A positively tuned voltage indicator for extended electrical recordings in the brain.

Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.

Mapping the neural dynamics of locomotion across the Drosophila brain.

Locomotion engages widely distributed networks of neurons. However, our understanding of the spatial architecture and temporal dynamics of the networks that underpin walking remains incomplete. We use volumetric two-photon imaging to map neural activity associated with walking across the entire brain of Drosophila. We define spatially clustered neural signals selectively associated with changes in either forward or angular velocity, demonstrating that neurons with similar behavioral selectivity are clustered. These signals reveal distinct topographic maps in diverse brain regions involved in navigation, memory, sensory processing, and motor control, as well as regions not previously linked to locomotion. We identify temporal trajectories of neural activity that sweep across these maps, including signals that anticipate future movement, representing the sequential engagement of clusters with different behavioral specificities. Finally, we register these maps to a connectome and identify neural networks that we propose underlie the observed signals, setting a foundation for subsequent circuit dissection. Overall, our work suggests a spatiotemporal framework for the emergence and execution of complex walking maneuvers and links this brain-wide neural activity to single neurons and local circuits.

BIFROST: a method for registering diverse imaging datasets.

Quantitative comparison of brain-wide neural dynamics across different experimental conditions often requires precise alignment to a common set of anatomical coordinates. While such approaches are routinely applied in functional magnetic resonance imaging (fMRI), registering in vivo fluorescence imaging data to ex vivo-derived reference atlases is challenging, given the many differences in imaging modality, microscope specification, and sample preparation. Moreover, in many systems, animal to animal variation in brain structure limits registration precision. Using the highly stereotyped architecture of the fruit fly brain as a model, we overcome these challenges by building a reference atlas based directly on in vivo multiphoton-imaged brains, called the Functional Drosophila Atlas (FDA). We then develop a novel two-step pipeline, BrIdge For Registering Over Statistical Templates (BIFROST), for transforming neural imaging data into this common space, and for importing ex vivo resources, such as connectomes. Using genetically labeled cell types to provide ground truth, we demonstrate that this method allows voxel registration with micron precision. Thus, this method provides a generalizable pipeline for registering neural activity datasets to one another, allowing quantitative comparisons across experiments, microscopes, genotypes, and anatomical atlases, including connectomes.

A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3.

A ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in the presence of sample motion. We performed combinatorial multi-site mutagenesis on a cyan-excitable red fluorescent protein to create the bright and monomeric mCyRFP3, which proved to be uniquely non-perturbing when fused to the GEVI ASAP3. The green/red ratio from ASAP3-mCyRFP3 (ASAP3-R3) reported voltage while correcting for motion artifacts, allowing the visualization of membrane voltage changes in contracting cardiomyocytes and throughout the cell cycle of motile cells.