Reconstruction of motor control circuits in adult Drosophila using automated transmission electron microscopy.

To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we studied neuronal networks that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. We show that a specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM more accessible and affordable to the scientific community.

Plasticity of perisynaptic astroglia during ischemia-induced spreading depolarization.

High astroglial capacity for glutamate and potassium clearance aids in recovering spreading depolarization (SD)-evoked disturbance of ion homeostasis during stroke. Since perisynaptic astroglia cannot be imaged with diffraction-limited light microscopy, nothing is known about the impact of SD on the ultrastructure of a tripartite synapse. We used serial section electron microscopy to assess astroglial synaptic coverage in the sensorimotor cortex of urethane-anesthetized male and female mice during and after SD evoked by transient bilateral common carotid artery occlusion. At the subcellular level, astroglial mitochondria were remarkably resilient to SD compared to dendritic mitochondria that were fragmented by SD. Overall, 482 synapses in `Sham' during `SD' and `Recovery' groups were randomly selected and analyzed in 3D. Perisynaptic astroglia was present at the axon-spine interface (ASI) during SD and after recovery. Astrocytic processes were more likely found at large synapses on mushroom spines after recovery, while the length of the ASI perimeter surrounded by astroglia has also significantly increased at large synapses. These findings suggest that as larger synapses have a bigger capacity for neurotransmitter release during SD, they attract astroglial processes to their perimeter during recovery, limiting extrasynaptic glutamate escape and further enhancing the astrocytic ability to protect synapses in stroke.

Localization of phosphorylated connexin 43 using serial section immunogold electron microscopy.

Gap junction turnover occurs through the internalization of both of the plasma membranes of a gap junction plaque, forming a double membrane-enclosed vesicle, or connexosome. Phosphorylation has a key role in regulation, but further progress requires the ability to clearly distinguish gap junctions and connexosomes, and to precisely identify proteins associated with them. We examined, by using electron microscopy, serial sections of mouse preovulatory ovarian follicles that had been collected with an automated tape collecting ultramicrotome (ATUM). We found that connexosomes can form from adjacent cell bodies, from thin cell processes or from the same cell. By immunolabeling serial sections, we found that residue S368 of connexin 43 (also known as GJA1) is phosphorylated on gap junctions and connexosomes, whereas connexin 43 residue S262 is phosphorylated only on some connexosomes. These data suggest that phosphorylation at S262 contributes to connexosome formation or processing, and they provide more precise evidence that phosphorylation has a key role in gap junction internalization. Serial section electron microscopy of immunogold-labeled tissues offers a new way to investigate the three-dimensional organization of cells in their native environment.

Versatile Undergraduate Neurobiology Course-Based Research Experiences Using Open Access 3D Electron Microscopy Image Volumes.

Course-based undergraduate research experiences (CUREs) using inquiry-based methodology provide a range of positive benefits to undergraduates and instructors. Yet, the required time and cost in designing and running CUREs with detailed data acquisition steps can lead to barriers in CURE implementation. This report describes an alternative approach to CUREs that utilizes free, open access 3D image volumes as data-rich resources for neurobiology CUREs. These open access image volume CUREs (ivCUREs) effectively combine the data acquisition and analysis steps within the course, allowing more time for students to critically evaluate their hypotheses and results, compare data with peers, and reflect on their experiences. Undergraduates in this 10-week ivCURE analyzed >670 excitatory synapses across two brain areas for the presence and origins of spinules within presynaptic boutons, and fully reconstructed 13 of these synapses in 3D. These data highlight the prevalence of these enigmatic synaptic features within excitatory presynaptic boutons, and their potential importance to neuronal function. Moreover, these results underscore key benefits to ivCURE implementation, including the (1) low-cost of experimental design and implementation, (2) ability to utilize the same data-rich image volume across multiple ivCUREs, (3) potential to generate publishable analyses, and (4) flexibility to scale projects and class sizes up at little to no cost. Opportunities for undergraduates to engage in inquiry-based ivCUREs that examine a host of unexplored questions in neurobiology will continue to grow, in parallel with rapid advances in 3D microscopy techniques and the increased availability and diversity of open access image volumes and analytical tools.

