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Biological specimens are intrinsically three dimensional; however, because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by “clearing” the tissue have been ongoing for over a century, there have been a large number of recent innovations. This Review introduces the physical basis for light scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue.

We describe a method to map the location of axonal arbors of many individual neurons simultaneously via the spectral properties of retrogradely transported dye-labeled vesicles. We inject overlapping regions of an axon target area with three or more different colored retrograde tracers. On the basis of the combinations and intensities of the colors in the individual vesicles transported to neuronal somata, we calculate the projection sites of each neuron's axon. This neuronal positioning system (NPS) enables mapping of many axons in a simple automated way. In our experiments, NPS combined with spectral (Brainbow) labeling of the input to autonomic ganglion cells showed that the locations of ganglion cell projections to a mouse salivary gland related to the identities of their preganglionic axonal innervation. NPS could also delineate projections of many axons simultaneously in the mouse central nervous system.

Electron-electron interactions and detector bandwidth limit the maximal imaging speed of single-beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers.

Accurate and complete neuronal wiring diagrams are necessary for understanding brain function at many scales from long-range interregional projections to microcircuits. Traditionally, light microscopy-based anatomical reconstructions use monochromatic labeling and therefore necessitate sparse labeling to eliminate tracing ambiguity between intermingled neurons. Consequently, our knowledge of neuronal morphology has largely been based on averaged estimations across many samples. Recently developed second-generation Brainbow tools promise to circumvent this limitation by revealing fine anatomical details of many unambiguously identifiable neurons in densely labeled samples. Yet, a means to quantify and analyze the information is currently lacking. Therefore, we developed nTracer, an ImageJ plugin capable of rapidly and accurately reconstructing whole-cell morphology of large neuronal populations in densely labeled brains.

Tissue clearing has become an important tool for the investigation of biological systems in three dimensions. However, many pioneering techniques were based on serendipitous discoveries. Next-generation clearing methods have been (re)designed with a better understanding of the chemistry and physics required to equalize the refractive index throughout a sample which prevents the random bending of light that clouds biological tissues.

Summary Neural integrators are involved in a variety of sensorimotor and cognitive behaviors. The oculomotor system contains a simple example, a hindbrain neural circuit that takes velocity signals as inputs and temporally integrates them to control eye position. Here we investigated the structural underpinnings of temporal integration in the larval zebrafish by first identifying integrator neurons using two-photon calcium imaging and then reconstructing the same neurons through serial electron microscopic analysis. Integrator neurons were identified as those neurons with activities highly correlated with eye position during spontaneous eye movements. Three morphological classes of neurons were observed: ipsilaterally projecting neurons located medially, contralaterally projecting neurons located more laterally, and a population at the extreme lateral edge of the hindbrain for which we were not able to identify axons. Based on their somatic locations, we inferred that neurons with only ipsilaterally projecting axons are glutamatergic, whereas neurons with only contralaterally projecting axons are largely GABAergic. Dendritic and synaptic organization of the ipsilaterally projecting neurons suggests a broad sampling from inputs on the ipsilateral side. We also observed the first conclusive evidence of synapses between integrator neurons, which have long been hypothesized by recurrent network models of integration via positive feedback.

The inner ear is a fluid-filled closed-epithelial structure whose normal function requires maintenance of an internal hydrostatic pressure and fluid composition by unknown mechanisms. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation every 1-3 hours. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of thin overlapping basal lamellae that dynamically extend over neighboring cells before rupturing under pressure leading to ES collapse. The unexpected discovery of a physical relief valve in the ear emphasizes the need for further study into how organs control fluid pressure, volume, flow, and ion homeostasis in development and disease.

Schwann cells (SCs) at neuromuscular junctions (NMJs) play active roles in synaptic homeostasis and repair. We have studied how SCs contribute to reinnervation of NMJs using vital imaging of mice whose motor axons and SCs are transgenically labeled with different colors of fluorescent proteins. Motor axons most commonly regenerate to the original synaptic site by following SC-filled endoneurial tubes. During the period of denervation, SCs at the NMJ extend elaborate processes from the junction, as shown previously, but they also retract some processes from territory they previously occupied within the endplate. The degree of this retraction depends on the length of the period of denervation. We show that the topology of the remaining SC processes influences the branching pattern of regenerating axon terminals and the redistribution of acetylcholine receptors (AChRs). Upon arriving at the junction, regenerating axons follow existing SC processes within the old synaptic site. Some of the AChR loss that follows denervation is correlated with failure of portions of the old synaptic site that lack SC coverage to be reinnervated. New AChR clustering is also induced by axon terminals that follow SC processes extended during denervation. These observations show that SCs participate actively in the remodeling of neuromuscular synapses following nerve injury by their guidance of axonal reinnervation.

