Ehud Y. Isacoff - US grants

DISCUSSION (Adapted from the investigator's application): A change in voltage across the plasma membrane represents the fundamental neural signal. Active propagation of this signal, and its coupling to synaptic transmission, depends on a superfamily of ion channels which open their conduction pathway in response to changes in membrane voltage. How a voltage change causes conformational rearrangements in these channels that open them is not understood. To address this question, the investigators have developed a novel method that reports conformational changes in real-time. This method uses fluorescent thiol reagents to tag cysteine-substituted channels in a site-specific manner, and combines measurement of fluorescent emission with voltage-clamping. In this way they can identify the gating transitions that correlate with the movement of the tagged protein segment. The investigators propose to use this and other methods to identify the structural components of gating in the voltage-gated Shaker potassium channel. They outline a plan for determining how gating elements interact, within and between subunits, and ask how such interactions act to stabilize gating conformations, to couple voltage-sensing to gating, and to confer cooperativity on certain conformational rearrangements. Abnormal function of members of the voltage-gated channel superfamily has been implicated in a variety of human diseases. Information obtained in the proposed project will help to elucidate the structural basis of gating for the entire superfamily of channels. This information will be valuable for the design of specific chemical and gene therapies to treat diseases which are caused by channel defects, or which are arneliorated by modulation of specific channel types.

DESCRIPTION (provided by applicant): Voltage-sensing domains (VSDs) confer voltage dependence onto the effector domains of membrane proteins. In the classical Na+, K+ and Ca++ channels that generate the action potential and control neural secretion and muscle contraction, four VSDs work in concert to control gates in a pore located at the interface between the pore domains of the four subunits. During the previous grant cycle we discovered that the situation is different in two recently isolated proteins: the sea squirt voltage-sensitive phosphatase, Ci-VSP and the human voltage- gated proton channel, Hv1. Both proteins have homologues across phyla, including in vertebrates. The VSPs provide a previously missing connection between membrane excitation and ion transport by PI(4,5)P2-sensitive channels and pumps, while the Hv channels play an important role in phagocytosis. We developed a new method of single molecule microscopy that enabled us to count the subunits of these proteins and combined these with electrophysiology to count pores and voltage clamp fluorometry to detect structural dynamics. We found that the two new members of the family break the tetrameric mold, with Ci-VSP being a monomer and Hv1 a dimer. In Hv1 we have shown that each subunit of the dimer has its own pore, voltage sensor and gate and we find now that, although the subunits can function on their own, they gate cooperatively in the dimer. We propose to determine how a single ion channel domain can combine sensor and effector functions that are separate in other channels and to understand the structural basis of the cooperativity. The work will provide insight into how a modular domain like a VSD has been shuffled around in evolution to confer voltage dependence onto a variety of membrane-associated proteins.

DESCRIPTION (Adapted from applicant's abstract): Two mechanisms (one activity-dependent, and the other activity-independent) are thought to regulate synaptic size and strength, and to function in parallel to expand the nerve terminal and its output to match the size of its target. Both mechanisms are proposed to depend on back and forth signaling between the pre- and postsynaptic cells. This study aims to identify new proteins that control synaptic growth, and to ask fundamental questions about the relationship between the structural and functional development of the synapse: How does the postsynaptic cell signal its size and activity to the presynaptic cell? By what means does the presynaptic cell respond? What is the relation between the growth of new synaptic release sites and the establishment of their function? Does development include the long-term modulation of transmission efficacy at release sites, as has been proposed to explain plasticity in adult? These studies will focus on a model genetic system, the glutamatergic neuromuscular synapse in the fruitfully Drosophila. The compelling reasons to use this system are the ability to conduct mutant screens, the ease of making transgenic animals, and the ready accessibility of the organism at many developmental stages to microscopic and physiological study. We will develop new protein-based optical reporters that will to visualize synaptic morphology, synaptic activity and the assembly of the protein signaling of the synapse. Transgenic methods will be used to generate stable lines of animals expressing these optical reporters in appropriate cells. These reporters will be used to follow synapse development with non-invasive time-lapse imaging, enabling us to elucidate the relationship between synapse formation and functional signaling in wild- type animals, as well as in mutants of synapse formation. The reporter expressing animals will also be used in large-scale mutant screens for new genes that control synaptic growth. The genes identified in the screen will be cloned, molecularly described, and placed into pre- and postsynaptic molecular signaling pathways. Synaptic growth genes discovered in Drosophila are likely to be highly conserved, allowing us to discover the I fundamental molecular machinery of synaptic growth in the human brain.

