Allen Institute

FUNCTIONALLY GUIDED ADULT WHOLE BRAIN CELL ATLAS IN HUMAN AND NHP - PROGRESS IN TREATING BRAIN DISORDERS HAS BEEN FRUSTRATINGLY SLOW, IN LARGE PART DUE TO THE EXTRAORDINARY COMPLEXITY OF THE HUMAN BRAIN AND ITS INACCESSIBILITY TO STUDY. REMARKABLE ADVANCES IN TECHNOLOGIES FOR STUDYING INDIVIDUAL CELLS, MOST NOTABLY SINGLE CELL GENOMICS, HAVE REVOLUTIONIZED THE STUDY OF COMPLEX NERVOUS TISSUES AND HAVE BEEN USED TO MAP CELLULAR DIVERSITY ACROSS THE ENTIRE MOUSE BRAIN WITH CELL TYPES DEFINED BY THEIR SPECIFIC PATTERNS OF GENE USAGE AND GENE REGULATORY MECHANISMS. THESE HIGHLY SCALABLE METHODS HAVE BEEN SUCCESSFULLY APPLIED TO BRAIN TISSUE FROM HUMAN AND OTHER SPECIES AND ARE READY TO BE APPLIED TO WHOLE BRAINS FROM HUMANS AND NON-HUMAN PRIMATES. A MAJOR CHALLENGE WITH STUDYING THE HUMAN BRAIN IS BRIDGING FIELDS AND SCALES FROM FUNCTIONAL MRI AND MACROSCALE CONNECTOMICS TO HISTOLOGICAL, CELLULAR AND MOLECULAR ANALYSES. BRIDGING THESE DOMAINS IS ESSENTIAL TO CREATING A TRANSFORMATIVE NEW CELL ATLAS THAT WILL DESCRIBE THE CELLULAR AND MOLECULAR UNDERPINNINGS OF THE FUNCTIONAL ORGANIZATION OF THE HUMAN BRAIN. AN IMPORTANT RECENT DEVELOPMENT FROM SINGLE CELL GENOMIC ANALYSIS IS THAT CELL TYPES CAN BE ALIGNED ACROSS SPECIES AND ARE HIGHLY CONSERVED ACROSS MAMMALS FROM MICE TO HUMANS, ALTHOUGH MORE SIMILAR IN EVOLUTIONARILY CLOSER PRIMATES THAN IN RODENTS. THIS FINDING AMPLIFIES THE VALUE OF PRIMATE SPECIES IN HELPING TO UNDERSTAND HUMAN BRAINS AND INFER CELLULAR PROPERTIES THAT CANNOT BE MEASURED IN HUMANS. THE CURRENT PROPOSAL BRINGS TOGETHER A UNIQUE TEAM OF WORLD LEADERS TO TACKLE THE CHALLENGE OF CREATING A NEW HUMAN AND NON-HUMAN PRIMATE CELL ATLAS LINKED TO FUNCTIONAL BRAIN ARCHITECTURE. SINGLE CELL TRANSCRIPTOMIC, EPIGENOMIC AND SPATIAL TRANSCRIPTOMICS WILL BE USED TO CLASSIFY AND SPATIALLY MAP CELL TYPES ACROSS THE ENTIRE HUMAN, MACAQUE AND MARMOSET BRAIN, SAMPLING BASED ON BRAIN MAPS DERIVED FROM STRUCTURAL AND FUNCTIONAL IMAGING. FUNCTION-LOCALIZING FMRI IN MACAQUES WILL ALLOW THE DIRECT ANALYSIS OF CELLULAR CORRELATES OF FUNCTIONAL TOPOGRAPHY. ADVANCES IN SPATIAL TRANSCRIPTOMICS WILL ALLOW AN UNPRECEDENTED WHOLE PRIMATE BRAIN MAP OF CELL TYPES. UNIQUE ACCESS TO MACAQUE TISSUES FOR ANALYSIS OF CELLULAR ANATOMY AND PHYSIOLOGY ALLOWS THE CHARACTERIZATION OF MOLECULARLY-DEFINED CELL TYPES IN MANY BRAIN REGIONS. SIMILAR TECHNIQUES WILL BE APPLIED TO LIVING NEUROSURGICALLY-DERIVED HUMAN BRAIN TISSUES, COUPLED WITH ENHANCER-AAV BASED TOOLS TO ALLOW SELECTIVE GENETIC LABELING OF CELL TYPES. FINALLY, PROFILING REGIONS CENTRAL TO PERCEPTION, BEHAVIOR AND MOOD ACROSS MANY INDIVIDUALS AND DIVERSE MAMMALS WILL LINK GENETIC, ENVIRONMENTAL AND EVOLUTIONARY FACTORS TO CELLULAR VARIATION. THE OUTCOME OF THESE EFFORTS WILL PRODUCE A NEW REFERENCE CLASSIFICATION FOR CELL TYPES ACROSS THE WHOLE HUMAN AND NHP BRAIN, SPATIAL MAPS OF MOLECULARLY DEFINED CELL TYPES, AND PHENOTYPIC CHARACTERIZATION OF FUNDAMENTAL BRAIN CELL TYPES. THE CLASSIFICATION WILL ALIGN HOMOLOGOUS CELL TYPES FROM MICE, MARMOSETS, MACAQUES AND HUMANS, ALLOWING INFERENCE AND COMPARISON OF CELLULAR PROPERTIES ACROSS SPECIES. FURTHERMORE, DATA WILL BE ALIGNED IN COMMON COORDINATE FRAMEWORKS, ALLOWING CREATION OF NEW ATLASES SPANNING STRUCTURAL, FUNCTIONAL, CELLULAR AND MOLECULAR INFORMATION. ALL DATA AND ANALYSES WILL BE DISTRIBUTED TO THE RESEARCH COMMUNITY, INCLUDING A FORMAL CELL ONTOLOGY OF CELL TYPES ACROSS SPECIES AND VISUALIZATION TOOLS FOR BROAD COMMUNITY ACCESS.

A COMPREHENSIVE WHOLE-BRAIN ATLAS OF CELL TYPES IN THE MOUSE

A PLATFORM FOR CELL TYPE-LEVEL TRANSCRIPTOMIC, EPIGENOMIC AND SPATIAL INTERROGATION OF ALZHEIMER'S DISEASE

A MULTIMODAL ATLAS OF HUMAN BRAIN CELL TYPES

THALAMUS IN THE MIDDLE: COMPUTATIONS IN MULTI-REGIONAL NEURAL CIRCUITS - SUMMARY, OVERALL (THALAMUS IN THE MIDDLE: COMPUTATIONS IN MULTI-REGIONAL NEURAL CIRCUITS) THIS COLLABORATIVE PROJECT AIMS TO UNCOVER THE LOGIC OF SIGNAL ROUTING FROM SUBCORTICAL AREAS TO THE FRONTAL CORTEX THROUGH THE THALAMUS. THE FRONTAL CORTEX DISPLAYS RICH PATTERNS OF NEURAL ACTIVITY, WHICH CAN BE DECOMPOSED INTO “ACTIVITY MODES” THAT CORRESPOND TO SPECIFIC ASPECTS OF BEHAVIOR. EXAMPLES INCLUDE THE PERSISTENT ACTIVITY CORRELATED WITH SHORT-TERM MEMORY AND MOTOR PLANNING, AND THE RAPIDLY OSCILLATING ACTIVITY DURING VOLUNTARY MOVEMENTS. IN THIS DYNAMICAL SYSTEMS PERSPECTIVE OF NEURAL COMPUTATION, COMPLEX BEHAVIORS CORRESPOND TO DISTINCT SEQUENCES OF CORTICAL ACTIVITY MODES. HOWEVER, THE CORTEX DOES NOT GENERATE THESE ACTIVITY MODES IN ISOLATION, BUT INSTEAD IS STRONGLY AND BIDIRECTIONALLY COUPLED TO THE THALAMUS, THE CENTRAL HUB OF THE FOREBRAIN. MOST OF THALAMUS IS NON- SENSORY (‘HIGHER-ORDER’), RECEIVING SUBCORTICAL INPUT FROM THE CEREBELLUM, MIDBRAIN, AND HIPPOCAMPUS. OUR CENTRAL THEORY IS THAT THESE SUBCORTICAL SIGNALS FLOW THROUGH HIGHER-ORDER THALAMUS TO REACH THE FRONTAL CORTEX, WHERE THEY ENABLE ACTIVITY MODES, UPDATE ACTIVITY MODES, AND CAUSE SWITCHING BETWEEN MODES, AKIN TO THE ‘UPDATE’ AND ‘RESET’ SIGNALS IN LONG SHORT-TERM MEMORY NETWORKS IN MACHINE LEARNING. HOWEVER, MOST OF WHAT WE KNOW ABOUT THALAMUS COMES FROM SENSORY SYSTEMS, AND OUR KNOWLEDGE OF SUBCORTEX THALAMUS FRONTAL CORTEX CIRCUITS IS NASCENT. WE STILL HAVE ONLY A RUDIMENTARY UNDERSTANDING OF THE INPUT AND OUTPUT CIRCUITS OF HIGHER-ORDER THALAMUS, THE MORPHOLOGY AND MOLECULAR PROPERTIES OF THALAMIC NEURONS, THE CIRCUIT MOTIFS THAT LINK SUBCORTICAL INPUT TO CORTICAL ACTIVITY, AND THE ENGAGEMENT OF THESE NETWORKS ACROSS THE FRONTAL CORTEX. WE BRING TOGETHER A TEAM WITH EXPERTISE IN MODERN HIGH-THROUGHPUT ANATOMY (PROJECT 1, 2), MOLECULAR NEUROSCIENCE (PROJECT 2, MOLECULAR SCIENCE CORE), CELLULAR AND SYNAPTIC NEUROPHYSIOLOGY (PROJECT 3), LARGE-SCALE NEUROPHYSIOLOGY IN MICE PERFORMING BEHAVIORS THAT REQUIRE SHORT-TERM MEMORY AND DECISION-MAKING (PROJECT 3, 4), AND THEORY AND COMPUTATION (PROJECT 5, DATA SCIENCE CORE). WE WILL COLLABORATE TO UNCOVER HOW INFORMATION FLOWS FROM SUBCORTICAL AREAS, THROUGH THALAMUS, TO CONTROL CORTICAL ACTIVITY MODES AND THEREBY SHAPE BEHAVIOR. INDIVIDUAL PROJECTS ARE GUIDED BY A CONCEPTUAL FRAMEWORK OF MULTI-REGIONAL NEURAL COMPUTATION, PLACING THE THALAMUS IN THE MIDDLE OF A MULTI-REGIONAL NEURAL NETWORK. TOGETHER, OUR WORK WILL HAVE BROAD IMPLICATIONS FOR THE UNDERSTANDING OF NEURAL COMPUTATION IN SUBCORTEX THALAMUS CORTEX CIRCUITS AND WILL PRODUCE ANATOMY- GUIDED MULTI-REGIONAL CIRCUIT MODELS OF COGNITIVE FUNCTION. WE WILL ALSO PRODUCE PARADIGM-SHIFTING COMMUNITY RESOURCES, INCLUDING QUANTITATIVE ANATOMY, NOVEL GENETIC REAGENTS, NEUROPHYSIOLOGICAL DATA, AND A RICH MODELING FRAMEWORK, UPON WHICH FUTURE STUDIES OF THALAMIC CIRCUITS WILL BE BUILT.

BRAIN CONNECTS: CENTER FOR A PIPELINE OF HIGH THROUGHPUT INTEGRATED VOLUMETRIC ELECTRON MICROSCOPY FOR WHOLE MOUSE BRAIN CONNECTOMICS - PROJECT SUMMARY: CENTER FOR WHOLE MOUSE BRAIN CONNECTOMICS USING HIGH-THROUGHPUT INTEGRATED VOLUMETRIC ELECTRON MICROSCOPY (HIVE) TWO FUNDAMENTAL COMPONENTS OF THE STRUCTURAL BASIS OF BRAIN FUNCTION ARE CELL TYPE COMPOSITION AND THE WIRING DIAGRAM BETWEEN THOSE CELLS. OVER THE PAST DECADE THERE HAS BEEN PARADIGM-SHIFTING PROGRESS IN UNDERSTANDING CELL TYPE COMPOSITION OF THE BRAIN. NOW IT’S TIME TO SYSTEMATICALLY UNCOVER THE BRAIN’S WIRING DIAGRAM AND PLACE IT INTO THE CONTEXT OF CELL TYPES. KNOWLEDGE ABOUT THE COMPLETE CONNECTOMES IN C. ELEGANS AND DROSOPHILA HAVE REVOLUTIONIZED THE UNDERSTANDING OF CELL TYPES AND CIRCUIT FUNCTION IN THOSE SYSTEMS. TRANSMISSION ELECTRON MICROSCOPY (TEM) HAS CONSISTENTLY LED PROGRESS IN THAT REVOLUTION AND HAS THE POTENTIAL TO SCALE UP TO THE ENTIRE MOUSE BRAIN WITH TECHNICAL IMPROVEMENTS IN CERTAIN AREAS. DURING THE IARPA MICRONS PROJECT, MEMBERS OF THE HIVE TEAM BUILT A COMPLETE PIPELINE TO SECTION AND IMAGE THE MM3 AND CREATED THE DATA PROCESSING, RECONSTRUCTION AND ANALYSIS INFRASTRUCTURE TO MAKE CELLS AND CONNECTIONS ANALYZABLE. THE RESULT WAS A DATASET WITH THE LARGEST EM LEVEL RECONSTRUCTIONS OF CELLS IN ANY SYSTEM, WITH NEURONS CONTAINING MORE THAN 14,000 INPUTS AND 15,000 OUTPUTS. WE ACCOMPLISHED THIS BY APPLYING A RIGOROUS STRUCTURED SCIENCE PROCESS THAT IS A HALLMARK OF THE ALLEN INSTITUTE’S TEAM SCIENCE APPROACH. IN THIS PROJECT WE AIM TO IMPROVE OUR PIPELINE, DEVELOPING CRITICAL TECHNOLOGIES TO TACKLE THE CHALLENGES OF SCALING UP TO THE WHOLE MOUSE BRAIN AND LINKING TO CELL TYPES. OUR PROPOSAL WILL PREPARE AND SECTION AN ENTIRE HEMISPHERE, IMAGE IT AT 120 NM RESOLUTION, AND IMAGE UP TO 10 MM3 AT SYNAPTIC RESOLUTION WITHIN THE CORTICAL BASAL GANGLIA THALAMIC LOOP TO PROVIDE KEY INSIGHTS INTO CIRCUIT MECHANISMS WITHIN THIS CIRCUIT. TO ACCOMPLISH THIS, WE WILL NEED TO IMPROVE ALL INDIVIDUAL ASPECTS OF THE PIPELINE, WHILE MAINTAINING CONSISTENT INTEGRATION TESTS THAT ENSURE THAT THE PIECES WORK TOGETHER. WE WILL STANDARDIZE A WHOLE MOUSE BRAIN STAINING PROTOCOL AND ADVANCE THE AUTOMATION OF SERIAL SECTIONING TO COLLECTION OF SERIAL TEM SECTIONS ACROSS A WHOLE MOUSE BRAIN. DEVELOPMENTS OF SERIAL SECTION TILT TEM TOMOGRAPHY WILL ALLOW TO SCALE EM IMAGING TO A WHOLE MOUSE BRAIN AT MULTIPLE SCALES, LEVERAGING THE RE-IMAGING CAPACITY OF SERIAL SECTION TEM. WE WILL DEVELOP OPEN SOURCE DATA PROCESSING TOOLS TO BRING DOWN THE COST OF SEGMENTATION AND WHILE IMPROVING ACCURACY AND INTEGRATING WITH A REAL-TIME GLOBALLY ACCESSIBLE PROOFREADING AND ANALYSIS PLATFORM. FINALLY, WE WILL INTEGRATE OUR DATA WITH FULL MORPHOLOGY RECONSTRUCTIONS LINKED TO GENE EXPRESSION, ALLOWING US TO CREATE AN INTEGRATED ATLAS OF CELL TYPES AND CONNECTIVITY. OUR DISSEMINATION STRATEGY WILL FURTHER AMPLIFY OUR IMPACT BY DEMOCRATIZING ACCESS FOR BOTH THE SCIENTIFIC AND EDUCATIONAL COMMUNITY.

SINGLE CELL TRANSCRIPTOMIC AND EPIGENOMIC CHANGES DURING CHRONIC HIV INFECTION AND COCAINE SELF-ADMINISTRATION - PROJECT SUMMARY THIS APPLICATION IS SUBMITTED IN RESPONSE TO RFA-D-21-019: SINGLE CELL OPIOID RESPONSES IN THE CONTEXT OF HIV (SCORCH) PROGRAM EXPANSION: CNS DATA GENERATION FOR CHRONIC OPIOID, METHAMPHETAMINE, AND/OR COCAINE EXPOSURES. WE HAVE ASSEMBLED AN OUTSTANDING TEAM OF INVESTIGATORS WITH WORLD-CLASS EXPERTISE IN HIV VIROLOGY, ADDICTION NEUROBIOLOGY, AND SINGLE CELL TRANSCRIPTOMICS. WE WILL LEVERAGE STATE-OF-THE-ART SINGLE-NUCLEI RNA SEQUENCING (SNRNA-SEQ) COUPLED TO SINGLE-NUCLEI EPIGENETICS (SNATAC-SEQ) TO DEFINE THE TRANSCRIPTIONAL AND EPIGENETIC LANDSCAPE OF NEURONAL AND NONNEURONAL CELLS IN ADDICTION-RELEVANT BRAIN CELLS OF HIV-INFECTED MICE THAT COMPULSIVELY SELF-ADMINISTER COCAINE. IN THIS MANNER WE WILL IDENTIFY CELL TYPES AND BRAIN SITES IN WHICH HIV AND COCAINE INTERACT TO EXACERBATE THE NEGATIVE IMPACT OF HIV INFECTION ON THE BRAIN AND CONTRIBUTE TO THE PERSISTENCE OF COCAINE USE DISORDER IN INFECTED INDIVIDUALS. TO FACILITATE THIS GOAL, WE HAVE DEVELOPED AN EXTENDED ACCESS INTRAVENOUS COCAINE SELF-ADMINISTRATION PROCEDURE FOR MICE THAT PRECIPITATES COMPULSIVE-LIKE RESPONSE FOR COCAINE THAT IS RESISTANT TO NEGATIVE OUTCOME (CONTINGENTLY DELIVERED NOXIOUS FOOT SHOCKS). WE WILL UTILIZE ECOHIV, WHICH IS A MODIFIED HIV STRAIN THAT CAN INFECT CONVENTIONAL IMMUNOCOMPETENT MICE. THE CHROMIUM SINGLE CELL MULTIOME ATAC AND GENE EXPRESSION ASSAY AND MULTIPLEXED ERROR-ROBUST FLUORESCENCE IN SITU HYBRIDIZATION (MERFISH) WILL BE USED TO IDENTIFY COCAINE AND/OR HIV-RESPONSIVE CELLS IN THE BRAIN WITH UNPRECEDENTED CELLULAR AND SPATIAL RESOLUTION. WE WILL CONFIRM OUR MAJOR FINDINGS USING POSTMORTEM BRAIN TISSUES FROM COCAINE- EXPERIENCED HIV-INFECTED INDIVIDUALS. OUR GOALS WILL BE ACCOMPLISHED THROUGH THE FOLLOWING FOUR SPECIFIC AIMS. IN AIM 1, WE WILL CHARACTERIZE COMPULSIVE-LIKE COCAINE INTAKE IN CONTROL AND HIV-INFECTED MICE AND DETERMINE THE IMPACT OF ANTIRETROVIRAL TREATMENT (ART) ON COCAINE RESPONSE IN THESE MICE. IN AIM 2, WE WILL USE SNRNA-SEQ AND MULITOME ASSAYS TO IDENTIFY THOSE CELLS THAT SHOW THE MOST ROBUST TRANSCRIPTIONAL AND EPIGENETIC PLASTICITY TO COCAINE CONSUMPTION AND HIV INFECTION. IN AIM 3, WE WILL USE MERFISH TO CONFIRM AND VALIDATE OUR MAJOR SNRNA- SEQ AND MULTIOME ASSAY FINDINGS, AND GENERATE MORE REFINED SPATIALLY RESOLVED MAPS OF THOSE CELLS MOST IMPACTED BY COCAINE AND/OR HIV. IN AIM 4, WE WILL CONFIRM OUR MAJOR FINDINGS USING POST-MORTEM HUMAN BRAIN TISSUES. THIS HIGHLY INNOVATIVE, COLLABORATIVE AND MULTIDISCIPLINARY PROGRAM OF RESEARCH PROMISES TO YIELD FUNDAMENTAL NEW INSIGHTS INTO DISEASE-RELATED INTERACTIONS BETWEEN HIV INFECTION AND COCAINE USE.

RESEARCH INFRASTRUCTURE: NSF MID-SCALE RI-2: OPEN MULTIMODAL AI INFRASTRUCTURE TO ACCELERATE SCIENCE -LANGUAGE MODELS WITH BILLIONS OF POSSIBLE ADJUSTMENTS AND TRAINED ON TRILLIONS OF WORDS ARE NOW POWERING THE FASTEST-GROWING COMPUTING APPLICATIONS IN HISTORY. LARGE LANGUAGE MODELS (LLM) ARE BUILT USING MASSIVE AMOUNTS OF TEXT, USUALLY OBTAINED BY PULLING DATA FROM MULTIPLE SOURCES ON THE INTERNET. RECENT ADVANCES ENABLE THESE MODELS TO PROCESS OTHER KINDS OF DATA, INCLUDING IMAGES, GRAPHS AND TABLES. MODELS WITH THESE ABILITIES ARE KNOWN AS MULTIMODAL LLMS. THE BEST-PERFORMING LLMS CURRENTLY DEPLOYED ARE PROPRIETARY, SO THEIR PARAMETERS, TRAINING DATA, CODE AND DOCUMENTATION ARE NOT OPENLY AVAILABLE. THUS, MOST ARTIFICIAL INTELLIGENCE (AI) SCIENTISTS CANNOT STUDY, EXPERIMENT DIRECTLY WITH, OR IMPROVE THESE STATE-OF-THE-ART MODELS. THIS PROJECT ? OPEN, MULTIMODAL ARTIFICIAL INTELLIGENCE (OMAI) - WILL PROVIDE INFRASTRUCTURE IN THE FORM OF A SUITE OF POWERFUL, WELL-DOCUMENTED, UP-TO-DATE, OPEN MODELS, AND OPEN-SOURCE INTERFACES DESIGNED FOR SCIENTIFIC WORK. SCIENTISTS WILL BE ABLE TO ACCESS THE MODELS, USE DISCIPLINE-SPECIFIC DATA AND OPTIMIZE THE MODELS. THE PROJECT EMPOWERS RESEARCHERS, PROVIDES DOCUMENTATION TO ACCELERATE RESEARCH AND EDUCATION, AND HAS AN ACTIVE PROGRAM IN EARLY-CAREER TRAINING TO ADVANCE US ECONOMIC AND SCIENTIFIC COMPETITIVENESS. IN ADDITION, OPPORTUNITIES PROVIDED THROUGH PARTNERSHIPS WITH A RANGE OF INSTITUTIONS AND PROGRAMS WILL ENHANCE TRAINING. THE LONG-TERM PLAN IS TO MAKE THE INFRASTRUCTURE AVAILABLE AS A LOW- OR ZERO-COST SERVICE TO THE RESEARCH COMMUNITY IN A MANNER LIKE OPEN-SOURCE CODE REPOSITORIES AND SCIENCE-FOCUSED DIGITAL LIBRARIES, TO MAXIMIZE USAGE. THE OMAI RESEARCH INFRASTRUCTURE CONSISTS OF A SERIES OF OPEN, MULTIMODAL LANGUAGE MODELS KEPT UP TO DATE WITH RECENT SCIENTIFIC PUBLICATIONS AND OPEN-SOURCE APPLICATION PROGRAMMING INTERFACES THAT ENABLE SCIENTISTS TO USE, EXPAND, AND MODIFY THOSE MODELS. IT ADDRESSES PRIORITIES SET FORTH IN THE WHITE HOUSE AI ACTION PLAN (HTTPS://WWW.WHITEHOUSE.GOV/WP-CONTENT/UPLOADS/2025/07/AMERICAS-AI-ACTION-PLAN.PDF) TO ACCELERATE AI-ENABLED SCIENCE AND ENSURE THE UNITED STATES IS PRODUCING THE LEADING OPEN MODELS. THE INFRASTRUCTURE AIMS TO ACCELERATE SCIENTIFIC DISCOVERY ACROSS DISCIPLINES RANGING FROM MATERIALS TO PROTEIN FUNCTION PREDICTION AND WEATHER MODELS. IT WILL ALSO ENABLE NEW UNDERSTANDING AND IMPROVEMENT OF FUTURE LLMS WHILE CONTRIBUTING TO THE DEVELOPMENT OF A WELL-TRAINED WORKFORCE CAPABLE OF BUILDING, CUSTOMIZING, AND MAINTAINING SUCH MODELS. IN ALLOWING RESEARCHERS TO FINE TUNE THE MODELS, RESEARCHERS CAN OPTIMIZE PERFORMANCE AND UNDERSTAND DESIGN DECISIONS THAT INFLUENCE TRAINING SPEED AND STABILITY, WHICH CAN IMPACT THE SHORT-TERM AND LONG-TERM ECONOMIC COSTS OF LLM DEVELOPMENT. THE PROJECT EMPHASIZES REPRODUCIBILITY, TRANSPARENCY, OPEN DATA, OPEN AND EVOLVING EVALUATIONS, MULTIMODALITY, AND SCIENTIFIC USE-CASES. IT WILL ENABLE A BROAD POPULATION OF SCIENTIST-USERS ACROSS ALL DISCIPLINES TO USE AND ADAPT ARTIFICIAL INTELLIGENCE MODELS TO THEIR OWN NEEDS AND LAYS THE FOUNDATION FOR FUTURE RESEARCH IN AI FOR SCIENCE. BY SUPPORTING WORK IN THESE NOVEL, CRITICAL RESEARCH AREAS, OMAI CAN ULTIMATELY BENEFIT BOTH SCIENCE AND THE PUBLIC. 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.- SUBAWARDS ARE PLANNED FOR THIS AWARD.