The overarching goal of the NIH BRAIN (Brain Research through Advancing Innovative Neurotechnologies) Initiative is to advance the understanding of healthy and diseased brain circuit function through technological innovation. Core principles for this goal include the validation and dissemination of the myriad innovative technologies, tools, methods, and resources emerging from BRAIN-funded research. Innovators, BRAIN funding agencies, and non-Federal partners are working together to develop strategies for making these products usable, available, and accessible to the scientific community. Here, we describe several early strategies for supporting the dissemination of BRAIN technologies. We aim to invigorate a dialogue with the neuroscience research and funding community, interdisciplinary collaborators, and trainees about the existing and future opportunities for cultivating groundbreaking research products into mature, integrated, and adaptable research systems. Along with the accompanying Society for Neuroscience 2019 Mini-Symposium, “BRAIN Initiative: Cutting-Edge Tools and Resources for the Community,” we spotlight the work of several BRAIN investigator teams who are making progress toward providing tools, technologies, and services for the neuroscience community. These tools access neural circuits at multiple levels of analysis, from subcellular composition to brain-wide network connectivity, including the following: integrated systems for EM- and florescence-based connectomics, advances in immunolabeling capabilities, and resources for recording and analyzing functional connectivity. Investigators describe how the resources they provide to the community will contribute to achieving the goals of the NIH BRAIN Initiative. Finally, in addition to celebrating the contributions of these BRAIN-funded investigators, the Mini-Symposium will illustrate the broader diversity of BRAIN Initiative investments in cutting-edge technologies and resources.

Summary: Reciprocal communication between neurons and oligodendrocytes is essential for the generation and localization of myelin, a critical feature of the CNS. In the neocortex, individual oligodendrocytes can myelinate multiple axons; however, the neuronal origin of the myelinated axons has remained undefined and, while largely assumed to be from excitatory pyramidal neurons, it also includes inhibitory interneurons. This raises the question of whether individual oligodendrocytes display bias for the class of neurons that they myelinate. Here, we find that different classes of cortical interneurons show distinct patterns of myelin distribution starting from the onset of myelination, suggesting that oligodendrocytes can recognize the class identity of individual types of interneurons that they target. Notably, we show that some oligodendrocytes disproportionately myelinate the axons of inhibitory interneurons, whereas others primarily target excitatory axons or show no bias. These results point toward very specific interactions between oligodendrocytes and neurons and raise the interesting question of why myelination is differentially directed toward different neuron types. : Zonouzi et al. demonstrate that a population of oligodendrocytes are biased for myelinating inhibitory interneurons in the neocortex and, further, that interneurons develop myelination profiles that are class-specific. The work highlights highly selective interactions between neurons and oligodendrocytes that help shape myelin distribution in vivo. Keywords: oligodendrocyte, myelination, interneuron, neocortex

Correlated electron microscopy and cathodoluminescence (CL) imaging using functionalized nanoparticles is a promising nanoscale probe of biological structure and function. Nanodiamonds (NDs) that contain CL-emitting color centers are particularly well suited for such applications. The intensity of CL emission from NDs is determined by a combination of factors, including particle size, density of color centers, efficiency of energy deposition by electrons passing through the particle, and conversion efficiency from deposited energy to CL emission. This paper reports experiments and numerical simulations that investigate the relative importance of each of these factors in determining CL emission intensity from NDs containing nitrogen-vacancy (NV) color centers. In particular, it is found that CL can be detected from NV-doped NDs with dimensions as small as ≈40 nm, although CL emission decreases significantly for smaller NDs.

