Search

Human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NSCs) hold promise for treating neurodegenerative and demyelinating disorders. However, comprehensive studies on their identity and safety remain limited. In this study, we demonstrate that hiPSC-NSCs adopt a radial glia-associated signature, sharing key epigenetic and transcriptional characteristics with human fetal neural stem cells (hfNSCs) while exhibiting divergent profiles from glioblastoma stem cells. Long-term transplantation studies in mice showed robust and stable engraftment of hiPSC-NSCs, with predominant differentiation into glial cells and no evidence of tumor formation. Additionally, we identified the Sterol Regulatory Element Binding Transcription Factor 1 (SREBF1) as a regulator of astroglial differentiation in hiPSC-NSCs. These findings provide valuable transcriptional and epigenetic reference datasets to prospectively define the maturation stage of NSCs derived from different hiPSC sources and demonstrate the long-term safety of hiPSC-NSCs, reinforcing their potential as a viable alternative to hfNSCs for clinical applications., (© 2024. The Author(s).)

The generation of human pluripotent stem cell (hPSC)-derived brain organoids is continuously refined, enhancing their reproducibility and complexity. Here, we present a guided differentiation protocol for generating cortical forebrain organoids and cortico-pericyte (CP)assembloids composed of a robust outer radial glia (oRG) population and an expanded outer subventricular zone (oSVZ). We describe the steps to generate hPSC-derived cortical organoids (COs), cortical pericytes, and CP assembloids. Moreover, we outline the procedures to characterize the organoids by immunostaining and to perform single-cell dissociation. For complete details on the use and execution of this protocol, please refer to Walsh et al. 1 ., Competing Interests: Declaration of interests L.S. is a scientific cofounder and paid consultant of BlueRock Therapeutics, Inc. L.S. is a scientific cofounder of DaCapo Brainscience., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The subventricular zone (SVZ) is one of the neurogenic regions of the adult mammalian brain. Neural stem cells (NSCs) in the SVZ have certain key features: they express glial fibrillary acidic protein (GFAP), proliferate slowly, have a radial glia-like (RG-L) morphology, and are in contact with the cerebrospinal fluid (CSF). NSCs have been isolated by FACS to analyse them, but their morphology has not been systematically examined. To address this knowledge gap, we sparsely labelled RG-L cells in the SVZ of neonatal mice by introducing via electroporation a plasmid expressing fluorescent protein under the control of the GFAP promoter. We then classified RG-L cells into three types (RG-L1, 2, and 3) based on their morphologies. RG-L1 cells had a basal process with some branches and numerous fine processes. RG-L2 cells had a basal process, but fewer branches and fine processes than RG-L1 cells. RG-L3 cells had one basal process that was almost free of branches and fine processes. Importantly, regardless of the cell type, about half of their somata resided on the basal side of the SVZ. Based on changes in their proportions during postnatal development and their expression of GFAP and cell proliferation markers at the adult stage, we speculated that NSCs change their morphologies during development/maturation and not all NSCs must always be in the apical SVZ or in contact with the CSF. Our results indicate that in addition to expression of markers for NSCs, the morphology is a critical feature to identify NSCs., (© 2024 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The primary supporting cell of the retina is the retinal glial Müller cell. They cover the entire retinal surface and are in close proximity to both the retinal blood vessels and the retinal neurons. Because of their growth, Müller cells perform several crucial tasks in a healthy retina, including the uptake and recycling of neurotransmitters, retinoic acid compounds, and ions (like potassium K+). In addition to regulating blood flow and maintaining the blood-retinal barrier, they also regulate the metabolism and the supply of nutrients to the retina. An established procedure for isolating primary mouse Müller cells is presented in this manuscript. To better understand the underlying molecular processes involved in the various mouse models of ocular disorders, Müller cell isolation is an excellent approach. This manuscript outlines a detailed procedure for Müller cell isolation from mice. From enucleation to seeding, the entire process lasts about a few hours. For 5-7 days after seeding, the media shouldn't be changed in order to allow the isolated cells to grow unhindered. Cell characterization using morphology and distinct immunofluorescent markers comes next in the process. Maximum passages for cells are 3-4 times.

