Spatiotemporal correlations in brain activity are functionally important and have been implicated in perception, learning and plasticity, exploratory behavior, and various aspects of cognition. Neurons in the cerebral cortex are strongly interacting. Their activity is temporally irregular and can exhibit substantial correlations. However, how the collective dynamics of highly recurrent and strongly interacting neurons can evolve into a state in which the activity of individual cells is highly irregular yet macroscopically correlated is an open question. Here, we develop a general theory that relates the strength of pairwise correlations to the anatomical features of networks of strongly coupled neurons. To this end, we investigate networks of binary units. When interactions are strong, the activity is irregular in a large region of parameter space. We find that despite the strong interactions, the correlations are generally very weak. Nevertheless, we identify architectural features, which if present, give rise to strong correlations without destroying the irregularity of the activity. For networks with such features, we determine how correlations scale with the network size and the number of connections. Our work shows the mechanism by which strong correlations can be consistent with highly irregular activity, two hallmarks of neuronal dynamics in the central nervous system.

The most fundamental choice an animal has to make when it detects a threat is whether to freeze, reducing its chances of being noticed, or to flee to safety. Here we show that Drosophila melanogaster exposed to looming stimuli in a confined arena either freeze or flee. The probability of freezing versus fleeing is modulated by the fly's walking speed at the time of threat, demonstrating that freeze/flee decisions depend on behavioral state. We describe a pair of descending neurons crucially implicated in freezing. Genetic silencing of DNp09 descending neurons disrupts freezing yet does not prevent fleeing. Optogenetic activation of both DNp09 neurons induces running and freezing in a state-dependent manner. Our findings establish walking speed as a key factor in defensive response choices and reveal a pair of descending neurons as a critical component in the circuitry mediating selection and execution of freezing or fleeing behaviors.

New methods in stem cell 3D organoid tissue culture, advanced imaging and big data image analytics now allow tissue scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice Light-Sheet Imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70µm x 60µm x 40µm xyz) at 5.7s/frame. We developed an open source data analysis package termed pyLattice to process the resulting large (∼60Gb) movie datasets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. Based on their localization in the organoid, we classified CME tracks into apical, lateral and basal events and found that CME dynamics are similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative, high temporal and spatial resolution analysis of subcellular events within tissues. Movie S1 Movie S1 Thresholded 3D AO-LLSM data of an intestinal epithelial organoid showing clathrin (red) and dynamin2 (green) puncta in surface depiction. The movie zooms out from a single clathrin mediated endocytosis event that shows both clathrin and dynamin2 at the same location to eventually show the whole AO-LLSM field of view. Nuclear envelopes and the outer membranes of the tissue are depicted in transparent white. Movie S2 Movie S2 Thresholded 3D AO-LLSM data of an intestinal epithelial organoid showing clathrin (red) and dynamin2 (green) puncta in surface depiction. The movie rotates the AO-LLSM field of view. Nuclear envelopes and the outer membranes of the tissue are depicted in transparent white. Movie S3 Movie S3 Thresholded 3D AO-LLSM data of an intestinal epithelial organoid. The curved surface is of the spherical organoid is visible as the movie rotates. Clathrin puncta are visible throughout the tissue (white). Movie S4 Movie S4 The detection step in the data processing pipeline retrieves all clathrin puncta in the volume. Detected puncta are marked with a cube (blue). Movie S5 Movie S5 Zoom on one clathrin puncta in the thresholded 3D dataset. The punctum of interest is marked with a blue cube. Other puncta are also visible. Movie S6 Movie S6 Zoom on the same clathrin puncta as in M3 in non-thresholded 3D data. The surrounding fluorescence is visible as a transparent cloud.