9987623
Daniel Rokhsar - University of California at Berkeley
IGERT: Physical Biosciences: From Molecular Machines to Neural Imaging

This Integrative Graduate Education and Research Training (IGERT) award supports the establishment of a multidisciplinary graduate training program of education and research on the development and application of physical and computational methods for the study of biological problems at the molecular, cellular, and systems levels. The program is a joint effort of 31 faculty and research scientists drawn from seven Departments at the University of California at Berkeley and three Divisions at the neighboring Lawrence Berkeley National Laboratory. It will transcend traditional academic boundaries to produce the next generation of physical bioscientists, equally conversant with physical and biological methods and problems. Research thrust areas include biomolecular structure, dynamics, and design, and cellular signaling networks and systems neuroscience, with an emphasis on the development and application of novel molecular microscopy and detection devices and theoretical and computational modeling approaches. Students enrolled in any of nine existing Ph.D. programs will participate in personalized training, including new courses on single-molecule methods, bioinformatics, molecular biophysics, and hand-on laboratory courses in physical bioscience. Research and career placement seminars, retreats, summer internships and intensive courses, and a dual mentoring program will provide a rich environment for multidisciplinary training.

IGERT is an NSF-wide program intended to meet the challenges of educating Ph.D. scientists and engineers with the multidisciplinary backgrounds and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing new, innovative models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries. In the third year of the program, awards are being made to nineteen institutions for programs that collectively span all areas of science and engineering supported by NSF. The intellectual foci of this specific award reside in the Directorates for Biological Sciences, Mathematical and Physical Sciences, Engineering, and Education and Human Resources.

A major research instrumentation grant has been awarded to Dr. Ehud Isacoff at the University of California at Berkeley for the purchase of a 2-photon scanning microscope. The research goal is to probe, non-invasively, by microscopic photometry and imaging, the distribution and structure of macromolecules (RNA, DNA and proteins), and their activity and interactions in living cells. The 2-photon microscope makes it possible to image cells and tissues with high spatial and reasonable temporal resolution, with penetrating excitation, but also with optically confined irradiation, thus limiting photo-damage to cells and photo-destruction of fluorescent tags. The instrument will form a centerpiece of a recently built Berkeley Imaging Center. The Imaging Center will provide access, training and technical support for use of the 2-photon microscope for students, postdocs and faculty in the biological sciences on campus and in the Lawrence Berkeley National Laboratory, and will make the instrument available to academic researchers at other Bay Area campuses.

The research projects conducted with the 2-photon microscope will include; 1) studies on synaptic proteins that have been engineered to be fluorescent and to change their fluorescence upon either activation or interaction with other proteins, 2) optical probes of neural activity to measure the function of neural circuits involved in visual information processing, 3) organic and protein-based indicator dyes to study the roles of calcium ions in synaptic transmission, short- and long-term plasticity, 4) the regulation of other second messenger systems and, 5) fluorescently labeled proteins to study the localization of postsynaptic receptors, and the changes in proteins involved in exocytosis during synaptic transmission.

The aim of the research using the 2-photon microscope is to employ innovative forms of microscopy that can characterize the dynamics of molecular machines in their physiological environments: inside intact cells and tissues.

We propose to create a Berkeley Nanomedicine Development Center in Membrane Signaling. The research focus of the Center will be on the biomolecular dynamics of signaling proteins and the operation of signaling networks. Our goal is to elucidate the mechanism of function of receptors in the plasma membrane, which sense signals in the external environment, and of the web of intracellular enzymes to which the membrane receptors relay their signals. Cellular signaling is fundamental to processes as varied as chemical sensation, information processing in the nervous and immune systems, and gene regulation. G-protein coupled receptors and protein kinases, on which much of the effort will focus, represent two of the largest protein families in the human genome. We propose a novel approach to the study of signaling, which we think will revolutionize how cell biology is done and how the function of individual proteins is assessed and controlled in tissues. This is an area of great significance for medicine because of the importance of receptors and kinases as therapeutic targets and because of the seriousness of illness caused by their mutation. Several innovative approaches developed at Berkeley will be deployed. This provide for unprecendented precision in the ability to monitor and manipulate the function of signaling proteins in their natural membrane and cell environments and in the context of complex signaling networks.