DNA methylation is extensively reconfigured during development, but the functional significance and cell type-specific dependencies of DNA demethylation in lineage specification remain poorly understood. Here, we demonstrate that developmental DNA demethylation, driven by ten-eleven translocation 1/2/3 (TET1/2/3) enzymes, is essential for establishment of neural stem cell (NSC) identity and gliogenic potential. We find that loss of all three TETs during NSC specification is dispensable for neural induction and neuronal differentiation but critical for astrocyte and oligodendrocyte formation, demonstrating a selective loss of glial competence. Mechanistically, TET-mediated demethylation was essential for commissioning neural-specific enhancers in proximity to master neurodevelopmental and glial transcription factor genes and for induction of these genes. Consistently, loss of all three TETs in embryonic NSCs in mice compromised glial gene expression and corticogenesis. Thus, TET-dependent developmental demethylation is an essential regulatory mechanism for neural enhancer commissioning during NSC specification and is a cell-intrinsic determinant of NSC identity and gliogenic potential.

Brain microphysiological systems (bMPS) recapitulate human brain cellular architecture and functionality more closely than traditional monolayer cultures and have become increasingly relevant for the study of neurological function in health and disease. Existing 3D brain models vary in reflecting the relative populations of different cell types present in the human brain. Most models consist mainly of neurons, while glial cells represent a smaller portion of the cell populations. Here, by means of a chemically defined glial-enriched medium (GEM), an improved method to expand the population of astrocytes and oligodendrocytes without compromising neuronal differentiation in bMPS, is presented. An important finding is that astrocytes also change in morphology when cultured in GEM, more closely recapitulating primary culture astrocytes. GEM bMPS are electro-chemically active and show different patterns of calcium staining and flux. Synaptic vesicles and terminals observed by electron microscopy are also present. No significant changes in neuronal differentiation are observed by gene expression, however, GEM enhanced neurite outgrowth and cell migration, and differentially modulated neuronal maturation in two different cell lines. These results have the potential to significantly improve functionality of bMPS for the study of neurological diseases and drug discovery, contributing to the unmet need for safe human models., (© 2023 The Authors. Advanced Biology published by Wiley‐VCH GmbH.)

The vagal ganglia, comprised of the superior (jugular) and inferior (nodose) ganglia of the vagus nerve, receive somatosensory information from the head and neck or viscerosensory information from the inner organs, respectively. Developmentally, the cranial neural crest gives rise to all vagal glial cells and to neurons of the jugular ganglia, while the epibranchial placode gives rise to neurons of the nodose ganglia. Crest-derived nodose glial progenitors can additionally generate autonomic neurons in the peripheral nervous system, but how these progenitors generate neurons is unknown. Here, we found that some Sox10+ neural crest-derived cells in, and surrounding, the nodose ganglion transiently expressed Phox2b, a master regulator of autonomic nervous system development, during early embryonic life. Our genetic lineage-tracing analysis in mice of either sex revealed that despite their common developmental origin and extreme spatial proximity, a substantial proportion of glial cells in the nodose, but not in the neighboring jugular ganglia, have a history of Phox2b expression. We used single-cell RNA-sequencing to demonstrate that these progenitors give rise to all major glial subtypes in the nodose ganglia, including Schwann cells, satellite glia, and glial precursors, and mapped their spatial distribution by in situ hybridization. Lastly, integration analysis revealed transcriptomic similarities between nodose and dorsal root ganglia glial subtypes and revealed immature nodose glial subtypes. Our work demonstrates that these crest-derived nodose glial progenitors transiently express Phox2b, give rise to the entire complement of nodose glial cells, and display a transcriptional program that may underlie their bipotent nature., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

