Mammalian brain is composed of vast numbers of intricately interconnected neurons with various molecular, anatomical and physiological identities. To understand the roles of these individual building blocks of the brain, it will be critical to develop spatio- temporally precise tools that will allow neuronal subtype specific single cell level analysis. In this project, we propose to develop molecular tools that will allow high throughput single cell genomic modifications and apply them in order to functionally and morphologically characterize cell type specific circuits within the mammalian brain. To achieve this, our approach will be to use light to trigger site-specific DNA modification enzymes. The site-specific DNA recombinases have been extremely useful tools for dissecting the functional and genetic components of the nervous system due to their ability to precisely modify individual genes within cells. However, they lack the ability to be regulated with high spatiotemporal accuracies to precisely target individual cells. We modified two of these enzymes -Cre and Dre- by combining them with a fungal-based light inducible protein in such a way that upon light induction the activity of the enzymes can be triggered. We propose to further apply the same strategy to the Flp recombinase in order to enrich the intersectional approach. We will then use these recombinases to generate Cre dependent mouse lines so that we can restrict the light inducible recombination to further subtypes of neurons either alone or in combination with existing Cre driver lines. Using these lines we will perform light induced focal recombination in order to perform sparse cell type specific genomic recombination of fluorescent reporters to fully reconstruct individual neuron subtypes in axonal and dendritic detail. Furthermore we will combine this approach with 2-photon excitation to perform single cell rabies tracing experiments. Our results will set the foundation for extremely detailed analysis of neuron type specific functional and anatomical circuits that will make it possible to link genetic identity, morphology, connectivity and function.

Public Health Relevance

In order to have a better understanding of the healthy and diseased brain a deep understanding of the underlying circuit working principles is necessary. Therefore dissecting the cell types that are the building blocks of such circuits is fundamental. In this project our aim is to develop light inducible single cell genetic modification methods and study the connections, morphologies, function and genetic identity of individual neurons within the brain.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Multi-Year Funded Research Project Grant (RF1)
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Special Emphasis Panel (ZMH1)
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Kim, Douglas S
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Stanford University
Schools of Arts and Sciences
United States
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