The brain plans and executes volitional movements. The underlying patterns of neural population activity have been explored in the context of movements of the eyes, limbs, tongue, and head in nonhuman primates and rodents. How do networks of neurons produce the slow neural dynamics that prepare specific movements and the fast dynamics that ultimately initiate these movements? Recent work exploits rapid and calibrated perturbations of neural activity to test specific dynamical systems models that are capable of producing the observed neural activity. These joint experimental and computational studies show that cortical dynamics during motor planning reflect fixed points of neural activity (attractors). Subcortical control signals reshape and move attractors over multiple timescales, causing commitment to specific actions and rapid transitions to movement execution. Experiments in rodents are beginning to reveal how these algorithms are implemented at the level of brain-wide neural circuits. Expected final online publication date for the , Volume 45 is July 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 μm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

Decisions are held in memory until enacted, which makes them potentially vulnerable to distracting sensory input. Gating of information flow from sensory to motor areas could protect memory from interference during decision-making, but the underlying network mechanisms are not understood. Here, we trained mice to detect optogenetic stimulation of the somatosensory cortex, with a delay separating sensation and action. During the delay, distracting stimuli lost influence on behavior over time, even though distractor-evoked neural activity percolated through the cortex without attenuation. Instead, choice-encoding activity in the motor cortex became progressively less sensitive to the impact of distractors. Reverse engineering of neural networks trained to reproduce motor cortex activity revealed that the reduction in sensitivity to distractors was caused by a growing separation in the neural activity space between attractors that encode alternative decisions. Our results show that communication between brain regions can be gated via attractor dynamics, which control the degree of commitment to an action.

Measuring the dynamics of neural processing across time scales requires following the spiking of thousands of individual neurons over milliseconds and months. To address this need, we introduce the Neuropixels 2.0 probe together with newly designed analysis algorithms. The probe has more than 5000 sites and is miniaturized to facilitate chronic implants in small mammals and recording during unrestrained behavior. High-quality recordings over long time scales were reliably obtained in mice and rats in six laboratories. Improved site density and arrangement combined with newly created data processing methods enable automatic post hoc correction for brain movements, allowing recording from the same neurons for more than 2 months. These probes and algorithms enable stable recordings from thousands of sites during free behavior, even in small animals such as mice.

The ability to probe the membrane potential of multiple genetically defined neurons simultaneously would have a profound impact on neuroscience research. Genetically encoded voltage indicators are a promising tool for this purpose, and recent developments have achieved a high signal-to-noise ratio in vivo with 1-photon fluorescence imaging. However, these recordings exhibit several sources of noise and signal extraction remains a challenge. We present an improved signal extraction pipeline, spike-guided penalized matrix decomposition-nonnegative matrix factorization (SGPMD-NMF), which resolves supra- and subthreshold voltages in vivo. The method incorporates biophysical and optical constraints. We validate the pipeline with simultaneous patch-clamp and optical recordings from mouse layer 1 in vivo and with simulated and composite datasets with realistic noise. We demonstrate applications to mouse hippocampus expressing paQuasAr3-s or SomArchon1, mouse cortex expressing SomArchon1 or Voltron, and zebrafish spines expressing zArchon1.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine novel spatially and molecularly defined subregions. EASI-FISH also facilitates iterative re-analysis of scRNA-Seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

Short-term memory is associated with persistent neural activity that is maintained by positive feedback between neurons. To explore the neural circuit motifs that produce memory-related persistent activity, we measured coupling between functionally characterized motor cortex neurons in mice performing a memory-guided response task. Targeted two-photon photostimulation of small (<10) groups of neurons produced sparse calcium responses in coupled neurons over approximately 100 μm. Neurons with similar task-related selectivity were preferentially coupled. Photostimulation of different groups of neurons modulated activity in different subpopulations of coupled neurons. Responses of stimulated and coupled neurons persisted for seconds, far outlasting the duration of the photostimuli. Photostimuli produced behavioral biases that were predictable based on the selectivity of the perturbed neuronal population, even though photostimulation preceded the behavioral response by seconds. Our results suggest that memory-related neural circuits contain intercalated, recurrently connected modules, which can independently maintain selective persistent activity.

Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.

Large scientific projects in genomics and astronomy are influential not because they answer any single question but because they enable investigation of continuously arising new questions from the same data-rich sources. Advances in automated mapping of the brain's synaptic connections (connectomics) suggest that the complicated circuits underlying brain function are ripe for analysis. We discuss benefits of mapping a mouse brain at the level of synapses.