Karl Deisseroth - US grants

DESCRIPTION (provided by applicant): In parts of the adult brain, proliferating cells continually give rise to new neurons. Adult neurogenesis may be involved in pathological conditions ranging from epilepsy to depression, and neurogenesis in the adult hippocampus may also have physiological significance in long-term memory storage. However, it is unknown if brain activity in the adult is meaningfully coupled to neurogenesis to modulate information storage, or other forms of adaptive plasticity at the cellular level. We have obtained data indicating that indeed, adult neural stem cells directly respond to activity using intrinsic calcium signaling that couples to neurogenesis. We next plan to delineate in vitro the molecular mechanisms involved in this excitation-neurogenesis coupling and the synaptic wiring of newborn neurons, focusing on specific channels and second messengers known to be involved in hippocampal calcium signaling. We will then extend these findings to the in vivo situation, and ultimately explore the behavioral consequences of adult excitation-neurogenesis coupling in intact animals. In the course of this research, Robert Malenka, MD PhD, will be the mentor, and Theo Palmer, PhD, will be a collaborator. Dr. Malenka is a world-renowned synaptic physiologist and psychiatrist with expertise in hippocampal activity-dependent plasticity extending to in vivo manipulations and behavior, and Dr. Palmer is an established neural stem cell expert also at Stanford. In my PhD training with Dr. Richard Tsien, I developed an extensive set of techniques in activity-dependent calcium signaling which can be applied to this new question; at the same time, the research proposed here will provide me with a critical opportunity to gain experience with in vivo manipulations, behavioral analysis, and stem cell techniques essential to my training. Furthermore, carrying out this proposal will allow me to develop an independent research program that can be transitioned to a tenure-track position in a psychiatry department, where I look forward to continuing with 75-80% research and 20-25% patient care, and opportunities to teach and mentor others

Abstract Not Provided

Adult neural circuits are in a state of constant remodeling. One of most striking mechanisms by which this occurs is the insertion of new neurons, a process that occurs normally in all adult mammals and has been hypothesized to underly aspects of physiological information storage. Abnormal adult neurogenesis, by contrast, has been linked to major neuropsychiatric diseases including drug addiction and depression. We have found that neural activity acts directly on proliferating progenitors to drive adult neurogenesis, and recent years have witnessed many other important new insights into the underlying molecular mechanisms. However, little or no insight has emerged into the impact of adult neurogenesis on neural circuit function. We propose to apply novel ion channel-based probes to study interactions between CMS circuits and calcium channel-dependent neurogenesis. We hypothesize that circuit activity acting through calcium channels is a crucial component of the cellular microenvironment controlling adult neurogenesis. We further hypothesize that this is a bidirectional interaction, as newborn neurons (once functionally inserted into the circuit as a result of local activity patterns) in turn directly modulate these local network activity patterns. Aim 1 will identify mechanisms by which activity and cellular microenvironment interact to drive calcium channel-dependent adult neurogenesis. Aims 2 and 3 will explore mechanisms by which newborn neurons in turn modulate circuit activity dynamics, using novel high-temporal resolution imaging (Aim 2) and stimulation (Aim 3) techniques. Aim 4 describes use of a new genetically-encoded light-responsive stimulation tool to provide physiological activity patterns to proliferating progenitor cells, in order to study molecular mechanisms of environmental control of excitation-neurogenesis coupling. Together, insights garnered from determining the reciprocal relationship between circuit activity and adult neurogenesis may profoundly illuminate core mechanisms of hippocampal neuropsychiatric disease and suggest novel therapeutic strategies, while deepening our understanding of the normal activity-dependent operation and plasticity of progenitor cells in the hippocampal circuit.

NIH Roadmap Initiative tag; bioengineering /biomedical engineering; bioimaging /biomedical imaging; brain imaging /visualization /scanning; cell cell interaction; molecular /cellular imaging

Understanding how brain cells give rise to complex processes like action, thought, and emotion is limited by the current availability of tools to control specific cells within neural tissue. Fast, genetically targeted control over brain cells would allow elucidation of the role of specific neuronal types, and greatly advance our understanding of brain function. To enable precise perturbation of living circuits, the light-activated ion channel, called channelrhodopsin-2 (ChR2), will be used for genetically targeted, millisecond-timescale optical excitation of neurons. Another protein, called NpHR, has been identified for temporally-precise optical inhibition of neural activity. Both proteins function in mammalian neurons, together forming a complete, complementary system for multimodal, high-speed, genetically-targeted, all-optical investigation of living neural circuits.

This project encompasses a broad technology development, testing, and dissemination plan that will yield generalizable, powerful tools for the physiology, neuroscience and biomedical engineering communities. Specifically, high-speed optical imaging reagents and technology for simultaneous imaging and optical stimulation/inhibition will be developed, in living animals. This is a fundamentally novel approach that could revolutionize the way circuits are probed in intact neural systems. These tools have an enormous range of applications, and the outcome of this work may be versatile enough to allow investigators to determine the contribution of a cell type of choice and relate it to circuit dynamics and behavior in virtually any brain region of interest. These new technologies also afford unique training opportunities for students, who will move on to other institutions and pass on expertise, leading over years to generation of a widespread cadre of scientists and engineers with intensive training in this powerful technology.

