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.

Structured cerebellar connectivity supports resilient pattern separation.

The cerebellum is thought to help detect and correct errors between intended and executed commands1,2 and is critical for social behaviours, cognition and emotion3-6. Computations for motor control must be performed quickly to correct errors in real time and should be sensitive to small differences between patterns for fine error correction while being resilient to noise7. Influential theories of cerebellar information processing have largely assumed random network connectivity, which increases the encoding capacity of the network's first layer8-13. However, maximizing encoding capacity reduces the resilience to noise7. To understand how neuronal circuits address this fundamental trade-off, we mapped the feedforward connectivity in the mouse cerebellar cortex using automated large-scale transmission electron microscopy and convolutional neural network-based image segmentation. We found that both the input and output layers of the circuit exhibit redundant and selective connectivity motifs, which contrast with prevailing models. Numerical simulations suggest that these redundant, non-random connectivity motifs increase the resilience to noise at a negligible cost to the overall encoding capacity. This work reveals how neuronal network structure can support a trade-off between encoding capacity and redundancy, unveiling principles of biological network architecture with implications for the design of artificial neural networks.

Local shape descriptors for neuron segmentation.

We present an auxiliary learning task for the problem of neuron segmentation in electron microscopy volumes. The auxiliary task consists of the prediction of local shape descriptors (LSDs), which we combine with conventional voxel-wise direct neighbor affinities for neuron boundary detection. The shape descriptors capture local statistics about the neuron to be segmented, such as diameter, elongation, and direction. On a study comparing several existing methods across various specimen, imaging techniques, and resolutions, auxiliary learning of LSDs consistently increases segmentation accuracy of affinity-based methods over a range of metrics. Furthermore, the addition of LSDs promotes affinity-based segmentation methods to be on par with the current state of the art for neuron segmentation (flood-filling networks), while being two orders of magnitudes more efficient-a critical requirement for the processing of future petabyte-sized datasets.

Automatic detection of synaptic partners in a whole-brain Drosophila electron microscopy data set.

We develop an automatic method for synaptic partner identification in insect brains and use it to predict synaptic partners in a whole-brain electron microscopy dataset of the fruit fly. The predictions can be used to infer a connectivity graph with high accuracy, thus allowing fast identification of neural pathways. To facilitate circuit reconstruction using our results, we develop CIRCUITMAP, a user interface add-on for the circuit annotation tool CATMAID.

Advanced Glycation End Products Are Retained in Decellularized Muscle Matrix Derived from Aged Skeletal Muscle.

Decellularized tissues are biocompatible materials that engraft well, but the age of their source has not been explored for clinical translation. Advanced glycation end products (AGEs) are chemical cross-links that accrue on skeletal muscle collagen in old age, stiffening the matrix and increasing inflammation. Whether decellularized biomaterials derived from aged muscle would suffer from increased AGE collagen cross-links is unknown. We characterized gastrocnemii of 1-, 2-, and 20-month-old C57BL/6J mice before and after decellularization to determine age-dependent changes to collagen stiffness and AGE cross-linking. Total and soluble collagen was measured to assess if age-dependent increases in collagen and cross-linking persisted in decellularized muscle matrix (DMM). Stiffness of aged DMM was determined using atomic force microscopy. AGE levels and the effect of an AGE cross-link breaker, ALT-711, were tested in DMM samples. Our results show that age-dependent increases in collagen amount, cross-linking, and general stiffness were observed in DMM. Notably, we measured increased AGE-specific cross-links within old muscle, and observed that old DMM retained AGE cross-links using ALT-711 to reduce AGE levels. In conclusion, deleterious age-dependent modifications to collagen are present in DMM from old muscle, implying that age matters when sourcing skeletal muscle extracellular matrix as a biomaterial.

Insights into the Autoproteolytic Processing and Catalytic Mechanism of the Chlamydia trachomatis Virulence-Associated Protease CPAF.