TRANSCRIPTIONAL ATLAS OF HUMAN BRAIN DEVELOPMENT

A COMMUNITY RESOURCE FOR SINGLE CELL DATA IN THE BRAIN

BRIDGING FUNCTION, CONNECTIVITY, AND TRANSCRIPTOMICS OF MOUSE CORTICAL NEURONS - BRIDGING FUNCTION, CONNECTIVITY, AND TRANSCRIPTOMICS OF MOUSE CORTICAL NEURONS THE VERSATILE AND POWERFUL FUNCTIONAL PROPERTIES OF THE BRAIN ARE REFLECTED IN THE NEURONAL ACTIVITY PATTERNS AND COMPUTATIONS, AND THEIR EVOLUTION OVER TIME DUE TO LEARNING, HOMEOSTATIC PLASTICITY, AND OTHER PROCESSES. THE COMPOSITION OF BRAIN CIRCUITS OUT OF A LARGE NUMBER OF CELL TYPES, WHICH MAY BE DEFINED BY THE CHARACTERISTIC PATTERNS OF GENE EXPRESSION, AND THE INTRICATE CONNECTIVITY OF THESE CIRCUITS ARE EXPECTED TO BE INTIMATELY RELATED TO THEIR FUNCTIONAL PROPERTIES. HOWEVER, THE EXACT NATURE OF THESE RELATIONSHIPS IS FAR FROM CLEAR. THE CONCEPT OF A CELL TYPE ITSELF, ESPECIALLY WHEN CONSIDERED AT A FINE-GRAINED LEVEL, WITH A HUNDRED OR MORE CELL TYPES IN ANY GIVEN BRAIN AREA, IS UNDER ACTIVE RESEARCH IN THE COMMUNITY. A CENTRAL QUESTION IS WHETHER AND HOW TRANSCRIPTOMICALLY-DEFINED CELL TYPES PROVIDE SPECIFIC UNDERPINNINGS FOR BROADER CIRCUIT PROPERTIES, SUCH AS THOSE EXPRESSED IN ANATOMY – DEFINED BY NEURON’S LOCATION, MORPHOLOGY, CONNECTIVITY – OR IN THE FUNCTIONAL TYPES OF NEURONAL ACTIVITY IN VIVO. THE PROPOSED PROJECT WILL ADDRESS THIS QUESTION BY INVESTIGATING THE LINKS BETWEEN MOLECULAR AND ANATOMICAL CELL TYPES TO CIRCUITS AND FUNCTION IN THE MOUSE PRIMARY VISUAL CORTEX (V1). WE WILL CONNECT THE TYPES OF FUNCTIONAL VISUAL RESPONSES IN VIVO WITH TRANSCRIPTOMIC TYPES VIA MULTIPLEXED FLUORESCENCE IN-SITU HYBRIDIZATION (MFISH). CALCIUM IMAGING OF NEURAL ACTIVITY WILL BE CARRIED OUT ACROSS THE FULL CORTICAL DEPTH IN V1, CO-REGISTERED WITH MFISH IMAGING OF THAT TISSUE, AND THE TRANSCRIPTOMIC TYPES OF THE NEURONS WILL BE DETERMINED, ESTABLISHING LINKS BETWEEN EACH NEURON’S FUNCTION AND ITS TYPE. IN PARALLEL, WE WILL USE A UNIQUE FUNCTIONAL CONNECTOMICS DATASET ALREADY OBTAINED AT THE ALLEN INSTITUTE, IN WHICH ELECTRON MICROSCOPY (EM) IMAGES ARE CO-REGISTERED WITH IN VIVO IMAGING DATA FROM V1. THESE DATA WILL PERMIT US TO MAP THE FUNCTIONAL PROPERTIES OF EACH NEURON TO ITS MORPHOLOGICAL TYPE AND CONNECTIVITY CHARACTERISTICS, RESOLVED IN THE EM VOLUME. THE MORPHOLOGICAL TYPE WILL, IN TURN, ALLOW US TO COMPARE THIS DATASET WITH THE TRANSCRIPTOMIC TYPES, USING OUR EARLIER PATCHSEQ DATASET, WHERE TRIPLE-MODALITY DATA OF MORPHOLOGY, INTRINSIC ELECTROPHYSIOLOGY, AND TRANSCRIPTOMICS WAS OBTAINED FOR INDIVIDUAL NEURONS. THESE DATA AND ANALYSES WILL BE FREELY SHARED WITH THE SCIENTIFIC COMMUNITY. WE WILL PROVIDE A WEB-BASED RESOURCE THROUGH THE ALLEN INSTITUTE CELL TYPE CARDS PORTAL, LINKING ACROSS TRANSCRIPTOMIC, MORPHOLOGICAL, CONNECTIVITY, AND FUNCTIONAL TYPES IN THESE DATASETS. THUS, THIS PROJECT WILL UNCOVER THE RELATIONS BETWEEN TRANSCRIPTOMIC TYPES, CORTICAL CIRCUIT STRUCTURE, AND ITS FUNCTION, WHILE PROVIDING A MAJOR RESOURCE FOR A BROAD SPECTRUM OF FUTURE STUDIES IN THIS AREA.

SYNAPTOMES OF MOUSE AND MAN

A COMMUNITY RESOURCE FOR SINGLE CELL DATA IN THE BRAIN - PROJECT SUMMARY THE BRAIN INITIATIVE CELL ATLAS NETWORK (BICAN) WILL GENERATE COMPREHENSIVE MOLECULAR TAXONOMIES AND ORGANIZATIONAL PRINCIPLES OF CELL TYPE DIVERSITY IN THE HUMAN AND NON-HUMAN PRIMATE EXTENDING COMPREHENSIVE BICCN WORK IN THE MOUSE. THIS DETAILED CENSUS OF THE STRUCTURE AND ROLE OF CELL TYPE SPECIFIC DATA IN THE BRAIN IS RECOGNIZED AS ONE OF THE MOST PROMISING AVENUES FOR ADVANCING OUR UNDERSTANDING OF THE HUMAN BRAIN IN HEALTH AND DISEASE. TO FACILITATE SUCCESSFUL OPERATIONS FOR BICAN, THIS PROJECT WILL ESTABLISH AN ENGAGEMENT, OUTREACH, AND COORDINATION UNIT FOR THE CONSORTIUM THAT WILL MANAGE OPERATIONS, ESSENTIAL STANDARDS DEVELOPMENT AND DOCUMENTATION, PROVIDE OPERATIONAL TRANSPARENCY, AND IDENTIFY AND COORDINATE SOLUTIONS TO KEY HURDLES FOR COLLABORATION AND ANALYSIS. IN ADDITION TO ORGANIZING VIRTUAL AND IN-PERSON MEETINGS OF THE BICAN NETWORK AND WORKING GROUPS THIS UNIT WILL IMPROVE INTEROPERABILITY AND ALIGNMENT WITH OTHER CONSORTIA AND PARTNERS AND AIM TO IMPROVE USAGE FOR A WIDE RANGE OF POTENTIAL DATA CONSUMERS. COMPLEMENTING THESE FUNDAMENTAL OPERATIONAL GOALS OF THE BICAN CONSORTIUM AND PARTNER CUBIE UNITS, THIS UNIT WILL PROVIDE A GOVERNANCE STRUCTURE FOR GENERATION AND OPERATIONAL TRANSPARENCY OF BICAN DATA PRODUCTION AND RELEASE AND STANDARDS DEVELOPMENT. ESSENTIAL ACTIVITIES WILL INCLUDE TECHNICAL MANAGEMENT AND FACILITATION OF COMMUNITY STANDARDS, SCHEMAS, AND ONTOLOGY DEVELOPMENT, AND CENTRALIZED CONSORTIUM DATA REGISTRATION AND DASHBOARD, AND COORDINATING AND ORGANIZING BICAN OUTREACH, TRAINING, AND EDUCATION ACTIVITIES TO ENHANCE INTERACTIONS WITH EXTERNAL RESEARCH PROGRAMS AND CONSORTIA. THE RESULTING CONSORTIUM GENERATED DATA AND TOOLS WILL BE MADE PUBLICLY ACCESSIBLE IN A COMMUNITY PORTAL SUPPORTING DATA RETRIEVAL, CONSORTIUM ACTIVITIES, AMPLIFY PUBLIC IMPACT THROUGH STRONG BICAN PARTNERSHIP MANAGEMENT, COMMUNICATION, STANDARDS COMMITTEES, AND ANALYSIS WORKING GROUPS, AND DEVELOP INFRASTRUCTURE FOR MAXIMIZING COMMUNITY USAGE, PUBLISHING STANDARDS AND TUTORIALS, AND COORDINATING EDUCATION AND OUTREACH.

AN EXTENSIBLE BRAIN KNOWLEDGE BASE AND TOOLSET SPANNING MODALITIES FOR MULTI-SPECIES DATA-DRIVEN CELL TYPES - PROJECT SUMMARY BRAIN INITIATIVE CELL CENSUS NETWORK (BICCN) IS COMPLETING A COMPREHENSIVE CELL CENSUS OF THE ADULT MOUSE BRAIN, AND BRAIN INITIATIVE CELL ATLAS NETWORK (BICAN) WILL EXTEND THIS WORK WITH EMPHASIS ON HUMAN AND NON- HUMAN PRIMATES. EFFECTIVELY ORGANIZING, SUMMARIZING, ACCESSING, AND REFINING THESE ATLASES IS CRITICAL TO MAXIMIZING THEIR IMPACT ON SCIENCE. THIS PROPOSAL IS TO DEVELOP AN EXTENSIBLE BRAIN CELL KNOWLEDGE BASE (BCKB) TO INGEST AND STANDARDIZE COMPREHENSIVE CELL TYPE INFORMATION FROM BICAN’S DEVELOPMENT OF A MULTIMODAL, MULTI-SPECIES BRAIN CELL ATLAS AND DISSEMINATE THAT ATLAS AS AN OPEN AND INTERACTIVE COMMUNITY RESOURCE FOR ADVANCING KNOWLEDGE OF THE BRAIN. THE BCKB WILL BE INITIALIZED DURING THIS PROJECT WITH MULTI- DIMENSIONAL BRAIN CELL TYPE CLASSIFICATIONS FROM BICCN AND WILL EXPAND AS DATA AND KNOWLEDGE ARE PRODUCED BY BICAN RESEARCHERS. UNDER AIM 1, WE WILL CREATE AN ADAPTIVE KNOWLEDGE GRAPH FOR LINKING BRAIN CELL INFORMATION. SPATIAL AGGREGATION WILL BE DONE USING COMMON COORDINATE FRAMEWORKS. A FLEXIBLE GRAPH-BASED DATA MODEL WILL CAPTURE DISCRETE AND CONTINUOUS CELL TYPE RELATIONSHIPS. THE WORK WILL START WITH CROSS-SPECIES MOP DATA AND COMPREHENSIVE WHOLE- BRAIN MOUSE DATASETS FROM BICCN AND LATER EXTEND INTO BICAN’S WHOLE-BRAIN MOLECULAR AND SPATIAL TRANSCRIPTOMICS DATA IN HUMAN AND NON-HUMAN PRIMATES AS SUCH DATA BECOMES AVAILABLE. ANCHORING OF TAXONOMIES IN SINGLE CELL MOLECULAR AND SPATIAL TRANSCRIPTOMICS PROVIDES A ROBUST FRAMEWORK FOR INTEGRATING MULTIMODAL DATA THAT IS SPATIALLY MAPPED AND/OR CELL TYPES MAPPED. AN ECOSYSTEM OF TOOLS FOR CURATING, ANNOTATING, MAPPING, AND VISUALIZATION OF CELL TYPE DATA WILL BE CREATED IN AIM 2. WE WILL BUILD AND EXTEND TOOLS, SUCH AS THE INITIAL CELL TYPES CARDS SHOWCASING BICCN’S MOP RESULTS, SO BICAN TEAMS AND PUBLIC LABS CAN SHARE AND REFINE BRAIN CELL TYPE TAXONOMIES AND ANATOMICAL INTEGRATION. THE TOOLS FRAMEWORK DEVELOPED FOR THIS AIM WILL PROVIDE A CENTRAL HUB, AKIN TO AN “APP STORE,” TO ACCESS OUR TOOLS AND OTHERS FROM THE COMMUNITY TO INTERACT WITH CELL TYPE DATA. THIS CONNECTED TOOLS FRAMEWORK WILL STREAMLINE SCIENTIFIC WORKFLOWS AND ENCOURAGE FAIR PRACTICES. AS PART OF AIM 3, WE WILL DEVELOP AN INFRASTRUCTURE TO LINK BRAIN CELL DATA AND KNOWLEDGE. THIS INFRASTRUCTURE WILL ENABLE INGESTING, STORING, SEARCHING, AND CURATING NEUROSCIENTIFIC INFORMATION FROM MULTIPLE SOURCES INTO A LINKED INFORMATION PLATFORM. THIS KNOWLEDGE INFRASTRUCTURE WILL HELP CONNECT DISPARATE PIECES OF CELL TYPE INFORMATION USING EXPERT ANNOTATIONS, MACHINE-LEARNING INFERENCES, AND DERIVATIONS USING PROVENANCE MECHANISMS. WE WILL USE ALLEN INSTITUTE’S BRAIN KNOWLEDGE PLATFORM FOR INITIAL IMPLEMENTATION. FINALLY, IN AIM 4 WE WILL GATHER, CURATE, AND INTEGRATE INFORMATION AND KNOWLEDGE FROM BICAN TEAMS BY CONDUCTING ANNUAL HANDS- ON TRAINING AND FEEDBACK WORKSHOPS. THESE EVENTS WILL CREATE ENGAGEMENT WITHIN AND OUTSIDE BICAN PROJECTS AND FOSTER COMMUNITY-BASED EVOLUTION, SUSTAINABILITY, AND GOVERNANCE.

OPEN-ACCESS AAV?TOOLBOX FOR BASAL GANGLIA CELL TYPES AND CIRCUITS - PROJECT SUMMARY WE PROPOSE TO LEVERAGE NEW AND EXISTING TRANSCRIPTOMIC AND EPIGENETIC DATASETS FROM MOUSE, MARMOSET, MACAQUE AND HUMAN BRAIN TO DEVELOP REFINED APPROACHES FOR BRAIN CELL TYPE ENHANCER SELECTION FOR CREATING CELL- TYPE SPECIFIC ENHANCER ADENO-ASSOCIATED VIRUSES (AAVS), AND TO MAKE INROADS TOWARD PREDICTION OF BRAIN-WIDE EXPRESSION SPECIFICITY. WE WILL FOCUS ON BASAL GANGLIA (BG) CELL TYPES FOR SEVERAL REASONS: 1) BG ENCOMPASS SEVERAL BRAIN AREAS WITH DIFFERENT EMBRYONIC ORIGINS AND CELLULAR COMPLEXITY, 2) BG ARE INTEGRAL FOR MOTOR OUTPUT AND REWARD PATHWAYS AND HAVE THUS BEEN HIGHLY CONSERVED THROUGH EVOLUTION, AND WE PREDICT MANY BG ENHANCERS CAN BE FOUND THAT ARE CONSERVED ACROSS SPECIES, 3) BG DYSFUNCTION IS A SHARED FEATURE OF MANY BRAIN DISORDERS, AND AAV VECTORS THAT TARGET BG CELL TYPES WITH HIGH SPECIFICITY WILL BE USEFUL FOR BASIC AND TRANSLATIONAL RESEARCH, AND 4) SUFFICIENT EPIGENETIC DATA ARE NOW AVAILABLE THAT COVER BG STRUCTURES IN MOUSE, MARMOSET, MACAQUE AND HUMAN, AND OUR PRELIMINARY ANALYSIS SUPPORTS THE FEASIBILITY OF UTILIZING THESE DATA SOURCES FOR DISCOVERY OF STRIATUM-SPECIFIC ENHANCERS WITH A HIGH SUCCESS RATE. TO CREATE A SUITE OF VIRAL TOOLS FOR BG CELL TYPES, WE PROPOSE TO CREATE AN END-TO-END SCREENING AND VALIDATION PIPELINE FOR DISCOVERY OF BRAIN CELL TYPE ENHANCER AAVS AT THE ALLEN INSTITUTE. MULTIPLE EPIGNOMIC AND TRANSCRIPTOMIC DATASETS WILL BE CONSOLIDATED AND INTERROGATED TO REVEAL CANDIDATE ENHANCERS THAT ARE SELECTIVELY ACCESSIBLE IN THE BG CELL POPULATIONS. ENHANCER AAVS WILL BE CONSTRUCTED AND SCREENED FOR DESIRABLE EXPRESSION EITHER SINGLY OR IN MULTIPLEXED APPROACHES. A SIZEABLE SUBSET OF ENHANCER AAVS WILL BE SUBJECTED TO MOLECULAR CHARACTERIZATION BY SINGLE CELL TRANSCRIPTOMICS AND/OR MULTIPLEXED FISH TO REVEAL SPECIFICITY AND COMPLETENESS OF LABELING FOR EACH VIRUS, AS WELL AS BRAIN-WIDE EXPRESSION MAPPING IN MOUSE WITH SERIAL TWO-PHOTON TOMOGRAPHY. WE WILL DELIVER A COLLECTION OF NOVEL VIRAL TOOLS FOR BG CELL TYPES THAT HAVE BEEN VALIDATED FOR HIGH SPECIFICITY AND COMPLETENESS OF LABELING IN VIVO IN BOTH MOUSE AND MACAQUE. FURTHERMORE, WE WILL CREATE AN OPEN-ACCESS ONLINE ATLAS THAT WILL DISPLAY DETAILED AAV CHARACTERIZATION DATA. LASTLY, WE WILL DEVELOP A PARTNERSHIP WITH ADDGENE AND LEVERAGE THEIR SELF-SUSTAINING AND SCALABLE MODEL FOR PLASMID DNA AND VIRUS DISTRIBUTION TO THE SCIENTIFIC COMMUNITY. WE WILL TRACK PLASMID AND VECTOR REQUESTS FROM OUR COLLECTION TO MEASURE THE IMPACT OF OUR VECTORS FOR THE WIDER RESEARCH COMMUNITY. IF SUCCESSFUL, THIS PILOT AWARD WILL ESTABLISH A ROADMAP FOR INTEGRATED ANALYSIS OF DIVERSE EPIGENETIC DATASETS FOR CELL TYPE ENHANCER DISCOVERY, SCALABLE ENHANCER AAV SCREENING METHODS IN THE MOUSE AND NON-HUMAN PRIMATE BRAIN, AND WIDESPREAD DISTRIBUTION OF KNOWLEDGE AND TOOLS TO THE RESEARCH COMMUNITY. ALTHOUGH WE FOCUS OUR EFFORTS ON BG CELLS TYPES AND CIRCUITS IN THIS PILOT U01 PROJECT, OUR GOAL IS TO BUILD A SCALABLE PIPELINE THAT IS GENERALIZABLE TO CELL TYPE ENHANCER DISCOVERY FOR THE WHOLE MOUSE BRAIN.

BRAIN CONNECTS: MAPPING BRAIN-WIDE CONNECTIVITY OF NEURONAL TYPES USING BARCODED CONNECTOMICS - PROJECT SUMMARY MAPPING THE BRAIN-WIDE CONNECTIONS OF NEURONS PROVIDES A FOUNDATION FOR UNDERSTANDING THE STRUCTURE AND FUNCTIONS OF A BRAIN. NEUROANATOMICAL TECHNIQUES BASED ON LIGHT-MICROSCOPY OR ELECTRON MICROSCOPY HAVE ADVANCED TREMENDOUSLY IN THROUGHPUT AND COST IN RECENT YEARS, BUT IT REMAINS CHALLENGING TO SCALE THEM UP TO SYSTEMATICALLY INTERROGATE LARGE NON-HUMAN PRIMATE (NHP) BRAINS. HERE WE PROPOSE TO DEVELOP SEQUENCING-BASED NEUROANATOMICAL APPROACHES TO ACHIEVE HIGH THROUGHPUT AND HIGHLY MULTIPLEXED BRAIN- WIDE MAPPING OF NEURONAL PROJECTIONS AND SYNAPTIC CONNECTIVITY IN NHPS AT CELLULAR RESOLUTION. UNLIKE MICROSCOPY-BASED TECHNIQUES, WHICH RELY ON VISUALLY TRACING INDIVIDUAL AXONS FROM THE SOMAS TO AXONAL TERMINI, SEQUENCING-BASED APPROACHES LABEL NEURONS WITH UNIQUE VIRALLY ENCODED RNA SEQUENCES, OR “BARCODES.” SEQUENCING AND MATCHING BARCODES THUS REVEALS THE PROJECTIONS AND/OR SYNAPTIC CONNECTIVITY OF NEURONS. THUS, BY TRANSFORMING PROJECTION AND CONNECTIVITY MAPPING INTO SEQUENCING PROBLEMS, SEQUENCING-BASED NEUROANATOMICAL APPROACHES ARE NOT CONSTRAINED BY THE SAME TRADEOFFS THAT PLAGUE MICROSCOPY-BASED TECHNIQUES. SPECIFICALLY, WE WILL DEVELOP AND OPTIMIZE THREE TECHNIQUES FOR BRAIN-WIDE MAPPING. FIRST, WE WILL OPTIMIZE BRICSEQ (BRAIN-WIDE INDIVIDUAL ANIMAL CONNECTOME SEQUENCING), WHICH CAN CURRENTLY MAP THE PROJECTIONS OF TENS TO HUNDREDS OF THOUSANDS OF NEURONS IN A SINGLE MOUSE BRAIN AT CELLULAR RESOLUTION. WE AIM TO ADAPT BRICSEQ FOR NHP BRAINS, FURTHER REDUCE COST AND INCREASE THROUGHPUT, TO ACHIEVE THE ABILITY TO MAP A MILLION NEURONS PER BRAIN AT CELLULAR RESOLUTION AT EXTREMELY LOW COST PER NEURON. SECOND, WE WILL OPTIMIZE BARSEQ (BARCODED ANATOMY RESOLVE BY SEQUENCING) FOR NHP BRAINS. BARSEQ USES IN SITU SEQUENCING OF THE SAME VIRAL BARCODES USED IN BRICSEQ TO ACHIEVE HIGHER RESOLUTION IN PROJECTION MAPPING AND TO ALSO READ OUT GENE EXPRESSION IN THE SAME NEURONS. THUS, BARSEQ CAN ASSOCIATE NEURONAL PROJECTIONS WITH CELL TYPES DEFINED BY GENE EXPRESSION IN INDIVIDUAL NEURONS. WE WILL AUTOMATE IN SITU SEQUENCING, REDUCE PROBE COST, AND SCALE UP BARSEQ TO ACHIEVE THE ABILITY TO MAP BRAIN- WIDE PROJECTIONS IN NHP BRAINS. FINALLY, WE WILL DEVELOP BARCODED RABIES VIRUS-BASED MONOSYNAPTIC TRACING TO ACHIEVE HIGHLY MULTIPLEXED MAPPING OF SYNAPTIC CONNECTIVITY OF NEURONAL TYPES AT CELLULAR RESOLUTION. DETERMINING THE SYNAPTIC CONNECTIVITY OF NEURONAL TYPES WILL POWERFULLY CONSTRAIN AND TEST COMPUTATIONAL MODELS OF CIRCUIT FUNCTION BEYOND WHAT KNOWING THE AXONAL PROJECTIONS ALLOWS. WE WILL APPLY ALL THREE TECHNIQUES TO GENERATE A MULTI-RESOLUTION PROJECTION AND SYNAPTIC CONNECTIVITY MAP OF THE MACAQUE VISUAL CORTEX. WITH THE ABILITY TO GENERATE MASSIVE SINGLE-NEURON DATASETS AND THE ABILITY TO LINK PROJECTIONS AND SYNAPTIC CONNECTIVITY TO NEURONAL TYPES, OUR PROPOSED TECHNIQUES COMPLEMENT MATURE TECHNIQUES DEPLOYED AT BRAIN CONNECTS CENTERS TO ACHIEVE AN UNPRECEDENTED VIEW OF NHP BRAINS.

OPENSCOPE: A PLATFORM FOR HIGH-THROUGHPUT AND REPRODUCIBLE NEUROPHYSIOLOGY OPEN TO EXTERNAL SCIENTISTS TO TEST IMPACTFUL THEORIES OF BRAIN FUNCTION - PROJECT SUMMARY OVER THE PAST FIVE YEARS, THE ALLEN INSTITUTE HAS BUILT TWO UNIQUE IN-HOUSE PIPELINES FOR IN VIVO ELECTRO- AND OPTICAL-PHYSIOLOGY: TOGETHER THEY FORM THE ALLEN BRAIN OBSERVATORY. WE USED THIS OBSERVATORY TO FREELY AND OPENLY SHARE CALCIUM IMAGING DATA FROM ~60,000 CELLS FROM 221 RUNNING MICE VIEWING STANDARD VISUAL STIMULI; IN OCTOBER 2019, WE WILL RELEASE NEUROPIXELS ELECTROPHYSIOLOGY RECORDINGS FROM ~100,000 CELLS FROM 100 MICE UNDER THE SAME CONDITIONS, ALL REGISTERED TO A COMMON ANATOMICAL COORDINATE SYSTEM (CCFV3). WE WISH TO OPEN THE BRAIN OBSERVATORY PIPELINE TO THE COMMUNITY, ENABLING THEORETICAL, COMPUTATIONAL, AND EXPERIMENTAL SCIENTISTS TO TEST SOPHISTICATED HYPOTHESES ON BRAIN FUNCTIONS IN A PROCESS ANALOGOUS TO ASTRONOMICAL OBSERVATORIES THAT SURVEY THE NIGHT SKY. OUR OBSERVATORY, CALLED OPENSCOPE, WILL ACCEPT, ONCE A YEAR, EXPERIMENTAL PROPOSALS FROM EXTERNAL SCIENTISTS, WHICH WILL BE REVIEWED BY A PANEL OF LEADING EXPERTS FOR THEIR FEASIBILITY AND SCIENTIFIC MERIT. THE ALLEN INSTITUTE WILL CARRY OUT THE SELECTED EXPERIMENTS BY FOLLOWING VERIFIED, REPRODUCIBLE, AND OPEN PROTOCOLS FOR IN VIVO SINGLE- AND MULTI-AREA TWO PHOTON CALCIUM IMAGING AND NEUROPIXELS ELECTROPHYSIOLOGY, MAKING THE DATA FREELY AVAILABLE TO THESE SCIENTISTS. FUNDING FOR THIS PROPOSAL WILL GIVE THE ENTIRE COMMUNITY ACCESS TO A MODERN EXPERIMENTAL PLATFORM THAT OPERATES UNDER STANDARDIZED, REPRODUCIBLE, AND HIGH-THROUGHPUT CONDITIONS. THIS RESOURCE WILL BE PARTICULARLY ATTRACTIVE TO THE COMPUTATIONAL AND THEORETICAL COMMUNITIES WITH LIMITED ACCESS TO EXPERIMENTAL FACILITIES. OUR PROJECT HAS THE POTENTIAL TO TRANSFORM SYSTEMS NEUROSCIENCE BY IMPLEMENTING AN OBSERVATORY MODEL THAT HAS BEEN TREMENDOUSLY SUCCESSFUL IN OTHER SCIENTIFIC FIELDS. BY REDUCING VARIABILITY IN TRAINING, IMAGING AND QUALITY CONTROL ACROSS ANIMALS AND EXPERIMENTS AND BY LOWERING THE BARRIER TO SELECT, TEST, AND VALIDATE THE BEST IDEAS, WE HOPE TO ACCELERATE PROGRESS TOWARD AN INTEGRATED VIEW OF NEURAL CIRCUITS.

NEUROPIXELS OPTO: INTEGRATED SILICON PROBES FOR CELL-TYPE-SPECIFIC ELECTROPHYSIOLOGY - PROJECT SUMMARY WITHIN EVERY BRAIN REGION, NEURONS CAN BE CLASSIFIED INTO DOZENS OR HUNDREDS OF DIFFERENT CELL TYPES, EACH WITH UNIQUE FUNCTIONAL ROLES AND UNIQUE IMPACTS ON DISEASE STATES. TRADITIONALLY, IN VIVO ELECTROPHYSIOLOGICAL RECORDINGS—WHICH HAVE MADE INVALUABLE CONTRIBUTIONS TO OUR UNDERSTANDING OF THE NEURAL BASIS OF BEHAVIOR— HAVE NOT BEEN ABLE TO DISTINGUISH THE ACTIVITY OF GENETICALLY DEFINED CELL TYPES. DESPITE RECENT ADVANCES IN OUR ABILITY TO MEASURE ACTION POTENTIALS FROM MANY NEURONS SIMULTANEOUSLY, IT REMAINS DIFFICULT TO CONNECT THESE SPIKE TRAINS TO UNDERLYING CELL TYPES AND ALL THAT IS KNOWN ABOUT THEIR MORPHOLOGY, CONNECTIVITY PATTERNS, AND INTRINSIC PROPERTIES. HERE, WE PROPOSE TO EXTEND THE WIDELY USED NEUROPIXELS PLATFORM BY CREATING A VERSION OF THESE PROBES THAT IS CAPABLE OF BOTH HIGH-DENSITY ELECTROPHYSIOLOGICAL RECORDING AND MULTI-COLOR LIGHT DELIVERY. THIS DEVICE, CALLED NEUROPIXELS OPTO, COULD BE USED TO IDENTIFY CELL TYPES THROUGH AN APPROACH KNOWN AS “OPTOTAGGING,” WHILE ALSO FACILITATING PRECISE MANIPULATIONS OF GENETICALLY DEFINED NEURAL POPULATIONS. WE WILL WORK WITH IMEC, A NANOELECTRONICS R&D ORGANIZATION WITH EXCLUSIVE ACCESS TO WORLD-CLASS FABRICATION FACILITIES, TO DESIGN AND BUILD A FULLY INTEGRATED IMPLANTABLE RECORDING DEVICE WITH 1280 ELECTRICAL READOUT SITES, UP TO 48 RED LIGHT EMITTERS, AND UP TO 62 BLUE LIGHT EMITTERS. A PROOF-OF-CONCEPT VERSION OF THIS PROBE HAS ALREADY BEEN DELIVERED TO THE ALLEN INSTITUTE, WHERE IT WAS SUCCESSFULLY USED IN AN OPTOTAGGING EXPERIMENT. HOWEVER, MORE WORK IS REQUIRED TO DEVELOP A DEVICE THAT CAN BE MANUFACTURED AT SCALE AND SOLD FOR AROUND $2500/PROBE, IN ORDER TO FACILITATE ITS DISSEMINATION THROUGHOUT THE SYSTEMS NEUROSCIENCE COMMUNITY. IN COLLABORATION WITH THREE EXTERNAL TEST SITES, WE WILL VALIDATE THE EFFICACY OF NEUROPIXELS OPTO FOR PERFORMING CELL-TYPE-SPECIFIC RECORDINGS AND MANIPULATIONS IN FOUR BRAIN REGIONS: THE VISUAL CORTEX, ENTORHINAL CORTEX, STRIATUM, AND THE VENTRAL TEGMENTAL AREA. IN ADDITION, WE WILL EXTEND POPULAR DATA ACQUISITION PACKAGES BY DEVELOPING MODULES FOR CONTROLLING THESE PROBES, AS WELL AS CREATE NEW TRANSGENIC MOUSE LINES THAT MAKE IT MUCH SIMPLER FOR USERS TO CARRY OUT DUAL-COLOR OPTOGENETIC MANIPULATIONS. TAKEN TOGETHER, THESE EFFORTS WILL MAKE NEUROPIXELS OPTO A POWERFUL, ACCESSIBLE, AND INDISPENSABLE TOOL FOR UNDERSTANDING THE ROLE THAT DIFFERENT CELL TYPES PLAY IN LIVING BRAINS.

