate in the hiring of technical experts working in the Modules (along with the Module Directors), chair the semi-annual Internal Review Committee Meetings (see policy # 2 below), and participate in the annual External Review Board meetings (policy # 6 below). Dr. Kramer will have the ultimate responsible for fair and equitable access to CORE resources and services and will have the ultimate responsible for budgetary decisions. Operation of the CORE grant will be supported by a 10% effort Administrative Assistant, who will compile information and assemble usage reports from each Module, and keep detailed records of budgets, expenditures, and balances. We have chosen highly accomplished faculty to be the directors of our 4 CORE Modules. Xiaohua Gong and John Flannery, co-directors of Gene Delivery are among the world¿s experts in molecular biology methods for eye research, with a particular emphasis on gene delivery. Maria Feller, director of Microscopic Imaging, did her PhD work in Physics with an emphasis in optics, and is he has maintained a strong interest in advanced microscopy approaches. She has excellent organizational skills and has run the Module quite successfully over the past 5 years. Marty Banks is an established researcher in computational visual neuroscience and a brilliant computer programmer, and has done a great job overseeing the Software Development over the past 5 years. Dennis Levi, is stepping down as the Dean of the UCBSO, and has the time and great desire to establish the new Translational Research Module. Meng Lin, the current director of the Clinical Research Center, will co direct this module. The pair are dedicating to encourage more people to directly translate their finding into clinical applications, starting with human subject studies facilitated by the Translational Research Module. All of the Module directors are putting great effort into the CORE, and all are committed to establishing an excellent group of Modules that will bring out the best in Vision Research at UC Berkeley. Policies of the UC Berkeley Vision Science CORE. To ensure fair and equitable access to CORE Modules by participating faculty, we have instituted a series of new general CORE policies. In addition, some of the individual Modules have instituted further use policies and governing procedures, and these can be found in the descriptive sections for each Module. The general CORE policies are as follows: 1. Access to resources and/or services generally will be on a first-come/first-served basis, but some restrictions apply. An investigator may only have two requests pending on the queue and jobs requiring more than three weeks for completion will be considered multiple requests. Special requests for multiple or extensive jobs may be made to the Module director or the Internal Review Committee (see below). 2. Investigator usage and Module expenditures will be evaluated by an Internal Review Committee, which will have regular meetings on a semi-annual basis, and additional ad hoc meetings, as necessary. The Internal Review Committee shall be composed ofthe Module directors, the PI, and a representative staff member from one ofthe Modules. The committee will evaluate semi-annual reports of usage by participating CORE faculty to ensure equitable access. The committee will consider special requests, including for large or especially time consuming projects, rush orders, and cases of financial hardship. 3. Module directors are responsible for promoting use of their Module by announcing new resources or services, and by publicizing openings in the job queue. Each Module has a website advertising available service and instructions about how to access these services. The Administrative Assistant will help the directors in this endeavor. Participating investigators who are moderate or extensive users of a module are encouraged to form a user group to discuss access, resources-, and services provided by the module and if desired, to propose changes to the Module Director and to the Internal Review Committee. 4. New investigators shall have priority access to Module resources and services. 5. Modules may charge an appropriate hourly fee for services to compensate for budget shortfalls. The fee structure for each module will be determined by the Internal Review Committee. CORE users shall be notified of the applicable recharge rate before work on a job has begun. 6. An External Review Board of three prominent local, but non-UC Berkeley, Vision Scientists will meet annually to evaluate the performance of the Modules, to assess the fairness of CORE faculty access, and to evaluate the CORE budget. This meeting will coincide with the Bay Area Vision Research Day (BAVRD), held in late August. The following investigators have already agreed to serve on the External Review Board for the next funding period (see enclosed letters in Appendix B): Dr. Marie Burns, UC Davis Ophthalmology & Vision Science Dr. Jonathan C. Horton, UCSF Ophthalmology ) Dr. Todd Margolis, UCSF Ophthalmology