DESCRIPTION (provided by applicant): In previous work we developed a light-activated ion channel channelrhodopsin-2 (ChR2) for genetically targeted, millisecond-timescale optical excitation of neurons. We now report that we have identified a high-speed optically-activated chloride pump (NpHR) from N. pharaonis for temporally-precise inhibition of neural activity. The action spectrum of NpHR is strongly red-shifted relative to ChR2, and like ChR2, NpHR functions in mammalian neurons without exogenous cofactors. Together NpHR and ChR2 form a complementary system for multimodal, high-speed, genetically-targeted, all-optical interrogation of intact neural circuits. Here we propose a broad effort for inter-institutional technology-development, capitalizing on the novel NpHR reagent and the unique skills of our Duke/Stanford collaborative team to develop multimodal high-speed optical tools for excitable cell physiology, and responding specifically to the NIMH call for neural technology development. Together these approaches will develop the general power of optogenetic control by targeting bidirectional photosensitivity to important neuronal subtypes and by defining precise optical methods for neural circuit activation. This proposal for developing next-generation optical technologies for precise control of living neural circuitry therefore squarely targets areas of fundamental importance to public health, and is directly responsive to the call of the National Institute of Mental Health for new technology development relevant to neuropsychiatric disease.

DESCRIPTION (provided by applicant): While great advances have been made in understanding the mechanisms of learning in the single synapse or cell, a large gap remains between this understanding and our knowledge of learning at the behavioral level. We know that the activity of large-scale neuronal circuits gives rise to behavior, yet we have little knowledge of what changes in those circuits during learning or how sensory feedback drives these changes. The biggest impediment to answering these questions is the inability to quantitatively measure large-scale circuit properties (e.g. connectivity between brain areas) or to precisely manipulate the activity patterns across these circuits. Optogenetics offers the potential to bridge this gap by allowing the direct control of neural activation in targeted cell types on the millisecond timescale. The development of these tools is progressing most rapidly in mouse, due to the relative ease of genetic manipulations in that species. In contrast, behavioral and circuit-level studies of learning are most practical and have been most successful in "non-genetic" species. Within our team, we have expertise in studying both the behavioral and neural bases of learning in rat, songbird, and nonhuman primate. We propose to develop the optogenetic tools and experimental techniques required to study the circuit-level mechanisms of learning in these species and to apply these to two specific scientific aims: Aim 1: Determine the functional connectivity of learning-related circuitry and how it is altered by experience. It is widely presumed that learning relies on the ability of instructive signals to drive functional modifications of connectivity in the circuits that underlie behavior. However, the tools for measuring functional connectivity in vivo have been limited. We will overcome this limitation using temporally and/or spatially precise optical activation of neurons within a circuit. Functional connectivity will be measured by recording optical-stimulation-triggered changes in activity in downstream neurons. We will assess how functional connectivity is dynamically altered by learning and by factors that may contribute crucially to learning. Connectivity changes will serve as a mechanistic index of the nature and sites of the plasticity that give rise to behavioral change. Aim 2: Test the causal contributions of patterned activity to learning in vivo. Prior research has generated specific and testable hypotheses about how and where patterned activity drives learning. Yet support for these hypotheses has derived primarily from correlative observations of activity during learning rather than causal tests of the proposed mechanisms. We will use optogenetics to causally test the contributions of patterned activity to learning. We will test the sufficiency of instructive signals by imposing precisely controlled patterns of activity at defined loci in a circuit and test their necessity by eliminating the putative signals for learning. PROJECT NARRATIVE This project is aimed at revolutionizing the study of the mechanisms of learning within large neural circuits in the brain by directly measuring large-scale properties of these circuits and precisely manipulating circuit activity. To accomplish this, we will make use of, and continue to develop, advanced new techniques that permit the control of specific population of neurons using optical stimulation (light). The knowledge and tools that we gain from these studies are likely to find broad application in the search for treatments of neurological disorders.

DESCRIPTION (provided by applicant): Neurons compute using a vast array of diverse signals, in which millisecond-scale electrical pulses are complemented by slower membrane potential changes and by slower neuromodulator-driven signals that operate over a broad range of timescales (from seconds to hours), to govern neuroplasticity and neural information processing. Optogenetics (the use of light to control genetically-defined cells within neural tissue) has enabled control over fast electrical events, but has left control over neuromodulatory and slower events relatively unexplored. This deficiency in optogenetics represents an enormous unmet need, as neuromodulator-driven plasticity is likely to be important in Parkinson's Disease, addiction, depression, and many other neuropsychiatric processes, while transient electrical events simply do not capture the full complexity of neural information processing. Uncaging strategies can release second messengers such as Ca2+ and cAMP (just as glutamate uncaging can control fast electrical events); however, uncaging involves bulk application of synthetic UV-releasable compounds that are neither suitable for in vivo use, nor useful for driving genetically-targeted cell types. Moreover, key neuromodulators such dopamine and norepinephrine (which the brain delivers in temporally precise, pulsed, phasic or tonic patterns depending on the situation) do not recruit a single messenger, but rather act on target cells to recruit a complex fabric of intracellular messengers that would be impossible to recapitulate with current technologies. Thus, there is no temporally-precise method to control neuromodulation in defined cells within living animals. In Aim 1, we will molecularly engineer novel versatile tools for optical recruitment of neuromodulatory signals, including those downstream of the G-protein coupled receptors linked to virtually every neuromodulator system. In Aim 2, we will engineer strategies for long-timescale electrical control, focusing on identification and molecular optimization of proteins that provide for generation of stably modulated electrical states. In Aim 3, we will adapt the novel tools from Aims 1 and 2 for targeting to specific locations in specific cell types, and in Aim 4, we will validate the novel tools, integrated with custom optical hardware in freely-moving mice, to test the causal roles of specific modulation patterns in behavioral conditioning. The new technologies, encompassing light sensor/effectors, devices, and targeting tools, will be 1) designed for versatile application across diverse fields; 2) distributed to the scientific community, and 3) applied to mammalian models in our laboratory. This approach leverages our work on optical control of electrical events, but opens the door to a much broader landscape. Indeed, the anticipated impact is movement toward a network engineering approach that spans timescales and modalities, in which complex excitable-tissue function is understood in terms of system properties emerging from interacting electrical and biochemical signals.

