Rapid Evaluation of Neuronal Activity in the Intact Whole-Brain at Single Cell Resolution

Project Summary New imaging tools are needed for brain wide visualization of the neural activity underlying defined behaviorsLive imaging techniques such as fMRI and MEG measure activity across the brainbut these methods cannot resolve single cell activityMoreoverin vivo electrophysiology and calcium imaging can reach cellular resolutionbut only simultaneously survey a few brain regions at bestCapturing 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 discoveryTo dateimmunohistochemical imaging of the activity dependent immediate early geneIEGcFos is the only established platform that visualizes neural activity linked to a defined behavior at cellular resolutionTo efficiently collect brain wide snapshots of neuronal activitytwo obstacles need to be overcomeFirstsince standard histological methods and modern imaging approachese gserial two photonrequire tissue slicingthese approaches to IEG imaging are time consuming and introduce anatomical distortions that prevent quantitative comparisons among brain regionsSecondsince traditional IEGs like cFos are also regulated by paracrine signaling factorsthe fidelity of IEG expression as a read out of neuronal activity is compromisedTo increase the throughput of whole brain histological imagingwe will take advantage of recent developments in tissue clearingfluorescence light sheet imaging and computational alignment and cell detectionFor tissue clearingwe optimized the iDISCO tissue clearing method for whole brain immunostainingFor imagingwe have customized a commercial light sheet micrscope for large format imaging of iDISCO cleared tissueWith our adapted technologywe are imaging a whole mouse brain in less than three hours at higher spatial resolution than in previous reportsUsing these methodswe have generated whole brainD images of genetically encoded fluorophoresWe will couple this technology to the use of a novelhigh quality monoclonal antibody against Npasa unique IEG that is neuron specific and exquisitely tuned to neuronal activityOur automated quantification pipeline aligns samples to a common reference atlas and generates brain wide cell counts of neuronal activity across andgtidentified brain areasThis new imaging platform will overcome the timeand labor intensive limitations of standard histologyand the use of Npasas a marker will provide the highest fidelitybrain wide read outs of neuronal activity to date Project Narrative To better establish the link between neural activity and behavior in health and disease statesnew tools must be developed to capture brain wide patterns of activity at cellular resolutionBy combining new advances in tissue clearingwhole brain light sheet imagingcomputational alignment of brain anatomy and a new molecular marker for neural activityour assay will provideD images of whole brain activity with unprecedented resolution and fidelity