Accurately predicting an outcome requires that animals learn supporting and conflicting evidence from sequential experience. In mammals and invertebrates, learned fear responses can be suppressed by experiencing predictive cues without punishment, a process called memory extinction. Here, we show that extinction of aversive memories in Drosophila requires specific dopaminergic neurons, which indicate that omission of punishment is remembered as a positive experience. Functional imaging revealed co-existence of intracellular calcium traces in different places in the mushroom body output neuron network for both the original aversive memory and a new appetitive extinction memory. Light and ultrastructural anatomy are consistent with parallel competing memories being combined within mushroom body output neurons that direct avoidance. Indeed, extinction-evoked plasticity in a pair of these neurons neutralizes the potentiated odor response imposed in the network by aversive learning. Therefore, flies track the accuracy of learned expectations by accumulating and integrating memories of conflicting events.

Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. Here, we show an animal model of PSE, in , owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase ()-null mutants and fail to encapsulate the neuronal cell bodies in the neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glial membranes. Further, we show the compromise in plasma membrane structure that underlies the glial cell membrane collapse in mutants and leads to the PSE phenotype.

The striatum shows general topographic organization and regional differences in behavioral functions. How corticostriatal topography differs across cortical areas and cell types to support these distinct functions is unclear. This study contrasted corticostriatal projections from two layer 5 cell types, intratelencephalic (IT-type) and pyramidal tract (PT-type) neurons, using viral vectors expressing fluorescent reporters in Cre-driver mice. Corticostriatal projections from sensory and motor cortex are somatotopic, with a decreasing topographic specificity as injection sites move from sensory to motor and frontal areas. Topographic organization differs between IT-type and PT-type neurons, including injections in the same site, with IT-type neurons having higher topographic stereotypy than PT-type neurons. Furthermore, IT-type projections from interconnected cortical areas have stronger correlations in corticostriatal targeting than PT-type projections do. As predicted by a longstanding model, corticostriatal projections of interconnected cortical areas form parallel circuits in the basal ganglia.

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. "Masking" the activity of small molecules using a general chemical modification and "unmasking" it only within target cells overcomes this limitation. To this end, we have developed a selective enzyme-substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2-nitro- N-methylimidazolyl (NM) masking group. To discover and optimize this NTR-NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.

The anterolateral motor cortex (ALM) and ventromedial (VM) thalamus are functionally linked to support persistent activity during motor planning. We analyzed the underlying synaptic interconnections using optogenetics and electrophysiology in mice (♀/♂). In cortex, thalamocortical (TC) axons from VM excited VM-projecting pyramidal-tract (PT) neurons in layer 5B of ALM. These axons also strongly excited layer 2/3 neurons (which strongly excite PT neurons, as previously shown) but not VM-projecting corticothalamic (CT) neurons in layer 6. The strongest connections in the VM→PT circuit were localized to apical-tuft dendrites of PT neurons, in layer 1. These tuft inputs were selectively augmented after blocking hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. In thalamus, axons from ALM PT neurons excited ALM-projecting VM neurons, located medially in VM. These axons provided weak input to neurons in mediodorsal nucleus, and little or no input either to neurons in the GABAergic reticular thalamic nucleus or to neurons in VM projecting to primary motor cortex (M1). Conversely, M1 PT axons excited M1- but not ALM-projecting VM neurons. Our findings indicate, first, a set of cell-type-specific connections forming an excitatory thalamo-cortico-thalamic (T-C-T) loop for ALM↔VM communication and a circuit-level substrate for supporting reverberant activity in this system. Second, a key feature of this loop is the prominent involvement of layer 1 synapses onto apical dendrites, a subcellular compartment with distinct signaling properties, including HCN-mediated gain control. Third, the segregation of the ALM↔VM loop from M1-related circuits of VM adds cellular-level support for the concept of parallel pathway organization in the motor system.Anterolateral motor cortex (ALM), a higher-order motor area in the mouse, and ventromedial thalamus (VM) are anatomically and functionally linked, but their synaptic interconnections at the cellular level are unknown. Our results show that ALM pyramidal tract neurons monosynaptically excite ALM-projecting thalamocortical neurons in a medial subdivision of VM, and vice versa. The thalamo-cortico-thalamic loop formed by these recurrent connections constitutes a circuit-level substrate for supporting reverberant activity in this system.