DESCRIPTION (provided by applicant): Studying intact systems with both local precision and global scope is a fundamental unmet goal in biology. For example, in the study of the brain, efforts to determine connectivity of small patches of brain, though pioneering, are challenged by the fact that resulting maps will be nearly impossible to interpret because both the brainwide sources and destinations of traced wiring connections, as well as the activity of corresponding cells during behaviorally significant events, will remain unknown. Conversely molecular, electrophysiological, or imaging methods to record population activity are agnostic with regard to brain-wide wiring patterns of recorded cells, creating an enormous gap in understanding of function. We have now laid foundations for addressing this challenge, by integrating chemical engineering, computational optics, and molecular genetics in an approach termed CLARITY. We will develop the approach in the behaving vertebrate CNS, challenging for speed and complexity, but CLARITY will become applicable across biology as we develop platforms for zebrafish, rodents, and primates. In Aim 1, we bring chemical engineering tools to bear, rapidly transforming scattering and impermeable tissues into intact but transparent and macromolecule-permeable (antibody-compatible) form. In Aim 2, we develop activity-readout technology designed to extract volumetric activity (even in freely-moving mammals) that can then be linked to the global wiring and molecular phenotypes. This transformative technology will allow rapid extraction of systems information (dynamics, history, wiring, and molecular phenotypes) from large and intact biological tissues or organs without disassembly, down to millisecond-scale and cellular resolution. In Aim 3, we directly apply CLARITY to behaving mice and zebrafish, rapidly obtaining brain-wide activity patterns of every cell involved in disease-relevant states of fear an reward. This alone will be a milestone achievement in biology, but furthermore these data will also be linked to full molecular and global wiring information of those cells in the same brains, al publicly accessible for mining/searching. Finally in Aim 4 we design and build online datasets for zebrafish, mouse, and primate to broadly serve the community. Computational infrastructure will address handling and public access to the massive amount of data collected (among the largest datasets in all of biology), including the records of activity in every neuron in vertebrate brains during specific experiences linked to molecular and global wiring information. We are experienced with technology outreach and education, and will leverage this experience to achieve the full transformative mission of this new technology.

This INSPIRE award is partially funded by the Modulation Program in the Divison of Integrative Organismal Systems in the Directorate for Biological Sciences, by the Graphics and Visualization Program in the Division of Information and Intelligent Systems in the Directorate for Computer Information in Science and Engineering, by the Advances in Biological Informatics in the Division of Biological Infrastructure in the Directorate for Biological Sciences, and by the Emerging Frontiers Program in the Directorate for Biological Sciences.

This interdisciplinary project will capitalize on a remarkable opportunity driven by new chemical-engineering technology to define the full brainwide activity patterns underlying complex mammalian behaviors. Achieving high-precision control and high-resolution information on a complex system, while maintaining intact a full global perspective on the same system, is especially difficult for neuroscience but extends to the study of all biology. The expected outcome of this work will be to enable biologists around the world to readily observe simultaneous activity of cells in 3D volumes of tissue and in real time. From the perspective of both society-at-large and NSF, the significance of this INSPIRE proposal may be very broad. This information will be crucial for understanding how complex behaviors can be tuned, and how complex biological systems operate, and will lead to development of what will be among the largest datasets in biology. This research program also will integrate new multidisciplinary approaches encompassing bioengineering, genetics, optics, chemical engineering, and computer science, which will capitalize upon and strengthen the rich local and nationwide multidisciplinary training and education environments. Computational tools will address data curation and free online public access to resulting information that will be accessible via the website (www.optogenetics.org), and the technology will be freely transferred to other U.S. academic or government laboratories. This Project meets the challenge of achieving high-resolution information on a complex system, while at the same time maintaining a global and functional perspective on the same system: an approach may be transformative, not only in neuroscience, but also throughout biology.