MOLECULAR AND ANATOMICAL CHARACTERIZATION OF CELL TYPES IN THE AGING MOUSE BRAIN

BRAIN CONNECTS: PATCHLINK, SCALABLE TOOLS FOR INTEGRATING CONNECTOMES, PROJECTOMES, AND TRANSCRIPTOMES - PROJECT SUMMARY / ABSTRACT UPCOMING BRAIN-WIDE DESCRIPTIONS OF SYNAPTIC CONNECTIVITY ARE POISED TO TRANSFORM OUR UNDERSTANDING OF BRAIN CIRCUITRY IN THE SAME WAY SINGLE-CELL GENOMICS HAS REVOLUTIONIZED OUR UNDERSTANDING OF CELL TYPE DIVERSITY. THE CHALLENGE OF RELATING WHOLE-BRAIN WIRING DIAGRAMS TO CELL-TYPE GENETIC PROPERTIES MUST BE OVERCOME IN ORDER TO FULLY REALIZE THE POTENTIAL OF THESE DATASETS. VERY FEW TECHNIQUES GENERATE MULTI-MODALITY, "ROSETTA STONE" DATASETS NEEDED TO LINK CELL TYPES TO CONNECTIVITY, AND NONE PRESENTLY HAVE THE THROUGHPUT TO DO SO ACROSS AN ENTIRE MAMMALIAN BRAIN. IN THIS PROPOSAL, WE ADDRESS KEY LIMITATIONS THAT CURRENTLY PREVENT SUCH TECHNIQUES FROM SCALING TO MEET THE THROUGHPUT OF WHOLE-MOUSE-BRAIN CONNECTIVITY INITIATIVES, AND DEVELOP THE COMPUTATIONAL FRAMEWORKS NEEDED TO BIND CELL TYPES TO WIRING DIAGRAMS. THE PATCH-SEQ METHOD LINKS THE FULL GENE EXPRESSION PROFILE OF SINGLE NEURONS WITH THEIR FUNDAMENTAL PROPERTIES, INCLUDING LOCAL MORPHOLOGY AND ELECTROPHYSIOLOGY1,2. IN AIM 1, WE WILL AUTOMATE THE PATCH-SEQ TECHNIQUE TO ALLOW PARALLELIZATION AND SCALING SUFFICIENT FOR WHOLE MOUSE BRAIN COVERAGE. THIS WILL BE ACHIEVED BY INTEGRATING AND OPTIMIZING RECENTLY DEVELOPED METHODS FOR PATCH CLAMP AUTOMATION, INCLUDING PIPETTE CLEANING, CELL DETECTION, AND MACHINE LEARNING APPROACHES TO CELL IDENTIFICATION AND TRACKING. DEVELOPMENTS WILL BE FULLY DOCUMENTED AND PACKAGED FOR DISSEMINATION TO LOWER BARRIERS TO ACCESS AND FURTHER IMPROVE THROUGHPUT VIA COLLABORATIVE DATA GENERATION. SIMILARLY, METHODS FOR RECONSTRUCTING THE BRAIN-WIDE FULL MORPHOLOGY OF SINGLE NEURONS PROVIDES SIMULTANEOUS ACCESS TO THEIR LOCAL MORPHOLOGY AND LONG-RANGE PROJECTION TARGETS. IN AIM 2, WE WILL IMPROVE AND EXTEND THE QUALITY, EFFICIENCY, AND CAPABILITY OF OUR AUTOMATIC MORPHOLOGICAL RECONSTRUCTION PIPELINE BY ADOPTING NEW APPROACHES TO RECONSTRUCTION (E.G., A HIERARCHY OF DEEP LEARNERS), AND TESTING ADVANCED METHODS FOR TISSUE PROCESSING AND IMAGING ACROSS OUR PATCH-SEQ AND FULL MORPHOLOGY DATA GENERATION PIPELINES. AUTOMATED RECONSTRUCTION METHODS WILL BE TRAINED AND TESTED ON GOLD STANDARD DATA. TOOLS AND DATA WILL BE COLLABORATIVELY GENERATED AND PUBLICLY SHARED. IN AIM 3, WE WILL DEVELOP NEW COMPUTATIONAL FRAMEWORKS TO LINK WHOLE-BRAIN CONNECTIVITY DATASETS TO MULTI- MODALITY CELL TYPE DATASETS. POWERED BY THE THROUGHPUT ACHIEVED IN AIMS 1 AND 2, WE WILL DEVELOP, APPLY, AND SHARE MACHINE LEARNING-BASED DATA ANALYSIS METHODS TO SYNTHESIZE THE OBSERVATIONS COLLECTED FROM INDIVIDUAL PLATFORMS TO ACHIEVE AN INTEGRATED AND PREDICTIVE UNDERSTANDING OF NEURONAL IDENTITY. THIS APPROACH, WHICH FACILITATES CELL TYPE ASSIGNMENT, CROSS-MODALITY INTEGRATION AND INFERENCE, AND CHARACTERIZATION OF THE DISCRETENESS AND CONTINUITY OF FUNDAMENTAL CELLULAR PROPERTIES WITHIN AND ACROSS TYPES, WILL BE SCALED TO ACHIEVE WHOLE MOUSE BRAIN COVERAGE.

ENHANCER AAV TOOLBOX FOR CELL CLASSES AND SUBCLASSES IN THE BRAINS OF MOUSE, MACAQUE AND MARMOSET. - PROJECT SUMMARY WE PROPOSE TO CREATE, SCREEN, CHARACTERIZE, AND DISTRIBUTE (THROUGH ONE OR MORE NON-PROFIT PARTNERS) A LARGE SET OF RECOMBINANT ADENO ASSOCIATED VIRUS (AAV) TOOLS TO TARGET THE CELL CLASSES, SUBCLASSES, AND SELECT TYPES IN THE MOUSE, MARMOSET AND MACAQUE BRAINS. WE PROPOSE TO LEVERAGE OUR NEW WHOLE MOUSE BRAIN (WMB) SINGLE- NUCLEUS MULTIOME DATASET TO NOMINATE MOUSE ENHANCERS FOR CLASSES AND SUBCLASSES IN THE MOUSE BRAIN. WE WILL EMPLOY A SECOND-GENERATION ENHANCER DISCOVERY, SCREENING AND CHARACTERIZATION PIPELINE TO EVALUATE AT LEAST 2000 ENHANCERS IN MOUSE, 100 IN MACAQUE AND 30 IN MARMOSET. THE CHARACTERIZATION OF ENHANCER VIRUS SPECIFICITY AND LABELING COMPLETENESS WILL BE CARRIED OUT IN MOUSE WITH A TRI-MODAL PIPELINE: WMB LIGHT SHEET IMAGING, WMB 10XFLEX SN-TRANSCRIPTOMICS, AND SPATIAL TRANSCRIPTOMICS ON SELECT SECTIONS WITH BARSEQ. THE ENHANCERS WITH DESIRABLE EXPRESSION PATTERNS WILL BE MODIFIED TO INCREASE EXPRESSION STRENGTH THROUGH ENHANCER CORE CONCATENATION AND TESTED FOR DRIVING DIFFERENT CARGOS (E.G., CRE RECOMBINASE). THE CHARACTERIZATION OF ENHANCER SPECIFICITY IN MACAQUE AND MARMOSET WILL RELY ON BARSEQ FOR THE INJECTED BRAIN REGIONS AT THE LEVEL OF CLASS AND SUBCLASS, AND WHERE FEASIBLE, SUPERTYPES. IN PARALLEL WITH THE BRAIN-WIDE ENHANCER AAV DISCOVERY, WE WILL CONDUCT A FOCUSED ENHANCER AAV DISCOVERY AT FINE CELL-TYPE GRANULARITY FOR MIDBRAIN DOPAMINERGIC NEURON TYPES AND SUBTYPES WITH EMPHASIS ON CROSS-SPECIES VALIDATION AND CONSERVATION. THESE CELL TYPES ARE KEY PLAYERS IN THE BRAIN CIRCUITS OF REWARD, MOTIVATION, AND MOTOR CONTROL AND THE TOOLS DISCOVERED HERE COULD BE APPLICABLE TO ADDRESSING CIRCUIT DYSFUNCTION IN BASAL GANGLIA DISORDERS. IF SUCCESSFUL, THIS AWARD WILL ESTABLISH AN UNPRECEDENTED MAMMALIAN BRAIN TOOLBOX SHOWCASING BOTH A BROAD BRAIN-WIDE APPROACH AND A FOCUSED CELL-TYPE APPROACH FOR THE NEUROSCIENCE COMMUNITY WITH REAGENTS, CHARACTERIZATION DATA AND PROTOCOLS FOR USE AVAILABLE TO ALL. THE DELIVERABLES FROM THIS AWARD COULD HAVE A TRANSFORMATIVE EFFECT ON CELL TYPE CHARACTERIZATION IN THE MAMMALIAN CENTRAL NERVOUS SYSTEMS, FROM MEASURING VARIOUS PROPERTIES OF CELL TYPES TO DEFINING THEIR FUNCTION AND THE DETAILED UNDERSTANDING OF STRUCTURE-FUNCTION RELATIONSHIPS IN MAMMALIAN BRAINS, AND POTENTIALLY, DEVELOPMENT OF CELL TYPE-SPECIFIC THERAPEUTICS FOR BRAIN DISORDERS.

BRAIN CONTROL OF INTERNAL ORGAN FUNCTION - ABSTRACT ADAPTIVE CONTROL OF BEHAVIOR IS CRITICAL FOR SURVIVAL. EVEN A SIMPLE MOVEMENT, LIKE EXTENDING THE ARM, REQUIRES THE ACTIVATION OF MANY NEURONAL POPULATIONS ACROSS THE NERVOUS SYSTEM. OUR LAB HAS USED A COMBINATION OF ANATOMICAL, GENETIC, OPTICAL AND BEHAVIORAL APPROACHES TO UNRAVEL HOW ANIMALS MOVE, AND LEARN TO CONTROL MOVEMENT. HOWEVER, ADAPTIVE RESPONSES ARE NOT EFFECTED ONLY THROUGH MUSCLES, BUT ALSO THROUGH OTHER ORGANS. FOR EXAMPLE, PLANNING TO PICK AN APPLE WILL TRIGGER NOT ONLY MUSCLE ACTIVITY BUT ALSO THE EXPECTATION OF FOOD, AND THE CONDITIONED RELEASE OF INSULIN. HENCE ADAPTIVE BEHAVIOR REQUIRES THE COORDINATION OF AN ORGANISM'S ACTIONS WITH ITS PHYSIOLOGICAL INTERNAL STATES. WE PROPOSE TO LEVERAGE OUR EXPERTISE TO DISSECT THE NEURAL CIRCUITS AND PRINCIPLES GOVERNING THE LEARNING AND ADAPTIVE “MOTOR” CONTROL OF INTERNAL ORGAN FUNCTION. WE WILL SPEARHEAD THIS NEW RESEARCH DIRECTION BY INVESTIGATING CONDITIONED INSULIN RELEASE AND CONDITIONED IMMUNOSUPPRESSION, MEDIATED BY THE INNERVATION OF THE PANCREAS AND SPLEEN, RESPECTIVELY. WE WILL LEVERAGE STATE OF THE ART VIRAL AND RNA-SEQ APPROACHES TO MAP WITH HIGH-RESOLUTION THE FIRST, SECOND AND THIRD-ORDER INNERVATION OF SPLEEN AND PANCREAS. OUR PRELIMINARY ANATOMICAL MAPPING OF THE INNERVATION OF THESE ORGANS REVEALED THAT DIFFERENT POPULATIONS OF CELIAC-MESENTERIC GANGLIA SYMPATHETIC NEURONS INNERVATE PANCREAS VERSUS SPLEEN. REMARKABLY, MOST INNERVATION OF THE THORACIC PREGANGLIONIC SPINAL CORD TARGETING THESE ORGANS EMERGES FROM THE CORTEX: MOTOR CORTEX, BUT ALSO SENSORY AND PREFRONTAL. WE THEREFORE HYPOTHESIZE THAT LEARNING TO SELECT THE APPROPRIATE RESPONSES IN INTERNAL ORGANS AFTER CONDITIONING IS MEDIATED BY HIGHER-ORDER BRAIN CIRCUITS, AND FOLLOWS PRINCIPLES SIMILAR TO THOSE USED FOR MOTOR RESPONSES. WE PROPOSE TO USE BOTH TARGETED AND UNBIASED APPROACHES TO IDENTIFY AND MANIPULATE THE ACTIVITY OF DESCENDING NEURAL POPULATIONS RESPONSIBLE FOR THE LEARNED CONTROL OF SPLEEN AND PANCREATIC FUNCTION. THIS NEW LINE OF RESEARCH IS INNOVATIVE BUT TRACKABLE WITH OUR EXPERTISE, AND THE PIONEER AWARD SUPPORT WILL HELP US ATTACK THIS NOVEL RESEARCH AREA. IMPORTANTLY, THE PROPOSED RESEARCH HAS THE POTENTIAL TO CONCEPTUALLY POSITION THE NERVOUS SYSTEM AS A “SMART” REGULATOR OF ORGANISM HOMEOSTASIS, AND HENCE IMPACT HEALTH IN UNEXPECTED WAYS - MENTAL DISORDERS LIKE ANXIETY AND DEPRESSION, OR NEUROLOGICAL PROBLEMS LIKE STROKE, ARE ASSOCIATED WITH ABNORMAL PHYSIOLOGICAL STATES LIKELY EMERGING FROM THESE BRAIN-INTERNAL ORGAN INTERACTIONS.

FUNCTIONAL AND CELL-TYPE SPECIFIC AXONAL PATHWAYS IN THE PRIMATE BRAIN - PROJECT SUMMARY/ABSTRACT OVER THE PAST DECADE, THERE HAVE BEEN TRANSFORMATIVE ADVANCES IN THREE AREAS OF MAMMALIAN NEUROSCIENCE. FIRST, OUR ABILITY TO RECORD FROM LARGE POPULATIONS OF NEURONS HAS DRAMATICALLY INCREASED WITH THE ADVENT OF NEW ELECTRODE TECHNOLOGIES AND IMPROVED MULTIPHOTON IMAGING. SECOND, THE STUDY OF BRAIN CONNECTIONS IN THEIR ENTIRETY, CONNECTOMICS, HAS COME INTO ITS OWN AS A FIELD. MOST RECENTLY, THERE HAS BEEN A REVOLUTION IN THE CLASSIFICATION OF NEURONAL TYPES, LARGELY BY PROBING WHICH GENES ARE TRANSLATED INTO RNA IN CELLS (TRANSCRIPTOMICS). WHAT IS LACKING IS A WAY TO BRING THESE THREE FIELDS TOGETHER, PARTICULARLY IN THE STUDIES OF NON-HUMAN PRIMATES AND HUMANS. THE GOAL OF THE CURRENT RFA IS TO CREATE INNOVATIVE TOOLS FOR USE IN HUMANS AND NON-HUMAN PRIMATES. IN PARTICULAR, TWO SUGGESTED TOPICS ARE TO DEVELOP: (1) “NOVEL METHODS FOR TAGGING INDIVIDUAL NEURONS SUCH THAT CELLULAR COMPONENTS OF A FUNCTIONAL CIRCUIT CAN BE EXPLORED” AND (2) “INNOVATIVE APPROACHES TO BRIDGE SCALES OF EXPERIMENTAL APPROACH. STUDIES THAT CAN EXPLORE MOLECULAR AND CELLULAR MECHANISMS OF NEURAL ACTIVITY IN BROADER CONTEXTS ARE ENCOURAGED.” TO ACHIEVE THESE GOALS, WE PRESENT AN INNOVATIVE APPROACH TO CHARACTERIZE NEURONAL CELL TYPES IN MACAQUES AND HUMANS, COMBINING TRANSCRIPTOMICS, INTER-AREAL CONNECTIVITY AND FUNCTIONAL STUDIES AT MULTIPLE SCALES, FROM INDIVIDUAL NEURONS TO ENTIRE BRAINS. WE WILL BUILD THE NECESSARY TOOLS TO CREATE INTEGRATED ATLASES OF INDIVIDUAL BRAINS THAT COMBINE SIX MODALITIES INTO A COMMON REFERENCE FRAME: (1) FUNCTIONAL MRI, TO MEASURE FUNCTIONAL PROPERTIES OF BRAIN AREAS AT 0.5-1 MM RESOLUTION, (2) WIDEFIELD OPTICAL IMAGING, TO MAP BULK NEURONAL ACTIVITY AT THE CORTICAL SURFACE WITH ~100 ΜM RESOLUTION, (3) MULTIPHOTON CALCIUM IMAGING, TO MAP NEURONAL ACTIVITY IN INDIVIDUAL NEURONS ACROSS MULTIPLE CORTICAL AREAS, (4) DIFFUSION TENSOR IMAGING (DTI), TO MAP AXONAL TRACTS IN THE WHITE MATTER WITH 0.5-1 MM RESOLUTION, (5) “AXONAL CONNECTOMICS”, TO MAP PROJECTIONS OF INDIVIDUAL MYELINATED AXONS FROM EFFERENT CELL BODIES TO THEIR POSTSYNAPTIC TARGETS, AND (6) MULTIPLEXED FISH, TO ASSAY TRANSCRIPTOMIC IDENTITIES OF THE SAME CELLS WHOSE PHYSIOLOGY AND PROJECTION TARGETS HAVE BEEN DEFINED. DATA FROM FIRST THREE MODALITIES WILL BE COLLECTED, STARTING WITH IN VIVO STUDIES OF MACAQUES AND CORRELATED WITH SUBSEQUENT ANALYSIS OF BRAIN TISSUE WITH THE LAST THREE MODALITIES. ONLY THE LAST THREE STEPS WILL BE USED IN THE STUDY OF HUMAN BRAIN TISSUE, ALTHOUGH FUNCTIONAL MRI COULD, IN PRINCIPLE, BE OBTAINED FROM RESEARCH INSTITUTIONS WITH APPROPRIATE PROGRAMS FOR PROSPECTIVE STUDIES.

ESTABLISHING A COMPREHENSIVE AND STANDARDIZED CELL TYPE CHARACTERIZATION PLATFORM

FUNCTIONAL MICRO-ARCHITECTURE OF THE VISUAL CORTEX

BRAIN CONNECTS: AN INTEGRATIVE CONNECTOMICS COORDINATION CENTER (IC3) - PROJECT SUMMARY THE UNIQUE CAPABILITIES OF MAMMALIAN BRAINS ARISE FROM THEIR EXTRAORDINARILY COMPLEX NETWORK STRUCTURE, WITH BILLIONS TO TRILLIONS OF SYNAPTIC CONNECTIONS AMONG MILLIONS TO BILLIONS OF NEURONS THAT FORM THOUSANDS OF CELL TYPES AND COUNTLESS FUNCTIONALLY SPECIFIC CIRCUIT PATHWAYS. UNRAVELING THIS NETWORK STRUCTURE, THE CONNECTOME, HOLDS THE KEY TO UNDERSTANDING HOW THE BRAIN GENERATES BEHAVIOR, THOUGHTS, EMOTION AND OTHER FUNCTIONS. THE NEWLY LAUNCHED BRAIN INITIATIVE CONNECTIVITY ACROSS SCALES (BRAIN CONNECTS) PROGRAM AIMS TO DEVELOP CUTTING- EDGE, HIGHLY SCALABLE TECHNOLOGY PLATFORMS THAT ENABLE THE GENERATION OF UNPRECEDENTED VOLUMES OF DATA IN DIFFERENT AND COMPLEMENTARY MODALITIES, TO CREATE COMPREHENSIVE BRAIN-WIDE CONNECTIVITY MAPS IN MOUSE, NON- HUMAN PRIMATE (NHP) AND HUMAN AT DIFFERENT RESOLUTIONS. THESE CONNECTIVITY MAPS WILL SERVE AS FOUNDATIONAL RESOURCES TO THE COMMUNITY TO DRAMATICALLY ACCELERATE AND TRANSFORM OUR UNDERSTANDING OF BRAIN FUNCTIONS AND DISEASES. THE INTEGRATIVE CONNECTOMICS COORDINATION CENTER (IC3) PROPOSED HERE WILL WORK CLOSELY WITH BRAIN CONNECTS DATA GENERATION TEAMS TO ENSURE THE SUCCESSFUL REALIZATION OF THE PROGRAM GOALS. THE IC3 BRINGS TOGETHER A TEAM OF INVESTIGATORS WITH EXTENSIVE EXPERIENCE IN VARIOUS CONNECTOMICS TECHNOLOGIES, LARGE-SCALE EXPERIMENTAL AND COMPUTATIONAL PLATFORM DEVELOPMENT, AND CONSORTIUM MANAGEMENT AND LEADERSHIP. WE WILL COORDINATE THE ACTIVITIES OF THE BRAIN CONNECTS NETWORK ACROSS COMPREHENSIVE CENTERS AND SPECIALIZED TECHNOLOGY GROUPS. COLLABORATING WITH DATA GENERATION GROUPS, WE WILL DEVELOP AND HARMONIZE THREE OF THE FOUR COMMON DATA PROCESSING PIPELINES IN THE NETWORK, INCLUDING ELECTRON MICROSCOPY, FLUORESCENCE LIGHT MICROSCOPY, AND BARCODED SEQUENCING DATASETS. WE WILL ORGANIZE AND CONDUCT RIGOROUS EVALUATION OF THE TECHNOLOGIES FOR IMPROVED PERFORMANCE AND SCALABILITY, TO ENABLE THE NEXT PHASE OF WHOLE-BRAIN SCALE DATA GENERATION. WE WILL DEVELOP CLOUD-BASED DATA PLATFORMS AND ANALYTIC TOOLS TO FACILITATE CONSORTIUM-WIDE DATA INTEGRATION AND JOINT ANALYSIS. WE WILL BROADLY DISTRIBUTE THESE TOOLS AND PRODUCTS FOR DEMOCRATIZED ACCESS AND ANALYSIS BY THE RESEARCH COMMUNITY. WE WILL ESTABLISH A BRAIN CONNECTS KNOWLEDGE BASE (CONNECTS-KB), ALONG WITH ADVANCED AI/ML BASED TOOLS, TO INCORPORATE CONNECTIVITY DATA INTO WHOLE BRAIN COMMON COORDINATE FRAMEWORKS (CCFS), ACHIEVE INTEGRATION BETWEEN MOLECULARLY DEFINED CELL TYPES AND CONNECTIVITY, AND CREATE A UNIFIED REPRESENTATION OF BRAIN CONNECTIVITY ACROSS DATA MODALITIES AND ACROSS SPECIES. WE WILL FACILITATE THE DEMONSTRATION OF SCIENCE AND HEALTH IMPACTS OF THE BRAIN CONNECTS PROGRAM TO THE BROAD RESEARCH COMMUNITY AND THE GENERAL PUBLIC, THROUGH PROOF-OF-CONCEPT CASE STUDIES, ONLINE TOOLS ALLOWING EXTERNAL RESEARCHERS TO ACCESS AND USE THE INTEGRATED DATA AND CONNECTIVITY MAPS, AND PUBLIC-FACING OUTREACH AND EDUCATION ACTIVITIES.

VIRTUAL OBSERVATORY OF THE CORTEX: ORGANELLES, CELLS, CIRCUITS, AND DYNAMICS - WE PROPOSE TO CREATE VORTEX, A VIRTUAL OBSERVATORY FOR THE CORTEX: SPANNING THE SCALES OF ORGANELLES, CELLS, CIRCUITS, AND DYNAMICS. THE OBSERVATORY WILL DISSEMINATE AN EXISTING DATASET: AN AUTOMATED RECONSTRUCTION OF ALL CELLS IN A CUBIC MILLIMETER OF MOUSE VISUAL CORTEX, ALONG WITH THE SYNAPTIC CONNECTIVITY OF THE NEURONS AND CALCIUM-IMAGED RESPONSES TO VIDEO STIMULI. THE CUBIC MILLIMETER VOLUME SPANS ALL LAYERS OF CORTEX AND FOUR VISUAL AREAS (V1, LM, AL, RL). A TEAM OF HUMAN PROOFREADERS WILL DETECT AND CORRECT THE REMAINING ERRORS IN THE AUTOMATED SEGMENTATION. SUCH SEMIAUTOMATED RECONSTRUCTION BY “PROOFREADING” IS MUCH FASTER THAN MANUAL TRACING OF NEURONS. CONSIDERABLE HUMAN PROOFREADING EFFORT IS STILL REQUIRED, HOWEVER, DUE TO THE ENORMOUS SCALE OF THE DATA. OUR CUBIC MILLIMETER VOLUME IS 35X LARGER THAN AN ENTIRE DROSOPHILA BRAIN, AND 70X LARGER THAN THE JANELIA FLY “HEMIBRAIN.” THE PROOFREADING TEAM CAN BE VIEWED AS A NEW KIND OF SCIENTIFIC INSTRUMENT. JUST AS A TELESCOPE IS DIRECTED BY THE ASTRONOMY COMMUNITY, PROOFREADING EFFORTS SHOULD BE DIRECTED BY THE NEUROSCIENCE COMMUNITY. BASED ON REQUESTS FROM USERS (TARGET CELLS, SCIENTIFIC RATIONALE), THE PROOFREADING SCHEDULE WILL BE DECIDED BY A SCIENTIFIC STEERING COMMITTEE. THE COMMITTEE WILL CONSIST OF REPRESENTATIVES FROM FIVE SUBFIELDS: NEURONAL CELL BIOLOGY, GLIAL CELL BIOLOGY, CORTICAL CELL TYPES, CORTICAL CIRCUITS, AND THEORY/SIMULATION. OUR NOVEL APPROACH OF CENTRALIZED LABOR, BUT DISTRIBUTED GOVERNANCE, COMBINED WITH FAST PUBLIC RELEASES OF PROOFREADING UPDATES, SHOULD ACCELERATE SCIENTIFIC DISCOVERY. THE PROOFREADING TEAM WILL FOLLOW ESTABLISHED QUALITY CONTROL PROCEDURES, AND QUANTITATIVE METRICS OF ACCURACY WILL BE MAINTAINED. UPDATES TO THE RECONSTRUCTED WIRING DIAGRAM WILL BE MADE ON AN AT LEAST QUARTERLY BASIS. INTERACTIVE VIEWING OF THE EM IMAGES AND AUTOMATED SEGMENTATION WILL BE POSSIBLE FROM ANY SITE WITH AN INTERNET CONNECTION. USERS MAY EXECUTE LARGE-SCALE COMPUTATIONAL ANALYSES ON THEIR LOCAL CLUSTERS OR COMMERCIAL CLOUD THROUGH PROGRAMMATIC DOWNLOADING OF THE DATA USING OUR CLOUDVOLUME SOFTWARE. A COMMUNITY MANAGER WILL SOLICIT PROOFREADING REQUESTS FROM THE USER COMMUNITY, DESIGN A PROOFREADING SCHEDULE THAT MAXIMIZES FULFILLMENT OF REQUESTS, AND PROPOSE THE SCHEDULE TO THE STEERING COMMITTEE. THE MANAGER WILL ALSO RECRUIT USERS AND HELP THEM WITH THE MECHANICS OF ACCESSING THE DATA. THE ALLEN INSTITUTE WILL CONTINUE TO EDUCATE IN THE USE OF THIS DATA AT THE SUMMER WORKSHOP ON THE DYNAMIC BRAIN. THE VORTEX WILL PROVIDE FUNDAMENTAL KNOWLEDGE ABOUT THE CORTEX. THE CELL BIOLOGY OF NEURONS AND GLIA IS RELEVANT FOR BRAIN DISEASES, AND THE NORMAL WIRING DIAGRAM OF THE CORTEX COULD BE RELEVANT TO THE SEARCH FOR CONNECTOPATHIES.

