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Creators/Authors contains: "Ellisman, Mark H."

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  1. Abstract

    Comparisons and linkage between multiple imaging scales are essential for neural circuit connectomics. Here, we report 20 new recombinant rabies virus (RV) vectors that we have developed for multi-scale and multi-modal neural circuit mapping tools. Our new RV tools for mesoscale imaging express a range of improved fluorescent proteins. Further refinements target specific neuronal subcellular locations of interest. We demonstrate the discovery power of these new tools including the detection of detailed microstructural changes of rabies-labeled neurons in aging and Alzheimer’s disease mouse models, live imaging of neuronal activities using calcium indicators, and automated measurement of infected neurons. RVs that encode GFP and ferritin as electron microscopy (EM) and fluorescence microscopy reporters are used for dual EM and mesoscale imaging. These new viral variants significantly expand the scale and power of rabies virus-mediated neural labeling and circuit mapping across multiple imaging scales in health and disease.

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    The Neuroscience domain stands out from the field of sciences for its dependence on the study and characterization of complex, intertwining structures. Understanding the complexity of the brain has led to widespread advances in the structure of large-scale computing resources and the design of artificially intelligent analysis systems. However, the scale of problems and data generated continues to grow and outpace the standards and practices of neuroscience. In this paper, we present an automated neuroscience reconstruction framework, called NeuroKube, for large-scale processing and labeling of neuroimage volumes. Automated labels are generated through a machine-learning (ML) workflow, with data-intensive steps feeding through multiple GPU stages and distributed data locations leveraging autoscalable cloud-native deployments on a multi-institution Kubernetes system. Leading-edge hardwareand storage empower multiple stages of machine-learning, GPU accelerated solutions. This demonstrates an abstract approach to allocating the resources and algorithms needed to elucidate the highly complex structures of the brain. We summarize an integrated gateway architecture, and a scalable workflowdriven segmentation and reconstruction environment that brings together image big data with state-of-the-art, extensible machinelearning methods. 
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    The biophysical properties of sensory neurons are influenced by their morphometric and morphological features, whose precise measurements require high-quality volume electron microscopy (EM). However, systematic surveys of these nanoscale characteristics for identified neurons are scarce. Here, we characterize the morphology of Drosophila olfactory receptor neurons (ORNs) across the majority of genetically identified sensory hairs.By analyzing serial block-face electron microscopy (SBEM) images of cryo fixed antennal tissues, we compile an extensive morphometric dataset based on 122reconstructed 3D models of 33 identifiedORN types.In addition, we observe multiple novel features—including extracellular vacuoles within sensillum lumen, intricate dendritic branching,mitochondria enrichment in select ORNs, novel sensillum types, and empty sensilla containing no neurons—which raise new questions pertinent to cell biology and sensory neurobiology.Our systematic survey is critical for future investigations into how the size and shape of sensory neurons influence their responses, sensitivity and circuit function. 
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    Dorsal Excitor motor neuron DE-3 in the medicinal leech plays three very different dynamical roles in three different behaviors. Without rewiring its anatomical connectivity, how can a motor neuron dynamically switch roles to play appropriate roles in various behaviors? We previously used voltage-sensitive dye imaging to record from DE-3 and most other neurons in the leech segmental ganglion during (fictive) swimming, crawling, and local-bend escape (Tomina and Wagenaar, 2017). Here, we repeated that experiment, then re-imaged the same ganglion using serial blockface electron microscopy and traced DE-3’s processes. Further, we traced back the processes of DE-3’s presynaptic partners to their respective somata. This allowed us to analyze the relationship between circuit anatomy and the activity patterns it sustains. We found that input synapses important for all the behaviors were widely distributed over DE-3’s branches, yet that functional clusters were different during (fictive) swimming vs. crawling. 
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  5. Abstract

    Cristae are high‐curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae‐shaping proteins have been defined, analogous lipid‐based mechanisms have yet to be elucidated. Here, we combine experimental lipidome dissection with multi‐scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the inner mitochondrial membrane against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein‐mediated curvatures. This model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that cardiolipin is essential in low‐oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of cardiolipin is dependent on the surrounding lipid and protein components of the IMM.

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    In the highly dynamic metabolic landscape of a neuron, mitochondrial membrane architectures can provide critical insight into the unique energy balance of the cell. Current theoretical calculations of functional outputs like ATP and heat often represent mitochondria as idealized geometries and therefore can miscalculate the metabolic fluxes. To analyze mitochondrial morphology in neurons of mouse cerebellum neuropil, 3D tracings of complete synaptic and axonal mitochondria were constructed using a database of serial TEM tomographyimages and converted to watertight meshes with minimal distortion of the original microscopy volumes with agranularity of 1.6 nanometer isotropic voxels. The resulting in silico representations were subsequently quantified by differential geometry methods in terms of the mean and Gaussian curvatures, surface areas, volumes, and membrane motifs, all of which can alter the metabolic output of the organelle. Finally, we identify structural motifs that are present across this population of mitochondria; observations which may contribute to future modeling studies of mitochondrial physiology and metabolism in neurons. 
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