DESCRIPTION (provided by applicant): Optogenetic technologies, designed for control of defined elements of neural circuitry with high temporal precision and within intact systems, have recently come into broad use. Key components of this approach include light-activated regulators of transmembrane ion conductance such as channelrhodopsins (ChRs) which can be temporally precise in action, and readily targeted to specific brain regions (e.g. by viral vectors. Now an unexpected alignment in results from our laboratory has opened up a path to optogenetic tools which will make defined circuit elements susceptible to potent control even in deep brain circuits and without genetics. We propose to develop, distribute, and apply (to anxiety research) these technologies, bringing to bear tools from biophysics and structural biology, through which brain circuits may be targeted for versatile inhibition or excitation. In Ai 1, we propose to leverage new structural information to create a potent inhibitory ChR. This is a long-sought goal of optogenetics, not yet achieved. Current fast optogenetic inhibitory tools are ion pumps, not potent enough to reliably interfere with nerve signaling in large tissue volumes or in illuminated axons. These pumps are limited in efficacy since only a single ion is moved across the membrane for each photon absorbed. But channels instead move many ions per photon and can also shunt and influence input resistance. To enable robust interception of spikes in deep-brain or axonal settings for loss-of-function experiments essential to the field, here we will integrate our novel structural information to create inhibitory optogenetic channels. In Aim 2, we extend this rational design of ChRs to include altered color-sensitivity in the settin of novel ion selectivity. Despite the fact that only a handful of ChRs have been identified, major advances have come from comparisons, comparative mutagenesis, and chimeras. We therefore will integrate structural information with a new set of ChRs to modulate photocurrent properties, creating and testing ChRs with diverse color-sensitivity properties in each class for versatile optogenetic control. Finally in Aim 3, we will validate and refine these novel tools for mammalian behavior in anxiety models. This technology cannot be developed optimally and distributed to the community without a phase of validation and refinement in a real-world setting. Now launching from our recently published work on anxiety, we will employ the tools developed here to probe behavioral consequences of deep-brain optogenetic control in experiments of high significance for understanding the generation and control of anxiety-related states. Together, these efforts will generate versatile and powerful new optogenetic tools, and provide fundamental insight into casual dynamics of anxiety- related behaviors based on precise control of distinct neural circuit pathways.

DESCRIPTION (provided by applicant): This proposal is designed to provide insights into the implementation of motivated behaviors by specific neural circuits, by testing and applying new tools that complement the control capability of optogenetics with novel structural and activity insights. Integrating our new chemical engineering- based technology (HETT: hydrogel-electrophoretic tissue transmutation) with imaging, behavioral, and optogenetic analysis, we propose to connect the wiring to the activity and causal impact of cell populations involved in mammalian motivated behavior, with simultaneous molecular precision and global scope. In brief, the opportunity has now emerged to measure, in models of mouse behavior relevant to motivation-linked clinical conditions: 1) brainwide neuronal activity patterns during specific motivated behaviors, 2) immunophenotypes of those activated cells, 3) global projection patterns of those same cells, and 4) causal impact of those cells on circuit activity and behavior. In Aim 1, we employ HETT to rapidly transform dense intact brain tissue into transparent and antibody-permeable form, and thereby rapidly and efficiently map in global (brainwide) fashion those neural populations with altered activity in settings of motivated behaviors (conditioned place preference, social interaction, sucrose consumption, and escape behavior); we have recently shown that in all of these behaviors we are able to bidirectionally modulate the key behavioral outcome with corresponding bidirectional modulation of dopamine neurons, providing substantial experimental leverage for Aims 2 and 3 below. The resulting brainwide activity maps will be made available online in rendered volumes as a community resource. In Aim 2, building on both our preliminary data as well as new data from Aim 1, in neural populations specifically and bidirectionally modulated during the four dopamine neuron-driven behaviors, we will bring to bear causal sufficiency and necessity tests using optogenetics in freely-moving mice to assess for modified behavior as a result of controlling activity in the implicated projections and populations of cells downstream of the dopamine neurons. We will track not only behavior but also local circuit activity in freely-moving mice, and HETT analysis will record on an individual-animal basis, linked to behavior, the extent and nature of circuit influences exerted in each experimental subject as well as the brainwide projection patterns of the causally implicated cell populations. Finally, in Aim 3 we seek circuit-dynamics insights into motivated behavior, applying high-speed volumetric activity readouts both in vivo and in acute slices from target brain regions implicated in Aims 1 and 2, during optogenetic drive of the dopamine neurons. The imaged tissues will then be processed for HETT, thereby transforming the very same 1) behaviorally tested and 2) live-imaged tissue into 3) a rigid and stable structure that can be interrogated for wiring and immunophenotype. Together, the approaches proposed here will integrate novel technology to probe fundamental causal underpinnings and mechanisms of circuit activity controlling motivated behaviors in freely-moving mammals.

Center PI: Malenka, Robert C. Principal Investigator (Project 4): Deisseroth, Karl/Malenka, Robert C. Project Summary Learning and memory must involve changes in neural circuit dynamics yet the mechanisms by which such changes occur remain largely unknown. This project will use a novel in vivo imaging modality, termed fiber photometry, which allows collection of activity patterns from genetically-targeted cells and processes in deep brain structures in freely-moving animals. Using fiber photometry, real-time activity in CA3 pyramidal cell axon projections and CA1 pyramidal cell bodies will be monitored during free behavior and while animals undergo hippocampus-dependent learning and memory tasks. Genetically encoded calcium indicators (GECIs) with different fluorescent properties will be expressed in CA3 and CA1 pyramidal cells so that the relationship between presynaptic activity in axonal projections and postsynaptic activity in their targets can be monitored simultaneously, thus allowing quantification of the relationship between pre- and postsynaptic activity at a defined set of synaptic connections. After validation of this novel method in anesthetized animals, it will be applied to well-established hippocampal-dependent memory tasks including contextual fear conditioning and one-trial avoidance learning with the goal of visualizing in awake behaving animals how CA3 to CA1 circuit dynamics change as learning occurs. In a final series of experiments, which will be entirely based on the results from the other three projects in the Conte Center, molecular interventions designed to modulate LTP or homeostatic synaptic plasticity at CA3-CA1 synapses will be performed to determine their effects on hippocampal circuit dynamics during learning and memory. Thus, this project has the potential to provide long- sought insight into the neural circuit changes that underlie learning and memory as well as elucidate the role of prominent forms of synaptic plasticity in these circuit adaptations. Relevance Learning and memory are due to long-lasting changes in specific circuits in the brain but it has not been possible to observe these changes occur. Using a new sophisticated method that allows visualization of neural circuit dynamics in behaving subjects, this project will define how a specific circuit changes during learning. The information collected will provide important insight into how the brain encodes memory and how this process can malfunction during brain disorders including mental illness.