VIRAL STRATEGIES FOR FUNCTIONAL CONNECTOMICS IN THE VISUAL SYSTEM

DEVELOPMENT OF TOOLS FOR CELL-TYPE SPECIFIC LABELING OF HUMAN AND MOUSE NEOCORTICAL NEURONS

STRUCTURE-FUNCTION CELL ATLAS FOR NOREPINEPHRINE AND SEROTONIN NEURONS - PROJECT ABSTRACT NOREPINEPHRINE (NE) AND SEROTONIN (5-HT) ARE NEUROTRANSMITTERS RELEASED BY A SMALL NUMBER OF NEURONS IN THE LOCUS COERULEUS (LC) AND DORSAL RAPHE (DR), RESPECTIVELY. EACH GROUP OF NEURONS PROVIDES EXTENSIVE INNERVATION OF MOST OF THE BRAIN. ALTHOUGH THESE GROUPS OF CELLS ARE OFTEN CONSIDERED TO BE HOMOGENOUS, RECENT DATA INDICATES THE EXISTENCE OF SUBCLASSES OF EACH NEURON. THIS PROPOSAL AIMS TO GENERATE MULTI-SCALE DATA ON EACH SYSTEM, COMPRISING MOLECULAR, GENETIC, ANATOMICAL, PHYSIOLOGICAL, AND BEHAVIORAL EXPERIMENTS. OUR TEAM IS IN A UNIQUE POSITION TO INTEGRATE ACROSS EXPERIMENTS AND DEVELOP A NEW UNDERSTANDING OF THE SUBCLASSES OF LC-NE AND DR-5-HT NEURONS AND THEIR FUNCTIONS DURING BEHAVIOR. THREE AIMS TEST SPECIFIC QUESTIONS ABOUT THE CELL TYPES AND FUNCTIONS OF LC-NE AND DR-5-HT NEURONS IN MICE: 1) LC-NE NEURON SUBCLASSES HAVE DISTINCT BIOPHYSICAL PROPERTIES AND AXONAL PROJECTIONS; 2) SUBCLASSES OF DR-5-HT NEURONS HAVE DISTINCT BIOPHYSICAL PROPERTIES AND AXONAL PROJECTIONS; AND 3) SUBCLASSES OF LC-NE AND DR-5-HT NEURONS HAVE DIFFERENT FUNCTIONS DURING DYNAMIC DECISION MAKING. OUR EXPERIMENTS WILL BE USED TO DEVELOP A COMPREHENSIVE CLASSIFICATION OF NE AND 5-HT NEURONAL SUBCLASSES AND WILL BE USED TO GENERATE REAGENTS TO TARGET THOSE CELLS.

SCALABLE TECHNOLOGIES FOR BRAIN-WIDE CONNECTOMICS OF TRANSCRIPTOMIC CELL TYPES: FOCUS ON BRAINSTEM - PROJECT SUMMARY, SCALABLE TECHNOLOGIES FOR BRAIN-WIDE CONNECTOMICS OF TRANSCRIPTOMIC CELL TYPES: FOCUS ON BRAINSTEM THIS PROPOSAL IS TO DEVELOP A SCALABLE PIPELINE TO COMBINE HIGH-RESOLUTION MORPHOLOGY AND MOLECULAR CLASSIFICATION OF INDIVIDUAL NEURONS TO DEFINE MORPHO-MOLECULAR CELL TYPES IN THE BRAIN. COMPLETE MORPHOLOGY OF INDIVIDUAL NEURONS PROVIDES INSIGHTS OF CONNECTIVITY AND INFORMATION PROCESSING IN THE BRAIN AND REVEALS HOW NEURONAL ACTIVITY IS ROUTED ACROSS BRAIN AREAS. LAYERING TRANSCRIPTOMIC INFORMATION ON TO MORPHOLOGICALLY DISTINCT TYPES PROVIDES THE BASIS TO ACCESS THESE DEFINED NEURON TYPES FOR FUNCTIONAL ANALYSIS. SUCH A COMBINED CLASSIFICATION OF THE BRAIN’S CELL TYPES IS FOUNDATIONAL FOR UNDERSTANDING THE ROLE OF DEFINED NEURON TYPES WITHIN NEURAL CIRCUITS AND HOW INFORMATION PROCESSING WITHIN MULTI-REGIONAL NEURAL CIRCUITS ORCHESTRATE COMPLEX BEHAVIORS. SEQUENCING-BASED APPROACHES HAVE BEEN USED TO CATEGORIZE THE BRAIN’S CELLS INTO TRANSCRIPTOMIC TYPES (T- TYPES) WITH HIGH THROUGHPUT. PARALLEL STRATEGIES FOR A COMPLETE DESCRIPTION OF THE MORPHOLOGICAL TYPES BRAIN-WIDE ARE TOO SLOW AND METHODS FOR A COMBINED ANALYSIS OF THESE TWO MODALITIES ARE LACKING. TO ADDRESS THIS, WE WILL CREATE A BRAIN-WIDE IMAGING AND NEURONAL RECONSTRUCTION PLATFORM THAT PROVIDES FASTER IMAGING USING SELECTIVE PLANE ILLUMINATION MICROSCOPY AND ACCELERATED RECONSTRUCTIONS WITH MODERN MACHINE LEARNING TOOLS BASED ON U-NETS AND REINFORCEMENT LEARNING. WE WILL COMBINE THIS WITH POST HOC TRANSCRIPTOMIC CHARACTERIZATION OF RECONSTRUCTED CELLS WITH MULTIPLEXED FLUORESCENT IN SITU HYBRIDIZATION TO DEFINE MORPHO-MOLECULAR TYPES. WE WILL CREATE A DATA SET OF 2,000 SUCH DUAL CATEGORIZED NEURON TYPES FROM A CRITICAL BRAIN AREA, THE MEDULLA, IN THE MOUSE. THE MEDULLA IS COMPRISED OF DIVERSE NEURONAL TYPES ORGANIZED IN NUMEROUS INTER-RELATED NUCLEI ESSENTIAL FOR AUTONOMIC FUNCTIONS SUCH AS BREATHING, VASOMOTOR CONTROL, INTEGRATION OF ASCENDING INPUTS FROM SENSORY AND INTEROCEPTIVE CHANNELS AND COORDINATION OF MOTOR ACTIONS SUCH AS CHEWING, LICKING AND SWALLOWING. OUR CENSUS OF MORPHO-MOLECULAR MEDULLARY NEURON TYPES WILL LAY THE FOUNDATION FOR A SYSTEMATIC CELL TYPE SPECIFIC FUNCTIONAL INTERROGATION OF THESE NEURONS WITHIN BRAINSTEM CIRCUITS AND IN THE LARGER CONTEXT OF MULTI- REGIONAL BRAIN CIRCUITS. FURTHERMORE, THIS WILL SERVE AS THE BLUEPRINT FOR CARRYING OUT SUCH STUDIES THROUGHOUT THE MOUSE BRAIN, AND OTHER BRAINS, INCLUDING THAT OF PRIMATES.

BRAIN CONNECTS: LIGHT MICROSCOPY FOR CELL-TYPE SPECIFIC MESOSCALE TO NANOSCALE DENSE CONNECTOMICS - PROJECT SUMMARY IN RECENT YEARS, THERE HAS BEEN REMARKABLE PROGRESS IN MAPPING THE CONNECTIVITY OF CELL TYPES IN THE BRAIN. WE NOW HAVE COMPLETE CONNECTOMES FOR INSECTS AND WORMS, AND IN MAMMALS, WE CAN MAP SYNAPSE CONNECTIVITY ACROSS MILLIMETER-SCALE REGIONS. AS THE BRAIN CONNECTS PROGRAM AIMS TO EXTEND THIS CAPABILITY TO ENTIRE MAMMALIAN BRAINS ACROSS MULTIPLE SCALES, IT IS ESSENTIAL TO LINK THESE CONNECTIVITY BREAKTHROUGHS WITH THE BRAIN'S MOLECULAR ARCHITECTURE. UNTIL NOW, THE CONNECTION BETWEEN FINE CONNECTIVITY OBTAINED THROUGH ELECTRON MICROSCOPY (EM) AND MOLECULAR CELL TYPES HAS BEEN ESTABLISHED THROUGH INDIRECT METHODS SUCH AS PATCH-SEQ. RECENT DEVELOPMENTS IN LIGHT-MICROSCOPY-BASED CONNECTOMICS (LICONN) HAVE SHOWN PROMISING RESULTS FOR DENSE CONNECTIVITY MAPPING. LICONN HAS THE POTENTIAL TO REGISTER CONNECTOMICS RESULTS TO MOLECULAR SUBCLASSES AT THE SINGLE-NEURON LEVEL AND AT SCALE. THIS PROJECT LEVERAGES LICONN AND OUR EXPERTISE IN CREATING AND ANALYZING EM DATA TO ADDRESS THREE MAIN OBJECTIVES OF THE BRAIN CONNECTS PROGRAM.THE FIRST OBJECTIVE IS TO DEMONSTRATE THAT LICONN CAN BE USED TO CREATE MOLECULARLY INFORMED CELL TYPE CONNECTIVITY PATTERNS BY INTEGRATING ITS RESULTS WITH LARGE-SCALE EM DATA. THIS APPROACH CAN PROVIDE A POWERFUL TOOL TO ESTABLISH A GROUND TRUTH LINK BETWEEN MOLECULAR AND CONNECTIVITY TYPES ACROSS DATASETS PLANNED WITH THE CONNECTS PORTFOLIO. THE SECOND OBJECTIVE IS TO COMBINE LIGHT-MICROSCOPY-BASED CONNECTOMICS WITH MESOSCALE EXPANSION MICROSCOPY IN A MULTISCALE FASHION. THIS INTEGRATION ALLOWS MAPPING MICROSCALE CONNECTIVITY IN GRAY MATTER AND LINKING IT WITH WHITE MATTER CONNECTIONS BETWEEN BRAIN REGIONS. THE THIRD OBJECTIVE IS TO TEST THE FEASIBILITY OF EXPANDING LICONN TO LARGER VOLUMES AND SCALE NANOSCALE EXPANSION MICROSCOPY. THE GOAL IS TO DEMONSTRATE THAT REGIONS LARGER THAN A CUBIC MILLIMETER CAN BE EXPANDED 16 TIMES TO ACHIEVE LICONN RESOLUTION, WHICH IS ABOUT TEN TIMES GREATER THAN THE CURRENT STATE-OF-THE-ART.

SCALABLE INTEGRATION OF CELL TYPES AND CONNECTIVITY IN THE MOTOR CORTEX OF RODENTS AND NON-HUMAN PRIMATES - PROJECT SUMMARY THE BICCN HAS RECENTLY COMPLETED A BROAD SURVEY OF THE CELLULAR COMPONENTS OF MOTOR CORTEX, INCLUDING TRANSCRIPTOMIC PROFILING, PATCH-SEQ, MULTIPLEXED FISH, INTER-AREAL CONNECTIVITY, AND SINGLE NEURON MORPHOLOGY. MISSING FROM THIS VIEW IS A DETAILED PICTURE OF HOW INDIVIDUAL NEURONS AND NEURONAL TYPES INTERCONNECT, IN LARGE PART BECAUSE ACQUIRING A COMPREHENSIVE PICTURE OF INDIVIDUAL NEURONAL CONNECTIONS IS BEST ACHIEVED WITH THE DIFFICULT METHODS OF LARGE-SCALE ELECTRON MICROSCOPIC RECONSTRUCTIONS. FURTHER, THERE ARE NO SCALABLE METHODS TO ASSIGN TRANSCRIPTOMIC CELL CLASSES TO EM RECONSTRUCTIONS, PARTICULARLY WHEN CONSIDERING THE MYRIAD SET OF NON- LOCAL INPUTS. IN ORDER TO FULFILL THE BICCN MANDATE, WE NEED SCALABLE METHODS FOR MEASURING CONNECTIVITY THAT CAN BE INTEGRATED WITH CROSS-MODAL DATA, SUCH AS FROM PATCH-SEQ, OR WITH TOOLS THAT TARGET SPECIFIC CELL CLASSES IN TRANSGENIC AND NON-TRANSGENIC SPECIES, SUCH AS NON-HUMAN PRIMATE. HERE, WE PROPOSE TO EXPLOIT BOTH APPROACHES—RETROSPECTIVELY LINKING EM DATA WITH PATCH-SEQ DATASETS OR THE PROSPECTIVE TARGETING OF CELL CLASSES LABELING IN EM RECONSTRUCTIONS—WITH OUR LARGE-SCALE ELECTRON MICROSCOPY PIPELINE, WHILE CONTINUING TO IMPROVE THROUGHPUT AND LOWER ITS OVERALL COST. WE PROPOSE TO DEPLOY THESE TOOLS TO EXAMINE THE MOTOR CORTEX OF MOUSE AND NON-HUMAN PRIMATE, TO DEMONSTRATE A SCALABLE APPROACH TO DELIVERING DATA THAT INTEGRATE LOCAL CELLULAR MORPHOLOGY, ULTRASTRUCTURAL DETAIL, AND SPECIFIC LOCAL CONNECTIVITY WITH TRANSCRIPTOMIC INFORMATION, FROM PATCH-SEQ, WHILE IDENTIFYING THE SOURCE AND CELL TYPE OF INDIVIDUAL AFFERENTS, WITH VIRAL GENETIC TOOLS.

CELL TYPE SELECTIVE VIRAL TOOLS TO INTERROGATE AND CORRECT NON-HUMAN PRIMATE AND HUMAN BRAIN CIRCUITRY

ADVANCING BIO-REALISTIC MODELING VIA THE BRAIN MODELING TOOLKIT AND SONATA DATA FORMAT - ADVANCING BIO-REALISTIC MODELING VIA THE BRAIN MODELING TOOLKIT AND SONATA DATA FORMAT ONE OF THE MAJOR GOALS OF THE BRAIN INITIATIVE IS TO DISTILL COMPLEX, MULTI-MODAL DATA INTO PREDICTIVE FRAMEWORKS VIA THEORY/MODELING. AS THE PLANNING DOCUMENT "BRAIN 2025: A SCIENTIFIC VISION" URGES, “THEORY AND MODELING SHOULD BE WOVEN INTO SUCCESSIVE STAGES OF ONGOING EXPERIMENTS, ENABLING BRIDGES TO BE BUILT FROM SINGLE CELLS TO CONNECTIVITY, POPULATION DYNAMICS, AND BEHAVIOR.” HOWEVER, DATA-DRIVEN, BIO-REALISTIC MODELING IS NOT WIDELY PRACTICED, IN PART BECAUSE THE FIELD NEEDS SOFTWARE SUPPORTING SUCH COMPLEX MODELING AND STANDARDS FOR MODEL SHARING AND REPRODUCIBILITY. THE ALLEN INSTITUTE HAS DEVELOPED TWO POWERFUL TOOLS ADDRESSING THESE NEEDS. ONE IS THE BRAIN MODELING TOOLKIT (BMTK) – A SOFTWARE SUITE FOR MODEL BUILDING AND SIMULATION AT MULTIPLE LEVELS OF RESOLUTION, FROM NETWORKS OF BIOPHYSICALLY DETAILED NEURONAL MODELS, TO POINT-NEURON NETWORKS, TO POPULATION-STATISTICS APPROACHES. THE OTHER ONE IS THE SONATA (SCALABLE OPEN NETWORK ARCHITECTURE TEMPLATE) DATA FORMAT, WHICH PROVIDES COMPUTATIONALLY EFFICIENT SOLUTIONS FOR STORING AND EXCHANGING DATA DESCRIBING ALL STAGES OF THE MODELING WORKFLOW (E.G., STRUCTURE OF MODEL NETWORKS, CONFIGURATION OF SIMULATIONS, SIMULATION OUTPUTS). THESE TOOLS WERE DEVELOPED IN COORDINATION WITH MANY INITIATIVES, SUCH AS NEURON, NEST, NEURODATA WITHOUT BORDERS, NEUROML, PYNN, NETPYNE, AND THE HUMAN BRAIN PROJECT. AS A RESULT, BMTK AND SONATA ENABLE MANY APPLICATIONS AND HAVE GENERATED SUBSTANTIAL INTEREST, WITH MANY USERS ALREADY EMPLOYING THESE TOOLS. MOST RECENTLY, BMTK AND SONATA WERE INSTRUMENTAL IN INTEGRATING DIVERSE DATA FROM THE ALLEN INSTITUTE AND FROM THE LITERATURE INTO SOME OF THE MOST SOPHISTICATED AND BIO-REALISTIC MODELS OF A BRAIN REGION TO DATE. WE PROPOSE TO BUILD A COMPREHENSIVE USER SUPPORT AND DISSEMINATION PLATFORM FOR BMTK AND SONATA AND HELP INTEGRATE THESE TOOLS INTO MODEL BUILDING AND SIMULATION PRACTICES IN THE COMMUNITY. IN ADDITION, THE ALLEN INSTITUTE TEAM JOINS FORCES WITH A UNIVERSITY OF ILLINOIS TEAM THAT DEVELOPED A WIDELY USED MOLECULAR VISUALIZATION SOFTWARE VMD. BY INTEGRATING THIS SOFTWARE WITH SONATA, WE WILL LEVERAGE ITS POWERFUL EXISTING CAPABILITIES TO OFFER A FREE, HIGHLY EFFICIENT VISUALIZATION TOOL FOR NEUROSCIENCE MODELING. TOGETHER, THESE TOOLS WILL FACILITATE FREE EXCHANGE AND REPRODUCIBILITY OF MODELS AND SUPPORT SOPHISTICATED MODELING WORK – ESPECIALLY IN CASES OF LARGE-SCALE BIOLOGICALLY REALISTIC MODELS RELYING ON SYSTEMATIC INTEGRATION OF EXPERIMENTAL DATA – FOR NOVICE AND EXPERT USERS ALIKE. THESE CONTRIBUTIONS WILL ADVANCE THE BRAIN INITIATIVE’S PRIORITY AREAS OF THEORY AND DATA ANALYSIS AND INTEGRATED APPROACHES AND WILL STRONGLY FACILITATE FAIRNESS (FINDABILITY, ACCESSIBILITY, INTEROPERABILITY, AND REUSE OF DIGITAL ASSETS) IN NEUROSCIENCE MODELING.

MOUSE CELL TYPE-SPECIFIC BRAIN MAPPING IN HEALTH AND DISEASE

CELL TYPE AND CIRCUIT MECHANISMS OF NON-INVASIVE BRAIN STIMULATION BY SENSORY ENTRAINMENT - CELL TYPE AND CIRCUIT MECHANISMS OF NON-INVASIVE BRAIN STIMULATION BY SENSORY ENTRAINMENT PATTERNED SENSORY STIMULATION (PSS) IS A NON-INVASIVE TECHNIQUE FOR MANIPULATING BRAIN ACTIVITY AND STATES, TYPICALLY EMPLOYING PERIODIC LIGHT FLICKER OR AUDITORY TONES PRESENTED AT REGULAR INTERVALS. WE AND OTHERS HAVE RECENTLY SHOWN THAT PSS AT CERTAIN FREQUENCIES (CENTERED AT 40 HZ) CAUSES WIDESPREAD NEURAL ENTRAINMENT AND STATE CHANGES IN NON-NEURONAL CELL POPULATIONS (INCLUDING, E.G., EFFECTS ON THE ACTIVITY OF MICROGLIA AND ON VASOMOTION), IMPROVEMENTS IN MEMORY AND COGNITIVE FUNCTION, AND CLEARANCE OF MARKERS OF NEURODEGENERATION IN ANIMAL MODELS OF BRAIN DISEASE. THESE OBSERVATIONS SUGGEST A STRONG POTENTIAL OF PSS FOR NON-INVASIVE BRAIN STIMULATION APPLICATIONS IN BASIC SCIENCE AND AS A THERAPEUTIC TOOL. TO ENABLE SUCH APPLICATIONS, HOWEVER, IT IS IMPORTANT TO KNOW THE MECHANISMS MEDIATING THE COMPLEX EFFECTS OF PSS ON NEURONS AND NON-NEURONAL CELLS. THESE MECHANISMS ARE POORLY UNDERSTOOD. IN THIS PROJECT, WE SYSTEMATICALLY INVESTIGATE MECHANISMS OF PSS BY DISSECTING HOW CELL TYPES AND CIRCUIT PROPERTIES IN THE BRAIN MEDIATE THE ENTRAINMENT OF NEURAL ACTIVITY AND MODIFICATIONS OF THE STATES OF NEURONAL AND NON-NEURONAL CELL POPULATIONS, WITH THE FOCUS ON THE MOUSE CORTEX AS A MODEL SYSTEM. THE CENTRAL COMPONENT OF THIS PROJECT IS A SYSTEMATIC MODELING EFFORT, RELYING ON OUR RECENT PROGRESS IN INTEGRATING DIVERSE STRUCTURAL AND FUNCTIONAL DATA INTO HIGHLY DETAILED, BIO-REALISTIC MODELS OF THE MOUSE CORTICAL CIRCUITS. THESE MODELS WILL BE APPLIED AND REFINED TO SIMULATE THE EFFECTS OF PSS AT THE LEVEL OF A SINGLE CORTICAL AREA (PRIMARY VISUAL CORTEX) AND THE WHOLE MOUSE CORTEX. WE WILL ALSO DEVELOP MODELS OF COUPLING FROM THE ACTIVITY OF DIFFERENT NEURON TYPES TO NON-NEURONAL CELLS, PROVIDING INSIGHTS INTO THE EFFECTS OF NEURONAL ENTRAINMENT TO PSS ON, E.G., MICROGLIA AND VASCULATURE. THESE MODELING EFFORTS WILL GO HAND-IN-HAND WITH ELECTROPHYSIOLOGY RECORDINGS IN AWAKE MICE, ACCOMPANIED BY CHRONIC AND ACUTE PERTURBATIONS (USING CHEMOGENETICS AND OPTOGENETICS). IN MULTIPLE ITERATIVE STAGES, MODELING PREDICTIONS REGARDING THE ROLES OF EXCITATORY AND INHIBITORY (E.G., PV, SST, VIP) CELL TYPES IN DIFFERENT CORTICAL LAYERS ON THE ENTRAINMENT TO PSS WILL BE TESTED EXPERIMENTALLY, AND MODELS WILL BE REFINED TO MATCH DATA. THE PROJECT WILL ALSO CHARACTERIZE TRANSCRIPTOMIC AND EPIGENETIC RESPONSES TO PSS IN DIFFERENT CELL TYPES, WHICH WILL BE CORRELATED WITH CIRCUIT EFFECTS REVEALED BY SIMULATIONS AND PERTURBATIVE EXPERIMENTS IN VIVO. THE RESULTS OF THESE STUDIES WILL PROVIDE A RICH DESCRIPTION OF MOLECULAR, CELL TYPE, AND CIRCUIT MECHANISMS MEDIATING THE PSS EFFECTS, WHICH WILL BE CRUCIAL FOR FUTURE RATIONAL DEVELOPMENT OF APPLICATIONS OF THIS BRAIN STIMULATION TECHNIQUE. BESIDES THE KNOWLEDGE, THIS PROJECT WILL ALSO PROVIDE HIGHLY BIOLOGICALLY REALISTIC, READY- TO-USE COMPUTATIONAL MODELS APPLICABLE FOR STUDIES OF PSS AND OTHER PHENOMENA, WHICH WE WILL FREELY SHARE WITH THE COMMUNITY.

SCIENTIFIC AND PUBLIC OUTREACH OF CELL TYPE TAXONOMIES (SPOCTT) INITIATIVE - PROJECT SUMMARY SINGLE CELL TRANSCRIPTOMICS HAS TRANSFORMED THE FIELD OF BRAIN CELL TYPE CLASSIFICATION, ALLOWING SIMULTANEOUS MEASUREMENT OF ENOUGH MOLECULAR FEATURES FROM ENOUGH CELLS TO CATEGORIZE NEURONS QUANTITIVELY AND WITH HIGH CONSERVATION ACROSS BRAIN AREAS AND SPECIES. FURTHERMORE, CELLS CLASSIFIED BY THEIR GENES HAVE LARGELY COHERENT SHAPES, ELECTRICAL PROPERTIES, SPATIAL LOCATIONS, AND PROJECTS TARGETS, BRINGING THE DREAM OF MULTIMODAL CELL TYPE DEFINITIONS EVER CLOSER TO REALITY, BUT ALSO INTRODUCING MAJOR CHALLENGES OF SIFTING THROUGH ALL THIS DISPARATE INFORMATION TO LEARN ANY GIVEN THING. TO ADDRESS THESE CHALLENGES THE ALLEN INSTITUTE HAS DEVELOPED A SERIES OF HIGH-QUALITY REFERENCE DATA SETS, A STANDARD FORMAT FOR ORGANIZING AND SHARING THEIR ASSOCIATED CELL TYPE CLASSIFICATIONS, AND A HOST OF WEB TOOLS FOR EXPLORING THESE CELL TYPES. THE CELL TYPE KNOWLEDGE EXPLORER (CTKE), RELEASED IN DECEMBER 2021, IS DESIGNED FOR EXPLORATION OF TRANSCRIPTOMICS, ELECTROPHYSIOLOGY, MORPHOLOGY, SPATIAL LOCALIZATION, AND EPIGENETICS OF INDIVIDUAL CELL TYPES IN THE PRIMARY MOTOR CORTEX OF MOUSE, MARMOSET, AND HUMAN. THE ALLEN BRAIN CELL (ABC) ATLAS, TO BE INITIALLY RELEASED IN LATE 2022, WILL ENABLE USERS TO EXPLORE A CELL TYPE TAXONOMY AND SPATIAL MAP OF THE WHOLE MOUSE BRAIN, DRAMATICALLY INCREASING THE BREADTH OF CELL TYPES COVERED BY THE CTKE, WHILE CONTINUING TO ENABLE USER-FRIENDLY DATA EXPLORATION. BOTH RESOURCES PRESENT CELL TYPE TAXONOMIES IN A MANNER UNIQUE TO THE FIELD OF NEUROSCIENCE. THESE DATA, STANDARDS, AND WEB TOOLS CONTINUE THE OPEN SCIENCE PRACTICES THAT HAVE BEEN A KEYSTONE OF THE ALLEN INSTITUTE SINCE THE RELEASE OF THE ALLEN MOUSE BRAIN ATLAS ALMOST TWO DECADES AGO. TO ENABLE USERS NOT JUST TO FIND, BUT ALSO TO SUCCESSFULLY UTILIZE THESE TOOLS, THE ALLEN INSTITUTE HAS AN ESTABLISHED TRAINING, EDUCATION, AND OUTREACH PROGRAM TO SUPPORT USERS OF ITS RESOURCES AT VARIOUS CAREER AND LEARNING STAGES. CELL TYPE AND TAXONOMY SCIENTIFIC RESOURCES, IN PARTICULAR, RELY ON SKILLS SPANNING NEUROBIOLOGY, GENETICS, APPLIED MATH, AND COMPUTER SCIENCE, POSING CHALLENGES FOR BOTH STUDENTS AND PROFESSIONALS, AND EXTENDING THIS PROGRAM TO SUPPORT SUCH TOOLS WOULD BE PARTICULARLY VALUABLE TO THE NEUROSCIENCE COMMUNITY. THE GOAL OF THE SCIENTIFIC AND PUBLIC OUTREACH OF CELL TYPE TAXONOMIES (SPOCTT) INITIATIVE IS TO MAKE THESE PUBLIC RESOURCES MORE WIDELY ACCESSIBLE TO THE GENERAL AND SCIENTIFIC COMMUNITIES, THROUGH A COMBINATION OF CONSOLIDATION, COLLABORATION, TRAINING, EDUCATION, AND OUTREACH EFFORTS. BY OFFERING BOTH SCALABLE TRAINING RESOURCES AND PERSONALIZED SUPPORT TO PROSPECTIVE USERS OF TAXONOMIES AND TOOLS, WE WILL (1) PROMOTE USE OF CELL TYPE TAXONOMIES AND ASSOCIATED TOOLS (SPECIFICALLY THE CTKE IN YEAR 1 AND THE ABC ATLAS IN YEAR 2), (2) SET BRAIN INITIATIVE CELL CENSUS NETWORK (BICCN) AND ALLEN INSTITUTE TAXONOMIES AS SCIENTIFIC STANDARDS FOR STUDYING THE BRAIN IN HEALTH AND DISEASE, AND (3) BROADEN THE REACH OF THE ALLEN INSTITUTE TO A YOUNGER AND MORE DIVERSE NEUROSCIENCE COMMUNITY.