A recent approach to promote central nervous system (CNS) regeneration after injury or disease is direct conversion of somatic cells to neurons. This is achieved by transduction of viral vectors that express neurogenic transcription factors. In this work we propose adult human mucosal olfactory ensheathing glia (hmOEG) as a candidate for direct reprogramming to neurons due to its accessibility and to its well-characterized neuroregenerative capacity. After induction of hmOEG with the single neurogenic transcription factor NEUROD1, the cells under study exhibited morphological and immunolabeling neuronal features, fired action potentials and expressed glutamatergic and GABAergic markers. In addition, after engraftment of transduced hmOEG cells in the mouse hippocampus, these cells showed specific neuronal labeling. Thereby, if we add to the neuroregenerative capacity of hmOEG cultures the conversion to neurons of a fraction of their population through reprogramming techniques, the engraftment of hmOEG and hmOEG-induced neurons could be a procedure to enhance neural repair after central nervous system injury., (© 2024. The Author(s).)

Glial cells are essential for the proper development and functioning of the peripheral nervous system (PNS). The ability to study the biology of glial cells is therefore critical for our ability to understand PNS biology and address PNS maladies. The genetic and proteomic pathways underlying vertebrate peripheral glial biology are understandably complex, with many layers of redundancy making it sometimes difficult to study certain facets of PNS biology. Fortunately, many aspects of vertebrate peripheral glial biology are conserved with those of the fruit fly, Drosophila melanogaster With simple and powerful genetic tools and fast generation times, Drosophila presents an accessible and versatile model for studying the biology of peripheral glia. We introduce here three techniques for studying the cell biology of peripheral glia of Drosophila third-instar larvae. With fine dissection tools and common laboratory reagents, third-instar larvae can be dissected, with extraneous tissues removed, revealing the central nervous system (CNS) and PNS to be processed using a standard immunolabeling protocol. To improve the resolution of peripheral nerves in the z -plane, we describe a cryosectioning method to achieve 10- to 20-µm thick coronal sections of whole larvae, which can then be immunolabeled using a modified version of standard immunolabeling techniques. Finally, we describe a proximity ligation assay (PLA) for detecting close proximity between two proteins-thus inferring protein interaction-in vivo in third-instar larvae. These methods, further described in our associated protocols, can be used to improve our understanding of Drosophila peripheral glia biology, and thus our understanding of PNS biology., (© 2024 Cold Spring Harbor Laboratory Press.)

The central nervous system (CNS) is a complicated neural network. The origin and evolution of functional neurons and glia cells remain unclear, as do the cellular alterations that occur during the course of cerebral disease rehabilitation. Lineage tracing is a valuable method for tracing specific cells and achieving a better understanding of the CNS. Recently, various technological breakthroughs have been made in lineage tracing, such as the application of various combinations of fluorescent reporters and advances in barcode technology. The development of lineage tracing has given us a deeper understanding of the normal physiology of the CNS, especially the pathological processes. In this review, we summarize these advances of lineage tracing and their applications in CNS. We focus on the use of lineage tracing techniques to elucidate the process CNS development and especially the mechanism of injury repair. Deep understanding of the central nervous system will help us to use existing technologies to diagnose and treat diseases., (© 2023. The Author(s).)