? DESCRIPTION (provided by applicant): This proposal is designed to provide circuit-dynamics understanding of anhedonia, a psychiatric symptom domain of enormous clinical significance that is well-suited for study in laboratory animals. In Aim 1, we generate high-resolution brainwide maps of endogenous and stimulus-triggered activity patterns in anhedonic states. We use our new readout technologies including ofMRI and COLM as described in the proposal, and our custom recombinase-driver rat lines to allow versatile mechanistic experiments determining if changes in dynamics are linked to altered activity in specific modulatory systems. In Aim 2, we employ another new technology (fiber photometry) to track and quantify local high-speed dynamical patterns corresponding to anhedonia, allowing observation during free behavior of relative balance and joint activity relationships across the brain (initially, we will test for evience of competition between prefrontal cortex and midbrain to exert influence over subcortical limbic pathways during behavior, following up our preliminary findings). In Aim 3, we test causal significance for anhedonia of the changes in dynamics identified in Aims 1 and 2, using new optogenetic tools to modulate coordinated activity relationships across the brain to induce anhedonia from baseline, and restore hedonic behavior in induced anhedonic states. We will also come full circle to Aim 1 measures, quantifying global activity patterns elicited by optical recruitment of the implicated circuit elements, in a final step toward identifying circuit-level phenotypes with causal explanatory power for anhedonic behavior. Together, the approaches proposed here will integrate novel technology to probe fundamental causal underpinnings and mechanisms of a key psychiatric symptom domain in freely-moving mammals.

Overall Summary We will develop a NIDA Center, Neural circuit dynamics of drug action, dedicated to the development, application, and dissemination of brainwide and cellular-resolution analyses of altered states elicited by drugs of abuse. Our science will focus on identifying the causal circuit-level actions of drugs of abuse in modulating behavior relevant to assessment of context, risk and reward. In a manner that brings together the collaborating groups of the Center, we focus on clinically significant drugs with different molecular profiles but shared significance for understanding behaviors and perceptions relevant to social and nonsocial risk and reward. Specific agents employed include methamphetamine, MDMA, and ketamine, in the setting of validated human, rat, and mouse social and nonsocial behaviors. We will both develop the brainwide technologies and engage in extensive outreach, training, and education to broaden impact, with the NIDA IRP and beyond. The Center includes four Research Projects (1: led by Dr. Karl Deisseroth, focusing on methamphetamine, MDMA and ketamine action in the cortex and across the brain of mice and rats; 2: led by Dr. Lisa Giocomo, focusing on methamphetamine and ketamine action in entorhinal cortex and hippocampal formation of mice and rats; 3: led by Dr. Robert Malenka, focusing on methamphetamine and MDMA action across the brain of mice; and 4: led by Dr. Leanne Williams and Brian Knutson, focusing on human structural and functional imaging relevant to methamphetamine, ketamine, MDMA, and risk/reward relationships. Broad and diverse interactions among these groups and external collaborators will be further enriched by the Center?s vital Training Core for disseminating these techniques to advance drug abuse research, a Technology Core for developing the next- generation technologies suitable for application to drug abuse research, and an Administrative Core for orchestrating these important interactions. This approach to the NIDA Center will allow us to capitalize on the unique strengths of our team, crossing scales from molecules and synapses, to circuits and behavior, reaching the scope of the intact human brain as we identify relevant structure-activity relationships within animal and human nervous systems.

This NeuroNex Technology Hub, based at Stanford and the Salk Institute in California, will profoundly advance the understanding of the brain by developing technologies to study the brain's structure and function. The investigators will develop new approaches to understand how the individual components that make up the nervous system operate during behavior, and indeed cause behavior. The team will merge principles from genetics, physics, optics, engineering, and biology, to build and disseminate methodology, instrumentation, and analytics that enable targeting and control of individual kinds of brain cells, and the technology developed will be taught via hands-on training available to the scientific community. The outcome will be a broadly-applicable platform for discovering how neural circuit activity gives rise to complex cognitions and behaviors in the brain, which is essential to understand how the nervous stem fails to operate well in neurological and psychiatric diseases. The structure of the NeuroNex Training Core is designed to drive the participation of investigators across the spectrum of background and demography, including junior investigators and students as well as women and other underrepresented groups in STEM.

Current understanding of brain function at the cellular network level is limited by the lack of integrative tools that (in the same individual organism) can be used for molecularly defining neural circuit components, for tracing local and global wiring of those same circuit components, and for observing and controlling activity in those same circuit components during precisely controlled and quantified behaviors. This integration, or "datastream linking", will fundamentally advance knowledge but is an enormous practical and intellectual challenge. This NeuroNex Technology Hub will 1) address this challenge with molecular, genetic and optical tools while also developing computational methods to discover the underlying natural and causal structural and dynamical motifs; 2) do so in a vertically-linked fashion so that all technologies built are mutually compatible in the same nervous system at the same time; 3) do so in a horizontally-linked fashion, so that the technologies built are suitable for primate rat, mouse, fly, and diverse fish species; 4) engage in outreach, training, and dissemination, open for broadest impact to the entire NSF community throughout the program. This teaching will leverage our current state-of-the-art methods and educational infrastructure, and will advance alongside the technology development and integration.