[unreadable] DESCRIPTION (provided by applicant): The pattern of activity in the circuits of the brain and their experience-dependent changes underlie the processing of sensory information, perception, and memory. Much has been learned about the anatomical wiring of brain circuits and about the receptive field properties of individual neurons in the intact brain, but considerable mystery remains about how the properties of individual neurons emerge from their connectivity and how their activity is assembled into coherent percepts. Part of the problem has been that high precision recording is usually obtained from only one or a few neurons at a time, when salient events are actually processed by large assemblies of neurons. The method with the best resolution in time and space currently for population recording is the optical recording of neural activity with voltage-sensitive chemical dyes. However, these optical reports are blurred by their lack of targeting to specific cell types or subcellular compartments. Glial signals are confounded with ones from neurons, distinct types of neurons contribute indistinguishably to the signal, and the neuronal component of the signal is dominated by the dendrites, which represent the major membrane area. What has been missing is a method of targeting these optical reporters. The goal of this grant is to create genetically-encoded optical reporters of neural activity to fill this gap. The advantage of this approach is that the reporters can be specifically expressed in desired cell types through the use of cell-type-specific promoters, and they can be targeted to specific subcellular compartments. Our emphasis is to develop reporters that reveal action potential firing, synaptic activity, second messenger signaling, and changes in the molecular assembly of presynaptic active zones and postsynaptic densities. These reporters will be expressed in cultured cells to optimize their properties and then delivered transgenically and via viruses to model systems (the Drosophila larval neuro-muscular junction, and the mouse and rat barrel cortex) in which they will be tested in vitro and in vivo. [unreadable] [unreadable]

Our overall goals are to develop new optogenetic tools, which sensitize to light the activity of signaling proteins and the cells in which they are expressed. We will employ these remote controls for basic research into understanding and manipulating the mast cell secretion in response to antigens and allergens (which trigger the inflammatory response), fluid absorption across the retinal pigment epithelium (which plays a role in cystoid macular edema), as well as efforts to re-engineer neurons for cell replacement therapy for models of CNS neurodegeneration. We have already made significant progress in two other directions, which represent our major preclinical work, namely efforts toward: a) the treatment of pain and the analysis of pain circuits, and b) the restoration of vision in retinal pathologies that lead to loss of photoreceptors and blindness. Our approach is to develop molecularly focused methods for dynamic manipulation of specific proteins in the complex environment of cells, which can be used in intact tissues, and, indeed, in the live animal. The logic is to use light as both input and output to probe and control protein function in cells. While there has been significant progress in optical detection of protein function over the last 2 decades, remote control has become only possible recently, partly from the efforts of our NDC. The NDC for the Optical Control of Biological Function has spent the first funding period developing methods for using light to rapidly switch on and off the function of select proteins in cells. We have demonstrated that the strategies are broadly applicable across protein classes, including ion channels, G-protein coupled receptors and enzymes: three of the largest families of signaling proteins in cells and major drug targets for the development of new pharmaceuticals.

This Major Research Instrumentation award funds the acquisition of a "High Resolution Optogenetic Microscope," incorporating a confocal microscope and a unique optical stimulation method which will greatly further optogenetic research, as well as other areas of research requiring highly sensitive confocal imaging. While there has been significant progress in the optical detection of protein function, remote optical control has become possible only recently with the emergence of the field of "optogenetics," which employs native or engineered light-sensitive proteins. The ability to combine well-controlled patterned light and sensitive confocal imaging greatly enhances efforts to understand neural circuit through improved spatiotemporal manipulation of protein function. The High Resolution Optogenetic Microscope will also be a large boon to the Molecular Imaging Center (MIC), which serves over 40 research groups on campus and at Lawrence Berkeley National Laboratory. The MIC organizes a yearly international workshop which focuses on new imaging techniques and applications, bringing together talented engineers, students, and researchers to exchange ideas and information. The MIC also conducts outreach programs with regional community colleges. The results of these research and teaching efforts will be broadly disseminated through abstracts and peer reviewed publications, as well as by active participation of students and faculty at professional meetings.