Shifting patterns of polyribosome accumulation at synapses over the course of hippocampal long-term potentiation.

Hippocampal long-term potentiation (LTP) is a cellular memory mechanism. For LTP to endure, new protein synthesis is required immediately after induction and some of these proteins must be delivered to specific, presumably potentiated, synapses. Local synthesis in dendrites could rapidly provide new proteins to synapses, but the spatial distribution of translation following induction of LTP is not known. Here, we quantified polyribosomes, the sites of local protein synthesis, in CA1 stratum radiatum dendrites and spines from postnatal day 15 rats. Hippocampal slices were rapidly fixed at 5, 30, or 120 min after LTP induction by theta-burst stimulation (TBS). Dendrites were reconstructed through serial section electron microscopy from comparable regions near the TBS or control electrodes in the same slice, and in unstimulated hippocampus that was perfusion-fixed in vivo. At 5 min after induction of LTP, polyribosomes were elevated in dendritic shafts and spines, especially near spine bases and in spine heads. At 30 min, polyribosomes remained elevated only in spine bases. At 120 min, both spine bases and spine necks had elevated polyribosomes. Polyribosomes accumulated in spines with larger synapses at 5 and 30 min, but not at 120 min. Small spines, meanwhile, proliferated dramatically by 120 min, but these largely lacked polyribosomes. The number of ribosomes per polyribosome is variable and may reflect differences in translation regulation. In dendritic spines, but not shafts, there were fewer ribosomes per polyribosome in the slice conditions relative to in vivo, but this recovered transiently in the 5 min LTP condition. Overall, our data show that LTP induces a rapid, transient upregulation of large polyribosomes in larger spines, and a persistent upregulation of small polyribosomes in the bases and necks of small spines. This is consistent with local translation supporting enlargement of potentiated synapses within minutes of LTP induction.

msemalign: a pipeline for serial section multibeam scanning electron microscopy volume alignment.

Serial section multibeam scanning electron microscopy (ssmSEM) is currently among the fastest technologies available for acquiring 3D anatomical data spanning relatively large neural tissue volumes, on the order of 1 mm3 or larger, at a resolution sufficient to resolve the fine detail of neuronal morphologies and synapses. These petabyte-scale volumes can be analyzed to create connectomes, datasets that contain detailed anatomical information including synaptic connectivity, neuronal morphologies and distributions of cellular organelles. The mSEM acquisition process creates hundreds of millions of individual image tiles for a single cubic-millimeter-sized dataset and these tiles must be aligned to create 3D volumes. Here we introduce msemalign, an alignment pipeline that strives for scalability and design simplicity. The pipeline can align petabyte-scale datasets such that they contain smooth transitions as the dataset is navigated in all directions, but critically that does so in a fashion that minimizes the overall magnitude of section distortions relative to the originally acquired micrographs.

Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector.

Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion, which are critical for a holistic understanding of the Leishmania life cycle.

Neuronal Morphological Model-Driven Image Registration for Serial Electron Microscopy Sections.

Registration of a series of the two-dimensional electron microscope (EM) images of the brain tissue into volumetric form is an important technique that can be used for neuronal circuit reconstruction. However, complex appearance changes of neuronal morphology in adjacent sections bring difficulty in finding correct correspondences, making serial section neural image registration challenging. To solve this problem, we consider whether there are such stable "markers" in the neural images to alleviate registration difficulty. In this paper, we employ the spherical deformation model to simulate the local neuron structure and analyze the relationship between registration accuracy and neuronal structure shapes in two adjacent sections. The relevant analysis proves that regular circular structures in the section images are instrumental in seeking robust corresponding relationships. Then, we design a new serial section image registration framework driven by this neuronal morphological model, fully utilizing the characteristics of the anatomical structure of nerve tissue and obtaining more reasonable corresponding relationships. Specifically, we leverage a deep membrane segmentation network and neural morphological physical selection model to select the stable rounded regions in neural images. Then, we combine feature extraction and global optimization of correspondence position to obtain the deformation field of multiple images. Experiments on real and synthetic serial EM section neural image datasets have demonstrated that our proposed method could achieve more reasonable and reliable registration results, outperforming the state-of-the-art approaches in qualitative and quantitative analysis.

Integrated Array Tomography for 3D Correlative Light and Electron Microscopy.

Volume electron microscopy (EM) of biological systems has grown exponentially in recent years due to innovative large-scale imaging approaches. As a standalone imaging method, however, large-scale EM typically has two major limitations: slow rates of acquisition and the difficulty to provide targeted biological information. We developed a 3D image acquisition and reconstruction pipeline that overcomes both of these limitations by using a widefield fluorescence microscope integrated inside of a scanning electron microscope. The workflow consists of acquiring large field of view fluorescence microscopy (FM) images, which guide to regions of interest for successive EM (integrated correlative light and electron microscopy). High precision EM-FM overlay is achieved using cathodoluminescent markers. We conduct a proof-of-concept of our integrated workflow on immunolabelled serial sections of tissues. Acquisitions are limited to regions containing biological targets, expediting total acquisition times and reducing the burden of excess data by tens or hundreds of GBs.

VAST (Volume Annotation and Segmentation Tool): Efficient Manual and Semi-Automatic Labeling of Large 3D Image Stacks.

Recent developments in serial-section electron microscopy allow the efficient generation of very large image data sets but analyzing such data poses challenges for software tools. Here we introduce Volume Annotation and Segmentation Tool (VAST), a freely available utility program for generating and editing annotations and segmentations of large volumetric image (voxel) data sets. It provides a simple yet powerful user interface for real-time exploration and analysis of large data sets even in the Petabyte range.

NeuroMorph: A Software Toolset for 3D Analysis of Neurite Morphology and Connectivity.

The geometries of axons, dendrites and their synaptic connections provide important information about their functional properties. These can be collected directly from measurements made on serial electron microscopy images. However, manual and automated segmentation methods can also yield large and accurate models of neuronal architecture from which morphometric data can be gathered in 3D space. This technical paper presents a series of software tools, operating in the Blender open source software, for the quantitative analysis of axons and their synaptic connections. These allow the user to annotate serial EM images to generate models of different cellular structures, or to make measurements of models generated in other software. The paper explains how the tools can measure the cross-sectional surface area at regular intervals along the length of an axon, and the amount of contact with other cellular elements in the surrounding neuropil, as well as the density of organelles, such as vesicles and mitochondria, that it contains. Nearest distance measurements, in 3D space, can also be made between any features. This provides many capabilities such as the detection of boutons and the evaluation of different vesicle pool sizes, allowing users to comprehensively describe many aspects of axonal morphology and connectivity.

Three-dimensional synaptic analyses of mitral cell and external tufted cell dendrites in rat olfactory bulb glomeruli.

Recent studies have suggested that the two excitatory cell classes of the mammalian olfactory bulb, the mitral cells (MCs) and tufted cells (TCs), differ markedly in physiological responses. For example, TCs are more sensitive and broadly tuned to odors than MCs and also are much more sensitive to stimulation of olfactory sensory neurons (OSNs) in bulb slices. To examine the morphological bases for these differences, we performed quantitative ultrastructural analyses of glomeruli in rat olfactory bulb under conditions in which specific cells were labeled with biocytin and 3,3'-diaminobenzidine. Comparisons were made between MCs and external TCs (eTCs), which are a TC subtype in the glomerular layer with large, direct OSN signals and capable of mediating feedforward excitation of MCs. Three-dimensional analysis of labeled apical dendrites under an electron microscope revealed that MCs and eTCs in fact have similar densities of several chemical synapse types, including OSN inputs. OSN synapses also were distributed similarly, favoring a distal localization on both cells. Analysis of unlabeled putative MC dendrites further revealed gap junctions distributed uniformly along the apical dendrite and, on average, proximally with respect to OSN synapses. Our results suggest that the greater sensitivity of eTCs vs. MCs is due not to OSN synapse number or absolute location but rather to a conductance in the MC dendrite that is well positioned to attenuate excitatory signals passing to the cell soma. Functionally, such a mechanism could allow rapid and dynamic control of OSN-driven action potential firing in MCs through changes in gap junction properties. J. Comp. Neurol. 525:592-609, 2017. © 2016 Wiley Periodicals, Inc.

Electron Microscopic Reconstruction of Functionally Identified Cells in a Neural Integrator.

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.

ON Bipolar Cells in Macaque Retina: Type-Specific Synaptic Connectivity with Special Reference to OFF Counterparts.

To date, 12 macaque bipolar cell types have been described. This list includes all morphology types first outlined by Polyak (1941) using the Golgi method in the primate retina and subsequently identified by other researchers using electron microscopy (EM) combined with the Golgi method, serial section transmission EM (SSTEM), and immunohistochemical imaging. We used SSTEM for the rod-dense perifoveal area of macaque retina, reconfirmed ON (cone) bipolar cells to be classified as invaginating midget bipolar (IMB), diffuse bipolar (DB)4, DB5, DB6, giant bipolar (GB), and blue bipolar (BB) types, and clarified their type-specific connectivity. DB4 cells made reciprocal synapses with a kind of ON-OFF lateral amacrine cell, similar to OFF DB2 cells. GB cells contacted rods and cones, similar to OFF DB3b cells. Retinal circuits formed by GB and DB3b cells are thought to substantiate the psychophysical finding of fast rod signals in mesopic vision. DB6 cell output synapses were directed to ON midget ganglion (MG) cells at 70% of ribbon contacts, similar to OFF DB1 cells that directed 60% of ribbon contacts to OFF MG cells. IMB cells contacted medium- or long-wavelength sensitive (M/L-) cones but not short-wavelength sensitive (S-) cones, while BB cells contacted S-cones but not M/L-cones. However, IMB and BB dendrites had similar morphological architectures, and a BB cell contacting a single S-cone resembled an IMB cell. Thus, both IMB and BB may be the ON bipolar counterparts of the OFF flat midget bipolar (FMB) type, likewise DB4 of DB2, DB5 of DB3a, DB6 of DB1, and GB of DB3b OFF bipolar type. The ON DB plus GB, and OFF DB cells predominantly contacted M/L-cones and their outputs were directed mainly to parasol ganglion (PG) cells but also moderately to MG cells. BB cells directed S-cone-driven outputs almost exclusively to small bistratified ganglion (SBG) cells. Some FMB cells predominantly contacted S-cones and their outputs were directed to OFF MG cells. Thus, two-step synaptic connections largely narrowed down the S-cone component to SBG and some OFF MG cells. The other OFF MG cells, ON MG cells, and ON and OFF PG cells constructed M/L-cone dominant pathways.

Large-scale automatic reconstruction of neuronal processes from electron microscopy images.

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 µm(3) volume of brain tissue over a cube of 30 µm 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.

OFF bipolar cells in macaque retina: type-specific connectivity in the outer and inner synaptic layers.

OFF bipolar cells in the macaque retina were recently classified into five types: flat midget bipolar (FMB) and diffuse bipolar (DB) 1, 2, 3a, and 3b. We examined all parallel pathways from cone photoreceptors via OFF bipolar cells to parasol and midget ganglion cells by serial section transmission electron microscopy. Basal contacts of OFF bipolar cells to cone pedicles were previously categorized as triad-associated (TA) and non-TA (NTA). The latter was further divided into two groups located in the middle and marginal areas of the pedicle at the present eccentricity of 15°. We then mapped the distributions of all three basal contacts of the five OFF bipolar cell types in the same area of cone pedicles. TA contacts were more numerous than NTA contacts in FMB (93%), DB1 (67%), and DB3a (81%) cells, but less in DB2 (30%) and DB3b (21%) cells. Cluster analysis of these contact parameters reconfirmed five distinct OFF bipolar cell types and showed these positional configurations of basal synapses to be cell type-specific. This architecture is thought to provide a spatial framework for the interstitial diffusion and local uptake of the neurotransmitter (glutamate) that spills over from ribbon synapses. All five OFF bipolar cell types formed ribbon-synaptic contacts to both parasol and midget ganglion cells. DB2 and 3a, DB1 and 3b, and FMB predominantly, moderately, and negligibly contacted parasol ganglion cells, respectively. FMB almost exclusively contacted midget ganglion cells, to which DB1 provided dominant output (58%), and DB2, 3a, and 3b provided between 3% and 10% of their output. Consequently, the cone signal sampling routes of a midget ganglion cell consisted of two substructures: the narrow (mainly 2-3 cones) FMB pathway and the wide (mainly 10 cones) DB pathway, where connection strength was four-fold greater in the FMB than DB pathway. The narrow and strong FMB pathway may confer the highest spatial resolution and sporadically may include blue cone signals.

Corrigendum: (1) Some OFF Bipolar Cell Types Make Contact With Both Rods and Cones in Macaque and Mouse Retinas; (2) OFF Bipolar Cells in Macaque Retina: Type-specific Connectivity in the Outer and Inner Synaptic Layers.

[This corrects the article on p. 105 in vol. 8, PMID: 25309346.][This corrects the article on p. 122 in vol. 9, PMID: 26500507.].

A new efficient method for synaptic vesicle quantification reveals differences between medial prefrontal cortex perforated and nonperforated synapses.

Communication between neurons is mediated by the release of neurotransmitter-containing vesicles from presynaptic terminals. Quantitative characterization of synaptic vesicles can be highly valuable for understanding mechanisms underlying synaptic function and plasticity. We performed a quantitative ultrastructural analysis of cortical excitatory synapses by mean of a new, efficient method, as an alternative to three-dimensional (3D) reconstruction. Based on a hierarchical sampling strategy and unequivocal identification of the region of interest, serial sections from excitatory synapses of medial prefrontal cortex (mPFC) of six Sprague-Dawley rats were acquired with a transmission electron microscope. Unbiased estimates of total 3D volume of synaptic terminals were obtained through the Cavalieri estimator, and adequate correction factors for vesicle profile number estimation were applied for final vesicle quantification. Our analysis was based on 79 excitatory synapses, nonperforated (NPSs) and perforated (PSs) subtypes. We found that total number of docked and reserve-pool vesicles in PSs significantly exceeded that in NPSs (by, respectively, 77% and 78%). These differences were found to be related to changes in size between the two subtypes (active zone area by 86%; bouton volume by 105%) rather than to postsynaptic density shape. Positive significant correlations were found between number of docked and reserve-pool vesicles, active zone area and docked vesicles, and bouton volume and reserve pool vesicles. Our method confirmed the large size of mPFC PSs and a linear correlation between presynaptic features of typical hippocampal synapses. Moreover, a greater number of docked vesicles in PSs may promote a high synaptic strength of these synapses.

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.

Some OFF bipolar cell types make contact with both rods and cones in macaque and mouse retinas.

This study compared the types of OFF bipolar cells found in the macaque retina with those found in the mouse retina and determined whether these OFF bipolar cells make direct contacts with both rods and cones by serial section transmission electron microscopy. We performed scatter plots and cluster analysis of the morphological variables of their axon terminals such as the stratification level, the arbor thickness, the arbor area, and the number of ribbons. Five OFF bipolar cell types, including the recently discovered DB3b type, were identified in the macaque retina. The macaque OFF bipolar cell types FMB, DB1, DB2, DB3a, and DB3b corresponded to the mouse OFF bipolar cell types 2, 1, 4, 3a, and 3b, respectively. In addition to contacting rod bipolar cells, ~7% of rods in the macaque retina made basal contacts exclusively with one cell type, DB3b, whereas 18% of rods in the mouse retina made basal contacts with one or two of types, 3a, 3b, and 4. Approximately 3% of mouse rods were divergently connected to two OFF bipolar cells of different types, but macaque rods were solely connected to one OFF bipolar cell. Rod-rod gap junctions were localized at rod cell bodies and axons in the outer nuclear layer in both macaque and mouse retinas. The direct rod-OFF bipolar connection system is slightly more developed in the mouse retina than in the macaque retina, possibly as a fine-tuned adaptation to nocturnal conditions. This one-step direct synaptic pathway from rods to OFF bipolar cells may enhance the response speed to OFF light stimuli compared with more indirect pathways via rod-cone gap junctions (a two-step pathway) and via rod bipolar and AII amacrine cells (a three-step pathway).