Connectomic reconstruction of a female Drosophila ventral nerve cord.

A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. Here, we apply automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC)1, which functions like the vertebrate spinal cord to sense and control the body. We find that the fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, we mapped the muscle targets of leg and wing motor neurons using genetic driver lines2 and X-ray holographic nanotomography3. With this motor neuron atlas, we identified neural circuits that coordinate leg and wing movements during take-off. We provide the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour.

Histological, Biomechanical, and Biological Properties of Genipin-Crosslinked Decellularized Peripheral Nerves.

Acellular nerve allografts (ANGs) represent a promising alternative in nerve repair. Our aim is to improve the structural and biomechanical properties of biocompatible Sondell (SD) and Roosens (RS) based ANGs using genipin (GP) as a crosslinker agent ex vivo. The impact of two concentrations of GP (0.10% and 0.25%) on Wistar rat sciatic nerve-derived ANGs was assessed at the histological, biomechanical, and biocompatibility levels. Histology confirmed the differences between SD and RS procedures, but not remarkable changes were induced by GP, which helped to preserve the nerve histological pattern. Tensile test revealed that GP enhanced the biomechanical properties of SD and RS ANGs, being the crosslinked RS ANGs more comparable to the native nerves used as control. The evaluation of the ANGs biocompatibility conducted with adipose-derived mesenchymal stem cells cultured within the ANGs confirmed a high degree of biocompatibility in all ANGs, especially in RS and RS-GP 0.10% ANGs. Finally, this study demonstrates that the use of GP could be an efficient alternative to improve the biomechanical properties of ANGs with a slight impact on the biocompatibility and histological pattern. For these reasons, we hypothesize that our novel crosslinked ANGs could be a suitable alternative for future in vivo preclinical studies.

Neural reconstruction of bone-eating Osedax spp. (Annelida) and evolution of the siboglinid nervous system.

BACKGROUND: Bone-devouring Osedax worms were described over a decade ago from deep-sea whale falls. The gutless females (and in one species also the males) have a unique root system that penetrates the bone and nourishes them via endosymbiotic bacteria. Emerging from the bone is a cylindrical trunk, which is enclosed in a transparent tube, that generally gives rise to a plume of four palps (or tentacles). In most Osedax species, dwarf males gather in harems along the female's trunk and the nervous system of these microscopic forms has been described in detail. Here, the nervous system of bone-eating Osedax forms are described for the first time, allowing for hypotheses on how the abberant ventral brain and nervous system of Siboglinidae may have evolved from a ganglionated nervous system with a dorsal brain, as seen in most extant annelids. RESULTS: The intraepidermal nervous systems of four female Osedax spp. and the bone-eating O. priapus male were reconstructed in detail by a combination of immunocytochemistry, CLSM, histology and TEM. They all showed a simple nervous system composed of an anterior ventral brain, connected with anteriorly directed paired palp and gonoduct nerves, and four main pairs of posteriorly directed longitudinal nerves (2 ventral, 2 ventrolateral, 2 sets of dorso-lateral, 2 dorsal). Transverse peripheral nerves surround the trunk, ovisac and root system. The nervous system of Osedax resembles that of other siboglinids, though possibly presenting additional lateral and dorsal longitudinal nerves. It differs from most Sedentaria in the presence of an intraepidermal ventral brain, rather than a subepidermal dorsal brain, and by having an intraepidermal nerve cord with several plexi and up to three main commissures along the elongated trunk, which may comprise two indistinct segments. CONCLUSIONS: Osedax shows closer neuroarchitectural resemblance to Vestimentifera + Sclerolinum (= Monilifera) than to Frenulata. The intraepidermal nervous system with widely separated nerve cords, double brain commissures, double palp nerves and other traits found in Osedax can all be traced to represent ancestral states of Siboglinidae. A broader comparison of the nervous system and body regions across Osedax and other siboglinids allows for a reinterpretation of the anterior body region in the group.