DESCRIPTION (provided by applicant): A major challenge for the advance of biology and medicine is to develop new ways of studying how proteins function in complex networks in cells and how cellular circuits operate to coordinate functions of the organism. Progress here requires molecularly focused methods for dynamic detection and manipulation that can be used in the living organism. An attractive approach is to use light as both input and output to probe signaling proteins in vivo. The UC Berkeley Nanomedicine Development Center for the Optical Control of Biological Function has been at the forefront of re-engineering proteins to be sensitive to light so that they can be rapidly switched on and off in select cells in vivo. We have developed light-gated ion channels and receptors and used these in cultured neurons, retina, and in vivo zebrafish and in the rodent eye. The development of optically controlled proteins opens the door for applications in medical research, including understanding the function and development of neural circuits, and their relation to behavior, and to re-engineering native cells to switch their state in response to light-a control that could be useful in cell replacement therapies and which could help restore vision in animal models of certain blinding diseases. For applications in both cell culture and in vivo, we require the ability to pinpoint optical stimulation in a microscope that can image fluorescent indicators of neural activity at high resolution. The optical stimulation needs to be spatially and temporally flexible, but at the same time well-focused in 3D and of sufficient intensity to enable us to mimic physiological patterns of activity in specific neurons, in specific regions of the brain, in some cases during behavioral assays. We request funds for an Optical Stimulation Microscope, which combines the high resolution high quality imaging of the Olympus FV1000 microscope with a novel approach to optical stimulation that uses LCOS-SLMs for 1P and 2P to achieve illumination that is shaped in 3D and which can stimulate structures as small as a piece of a dendrite, as large as a whole cell or nearby group of cells and ranging up to cells that are dispersed in the field of view. This unique Optical Stimulation Microscope will fill a void at UC Berkeley. The Optical Stimulation Microscope will enable us to study the development and function of neural circuits, the integration into the adult brain of transplanted neurons for cell replacement therapy and the restoration of light sensitivity to retinas that have lost their photoreceptor cells in attempts to restore vision to models of blindness. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 1 Continuation Format Page PUBLIC HEALTH RELEVANCE: A major challenge for the advance of biology and medicine is to develop new ways of probing proteins in intact cellular circuits to learn how they operate and to repair their function. We have re-engineered proteins to be sensitive to light so that they can be remote-controlled and request funding for a unique Optical Stimulation Microscope that can point light to selectively manipulate the activity of select neurons in the living organism. The Optical Stimulation Microscope will enable us to study the development and function of neural circuits, the integration into the adult brain of transplanted neurons for cell replacement therapy and the restoration of light sensitivity to retinas that have lost their photoreceptor cells in attempts to restore vision to models of blindness.

? 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.

The connectivity of brain circuits is central to how sensory information is processed, motor activity coordinated, memories stored and retrieved, and how behaviors emerge. While new physical connections can form and old ones be eliminated, much of brain processing depends on how synaptic inputs are integrated and how their strengths are dynamically adjusted. To understand neural connectivity one must develop the means to directly manipulate synaptic transmission by controlling the neurotransmitter-gated receptors of synapses. We propose to do this by engineering optical control into neurotransmitter-gated G-protein coupled receptors (GPCRs). These light-controlled receptors can answer questions at every scale of analysis of the nervous system, from elucidating the contribution of a specific subtype of a neurotransmitter receptor to transmission and plasticity, to the events in a single dendritic spine, to understanding the role of pre- and postsynaptic receptors in neural computations, to understanding the synaptic basis of circuit operations and the role of those circuits in behavior and sensation. An additional and critical part of the project is tool dissemination and web-based tutorials, which will be a high priority for the researchers. Training will be provided to the broader scientific community during the annual Berkeley Advanced Imaging and Microscopy workshop. The proposal involves an international collaboration in research and training with Israel, which will expose students from the US and Israel to the expertise of the labs in the collaborating countries.