NEURONAL ENSEMBLES IN THE RODENT VISUAL CORTEX

DISSEMINATION OF 3-PHOTON IMAGING FOR CHRONIC CELLULAR IMAGING ACROSS SPECIES

MULTIMODAL ANALYSIS OF PRIMATE INFRAGRANULAR PYRAMIDAL NEURONS AND THEIR MODULATION - THE LONG-TERM GOAL OF THIS PROJECT IS TO DETERMINE THE CONSEQUENCES OF CELL-TYPE SPECIFIC EXPRESSION OF ION CHANNEL AND NEUROMODULATOR RECEPTOR GENES ON PRIMATE NEOCORTICAL FUNCTION. THE HUMAN BRAIN IS COMPOSED OF AN ASTONISHING NUMBER OF CELL TYPES. MOLECULAR PROFILING SUGGESTS THAT UPWARDS OF ~75 UNIQUE NEURONAL CELL TYPES RESIDE IN A GIVEN NEOCORTICAL AREA, AND THAT EACH AREA HAS EXCLUSIVE TYPES. HOW DO DIFFERENCES IN GENE EXPRESSION TRANSLATE INTO A NEURON’S PHENOTYPIC IDENTITY? SOLVING THIS PROBLEM IS CRUCIAL BECAUSE SEVERAL EMERGING LINES OF EVIDENCE SUGGEST THAT HUMAN BRAIN DISORDERS MAY HAVE CELL TYPE-SPECIFIC ETIOLOGIES, WHEREIN DIFFERENT CLASSES OF NEURONS MAKE DISTINCT CONTRIBUTIONS TO THE PATHOPHYSIOLOGY OF THE DISEASE. WE PROPOSE TO EXAMINE IN HUMAN AND NONHUMAN PRIMATES HOW MRNA EXPRESSION IN TWO BROAD CATEGORIES OF NEOCORTICAL INFRAGRANULAR PYRAMIDAL NEURONS TRANSLATES INTO THEIR UNIQUE PHYSIOLOGY, MORPHOLOGY AND RESPONSE TO NEUROMODULATION. EMPLOYING A STATE-OF-THE-ART PATCH CLAMPING TECHNIQUE, PATCH-SEQ, WE CAN GENETICALLY IDENTIFY PHYSIOLOGICALLY PROBED NEURONS FROM HUMAN AND NON-HUMAN PRIMATE NEOCORTEX. WE TEST HYPOTHESES ABOUT HOW SPECIFIC ION CHANNELS AND NEUROMODULATOR RECEPTORS SHAPE THE UNIQUE INPUT-OUTPUT PROPERTIES OF THESE NEURONS. WE ALSO UTILIZE VIRAL TOOLS TO PROSPECTIVELY LABEL NEURONS, IN PARTICULAR THE LAYER 5 (L5) EXTRATELENEPHALIC (ET)-PROJECTING NEURONS (WHICH SEND AXONAL PROJECTIONS TO SUBCEREBRAL REGIONS). SEVERAL TYPES OF L5 ET NEURONS ARE NOT FOUND IN THE RODENT BRAIN (E.G., BETZ CELLS OF MOTOR CORTEX). THREE FACTORS MAKE THIS PROPOSAL ESPECIALLY RELEVANT FOR HUMAN HEALTH AND DISEASE. FIRST, L5 ET NEURONS REPRESENT THE SOLE DIRECT OUTPUT OF THE NEOCORTEX TO MANY SUBCEREBRAL STRUCTURES AND ARE IMPLICATED IN SEVERAL NEUROLOGICAL DISORDERS INCLUDING ALZHEIMER’S DISEASE AND AMYOTROPHIC LATERAL SCLEROSIS (ALS). SECOND, WE WILL BE DIRECTLY WORKING IN NON-HUMAN PRIMATE AND HUMAN BRAIN SLICES RATHER THAN THE TRADITIONAL RODENT MODELS. THE LATTER POINT IS ESPECIALLY PERTINENT GIVEN RECENT PUBLISHED FINDINGS OF MAJOR DIFFERENCES IN MURINE AND HUMAN PYRAMIDAL NEURON PHYSIOLOGY. EXPERIMENTS WITH MONKEY TISSUE WILL PROVIDE DIRECT ACCESS TO LONG-RANGE AXONAL PROJECTION TARGETS IN VIVO (WHICH ISN’T FEASIBLE FOR HUMAN BRAIN SLICES), AS WELL AS THE ABILITY TO STUDY BRAIN AREAS RARELY AVAILABLE IN THE HUMAN FROM SURGICAL SPECIMENS (E.G., PRIMARY MOTOR CORTEX). FINALLY, THIS PROPOSAL LAYS THE FOUNDATIONAL KNOWLEDGE NECESSARY FOR EVENTUAL DEVELOPMENT OF CELL TYPE-SPECIFIC GENETIC AND PHARMACOLOGICAL TREATMENT OF DISEASE.

A ROBUST, LOW-COST PLATFORM FOR EM CONNECTOMICS - PROJECT SUMMARY/ABSTRACT OVER THE PAST DECADE, SERIAL-SECTION ELECTRON MICROSCOPY HAS COME INTO ITS OWN AS A METHOD TO STUDY THE CONNECTIVITY OF NEURAL CIRCUITS, FROM LOCAL CIRCUITS IN MAMMALS TO ENTIRE INVERTEBRATE BRAINS. RECENTLY, THE EMPHASIS IN THE FIELD HAS BEEN TO CREATE INCREASINGLY LARGE DATA SETS, WHILE COMPARATIVELY LITTLE EFFORT HAS BEEN SPENT ON MAKING THE TOOLS OF EM CONNECTOMICS AVAILABLE TO A LARGE NUMBER OF CIRCUIT NEUROSCIENTISTS. OBSTACLES EXIST AT MULTIPLE LEVELS. MANUAL APPROACHES TO SERIAL SECTIONING ARE PROHIBITIVELY DIFFICULT, WHILE AUTOMATED APPROACHES REQUIRE COMPLEX, EXPENSIVE EQUIPMENT THAT IS DIFFICULT TO DEPLOY. HIGH THROUGHPUT SCANNING EM IS LIMITED TO MULTI- BEAM APPROACHES THAT ARE EXTREMELY EXPENSIVE. TRANSMISSION EM IS FAR LESS EXPENSIVE, BUT AUTOMATED APPROACHES TO SECTIONING REMAIN CHALLENGING AND REQUIRE EXPENSIVE SUBSTRATES THAT ARE HARD TO MANUFACTURE AND DIFFICULT TO USE. WE PROPOSE TO DEVELOP A NEW APPROACH, ALREADY PROTOTYPED BY OUR GROUP AND OUR INDUSTRY PARTNER, TO ESTABLISH A ROBUST PLATFORM OPTIMIZED TO ACHIEVE THE WIDEST POSSIBLE ADOPTION. THE SYSTEM WILL CENTER ON AN OPEN SOURCE SERIAL SECTIONING ROBOT IMPLEMENTING A NOVEL COLLECTION APPROACH. THE GOAL IS TO CREATE A SYSTEM THAT CAN BE USED AT A VARIETY OF SCALES, FROM THE CURRENT STATE OF THE ART (1 MM3 OR GREATER), TO SMALL VOLUMES THAT CAN BE SECTIONED AND IMAGED ROUTINELY. UP TO NOW, EACH PUBLISHED EM VOLUME FOR CONNECTOMICS HAS REQUIRED A MULTI-YEAR EFFORT. INSTEAD, OUR GOAL IS TO USE VOLUME RECONSTRUCTION AS AN ASSAY, RATHER THAN AN END UNTO ITSELF, IN THE CONTEXT OF OTHER EXPERIMENTS. IN THE FINAL YEAR, WE WILL CREATE DATA SETS THAT TEST THE FLEXIBILITY AND ROBUSTNESS OF THE APPROACH BY CREATING EM VOLUMES RANGING FROM 50ΜM ON A SIDE TO VERY LARGE VOLUMES ENCOMPASSING >1 MM3.

MULTI-MODAL, LARGE-SCALE CHARACTERIZATION OF CELLULAR AND CELL-TYPE-SPECIFIC EFFECTS WITH ELECTRIC STIMULATION IN RODENT AND HUMAN BRAIN

A COMMUNITY FRAMEWORK FOR DATA-DRIVEN BRAIN TRANSCRIPTOMIC CELL TYPE DEFINITION, ONTOLOGY, AND NOMENCLATURE

UNRAVELING THE DEVELOPMENTAL LOGIC OF CORTICAL LONG-RANGE PROJECTIONS USING IN SITU SEQUENCING-BASED NEUROANATOMY - PROJECT SUMMARY THE CONNECTIVITY OF NEURONS ALLOWS COMPLEX FUNCTIONS TO EMERGE FROM A CIRCUIT COMPOSED OF DIVERSE NEURONAL TYPES. IN THE MAMMALIAN NERVOUS SYSTEM, RECENT ADVANCES IN SINGLE-CELL TRANSCRIPTOMICS MAKE IT APPEALING TO DEFINE NEURONAL TYPES BY THEIR GENE EXPRESSION PATTERNS (I.E. TRANSCRIPTOMIC TYPES). AT A HIGH LEVEL, CLASSES OF NEURONS DEFINED BY TRANSCRIPTOMICS ARE ALSO DISTINCT IN OTHER NEURONAL PROPERTIES, INCLUDING THEIR LONG-RANGE PROJECTION PATTERNS. FINER-LEVEL TRANSCRIPTOMIC TYPES, HOWEVER, DO NOT CORRESPOND TO PROJECTION PATTERNS: NEURONS OF DIFFERENT TRANSCRIPTOMIC TYPES MAY SHARE SIMILAR PROJECTIONS, AND NEURONS OF THE SAME TRANSCRIPTOMIC TYPE CAN PROJECT DIVERSELY. THIS LACK OF CORRESPONDENCE AT A FINE LEVEL RAISES THE QUESTION OF HOW NEURONAL TYPES ARE WIRED INTO COMPLEX CIRCUITS AND, FURTHERMORE, HOW CELL TYPES CAN BE DEFINED BY BOTH GENE EXPRESSION AND CONNECTIVITY. KNOWING THE DEVELOPMENTAL RELATIONSHIP BETWEEN GENE EXPRESSION AND PROJECTIONS MAY HELP UNDERSTAND THIS COMPLEX RELATIONSHIP, BECAUSE THE PROJECTION PATTERN OF AN ADULT NEURON IS THE CUMULATIVE RESULT OF MANY DEVELOPMENTAL PROCESSES. HOWEVER, INTERROGATING THE DEVELOPMENTAL RELATIONSHIP BETWEEN GENE EXPRESSION AND PROJECTIONS IS CHALLENGING, BECAUSE CONVENTIONAL SINGLE-CELL ANATOMICAL APPROACHES CAN ONLY MAP THE PROJECTION PATTERNS OF A SMALL NUMBER OF NEURONS AND ARE DIFFICULT TO ASSOCIATE PROJECTIONS WITH GENE EXPRESSION MEASURED IN THE SAME CELLS. HERE I PROPOSE TO OVERCOME THIS CHALLENGE BY MASSIVELY IMPROVING THE RESOLUTION AND SCALE OF IN SITU SEQUENCING-BASED NEUROANATOMICAL APPROACHES. IN SITU SEQUENCING-BASED NEUROANATOMY ACHIEVES HIGH THROUGHPUT AND CELLULAR RESOLUTION IN MAPPING PROJECTIONS BY LABELING EACH NEURON WITH A UNIQUE RNA BARCODE. THESE RNA BARCODES AND ENDOGENOUS MRNAS CAN BOTH BE SEQUENCED IN SITU TO ASSOCIATE PROJECTION PATTERNS WITH GENE EXPRESSION FOR MANY NEURONS IN PARALLEL. BY IMPROVING BOTH THE RESOLUTION AND THE THROUGHPUT OF IN SITU BARCODE SEQUENCING, I WILL GENERATE AN UNPRECEDENTED VIEW OF THE RELATIONSHIP BETWEEN GENE EXPRESSION AND THE COMPLETE BRAIN-WIDE PROJECTION PATTERNS OF NEURONS IN THE PRIMARY AND HIGHER VISUAL CORTEX OVER THE COURSE OF POST-NATAL DEVELOPMENT. I WILL COMPLEMENT THIS SYSTEMATIC BUT CORRELATIONAL APPROACH WITH PERTURBATION EXPERIMENTS TO ESTABLISH CAUSAL RELATIONSHIP BETWEEN KEY GENES AND PROJECTIONS. BY UNRAVELING HOW THE COMPLEX RELATIONSHIP BETWEEN GENE EXPRESSION AND PROJECTIONS IS ESTABLISHED STEP-BY-STEP IN DEVELOPMENT, THIS COMBINED APPROACH WILL PROVIDE INSIGHTS INTO THE WIRING RULES OF CORTICAL NEURONAL TYPES. THE DATASET GENERATED WILL PROVIDE A REFERENCE FOR FUTURE RESEARCH INTO LONG-RANGE CONNECTIVITY DEFECTS IN NEURODEVELOPMENTAL DISEASE MODELS. FINALLY, THE IMPROVEMENT IN IN SITU SEQUENCING-BASED NEUROANATOMY WILL ACHIEVE BROAD IMPACT BEYOND THE DEVELOPMENTAL FOCUS OF THIS PROPOSAL BY ENABLING SIMILAR SYSTEMATIC APPROACHES IN UNDERSTANDING LONG-RANGE PROJECTIONS DURING AGING, ACROSS INDIVIDUAL ANIMALS, AND ACROSS SPECIES.

CELL CLASS- OR TYPE-SPECIFIC VIRUSES FOR BRAIN-WIDE LABELING AND NEURAL CIRCUIT EXAMINATION

AXONAL CONNECTOMICS: DENSE MAPPING OF PROJECTION PATTERNS BETWEEN CORTICAL AREAS

EXPANDING ACCESS TO OPEN-SOURCE DATA ACQUISITION SOFTWARE FOR NEXT-GENERATION SILICON PROBES

TEMPORAL, CELL TYPE- AND LOCUS-SPECIFIC EPIGENETIC CONTROL IN TRANSGENIC MICE

A TRANSGENIC PLATFORM TO PRODUCE GENETIC TOOLS FOR MONITORING & MANIPULATING PLAS

CCRI: RESEARCH INFRASTRUCTURE: NEW: SEMANTIC SCHOLAR OPEN DATA PLATFORM: ENABLING RESEARCH INTO SCIENTIFIC SEARCH AND DISCOVERY -THE EXPONENTIAL GROWTH OF SCIENTIFIC PUBLICATION MAKES IT DIFFICULT FOR SCIENTISTS TO TRACK DEVELOPMENTS IN THEIR FIELD AND MAKE CONNECTIONS BETWEEN DIFFERENT ADVANCES. IN RESPONSE, ARTIFICIAL-INTELLIGENCE RESEARCHERS HAVE STARTED TO DEVELOP TECHNIQUES THAT ALLOW COMPUTERS TO ?READ? SCIENTIFIC PAPERS AND AUTOMATICALLY CLASSIFY TOPICS, EXTRACT KEY RESULTS, SUMMARIZE CONTRIBUTIONS, IDENTIFY CONNECTIONS, AND SELECT A PERSONALIZED SET OF PAPERS THAT MAY BE OF SPECIAL INTEREST TO EACH SCIENTIST. THE ENDURING VISION IS TO BUILD AI SYSTEMS THAT CAN PROCESS AN IMMENSE CORPUS OF SCHOLARLY DOCUMENTS AND AUGMENT THE CAPABILITIES OF HUMAN SCIENTISTS ? ACCELERATING SCIENTIFIC DISCOVERY AND HELPING HUMANITY QUICKLY CONFRONT DISASTERS SUCH AS THE COVID-19 PANDEMIC. THE PROPOSED SEMANTIC SCHOLAR OPEN DATA PLATFORM BUILDS INFRASTRUCTURE TO SUPPORT THIS RESEARCH BY FIRST GATHERING A COMPREHENSIVE SET OF PAPERS AND ARRANGING FOR EFFICIENT INDEXING. THE SYSTEM PROCESSES PDF-FORMATTED PAPERS TO EXTRACT INFORMATION AND USE ADVANCED ANALYTIC PROCESSING APPROACHES TO PROVIDE RESEARCHERS ACCESS TO RESULTS. THE INFRASTRUCTURE WILL DRAMATICALLY LOWER THE BARRIER TO ENTRY FOR NEWCOMERS TO THE FIELD OF SCHOLARLY DOCUMENT PROCESSING, IMPROVE REPRODUCIBILITY OF EXPERIMENTS, AND ACCELERATE INNOVATION IN THE IMPORTANT AREA OF AI-AUGMENTED SCIENTIFIC DISCOVERY THE INFRASTRUCTURE PROPOSED IS UNIQUE, BECAUSE ALTERNATIVE SOURCES OF ACADEMIC PAPERS ARE EITHER CLOSED, INCOMPLETE, HAVE LIMITED PROGRAMMATIC ACCESS, OR HAVE BEEN RETIRED. THE PROPOSED SEMANTIC SCHOLAR OPEN DATA PLATFORM HAS THREE PARTS: 1) A COMPREHENSIVE SET OF ONLINE SERVICES ENABLING RESEARCHERS TO PROGRAMMATICALLY SEARCH, FILTER, EXTRACT, SUMMARIZE, AND ANALYZE A LARGE AND CONTINUALLY-UPDATED CORPUS OF DOCUMENTS; 2) A NEW MECHANISM THAT ENABLES RESEARCHERS TO CURATE THEIR OWN DOMAIN-SPECIFIC TEXT CORPORA, AS THE TEAM PREVIOUSLY CREATED THE CORD-19 DATASET FOR CORONAVIRUS RESEARCH; 3) OPEN SOURCE SOFTWARE, INCLUDING PRETRAINED LANGUAGE MODELS AND USER INTERFACE TEMPLATES TO SERVE AS RESEARCH BUILDING BLOCKS. TOGETHER THE INFRASTRUCTURE WILL DRAMATICALLY LOWER THE BARRIER TO ENTRY FOR NEWCOMERS TO THE FIELD OF SCHOLARLY DOCUMENT PROCESSING, IMPROVE REPRODUCIBILITY OF EXPERIMENTS, AND ACCELERATE INNOVATION IN THE IMPORTANT AREA OF AI-AUGMENTED SCIENTIFIC DISCOVERY. FORTUNATELY, THE RECENT INCREASE IN RESEARCH IN SCHOLARLY DOCUMENT PROCESSING (E.G., THE RAPID UPTAKE OF OUR CORD-19 DATASET) SHOWS THAT THE COMPUTER AND INFORMATION SCIENCE COMMUNITY HAS THE INTEREST AND CAPABILITY TO DEVELOP NEW TECHNOLOGIES THAT ACCELERATE SCIENCE AND HELP MEET GLOBAL SOCIETAL CHALLENGES, SUCH AS PANDEMICS AND CLIMATE CHANGE. THE RESULTING ADVANCES IN AI-AUGMENTED SCIENTIFIC DISCOVERY WILL BENEFIT ALL AREAS OF SCIENCE, SPURRING MEDICAL ADVANCES, CREATING NEW JOBS, AND IMPROVING ACCESS FOR BLIND RESEARCHERS. WE WILL IMPROVE GLOBAL INFRASTRUCTURE BY PROVIDING OPEN SERVICES, DATA SETS, CODE, AND ASSOCIATED EDUCATIONAL MATERIALS. THE TEAM WILL ALSO ENGAGE WITH UNDERREPRESENTED STEM STUDENTS AND THROUGH K-12 OUTREACH. 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.

RESOLVING CELLULAR AND ANATOMICAL COMPLEXITY OF THE BRAINSTEM USING SINGLE-CELL GENOMICS - PROJECT SUMMARY THE BRAIN IS AN EXTRAORDINARILY COMPLEX ORGAN CONTAINING MANY MILLIONS OF NEURONS AND NON-NEURONAL CELLS THAT ARE ORGANIZED INTO HIGHLY SPECIALIZED YET INTRICATELY INTEGRATED CIRCUITS CONTROLLING VARIOUS ACTIVITIES, SUCH AS SENSORY PERCEPTION, MOTOR CONTROL, AND COGNITIVE PROCESSES. THE BRAINSTEM IS A CRITICAL REGION RESPONSIBLE FOR REGULATING ESSENTIAL BODILY FUNCTIONS, LIKE HEART RATE, BLOOD PRESSURE, AND DIGESTION, AND THUS MAINTAINING BODY HOMEOSTASIS. THE INDIVIDUAL NEURONS IN BRAINSTEM CAN BE CLASSIFIED INTO TYPES BASED ON SHARED CHARACTERISTICS LIKE GENE EXPRESSION. BINNING INDIVIDUAL CELLS INTO CELL TYPES IS FUNDAMENTAL FOR ADVANCING OUR UNDERSTANDING OF COMPLEX BIOLOGICAL SYSTEMS, LIKE THE BRAIN, FROM NORMAL TISSUE FUNCTION TO DISEASE PROCESSES. THE CHARACTERIZATION OF CELL TYPES ENABLES CREATION OF TOOLS TO GAIN GENETIC ACCESS TO GROUPS OF CELLS, IT ENABLES DISSECTION OF CELLULAR HETEROGENEITY, IDENTIFY KEY PLAYERS IN VARIOUS CONTEXTS LIKE DISEASE AND AGING, AND LAY THE GROUNDWORK FOR TARGETED INTERVENTIONS AND THERAPIES. WE RECENTLY BUILT A COMPREHENSIVE, HIGH-RESOLUTION ATLAS OF CELL TYPES ACROSS THE ENTIRE ADULT MOUSE BRAIN AND THE CELL TYPE DIVERSITY IN BRAINSTEM EXCEEDED OUR EXPECTATIONS. BRAINSTEM IS HOME TO A HIGHLY HETEROGENEOUS GROUP OF NEURONS THAT DOES NOT SHARE A SPECIFIC GENE MODULE, YET THESE NEURONS ARE HIGHLY SIMILAR TO ONE ANOTHER. IN ADDITION, THESE CELL TYPES INTERMINGLE IN VARIOUS REGIONS AND THEIR FUNCTION IS STRONGLY DETERMINED BY THEIR INPUT/OUTPUT RELATIONSHIP. THIS SUGGESTS THAT A HIGH-DIMENSIONAL COMBINATORIAL GENE EXPRESSION CODE IS NEEDED TO RESOLVE THE UNIQUE TRANSCRIPTOMIC CELL TYPES IN THIS REGION. OUR GOAL IS TO CREATE A REFINED ATLAS OF CELL TYPES IN BRAINSTEM USING A COMBINATION OF SINGLE CELL TRANSCRIPTOMIC PROFILING, SPATIAL TRANSCRIPTOMIC PROFILING, AND MAPPING OF PROJECTION PATTERNS TO TRANSCRIPTOMIC CELL TYPES IN BRAINSTEM. IN ADDITION, WE WILL COMPUTATIONALLY ALIGN BRAIN STEM CELL TYPES FROM MOUSE, NON-HUMAN PRIMATE, AND HUMAN TO DEFINE A CROSS-SPECIES CONSENSUS ATLAS OF BRAIN STEM. CELL TYPE HOMOLOGIES ACROSS SPECIES CAN BE ESTABLISHED BASED ON CONSERVED MARKER EXPRESSION. THIS ENABLES INFERENCE OF CELLULAR PROPERTIES, SUCH AS LONG- RANGE PROJECTION TARGETS, THAT ARE DIFFICULT TO MEASURE IN HUMANS. THE PROPOSED EFFORTS WILL LEAD TO A SIGNIFICANTLY IMPROVED UNDERSTANDING OF BRAINSTEM CELL TYPES AND THEIR FUNCTION AND LAY THE FOUNDATION FOR A BETTER UNDERSTANDING OF DISEASE PROCESSES RELATED TO THAT REGION.