? DESCRIPTION (provided by applicant): Optogenetics has revolutionized neuroscience by making it possible to use heterologously expressed light-gated ion channels and pumps to stimulate or inhibit action potential firing of genetically selected neurons in order to define ther roles in brain circuits and behavior. Since the flow of information through neural circuits depends on synaptic transmission between cells, an important next technological step is to bring optogenetic control to the neurotransmitter receptors of the synapse. The Optogenetic Pharmacology that we propose makes this possible. In this approach genetically-engineered neurotransmitter receptor channels and G protein coupled receptors (GCPRs) from synapse are derivatized with synthetic Photoswitched Tethered Ligands (PTLs) and thereby made controllable by light. Our goal is to develop this new technology to gain optical control over synaptic transmission and plasticity in the living brain for studies of neural circuits and behavio. We focus on the two fundamental synapses of the brain: the excitatory glutamatergic synapse and inhibitory GABAergic synapse. An initial series of light-regulated glutamate and GABA receptors has already been made. This series will be optimized for in vivo use and expanded to obtain comprehensive control of these synapses. The receptors are minimally-modified, with a single point mutation enabling PTL attachment. Thus they retain their normal ability to respond to neurotransmitters. However, they can be blocked to prevent normal synaptic transmission or the induction of certain forms of plasticity, or they can be activated to mimic transmission or trigger plasticity changes, with cell and subtype specificity as well as high spatial and temporal precision. The receptors integrate into synapses, and control can be exerted across broad spatial scales, from individual pre- or postsynaptic terminals, to one or more dendritic branches, to individual or groups of cells, to entire brain regions. New methods for genetic manipulation allow the modified receptors to be genomically substituted for their wild-type counterparts, exactly replicating the number and distribution of endogenous receptors in the brain. Optogenetic Pharmacology provides a powerful approach for understanding brain circuits and behavior in health and disease.

? DESCRIPTION (provided by applicant): Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are blinding diseases caused by the degeneration of rods and cones, leaving the rest of the visual system intact but unable to respond to light. A synthetic chemical photoswitch, named DENAQ, can restore visual responses in blind mouse models of RP. Previous studies showed that DENAQ imparts light-sensitivity on action potential firing in retinal ganglion cells (RGC), but how this occurs is unclear. The goal of this project is to elucidate the mechanism of DENAQ photosensitization, crucial for enabling discovery of improved drug candidates and for optimizing photo- stimulation strategies for vision restoration. The first aim i to understand why DENAQ selectively photosensitizes retinas from mice with dead rods and cones while having no effect on healthy retinas with intact rods and cones. We will test the hypothesis that degeneration leads to enhanced entry of DENAQ into RGCs and/or enhanced action on ion channels underlying spontaneous firing in RGCs. The second aim is to identify which RGCs are photosensitized by DENAQ. In the healthy retina, some RGCs fire at light onset, some at offset, and some at onset and offset. Studies will determine which are photosensitized by DENAQ, and whether local degeneration of rods and cones leads to spatially constrained RGC photosensitization, of particular relevance for AMD, a localized degenerative disease. Other studies will reveal whether DENAQ photosensitization applies to human RGCs in tissue samples obtained during surgical retinectomy. The third aim is to exploit our findings to optimize vision restoration. Information about the ion channels targeted by DENAQ will enable development of more specific photoswitches. Subcellular localization of these channels in RGCs will enable more spatially-precise photo-control. Finally imaging studies in vivo will reveal signals transmitted from the DENAQ-treated retina to the brain of blind mice, validating the functional integrity of the visual system and providing a platform for optimizing retinal stimulatin patterns to best recapitulate normal visual responses.

? DESCRIPTION (provided by applicant): Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are blinding diseases caused by the degeneration of rods and cones, leaving the rest of the visual system intact but unable to respond to light. A synthetic chemical photoswitch, named DENAQ, can restore visual responses in blind mouse models of RP. Previous studies showed that DENAQ imparts light-sensitivity on action potential firing in retinal ganglion cells (RGC), but how this occurs is unclear. The goal of this project is to elucidate the mechanism of DENAQ photosensitization, crucial for enabling discovery of improved drug candidates and for optimizing photo- stimulation strategies for vision restoration. The first aim i to understand why DENAQ selectively photosensitizes retinas from mice with dead rods and cones while having no effect on healthy retinas with intact rods and cones. We will test the hypothesis that degeneration leads to enhanced entry of DENAQ into RGCs and/or enhanced action on ion channels underlying spontaneous firing in RGCs. The second aim is to identify which RGCs are photosensitized by DENAQ. In the healthy retina, some RGCs fire at light onset, some at offset, and some at onset and offset. Studies will determine which are photosensitized by DENAQ, and whether local degeneration of rods and cones leads to spatially constrained RGC photosensitization, of particular relevance for AMD, a localized degenerative disease. Other studies will reveal whether DENAQ photosensitization applies to human RGCs in tissue samples obtained during surgical retinectomy. The third aim is to exploit our findings to optimize vision restoration. Information about the ion channels targeted by DENAQ will enable development of more specific photoswitches. Subcellular localization of these channels in RGCs will enable more spatially-precise photo-control. Finally imaging studies in vivo will reveal signals transmitted from the DENAQ-treated retina to the brain of blind mice, validating the functional integrity of the visual system and providing a platform for optimizing retinal stimulatin patterns to best recapitulate normal visual responses.

