Rapid circuit level dendritic spine morphometric analysis for in vivo pharmacology in CNS drug discovery

In vivo phenotypic CNS assays that successfully identify hits and leads in academic translational research are often challenging to scale up and standardize for commercial SAR and MedChem Afraxisandapos Enhanced Spine Profiling platform quantifies brain wide dendritic spine morphometry and plasticity providing insights into neural circuits and changes resulting from disease pharmacological and behavioral manipulations ESP enables dendritic spine analysis for drug discovery using patented methods to obtain redundant human observer based analyses Expert Crowdsourcing via automated processes that demonstrate exceptional repeatability and require no intervention from investigators and analysts We proposes to further align ESP with the needs of medium high throughput compound screens and optimization through three key improvements a implementation of superresolution microscopy b design novel implementations of image recognition software to aid observer based spine quantification and c development of more robust hierarchical multi dimensional statistical methods appropriate to multi observation image and feature analysis for the larger and more complex datasets produced by superresolution imaging We will complete a series of validation studies to empirically refine the optimizations and demonstrate reliable and stable signal Due to time limits of this initial project we chose ketamine as a fast turnaround validation tool compound because of its rapid well studied structural plasticity effects in rodents and clinical relevance to cognition and disease We will next profile five procognitive drugs as reference compounds to verify multivariate signal dynamic ranges across multiple compounds Following these evaluations ESP will be a robust commercially validated phenotypic tool for batch compound analysis to identify hits and guide SAR and MedChem optimization Specific Aim Optimize the Afraxis dendritic spine analysis platform for Lead Identification criteria We propose to optimize the performance of the Afraxis ESP platform to meet the fundamental criteria for inclusion in traditional structure activity relationship SAR paradigms rendering the assay viable for lead identification and lead optimization in CNS drug discovery programs We propose to meet these operational criteria through implementation superresolution microscopy adaptation of our novel distributed corroborative human observer analytical paradigm to facilitate novel image segmentation spine morphometric quantification and novel hierarchical statistical methodologies Specific Aim Validate Dendritic Spine analysis using the rapid anti depressant ketamine Considering the long lead times required for investigating aged animals we propose to validate the optimized version of the assay using the rapid anti depressant ketamine Ketamine rapidly triggers structural modifications that are relevant to aging and age related diseases and Afraxis has previously and multiply replicated from numerous published studies the effect whereby low doses of ketamine induce increased cortical spine density Afraxis has expanded on the original work to show diverse responses across doses dosing paradigms cortical regions and cortical lamina Here we will validate the optimized assay across these contexts by evaluating ketamineandapos s effect in independent pathways within brain regions In this manner we will attempt to characterize a high resolution multivariate dose response for ketamineandapos s anti depressant effect on dendritic spines Central to this effort is empirical establishment of statistical tools optimized for this novel evaluation design see discussion under Approach Specific Aim Specific Aim Validate Dendritic Spine Analysis across known clinical compounds We propose to build on our understanding of dendritic spine responses in SA by testing compounds classified as pro cognitive from clinical evaluations This panel will consist of clinically tested compounds of mixed efficacious results across cognitive domains e g working memory reversal learning The statistical procedures developed in SA and validated in SA will be broadened to evaluate hits within a screening panel based on response profiles across the diverse set of neuroanatomical pathways tested in SA Project Narrative Afraxisandapos Enhanced Spine Profiling platform quantifies brain wide dendritic spine morphometry and plasticity for drug discovery using patented methods to obtain redundant human observer based analyses Expert Crowdsourcing via automated processes that with exceptional repeatability and no intervention from investigators We will further develop ESP for medium high throughput compound screens and optimization by implementing superresolution microscopy novel image recognition software for spine quantification and develop robust hierarchical multi dimensional statistical methods appropriate to multi observation image and feature analysis We will empirically refine the optimizations to validate the multivariate signal dynamic ranges of procognitive reference compounds