U01NS132353

Brain Connects: Synaptic Resolution Whole-Brain Circuit Mapping of Molecularly Defined Cell Types Using a Barcoded Rabies Virus - Abstract

Single-cell transcriptomics has revolutionized our understanding of neuronal diversity and enabled high-throughput characterization of molecular cell types across brain areas and species. We and others have pioneered multi-modal technologies such as Patch-seq and spatial transcriptomics to link molecularly-defined cell types with their physiology, cytomorphology, and anatomical features. But we still lack high-throughput, cost-effective methods that can provide comprehensive synaptic resolution wiring diagrams of entire mammalian brains and integrate these connectomes with molecularly-defined cell types.

We propose to further develop and validate Rabies Barcode Interaction Detection followed by Sequencing (RABID-SEQ) to enable high-throughput, scalable, and cost-effective mapping of brain-wide synaptic-level connectivity and transcriptomic profiling of the mapped neurons. We have optimized Rabies virus production and packaging to achieve barcoded libraries containing more than 1.7 million unique barcodes, two orders of magnitude higher compared to prior studies, enough to map the inputs to thousands of post-synaptic neurons in a single animal. However, this technology still faces several experimental and computational challenges to realize its full potential.

In Aim 1, we will address three potential challenges that may arise when scaling RABID-SEQ to study brain-wide, densely labeled circuits: stochasticity of initial infection and spread, toxicity, and the potential for polysynaptic events when many founder cells are labeled. In addition, we will develop a new variant of Rabies featuring an evolvable barcode that can disambiguate monosynaptic vs polysynaptic spread in the setting of dense labeling.

In Aim 2, we will benchmark RABID-SEQ connectomes against other gold standard techniques measuring connectivity using multipatch-seq and spatial transcriptomics.

In Aim 3, we will develop new algorithms using graph neural networks to reconstruct monosynaptic connectomes from barcoded viral datasets, assess the robustness of these algorithms under different experimental parameters in silico, and test whether an evolvable barcode can improve monosynaptic circuit reconstruction.

If successful, these studies will establish RABID-SEQ as a scalable, cost-effective tool for brain-wide connectivity mapping that can integrate transcriptomic cell types with their synaptic-level wiring diagram at single-cell resolution. By reducing the problem of synaptic connectivity into a problem of barcode sequencing, our approach has the potential to dramatically increase throughput, decrease costs, and provide a direct link to the transcriptome of each mapped cell.

RABID-SEQ will transform brain-wide circuit mapping into a routine experiment that can be performed in any lab with modest resources, making it possible to explore how circuits differ between treatment conditions, in disease states, between the sexes, and across the lifespan. We will also generate pilot data in both mice and human slice cultures to demonstrate the utility of this tool across species.

The Leland Stanford Junior University

(1) TO SUPPORT EXTRAMURAL RESEARCH FUNDED BY THE NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE (NINDS) INCLUDING: BASIC RESEARCH THAT EXPLORES THE FUNDAMENTAL STRUCTURE AND FUNCTION OF THE BRAIN AND THE NERVOUS SYSTEM, RESEARCH TO UNDERSTAND THE CAUSES AND ORIGINS OF PATHOLOGICAL CONDITIONS OF THE NERVOUS SYSTEM WITH THE GOAL OF PREVENTING THESE DISORDERS, RESEARCH ON THE NATURAL COURSE OF NEUROLOGICAL DISORDERS, IMPROVED METHODS OF DISEASE PREVENTION, NEW METHODS OF DIAGNOSIS AND TREATMENT, DRUG DEVELOPMENT, DEVELOPMENT OF NEURAL DEVICES, CLINICAL TRIALS, AND RESEARCH TRAINING IN BASIC, TRANSLATIONAL AND CLINICAL NEUROSCIENCE. THE INSTITUTE IS THE LARGEST FUNDER OF BASIC NEUROSCIENCE IN THE US AND SUPPORTS RESEARCH ON TOPICS INCLUDING BUT NOT LIMITED TO: DEVELOPMENT OF THE NERVOUS SYSTEM, INCLUDING NEUROGENESIS AND PROGENITOR CELL BIOLOGY, SIGNAL TRANSDUCTION IN DEVELOPMENT AND PLASTICITY, AND PROGRAMMED CELL DEATH, SYNAPSE FORMATION, FUNCTION, AND PLASTICITY, LEARNING AND MEMORY, CHANNELS, TRANSPORTERS, AND PUMPS, CIRCUIT FORMATION AND MODULATION, BEHAVIORAL AND COGNITIVE NEUROSCIENCE, SENSORIMOTOR LEARNING, INTEGRATION AND EXECUTIVE FUNCTION, NEUROENDOCRINE SYSTEMS, SLEEP AND CIRCADIAN RHYTHMS, AND SENSORY AND MOTOR SYSTEMS. IN ADDITION, THE INSTITUTE SUPPORTS BASIC, TRANSLATIONAL AND CLINICAL STUDIES ON A NUMBER OF DISORDERS OF THE NERVOUS SYSTEM INCLUDING (BUT NOT LIMITED TO): STROKE, TRAUMATIC INJURY TO THE BRAIN, SPINAL CORD AND PERIPHERAL NERVOUS SYSTEM, NEURODEGENERATIVE DISORDERS, MOVEMENT DISORDERS, BRAIN TUMORS, CONVULSIVE DISORDERS, INFECTIOUS DISORDERS OF THE BRAIN AND NERVOUS SYSTEM, IMMUNE DISORDERS OF THE BRAIN AND NERVOUS SYSTEM, INCLUDING MULTIPLE SCLEROSIS, DISORDERS RELATED TO SLEEP, AND PAIN. PROGRAMMATIC AREAS, WHICH ARE PRIMARILY SUPPORTED BY THE DIVISION OF NEUROSCIENCE, ARE ALSO SUPPORTED BY THE DIVISION OF EXTRAMURAL ACTIVITIES, THE DIVISION OF TRANSLATIONAL RESEARCH, THE DIVISION OF CLINICAL RESEARCH, THE OFFICE OF TRAINING AND WORKFORCE DEVELOPMENT, THE OFFICE OF PROGRAMS TO ENHANCE NEUROSCIENCE WORKFORCE DEVELOPMENT, AND THE OFFICE OF INTERNATIONAL ACTIVITIES. (2) TO EXPAND AND IMPROVE THE SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, TO INCREASE SMALL BUSINESS PARTICIPATION IN FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. TO UTILIZE THE SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM, TO STIMULATE AND FOSTER SCIENTIFIC AND TECHNOLOGICAL INNOVATION THROUGH COOPERATIVE RESEARCH AND DEVELOPMENT CARRIED OUT BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO FOSTER TECHNOLOGY TRANSFER BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION.

93.853 - Extramural Research Programs in the Neurosciences and Neurological Disorders

National Institute of Neurological Disorders and Stroke (NNDS) [HHS - NIH]

Stanford, California 94305 United States

Single Zip Code

BRAIN Initiative Connectivity across Scales (BRAIN CONNECTS): Specialized Projects for Scalable Technologies (U01 Clinical Trial Not Allowed) (RFA-NS-22-049)

Amendment Since initial award the total obligations have increased 178% from $2,189,013 to $6,077,623.