DIRECT NICOTINIC EXCITATION OF LAYER 5 NEOCORTICAL PYRAMIDAL NEURONS

REVEALING THE TRANSCRIPTIONAL BASIS OF CORTICOTHALAMIC PROJECTIONS USING IN SITU SEQUENCE-BASED NEUROANATOMY - PROJECT SUMMARY IN NEURONAL CIRCUITS, THE RELATIONSHIP BETWEEN GENE EXPRESSION AND LONG-RANGE NEURONAL PROJECTIONS DEFINES THE STRUCTURE OF CIRCUITS AND CONSTRAINS COMPUTATIONAL MODELS OF THEIR FUNCTIONS. IN THE PAST FEW YEARS, SYSTEMATIC STUDIES ON THE GENE EXPRESSION AND PROJECTIONS OF CORTICAL NEURONS ACROSS THE WHOLE CORTEX HAVE REVEALED TREMENDOUS DIVERSITY IN CELL TYPES DEFINED BY GENE EXPRESSION, I.E. TRANSCRIPTOMIC TYPES, AND IN LONG-RANGE PROJECTIONS. THESE PREVIOUS STUDIES HAVE LAID THE FOUNDATION FOR UNDERSTANDING THE COMPLEX RELATIONSHIP AMONG GENE EXPRESSION, PROJECTIONS, AND CORTICAL AREAS AT CELLULAR RESOLUTION. UNRAVELING THIS RELATIONSHIP, HOWEVER, IS CHALLENGING, BECAUSE IT REQUIRES ASSOCIATING GENE EXPRESSION AND PROJECTIONS ACROSS LARGE AREAS OF THE CORTEX AT CELLULAR RESOLUTION. FURTHERMORE, GENE EXPRESSION THAT ARE ASSOCIATED WITH THE DEVELOPMENT OF PROJECTIONS MAY BE TRANSIENTLY EXPRESSED IN DEVELOPMENT. HERE WE OVERCOME THESE CHALLENGES BY APPLYING TWO HIGH-THROUGHPUT IN SITU SEQUENCING-BASED NEUROANATOMICAL APPROACHES, BARSEQ AND RETRO-BARSEQ, TO COMPREHENSIVELY INTERROGATE BOTH GENE EXPRESSION AND PROJECTIONS IN THE CORTEX. THESE TECHNIQUES RELY ON IN SITU SEQUENCING OF RNA BARCODES TO DETERMINE THE PROJECTIONS OF THOUSANDS OF NEURONS TOGETHER AT CELLULAR RESOLUTION. BECAUSE THESE TECHNIQUES USE IN SITU SEQUENCING, THEY CAN ALSO SIMULTANEOUSLY INTERROGATE THE EXPRESSION OF HUNDREDS OF GENES IN THE SAME CELLS. THUS, THE HIGHLY MULTIPLEXED NATURE AND THE ABILITY TO ASSOCIATE PROJECTIONS WITH GENE EXPRESSION IN THE SAME CELLS MAKE BARSEQ AND RETRO-BARSEQ UNIQUELY SUITED FOR UNRAVELING HOW GENE EXPRESSION ASSOCIATE WITH PROJECTIONS. WE WILL COMBINE THESE HIGH-THROUGHPUT TECHNIQUES WITH ROBUST META-ANALYSIS TO TAKE ADVANTAGE OF EXISTING SYSTEMATIC SINGLE-CELL RNASEQ DATASETS IN THE CORTEX TO IDENTIFY ASSOCIATION BETWEEN GENE EXPRESSION AND PROJECTIONS WITH UNPRECEDENTED DETAILS. WE WILL FOCUS ON THE CORTICOTHALAMIC CIRCUIT BECAUSE ITS PROJECTIONS ARE HIGHLY DIVERSE AND ORGANIZED ACROSS BOTH CORTICAL AREAS AND TRANSCRIPTOMIC TYPES. WE WILL START BY BUILDING A WHOLE-CORTEX TO WHOLE-THALAMUS PROJECTION MAP OF TRANSCRIPTOMIC TYPES. WE WILL MATCH TRANSCRIPTOMIC TYPES IN OUR DATA TO CELL TYPES IN REFERENCE TRANSCRIPTOMIC DATASETS TO BROADEN OUR IMPACT. WE WILL THEN FOCUS ON A PORTION OF THE CORTEX ENCOMPASSING MULTIPLE NEIGHBORING CORTICAL AREAS TO IDENTIFY GENE CORRELATES OF PROJECTIONS WITHIN THE SAME TRANSCRIPTOMIC TYPES ACROSS NEIGHBORING AREAS. FINALLY, WE WILL APPLY THE SAME APPROACH TO THE DEVELOPING BRAIN TO ASSESS THE DEVELOPMENTAL ASSOCIATION BETWEEN GENE EXPRESSION AND PROJECTIONS. THE DATASETS GENERATED IN THIS STUDY WILL NOT ONLY PROVIDE A FOUNDATIONAL RESOURCE THAT ENABLES FUTURE STRUCTURAL AND FUNCTIONAL STUDIES AT THE CELL TYPE RESOLUTION, BUT ALSO PROVIDE A UNIQUE VIEW OF THE RELATIONSHIP BETWEEN CELLULAR PROGRAMS ENCODED BY GENE EXPRESSION AND WIRING DIAGRAMS ENCODED BY PROJECTIONS.

DEDUCING THE ORIGIN AND EFFECT OF EXTRACELLULAR ELECTRIC FIELDS IN HIPPOCAMPUS

CELL TYPE SELECTIVE VIRAL TOOLS TO INTERROGATE AND CORRECT NON-HUMAN PRIMATE AND HUMAN BRAIN CIRCUITRY - ABSTRACT: MANY CELL TYPES TOGETHER ASSEMBLE THE FUNCTIONAL CIRCUITRY OF THE HUMAN BRAIN. FOR OVER A CENTURY, NEUROSCIENTISTS HAVE CATEGORIZED BRAIN CELL TYPES BY THEIR FEATURES, INCLUDING SHAPE, POSITION, PHYSIOLOGY, MOLECULES, AND FUNCTION. SINGLE CELL TRANSCRIPTOMICS STUDIES ARE NOW DEFINING MOLECULAR CELL TYPES AT A RESOLUTION NOT PREVIOUSLY POSSIBLE, UNCOVERING A TAXONOMY OF HUNDREDS TO THOUSANDS OF BRAIN CELL TYPES. THESE STUDIES HAVE ALSO REVEALED DRAMATIC DIFFERENCES IN MOLECULAR SIGNATURES OF HOMOLOGOUS CELL TYPES ACROSS SPECIES, SHOWING DECISIVELY THAT THE DIFFERENCE BETWEEN MOUSE AND HUMAN BRAIN IS NOT SIMPLY THE TOTAL NUMBER OF NEURONS. HOWEVER, THE FUNCTION OF EACH CELL CLASS OR TYPE IN BRAIN CIRCUITRY, AND DYSFUNCTION IN DISEASE, IS ONLY BEGINNING TO BE EVALUATED. TO CHARACTERIZE THE ROLES OF HUMAN BRAIN CELL CLASSES IN NORMAL FUNCTION AND DISEASE, IT IS CRITICAL THAT TOOLS BE DEVELOPED TO ALLOW GENETIC ACCESS TO CELL CLASSES IN VIVO. SUCH TOOLS WOULD ENABLE PRECISE THERAPEUTIC GENE DELIVERY TO BRAIN CELL CLASSES, PERMITTING TARGETED TREATMENT FOR CLASS-SPECIFIC ETIOLOGIES LIKE SOME EPILEPSIES. FEW GENETIC TOOLS ARE AVAILABLE TO MARK AND MANIPULATE CELL CLASSES AND TYPES IN NON-GENETICALLY TRACTABLE SPECIES LIKE HUMAN AND NON-HUMAN PRIMATE (NHP). VIRUSES INCLUDING ADENO-ASSOCIATED VIRUSES (AAVS), CONTAINING CELL CLASS AND TYPE SELECTIVE ENHANCERS CAN BE LEVERAGED TO GAIN GENETIC ACCESS TO, AND DRIVE GENE EXPRESSION IN SPECIFIC BRAIN CELL CLASSES IN THESE SPECIES. WE HAVE INITIATED A PROJECT THROUGH THE BRAIN INITIATIVE TO GENERATE AND VALIDATE REPORTER AAVS TO MARK SPECIFIC CELL CLASSES IN THE MOUSE CORTEX IN VIVO AND IN HUMAN NEOCORTICAL TISSUE EX VIVO. OUR GROUPS HAVE ENGINEERED AAV VECTORS AND OPTIMIZED CAPSIDS TO ACCESS NEURONS AND EXPRESS TRANSGENES IN MANY DISCRETE CELL CLASSES AND TYPES IN MOUSE AND PRIMATE. NEW AND IMPROVED AAV TOOLS PROMISE TO FUEL HUMAN BRAIN SCIENTIFIC DISCOVERY AND CLINICAL PROGRESS, BUT ONE IMPEDIMENT HAS BEEN THE COSTLY AND TIME-CONSUMING PROCESS OF VALIDATING NEW VECTORS IN PRIMATES. WE PRESENT THREE AIMS TO TRANSLATE THESE PROMISING NEW AAV VECTORS INTO A HIGH-VALUE SET OF PRIMATE-OPTIMIZED TOOLS THAT COULD EVENTUALLY BE USED FOR GENE THERAPIES IN HUMANS. FIRST, WE WILL DEVELOP A PLATFORM FOR SCREENING AAV VECTORS IN NHP EX VIVO BRAIN SLICES, FOLLOWED BY INDIVIDUAL VALIDATION OF PROMISING VECTORS IN NHP IN VIVO AND HUMAN EX VIVO BRAIN SLICE CULTURES. SECOND, WE WILL IDENTIFY OPTIMAL AAV CAPSIDS TO: A) SUPPORT WIDESPREAD NHP NEURONAL TRANSDUCTION IN VIVO WHEN APPLIED INTRAVENOUSLY OR TO CEREBROSPINAL FLUID (CSF), TWO PREFERRED ROUTES OF DELIVERY FOR HUMAN CNS GENE THERAPY, AND B) SUPPORT AAV TRANSDUCTION OF HUMAN PRIMARY BRAIN TISSUE EX VIVO. THIRD, WE WILL PERFORM PROOF-OF-CONCEPT EXPERIMENTS USING CELL CLASS-SELECTIVE VECTORS TO EXPRESS A THERAPEUTIC TRANSGENE IN DEFINED CLASSES TO TREAT A SEVERE AND INTRACTABLE FORM OF CHILDHOOD EPILEPSY CALLED DRAVET SYNDROME (DS). THESE EXPERIMENTS REPRESENT A SIGNIFICANT STEP TOWARDS CONVERTING CELL CLASS-SELECTIVE AAVS INTO FIRST-IN-CLASS VIRAL TOOLS OPTIMIZED FOR IN VIVO NHP BRAIN STUDIES AND HUMAN GENE THERAPY APPLICATIONS.

FROM SYNAPSES TO GENES THROUGH MORPHOLOGY: AN INTEGRATED CHARACTERIZATION OF CELL TYPES BASED ON CONNECTOMICS AND TRANSCRIPTOMICS DATA - PROJECT SUMMARY THE GOAL OF THIS PROJECT IS TO CREATE A UNIFIED FRAMEWORK FOR UNDERSTANDING THE RELATIONSHIP BETWEEN NEURONAL GENE EXPRESSION AND CONNECTIVITY IN MOUSE VISUAL CORTEX, BY USING MORPHOLOGY AS A KEY LINKING MODALITY. THERE NOW EXIST PUBLICLY AVAILABLE LARGE-SCALE DATA SETS THAT MEASURE BOTH THESE MODALITIES IN MOUSE VISUAL CORTEX. ONE DATASET IS A LARGE SET OF PATCH-SEQ EXPERIMENTS FROM SINGLE CELLS, WHICH PROVIDE MEASUREMENTS OF GENE EXPRESSION, ELECTROPHYSIOLOGICAL PROPERTIES AND MORPHOLOGY FOR INDIVIDUAL CELLS. A SECOND DATASET IS FROM LARGE SCALE CONNECTOMICS USING ELECTRON MICROSCOPY, WHICH PROVIDES NEURONAL MORPHOLOGY; FINE-SCALE AND DETAILED CELLULAR AND ULTRASTRUCTURAL PROPERTIES; AND MEASUREMENTS OF THE CONNECTIVITY BETWEEN INDIVIDUAL NEURONS. OUR FIRST AIM IS TO ANALYZE THESE TWO DATASETS IN AN EXPLICITLY INTEGRATIVE FASHION, IN ORDER TO BUILD BETTER CLASSIFIERS OF NEURONAL TYPES, ALONG WITH TOOLS TO TRANSLATE TYPE PREDICTIONS BETWEEN EACH DATA MODALITY. OUR SECOND AIM BUILDS ON THE FIRST, BY USING THOSE TOOLS TO CHARACTERIZE CELL-TYPE SPECIFIC CONNECTIVITY OF MOUSE VISUAL CORTEX. THIS WILL ALLOW US TO DESCRIBE HOW THAT CONNECTIVITY RELATES TO THE LIKELY MOLECULAR COMPOSITION OF INDIVIDUAL CELLS AND PROVIDE INSIGHT INTO WHICH MOLECULAR DISTINCTIONS DRIVE DIFFERENCES IN CELL TYPE SPECIFIC CONNECTIVITY PATTERNS. OUR THIRD AIM IS TO REDISTRIBUTE THE RESULTS OF OUR ANALYSIS BACK INTO PUBLICLY AVAILABLE DATA REPOSITORIES AND CREATE TOOLS THAT ALLOW OTHER RESEARCHERS TO QUERY THE GENE EXPRESSION, CONNECTIVITY, ELECTROPHYSIOLOGICAL AND MORPHOLOGICAL DESCRIPTORS OF NEURONS IN THE DATASETS, AS WELL AS APPLY THOSE SAME TOOLS TO THEIR OWN DATA. LIKE A ROSETTA STONE FOR CELL TYPES, THIS WILL ENABLE RESEARCHERS USING DISPARATE METHODS TO INTEGRATE THEIR DATA WITH OTHER MODALITIES AND FOSTER A RICH ENVIRONMENT FOR UNDERSTANDING THE ROLE OF CELL TYPES IN BRAIN FUNCTION AND DISEASE.

MEASURING INPUT-OUTPUT OPERATIONS OF CORTICAL NEURONS WITH LARGE-SCALE NEUROTRANSMITTER IMAGING - PROJECT SUMMARY/ABSTRACT SATISFYING EXPLANATIONS OF THE PHYSIOLOGICAL FUNCTION OF A TISSUE, WHICH HELP GUIDE MEDICAL INTERVENTIONS, FRAME THAT FUNCTION IN TERMS OF THE INPUTS OF COMPONENT CELLS AND AN ALGORITHM FOR HOW THOSE CELLS TRANSFORM THEIR INPUTS INTO OUTPUTS. BRAIN FUNCTIONS HAVE SO FAR ELUDED SUCH MECHANISTIC EXPLANATION, IN PART BECAUSE 1) THE COMPONENT CELLS – NEURONS – EACH COMBINE UP TO THOUSANDS OF SYNAPTIC INPUTS TO GENERATE THEIR OUTPUT, AND BECAUSE 2) IT IS DIFFICULT TO DETERMINE HOW ANY GIVEN NEURON CONTRIBUTES TO THE FUNCTION OF THE BRAIN AS A WHOLE. AS A RESULT, WE DO NOT HAVE EXPLANATIONS IN THE ABOVE TERMS FOR MAMMALIAN BRAIN CIRCUITS, NOR ARE WE ABLE TO MEASURE THE INPUT-OUTPUT OPERATIONS OF EVEN A SINGLE NEURON IN THE MAMMALIAN BRAIN. ADDRESSING THE ABOVE CHALLENGES WILL AID DESIGN OF MEDICAL INTERVENTIONS IN THE BRAIN, ESPECIALLY OF THERAPEUTIC DEVICES THAT MUST DIRECTLY INTERFACE WITH NEURONS – SO-CALLED BRAIN-MACHINE INTERFACES (BMIS). I WILL ADDRESS THE FIRST CHALLENGE BY USING SENSITIVE NEW GENETICALLY ENCODED NEUROTRANSMITTER INDICATORS (GETIS) AND A NOVEL HIGH-BANDWIDTH IN VIVO MICROSCOPE TO SIMULTANEOUSLY RECORD THE ACTIVITY OF THOUSANDS OF SYNAPTIC INPUTS AND OUTPUTS WITHIN INDIVIDUAL NEURONS IN THE CORTEX OF BEHAVING MICE. I WILL BUILD ON MY RECENT WORK DEVELOPING A HIGH-SENSITIVITY GETI FOR GLUTAMATE BY DEVELOPING A SPECTRALLY-COMPATIBLE PAIR OF GETIS FOR GLUTAMATE AND GABA. I WILL COMPLETE THE DEVELOPMENT OF THE 2ND GENERATION SCANNED LINE PROJECTION MICROSCOPE (SLAP2), AN IN VIVO MICROSCOPE THAT WILL ACCURATELY AND EFFICIENTLY RECORD FROM THOUSANDS OF SYNAPSES IN 3D AT >100 HZ. TOGETHER THESE TOOLS WILL MAKE IT POSSIBLE TO DIRECTLY SEE, AT HIGH SPEED, THE PRECISE TIMING AND LOCATION OF MYRIAD NEUROTRANSMITTER INPUTS TO A NEURON, OBSERVE HOW THOSE INPUTS LINE UP TO DRIVE FIRING, AND WATCH IN REAL-TIME AS INPUTS CHANGE WITH LEARNING. TO OVERCOME THE SECOND CHALLENGE AND ENABLE RELIABLE ACCESS TO NEURONS WITH A KNOWN CONTRIBUTION TO A BEHAVIOR, I WILL ADOPT A RAPIDLY-TRAINED BMI- BASED LEARNING TASK IN WHICH A MOUSE LEARNS TO ACTIVATE A SINGLE TARGET CORTICAL NEURON IN A SPECIFIC CONTEXT. I WILL USE HIGH-BANDWIDTH GETI IMAGING TO STUDY HOW THE TARGET NEURON’S SYNAPTIC INPUTS AND INPUT-OUTPUT OPERATIONS CHANGE WITH LEARNING. MOREOVER, I WILL ADAPT THE BMI TASK TO INSTEAD TRAIN NEURONS TO PERFORM AN EXPERIMENTER-SELECTED INPUT-OUTPUT OPERATION, TO THEREBY INVESTIGATE WHAT TYPES OF INPUT-OUTPUT OPERATIONS INDIVIDUAL NEURONS CAN LEARN. THESE TECHNOLOGIES COMBINED WILL ESTABLISH A NEW EXPERIMENTAL PARADIGM WITH NEARLY LIMITLESS POSSIBILITIES FOR STUDYING NEURAL COMPUTATION AND LEARNING. I WILL USE THESE TOOLS TO ASK: 1) HOW ARE BEHAVIORALLY- RELEVANT INPUT-OUTPUT OPERATIONS - THE INDIVIDUAL STEPS OF NEURAL ALGORITHMS - IMPLEMENTED WITHIN THE CORTEX? 2) HOW DO CORTICAL NEURONS LEARN TO PERFORM A SPECIFIC INPUT-OUTPUT OPERATION? 3) WHAT OPERATIONS CAN INDIVIDUAL CORTICAL NEURONS LEARN TO PERFORM? AND 4) CAN WE USE THE RESULTING KNOWLEDGE TO DEVELOP MORE EFFECTIVE BMIS?

MODELING THE STRUCTURE-FUNCTION RELATION IN A RECONSTRUCTED CORTICAL TISSUE

EXA-SCALE TISSUE READOUT METHODS - PROJECT ABSTRACT OUR BRAIN IS THE MOST COMPLEX SYSTEM UNDER STUDY. IT RELIES ON PRECISE WIRING AND SIGNALING ACROSS BILLIONS OF NEURONS. OUR NEURONS SPAN CENTIMETERS, COMMUNICATING WITH ONE ANOTHER VIA A SEA OF SUB-MICROMETER SYNAPSES AND NANOSCALE PROTEINS. AT THE SAME TIME, EACH OF OUR NEURONS HAS A DISTINCT TYPE, STEMMING FROM ITS UNIQUE EXPRESSION OF THOUSANDS OF GENES, AND CAN ASSEMBLE INTO SPATIAL GRADIENTS WITH OTHER NEURONS THAT SPAN ENTIRE REGIONS OF OUR BRAIN. CHARTING VAST MULTI-DIMENSIONAL MAPS OF OUR BRAIN IS ONE OF THE GREATEST CHALLENGES OF MODERN NEUROSCIENCE, WITH FAR-REACHING IMPLICATIONS FOR SHEDDING LIGHT ON THE MECHANISMS OF BRAIN FUNCTION AND IMPROVING OUR ABILITY TO DIAGNOSE AND TREAT NEUROLOGICAL DISEASES. HOWEVER, THE SIZE AND SCALE OF THESE MAPS IS UNPRECEDENTED, AS THEY REQUIRE US TO SEE THE BIG (I.E., THE ENTIRE BRAIN) AND THE SMALL (I.E., INDIVIDUAL SYNAPSES, PROTEINS, AND RNA TRANSCRIPTS) IN A HIGHLY MULTIPLEXED MANNER (I.E., MANY MOLECULAR MARKERS). A GROWING NUMBER OF RECENT INNOVATIONS IN TISSUE PROCESSING, INCLUDING TISSUE EXPANSION FOR MICROSCOPY (PHYSICALLY ENLARGING TISSUE), MULTIPLEXED ANTIBODY LABELING (PROTEIN MAPPING), AND IN SITU SEQUENCING (READING OUT MRNA IN TISSUE USING SEQUENCING BY SYNTHESIS), NOW MAKE IT POSSIBLE TO VISUALIZE HUNDREDS OF MOLECULAR ENTITIES IN THREE DIMENSIONS ACROSS CENTIMETER-SCALE TISSUE VOLUMES AT RESOLUTIONS THAT EXCEED THE DIFFRACTION LIMIT. AT THE SAME TIME, THE COST OF DATA STORAGE AND COMPUTATION CONTINUES TO DECREASE ACCORDING TO MOORE’S LAW. THESE BREAKTHROUGHS OPEN THE DOOR TO A NEW FRONTIER OF SCIENTIFIC DISCOVERY THAT IS NOT BOUNDED BY OUR ABILITY TO INTERROGATE THE MOLECULAR CONTENTS OF TISSUE, OR STORE AND PROCESS THE RESULTING IMAGING DATA. THIS FRONTIER IS INSTEAD SEVERELY BOUNDED BY OUR CURRENT IMAGING TECHNOLOGIES AND THE RATE AT WHICH WE CAN COLLECT HIGH- RESOLUTION, HIGHLY MULTIPLEXED DATA FROM LARGE TISSUE VOLUMES. HERE I PROPOSE TO DEVELOP A PAIR OF EXA-SCALE TISSUE READOUT METHODS (EXTREME) THAT OVERCOME THIS BOUNDARY AND IMPROVE IMAGING THROUGHPUT BY ORDERS OF MAGNITUDE OVER CURRENT STATE-OF-THE-ART APPROACHES. I WILL DEVELOP A FIRST EXTREME PLATFORM TO ENABLE A NEW TYPE OF MOLECULAR INTERROGATION OF NEURAL CIRCUITS, MAPPING INDIVIDUAL PROTEINS AT MOLECULAR RESOLUTIONS, IN A HIGHLY MULTIPLEXED MANNER, ACROSS ENTIRE BRAINS. I WILL DEVELOP A SECOND EXTREME PLATFORM TO SCALE IN SITU SEQUENCING METHODS TO LARGER MAMMALIAN BRAINS. MAPPING NEURON TYPES ACROSS THE MACAQUE OR HUMAN BRAIN WILL BE ACCOMPLISHED IN SEVERAL MONTHS OR YEARS, AS OPPOSED TO SEVERAL DECADES OR CENTURIES. THESE NEW IMAGING TOOLS WILL TRANSFORM OUR UNDERSTANDING OF THE BRAIN’S CELL TYPES AND THEIR CONNECTIONS AND PROVIDE DETAILED MOLECULAR FINGERPRINTS OF NEUROLOGICAL AND NEUROPSYCHIATRIC DISORDERS. ALTHOUGH THIS PROJECT FOCUSES ON APPLICATIONS IN BRAIN RESEARCH, BOTH EXTREME PLATFORMS WILL HAVE BROAD IMPACT IN OTHER FIELDS, INCLUDING BUT NOT LIMITED TO ONCOLOGY, IMMUNOLOGY, AND DEVELOPMENTAL BIOLOGY.

LARGE-SCALE CONNECTIVITY AND FUNCTION IN A CORTICAL CIRCUIT

GENERATION AND CHARACTERIZATION OF NOVEL AND HIGHLY SPECIFIC NERUONAL SUBTYPE TRA

FROM DIVERSE DYNAMICS TO DIVERSE COMPUTATION VIA NEURAL CELL TYPES - PROJECT SUMMARY A PROMINENT FEATURE OF BIOLOGICAL NEURONAL NETWORKS IS THE ASTONISHING DIVERSITY OF THEIR CELL TYPES. MAJOR, NATIONALLY COORDINATED EXPERIMENTAL EFFORTS, INCLUDING THE BRAIN INITIATIVE’S CELL CENSUS NETWORK (BICCN) AND THE ALLEN INSTITUTE CELL TYPES PROGRAMS, ARE CURRENTLY REVEALING THIS CELLULAR DIVERSITY AT NEW AND VERY HIGH LEVELS OF RESOLUTION. FOR EXAMPLE, JUST ACROSS TWO AREAS OF MOUSE CORTEX 133 CELL TYPES HAVE BEEN CHARACTERIZED, WITH MANY TYPES SHARED ACROSS AREAS! SIMILAR CELL CLASSES HAVE BEEN OBSERVED ACROSS SPECIES. THESE TYPES SHOW MARKED DIFFERENCES NOT ONLY GENE EXPRESSION AND CONNECTIVITY, BUT ALSO MEMBRANE, SPIKING, AND SYNAPTIC DYNAMICS. THIS IS IN SHARP CONTRAST TO MOST COMPUTATIONAL AND THEORETICAL MODELS OF LEARNING IN NEURAL NETWORKS, WHICH GENERALLY MAKE USE OF ONLY ONE OR A SMALL NUMBER OF CELL TYPES. THE GOAL OF THIS PROJECT IS TO PRODUCE NEW COMPUTATIONAL AND THEORETICAL TOOLS TO HELP CLOSE THIS GAP. THIS WILL ENABLE US, AND THE BROADER COMMUNITY, TO TEST A HYPOTHESIS FOR THE FUNCTIONAL ROLE OF CELL-TYPE SPECIFIC, HETEROGENEOUS CELLULAR AND SYNAPTIC DYNAMICS: THAT THEY CAN BE HARNESSED TO GENERATE COMPLEX NETWORK DYNAMICS WHICH ALLOWS FASTER OR MORE ACCURATE LEARNING OF TASKS WHICH THEMSELVES HAVE INPUTS OR OBJECTIVES WHICH HAVE COMPLEX DYNAMICS. SUCH TASKS ABOUND IN NATURAL ENVIRONMENTS. TESTING THIS HYPOTHESIS REQUIRES NEW HIGH-THROUGHPUT COMPUTATIONAL TOOLS TO TRAIN NEURAL NETWORKS WITH BIOLOGICALLY REALISTIC DYNAMICS AND CONNECTIVITY TO SOLVE TASKS, NEW THEORETICAL TOOLS TO UNDERSTAND HOW DIVERSE CELLULAR DYNAMICS CONTRIBUTE TO NETWORK COMPUTATION, AND NEW APPLICATION TO LARGE-SCALE, CELLULAR DATA-DRIVEN MODELS. FIRST, WITH THE EXPERTISE OF A GRANT-SUPPORTED SCIENTIFIC SOFTWARE ENGINEER, WILL BUILD, TEST, AND DISSEMINATE A SOFTWARE PACKAGE THAT FLEXIBLY IMPLEMENTS HETEROGENEOUS DYNAMICS OF SINGLE CELLS AND SHORT-TERM SYNAPTIC DYNAMICS. WE PLAN TO USE A VERY POPULAR, FREELY AVAILABLE AND OPEN SOURCE SOFTWARE FRAMEWORK FOR MACHINE LEARNING (PYTORCH). NEXT, WE WILL ESTABLISH METRICS OF NETWORK DYNAMICS THAT HELP TO MECHANISTICALLY EXPLAIN WHAT DOES -- AND DOES NOT -- MATTER ABOUT CELLULAR AND SYNAPTIC HETEROGENEITY IN IMPACTING LEARNING PERFORMANCE. FINALLY, WE WILL INTEGRATE THESE TOOLS WITH A PRIOR CELL-TYPE SPECIFIC COMPUTATIONAL MODEL OF THE MOUSE PRIMARY VISUAL CORTEX, BASED ON LARGE SCALE ALLEN INSTITUTE DATABASES, TO TEST THE HYPOTHESIS STATED ABOVE. SPECIFICALLY, WE WILL NEWLY DETERMINE WHETHER EXPERIMENTALLY OBSERVED LEVELS OF HETEROGENEITY IN CELLULAR AND SYNAPTIC DYNAMICS CONTRIBUTE TO THE ABILITY OF VISUAL MICROCIRCUITS TO PERFORM VISUAL COMPUTATION.

