Large-scale voltage imaging in behaving mice using targeted illumination.

Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold transmembrane voltage in vivo. To perform high-speed voltage imaging of many neurons simultaneously over a large anatomical area, widefield microscopy remains an essential tool. However, the lack of optical sectioning makes widefield microscopy prone to background cross-contamination. We implemented a digital-micromirror-device-based targeted illumination strategy to restrict illumination to the cells of interest and quantified the resulting improvement both theoretically and experimentally with SomArchon expressing neurons. We found that targeted illumination increased SomArchon signal contrast, decreased photobleaching, and reduced background cross-contamination. With the use of a high-speed, large-area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in behaving mice. Thus, the targeted illumination strategy described here offers a simple solution for widefield voltage imaging of many neurons over a large field of view in behaving animals.

A Viral Toolbox of Genetically Encoded Fluorescent Synaptic Tags.

Fibronectin intrabodies generated with mRNA display (FingRs) are a recently developed tool for labeling excitatory or inhibitory synapses, with the benefit of not altering endogenous synaptic protein expression levels or synaptic transmission. Here, we generated a viral vector FingR toolbox that allows for multi-color, neuron-type-specific labeling of excitatory or inhibitory synapses in multiple brain regions. We screened various fluorophores, FingR fusion configurations, and transcriptional control regulations in adeno-associated virus (AAV) and retrovirus vector designs. We report the development of a red FingR variant and demonstrated dual labeling of excitatory and inhibitory synapses in the same cells. Furthermore, we developed cre-inducible FingR AAV variants and demonstrated their utility, finding that the density of inhibitory synapses in aspiny striatal cholinergic interneurons remained unchanged in response to dopamine depletion. Finally, we generated FingR retroviral vectors, which enabled us to track the development of excitatory and inhibitory synapses in hippocampal adult-born granule cells.