Population activity measurement by calcium imaging can be combined with cellular resolution optogenetic activity perturbations to enable the mapping of neural connectivity in vivo. This requires accurate inference of perturbed and unperturbed neural activity from calcium imaging measurements, which are noisy and indirect, and can also be contaminated by photostimulation artifacts. We have developed a new fully Bayesian approach to jointly inferring spiking activity and neural connectivity from in vivo all-optical perturbation experiments. In contrast to standard approaches that perform spike inference and analysis in two separate maximum-likelihood phases, our joint model is able to propagate uncertainty in spike inference to the inference of connectivity and vice versa. We use the framework of variational autoencoders to model spiking activity using discrete latent variables, low-dimensional latent common input, and sparse spike-and-slab generalized linear coupling between neurons. Additionally, we model two properties of the optogenetic perturbation: off-target photostimulation and photostimulation transients. Our joint model includes at least two sets of discrete random variables; to avoid the dramatic slowdown typically caused by being unable to differentiate such variables, we introduce two strategies that have not, to our knowledge, been used with variational autoencoders. Using this model, we were able to fit models on 30 minutes of data in just 10 minutes. We performed an all-optical circuit mapping experiment in primary visual cortex of the awake mouse, and use our approach to predict neural connectivity between excitatory neurons in layer 2/3. Predicted connectivity is sparse and consistent with known correlations with stimulus tuning, spontaneous correlation and distance.

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of >10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a "waterfall" mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

Neurons in motor cortex and connected brain regions fire in anticipation of specific movements, long before movement occurs. This neural activity reflects internal processes by which the brain plans and executes volitional movements. The study of motor planning offers an opportunity to understand how the structure and dynamics of neural circuits support persistent internal states and how these states influence behavior. Recent advances in large-scale neural recordings are beginning to decipher the relationship of the dynamics of populations of neurons during motor planning and movements. New behavioral tasks in rodents, together with quantified perturbations, link dynamics in specific nodes of neural circuits to behavior. These studies reveal a neural network distributed across multiple brain regions that collectively supports motor planning. We review recent advances and highlight areas where further work is needed to achieve a deeper understanding of the mechanisms underlying motor planning and related cognitive processes.

Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.

In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact Escherichia coli class I TAC containing a CAP dimer, a σ(70)-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.

The GFP-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds. Here we describe "semisynthetic" pH-sensitive protein conjugates with organic fluorophores, carbofluorescein, and Virginia Orange that match the properties of SEP. Conjugation to genetically encoded self-labeling tags or antibodies allows visualization of both exocytosis and endocytosis, constituting new bright sensors for these key steps of synaptic transmission.

Sensory, motor and cognitive operations involve the coordinated action of large neuronal populations across multiple brain regions in both superficial and deep structures. Existing extracellular probes record neural activity with excellent spatial and temporal (sub-millisecond) resolution, but from only a few dozen neurons per shank. Optical Ca(2+) imaging offers more coverage but lacks the temporal resolution needed to distinguish individual spikes reliably and does not measure local field potentials. Until now, no technology compatible with use in unrestrained animals has combined high spatiotemporal resolution with large volume coverage. Here we design, fabricate and test a new silicon probe known as Neuropixels to meet this need. Each probe has 384 recording channels that can programmably address 960 complementary metal-oxide-semiconductor (CMOS) processing-compatible low-impedance TiN sites that tile a single 10-mm long, 70 × 20-μm cross-section shank. The 6 × 9-mm probe base is fabricated with the shank on a single chip. Voltage signals are filtered, amplified, multiplexed and digitized on the base, allowing the direct transmission of noise-free digital data from the probe. The combination of dense recording sites and high channel count yielded well-isolated spiking activity from hundreds of neurons per probe implanted in mice and rats. Using two probes, more than 700 well-isolated single neurons were recorded simultaneously from five brain structures in an awake mouse. The fully integrated functionality and small size of Neuropixels probes allowed large populations of neurons from several brain structures to be recorded in freely moving animals. This combination of high-performance electrode technology and scalable chip fabrication methods opens a path towards recording of brain-wide neural activity during behaviour.