Understanding the human brain remains one of the great challenges of modern science. The scope of disciplines required to understand brain structure and function - chemistry, molecular biology, structural biology, biophysics, electrical engineering, computational science, cognitive science and psychology - to say nothing of the fields of inquiry and exploration that are influenced by this understanding, such as religion, art, music, philosophy, sociology and literature, is far-reaching. The sheer scale of the cells contained in the human brain, in contemplating the vast number of neurons, some 80 billion, and the hundreds to thousands of connections that each neuron forms with other neurons, along with the additional 80 billion non-neuronal support cells, makes for a daunting parts list to catalog. And yet, beyond just a static picture of the arrangement of these various cells into ensembles and networks, the dynamic information flow between these cells, the electrical and chemical impulses that underpin the very essence of human existence - sensation, thought, emotion, cognition - represent not just an additional layer of complexity, but, at its core, a deep mystery to be unraveled and explored. To push back at this frontier requires new thoughts, new tools, new techniques, and new interpretations that will almost certainly come from teams of scientists working across disciplines to bring new approaches that are more than the sum of their parts. This project will develop and apply new methods for non-invasively measuring electrical signals underlying brain cell communication.

This award establishes a NeuroNex Innovation Project at the University of California, Berkeley, which will develop chemical-genetic methods to measure neuronal activity in a non-invasive, high-throughput, high-fidelity manner across multiple length scales, at high speed, and in multiple species with molecular precision. The team will optically read-out neuronal activity by directly imaging changes in membrane voltage with bright, sensitive, chemically-synthesized voltage-sensitive fluorophores. The voltage-sensitive fluorophore make use of photoinduced electron transfer (PeT) as a voltage-sensing trigger to provide fast, sensitive, non-disruptive optical recordings in neurons. In this project, pairing of PeT-based voltage-sensitive dyes with genetic targeting methods to enable optical voltage sensing with sub-cellular and sub-millisecond resolution in intact animal brains will be conducted. This NeuroNex Innovation Award is part of the BRAIN Initiative and NSF's Understanding the Brain activities.

GABA and glutamate are the main inhibitory and excitatory neurotransmitters in the brain. Activity-dependent redistribution of glutamate receptors contributes to excitatory long-term synaptic plasticity, learning and memory. A similar redistribution of GABAA receptors (GABAARs) has been shown to occur in neuronal culture, but the functional significance of this process in intact brain circuits remains unclear. Our goal is to better understand the role that GABAA receptors play in synaptic plasticity in the hippocampus, a brain region crucial for learning and memory. We have developed photoswitch chemicals that allow light to reversibly block GABAA receptors with high spatial and temporal precision and absolute specificity for a specified mutant ?-subunit. Here we focus on receptors containing either the broadly- expressed ?1-subunit, or the hippocampus-enriched ?5-subunit. To enable photo-control of endogenous receptors, we have developed knock-in mice with photoswitch-ready version of ?1 or ?5 replacing their wild-type counterparts. Using these mice and novel intrabody probes that light-up or disrupt GABAARs scaffolds, we will evaluate where ?1- and ?5-GABAARs are in neurons, how they contribute to synaptic function, and what role they play in synaptic plasticity. Aim 1 is to map the distribution of the two GABAA types in hippocampal neurons. This includes determining whether they are expressed in proximal to distal dendritic gradients, determining whether they are aggregated at synapses or dispersed extrasynapatically, and determining their individual contributions to inhibitory synaptic transmission from different identified inhibitory interneurons. Aim 2 is to investigate how neuronal activity alters the distribution of GABAARs. This includes using the photoswitch as a stable tag to evaluate whether activity alters the lifespan and lateral mobility of the receptors on the plasma membrane. Aim 3 is to investigate how GABAARs alter excitatory long-term synaptic plasticity. This includes investigating the impact of the GABAARs on induction of long-term potentiation and depression and NMDA receptor signaling. The knowledge gained from this work will fill a gaping hole in our understanding of brain function and may reveal new treatment strategies for memory disorders and other neurological diseases. !