GPCRs form the largest class of membrane signaling proteins. They respond to a wide-array of stimuli. The roles of many GPCRs in neural circuits and behavior are not well-understood. Part of the problem is that the same GPCR may be found on presynaptic excitatory and inhibitory nerve terminals, postsynaptic dendritic spines and on associated glial processes. Another is that even though multiple GPCRs in a cell may couple to the same G-protein they often activate distinct targets due to molecular interactions that localize them to specific protein complexes. Thus, to determine the function of a GPCR one needs tools for that are subtype and cell-type specific, that are spatially precise, and that are rapid and reversible. In this project, individual full-length GPCRs that can be activated or blocked by light will be developed. Specifically, light-gated versions of the group I metabotropic glutamate receptors (mGluRs) will be engineered and then used to study synaptic plasticity.

? DESCRIPTION (provided by applicant): Fundamental to understanding brain function is the ability to relate the spatio-temporal firing patterns of specific neurons to their functional connectivity and determine the strength and experience-dependent regulation of those connections. Genetically-encoded optical indicators have revolutionized both endeavors, enabling action potentials and synaptic transmission to be detected, but these approaches face two major hurdles: 1) overly dense expression often makes it impossible to trace the morphology and hence connectivity of specific neurons, and 2) there has been no method to measure the probability of synaptic transmission (Pr) and quantal size of synapses in vivo, leading synaptic strength connections and the mechanism of experience- dependent change unresolved. The first problem emerges from a lack of ability to target activity indicators to specific cells that are few enough in number so that their morphology and physical connectivity could be determined in a densely packed environment, to then permit the physical picture to be related to activity and connectivity. To meet this challenge, we propose a generalizable strategy for the creation of turn-on genetically-encoded activity indicators. These indicators are rationally modified from some of the best existing indicators of neural activity and synaptic transmission. The re-engineering enables the indicators to be activated by light to provide a Golgi-like view of cell morphology and report on action potentials and synaptic input. Because the indicators are turned on by light (unlike in the random labeling methods like the Golgi stain), one can select specific target cells and functionally image densely packed cells whose processes heavily overlap while knowing which process belongs to which cell, thereby permitting a simple form of elegant connectivity mapping. The second problem emerges from the lack of a method for synapse-specific quantal analysis. Large-scale quantal resolution imaging of synaptic responses would represent a powerful addition to the experimental neuroscience toolkit to help address how dynamic changes in synaptic strength contribute to sensation, action, learning and memory. Despite a wealth of knowledge on synaptic function in reduced ex vivo preparations, such as brain slices, due to the lack of effective tools, our knowledge of synaptic function in vivo during learning and behavior is extremely limited. A new approach that overcomes this technical gap would bridge the divide between synaptic and circuit level analyses in awake, behaving animals. High signal-to-noise spine level calcium imaging in behaving animals could address this gap. To meet this challenge we propose to develop synaptically-targeted calcium indicators that enable excitatory synaptic transmission to be imaged with quantal resolution simultaneously at hundreds to thousands of connections. Because this is an imaging method, it provides synapse-specific information that one cannot readily obtain from electrophysiological recordings that lump together measurements from a large number of inputs distributed over a neuron's dendritic tree. Optical quantal analysis in behaving animals would permit direct assessment of the dynamic fluctuations in synaptic efficacy that may underlie learning. It will also open whole new avenues of research that could explore how changes in synaptic efficacy contribute to fundamental aspects of sensation, action, and higher cognitive function. The collaboration between Isacoff, Scott and Adesnik enables these new tools to be validated for in vivo applications in brain circuit analysis and behavior in three model organisms: zebrafish, fruitfly and mouse.