Schwann-like cells seeded in acellular nerve grafts improve nerve regeneration.

BACKGROUND: This study evaluated whether Schwann-like cells (SLCs) induced from bone marrow-derived mesenchymal stem cells (BM-MSCs) transplanted into acellular nerve grafts (ANGs) could repair nerve defects compared with nerve isografts and ANGs with BM-MSCs. METHODS: BM-MSCs extracted, separated and purified from the bone marrow of rats, and some of the BM-MSCs were cultured with mixed induction agents that could induce BM-MSCs into SLCs. Either SLCs or BM-MSCs were seeded onto 10-mm ANGs, and the isografts were chosen as the control. The walking-track test, tibialis anterior muscle weight measurement, electrophysiological examination, toluidine blue staining, transmission electron micrographs and immunostaining of S-100 and VEGF in these three groups were evaluated in a 10-mm rat sciatic injury-repair model. RESULTS: The walking-track test, tibialis anterior muscle weight measurement and electrophysiological examination of the sciatic nerve suggested the groups of ANGs with SLCs and isografts obtained better results than the BM-MSC group (P<0.05). Meanwhile, the results of the SLCs and isograft groups were similar (P>0.05). All the histomorphometric analyses (toluidine blue staining, transmission electron micrographs and immunostaining of S-100 and VEGF) showed that there were more regenerating nerve fibers in the group of ANGs with SLCs than the BM-MSCs (P<0.05), but there was no significant difference between the SLC and isograft groups (P>0.05). CONCLUSIONS: SLCs seeded in ANGs and isografts show better functional regeneration compared with BM-MSCs seeded in ANGs. Additionally, SLCs combined with ANGs present almost the same outcome as the isografts. Therefore, SLCs with ANGs can be a good choice in nerve defect repairs.

Unsupervised segmentation of noisy electron microscopy images using salient watersheds and region merging.

BACKGROUND: Segmenting electron microscopy (EM) images of cellular and subcellular processes in the nervous system is a key step in many bioimaging pipelines involving classification and labeling of ultrastructures. However, fully automated techniques to segment images are often susceptible to noise and heterogeneity in EM images (e.g. different histological preparations, different organisms, different brain regions, etc.). Supervised techniques to address this problem are often helpful but require large sets of training data, which are often difficult to obtain in practice, especially across many conditions. RESULTS: We propose a new, principled unsupervised algorithm to segment EM images using a two-step approach: edge detection via salient watersheds following by robust region merging. We performed experiments to gather EM neuroimages of two organisms (mouse and fruit fly) using different histological preparations and generated manually curated ground-truth segmentations. We compared our algorithm against several state-of-the-art unsupervised segmentation algorithms and found superior performance using two standard measures of under-and over-segmentation error. CONCLUSIONS: Our algorithm is general and may be applicable to other large-scale segmentation problems for bioimages.

Targeted neuromodulation of pelvic floor nerves in aging and multiparous rabbits improves continence.

Pelvic floor muscle stretch injury during pregnancy and birth is associated with the incidence of stress urinary incontinence (SUI), a condition that affects 30-60% of the female population and is characterized by involuntary urine leakage during physical activity, further exacerbated by aging. Aging and multiparous rabbits suffer pelvic nerve and muscle damage, resulting in alterations in pelvic floor muscular contraction and low urethral pressure, resembling SUI. However, the extent of nerve injury is not fully understood. Here, we used electron microscopy analysis of pelvic and perineal nerves in multiparous rabbits to describe the extent of stretch nerve injury based on axon count, axon size, myelin-to-axon ratio, and elliptical ratio. Compared to young nulliparous controls, mid-age multiparous animals showed an increase in the density of unmyelinated axons and in myelin thickness in both nerves, albeit more significant in the bulbospongiosus nerve. This revealed a partial but sustained damage to these nerves, and the presence of some regenerated axons. Additionally, we tested whether electrical stimulation of the bulbospongiosus nerve would induce muscle contraction and urethral closure. Using a miniature wireless stimulator implanted on this perineal nerve in young nulliparous and middle age multiparous female rabbits, we confirmed that these partially damaged nerves can be acutely depolarized, either at low (2-5 Hz) or medium (10-20 Hz) frequencies, to induce a proportional increase in urethral pressure. Evaluation of micturition volume in the mid-age multiparous animals after perineal nerve stimulation, effectively reversed a baseline deficit, increasing it 2-fold (p = 0.02). These results support the notion that selective neuromodulation of pelvic floor muscles might serve as a potential treatment for SUI.