CO-EXPRESSION NETWORKS OF ADDICTION-RELATED GENES IN THE MOUSE AND HUMAN BRAIN

SCALABLE TOOLS FOR CONSISTENT IDENTIFICATION OF NEURONAL CELL TYPES IN MOUSE AND HUMAN - PROJECT SUMMARY THE PROPOSED WORK WILL ADDRESS A CRITICAL GAP IN OUR UNDERSTANDING OF NEURONAL PHENOTYPES AND CELL TYPES BY DEVELOPING MACHINE LEARNING ALGORITHMS AND CLOUD-BASED SOFTWARE FOR THE INTEGRATION OF MULTIPLE MODALITY CHARACTERIZATIONS LARGE AND GROWING DATASETS OF CORTICAL NEURONS IN MOUSE AND HUMAN. THROUGH OPTIMAL AND INNOVATIVE USE OF POTENTIALLY INCOMPLETE DATA AND EMPHASIS ON AUTOMATED MORPHOLOGICAL CHARACTERIZATION, THE PROPOSED TOOLS WILL ENABLE RICHER AND CONSISTENT CHARACTERIZATIONS OF NEURONS FROM TRANSCRIPTOMIC, ANATOMICAL, OR ELECTROPHYSIOLOGICAL PROFILING. WHILE LARGE-SCALE, BICCN-FUNDED CELL TYPE RESEARCH PROGRAMS RELY ON THE NOTION OF UNIQUE NEURONAL IDENTITY WHICH DETERMINES THE CELL’S PHENOTYPE ACROSS DIFFERENT OBSERVATION MODALITIES, OVERARCHING AGREEMENTS ACROSS PHYSIOLOGICAL, ANATOMICAL AND MOLECULAR CHARACTERIZATIONS REMAIN ELUSIVE. ALTHOUGH THESE LARGE-SCALE PROGRAMS SUCCEEDED IN GENERATING EXTENSIVE MULTIPLE MODALITY DATASETS, THE LACK OF PRINCIPLED, ACCURATE AND WIDELY AVAILABLE COMPUTATIONAL ALIGNMENT AND INFERENCE TOOLS PRESENTS A ROADBLOCK TO THE SUCCESS OF THE OVERALL PROGRAM. A SECOND ISSUE IS THAT ANATOMICAL CHARACTERIZATION, DESPITE BEING THE CLASSICAL APPROACH TO UNDERSTANDING CELL TYPES, LAGS SIGNIFICANTLY BEHIND MOLECULAR AND PHYSIOLOGICAL METHODS IN TERMS OF THROUGHPUT. THE RESEARCH PROPOSED HERE AIMS TO ADDRESS THE ALIGNMENT PROBLEM BY BUILDING ON THE COUPLED AUTOENCODER APPROACH, WHICH PRESENTS AN EFFICIENT OPTIMIZATION FRAMEWORK CENTERED ON THE UBIQUITY OF NEURONAL IDENTITY. IMPORTANTLY, THE PROPOSED SOFTWARE CAN UTILIZE INCOMPLETELY CHARACTERIZED DATA POINTS, WHICH IS COMMON IN PRACTICE, TO PRODUCE UNIFIED VISUALIZATION AND ANALYSIS OF ABSTRACT NEURONAL IDENTITY. THIS TOOL WILL BE BOTH FLEXIBLE (E.G., THE FEATURE SET CAN BE CHANGED) AND EXTENSIBLE (E.G., MORE OBSERVATION MODALITIES CAN BE ADDED FOR JOINT ALIGNMENT). THE ALIGNED REPRESENTATIONS ENABLE CONSISTENT CLUSTERING OF THE NEURONAL POPULATION ACROSS THE DIFFERENT OBSERVATION MODALITIES, WHICH IS A PRESSING PROBLEM IN MODERN NEUROSCIENCE. WE PROPOSE TO ADDRESS THE ANATOMICAL THROUGHPUT ISSUE WITH AN END-TO-END COMPUTATIONAL PIPELINE, FROM THE RAW IMAGE OF LOCAL NEURONAL ARBORS TO THE ANATOMICAL DESCRIPTOR THAT CAN BE READILY ALIGNED AND INTERPRETED BY THE COUPLED AUTOENCODER SOFTWARE. BY UTILIZING OUR EXTENSIVE GOLD-STANDARD MANUAL RECONSTRUCTIONS, WE WILL TRAIN SUPERVISED DEEP ARTIFICIAL NEURAL NETWORKS TO SEGMENT NEURONAL ARBORS IN SPARSE LABELING SCENARIOS. THE RICH SET OF TRAINING EXAMPLES, TOGETHER WITH ALGORITHMIC INNOVATIONS, WILL ENDOW SUPERIOR GENERALIZABILITY OF THIS AUTOMATED SEGMENTATION TOOL, ACCELERATING SCIENCE FOR LIGHT MICROSCOPY-BASED STUDIES.

A MULTI-PLANE 3-PHOTON MICROSCOPE FOR VOLUME IMAGING IN NHP CORTEX - PROJECT ABSTRACT RECENT ADVANCES IN MICROSCOPY PERMIT VOLUME IMAGING - THE NEAR-SIMULTANEOUS IMAGING OF MANY NEURONS IN A CIRCUIT - WITH 2-PHOTON (2P) EXCITATION, YIELDING NEW INSIGHTS INTO THE CIRCUITS UNDERLYING RELATIVELY SIMPLE BEHAVIORS. VOLUME IMAGING TECHNIQUES AND 2P EXCITATION HAVE BEEN LIMITED MAINLY TO SMALL ANIMALS SUCH AS RODENTS, INCLUDING STUDIES OF MOUSE CORTEX. MOUSE CORTEX DIFFERS FROM NEOCORTEX IN NON-HUMAN PRIMATES AND HUMANS AND IT IS UNLIKELY THAT STUDIES OF MOUSE CORTEX WILL BE SUFFICIENT TO UNDERSTAND THE ROLE OF CORTEX IN HUMANS. IN THIS R34 APPLICATION, WE PROPOSE TO BUILD A MULTI-PLANE 3-PHOTON (3P) FLUORESCENCE MICROSCOPE TO PERFORM VOLUME IMAGING IN MACAQUES. UNLIKE 2P EXCITATION, 3P EXCITATION SUPPORTS IMAGING DEEP INTO MACAQUE CORTEX. IN A FUTURE R01 APPLICATION, WE WILL USE THIS 3P MICROSCOPE TO IMAGE LARGE VOLUMES OF MACAQUE PRIMARY VISUAL CORTEX, STUDYING HOW MOTION INFORMATION IS POOLED ACROSS NEURONS IN LAYER 4B AND PAIRING 3P MICROSCOPY WITH SERIAL-SECTION ELECTRON MICROSCOPY TO STUDY THE PROCESSING OF COLOR INFORMATION, PARTICULARLY IN LAYER 4CSS. WE WILL PURSUE FOUR SPECIFIC AIMS: - SPECIFIC AIM 1: CONSTRUCT AND TEST A MULTI-PLANE 3P MICROSCOPE - SPECIFIC AIM 2: MEASURE VISUAL TUNING OF ~50,000 NEURONS IN LAYERS 1-4 IN A MACAQUE. - SPECIFIC AIM 3: EXTEND MULTI-PLANE 3P IMAGING THROUGH LAYERS 5 AND 6 OF MACAQUE CORTEX. - SPECIFIC AIM 4: MAKE HARDWARE DESIGNS, SOFTWARE AND PROTOCOLS FREELY AVAILABLE.

DEVELOPMENT OF AN OLIGONUCLEOTIDE THERAPEUTIC FOR DRAVET SYNDROME - PROJECT SUMMARY DRAVET SYNDROME (DS) IS A SEVERE EPILEPTIC ENCEPHALOPATHY MANIFESTING IN THE FIRST YEAR OF LIFE, CHARACTERIZED BY FEBRILE SEIZURES, INTELLECTUAL DISABILITY, AND HIGH RISK OF SUDDEN UNEXPECTED DEATH IN EPILEPSY (SUDEP). MOST DS PATIENTS LACK ONE FUNCTIONAL COPY OF SCN1A, THE GENE ENCODING THE VOLTAGE-GATED SODIUM CHANNEL NAV1.1. THIS MONOGENIC NATURE IN DS SUGGESTS SCN1A UPREGULATION OR REPLACEMENT AS OBVIOUS TREATMENT STRATEGIES; YET DESPITE CONSIDERABLE EFFORTS, DS STILL LACKS AN FDA-APPROVED DISEASE-MODIFYING DRUG. HERE WE PROPOSE TO DEVELOP A NEW THERAPEUTIC TARGET STRATEGY FOR SCN1A UPREGULATION, EMBODIED BY A NEW ANTISENSE OLIGONUCLEOTIDE (ASO) THAT ELEVATES NAV1.1 PROTEIN EXPRESSION. THIS ASO BLOCKS TRANSLATION INITIATION OF AN UPSTREAM OPEN READING FRAME (UORF), THEREBY DIRECTLY INCREASING NAV1.1 PROTEIN PRODUCTION AND ELEVATING THE STEADY STATE LEVEL OF NAV1.1. IMPORTANTLY, THIS ASO TARGETS A DIFFERENT MECHANISM THAN CURRENT ASOS UNDER TESTING THAT MODULATE A POISON EXON OR AN ANTISENSE REGULATORY GENE. IN SIDE-BY-SIDE MATCHED-DOSE COMPARISONS TO A POISON EXON-BLOCKING ASO, THIS ASO PROVIDES SUPERIOR RESCUE OF MORTALITY AND HEAT-INDUCED SEIZURES IN A DS MOUSE MODEL. IN THIS PROPOSED PROJECT, WE WILL CHARACTERIZE THE PHARMACOKINETICS AND PHARMACODYNAMICS OF THIS PROMISING NEW DS-TARGETING ASO AND CLOSELY RELATED DERIVATIVES. THE PROPOSAL IS DIVIDED INTO TWO PHASES, R61 AND R33. IN THE TWO-YEAR R61 PHASE, WE WILL ESTABLISH OUR LEAD ASO COMPOUND. ACTIVITIES IN THE R61 PHASE INCLUDE: OPTIMIZATION OF ASOS BY VARYING THE POSITION ALONG THE TRANSCRIPT AND THE ASO CHEMISTRY, ESTABLISHMENT OF METHODS FOR BIODISTRIBUTION, DESCRIPTION OF ASO BIODISTRIBUTION AND HALF-LIFE, DEMONSTRATION OF NAV1.1 UPREGULATION IN MOUSE BRAINS AND HUMAN NEURONS, IDENTIFICATION OF THE OPTIMAL DOSE FOR NAV1.1 UPREGULATION, AND LAST, GENERATION AND VALIDATION A LARGE-SCALE BATCH OF THE LEAD ASO. IN THE R33 PHASE, WE WILL APPLY OUR LEAD ASO TO DEMONSTRATE EFFICACY AND SAFETY. R33 ACTIVITIES WILL INCLUDE: EVALUATION OF LEAD ASO IN TWO DS ANIMAL MODELS AFTER ADMINISTRATION IN NEONATAL MICE TO TEST FOR DISEASE PREVENTION, EVALUATION OF EFFICACY AFTER ADMINISTRATION INTO ONE ANIMAL MODEL AT P28 TO TEST DISEASE REVERSIBILITY, AND EVALUATION OF THE LEAD ASO BIODISTRIBUTION AND SAFETY. THE ULTIMATE GOAL OF THIS PROPOSAL IS TO GENERATE AN INDUSTRY-LEADING ASO THAT UPREGULATES NAV1.1 THROUGH SCN1A UORF BLOCKADE AND CONFERS STRONG AND SAFE RESCUE IN MULTIPLE MOUSE MODELS OF DS. FURTHERMORE, WE HOPE THIS GRANT WILL HELP CREATE A DIRECT PATH TO THE CLINIC FOR THE LEAD ASO EITHER THROUGH THE NIH BLUEPRINT NEUROTHERAPEUTICS NETWORK-BIOLOGIC “DRUG DISCOVERY AND DEVELOPMENT FOR DISORDERS OF THE NERVOUS SYSTEM” GRANT MECHANISM, OR THOUGH AN INDUSTRIAL PARTNERSHIP.

LIGHT-SHEET MICRO-ASPIRATION MICROSCOPY FOR THE ISOLATION AND ANALYSIS OF RARE TUMOR CELLS FROM INTACT CLINICAL SPECIMENS - PROJECT SUMMARY THIS GRANT APPLICATION RESPONDS TO PROGRAM ANNOUNCEMENT NUMBER PA-18-398 AND OUTLINES A TRAINING AND RESEARCH PROGRAM WHICH WILL PREPARE ME FOR A LONG-TERM RESEARCH CAREER FOCUSED ON THE DEVELOPMENT OF OPTICAL IMAGING METHODS TO BETTER UNDERSTAND, DIAGNOSE, AND TREAT HUMAN PROSTATE CANCER. THE PROPOSAL OUTLINES A MENTORED-TRAINING PROGRAM WHICH PLACES ME UNDER THE DIRECT GUIDANCE OF A TEAM OF NIH FUNDED RESEARCHERS FROM THE UNIVERSITY OF WASHINGTON (UW) AND THE NEIGHBORING FRED HUTCHINSON CANCER RESEARCH CENTER (FHCRC). MY TRAINING PLAN INVOLVES THREE COMPLIMENTARY AREAS OF FOCUS: (1) RECEIVING FURTHER TRAINING IN TISSUE CLEARING AND LABELING, (2) OBTAINING EDUCATIONAL AND HANDS-ON TRAINING IN SINGLE-CELL ISOLATION AND ANALYSIS, AND (3) DEVELOPING A KNOWLEDGE-BASE IN MOLECULAR ASSAYS AND PROSTATE CANCER TO FACILITATE MEANINGFUL FUTURE COLLABORATIONS WITH EXPERTS IN THESE FIELDS. TO ACHIEVE THESE GOALS, I WILL USE THE FUNDING TO DEDICATE TIME FOR ATTENDING GRADUATE COURSES, WORKSHOPS, TRAINING SEMINARS, GRAND ROUNDS LECTURES, AND MOST IMPORTANTLY REGULAR MEETINGS WITH MY ENTIRE CAREER AWARD TEAM, AS OUTLINED IN MY CAREER DEVELOPMENT PLAN. THE RESEARCH PROJECT COMPONENT OF THE PROPOSAL IS DRIVEN BY THE FACT THAT RARE CELL POPULATIONS ARE KNOWN TO PLAY AN OUTSIZED ROLE IN MANY OF THE MOST CHALLENGING PROBLEMS IN CANCER, AND DOWNSTREAM PROFILING (I.E., GENOMICS, PROTEOMICS, AND TRANSCRIPTOMICS) OF THESE INDIVIDUAL CELLS CAN THEREFORE ALLOW FOR MORE-EFFECTIVE THERAPIES TARGETED AGAINST THESE OFTEN-HIDDEN LYNCHPINS OF THE DISEASE. DESPITE THIS, SINGLE-CELL ISOLATION (THE PROCESS BY WHICH INDIVIDUAL CELLS ARE TARGETED AND COLLECTED FOR FURTHER STUDY) IS STILL TECHNICALLY CHALLENGING WITH CURRENT METHODS, PARTICULARLY FOR RARE TUMOR CELLS. THIS IS PARTICULARLY PROBLEMATIC, AS THESE RARE TUMOR CELLS OFTEN DICTATE METASTASIS, TREATMENT RESISTANCE, AND RECURRENCE, ALL OF WHICH ULTIMATELY RESULT IN PATIENT MORTALITY. WHILE THIS CHALLENGE EXISTS FOR NEARLY ALL FORMS OF CANCER, THIS PROPOSAL WILL FOCUS SPECIFICALLY ON METASTATIC TUMOR CELLS WHICH HAVE INVADED THE LYMPHO-VASCULAR SPACE IN PROSTATE CANCER, AS THE ISOLATION AND ANALYSIS OF THESE RARE CELLS CAN ENABLE THE DEVELOPMENT OF NEW DIAGNOSTIC AND TREATMENT STRATEGIES. TO ACHIEVE THIS, I PROPOSE TO BUILD UPON MY POST- DOCTORAL RESEARCH, AND WITH THE NECESSARY TRAINING ABOVE, DEVELOP A NOVEL LIGHT-SHEET MICRO-ASPIRATION MICROSCOPE WHICH WILL ENABLE THE ISOLATION OF THESE RARE TUMOR CELLS FROM INTACT CLINICAL SPECIMENS FOR THE FIRST TIME. THE PROJECT IS COMPRISED OF THREE SPECIFIC AIMS: (1) DEVELOPMENT OF A MULTI-RESOLUTION OPEN-TOP LIGHT-SHEET MICROSCOPE SYSTEM FOR IDENTIFYING RARE TUMOR CELLS; (2) COMBINED DEMONSTRATION OF LIGHT-SHEET MICROSCOPY WITH MICRO-ASPIRATION FOR ISOLATING RARE TUMOR CELLS; AND (3) PROOF-OF-CONCEPT CLINICAL VALIDATION OF THE SYSTEM FOR ISOLATING RARE INVADING TUMOR CELLS FROM HUMAN PROSTATE TISSUES WITH LYMPHO-VASCULAR INVASION. THE PROSTATE CANCER RESEARCH ENVIRONMENT AT UW AND THE FHCRC IS AN IDEAL PLACE FOR ME TO TRAIN AND PURSUE THIS RESEARCH PROJECT, AND WITH THE GUIDANCE OF MY MENTORSHIP TEAM, I WILL SUCCESSFULLY COMPLETE THIS CAREER DEVELOPMENT AWARD AND PURSUE MY LONG-TERM CAREER GOAL OF BECOMING AN INDEPENDENT INVESTIGATOR. !

CRCNS US-GERMAN RESEARCH PROPOSAL: COMPUTATIONAL MODELS OF REPRESENTATION AND INTER-AREAL COMMUNICATION IN MOUSE VISUAL CORTEX -LARGE NEUROSCIENCE DATASETS HAVE BEEN PRODUCED THAT CHARACTERIZE THE PROPERTIES OF INDIVIDUAL NEURONS AND THEIR CONNECTIONS, AND NEURAL ACTIVITY DURING BEHAVIOR. EVEN WITH THIS WEALTH OF DATA, MECHANISTIC UNDERSTANDING OF HOW FUNCTION ARISES FROM THE BRAIN IS STILL EXTREMELY LIMITED. HOW DO DIFFERENT BRAIN AREAS WORK TOGETHER TO REPRESENT THE WORLD THAT IS PERCEIVED? HOW ARE COMMUNICATIONS AMONG DIFFERENT AREAS -- WHICH DEPEND ON STIMULI IN THE ENVIRONMENT AND THE STATE OF THE OBSERVER -- IMPLEMENTED BY A CIRCUIT WHICH IS FIXED? THIS PROJECT WILL INTEGRATE VAST DATASETS INTO A SET OF MODELS WHICH WILL BE PROBED TO ADDRESS MECHANISTIC QUESTIONS INVOLVING MULTI-AREA INTERACTIONS SUCH AS THESE. MORE SPECIFICALLY, A SET OF MODELS WHICH ARE SIMILAR IN NATURE WILL BE CONSTRUCTED. THIS SET WILL START SIMPLE, BUT GRADUALLY SIMPLIFYING ASSUMPTIONS ABOUT MODEL PARAMETERS WILL BE REPLACED BY OPTIMIZATIONS BASED ON DYNAMICAL AND FUNCTIONAL DATA. THE MODELS WILL BE USED TO TEST THE HYPOTHESES THAT: 1. THE CELL-TYPE SPECIFIC PATTERNS OF CONNECTIVITY BETWEEN AREAS ARE IMPORTANT IN DETERMINING TEMPORAL RESPONSES TO SIMPLE STIMULI AND OBSERVED FREQUENCY-SPECIFIC INTER-AREA INTERACTIONS. 2. THE FUNCTIONAL SPECIFICITY OF INTER-AREAL CONNECTIONS IS IMPORTANT IN DETERMINING THE RESPONSES AND INTER-AREA COMMUNICATION IN RESPONSE TO COMPLEX STIMULI. 3. VARIATIONS IN SINGLE-CELL PROPERTIES EXPLAIN AROUSAL-RELATED CHANGES IN VISUAL RESPONSES AND OSCILLATIONS. THIS PROJECT COMBINES THE UNIQUE EXPERIMENTAL DATASETS AND EXPERTISE OF THE ALLEN INSTITUTE WITH THE J?LICH EXPERTISE AND RESOURCES FOR LARGE-SCALE SIMULATION, AND THE EXPERTISE OF BOTH PARTNERS IN DATA-BASED NEURAL NETWORK MODELING. TO FACILITATE FURTHER SCIENTIFIC INQUIRY, THE PROJECT WILL CONSTRUCT A SET OF DETAILED SYSTEM-WIDE MODELS TO STUDY HOW PROPERTIES OF INTER-AREA CONNECTIVITY INFLUENCE INTER-AREA INTERACTIONS, COMMUNICATION, AND STIMULUS REPRESENTATIONS. BEYOND THE SPECIFIC RESULTS FOR THE VISUAL SYSTEM, ADDRESSING THE MECHANISMS OF INTER-AREA COMMUNICATION IS LIKELY IMPORTANT FOR A MECHANISTIC UNDERSTANDING OF COGNITION. A COMPANION PROJECT IS BEING FUNDED BY THE FEDERAL MINISTRY OF EDUCATION AND RESEARCH, GERMANY (BMBF). 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.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.

DYSREGULATION OF NAIVE T CELL QUIESCENCE DURING AGING.

EVALUATING THE IMPACT OF AN OPEN-DATA-BASED LESSONS AND COURSE-BASED RESEARCH EXPERIENCES ON STUDENT KNOWLEDGE, SKILLS, AND ATTITUDES -THIS PROJECT AIMS TO SERVE THE NATIONAL INTEREST BY STUDYING LEARNING OUTCOMES WHEN STUDENTS USE OPEN DATA AND SCIENTIFIC TOOLS IN NOVEL CURRICULUM UNITS THAT TEACH MITOSIS, A FOUNDATIONAL CONCEPT IN BIOLOGY. PRIOR STUDIES HAVE INDICATED THAT COURSE-BASED UNDERGRADUATE RESEARCH EXPERIENCES (CURES) AND OTHER FORMS OF ACTIVE LEARNING LED TO DEEPER UNDERSTANDING OF SCIENTIFIC TOPICS, IMPROVED RETENTION IN SCIENCE CAREERS, AND OTHER POSITIVE OUTCOMES FOR STUDENTS. HOWEVER, ACTIVE LEARNING AND CURES ARE CHALLENGING TO SCALE, AND THE LATTER USUALLY REQUIRE ACCESS TO LAB FACILITIES AND MATERIALS WHICH ARE OFTEN INEQUITABLY DISTRIBUTED. TO ADDRESS THESE CHALLENGES, THIS PROJECT WILL DEVELOP ACTIVE LEARNING-BASED AND CURE CURRICULUM UNITS THAT USE FREE, ONLINE OPEN DATA AND TOOLS FROM THE ALLEN INSTITUTE. IN ADDITION, THE PROJECT TEAM WILL DEVELOP A STANDARDIZED CONCEPT INVENTORY FOR MEASURING STUDENT UNDERSTANDING OF MITOSIS, INVESTIGATE THE IMPACTS OF THOSE CURRICULA ON STUDENT LEARNING OUTCOMES USING THE CONCEPT INVENTORY, AND ULTIMATELY RELEASE BOTH THE TEACHING TOOLS AS WELL AS THE RESEARCH RESULTS. THE KEY INNOVATIONS OF THIS PROJECT WILL BE A) TO ADVANCE UNDERSTANDING OF THE IMPACTS OF OPEN DATA AS A TEACHING TOOL ON STUDENT ATTITUDES AND KNOWLEDGE, B) TO CREATE AND RELEASE VALIDATED OPEN-DATA-BASED LESSONS, AND C) TO DEVELOP AND RELEASE A VALIDATED CONCEPT INVENTORY (CI) ON MITOSIS, A TOOL USED TO CAPTURE STUDENT UNDERSTANDING OF CORE CONCEPTS ON A TOPIC. IF USE OF OPEN DATA FOR ACTIVE LEARNING OR A FULL VIRTUAL CURE GENERATES SIGNIFICANT LEARNING AND ATTITUDE BENEFITS FOR STUDENTS, THIS INDICATES THAT OPEN DATA MAY BE USED IN TEACHING SETTINGS TO LOWER SOME OF THE SOCIAL AND ECONOMIC BARRIERS TO ENTRY AND RETENTION IN SCIENTIFIC RESEARCH AND INCREASE ACCESS TO IMPACTFUL EXPERIENCES IN THE FIELD. THE FINDINGS FROM THIS STUDY WOULD INFORM TEACHING ACROSS TOPICS ON THE IMPACT OF OPEN DATA ON STUDENT SCIENTIFIC THINKING, ENGAGEMENT WITH LEARNING, RETENTION OF KNOWLEDGE, AND PERSISTENCE IN THE FIELD. THE GOAL OF THIS PROJECT IS TO SYSTEMATICALLY EVALUATE THE IMPACT OF USING OPEN DATA AND RESEARCH TOOLS IN A CLASSROOM SETTING ON STUDENT UNDERSTANDING, SCIENTIFIC THINKING, CONFIDENCE, AND ATTITUDES, SPECIFICALLY IN THE CONTEXT OF LEARNING THE BIOLOGICAL PROCESS OF MITOSIS. THE OPEN DATA AND TOOLS USED IN THIS PROJECT HAVE BEEN PREVIOUSLY PRODUCED BY THE ALLEN INSTITUTE FOR CELL SCIENCE, A DIVISION OF THE ALLEN INSTITUTE, AND INCLUDE THOUSANDS OF HIGH-RESOLUTION, FLUORESCENTLY-TAGGED 3D IMAGES OF LIVE HUMAN CELLS WITH ACCOMPANYING VISUALIZATION AND QUANTITATIVE ANALYSIS TOOLS. THIS STUDY WOULD EXTEND THE VALIDATED USE CASES AND IMPACT OF THESE OPEN RESOURCES INTO THE WORLD OF EDUCATION. THIS PROJECT WILL PRODUCE TWO PRIMARY PRODUCTS: A) TWO CURRICULA ON MITOSIS USING OPEN DATA FOR ACTIVE LEARNING OR FOR A CURE, WHICH WILL ALSO BE RELEASED AS OPEN EDUCATIONAL RESOURCES (OERS) AT THE END OF THE STUDY, AND B) A VALIDATED CI ON MITOSIS. THESE PRODUCTS WILL BE USED TO QUANTIFY THE IMPACT OF THESE VARIANT CURRICULA ON STUDENTS? SCIENTIFIC THINKING SKILLS, KNOWLEDGE OF MITOSIS (MEASURED WITH THE CI), CONFIDENCE IN THEIR OWN KNOWLEDGE, INTEREST IN CONTINUING STUDY, AND OTHER ATTITUDINAL OUTCOMES. THIS STUDY AIMS TO EXTEND THE BENEFITS OF CURES TO SETTINGS WHERE A LABORATORY-BASED OPTION IS NOT AVAILABLE, AND TO PROVIDE NEW TOOLS AND OPTIONS TO EDUCATORS INTERESTED IN IMPLEMENTING CURES. THE NSF IUSE: EHR PROGRAM SUPPORTS RESEARCH AND DEVELOPMENT PROJECTS TO IMPROVE THE EFFECTIVENESS OF STEM EDUCATION FOR ALL STUDENTS. THROUGH ITS ENGAGED STUDENT LEARNING TRACK, THE PROGRAM SUPPORTS THE CREATION, EXPLORATION, AND IMPLEMENTATION OF PROMISING PRACTICES AND TOOLS. 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.