Administrative Core Summary The Administrative Core of the Center (directed by Dr. Deisseroth) will shoulder the complex burden of coordination and communication across projects, cores, collaborators, and visiting scientists; will manage financial, administrative, approval, and safety issues; and will oversee and implement data sharing and dissemination. For a Center seeking to break technological barriers, while also teaching and training in existing technologies, and using all of these technologies to discover new principles regarding drug action on the brain, a robust and well-integrated Administrative Core will be fundamental to rigorous, safe, stable, and effective implementation. Monthly full meetings and weekly subgroup meetings will require steady and experienced staff to coordinate. The Center by necessity also includes substantial complexity in terms of personnel, finances, and safety. The diverse network of staff scientists, graduate students, postdoctoral fellows, collaborators, and training activity is essential to the Center and requires an able Administrative Core to implement, track, manage, and report. These efforts will include managing compliance and approval on human subjects research, animal protocols, safety inspections, DEA and drug use regulations, progress reports, publication management; financial projections, reimbursements, auditing, and accounting; and interactions with University resources that will be leveraged. Finally, the greatest opportunity of the Center?sharing of data and technology-- is also its greatest challenge. The Center web pages, wikis, and online forum (next-generation implementations of our existing small efforts in this direction) will be another high-impact and challenging administrative element. As with the other Cores and the Projects, this Core will stand in part upon well- established human and other resources, but will expand and diversify in fundamentally new directions under the auspices of the P50 and of NIDA. Success of the Center ultimately will be measured in terms of the both the science and the outreach; the Administrative Core is absolutely crucial for both. To achieve its stated goals in the most efficient and cost-effective manner, the Center will require an Administrative Core that will provide administrative support to all Center investigators and assist in the dissemination of reagents and findings generated by the Center.

Project 1 Summary Here we implement a technology-driven approach to identify and control circuit dynamics underlying drug- modulated social and nonsocial behaviors, in contexts carrying reward and risk. We apply technology representing a major alignment of opportunity for drug abuse research (optoencephalography or OEG, frame- projected independent-fiber photometry or FIP, and CLARITY-optimized lightsheet microscopy or COLM) which allow not only observation and control of genetically-defined circuitry, but multiple circuit elements simultaneously and independently-- before, during and after behaviors in the drug-altered state. In Aim 1, we begin at the broadest (brainwide) scale in awake rodents, to identify key players and principles in unbiased fashion, while maintaining circuit element-specificity for observation and control of activity. Technologically, Aim 1 experiments will include optogenetically-driven precise pulse patterns targeted to specific circuits and projections, as well as rapid and quantitative COLM- or ofMRI- based assessment of brainwide activity patterns. These initial unbiased global assessments will powerfully inform and focus more spatially-restricted investigations in Aims 2 and 3 that resolve essential features of acutely altered states. In Aim 2 we operate at much higher spatial and temporal resolution, the next step toward detailed elucidation of causal circuit dynamics in the acutely drug-altered state. For the same drug and behavioral conditions in Aim 1, now quantitatively guided at the individual-animal level in terms of neuronal activity levels to be targeted using the population and projection-specific recording capability of FIP, we play-in patterns of population and projection activity to test causal impact on behavior. And in Aim 3, we leverage the highest-resolution of our new methods, that nonetheless maintain broad perspective. We are now able to image (with OEG as well as resonant-scanning two-photon microscopy) large volumes of brain and quantify high-speed dynamical activity patterns that are cell type-specific, approaching single-cell resolution while maintaining map-like brainwide perspective, during behavior and during exposure to drugs of abuse. Interventional tests for causality will be directly guided by these observations. As described below, we have already observed highly specific cortical activity patterns in response to subanaesthetic ketamine doses, which here will be extended to the same drug and behavioral conditions pertaining to Aims 1 and 2. This precise observation and control of distinct neural circuit pathways is tightly intertwined with the research programs described in Projects 2-4, and the Technology and Training Cores. Together, these experiments in Project 1 will test versatile, powerful new circuit-dynamics tools for use in the NIDA Center and for the drug abuse community more broadly, and will also apply these tools to deepen our understanding of acute or chronically-altered drug altered states, and of the brain itself as a dynamical system.

Project Summary/Abstract This proposal requests funding for a Lavision Biotec Ultramicroscope II. This will be the first lightsheet microscope to be made available at a core facility at Stanford University. The microscope will fill a demonstrated unmet need for large-scale imaging of cleared tissue at single-cell resolution. It will be installed at the Neuroscience Microscopy Service, a university- wide service center open to all researchers at Stanford University. The investigators that constitute the Major and Minor User groups in this application are poised to apply this instrument along with the latest tissue clearing protocols and cell-specific fluorescent-labeling tools in their NIH-funded research programs. Together, they are striving to make significant advances across a broad range of biomedical research, including: ? neuroscience, with investigations of: o the functional role of microglia in brain development. o mapping of cell-type-specific hypothalamic afferents. o regeneration of the optic nerve and optic tract after injury. o mapping recovery from the effects of early visual deprivation in mouse models by re-induction of molecular drivers of synaptic plasticity in adulthood. o post-stroke recovery mechanisms in the cortex. o mapping of activity perturbations in mouse models of neuro-degenerative disease and neurodevelopmental disorders. o detailed correlation of MRI diffusion-tensor imaging signals with neuronal fiber anatomy. ? stem cell biology: o with a study to characterize the bone marrow niche of long-term hematopoietic stem cells. ? developmental biology: o with studies of the growth and development of blood vessels in the lung. The placement of this instrument in a well-established, highly successful service center open to all researchers at Stanford insures that, over the life of this instrument, it will be used by hundreds of researchers from many tens of labs to support similarly excellent research to better understand and combat disease.

