Damian G. Wheeler, Ph.D. - US grants

Project Summary New imaging tools are needed for brain-wide visualization of the neural activity underlying defined behaviors. Live imaging techniques such as fMRI and MEG measure activity across the brain, but these methods cannot resolve single cell activity. Moreover, in vivo electrophysiology and calcium imaging can reach cellular resolution, but only simultaneously survey a few brain regions at best. Capturing a brain-wide snapshot of recent neural activity at cellular resolution would provide an incredible tool for hypothesis generation and assay development for CNS drug discovery. To date, immunohistochemical imaging of the activity-dependent immediate-early gene (IEG) cFos is the only established platform that visualizes neural activity linked to a defined behavior at cellular resolution. To efficiently collect brain-wide snapshots of neuronal activity, two obstacles need to be overcome. First, since standard histological methods and modern imaging approaches (e.g., serial two-photon) require tissue slicing, these approaches to IEG imaging are time consuming and introduce anatomical distortions that prevent quantitative comparisons among brain regions. Second, since traditional IEGs like cFos are also regulated by paracrine signaling factors, the fidelity of IEG expression as a read-out of neuronal activity is compromised. To increase the throughput of whole-brain histological imaging, we will take advantage of recent developments in tissue clearing, fluorescence light sheet imaging and computational alignment and cell detection. For tissue clearing, we optimized the iDISCO tissue clearing method for whole brain immunostaining. For imaging, we have customized a commercial light sheet micrscope for large format imaging of iDISCO cleared tissue. With our adapted technology, we are imaging a whole mouse brain in less than three hours at higher spatial resolution than in previous reports. Using these methods, we have generated whole brain 3D images of genetically-encoded fluorophores. We will couple this technology to the use of a novel, high quality monoclonal antibody against Npas4, a unique IEG that is neuron-specific and exquisitely tuned to neuronal activity. Our automated quantification pipeline aligns samples to a common reference atlas and generates brain-wide cell counts of neuronal activity across >1,000 identified brain areas. This new imaging platform will overcome the time- and labor-intensive limitations of standard histology, and the use of Npas4 as a marker will provide the highest fidelity, brain-wide read-outs of neuronal activity to date.

Project Summary Here we seek to develop a ?Neuronal Activity Quantitative Diagnostic? platform and reagent kits to disseminate accessible products for measuring recent neural activity across the entire mouse brain to the neuroscience community. Previously we have shown that the activity-dependent immediate-early gene protein products Npas4 and cFos can be clearly detected, imaged, and quantified using developmental versions of our platform tools. Immediate early genes are genes that are activated transiently and rapidly in response to a wide variety of cellular stimuli. The three overlapping aims to be investigated in this Phase II project are: 1) Demonstration of the fidelity of our assay for our customers. To demonstrate the robustness and accuracy of our neuronal activity assay with another well-known assay for activity, we will perform two photon calcium fluorescence imaging in the primary visual cortex of mice presented with repeated visual stimuli. We will clear and stain the brains for cFos and Npas4 and demonstrate the fidelity of these markers for detecting recently active neurons. 2) Develop and beta test robust versions of our software tools useable by researchers with no specialized coding or statistics knowledge. We will develop Stitchy and BrainSnap into robust, user-friendly software products that remove the current analysis bottleneck for neuroscientists interested in whole-brain imaging. Stitchy will be an improved browser-based version of our internal software that simplifies and speeds the process of stitching together light sheet microscope images. BrainSnap software will integrate our high-performance 3TK- Quant framework for machine learning-enabled quantification and our statistical framework for whole-brain anatomics, 3TK-Stats, in a browser-based interface. We have recruited end users who will test this software in a head-to-head comparison with our in-house testing. 3) Develop and test a brain clearing and staining kit for neuronal activity. To help researchers produce high quality cleared brains stained with neuronal activity markers, we will generate Neuronal Activity clearing and staining reagents kits for cFos and Npas4. These will be tested by collaborators in head-to-head comparisons with our in-house procedures on brains prepared in parallel. At the end of Phase II, Translucence will have a market-ready Neuronal Activity Quantitative Diagnostic platform including reagent kits, user-friendly software, and service options.