CPAF (chlamydial protease-like activity factor) is a Chlamydia trachomatis protease that is translocated into the host cytosol during infection. CPAF activity results in dampened host inflammation signaling, cytoskeletal remodeling, and suppressed neutrophil activation. Although CPAF is an emerging antivirulence target, its catalytic mechanism has been unexplored to date. Steady state kinetic parameters were obtained for recombinant CPAF with vimentin-derived peptide substrates using a high-performance liquid chromatography-based discontinuous assay (kcat = 45 ± 0.6 s-1; kcat/Km = 0.37 ± 0.02 µM-1 s-1) or a new fluorescence-based continuous assay (kcat = 23 ± 0.7 s-1; kcat/Km = 0.29 ± 0.03 µM-1 s-1). Residues H105, S499, E558, and newly identified D103 were found to be indispensable for autoproteolytic processing by mutagenesis, while participation of C500 was ruled out despite its proximity to the S499 nucleophile. Pre-steady state kinetics indicated a burst kinetic profile, with fast acylation (kacyl = 110 ± 2 s-1) followed by slower, partially rate-limiting deacylation (kdeacyl = 57 ± 1 s-1). Both kcat- and kcat/Km-pH profiles showed single acidic limb ionizations with pKa values of 6.2 ± 0.1 and 6.5 ± 0.1, respectively. A forward solvent deuterium kinetic isotope effect of 2.6 ± 0.1 was observed for D2Okcatapp, but a unity effect was found for D2Okcat/Kmapp. The kcat proton inventory was linear, indicating transfer of a single proton in the rate-determining transition state, most likely from H105. Collectively, these data provide support for the classification of CPAF as a serine protease and provide a mechanistic foundation for the future design of inhibitors.

Anti-NMDA-receptor encephalitis during pregnancy: A case report and literature review.

Anti-N-methyl-d-aspartate receptor (anti-NMDA-R) encephalitis is an autoimmune disorder that was first described by Dr Vitaliani in 2005. In 2007, Dalmau et al. found anti-NMDA-R antibody expressed both in the hippocampus and prefrontal nerve cell membrane, finally proposing the diagnosis of autoimmune anti-NMDA-R encephalitis. Most of the patients are female (91%), with ages ranging from 4 to 76 years. The average age is 23 years, a birth peak age, although anti-NMDA-R encephalitis is rare during pregnancy. The disorder is characterized by prominent psychosis, dyskinesias, seizures, autonomic disturbance, and central hypoventilation. We report a 24-year-old woman hospitalized at 28 gestational weeks with acute-onset psychosis. Over the course of 3 weeks, her mental status worsened until she fell into a coma. Both serum and cerebrospinal fluid anti-NMDA-R antibodies were found to be positive. At cesarean section, a healthy baby boy was born and a wedge-shaped bilateral ovarian resection was performed. Treatment with corticosteroids, intravenous immunoglobulin, and plasmapheresis can lead to improved outcomes for both mother and baby.

Dichlorvos exposure results in large scale disruption of energy metabolism in the liver of the zebrafish, Danio rerio.

BACKGROUND: Exposure to dichlorvos (DDVP), an organophosphorus pesticide, is known to result in neurotoxicity as well as other metabolic perturbations. However, the molecular causes of DDVP toxicity are poorly understood, especially in cells other than neurons and muscle cells. To obtain a better understanding of the process of non-neuronal DDVP toxicity, we exposed zebrafish to different concentrations of DDVP, and investigated the resulting changes in liver histology and gene transcription. RESULTS: Functional enrichment analysis of genes affected by DDVP exposure identified a number of processes involved in energy utilization and stress response in the liver. The abundance of transcripts for proteins involved in glucose metabolism was profoundly affected, suggesting that carbon flux might be diverted toward the pentose phosphate pathway to compensate for an elevated demand for energy and reducing equivalents for detoxification. Strikingly, many transcripts for molecules involved in ß-oxidation and fatty acid synthesis were down-regulated. We found increases in message levels for molecules involved in reactive oxygen species responses as well as ubiquitination, proteasomal degradation, and autophagy. To ensure that the effects of DDVP on energy metabolism were not simply a consequence of poor feeding because of neuromuscular impairment, we fasted fish for 29 or 50 h and analyzed liver gene expression in them. The patterns of gene expression for energy metabolism in fasted and DDVP-exposed fish were markedly different. CONCLUSION: We observed coordinated changes in the expression of a large number of genes involved in energy metabolism and responses to oxidative stress. These results argue that an appreciable part of the effect of DDVP is on energy metabolism and is regulated at the message level. Although we observed some evidence of neuromuscular impairment in exposed fish that may have resulted in reduced feeding, the alterations in gene expression in exposed fish cannot readily be explained by nutrient deprivation.

