High-throughput methods for measuring cortical synaptic connectivity at single-cell resolution

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Project Summary Information processing in the brain is accomplished by integrating neuronal activity via specific patterns of synaptic connectivity between diverse neuronal subtypes, and changes in connectivity are hypothesized to cause a range of neuropsychiatric disorders. However, current methods to measure synaptic connectivity with single-cell precision are laborious, time-consuming, and costly. The main goal of my research program is to develop high-throughput methods for measuring the patterns of synaptic connectivity in the cerebral cortex. To accomplish this, I will pursue three separate approaches using viral, functional, and molecular strategies. First, I will optimize rabies-based trans-synaptic tracing to enable labeling of local cortical circuitry and classify connected neurons with multiplexed, error- robust fluorescent in situ hybridization (merFISH). Next, I will screen for functional connectivity by combining holographic optogenetic stimulation of individual pre-synaptic neurons with multiplexed whole-cell automated patch-clamp to increase the number of synaptic connections that can be probed from a single animal. Finally, I will design molecular barcodes targeted to pre- and post-synaptic sites that can be imaged with merFISH to identify synaptic connections in concert with transcriptional information for cell-type classification. I will first apply these methods towards high-confidence risk genes associated with schizophrenia and bipolar disorder, as disordered cortical circuitry is thought to cause these diseases. This proposal requires the innovative integration of multiple cutting-edge technologies and the development of new, non-existing techniques. Given my training in electrophysiology, optogenetics, advanced microscopy, and imaging analysis as well as my current environment at the Stanley Center at the Broad Institute, I am ideally positioned to develop and validate these techniques. Successful completion of this proposal will result in new methods for analyzing synaptic connectivity applicable to a wide range of neuroscientific questions and provide insight into the pathophysiological mechanisms of psychiatric disorders.