An unbiased template of the Drosophila brain and ventral nerve cord.

The fruit fly Drosophila melanogaster is an important model organism for neuroscience with a wide array of genetic tools that enable the mapping of individual neurons and neural subtypes. Brain templates are essential for comparative biological studies because they enable analyzing many individuals in a common reference space. Several central brain templates exist for Drosophila, but every one is either biased, uses sub-optimal tissue preparation, is imaged at low resolution, or does not account for artifacts. No publicly available Drosophila ventral nerve cord template currently exists. In this work, we created high-resolution templates of the Drosophila brain and ventral nerve cord using the best-available technologies for imaging, artifact correction, stitching, and template construction using groupwise registration. We evaluated our central brain template against the four most competitive, publicly available brain templates and demonstrate that ours enables more accurate registration with fewer local deformations in shorter time.

An ultrastructural examination of the role of cell membrane surface coat material during neurulation.

Data from neural crest cultures indicate that cell surface coat material (CSM) is directly involved in cellular migration and events surrounding differentiation. To investigate whether the CSM also has a morphogenetic role, embryos of the amphibian Ambystoma maculatum were examined ultrastructurally throughout the stages of neurulation. Segments of the neural axis were fixed in glutaraldehyde-containing Alcian blue 8GX, which reportedly enhances preservation of CSM, and were postfixed in OsO4 containing 1 percent lanthanum nitrate, which stains the CSM. The medial groove formed by the appearance of the neural ridges contains a large amount of CSM and numerous vesicles coated with lanthanum-positive material. In contrast, the lateral ridge surfaces are covered by a small amount of uniformly distributed CSM and a paucity of vesicles. As the ridges begin to fold there is a progressive increase in the amount of CSM within the presumptive neural tube region. Further convergence of the neural folds is accompanied by an increase of CSM at their leading edges. As the folds approximate each other, lanthanum-positive material physically bridges the gap. However, as the apposing tissue actually abuts to form the neural tube, no CSM is observed in the remaining interspace. The specific distribution and sequential accumulation of cell CSM during the events of neurulation strongly suggest its direct participation in the morphogenetic process.

Methods for the freeze-fracturing of nerve tissue cultures and cell monolayers.

Two methods for freeze-cleaving of thin tissue layers are presented. Whereas a simple technique can be employed to fracture continuous, relatively firm tissue layers, a more sophisticated technique employing special carriers is needed to fracture very thin and incomplete layers, e.g., the fiber outgrowth of cultured nerve tissue or sparsely seeded isolated cells. Both methods basically consist of freezing the specimens sandwiched between two small metal carriers which are then fractured apart so that the cleavage plane runs through the tissue. In the resulting replicas of such thin specimens, large membrane areas are exposed, and new information is provided on the topography of membrane properties in entire cells or cell processes. The technique should also be useful for studies on the interactions of cells grown in culture.

Tannic acid-stained microtubules with 12, 13, and 15 protofilaments.

