Cell-Resolution Imaging of Synapses and Circuits in Post-Mortem Specimens

PROJECT SUMMARY Our brain's ~90 billion neurons allow us to think, move, and respond to stimuli. The brain's remarkable properties stem from neural circuits, which perform logical operations based on both the temporal patterns of neural activity and the spatial connectivity of neurons and astrocytes. Gaining a better understanding of the brain's circuitry will have wide ranging implications for human health. Strategies for visualizing and controlling neural activity abound, but there are few methods that enable direct imaging of neural circuit connectivity, and those that exist rely of functional cell physiology, which makes them not applicable to systems that require analysis using fixed post-mortem samples. In this application, we propose a chemical strategy that will enable light microscopy-based imaging of neural synapses and circuit connectivity in post-mortem specimens, including, potentially, the human brain. Our strategy for imaging synapses and circuits employs reactive chemical groups that we target to the neural synapse where they trigger fluorescence on connected cells. Fundamentally, this strategy is composed of two parts: (1) the molecules that become fluorescent to reveal the connected neurons, and (2) the molecules that are targeted to the synapse in neurons of interest to activate fluorescence in connected neurons. The first molecules are pro-fluorescent lipids that we will use to label neuron populations of interest, similar to the popular lipophilic neural tracers DiO and DiI. These molecules are non-fluorescent until they react with the second component, at which point they become fluorescent. The second component is the reactive fluorescence activator that we deliver uniformly to the membranes of the sample but is designed to only become reactive at the neural synapse after exposure to an enzyme localized to the synapse by an antibody. Importantly, we have recently developed the critical enabling technology: a chemical ligation reaction that can be activated by the presence of an enzyme to produce a fluorescent product. Overall, this strategy we describe merges aspects of immunochemistry and the classic lipophilic tracers used to chart neuron morphology, two methods that excel in post-mortem samples. The result is a reactive neural tracer that will enable synapse detection and circuit imaging with light microscopy in post-mortem specimens. The ability to interrogate neural circuitry in post-mortem specimens with light microscopy will enable the field to directly assess the validity of circuit level findings from lower organism disease models to humans.