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2529 Janelia Publications
Showing 331-340 of 2529 resultsMany animals maintain an internal representation of their heading as they move through their surroundings. Such a compass representation was recently discovered in a neural population in the Drosophila melanogaster central complex, a brain region implicated in spatial navigation. Here, we use two-photon calcium imaging and electrophysiology in head-fixed walking flies to identify a different neural population that conjunctively encodes heading and angular velocity, and is excited selectively by turns in either the clockwise or counterclockwise direction. We show how these mirror-symmetric turn responses combine with the neurons' connectivity to the compass neurons to create an elegant mechanism for updating the fly's heading representation when the animal turns in darkness. This mechanism, which employs recurrent loops with an angular shift, bears a resemblance to those proposed in theoretical models for rodent head direction cells. Our results provide a striking example of structure matching function for a broadly relevant computation.
The field of connectomics has recently produced neuron wiring diagrams from relatively large brain regions from multiple animals. Most of these neural reconstructions were computed from isotropic (e.g., FIBSEM) or near isotropic (e.g., SBEM) data. In spite of the remarkable progress on algorithms in recent years, automatic dense reconstruction from anisotropic data remains a challenge for the connectomics community. One significant hurdle in the segmentation of anisotropic data is the difficulty in generating a suitable initial over-segmentation. In this study, we present a segmentation method for anisotropic EM data that agglomerates a 3D over-segmentation computed from the 3D affinity prediction. A 3D U-net is trained to predict 3D affinities by the MALIS approach. Experiments on multiple datasets demonstrates the strength and robustness of the proposed method for anisotropic EM segmentation.
Full reconstruction of neuron morphology is of fundamental interest for the analysis and understanding of their functioning. We have developed a novel method capable of automatically tracing neurons in three-dimensional microscopy data. In contrast to template-based methods, the proposed approach makes no assumptions about the shape or appearance of neurite structure. Instead, an efficient seeding approach is applied to capture complex neuronal structures and the tracing problem is solved by computing the optimal reconstruction with a weighted graph. The optimality is determined by the cost function designed for the path between each pair of seeds and by topological constraints defining the component interrelations and completeness. In addition, an automated neuron comparison method is introduced for performance evaluation and structure analysis. The proposed algorithm is computationally efficient and has been validated using different types of microscopy data sets including Drosophila’s projection neurons and fly neurons with presynaptic sites. In all cases, the approach yielded promising results.
Many different types of functional non-coding RNAs participate in a wide range of important cellular functions but the large majority of these RNAs are not routinely annotated in published genomes. Several programs have been developed for identifying RNAs, including specific tools tailored to a particular RNA family as well as more general ones designed to work for any family. Many of these tools utilize covariance models (CMs), statistical models of the conserved sequence, and structure of an RNA family. In this chapter, as an illustrative example, the Infernal software package and CMs from the Rfam database are used to identify RNAs in the genome of the archaeon Methanobrevibacter ruminantium, uncovering some additional RNAs not present in the genome’s initial annotation. Analysis of the results and comparison with family-specific methods demonstrate some important strengths and weaknesses of this general approach.
Reconstructing neuronal circuits at the level of synapses is a central problem in neuroscience and becoming a focus of the emerging field of connectomics. To date, electron microscopy (EM) is the most proven technique for identifying and quantifying synaptic connections. As advances in EM make acquiring larger datasets possible, subsequent manual synapse identification ({\em i.e.}, proofreading) for deciphering a connectome becomes a major time bottleneck. Here we introduce a large-scale, high-throughput, and semi-automated methodology to efficiently identify synapses. We successfully applied our methodology to the Drosophila medulla optic lobe, annotating many more synapses than previous connectome efforts. Our approaches are extensible and will make the often complicated process of synapse identification accessible to a wider-community of potential proofreaders.
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.
Epithelial tissues can elongate in two dimensions by polarized cell intercalation, oriented cell division, or cell shape change, owing to local or global actomyosin contractile forces acting in the plane of the tissue. In addition, epithelia can undergo morphogenetic change in three dimensions. We show that elongation of the wings and legs of Drosophila involves a columnar-to-cuboidal cell shape change that reduces cell height and expands cell width. Remodeling of the apical extracellular matrix by the Stubble protease and basal matrix by MMP1/2 proteases induces wing and leg elongation. Matrix remodeling does not occur in the haltere, a limb that fails to elongate. Limb elongation is made anisotropic by planar polarized Myosin-II, which drives convergent extension along the proximal-distal axis. Subsequently, Myosin-II relocalizes to lateral membranes to accelerate columnar-to-cuboidal transition and isotropic tissue expansion. Thus, matrix remodeling induces dynamic changes in actomyosin contractility to drive epithelial morphogenesis in three dimensions.
Tracing of neuron morphology is an essential technique in computational neuroscience. However, despite a number of existing methods, few open-source techniques are completely or sufficiently automated and at the same time are able to generate robust results for real 3D microscopy images.
View Publication PageProper brain function requires the precise localization of proteins and signaling molecules on a nanometer scale. The examination of molecular organization at this scale has been difficult in part because it is beyond the reach of conventional, diffraction-limited light microscopy. The recently developed method of superresolution, localization-based fluorescent microscopy (LBM), such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), has demonstrated a resolving power at a 10 nm scale and is poised to become a vital tool in modern neuroscience research. Indeed, LBM has revealed previously unknown cellular architectures and organizational principles in neurons. Here, we discuss the principles of LBM, its current applications in neuroscience, and the challenges that must be met before its full potential is achieved. We also present the unpublished results of our own experiments to establish a sample preparation procedure for applying LBM to study brain tissue. Synapse, 69:283-294, 2015. © 2015 Wiley Periodicals, Inc.
The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics.