Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals.

The resolution and dimensionality with which biologists can characterize cell types have expanded dramatically in recent years, and intersectional consideration of such features (e.g., multiple gene expression and anatomical parameters) is increasingly understood to be essential. At the same time, genetically targeted technology for writing in and reading out activity patterns for cells in living organisms has enabled causal investigation in physiology and behavior; however, cell-type-specific delivery of these tools (including microbial opsins for optogenetics and genetically encoded Ca2+ indicators) has thus far fallen short of versatile targeting to cells jointly defined by many individually selected features. Here, we develop a comprehensive intersectional targeting toolbox including 39 novel vectors for joint-feature-targeted delivery of 13 molecular payloads (including opsins, indicators, and fluorophores), systematic approaches for development and optimization of new intersectional tools, hardware for in vivo monitoring of expression dynamics, and the first versatile single-virus tools (Triplesect) that enable targeting of triply defined cell types.

Cortical Observation by Synchronous Multifocal Optical Sampling Reveals Widespread Population Encoding of Actions.

To advance the measurement of distributed neuronal population representations of targeted motor actions on single trials, we developed an optical method (COSMOS) for tracking neural activity in a largely uncharacterized spatiotemporal regime. COSMOS allowed simultaneous recording of neural dynamics at ∼30 Hz from over a thousand near-cellular resolution neuronal sources spread across the entire dorsal neocortex of awake, behaving mice during a three-option lick-to-target task. We identified spatially distributed neuronal population representations spanning the dorsal cortex that precisely encoded ongoing motor actions on single trials. Neuronal correlations measured at video rate using unaveraged, whole-session data had localized spatial structure, whereas trial-averaged data exhibited widespread correlations. Separable modes of neural activity encoded history-guided motor plans, with similar population dynamics in individual areas throughout cortex. These initial experiments illustrate how COSMOS enables investigation of large-scale cortical dynamics and that information about motor actions is widely shared between areas, potentially underlying distributed computations.