? DESCRIPTION (provided by applicant): Voltage-gated ion channels have evolved to open and close in response to changes in the membrane potential and rapidly conduct ions selectively. The members of the family that longest eluded isolation were the voltage-gated proton channel, Hv1, and its relatively close relative, the voltage sensing phosphatase (VSP). Hv1 plays a central role in innate immunity and other physiological processes. The biological function of VSP is not known. This proposal focuses on 3 fundamental aspects to the function of these VSD proteins, which, despite their similarities, differ radically in their effectors: with Hv1 having is channel effector uniquely situated within its VSD, while VSP's effector is the only one so far to have its effector outside of the membrane, in this case on the internal side. Our aims for Hv1 are to elucidate its pore pathway, understand how it is gated, how the gating apparatus in one subunit influences that of the dimeric partner and elucidate the mechanism by which Hv1 detects the absolute transmembrane gradient of pH and uses it to regulate gating. Our aim for VSP is to understand how conformational sequences in the VSD induce conformational sequences in the enzyme domain to alter the choice of substrate. Our goal is to arrive at mechanistic molecular models of gating, cooperativity and modulation of the VSD by pH and modulation by the VSD of the effector. The proposed studies should provide insight into the function of VSDs across voltage-gated proteins and the new methods should be applicable to a range of other channels and receptors whose protein motions, subunit interactions and modulation by ligands are of interest. The proposed work is designed to elucidate the mechanism of function of the voltage-gated proton channel (which is fundamental to innate immunity, reproduction and epithelial transport, and appears to have a role in stroke and cancer) and its relative, the voltage sensing phosphatase. The approach employs several novel optical methods that should prove to be applicable to the study of a broad set of ion channels and receptors.

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.

Over 100,000 Americans of all ages suffer from inherited retinal diseases (IRD), which cause a progressive loss of vision. In most IRDs, disease begins in the rods, causing vision loss from the periphery to the center, leaving patients unable to navigate their surroundings. Electronic retinal prosthesis restore useful vision in patients affected by IRDs, and optogenetics is an alternative therapeutic. A major limitation of microbial opsins for restoration of retinal light sensitivity is the high light intensity required for activating channelrhodopsins. A solution to this caveat is the use of opsins with higher light sensitivity but sufficiently fast kinetics for useful motion vision. We propose a novel approach to restore vision to patients using a virus to express a light sensitive protein in specific, second-order retina neurons to make them light sensitive. Our approach uses a common neuronal receptor, modified to add a light receptive function to the remaining light-insensitive retinal neurons that survive after photoreceptor degeneration. The receptor uses either retinal, which is available in the eye, or a synthetic chemical photoswitch delivered by intravitreal injection. In this way, the cells in which the receptor is located respond to light with a change in neural firing. This compensates for their loss of input from photoreceptors, restoring light responsiveness to the retina and sending information to the brain to restore vision. In most cases, this approach is independent of the mutation that caused the photoreceptor degeneration. Exceptions to this approach may be diseases that cause RPE cell death, such as choroideremia. To date, versions of this approach, developed by Co-PIs Isacoff and Flannery, and others in the field, have employed receptors that are rather insensitive to light or very slow in response and so could not support normal vision. We now propose a new strategy that uses the natural amplification properties of GPCR signaling to increase sensitivity (by 1000 times) and speed. GPCR signaling cascades are intrinsic to rods and cones, as well as bipolar, ganglion cells and other cells in the retina. We also pursue a new discovery, emerging from our preliminary experiments, which enables a combinatorial approach that uses more than one optical sensor molecule at a time in order to recreate the natural diversity of natural signaling in the retina that had earlier been missing. Finally, we employ sophisticated behavioral analysis to test not only the restoration of the ability to tell light from dark or flashing from steady light, but to determine if the animal is able to see images. Success of this program would represent a major step in the creation of a retinal prosthetic based on gene therapy.