Abstract A core criterion of substance use disorder is continuing use of the rewarding substance despite clear consequences that would otherwise be highly aversive and thus behaviorally powerful in driving avoidance: harmful physical sequelae, negative social effects, and/or placement of the user in dangerous situations. Modern models of drug addiction invoke many possible sources of positive and negative reinforcement, but it is not known which specific circuitry is actually operative (causal) in allowing actions that cause normally-aversive physical harm, and it is not fully understood from the perspective of organismal-survival mechanisms how the destructive consequences of substance use could become entirely unable to deter drug use behavior. This remarkable conditionality of aversion, central to drug abuse, is also of fundamental significance in non- drug-related behavior; normally-aversive experiences can manifest with altered (neutral, or even positive) valence for a variety of adaptive and maladaptive reasons. In some cases, these effects might be understood by relatively simple neuroeconomic risk/benefit or gain/loss considerations; for example, sufficient reward (e.g. rare delivery of large food/water resources) may adaptively drive tolerance of same-class aversive events (e.g. temporary loss of food/water). We and others have studied and described these circuit computations extensively, including in the prior period of support from the present grant, by meticulous construction of carefully-balanced, defined-value, same-category reward/aversion stimuli. However, circuitry implementing this seemingly straightforward adaptive behavior (in which rewarding and aversive consequences are experienced, but expected-value of one is simply of greater magnitude) may be of insufficient complexity for the large majority of naturalistic situations, wherein reward and aversion are categorically different. In this proposal, we build upon our insights into casual, cell-specific reward circuitry, leveraging next-generation circuit-interrogation technology to identify (in brainwide fashion) the structurally- and molecularly-defined circuit elements at the intersection of reward and aversion, by which the vertebrate brain alters behavioral responses to aversive stimuli. In Aim 1, we develop next-gen CLARITY adapted to these paradigms to obtain brainwide wiring and molecular identification of all cells that are specifically recruited (active) in this key behavior (all registered to genetically-encoded Ca2+ sensor-derived activity data collected during the aversion- suppression behaviors). In Aim 2, using the tools from Aim 1 in combination with our latest optogenetic control methods, we will test specific circuit-activity hypotheses for causality in implementing cross-category modulation of aversion. In addition to the novel circuit targets that will emerge from the brainwide unbiased investigation of Aim 1, we already have specific circuit-level hypotheses to test based upon our existing preliminary data, ensuring that the later Aims of the proposal stand on an already-solid foundation. We hypothesize that this candidate circuit activity (in a tunable subset of projections from mPFC to specific subcortical structures) causes diminished behavioral impact of negative-valence stimuli via disrupted internal representation (and experience) of aversive stimuli. And in Aim 3, building on (and guided by) our identification of circuit elements that are naturally and causally involved in suppression of aversive responses, in this Aim we achieve single-cell real-time resolution during behavior. In the fiber-readout strategy of Aim 1 and 2; activity dynamics emerge as a single time-series from the fiber; as valuable as these data are, it is possible that signal increases could arise from altered synchrony or spread of activity in the region rather than from increased activity in a specific subset of target neurons. Single-cell resolution in causally implicated ensembles here will not only resolve this fundamental question, but also allow deep molecular profiling of the specific cells causally involved, with clear basic and translational implications. Identifying this circuitry will not only provide insight into the basic science of reward and aversion, but also will advance potentially revolutionary understanding and targeting of circuit elements that may be causal (or therapeutic) in human substance-use and neuropsychiatric disorders.

Summary Aging is a gradual process that results in the loss of cellular function across the body, leading to numerous chronic diseases that promote mortality. Elucidating the precise mechanisms of aging is critical for reducing illness and extending healthy lifespan. However, almost every tissue in the body is modified by aging, making it difficult to pinpoint the principal controller of aging. The goal of this proposal is to determine whether the brain modulates aging through coordinated activity patterns within discrete neuronal networks. We will use one of the shortest-living vertebrates, the African turquoise killifish, as a rapid, high-throughput model of aging to uncover genetically- defined neurons that regulate cellular metabolism and lifespan. Employing large-scale light-sheet imaging in killifish, we will visualize brain-wide calcium activity dynamics to unbiasedly identify neurons that respond to longevity interventions. We will characterize the genetic profiles of the identified neurons via a combination of immunohistochemical, single cell, and phosphorylated ribosome capture approaches. To examine whether these neurons play a causal role to control overall cellular function in the brain and other tissues, we will optogenetically activate these neurons and measure molecular signatures of youth and in vivo metabolic activity in the brain and peripheral tissues. We will monitor and manipulate neural activity throughout the short lifespan of killifish using fiber photometry to determine if this ?neural pacemaker? dictates the tempo of aging and youthful behavior. These approaches will then be extended to longer-lived species ? zebrafish and mice. Knowledge resulting from these studies should be transformative to understand the fundamental mechanisms that regulate and synchronize aging and longevity. As age is the prime risk factor for many diseases, including neurodegenerative diseases, this proposal should provide new, circuit-based approaches to treat these diseases. !