In many parts of the nervous system, signals pass across multiple synaptic relays on their way to a destination, but little is known about how these relays form and the function they serve. To get some insight into this question we ask how the connectivity patterns are organized at two successive synaptic relays in a simple, cholinergic efferent pathway. We found that the organization at successive relays in the parasympathetic nervous system strongly resemble each other despite the different embryological origin and physiological properties of the pre- and postsynaptic cells. Additionally, we found a similar developmental synaptic pruning and elaboration strategy is used at both sites to generate their adult organizations. The striking parallels in adult innervation and developmental mechanisms at the relays argue that a general strategy is in operation. We discuss why from a functional standpoint this structural organization may amplify central signals while at the same time maintaining positional targeting. DOI: http://dx.doi.org/10.7554/eLife.23193.001

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However, the cells generated within organoids and the extent to which they recapitulate the regional complexity, cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells, which are related to endogenous classes, including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months), allowing for the establishment of relatively mature features, including the formation of dendritic spines and spontaneously active neuronal networks. Finally, neuronal activity within organoids could be controlled using light stimulation of photosensitive cells, which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli.

Summary Background The cleavage-stage mouse embryo is composed of superficially equivalent blastomeres that will generate both the embryonic inner cell mass (ICM) and the supportive trophectoderm (TE). However, it remains unsettled whether the contribution of each blastomere to these two lineages can be accounted for by chance. Addressing the question of blastomere cell fate may be of practical importance, because preimplantation genetic diagnosis requires removal of blastomeres from the early human embryo. To determine whether blastomere allocation to the two earliest lineages is random, we developed and utilized a recombination-mediated, noninvasive combinatorial fluorescent labeling method for embryonic lineage tracing. Results When we induced recombination at cleavage stages, we observed a statistically significant bias in the contribution of the resulting labeled clones to the trophectoderm or the inner cell mass in a subset of embryos. Surprisingly, we did not find a correlation between localization of clones in the embryonic and abembryonic hemispheres of the late blastocyst and their allocation to the TE and ICM, suggesting that TE-ICM bias arises separately from embryonic-abembryonic bias. Rainbow lineage tracing also allowed us to demonstrate that the bias observed in the blastocyst persists into postimplantation stages and therefore has relevance for subsequent development. Conclusions The Rainbow transgenic mice that we describe here have allowed us to detect lineage-dependent bias in early development. They should also enable assessment of the developmental equivalence of mammalian progenitor cells in a variety of tissues.

Cortical sensory maps are remodeled during early life to adapt to the surrounding environment. Both sensory and contextual signals are important for induction of this plasticity, but how these signals converge to sculpt developing thalamocortical circuits remains largely unknown. Here we show that layer 1 (L1) of primary auditory cortex (A1) is a key hub where neuromodulatory and topographically organized thalamic inputs meet to tune the cortical layers below. Inhibitory interneurons in L1 send narrowly descending projections to differentially modulate thalamic drive to pyramidal and parvalbumin-expressing (PV) cells in L4, creating brief windows of intracolumnar activation. Silencing of L1 (but not VIP-expressing) cells abolishes map plasticity during the tonotopic critical period. Developmental transitions in nicotinic acetylcholine receptor (nAChR) sensitivity in these cells caused by Lynx1 protein can be overridden to extend critical-period closure. Notably, thalamocortical maps in L1 are themselves stable, and serve as a scaffold for cortical plasticity throughout life.

One goal of systems neuroscience is a structure-function model of nervous system organization that would allow mechanistic linking of mind, brain, and behavior. A necessary but not sufficient foundation is a connectome, a complete matrix of structural connections between the nodes of a nervous system. Connections between two nodes can be described at four nested levels of analysis: macroconnections between gray matter regions, mesoconnections between neuron types, microconnections between individual neurons, and nanoconnections at synapses. A long history of attempts to understand how the brain operates as a system began at the macrolevel in the fifth century, was revolutionized at the meso- and microlevels by Cajal and others in the late nineteenth century, and reached the nanolevel in the mid-twentieth century with the advent of electron microscopy. The greatest challenge today is extracting knowledge and understanding of nervous system structure-function architecture from vast amounts of data.

Resolving patterns of synaptic connectivity in neural circuits currently requires serial section electron microscopy. However, complete circuit reconstruction is prohibitively slow and may not be necessary for many purposes such as comparing neuronal structure and connectivity among multiple animals. Here, we present an alternative strategy, targeted reconstruction of specific neuronal types. We used viral vectors to deliver peroxidase derivatives, which catalyze production of an electron-dense tracer, to genetically identify neurons, and developed a protocol that enhances the electron-density of the labeled cells while retaining the quality of the ultrastructure. The high contrast of the marked neurons enabled two innovations that speed data acquisition: targeted high-resolution reimaging of regions selected from rapidly-acquired lower resolution reconstruction, and an unsupervised segmentation algorithm. This pipeline reduces imaging and reconstruction times by two orders of magnitude, facilitating directed inquiry of circuit motifs. DOI: http://dx.doi.org/10.7554/eLife.15015.001, eLife digest Neurons connect with each other to form complex circuits that underlie mental activities. Mapping these connections to obtain a so-called wiring diagram is an essential step in learning how the brain works. The only way to do this precisely enough is by using electron microscopy. However, this technique is so time-consuming that thousands of hours of work are typically required to image even the smallest of tissue samples. Electron microscopes fire beams of electrons at tissue samples, and detect the scattering of the electrons. Stains are used to make specific neurons less permeable to electrons, or more “electron dense”. Labeled cells scatter more electrons, which increases the contrast of the images. In an approach called serial-section electron microscopy, a tissue sample is first cut into extremely thin sections. These are imaged individually, and the images are then pieced together to reconstruct the sample. Joesch et al. have now developed a new procedure – named ARTEMIS – that uses a combination of multiple techniques to speed up the mapping of neurons and their connections. ARTEMIS makes use of genetic engineering, serial-scanning electron microscopy, an enhanced chemical staining procedure and a new image processing approach. First, gene technology is used to selectively stain specific types of neurons in mice and flies. Then, a tissue sample is collected and treated with a chemical that enhances the electron density of the stained neurons, without disrupting the tissue’s structure. Next, a labeled target neuron is imaged at relatively low resolution to reveal its overall structure. Small areas of that neuron are then re-imaged at higher resolution to map the connections between neurons. Lastly, an algorithm pieces together the individual images to produce a reconstruction of the cell. This pipeline of steps reduces the time required to map the shapes and connectivity of neurons with electron microscopy by some two orders of magnitude. This should enable neuroscientists to obtain more rapid insights into the roles of specific neural circuits in the brains of healthy animals, and to identify cases where this wiring goes awry and leads to disease. DOI: http://dx.doi.org/10.7554/eLife.15015.002

Summary Using 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 Abstract

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.

Using neuroimaging, researchers are succesfully mapping neural connectivity and in the process creating vivid "brainbows."

Spatial competition between glial cells causes them to partition neuromuscular junctions into discrete domains within a synapse., Schwann cells (SCs), the glial cells of the peripheral nervous system, cover synaptic terminals, allowing them to monitor and modulate neurotransmission. Disruption of glial coverage leads to axon degeneration and synapse loss. The cellular mechanisms that establish and maintain this coverage remain largely unknown. To address this, we labeled single SCs and performed time-lapse imaging experiments. Adult terminal SCs are arranged in static tile patterns, whereas young SCs dynamically intermingle. The mechanism of developmental glial segregation appears to be spatial competition, in which glial–glial and axonal–glial contacts constrain the territory of single SCs, as shown by four types of experiments: (1) laser ablation of single SCs, which led to immediate territory expansion of neighboring SCs; (2) axon removal by transection, resulting in adult SCs intermingling dynamically; (3) axotomy in mutant mice with blocked axon fragmentation in which intermingling was delayed; and (4) activity blockade, which had no immediate effects. In summary, we conclude that glial cells partition synapses by competing for perisynaptic space.

Potassium fluxes integrate mitochondria into cellular activities, controlling their volume homeostasis and structural integrity in many pathophysiological mechanisms. The outer mitochondrial membrane (OMM) is thought to play a passive role in this process because K(+) is believed to equilibrate freely between the cytosol and mitochondrial intermembrane space. By patch clamping mitochondria isolated from the central nervous systems of adult mitoCFP transgenic mice, we discovered the existence of I(OMMKi), a novel voltage-dependent inwardly rectifying K(+) conductance located in the OMM. I(OMMKi) is regulated by osmolarity, potentiated by cAMP, and activated at physiological negative potentials, allowing K(+) to enter the mitochondrial intermembrane space in a controlled regulated fashion. The identification of I(OMMKi) in the OMM supports the notion that a membrane potential could exist across this membrane in vivo and suggests that the OMM possesses regulated pathways for K(+) uptake.

In the version of this article initially published online, the wrong version of Fig. 5 was used. There were errors in the statistical comparison brackets in Fig. 5c and the left-hand error bar in Fig. 5f. The errors have been corrected in the print, PDF and HTML versions of this article. In the version of this article initially published online and in print, the wrong version of Fig. 3h was used. There was a slight error in the alignment of the traces in the top right panel. The error has been corrected in the PDF and HTML versions of this article. The original and corrected figures are shown in the accompanying Publisher Correction.

Micropatterned poly(dimethylsiloxane) substrates fabricated by soft lithography led to large-scale orientation of myoblasts in culture, thereby controlling the orientation of the myotubes they formed. Fusion occurred on many chemically identical surfaces in which varying structures were arranged in square or hexagonal lattices, but only a subset of patterned surfaces yielded aligned myotubes. Remarkably, on some substrates, large populations of myotubes oriented at a reproducible acute angle to the lattice of patterned features. A simple geometrical model predicts the angle and extent of orientation based on maximizing the contact area between the myoblasts and patterned topographic surfaces. Micropatterned substrates also provided short-range cues that influenced higher-order functions such as the localization of focal adhesions and accumulation of postsynaptic acetylcholine receptors. Our results represent what we believe is a new approach for musculoskeletal tissue engineering, and our model sheds light on mechanisms of myotube alignment in vivo.

Developmental neurobiology has been greatly invigorated by a recent string of breakthroughs in molecular biology and optical physics that permit direct in vivo observation of neural circuit assembly. The imaging done thus far suggests that as brains are built, a significant amount of unbuilding is also occurring. We offer the view that this tumult is the result of the intersecting behaviors of the many single-celled creatures (i.e., neurons, glia, and progenitors) that inhabit brains. New tools will certainly be needed if we wish to monitor the myriad cooperative and competitive interactions at play in the cellular society that builds brains.

We provide a protocol that describes an explant system that allows the dynamics of motor axons to be imaged. This method is based on nerve–muscle explants prepared from the triangularis sterni muscle of mice, a thin muscle that covers the inside of the thorax. These explants, which can be maintained alive for several hours, contain long stretches of peripheral motor axons including their terminal arborizations and neuromuscular junctions. Explants can be prepared from transgenic mouse lines that express fluorescent proteins in neurons or glial cells, which enables direct visualization of their cellular and subcellular morphology by fluorescence microscopy. Time-lapse imaging then provides a convenient and reliable approach to follow the dynamic behavior of motor axons, their surrounding glial cells and their intracellular organelles with high temporal and spatial resolution. Triangularis sterni explants can be prepared in 15 min, imaged ex vivo for several hours and processed for immunohistochemistry in about 2 h.

Some neuroscientists argue that detailed maps of synaptic connectivity - wiring diagrams - will be needed if we are to understand how the brain underlies behavior and how brain malfunctions underlie behavioral disorders. Such large-scale circuit reconstruction, which has been called connectomics, may soon be possible, owing to numerous advances in technologies for image acquisition and processing. Yet, the community is divided on the feasibility and value of the enterprise. Remarkably similar objections were voiced when the Human Genome Project, now widely viewed as a success, was first proposed. We revisit that controversy to ask if it holds any lessons for proposals to map the connectome.

A central aim of neuroscience is to map neural circuits, in order to learn how they account for mental activities and behaviours and how alterations in them lead to neurological and psychiatric disorders. However, the methods that are currently available for visualizing circuits have severe limitations that make it extremely difficult to extract precise wiring diagrams from histological images. Here we review recent advances in this area, along with some of the opportunities that these advances present and the obstacles that remain.

We present an interactive approach to train a deep neural network pixel classifier for the segmentation of neuronal structures. An interactive training scheme reduces the extremely tedious manual annotation task that is typically required for deep networks to perform well on image segmentation problems. Our proposed method employs a feedback loop that captures sparse annotations using a graphical user interface, trains a deep neural network based on recent and past annotations, and displays the prediction output to users in almost real-time. Our implementation of the algorithm also allows multiple users to provide annotations in parallel and receive feedback from the same classifier. Quick feedback on classifier performance in an interactive setting enables users to identify and label examples that are more important than others for segmentation purposes. Our experiments show that an interactively-trained pixel classifier produces better region segmentation results on Electron Microscopy (EM) images than those generated by a network of the same architecture trained offline on exhaustive ground-truth labels.