Detection of dichlorvos adducts in a hepatocyte cell line.

The toxicity of dichlorvos (DDVP), an organophosphate (OP) pesticide, classically results from modification of the serine in the active sites of cholinesterases. However, DDVP also forms adducts on unrelated targets such as transferrin and albumin, suggesting that DDVP could cause perturbations in cellular processes by modifying noncholinesterase targets. Here we identify novel DDVP-modified targets in lysed human hepatocyte-like cells (HepaRG) using a direct liquid chromatography-mass spectrometry (LC-MS) assay of cell lysates incubated with DDVP or using a competitive pull-down experiments with a biotin-linked organophosphorus compound (10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide; FP-biotin), which competes with DDVP for similar binding sites. We show that DDVP forms adducts to several proteins important for the cellular metabolic pathways and differentiation, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. We validated the results using purified proteins and enzymatic assays. The study not only identified novel DDVP-modified targets but also suggested that the modification directly inhibits the enzymes. The current approach provides information for future hypothesis-based studies to understand the underlying mechanism of toxicity of DDVP in non-neuronal tissues. The MS data have been deposited to the ProteomeXchange with identifier PXD001107.

Specialized connectivity of molecular layer interneuron subtypes leads to disinhibition and synchronous inhibition of cerebellar Purkinje cells.

Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex and are vital to cerebellar processing. MLIs are thought to primarily inhibit Purkinje cells (PCs) and suppress the plasticity of synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs, but the functional significance of these connections is not known. Here, we find that two recently recognized MLI subtypes, MLI1 and MLI2, have a highly specialized connectivity that allows them to serve distinct functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond timescale in vivo, and synchronously pause PC firing. MLI2s are not electrically coupled, primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent behavior and learning. The synchronous firing of electrically coupled MLI1s and disinhibition provided by MLI2s require a major re-evaluation of cerebellar processing.

Permanent deconstruction of intracellular primary cilia in differentiating granule cell neurons.

Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While essential during early developmental stages, the fate of granule cell cilia is unknown. Here, we provide nanoscopic resolution of ciliary dynamics in situ by studying developmental changes in granule cell cilia using large-scale electron microscopy volumes and immunostaining of mouse cerebella. We found that many granule cell primary cilia were intracellular and concealed from the external environment. Cilia were disassembed in differentiating granule cell neurons in a process we call cilia deconstruction that was distinct from pre-mitotic cilia resorption in proliferating progenitors. In differentiating granule cells, ciliary loss involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Cilia did not reform from the docked centrioles, rather, in adult mice granule cell neurons remained unciliated. Many neurons in other brain regions require cilia to regulate function and connectivity. In contrast, our results show that granule cell progenitors had concealed cilia that underwent deconstruction potentially to prevent mitogenic hedgehog responsiveness. The ciliary deconstruction mechanism we describe could be paradigmatic of cilia removal during differentiation in other tissues.

Cerebellar circuits for disinhibition and synchronous inhibition.

The cerebellar cortex contributes to diverse behaviors by transforming mossy fiber inputs into predictions in the form of Purkinje cell (PC) outputs, and then refining those predictions1. Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex2, and are vital to cerebellar processing1,3. MLIs are thought to primarily inhibit PCs and suppress the plasticity of excitatory synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs4-7, but the functional significance of these connections is not known1,3. Behavioral studies suggest that cerebellar-dependent learning is gated by disinhibition of PCs, but the source of such disinhibition has not been identified8. Here we find that two recently recognized MLI subtypes2, MLI1 and MLI2, have highly specialized connectivity that allows them to serve very different functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond time scale in vivo, and synchronously pause PC firing. MLI2s are not electrically coupled, they primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent learning8. These findings require a major reevaluation of processing within the cerebellum in which disinhibition, a powerful circuit motif present in the cerebral cortex and elsewhere9-17, greatly increases the computational power and flexibility of the cerebellum. They also suggest that millisecond time scale synchronous firing of electrically-coupled MLI1s helps regulate the output of the cerebellar cortex by synchronously pausing PC firing, which has been shown to evoke precisely-timed firing in PC targets18.

Three-dimensional reconstructions of mechanosensory end organs suggest a unifying mechanism underlying dynamic, light touch.