What sets the transmission strength of synapses? What determines their plasticity properties? How do synapses homeostatically adjust synaptic weight to accommodate to changing conditions and ensure robust behavior? What happens if synaptic homeostasis is insufficient to compensate for a disruption or a change in demand, are other backup mechnisms of compensation recruited and, if so, how do they work? We combine in vivo super-resolution quantal imaging of synaptic transmission and behavioral analysis with focused RNAi knockdown in one cell type and single cell transcriptome analysis to address these questions. Our preparation is the Drosophila larval neuromuscular junction?an ideal system for imaging and genetics, which shares synaptic signaling machinery and functional properties with vertebrate central excitatory synapses. Our in vivo quantal analysis has revealed that two converging glutamatergic motor neuron (MN) inputs have great heterogeneity in evoked release probability (Pr) and short-term plasticity and that only Ib undergoes ?synaptic homeostasis,? whereby transmitter release changes to compensate for altered postsynaptic sensitivity. Our goal is to identify the molecules responsible for the synapse to synapse and input to input differences. Equally exciting, preliminary work suggests the existence of a novel layer of gain control: ?circuit homeostasis,? which is recruited when synaptic transmission is so compromised that ?synaptic homeostasis? cannot compensate sufficiently. The circuit homeostasis system adjusts neural firing pattern in the presynaptic cell and upstream circuit to preserve locomotor behavior when synaptic transmission is inadequate. Our goal is to define the mechanisms that assure neural output by setting and adjusting transmitter release and firing dynamics. Progress will provide fundamental insight into the robustness of the nervous system that preserves health and which may cause disease when it goes awry.

SUMMARY/ABSTRACT A major goal of neuroscience is to understand how neuromodulatory systems regulate core processes of brain and behavior, from motor function and learning to reward, aversion, attention, and sleep. These systems go awry in schizophrenia and disorders of mood, motor control and cognition. Treatment for these conditions often turns to pharmacological manipulation of neuromodulators and their receptors. Understanding of neuromodulatory circuits has advanced considerably thanks to optogenetics and chemogenetics. But neuromodulation is difficult to crack. A major obstacle is that a single neuromodulator may play many diverse roles because it has multiple receptors, with different functions in different cell locations and different cells within a circuit. Unfortunately, drugs and genetic manipulations cannot typically be targeted or controlled with sufficient spatio-temporal precision to unravel these different functions. We are developing two approaches that provide the needed precision by controlling native, full-length neuromodulatory receptors with photoswitchable tethered ligands (PTLs). Preliminary Iwork demonstrates feasibility of both approaches as applied to two core neuromodulatory receptor families: metabotropic glutamate receptors (mGluRs) and dopamine receptors (DARs). In this proposal, we optimize and expands an approach in which we directly attach PTLs to a specific receptor subtype and develop a new approach that does not touch those receptors, but deliver the PTL to them on a membrane anchor. Because the membrane anchor can be targeted to specific locations within the cell, it can selectively control receptors only at that location, and it can use either broad ligands (which hit all local receptors of that type) or highly selective ligands (to control only one subtype at a time). The tools will be demonstrated in mice and provided to the community as gene delivery vectors, PTLs and transgenic animal lines.

SUMMARY/ABSTRACT G-protein?coupled receptors (GPCRs), the largest class of membrane signaling proteins, respond to a wide array of extracellular stimuli to initiate intracellular signaling via G proteins and arrestins. Recent studies have provided snapshots of GPCR structures in distinct conformations and revealed that they are extremely dynamic. The conformational dynamics appear to be central to ligand recognition, activation and signaling. Membrane receptors have evolved to respond to precise spatio-temporal concentration profiles of extracellular ligands. In the nervous system, neurotransmitter receptors encounter a wide range of neurotransmitter concentrations and spatio-temporal profiles. Key factors are the small extracellular volume of the synaptic cleft, pumps and/or enzymes that remove neurotransmitter, and diffusion. Additionally, neurotransmitter receptors can be localized within the synapse both pre- and postsynaptically, as well as extrasynaptically where they can encounter neurotransmitter released either locally, which briefly reaches low millimolar levels within the cleft, and spillover from nearby synapses, which reaches lower concentrations. Metabotropic glutamate receptors (mGluRs) are found pre- and postsynaptically at excitatory glutamatergic synapses, as well as on glia and at inhibitory GABAergic presynaptic nerve terminals, meaning that they are activated by both high local concentrations near the site of release and spillover. mGluRs of various kinds can be found together in presynaptic nerve terminals, even when they are all coupled to the same G protein. And they can dimerize, generating hybrid or in some cases totally unique properties and pharmacological profiles. To understand what each mGluR subtype does and develop effective drugs to treat the neurological disorders in which they are implicated, we need to understand how they function and how they are regulated. Our goal here is to define the molecular mechanisms that set and regulate the functional properties of homo- and heteromeric mGluRs at synapses and put into place assays that can be used to screen modulation in the nervous system.