Subunit structure in the walls of sectioned microtubules was first noted by Ledbetter and Porter (6), who clearly showed that certain microtubules of plant meristematic cells have 13 wall protofilaments when seen in cross section. Earlier, protofilaments of microtubular elements had been described in negatively stained material, although exact counts of their number were difficult to obtain. In microtubular elements of axonemes, some success has been achieved in visualizing protofilaments in conventionally fixed and sectioned material (8, 10); much less success has been achieved in identifying and counting protofilaments of singlet cytoplasmic microtubules. By using glutaraldehyde-tannic acid fixation, as described by Misuhira and Futaesaku (7), Tilney et al. (12) studied microtubules from a number of sources and found that all have 13 protofilaments comprising their walls. These authors note that "...the number of subunits and their arrangement as protofilaments appear universal...". Preliminary studies of ventral nerve cord of crayfish fixed in glutaraldehyde-tannic acid indicated that axonal microtubules in this material possess only 12 protofilaments (4). On the basis of this observation, tannic acid preparations of several other neuronal and non-neuronal systems were examined. Protofilaments in microtubules from these several cell types are clearly demonstrated, and counts have been made which show that some kinds of microtubules have more or fewer protofilaments than the usual 13 and that at least one kind of microtubule has an even rather than an odd number.

The effects of halothane on cultured mouse neuroblastoma cells. I. Inhibition of morphological differentiation.

Mouse neuroblastoma cells (clone NB2a) were cultured in the presence of 0.3-2.1% halothane in the gas phase for up to 72 h. Halothane inhibited neurite extension dose dependently and virtually abolished microspike formation even at the lowest concentration tested. These effects were completely reversible. Electron microscopy demonstrated that microfilaments measuring 40-80 A in diameter are the only fibrous organelles visible within microspikes. When the cells were exposed to halothane, no microfilamentous complexes could be identified in any cells and the subcortical regions of neurites often appeared devoid of individual microfilaments. Microtubules were still present in neurites after exposure to halothane concentrations at which microfilaments disappeared. However, at concentrations above 1.0%, microtubules gradually appeared to decrease in number. Short-term experiments showed that existing neurites and microspikes rapidly retracted when suddenly exposed to culture medium equilibrated with 1.0% halothane and quickly reformed when the halothane was removed. The inhibition of neuroblastoma cell differentiation by halothane appears to be mediated by disruption of 40-80 A diameter microfilaments.

Neural probe design for reduced tissue encapsulation in CNS.

This study investigated relationships between a microscale neural probe's size and shape and its chronic reactive tissue response. Parylene-based probes were microfabricated with a thick shank (48 microm by 68 microm) and an integrated thin lateral platform (5 microm by 100 microm, either solid or one of three lattice sizes). Devices were implanted in rat cerebral cortex for 4 weeks before immunostaining for neurons, astrocytes, microglia, fibronectin, laminin, and neurofilament. While nonneuronal density (NND) generally increased and neuronal density decreased within 75 microm of a probe interface compared to unimplanted control regions, there were significant differential tissue responses within 25 microm of the platform's lateral edge compared to the shank. The NND in this region of the lateral edge was less than one-third of the corresponding region of the shank (129% and 425% increase, respectively). Moreover, neuronal density around the platform lateral edge was about one-third higher than at the shank (0.70 and 0.52 relative to control, respectively). Also, microglia reactivity and extracellular protein deposition was reduced at the lateral edge. There were no significant differences among platform designs. These results suggest that neural probe geometry is an important parameter for reducing chronic tissue encapsulation.

Genomic and morphological changes of neuroblastoma cells in response to three-dimensional matrices.

Advances in neural tissue engineering require a comprehensive understanding of neuronal growth in 3 dimensions. This study compared the gene expression of SH-SY5Y human neuroblastoma cells cultured in 3-dimensional (3D) with those cultured in 2-dimensional (2D) environments. Microarray analysis demonstrated that, in response to varying matrix geometry, SH-SY5Y cells exhibited differential expression of 1,766 genes in collagen I, including those relevant to cytoskeleton, extracellular matrix, and neurite outgrowth. Cells extended longer neurites in 3D collagen I cultures than in 2D. Real-time reverse transcriptase polymerase chain reaction experiments and morphological analysis comparing collagen I and Matrigel tested whether the differential growth and gene expression reflected influences of culture dimension or culture material. SH-SY5Y neuroblastoma cells responded to geometry by differentially regulating cell spreading and genes associated with actin in similar patterns for both materials; however, neurite outgrowth and the expression of the gene encoding for neurofilament varied with the type of material. Electron microscopy and mechanical analysis showed that collagen I was more fibrillar than Matrigel, with larger inter-fiber distance and higher stiffness. Taken together, these results suggest complex cell-material interactions, in which the dimension of the culture material influences gene expression and cell spreading and the structural and mechanical properties of the culture material influence gene expression and neurite outgrowth.

