Project Grant R43ES036389

INTEGRATING DEVELOPMENTAL NEUROTOXICITY SCREENING USING AUTOMATED NEURO-BEHAVIORAL, MORPHOLOGICAL AND NEURO-DEVELOPMENTAL TESTS IN THE LARVAL ZEBRAFISH - INTEGRATING DEVELOPMENTAL NEUROTOXICITY SCREENING USING AUTOMATED NEURO-BEHAVIORAL, MORPHOLOGICAL AND NEURO-DEVELOPMENTAL TESTS IN THE LARVAL ZEBRAFISH ABSTRACT: ZEBRAFISH IS A MODEL THAT HOLDS IMMENSE PROMISE FOR SCREENING EFFECTS OF ENVIRONMENTAL CHEMICALS ON NERVOUS SYSTEM DEVELOPMENT AND HAS THE POTENTIAL TO REVEAL PATHWAYS OF DEVELOPMENTAL NEUROTOXICITY (DNT) IN ORDER TO PROVIDE A SOUND BASIS FOR HUMAN-RISK ASSESSMENT. EXPERIMENTS USING THE ZEBRAFISH FOR DNT CAN BE BROADLY CATEGORIZED AS GENE EXPRESSION PROFILING, NEUROIMAGING AND NEUROBEHAVIORAL ASSESSMENTS. GENE EXPRESSION PROFILING STUDIES CAN BE SCALED IN THROUGHPUT BUT NEUROIMAGING AND NEUROBEHAVIORAL EXPERIMENTS ARE ASSESSED USING IMAGING SYSTEMS WHERE THROUGHPUT, ANALYSIS AUTOMATION AND STANDARDIZATION ARE KNOWN BOTTLENECKS. CURRENTLY, SINGLE-CAMERA SYSTEMS USED FOR NEUROBEHAVIORAL ASSESSMENTS CAN PROVIDE 2D BEHAVIOR METRICS BUT DON'T HAVE SUFFICIENT RESOLUTION TO SCREEN FOR MORPHOLOGY. SIMILARLY, FOR NEUROIMAGING ASSESSMENTS, CURRENT SINGLE OBJECTIVE IMAGERS STEP-AND-SCAN OVER EVERY WELL TO SCREEN AN ENTIRE WELL PLATE IN SEVERAL MINUTES, BUT CAN'T PERFORM LONGITUDINAL ASSAYS (FOR E.G. TIMELAPSE). MOREOVER, NO IMAGING SYSTEM CURRENTLY EXISTS TO OUR KNOWLEDGE THAT CAN ASSESS 3D BEHAVIOR AND LATERAL MORPHOLOGY OF ZEBRAFISH AT A SUFFICIENTLY HIGH THROUGHPUT (> 40 FISH AT A TIME). HERE WE PROPOSE TO USE OUR NOVEL MULTI-CAMERA ARRAY MICROSCOPE (MCAM) TECHNOLOGY STACK TO DEVELOP AN INTEGRATED HIGH-THROUGHPUT IMAGING SYSTEM (MCAM-DNT), AN INNOVATIVE "PERISCOPIC"MULTI-WELL ASSAY PLATE AND ASSOCIATED ANALYSIS SOFTWARE FOR INTEGRATED ASSESSMENTS OF NEUROBEHAVIORAL AND NEUROIMAGING EXPERIMENTS ON THE SAME SYSTEM. FOR NEUROIMAGING, OUR PROPOSED SYSTEM WILL ENABLE NON-INVASIVE MONITORING AND PROFILING OF CHANGES IN REGIONS OF EXPRESSION THROUGH DEVELOPMENT ACROSS AN ENTIRE WELL-PLATE WITHIN SECONDS. FOR NEUROBEHAVIOR ASSESSMENTS, WE WILL OBTAIN LATERAL MORPHOLOGY AS WELL AS DORSAL MORPHOLOGY, 2D BEHAVIOR AND 3D BEHAVIOR IN AN AUTOMATED FASHION FOR UNMOUNTED AND FREELY SWIMMING FISH UPTO 48 AT A TIME. LASTLY, A USER FACING SOFTWARE WILL COLLATE ACTIONABLE INSIGHTS FROM NEUROBEHAVIOR AND NEUROIMAGING ASSESSMENTS TO PRESENT TO THE USER AS WELL AS FOR DOWNSTREAM ANALYSIS. THIS PHASE-1 GRANT IS THE FIRST STEP TOWARDS ESTABLISHING THE UTILITY OF THE MCAM-DNT IMAGING SYSTEM AND ANALYSIS SOFTWARE FOR DNT SCREENING IN MODEL ORGANISMS (PLANARIA, MEDAKA) BEYOND THE ZEBRAFISH.