Contacts between the endoplasmic reticulum and other membranes in neurons

The cytoplasm of eukaryotic cells is compartmentalized by intracellular membranes that define subcellular organelles. One of these organelles, the endoplasmic reticulum, forms a continuous network of tubules and cisternae that extends throughout all cell compartments, including neuronal dendrites and axons. This network communicates with most other organelles by vesicular transport, and also by contacts that do not lead to fusion but allow cross-talk between adjacent bilayers. Though these membrane contacts have previously been observed in neurons, their distribution and abundance has not been systematically analyzed. Here, we have carried out such analysis. Our studies reveal new aspects of the internal structure of neurons and provide a critical complement to information about interorganelle communication emerging from functional and biochemical studies

Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits

The automated tape-collecting ultramicrotome (ATUM) makes it possible to collect large numbers of ultrathin sections quickly—the equivalent of a petabyte of high resolution images each day. However, even high throughput image acquisition strategies generate images far more slowly (at present ~1 terabyte per day). We therefore developed WaferMapper, a software package that takes a multi-resolution approach to mapping and imaging select regions within a library of ultrathin sections. This automated method selects and directs imaging of corresponding regions within each section of an ultrathin section library (UTSL) that may contain many thousands of sections. Using WaferMapper, it is possible to map thousands of tissue sections at low resolution and target multiple points of interest for high resolution imaging based on anatomical landmarks. The program can also be used to expand previously imaged regions, acquire data under different imaging conditions, or re-image after additional tissue treatments

Pervasive Synaptic Branch Removal in the Mammalian Neuromuscular System at Birth

SummaryUsing light and serial electron microscopy, we show profound refinements in motor axonal branching and synaptic connectivity before and after birth. Embryonic axons become maximally connected just before birth when they innervate ∼10-fold more muscle fibers than in maturity. In some developing muscles, axons innervate almost every muscle fiber. At birth, each neuromuscular junction is coinnervated by approximately ten highly intermingled axons (versus one in adults). Extensive die off of terminal branches occurs during the first several postnatal days, leading to much sparser arbors that still span the same territory. Despite the extensive pruning, total axoplasm per neuron increases as axons elongate, thicken, and add more synaptic release sites on their remaining targets. Motor axons therefore initially establish weak connections with nearly all available postsynaptic targets but, beginning at birth, massively redistribute synaptic resources, concentrating many more synaptic sites on many fewer muscle fibers. Analogous changes in connectivity may occur in the CNS.Video Abstrac

Ultrastructurally-smooth thick partitioning and volume stitching for larger-scale connectomics

FIB-SEM has become an essential tool for studying neural tissue at resolutions below 10×10×10 nm, producing datasets superior for automatic connectome tracing. We present a technical advance, ultrathick sectioning, which reliably subdivides embedded tissue samples into chunks (20 µm thick) optimally sized and mounted for efficient, parallel FIB-SEM imaging. These chunks are imaged separately and then ‘volume stitched’ back together, producing a final 3D dataset suitable for connectome tracing

En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics

Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila, in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature en bloc staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the Drosophila brain. These requirements include: good preservation of ultrastructural detail, high level of en bloc staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly’s delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging

A connectome and analysis of the adult Drosophila central brain.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain

Neural evidence for intermediate representations in object recognition

AbstractThe lateral occipital complex (LOC), a cortical region critical for human object recognition, has been shown to primarily code the shape, rather than the surface properties, of an object. But what aspects of shape? Using an fMRI-adaptation (fMRI-a) paradigm in which subjects judged whether two contour-deleted images of objects were the same or different exemplars, virtually all the adaptation in LOC [especially in LOC’s most anterior portion (pFs)] could be attributed to repetition of the parts, almost none to the repetition of local image features, such as lines or vertices, templates, or basic- or subordinate-level concepts of the object. These results support the hypothesis that the neural representation of shape in LOC is an intermediate one, encoding the parts of an object

Automating Sheet-Based Fabrication: The Conveyed-Adherent Process

A new automated fabrication technology is described which breaks the fabrication process into spatially separate layer-formation and layer-bonding stages. The technique uses sheet material on a substrate as feedstock and cuts cross-section contours into the material before conveying the material on the substrate to a stacking station. Advantages include (a) speed, (b) versatility in fabrication materials, and (c) ability to fabricate hollows, embed or cast secondary materials, and selectively enhance or degrade material properties on a regional basis. A prototype fabricator has been built which automates all aspects ofthis process except weeding. Applications demonstrated using this machine include traditional rapid prototyping and visualization-model creation as well as functional machines taking advantage of embedding and cast-in-place techniques.Mechanical Engineerin

Adaptation to objects in the lateral occipital complex (LOC): Shape or semantics?

AbstractA change in the basic-level class when viewing a sequence of two objects produces a large release from adaptation in LOC compared to when the images are identical. Is this due to a change in semantics or shape? In an fMRI-adaptation experiment, subjects viewed a sequence of two objects and judged whether the stimuli were identical in shape. Different-shaped stimuli could be from the same or different basic-level classes, where the physical similarities of the pairs in the two conditions were equated by a model of simple cell similarity. BOLD responses in LOC for the two conditions were equivalent, and higher than that of the identical condition, indicating that LOC is sensitive to shape rather than to basic-level semantics