Aragonite crystalline biomatrices support astrocytic tissue formation in vitro and in vivo.

Astrocytes play a pivotal role in the development and function of the central nervous system by regulating synaptic activity and supporting and guiding growing axons. It is therefore a central therapeutic and scientific challenge to develop means to control astrocytic survival and growth. We cultured primary hippocampal astrocytes on a crystalline three-dimensional (3D) aragonite biomatrix prepared from the exoskeleton of the coral Porites lutea. Such culturing led to the formation of astrocytic tissue-like 3D structures in which the cells had a higher survival rate than astrocytes grown in conventional cell culture. Within the pore void areas, multiple layers of astrocytic processes formed concave sheet structures that had no physical contact with the surface. The astrocytes attached to the crystalline perpendicular edges of the crystalline template surface extended processes in 3D and expressed glial fibrillary acidic protein. The astrocytes also expressed gap junctions and developed partly synchronized cytosolic Ca2+ oscillations. Preliminary in vivo models showed that astrocytic networks were also developed when the matrices were implanted into cortical areas of postnatal rat brains. Hence, we suggest that the biomatrix is a biocompatible supportive scaffold for astrocytes and may be exploited in applications for neuronal tissue restoration in injured or diseased central nervous system.

Reversal of a hallmark of brain ageing: lipofuscin accumulation.

The prospect of removing cellular deposits of lipofuscin is of considerable interest because they may contribute to age related functional decline and disease. Here, we use a decapod crustacean model to circumvent a number of problems inherent in previous studies on lipofuscin loss. We employ (a) validated lipofuscin quantification methods, (b) an in vivo context, (c) essentially natural environmental conditions and (d) a situation without accelerated production of residual material or (e) application of pharmacological compounds. We use a novel CNS biopsy technique that produces both an anti-ageing effect and also permits longitudinal sampling of individuals, thus (f) avoiding conventional purely cross-sectional population data that may suffer from selective mortality biases. We quantitatively demonstrate that lipofuscin, accrued through normal ageing, can be lost from neural tissue. The mechanism of loss probably involves exocytosis and possibly blood transport. If non-disruptive ways to accelerate lipofuscin removal can be found, our results suggest that therapeutic reversal of this most universal manifestation of cellular ageing may be possible.

[Nervous component of mature cystic ovarian teratomas]. / Nervová slozka zralých cystických teratomu ovaria.

In 30-50 percent of cases mature cystic ovarian teratomas contain a nervous tissue which can be highly differentiated. This study was focused on research of the nervous component of mature cystic ovarian teratomas with generally available methods to pathologists, including impregnation techniques, immunohistochemistry and electron microscopy. From the total number of 212 mature cystic ovarian teratomas, the nervous tissue was found in 72 cases (34%), which corresponds to the literature data. According to its differentiation, it was possible to distinguish five categories of nervous tissue by light microscopy: 0 peripheral nervous tissue only, 1--solid glial nodules, 2--glial cysts, 3--glial tissue with abundant scattered neurons and, finally, 4--organoid nervous tissue similar to certain CNS structures. Apart from the foci similar to grey matter of the spinal cord and cerebral cortex, those of differentiated cerebellar cortex were present as well. Astrocytes mostly predominated in the nervous tissue, and they sometimes showed reactive changes including gemistocytes and formation of Rosenthal fibres. Neuronal elements also showed degenerative changes quite frequently, especially in a less differentiated nervous component. These changes might have developed due to an abnormal location of the nervous tissue or its hypoxia in the teratoma. Contrary to some literature data, oligodendrocytes and myelin were present in the nervous tissue of most of our cases. Ultrastructurally, neurons with fully developed synapses were observed in the nervous component, and dendritic spines were present on dendrites of Purkinje cells of cerebellar cortex. The results obtained from the examination of teratomas in this study confirmed and enriched the literature data concerning the high degree of differentiation of their nervous components. We suggest that the differentiated nervous tissue of teratoma represents a unique natural model suitable for research of some aspects of neurohistology and neuropathology, e.g. synaptogenesis or myelinogenesis.