COLLABORATIVE RESEARCH: FRAMEWORKS: CYBERSHUTTLE: AN END-TO-END CYBERINFRASTRUCTURE CONTINUUM TO ACCELERATE DISCOVERY IN SCIENCE AND ENGINEERING -SCIENCE DEPENDS CRITICALLY ON ACCESSING SCIENTIFIC AND ENGINEERING SOFTWARE, DATA REPOSITORIES, STORAGE RESOURCES, ANALYTICAL TOOLS, AND A WIDE RANGE OF ADVANCED COMPUTING RESOURCES, ALL OF WHICH MUST BE INTEGRATED INTO A COHESIVE SCIENTIFIC RESEARCH ENVIRONMENT. THE CYBERSHUTTLE PROJECT IS CREATING A SEAMLESS, SECURE, AND HIGHLY USABLE SCIENTIFIC RESEARCH ENVIRONMENT THAT INTEGRATES ALL OF A SCIENTIST?S RESEARCH TOOLS AND DATA, WHICH MAY BE ON THE SCIENTIST?S LAPTOP, A COMPUTING CLOUD, OR A UNIVERSITY SUPERCOMPUTER. THESE RESEARCH ENVIRONMENTS CAN FURTHER SUPPORT SCIENTIFIC RESEARCH BY ENABLING SCIENTISTS TO SHARE THEIR RESEARCH WITH COLLABORATORS AND THE BROADER SCIENTIFIC COMMUNITY, SUPPORTING REPLICABILITY AND REUSE. THE CYBERSHUTTLE TEAM INTEGRATES BIOPHYSICISTS, NEUROSCIENTISTS, ENGINEERS, AND COMPUTER SCIENTISTS INTO A SINGLE TEAM PURSUING THE PROJECT GOALS WITH A GROUNDING IN CUTTING-EDGE RESEARCH PROBLEMS SUCH AS UNDERSTANDING HOW SPIKE PROTEINS IN VIRUSES WORK, HOW THE BRAIN FUNCTIONS DURING SLEEP, AND HOW ARTIFICIAL INTELLIGENCE TECHNIQUES CAN BE APPLIED TO MODELING ENGINEERING MATERIALS. TO MEET ITS AMBITIOUS GOALS, THE PROJECT IS BUILDING ON OVER A DECADE OF EXPERIENCE IN DEVELOPING AND OPERATING THE OPEN-SOURCE APACHE AIRAVATA SOFTWARE FRAMEWORK FOR CREATING SCIENCE-CENTRIC DISTRIBUTED SYSTEMS. CYBERSHUTTLE IS PROVIDING A SYSTEM THAT CAN BE USED AS A TRAINING GROUND TO EDUCATE STUDENTS IN CONCEPTS OF OPEN-SOURCE SOFTWARE DEVELOPMENT AND APPLIED DISTRIBUTED SYSTEMS, FOSTERING A GLOBALLY COMPETITIVE WORKFORCE WHO CAN MOVE EASILY BETWEEN ACADEMIC AND NON-ACADEMIC CAREERS. CYBERSHUTTLE IS CREATING A NEW TYPE OF USER-FACING CYBERINFRASTRUCTURE THAT WILL ENABLE SEAMLESS ACCESS TO A CONTINUUM OF CI RESOURCES USABLE FOR ALL RESEARCHERS, INCREASING THEIR PRODUCTIVITY. THE CORE OF THE CYBERSHUTTLE FRAMEWORK IS A HYBRID DISTRIBUTED SYSTEM, BASED ON OPEN-SOURCE APACHE AIRAVATA SOFTWARE. THIS SYSTEM INTEGRATES LOCALLY DEPLOYED AGENT PROGRAMS WITH CENTRALLY HOSTED MIDDLEWARE TO ENABLE AN END-TO-END INTEGRATION OF COMPUTATIONAL SCIENCE AND ENGINEERING RESEARCH ON RESOURCES THAT SPAN USERS? LOCAL RESOURCES, CENTRALIZED UNIVERSITY COMPUTING AND DATA RESOURCES, COMPUTATIONAL CLOUDS, AND NSF-FUNDED, NATIONAL-SCALE COMPUTING CENTERS. SCIENTISTS AND ENGINEERS ACCESS THIS SYSTEM USING SCIENTIFIC USER ENVIRONMENTS DESIGNED FROM THE BEGINNING WITH THE BEST USER-CENTERED DESIGN PRACTICES. CYBERSHUTTLE USES A SPIRAL APPROACH FOR DEVELOPING, DEPLOYING, AND INCREASING USAGE AND USABILITY, BEGINNING WITH ON-TEAM SCIENTISTS AND EXPANDING TO LARGER SCIENTIFIC COMMUNITIES. THE PROJECT ENGAGES THE LARGER COMMUNITY OF SCIENTISTS, CYBERINFRASTRUCTURE EXPERTS, AND OTHER STAKEHOLDERS IN THE CREATION AND ADVANCEMENT OF CYBERSHUTTLE THROUGH A STAKEHOLDER ADVISORY BOARD. CYBERSHUTTLE'S TEAM INCLUDES RESEARCHERS FROM INDIANA UNIVERSITY, THE UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN, THE UNIVERSITY OF CALIFORNIA SAN DIEGO, THE SAN DIEGO SUPERCOMPUTER CENTER, AND THE ALLEN INSTITUTE. 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.

HEALTH CARE AND OTHER FACILITIES

CO-EXPRESSION NETWORKS OF ADDICTION-RELATED GENES IN THE MOUSE AND HUMAN BRAIN

THE LS2 INITIATIVE: AN OPEN-SOURCE LIGHT-SHEET MICROSCOPE FOR IMAGING ACROSS SCALES - PROJECT ABSTRACT BIOLOGICAL TISSUE IS ORGANIZED OVER SCALES OF NANOMETERS TO CENTIMETERS. UNDERSTANDING INDIVIDUAL CELLS AND MULTI- CELLULAR ORGANIZATION REQUIRES SIMULTANEOUSLY PROBING THE ARCHITECTURE OF TISSUE ACROSS THESE SPATIAL SCALES. RECENT ADVANCES IN TISSUE CLEARING, EXPANSION, AND FLUORESCENCE LABELING ARE PROVIDING UNPRECEDENTED ACCESS TO STRUCTURAL AND MOLECULAR INFORMATION FROM INTACT TISSUES. THESE METHODS HAVE THE POTENTIAL TO ACCELERATE NEW DISCOVERIES ACROSS DIVERSE FIELDS OF RESEARCH. WITHIN NEUROSCIENCE, THESE TECHNIQUES OFFER NEW POSSIBILITIES TO MAP NEURAL CIRCUITS FROM SYNAPSES TO THE WHOLE MOUSE, PRIMATE, AND HUMAN BRAIN. HOWEVER, FULLY HARNESSING MOLECULAR IMAGING ACROSS THREE-DIMENSIONAL SAMPLES AT SCALE REQUIRES NEW ADVANCES IN MICROSCOPY. STANDARD MICROSCOPE OBJECTIVES HAVE LIMITED WORKING DISTANCES (< 1 MM) AND FIELDS OF VIEW (< 1 MM). THEREFORE, IMAGING LARGE TISSUE VOLUMES AT HIGH RESOLUTION TYPICALLY REQUIRES PHYSICAL SECTIONING AND EXTENSIVE IMAGE TILING, BOTH OF WHICH SEVERELY COMPLICATE LARGE-SCALE VOLUMETRIC IMAGING. THESE TECHNICAL CONSTRAINTS OFTEN LIMIT CURRENT IMAGING TECHNOLOGIES TO IMAGING SMALL (MM3) VOLUMES AT HIGH RESOLUTION (<1 ΜM) OR LARGE (CM3) VOLUMES AT LOWER RESOLUTION (>1 ΜM). TO ADDRESS THIS NEED, WE HAVE DEVELOPED A NEW LARGE-SCALE LIGHT-SHEET (LS2) MICROSCOPE. OUR LS2 SYSTEM ENABLES DIFFRACTION-LIMITED AND ABERRATION-FREE IMAGING OF CENTIMETER-SCALE TISSUES WITH HIGH CONTRAST, RESOLUTION, SPEED, AND EASE-OF-USE (I.E., NO TISSUE SECTIONING AND MINIMAL IMAGE TILING) - ATTRIBUTES THAT ARE CURRENTLY UNATTAINABLE WITH EXISTING IMAGING TECHNOLOGIES. BY LEVERAGING HIGHLY ENGINEERED LENSES AND CAMERA SENSORS FROM THE ELECTRONICS METROLOGY INDUSTRY, THE LS2 SYSTEM ACHIEVES A FIELD OF VIEW OF 10.6 X 8.0 MM (30X LARGER THAN PREVIOUS SYSTEMS) AND 35 MM WORKING DISTANCE (10X LARGER) WITH A NEAR-ISOTROPIC RESOLUTION OF 1.0 X 1.0 X 2.5 ΜM. INCLUDING STAGE MOVEMENT, THE SYSTEM CAN ACCESS A 200 X 52 X 35 MM3 VOLUME WITHOUT SECTIONING, AT UP TO 1 GIGAVOXEL/SEC. WHILE THE LS2 SYSTEM WAS DESIGNED AT THE ALLEN INSTITUTE FOR NEURAL DYNAMICS FOR BRAIN-WIDE IMAGING AND RECONSTRUCTION OF NEURAL CIRCUITS IN THE MOUSE BRAIN, IT WILL ALSO BE TRANSFORMATIVE FOR AN ARRAY OF SCIENTIFIC QUESTIONS BEING PURSUED BY MANY OTHER INVESTIGATORS AND INSTITUTIONS. HOWEVER, FOR THE LS2 SYSTEM TO DRIVE SCIENTIFIC DISCOVERY, IT IS IMPERATIVE TO OFFER WELL-DOCUMENTED, SUPPORTED HARDWARE AND SOFTWARE DESIGNS, ALONGSIDE EDUCATIONAL RESOURCES—ELEMENTS CRUCIAL TO LOWERING THE ADOPTION BARRIER. THE WORK PROPOSED HERE WILL SUPPORT THESE EFFORTS, THEREBY ALLOWING US TO MAXIMIZE THE SCIENTIFIC IMPACT OF A RECENTLY OPTIMIZED AND RE-ENGINEERED AND OPTIMIZED LS2 SYSTEM.

HEALTH CARE AND OTHER FACILITIES

HOW NEURONAL ENSEMBLES CONTROL BRAIN NETWORK DYNAMICS TO DRIVE ACTION - PROJECT ABSTRACT IF AN ORGANISM PERFORMS AN ACTION THAT LEADS TO A DESIRED OUTCOME, IT IS ABLE TO PERFORM THAT ACTION AGAIN IN THE FUTURE IN ORDER TO OBTAIN THAT SAME OUTCOME. WHILE WORK ON THE MECHANISMS OF REINFORCEMENT LEARNING HAS EXTENSIVELY STUDIED HOW THE BRAIN LEARNS CERTAIN ACTIONS ARE MORE VALUABLE THAN OTHERS, THERE IS LITTLE KNOWLEDGE ABOUT HOW THE BRAIN ACTUALLY RE-ENTERS NEURAL STATES ON-DEMAND TO PRODUCE THE BEHAVIOR THAT LEADS TO THE DESIRED OUTCOME. THIS IS A CENTRAL QUESTION IN NEUROSCIENCE WHICH UNDERLIES LEARNING, MEMORY, AND MOVEMENT AND HAS IMPLICATIONS FOR THERAPIES TO RESTORE THESE ABILITIES INCLUDING BRAIN-MACHINE INTERFACES. IT IS BELIEVED THAT CONNECTIVITY BETWEEN NEURONS GIVES RISE TO DYNAMICS—RULES FOR HOW THE BRAIN TRANSITIONS BETWEEN NEURAL STATES—AND THAT MODIFICATION OF CONNECTIVITY ENABLES LEARNING TO RE-ENTER NEURAL STATES. HOWEVER, TWO MAIN EXPERIMENTAL CHALLENGES HAVE IMPEDED DIRECT INVESTIGATION: 1) MEASURING AND MANIPULATING CONNECTIVITY BETWEEN NEURONS IN VIVO, AND 2) IDENTIFYING THE NEURONS AND ACTIVITY PATTERNS GENERATING A BEHAVIOR. IN THIS PROPOSAL, I WILL OVERCOME THESE CHALLENGES USING 1) 2-PHOTON MICROSCOPY TO MEASURE AND MANIPULATE FUNCTIONAL CONNECTIVITY IN VIVO BY PHOTOSTIMULATING INDIVIDUAL TARGETED NEURONS AND MEASURING THE NETWORK’S RESPONSE, AND 2) A BRAIN-MACHINE INTERFACE (BMI) PARADIGM TO DEFINE HOW NEURAL ACTIVITY IS TRANSFORMED INTO BEHAVIOR AND REINFORCEMENT. THROUGH EXPERIMENTS THAT APPLY THESE TECHNIQUES BASED ON NOVEL MODELS OF NETWORK DYNAMICS, MY PROPOSAL SEEKS PRINCIPLES FOR HOW FUNCTIONAL CONNECTIVITY UNDERLIES NETWORK DYNAMICS AND ENABLES LEARNING IN MOTOR CORTEX, A CRITICAL REGION FOR GENERATING MOVEMENT. IN THE FIRST AIM (K99), I WILL DETERMINE WHETHER A MODEL OF NETWORK DYNAMICS PREDICTS FUNCTIONAL CONNECTIVITY AND HOW PATTERNED PHOTOSTIMULATION PROPAGATES THROUGH CONNECTIVITY TO MODIFY THE NETWORK STATE. IN AIM 2 (K99/R00), I WILL DESIGN A BMI TO STUDY WHETHER FUNCTIONAL CONNECTIVITY CONSTRAINS LEARNING. THE BMI WILL TEST WHETHER IT IS EASIER TO LEARN NETWORK STATES THAT CAN BE ENTERED THROUGH PHOTOSTIMULATION PROPAGATION. I WILL ALSO DETERMINE WHETHER CHANGES IN FUNCTIONAL CONNECTIVITY SUPPORT LEARNING BY TESTING WHETHER PHOTOSTIMULATION MORE EASILY PROPAGATES TO ENTER LEARNED NETWORK STATES. FINALLY, IN AIM 3 (R00), I WILL REVEAL PRINCIPLES FOR HOW NETWORK ACTIVITY CAN CHANGE NETWORK CONNECTIVITY AND DYNAMICS. I WILL TEST DIFFERENT PROTOCOLS FOR STIMULATING SPATIOTEMPORAL PATTERNS AND REVEAL PRINCIPLES OF STIMULATION PROTOCOLS THAT CHANGE THE NETWORK. DURING THE K99, THIS WORK WILL BE CONDUCTED IN THE COLLABORATIVE ZUCKERMAN INSTITUTE FOR BRAIN AND BEHAVIOR AT COLUMBIA UNIVERSITY WITH THE MENTORSHIP OF DR. RUI COSTA - EXPERT IN THE NEUROBIOLOGY OF ACTION AND DR. LIAM PANINSKI – EXPERT IN COMPUTATIONAL MODELING, AND WITH THE COLLABORATION OF DR. DARCY PETERKA – EXPERT IN OPTICS AND 2-PHOTON MICROSCOPY WITH PHOTOSTIMULATION. I BELIEVE THEIR TECHNICAL AND PROFESSIONAL MENTORSHIP WILL POSITION ME TO LEAD AN INDEPENDENT GROUP STUDYING PRINCIPLES FOR HOW NETWORKS GENERATE AND LEARN DYNAMICS DRIVING BEHAVIOR. THIS WORK WILL HAVE IMPORTANT THERAPEUTIC APPLICATIONS, INCLUDING FOR BRAIN-MACHINE INTERFACES.

INTERROGATING THE FGL2-FCGRIIB AXIS: A NOVEL MECHANISM MEDIATING APOPTOSIS OF TUMOR-SPECIFIC MEMORY CD8+ T CELLS - PROJECT SUMMARY ONE IN FIFTY AMERICANS WILL BE DIAGNOSED WITH MELANOMA IN THEIR LIFETIME AND SKIN CUTANEOUS MELANOMA IS THE DEADLIEST SKIN CANCER. CANCER IMMUNOTHERAPY IS A BREAKTHROUGH APPROACH TO TREAT THIS DISEASE AND CYTOTOXIC CD8+ T-CELL TUMOR INFILTRATION IS A CRITICAL FACTOR TO IMMUNOTHERAPEUTIC SUCCESS. AS SUCH, IDENTIFYING EFFECTIVE STRATEGIES TO INCREASE THE MAGNITUDE AND FUNCTIONALITY OF THE PATIENT’S TUMOR-SPECIFIC CD8+ T-CELL RESPONSE REMAINS AN IMPORTANT GOAL. INHIBITORY MOLECULES ON CD8+ T CELLS ARE IMPERATIVE TO T-CELL SIGNALING AND IMMUNE HOMEOSTASIS. HOWEVER, ELEVATED EXPRESSION OF THESE MOLECULES IS CORRELATED WITH DAMPENED ANTITUMOR EFFECTOR RESPONSE AS WELL AS POORER PATIENT SURVIVAL. FCΓRIIB IS AN INHIBITORY FC RECEPTOR RECENTLY DISCOVERED ON A SUBSET OF CD8+ T CELLS. FCΓRIIB+ CD8+ T CELLS EXHIBIT INCREASED EXPRESSION OF ACTIVATION MARKERS, HIGHER PROLIFERATIVE ABILITY, AND SECRETE MORE PROINFLAMMATORY CYTOKINES THAN THEIR FCΓRIIB- COUNTERPARTS IN MICE AND HUMANS, MAKING THEM IMPERATIVE TO THE ANTITUMOR RESPONSE. RECENTLY, WE DISCOVERED THAT AN IMMUNOSUPPRESSIVE CYTOKINE, FIBRINOGEN-LIKE PROTEIN 2 (FGL2), IS A LIGAND THAT BINDS FCΓRIIB ON CD8+ T CELLS AND INDUCES FCΓRIIB- MEDIATED APOPTOSIS OF CD8+ T CELLS. THE GOAL OF THIS RESEARCH IS TO INTERROGATE THE MECHANISM BY WHICH FGL2 REGULATES TUMOR-SPECIFIC FCΓRIIB+ CD8+ T CELLS USING SYNGENEIC MOUSE MODELS VIA THE FOLLOWING AIM. AIM 1 (F99): DETERMINE THE CELLULAR AND MOLECULAR MECHANISM BY WHICH FGL2 CRITICALLY REGULATES TUMOR-SPECIFIC CD8+ T CELLS. OUR STUDIES SHOW THAT BOTH FOXP3+ REGULATORY T CELLS AND CD8+ T CELLS EXPRESS FGL2 AT THE TUMORS OF MICE AND HUMANS. THUS, WE WILL DETERMINE IF FGL2 SECRETED BY THESE CELL TYPES IS NECESSARY AND/OR SUFFICIENT FOR FCΓRIIB-MEDIATED CD8+ T-CELL APOPTOSIS, FINDINGS WHICH WOULD PROVIDE THE IMPETUS FOR SUBSEQUENT THERAPEUTIC TARGETING OF THIS CELL TYPE. ADDITIONALLY, AS WE HAVE DISCOVERED THAT FCΓRIIB-FGL2 BINDING INDUCES APOPTOSIS, THE UPSTREAM REQUIREMENTS OF APOPTOSIS (E.G. T-CELL RECEPTOR STIMULATION, PROTEINS RECRUITED TO THE INTRACELLULAR DOMAIN OF FCΓRIIB) ARE PROXIMAL ITEMS OF INVESTIGATION IN THE LATTER PART OF AIM 1. PIECING TOGETHER THE PATHWAY BY WHICH FCΓRIIB INDUCES APOPTOSIS VIA FGL2 COULD UNCOVER A NEW CD8+ T CELL PATHWAY READILY HARNESSED FOR FUTURE IMMUNOTHERAPIES. AIM 2 (K00): IDENTIFY NOVEL MECHANISMS OF T CELL RESISTANCE TO CANCER IMMUNOTHERAPY. AFTER THE F99 STAGE, I INTEND TO TRANSITION TO THE K00 STAGE TO BEGIN POSTDOCTORAL STUDIES. NUMEROUS STUDIES HIGHLIGHT THE ROLE OF ELEVATED CHECKPOINT MOLECULE EXPRESSION (PD-1, TIM-3) AS WELL AS DECREASED PROINFLAMMATORY CYTOKINE PRODUCTION (IFNΓ, TNF) IN MEDIATING RESISTANCE TO ICB. THE CURRENT PARADIGM IN CANCER IMMUNOTHERAPY REVOLVES AROUND THE SUPPRESSIVE IMPACT OF THE TUMOR MICROENVIRONMENT ON T CELLS, BUT THE EXISTENCE AND IMPACT OF IMMUNOSUPPRESSIVE FACTORS SECRETED BY EFFECTOR CD8+ T CELLS THEMSELVES IS INCOMPLETELY UNDERSTOOD. THE IMPACT OF THE PROPOSED AIMS IS CONSIDERABLE AS THEY WILL IDENTIFY NOVEL TARGETS, THAT COULD RESCUE A POPULATION OF MEMORY CD8+ T CELLS THAT ARE CRUCIAL TO THE IMMUNE RESPONSE TO TUMOR.

MILLISECOND RESOLUTION STATISTICS OF CORTICAL POPULATIONS

CONVERTING VALUE INTO ACTION: COMPUTATIONS IN CORTICOSTRIATAL CIRCUITS FOR FLEXIBLE DECISION MAKING - PROJECT SUMMARY TO FLEXIBLY EXECUTE BEHAVIOR, CHOICES ARE MADE BASED ON PREVIOUS OUTCOMES THAT WILL MAXIMIZE REWARD. CRUCIALLY, LEARNING THE VALUE OF EACH ACTION TO OBTAIN A REWARD IS THOUGHT TO DRIVE THIS DECISION MAKING PROCESS. IN A VALUE-BASED DECISION MAKING FRAMEWORK, THESE VALUES ARE FIRST COMPUTED AND THEN USED TO SELECT AND EXECUTE ACTIONS. DYSFUNCTION IN THIS DECISION MAKING PROCESS IS EVIDENT IN MANY NEUROPSYCHIATRIC DISORDERS INCLUDING ADDICTION AND IN PATIENTS WITH FRONTAL CORTICAL DAMAGE WHO SHOW AN INABILITY TO FLEXIBLY ADJUST OR ADAPT THEIR BEHAVIOR. THE CORTICOSTRIATAL PATHWAY, FROM THE MEDIAL PREFRONTAL CORTEX (MPFC) TO ITS DOWNSTREAM TARGET, DORSOMEDIAL STRIATUM (DMS), IS A CANDIDATE CIRCUIT IMPLICATED IN PROMOTING FLEXIBLE BEHAVIOR. NEURAL CORRELATES OF VALUE HAVE BEEN FOUND IN MPFC ACROSS SPECIES, AND DMS IS TRADITIONALLY THOUGHT TO BE INVOLVED IN ACTION SELECTION, WHERE REPRESENTATIONS OF ACTION VALUES HAVE BEEN FOUND IN DMS NEURONS. HOWEVER, IT IS UNCLEAR EXACTLY HOW REPRESENTATIONS OF VALUE ARE THEN TRANSFORMED INTO ACTUAL MOVEMENTS FOR ADAPTIVE BEHAVIOR. SIMILARLY, MANY STUDIES THAT HAVE DESCRIBED REPRESENTATIONS OF VALUE IN EITHER REGION HAVE DONE SO USING DISCRETE BINARY MEASURES OF BEHAVIOR AND CORRELATE NEURAL ACTIVITY ONLY TO THE ACTION'S ENDPOINT (E.G. EMITTING A LICK AT A LEFT OR RIGHT SPOUT, OR MAKING A SACCADE TO A TARGET). THIS PROJECT WILL INVESTIGATE THE TRANSFORMATION OF VALUE INTO ACTIONS IN MICE BY INVESTIGATING HOW MPFC AND DMS NEURAL ACTIVITY REPRESENT VALUE DURING ONGOING MOVEMENTS (AIM 1), AND WILL REVEAL THE SPECIFIC CELL TYPES AND PATHWAYS WITHIN THE CORTICOSTRIATAL PATHWAY THAT ARE NECESSARY FOR VALUE REPRESENTATION AND ACTION SELECTION (AIM 2). IN BOTH AIMS, ACTION POTENTIALS FROM MPFC AND DMS WILL BE RECORDED AS THIRSTY HEAD-FIXED MICE PERFORM IN A DYNAMIC FORAGING TASK THAT REQUIRES A JOYSTICK MOVEMENT TO SEARCH FOR THE HIGH-PROBABILITY REWARD, WHERE THE LOCATION OF THE HIGH-PROBABILITY REWARD CHANGES OVER TIME. VALUE-BASED DECISION VARIABLES, SUCH AS ACTION VALUES AND RELATIVE VALUE, WILL BE QUANTIFIED BY CORRELATING THE ANIMAL'S JOYSTICK MOVEMENT BEHAVIOR TO NEURAL ACTIVITY IN BOTH AREAS (AIM 1). THIS WILL REVEAL HOW MPFC AND DMS COMPUTE DECISION VARIABLES DURING ONGOING MOVEMENTS. AIM 2 WILL REVEAL THE SPECIFIC NEURAL CIRCUITRY BETWEEN MPFC AND DMS; PERTURBATIONS OF MPFC LAYER 5 PYRAMIDAL TRACT NEURONS WILL REVEAL HOW VALUE-BASED DECISION VARIABLES ARE REPRESENTED IN THE CORTICOSTRIATAL PATHWAY, AND BY USING TRANSGENIC MOUSE LINES PAIRED WITH ANTIDROMIC STIMULATION, DIRECT AND INDIRECT PATHWAY NEURONS IN DMS WILL BE IDENTIFIED TO DETERMINE EACH CELL TYPE'S ROLE IN REPRESENTING VALUE AND TRANSFORMING VALUE INTO ACTION. THIS PROJECT OVERALL WILL PROVIDE A BETTER UNDERSTANDING OF HOW VALUES ARE ATTRIBUTED TO ACTIONS DURING THE DECISION MAKING PROCESS, AND WILL IDENTIFY HOW SPECIFIC CELLS IN THE CORTICOSTRIATAL PATHWAY COMPUTE AND TRANSFORM INFORMATION ABOUT VALUE TO MAKE OPTIMAL CHOICES.

TRAVEL: NSF STUDENT GRANT FOR 2025 IEEE/CVF INTERNATIONAL CONFERENCE ON COMPUTER VISION (ICCV) -THIS AWARD PROVIDES TRAVEL SUPPORT FOR STUDENTS ENROLLED AT U.S. INSTITUTIONS TO PARTICIPATE IN THE DOCTORAL CONSORTIUM AT THE 2025 IEEE/CVF INTERNATIONAL CONFERENCE ON COMPUTER VISION (ICCV), TO BE HELD IN HONOLULU, HAWAII. NOW IN ITS 20TH YEAR, ICCV IS A PREMIER INTERNATIONAL CONFERENCE THAT CONVENES LEADING RESEARCHERS AND STUDENTS WORKING ON COMPUTER VISION, A FOUNDATIONAL AREA OF ARTIFICIAL INTELLIGENCE (AI) WITH BROAD APPLICATIONS IN FIELDS SUCH AS HEALTHCARE, SECURITY, ROBOTICS, AND AUTONOMOUS SYSTEMS. THE DOCTORAL CONSORTIUM HELPS CULTIVATE FUTURE LEADERS IN AI AND COMPUTER VISION BY PROVIDING EARLY-CAREER RESEARCHERS WITH OPPORTUNITIES TO ENGAGE IN TECHNICAL DISCOURSE, REFINE THEIR RESEARCH DIRECTIONS, AND BUILD PROFESSIONAL NETWORKS. BY ENABLING APPROXIMATELY 20 PH.D. STUDENTS FROM A DIVERSE RANGE OF ACADEMIC INSTITUTIONS TO ATTEND THE CONSORTIUM, THE AWARD PROMOTES ACCESS TO MENTORSHIP, CAREER DEVELOPMENT, AND SCHOLARLY EXCHANGE. IN DOING SO, THIS AWARD DIRECTLY SUPPORTS THE PROGRESS OF SCIENCE AND THE DEVELOPMENT OF THE U.S. SCIENTIFIC WORKFORCE. THE AWARD PROVIDES TRAVEL SUPPORT FOR 20 STUDENTS FROM US INSTITUTIONS TO PARTICIPATE IN A STRUCTURED PROGRAM HELD IN CONJUNCTION WITH ICCV 2025. THE DOCTORAL CONSORTIUM INCLUDES POSTER SESSIONS, MENTORSHIP SESSIONS WITH SENIOR RESEARCHERS, AND FOCUSED DISCUSSIONS ON CAREER PATHWAYS IN ACADEMIA AND INDUSTRY. STUDENT APPLICANTS WILL BE EVALUATED BY THE CONSORTIUM CHAIRS BASED ON THE QUALITY AND RELEVANCE OF THEIR RESEARCH, THEIR POTENTIAL FOR LEADERSHIP IN THE FIELD, AND THE EXPECTED IMPACT OF THEIR PARTICIPATION. THE TRAVEL AWARDS WILL HELP STUDENTS COVER KEY EXPENSES SUCH AS AIRFARE, LODGING, AND LOCAL TRANSPORTATION. BY PROVIDING THIS SUPPORT, THE AWARD ENSURES THAT PROMISING YOUNG RESEARCHERS CAN CONTRIBUTE TO AND BENEFIT FROM ONE OF THE MOST INFLUENTIAL VENUES IN COMPUTER VISION AND ARTIFICIAL INTELLIGENCE 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.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.

GOVERNING COOPERATIVE AGREEMENT: NSF MID-SCALE RI-2: OPEN MULTIMODAL AI INFRASTRUCTURE TO ACCELERATE SCIENCE -- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.