In the field of connectomics, neuroscientists acquire electron microscopy volumes at nanometer resolution in order to reconstruct a detailed wiring diagram of the neurons in the brain. The resulting image volumes, which often are hundreds of terabytes in size, need to be segmented to identify cell boundaries, synapses, and important cell organelles. However, the segmentation process of a single volume is very complex, time-intensive, and usually performed using a diverse set of tools and many users. To tackle the associated challenges, this paper presents NeuroBlocks, which is a novel visualization system for tracking the state, progress, and evolution of very large volumetric segmentation data in neuroscience. NeuroBlocks is a multi-user web-based application that seamlessly integrates the diverse set of tools that neuroscientists currently use for manual and semi-automatic segmentation, proofreading, visualization, and analysis. NeuroBlocks is the first system that integrates this heterogeneous tool set, providing crucial support for the management, provenance, accountability, and auditing of large-scale segmentations. We describe the design of NeuroBlocks, starting with an analysis of the domain-specific tasks, their inherent challenges, and our subsequent task abstraction and visual representation. We demonstrate the utility of our design based on two case studies that focus on different user roles and their respective requirements for performing and tracking the progress of segmentation and proofreading in a large real-world connectomics project.

Proofreading refers to the manual correction of automatic segmentations of image data. In connectomics, electron microscopy data is acquired at nanometer-scale resolution and results in very large image volumes of brain tissue that require fully automatic segmentation algorithms to identify cell boundaries. However, these algorithms require hundreds of corrections per cubic micron of tissue. Even though this task is time consuming, it is fairly easy for humans to perform corrections through splitting, merging, and adjusting segments during proofreading. In this paper we present the design and implementation of Mojo, a fully-featured single-user desktop application for proofreading, and Dojo, a multi-user web-based application for collaborative proofreading. We evaluate the accuracy and speed of Mojo, Dojo, and Raveler, a proofreading tool from Janelia Farm, through a quantitative user study. We designed a between-subjects experiment and asked non-experts to proofread neurons in a publicly available connectomics dataset. Our results show a significant improvement of corrections using web-based Dojo, when given the same amount of time. In addition, all participants using Dojo reported better usability. We discuss our findings and provide an analysis of requirements for designing visual proofreading software.

Automated sample preparation and electron microscopy enables acquisition of very large image data sets. These technical advances are of special importance to the field of neuroanatomy, as 3D reconstructions of neuronal processes at the nm scale can provide new insight into the fine grained structure of the brain. Segmentation of large-scale electron microscopy data is the main bottleneck in the analysis of these data sets. In this paper we present a pipeline that provides state-of-the art reconstruction performance while scaling to data sets in the GB-TB range. First, we train a random forest classifier on interactive sparse user annotations. The classifier output is combined with an anisotropic smoothing prior in a Conditional Random Field framework to generate multiple segmentation hypotheses per image. These segmentations are then combined into geometrically consistent 3D objects by segmentation fusion. We provide qualitative and quantitative evaluation of the automatic segmentation and demonstrate large-scale 3D reconstructions of neuronal processes from a 27 , 000 μ m 3 volume of brain tissue over a cube of 30 μ m in each dimension corresponding to 1000 consecutive image sections. We also introduce Mojo, a proofreading tool including semi-automated correction of merge errors based on sparse user scribbles.

Although fluorescence microscopy permeates all of cell and molecular biology, most biologists have little experience with the underlying photophysical phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Additionally, fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.

The poor response of central axons to transection underlies the bleak prognosis following spinal cord injury. Here, we monitor individual fluorescent axons in the spinal cords of living transgenic mice over several days after spinal injury. We find that within 30 min after trauma, axons die back hundreds of micrometers. This acute form of axonal degeneration is similar in mechanism to the more delayed Wallerian degeneration of the disconnected distal axon, but acute degeneration affects the proximal and distal axon ends equally. In vivo imaging further shows that many axons attempt regeneration within 6-24 h after lesion. This growth response, although robust, seems to fail as a result of the inability of axons to navigate in the proper direction. These results suggest that time-lapse imaging of spinal cord injury may provide a powerful analytical tool for assessing the pathogenesis of spinal cord injury and for evaluating therapies that enhance regeneration.

Little is known about the effects of aging on synapses in the mammalian nervous system. We examined the innervation of individual mouse submandibular ganglion (SMG) neurons for evidence of age-related changes in synapse efficacy and number. For approximately 85% of adult life expectancy (30 months) the efficacy of synaptic transmission, as determined by excitatory postsynaptic potential (EPSP) amplitudes, remains constant. Similarly, the number of synapses contacting individual SMG neurons is also unchanged. After 30 months of age, however, some neurons (23%) dramatically lose synaptic input exhibiting both smaller EPSP amplitude and fewer synaptic boutons. Attenuation of both the amplitude and frequency of miniature EPSPs was also observed in neurons from aged animals. Electron micrographs revealed that, although there were many vesicle-laden preganglionic axonal processes in the vicinity of the postsynaptic membrane, the number of synaptic contacts was significantly lower in old animals. These results demonstrate primary, age-associated synapse elimination with functional consequences that cannot be explained by pre- or postsynaptic cell death.

On the thirtieth birthday of long-term potentiation (LTP), it is appropriate to step back from the fray, evaluate what has been learned and look to the future. Two of us (J.W.L. and J.R.S.) published such an evaluation a few years ago in the hope that the ignorance conferred to us by our status as outsiders to the field might be offset, at least in part, by a freshness of perspective. The responses to that article have been numerous and fascinating, with experts and non-experts raising points in private that would benefit from open discussion. Here, joined by an LTP 'insider' (J.L.), we initiate such a discussion, restating, clarifying and debating some of the points that were raised in the original article.

In developing mammalian muscle, axon branches of several motor neurons co-innervate the same muscle fibre. Competition among them results in the strengthening of one and the withdrawal of the rest. It is not known why one particular axon branch survives or why some competitions resolve sooner than others. Here we show that the fate of axonal branches is strictly related to the identity of the axons with which they compete. When two neurons co-innervate multiple target cells, the losing axon branches in each contest belong to the same neuron and are at nearly the same stage of withdrawal. The axonal arbor of one neuron engages in multiple sets of competitions simultaneously. Each set proceeds at a different rate and heads towards a common outcome based on the identity of the competitor. Competitive vigour at each of these sets of local competitions depends on a globally distributed resource: neurons with larger arborizations are at a competitive disadvantage when confronting neurons with smaller arborizations. An accompanying paper tests the idea that the amount of neurotransmitter released is this global resource.

α-Dystrobrevin (DB), a cytoplasmic component of the dystrophin–glycoprotein complex, is found throughout the sarcolemma of muscle cells. Mice lacking αDB exhibit muscular dystrophy, defects in maturation of neuromuscular junctions (NMJs) and, as shown here, abnormal myotendinous junctions (MTJs). In normal muscle, alternative splicing produces two main αDB isoforms, αDB1 and αDB2, with common NH2-terminal but distinct COOH-terminal domains. αDB1, whose COOH-terminal extension can be tyrosine phosphorylated, is concentrated at the NMJs and MTJs. αDB2, which is not tyrosine phosphorylated, is the predominant isoform in extrajunctional regions, and is also present at NMJs and MTJs. Transgenic expression of either isoform in αDB−/− mice prevented muscle fiber degeneration; however, only αDB1 completely corrected defects at the NMJs (abnormal acetylcholine receptor patterning, rapid turnover, and low density) and MTJs (shortened junctional folds). Site-directed mutagenesis revealed that the effectiveness of αDB1 in stabilizing the NMJ depends in part on its ability to serve as a tyrosine kinase substrate. Thus, αDB1 phosphorylation may be a key regulatory point for synaptic remodeling. More generally, αDB may play multiple roles in muscle by means of differential distribution of isoforms with distinct signaling or structural properties.

Activity-dependent and -independent signals collaborate to regulate synaptogenesis, but their relative contributions are unclear. Here, we describe the formation of neuromuscular synapses at which neurotransmission is completely and specifically blocked by mutation of the neurotransmitter-synthesizing enzyme choline acetyltransferase. Nerve terminals differentiate extensively in the absence of neurotransmitter, but neurotransmission plays multiple roles in synaptic differentiation. These include influences on the numbers of pre- and postsynaptic partners, the distribution of synapses in the target field, the number of synaptic sites per target cell, and the number of axons per synaptic site. Neurotransmission also regulates the formation or stability of transient acetylcholine receptor-rich processes (myopodia) that may initiate nerve-muscle contact. At subsequent stages, neurotransmission delays some steps in synaptic maturation but accelerates others. Thus, neurotransmission affects synaptogenesis from early stages and coordinates rather than drives synaptic maturation.

We show that fluorescently tagged ligands with high affinity for their targets can be reversibly unbound by focused laser excitation. By sequential unbinding and relabeling with different colors of alpha-bungarotoxin, we selectively labeled adjacent pools of acetylcholine receptors (AChRs) at neuromuscular junctions of adult mice. Timelapse imaging in vivo revealed that synaptic AChRs completely intermingle over approximately 4 days and many extrasynaptic AChRs are incorporated into the synapse each day. In mice that lacked alpha-dystrobrevin, a component of the dystrophin-glycoprotein complex, rates of AChR turnover, and intermingling were increased approximately 4- to 5-fold. These results demonstrate remarkable molecular dynamism underlying macroscopic stability of the postsynaptic membrane, and establish alpha-dystrobrevin as a key control point for regulation of mobility and turnover.

Focused-ion-beam scanning electron microscopy (FIB-SEM) has become an essential tool for studying neural tissue at resolutions below 10 nm x 10 nm x 10 nm, producing data sets optimized for automatic connectome tracing. We present a technical advance, ultrathick sectioning, which reliably subdivides embedded tissue samples into chunks (20 mu m 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 three-dimensional data set suitable for connectome tracing.

The structure of the nervous system is extraordinarily complicated because individual neurons are interconnected to hundreds or even thousands of other cells in networks that can extend over large volumes. Mapping such networks at the level of synaptic connections, a field called connectomics, began in the 1970s with a the study of the small nervous system of a worm and has recently garnered general interest thanks to technical and computational advances that automate the collection of electron-microscopy data and offer the possibility of mapping even large mammalian brains. However, modern connectomics produces 'big data', unprecedented quantities of digital information at unprecedented rates, and will require, as with genomics at the time, breakthrough algorithmic and computational solutions. Here we describe some of the key difficulties that may arise and provide suggestions for managing them., National Science Foundation (U.S.) (CCF-1217921), National Science Foundation (U.S.) (CCF-1301926), National Science Foundation (U.S.) (IIS-1447786), United States. Dept. of Energy (Advanced Scientific Computing Research Grant ER26116/DE-SC0008923), Oracle Corporation, Intel Corporation

Tissue reinnervation following trauma, disease, or transplantation often presents a significant challenge. Here, we show that the delivery of vascular endothelial growth factor (VEGF) from alginate hydrogels ameliorates loss of skeletal muscle innervation after ischemic injury by promoting both maintenance and regrowth of damaged axons in mice. Nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) mediated VEGF-induced axonal regeneration, and the expression of both is induced by VEGF presentation. Using both in vitro and in vivo modeling approaches, we demonstrate that the activity of NGF and GDNF regulates VEGF-driven angiogenesis, controlling endothelial cell sprouting and blood vessel maturation. Altogether, these studies produce evidence of new mechanisms of VEGF action, further broaden the understanding of the roles of NGF and GDNF in angiogenesis and axonal regeneration, and suggest approaches to improve axonal and ischemic tissue repair therapies.

Patchy Insulation Myelin insulates neuronal axons such that their electrical signals travel faster and more efficiently. However, not all axons are myelinated equally. Tomassy et al. (p. 319 ; see the Perspective by Fields ) obtained detailed images from two snippets of the adult mouse brain and generated three-dimensional reconstructions of individual neurons and their myelination patterns. The images show that some axons have long, unmyelinated stretches, which might offer sites for building new connections. Thus, myelination is not an all-or-none phenomenon but rather is a characteristic of what may be a specific dialogue between the neuron and the surrounding myelin-producing cells.

Dynamic processes of neural development, such as migrations of precursor cells, growth of axons and dendrites, and formation and modification of synapses, can be fully analyzed only with techniques that monitor changes over time. Although there has been long-standing motivation for following cellular and synaptic events in vivo (intravital microscopy), until recently few preparations have been studied, and then often only with great effort. Innovations in low-light and laser-scanning microscopies, coupled with developments of new dyes and of genetically encoded indicators, have increased both the breadth and depth of in situ imaging approaches. Here we present the motivations and challenges for dynamic imaging methods, offer some illustrative examples and point to future opportunities with emerging technologies.

In developing muscle, synapse elimination reduces the number of motor axons that innervate each postsynaptic cell. This loss of connections is thought to be a consequence of axon branch trimming. However, branch retraction has not been observed directly, and many questions remain, such as: do all motor axons retract branches, are eliminated branches withdrawn synchronously, and are withdrawing branches localized to particular regions? To address these questions, we used transgenic mice that express fluorescent proteins in small subsets of motor axons, providing a unique opportunity to reconstruct complete axonal arbors and identify all the postsynaptic targets. We found that, during early postnatal development, each motor axon loses terminal branches, but retracting branches withdraw asynchronously and without obvious spatial bias, suggesting that local interactions at each neuromuscular junction regulate synapse elimination.