The broad vision for this project is to develop new tools for future biomanufacturing through cross-cutting collaborations of scientists?chemists, biologists, physicians, and engineers?united by the immense opportunity of building functional materials with, and within, biological life. The proposed methodologies establish the biomanufacturing toolbox for genetically-targeted chemical synthesis of a variety of functional materials within living cells, tissues and animals. Diverse cell-specific chemical syntheses enable a broad array of functional characteristics and assembled structures. In the long term, such techniques enable building electronics directly within biological systems by harnessing the complex assembly structures within cells. The application of these techniques to develop the capability to create new conductive pathways within the brain may lead to rewiring of neural circuits. Moreover, genetically-targeting the peripheral nerve may allow cell-specific nerve stimulation and recording for neuroprosthesis. Further investigation of the deposited material on neural activity may lead to treatments for diseases such as Alzheimer?s disease (AD) and amyotrophic lateral sclerosis (ALS), or selectively repair demyelinated areas for treatment of multiple sclerosis (MS). Even though current work only focuses on basic tool development and initial understanding of the impact of the modifications on neural activities, the tools can be potentially expanded to diverse cell types for therapeutics and creation of new materials and assemblies. This project offers direct training opportunities for the students and postdocs involved in terms of research as well as important skills for interdisciplinary collaboration. These trainees subsequently further the development of biomanufacturing and their method of collaboration by running their own independent research groups in academia or by incorporating their knowledge into future industrial developments. A Training Core program in this project provides hands-on training on basic biomanufacturing techniques for hundreds of students, instructors and researchers.

Despite existing ability to engineer materials with diverse form and functionality, a high-level of structural and functional complexity found in multicellular living systems are still challenging to realize. The capability of genetically targeting enzymes and other proteins to specific cell types has yet to be harnessed to direct complex assembly of functional structures instructed by biological systems. This project integrates the fields of molecular genetics, tissue biology, chemistry, and materials science in unprecedented ways to transform the biomanufacturing of complex structures. The project focuses on building novel structures in vivo, creating natural and unnatural polymers within targeted cell-types of living organisms. This approach is extended to the development of a universal shared methodology for targeted chemistry within living beings. The work proposed focuses on developing and applying novel toolboxes for diverse genetically-targeted synthetic processes while engineering for biocompatibility, characterizing the synthesized molecules/materials, and understanding the mechanisms and implications of forming synthetic materials for eliciting natural and novel biological functions.

This award is co-funded by the Division of Molecular and Cellular Biosciences, the Division of Chemical, Bioengineering, Environmental and Transport Systems and the Division of Chemistry, and also by the Division of Industrial Innovation and Partnerships, the Division of Engineering Education and Centers, and the Division of Materials Research.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

This proposal is designed to provide circuit-dynamics understanding of anhedonia, a psychiatric symptom domain of enormous clinical significance that is well-suited for study in laboratory animals. This work, alongside our recently-developed methods for obtaining brainwide cellular-resolution activity readout and control, has created a powerful and fortuitous alignment enabling us to bridge local and global neuronal dynamics, and to identify brain-spanning circuitry mediating behavioral drives, conflicts, and resolutions. In Aim 1, we identify single-cell-resolved orbitofrontal (OFC) dynamics underlying distinct consummatory behaviors. We have developed a temporally-precise alternative-choice mouse-behavioral paradigm, crucially designed for compatibility with our wide-field cellular-resolution imaging/recording methods, in which mice select among multiple motivational drives, and adjust action planning in light of internal or external context. We apply this paradigm along with our cellular-resolution readouts and analyses, beginning with addressing both hunger and thirst in OFC. We identify dynamics of motivational drive resolution both in the presence or absence of controlled internal states, and in the presence or absence of external (social) context, using our new methods; we hypothesize from prior work (Jennings et al., Nature 2019) that resolution of these conflicts will depend upon not only the motivational (internal) state of the animal but also the external context. In Aim 2, we map causal global dynamics of motivational drive conflict and resolution, quantifying the high-speed cellular-resolution brainwide circuit dynamics underlying these motivational drive interactions (drives naturally-occurring; or, to leverage our fast electrophysiological readout, instead induced in temporally- precise fashion by optogenetically driving AGRP neurons in the case of hunger, and/or SFO inputs to the MnPO in the case of thirst, using our established models and methods; Allen et al., Science 2019; Jennings et al., Nature 2019; Marshel et al., Science 2019). Identification of novel region-specific dynamics in conditions of varying motivational drive and social context will feed back to inform Aim 1 imaging workflow, already with a firm foundation from our prior work imaging OFC states corresponding to social and thirst drive interaction. In Aim 3, we define cells underlying inter-drive competition and corresponding brainwide dynamics. Multiple single cells identified by natural activity will be optogenetically targeted with our unique wide-field and high-resolution spatial light-guidance technology. We register cellular ensembles observed to be naturally and causally involved, to detailed 3D intact-tissue (STARmap) transcriptomic information from the same cells in the same organism. Alignment with wiring-based anatomy and deep molecular datastreams allow cell-type- resolved and single cell-level insight into, and targeting of, survival drive competition and resolution processes, with both basic significance and relevance to brain disease. Together, the approaches proposed here will integrate novel technology to probe causal underpinnings of key symptom domains in freely-moving mammals.