Specialized mechanosensory end organs within mammalian skin-hair follicle-associated lanceolate complexes, Meissner corpuscles, and Pacinian corpuscles-enable our perception of light, dynamic touch 1 . In each of these end organs, fast-conducting mechanically sensitive neurons, called Aß low-threshold mechanoreceptors (Aß LTMRs), associate with resident glial cells, known as terminal Schwann cells (TSCs) or lamellar cells, to form complex axon ending structures. Lanceolate-forming and corpuscle-innervating Aß LTMRs share a low threshold for mechanical activation, a rapidly adapting (RA) response to force indentation, and high sensitivity to dynamic stimuli 1-6 . How mechanical stimuli lead to activation of the requisite mechanotransduction channel Piezo2 7-15 and Aß RA-LTMR excitation across the morphologically dissimilar mechanosensory end organ structures is not understood. Here, we report the precise subcellular distribution of Piezo2 and high-resolution, isotropic 3D reconstructions of all three end organs formed by Aß RA-LTMRs determined by large volume enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging. We found that within each end organ, Piezo2 is enriched along the sensory axon membrane and is minimally or not expressed in TSCs and lamellar cells. We also observed a large number of small cytoplasmic protrusions enriched along the Aß RA-LTMR axon terminals associated with hair follicles, Meissner corpuscles, and Pacinian corpuscles. These axon protrusions reside within close proximity to axonal Piezo2, occasionally contain the channel, and often form adherens junctions with nearby non-neuronal cells. Our findings support a unified model for Aß RA-LTMR activation in which axon protrusions anchor Aß RA-LTMR axon terminals to specialized end organ cells, enabling mechanical stimuli to stretch the axon in hundreds to thousands of sites across an individual end organ and leading to activation of proximal Piezo2 channels and excitation of the neuron.

Three-dimensional reconstructions of mechanosensory end organs suggest a unifying mechanism underlying dynamic, light touch.

Across mammalian skin, structurally complex and diverse mechanosensory end organs respond to mechanical stimuli and enable our perception of dynamic, light touch. How forces act on morphologically dissimilar mechanosensory end organs of the skin to gate the requisite mechanotransduction channel Piezo2 and excite mechanosensory neurons is not understood. Here, we report high-resolution reconstructions of the hair follicle lanceolate complex, Meissner corpuscle, and Pacinian corpuscle and the subcellular distribution of Piezo2 within them. Across all three end organs, Piezo2 is restricted to the sensory axon membrane, including axon protrusions that extend from the axon body. These protrusions, which are numerous and elaborate extensively within the end organs, tether the axon to resident non-neuronal cells via adherens junctions. These findings support a unified model for dynamic touch in which mechanical stimuli stretch hundreds to thousands of axon protrusions across an end organ, opening proximal, axonal Piezo2 channels and exciting the neuron.

Human Adipose-Derived Stromal Cells Delivered on Decellularized Muscle Improve Muscle Regeneration and Regulate RAGE and P38 MAPK.

Volumetric muscle loss (VML) is the acute loss of muscle mass due to trauma. Such injuries occur primarily in the extremities and are debilitating, as there is no clinical treatment to restore muscle function. Pro-inflammatory advanced glycation end-products (AGEs) and the soluble receptor for advanced glycation end-products (RAGE) are known to increase in acute trauma patient's serum and are correlated with increased injury severity. However, it is unclear whether AGEs and RAGE increase in muscle post-trauma. To test this, we used decellularized muscle matrix (DMM), a pro-myogenic, non-immunogenic extracellular matrix biomaterial derived from skeletal muscle. We delivered adipose-derived stromal cells (ASCs) and primary myoblasts to support myogenesis and immunomodulation (N = 8 rats/group). DMM non-seeded and seeded grafts were compared to empty defect and sham controls. Then, 56 days after surgery muscle force was assessed, histology characterized, and protein levels for AGEs, RAGE, p38 MAPK, and myosin heavy chains were measured. Overall, our data showed improved muscle regeneration in ASC-treated injury sites and a regulation of RAGE and p38 MAPK signaling, while myoblast-treated injuries resulted in minor improvements. Taken together, these results suggested that ASCs combined with DMM provides a pro-myogenic microenvironment with immunomodulatory capabilities and indicates further exploration of RAGE signaling in VML.