ABSTRACT This proposal requests funds to support ?The Optical Revolution in Physiology: From Membrane to Brain?, the 71st Annual Symposium of the Society of General Physiologists (SGP). The meeting will be held on September 6 ? 10, 2017, at the Marine Biological Laboratory (MBL) in Woods Hole, MA. The SGP annual symposium has an established reputation as a world-class meeting for physiologists, cell biologists, and neuroscientists, spanning across all career stages and professional arenas. Each year the meeting topic is unique, chosen to highlight emerging fields. The 2017 conference will bring together ~175 scientists who are developers and users of new optical tools for understanding biological function, both in normal physiology and in disease. Optical methods are having an increasingly huge impact on physiology, particularly pertaining to the nervous system. From the invention of new probes for visualizing and manipulating cell physiology, to the invention of new types of microscopy, the optical revolution is transforming physiological study at every level of complexity, from membranes to cells to intact organisms. The significance of the optical revolution is clear: today?s breakthrough optical technologies will turn into tomorrow?s cutting-edge tools for diagnosing and treating human disease. This includes the possibility of optical high-throughput drug screening on cells from individual patients with rare diseases. One major theme for this meeting is the focus optical imaging at an increasingly fine resolution. Nobel Laureate Eric Betzig (HHMI, Janelia Farms) will deliver the ?Friends of Physiology' keynote lecture, highlighting his invention of super-resolution microscopy, a technology that allows visualization of molecules in cells at a resolution higher than the diffraction limit of light. SGP symposia are large enough to cover a range of speakers, while being small enough to maximize individual discussions and foster collaborative interactions between students, postdoctoral fellows, new investigators, and established leaders. The organizers, Dr. Richard H Kramer (UC Berkeley) and Edwin Levitan (U Pitt.), are pioneers of optical techniques in physiology. They have assembled a star-studded group of 21 platform speakers, whose presentations focus on 4 aspects of the optical revolution: (1) new optical technologies for imaging and manipulating individual protein molecules, (2) new optical probes for imaging and manipulating signal transduction and membrane trafficking, (3) new optochemical and optogenetic tools for sensing and manipulating electrical activity, and (4) implementation of optical approaches for in vivo imaging and manipulation. The Society of General Physiologists provides excellent administrative support to ensure the success of the meeting. The overarching goal of the meeting is to inspire young investigators and to communicate new and significant ideas essential to the advancement of human health and treatment of disease.

OVERALL ABSTRACT The vision science community at UC Berkeley has a long and distinguished history, having contributed manyseminal discoveries in the fields of visual system development, physiology, psychophysics, and pathology over the past 50 years. UC Berkeley vision scientists come from diverse academic disciplines, increasing our understanding of vision at many different levels. Our group consists of 18 CORE investigators and more than 20 other labs studying vision, ranging in interests from molecular mechanisms of retinal physiology and pathology to human visual perception. We request continued support for our CORE grant to ensure further success in vision research through shared resources and services. We seek funding for three modules which will support current faculty and attract new faculty to investigate the visual system. The modules are: (1) Gene Delivery (Xiaohua Gong & John Flannery, co-directors), designed to provide molecular biology expertise and support in the use of viral vectors for delivering genes into tissues of the visual system and for creating transgenic animal models of ocular disease. (2) Microscopic Imaging (Maria Feller and Austin Roorda, co-directors), which will apply and develop advanced imaging methods for visualizing cells in both animal and human eyes ? designing, building, and facilitating the use of customized microscopes in individual labs and the Microscopic Imaging Center, and (3) Software Development (Marty Banks & Michael Silver, co- directors), which will provide custom software solutions for shared use by visual system investigators using psychophysical and physiological methods. UC Berkeley has demonstrated its strong commitment with 5 new faculty whose research focus is on vision, with 5 more Vision Science hires planned for the next several years. The UC Berkeley central administration, and the academic centers for Vision Science on campus (Departments of Molecular & Cell Biology, Optometry, Psychology, and the Helen Wills Neuroscience Institute) have all demonstrated their continued dedication to Vision Science by committing resources equivalent to $400,000 over the next 5 years to support the Vision Science CORE.