[Gap junctions--major structures promoting intercellular communication].

Gap junctions provide humoral and electric communication between the cells, thus contributing to their morpho-functional cooperation. Gap junction is formed by multiple intercellular channels, each of them being made by two closed hemichannels--connexons, that are oligomeric transmembrane proteins built by 6 subunits, belonging to connexin family. Permeability and electric conductivity of gap junction channels is determined by molecular peculiarities of connexins, their capacity for phosphorilation and by some extra- and intracellular factors. According to the current data, gap junctions in both cell cultures and tissues are dynamic structures with a short half-life period. Main mechanisms responsible for gap junction assembly and destruction have been discovered. These mechanisms were shown to depend upon peculiarities of differential genome activity and to be controlled by extra- and intracellular factors. The data on the gap junctions in the nervous system, heart and epidermis are presented.

Ventral nerve cord remodeling in a stingless bee (Melipona quadrifasciata anthidioides, Hymenoptera, Apidae) depends on ecdysteroid fluctuation and programmed cell death.

The reorganization of the ventral nerve cord (VNC) during metamorphosis of M. quadrifasciata was observed to be characterized by shortening of connectives and subsequent fusion of the 2nd and 3rd thoracic and the 1st abdominal ganglia. Also, the 5th to 7th abdominal ganglia came into very close contact. These changes were accompanied by increasing levels of endogenous ecdysteroids, as determined by a radioimmunoassay. Incubation of VNC in the presence of 5 microg 20-hydroxyecdysone, caused significant shortening of connectives in the thoracic region, but not in the abdomen, evidencing a segment-specific response to this hormone. Cell death in the ventral ganglia was revealed by transmission electron microscopy and TUNEL-reaction. Detection of labeled cells in the region where contiguous ganglia come into close contact suggests that programmed cell death is involved in ganglionic fusion.

DLA based compressed sensing for high resolution MR microscopy of neuronal tissue.

In this work we present the implementation of compressed sensing (CS) on a high field preclinical scanner (17.2 T) using an undersampling trajectory based on the diffusion limited aggregation (DLA) random growth model. When applied to a library of images this approach performs better than the traditional undersampling based on the polynomial probability density function. In addition, we show that the method is applicable to imaging live neuronal tissues, allowing significantly shorter acquisition times while maintaining the image quality necessary for identifying the majority of neurons via an automatic cell segmentation algorithm.

Discovering the structure of nerve tissue: part 1: from Marcello Malpighi to Christian Berres.

The invention of the microscope at the beginning of the seventeenth century was a pivotal event for subsequent studies of the microscopic structure of nerve tissue. The present article, using translations of the original texts, presents a recollection of the discoveries made during the second half of the seventeenth century up to the beginning of the nineteenth century by prominent scholars as well as those nearly forgotten today. The findings in the field of neuroanatomy are collected together into a coherent form and in chronological order, showing the progress of the discoveries from a historical perspective. The early scientists discovered, and then repeatedly confirmed, that nerve tissue was remarkably similar over a wide range of animal forms. While they offered little detail, and much of what was described was flawed because of various technical restraints of the time, what they did report was very similar from animal to animal. Their studies, however, in parallel with the improvement of microscopic techniques as well as the processing and fixation of animal tissues, helped to create fertile ground for a number of important neurohistological discoveries in the first half of the nineteenth century.