OVERALL ABSTRACT The vision science community at UC Berkeley has a long and distinguished history, having contributed manyseminal discoveries in the fields of visual system development, physiology, psychophysics, and pathology over the past 50 years. UC Berkeley vision scientists come from diverse academic disciplines, increasing our understanding of vision at many different levels. Our group consists of 18 CORE investigators and more than 20 other labs studying vision, ranging in interests from molecular mechanisms of retinal physiology and pathology to human visual perception. We request continued support for our CORE grant to ensure further success in vision research through shared resources and services. We seek funding for three modules which will support current faculty and attract new faculty to investigate the visual system. The modules are: (1) Gene Delivery (Xiaohua Gong & John Flannery, co-directors), designed to provide molecular biology expertise and support in the use of viral vectors for delivering genes into tissues of the visual system and for creating transgenic animal models of ocular disease. (2) Microscopic Imaging (Maria Feller and Austin Roorda, co-directors), which will apply and develop advanced imaging methods for visualizing cells in both animal and human eyes ? designing, building, and facilitating the use of customized microscopes in individual labs and the Microscopic Imaging Center, and (3) Software Development (Marty Banks & Michael Silver, co- directors), which will provide custom software solutions for shared use by visual system investigators using psychophysical and physiological methods. UC Berkeley has demonstrated its strong commitment with 5 new faculty whose research focus is on vision, with 5 more Vision Science hires planned for the next several years. The UC Berkeley central administration, and the academic centers for Vision Science on campus (Departments of Molecular & Cell Biology, Optometry, Psychology, and the Helen Wills Neuroscience Institute) have all demonstrated their continued dedication to Vision Science by committing resources equivalent to $400,000 over the next 5 years to support the Vision Science CORE.

ABSTRACT Light responses are initiated in rod and cone photoreceptors, processed by retinal interneurons, and synaptically transmitted to retinal ganglion cells (RGCs), which send information, in the form of spike trains, to the brain. In degenerative retinal disorders, including Age-related Macular Degeneration (AMD) and Retinitis Pigmentosa (RP), the photoreceptors gradually die off, depriving downstream neurons of light-sensitive input. However, recent evidence suggests that losing photoreceptors is only part of the problem in these disorders. Downstream retinal neurons become hyperactive, with retinal ganglion cells (RGCs) firing spontaneously in darkness at up to 10 times faster than in healthy retina, corrupting the proper encoding of visual information. We recently reported that retinoic acid (RA), a small molecule that activates gene transcription, is the signal that triggers RGC hyperactivity. Blocking the receptor for RA in vivo can reverse hyperactivity, unmasking light responses that would otherwise be obscured by spontaneous RGC firing. Blocking RA receptors in the retina also augments the contrast-sensitivity of learned visual behaviors in a mouse model of RP. Our goal in this project is to assess whether drugs or gene therapies that inhibit RA signaling can improve vision in mouse models of RP, with the hope of extending useful vision for years in humans with degenerative retinal disorders. First, we will ask whether inhibiting RA signaling not only improves light-sensitivity, but actually improves conscious visual function in vision-impaired mice, assessed with behavioral tests of contrast sensitivity and spatial frequency threshold. We will determine how when during the degeneration process RA inhibitors are most effective, revealing the optimal time for beginning treatment. Second, we will investigate retinal neurons that lie upstream of RGCs, namely bipolar cells and amacrine cells. We will ask whether pathophysiological changes in these presynaptic neurons are also induced by elevated RA signaling and whether inhibiting RAR can reverse these changes, providing critical information for effective cellular targeting of gene therapy. Third, we will test whether vision can be improved by inhibiting the enzyme that synthesizes RA, with a re-purposed drug that is already FDA-approved for other indications, paving the way for human clinical studies. Taken together, this project will establish the proof-of-principle behind a new treatment paradigm for augmenting vision in retinal degenerative disorders.