Here, we present our protocol to culture enteric glial cells from the submucosal and myenteric plexus of neonatal and juvenile pig colons. We describe steps for colon isolation, microdissection, and enzymatic and mechanical dissociation. We include procedures for passaging and analyzing cell yield, freeze/thaw efficiency, and purity. This protocol allows for the generation of primary cultures of enteric glial cells from single-cell suspensions of microdissected layers of the colon wall and can be used to culture enteric glia from human colon specimens. For complete details on the use and execution of this protocol, please refer to Ziegler et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Nervous systems of bilaterian animals generally consist of two cell types: neurons and glial cells. Despite accumulating data about the many important functions glial cells serve in bilaterian nervous systems, the evolutionary origin of this abundant cell type remains unclear. Current hypotheses regarding glial evolution are mostly based on data from model bilaterians. Non-bilaterian animals have been largely overlooked in glial studies and have been subjected only to morphological analysis. Here, we provide a comprehensive overview of conservation of the bilateral gliogenic genetic repertoire of non-bilaterian phyla (Cnidaria, Placozoa, Ctenophora, and Porifera). We overview molecular and functional features of bilaterian glial cell types and discuss their possible evolutionary history. We then examine which glial features are present in non-bilaterians. Of these, cnidarians show the highest degree of gliogenic program conservation and may therefore be crucial to answer questions about glial evolution., (© 2024. The Author(s).)

CNS injuries are associated with profound changes in cell organization. This protocol presents a stepwise approach to quantitatively describe the spatiotemporal changes in glial cell rearrangement in the injured murine brain, which is applicable to other biological contexts. Herein, we apply common immunolabeling of neurons and glial cells and wide-field microscopy imaging. Then, we employ computational tools for alignment to the Allen Brain Atlas, unbiased/automatic detection of cells, generation of point patterns, and data analysis. For complete details on the use and execution of this protocol, please refer to Manrique-Castano et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The brain is consisted of diverse neurons arising from a limited number of neural stem cells. Drosophila neural stem cells called neuroblasts (NBs) produces specific neural lineages of various lineage sizes depending on their location in the brain. In the Drosophila visual processing centre - the optic lobes (OLs), medulla NBs derived from the neuroepithelium (NE) give rise to neurons and glia cells of the medulla cortex. The timing and the mechanisms responsible for the cessation of medulla NBs are so far not known. In this study, we show that the termination of medulla NBs during early pupal development is determined by the exhaustion of the NE stem cell pool. Hence, altering NE-NB transition during larval neurogenesis disrupts the timely termination of medulla NBs. Medulla NBs terminate neurogenesis via a combination of apoptosis, terminal symmetric division via Prospero, and a switch to gliogenesis via Glial Cell Missing (Gcm); however, these processes occur independently of each other. We also show that temporal progression of the medulla NBs is mostly not required for their termination. As the Drosophila OL shares a similar mode of division with mammalian neurogenesis, understanding when and how these progenitors cease proliferation during development can have important implications for mammalian brain size determination and regulation of its overall function., Competing Interests: PN, LC No competing interests declared, (© 2024, Nguyen and Cheng.)

Neural precursor cells (NPCs) that persist in the postnatal/adult subventricular zone (SVZ) express connexins that form hemichannels and gap junctions. Gap junctional communication plays a role in NPC proliferation and differentiation during development, but its relevance on postnatal age remains to be elucidated. In this work we aimed to evaluate the effect of the blockade of gap junctional communication on proliferation and cell fate of NPCs obtained from the SVZ of postnatal rats. NPCs were isolated and expanded in culture as neurospheres. Electron microscopy revealed the existence of gap junctions among neurosphere cells. Treatment of cultures with octanol, a broad-spectrum gap junction blocker, or with Gap27, a specific blocker for gap junctions formed by connexin43, produced a significant decrease in bromodeoxyuridine incorporation. Octanol treatment also exerted a dose-dependent antiproliferative effect on glioblastoma cells. To analyze possible actions on NPC fate, cells were seeded in the absence of mitogens. Treatment with octanol led to an increase in the percentage of astrocytes and oligodendrocyte precursors, whereas the percentage of neurons remained unchanged. Gap27 treatment, in contrast, did not modify the differentiation pattern of SVZ NPCs. Our results indicate that general blockade of gap junctions with octanol induces significant effects on the behavior of postnatal SVZ NPCs, by reducing proliferation and promoting glial differentiation.

Astrocyte-secreted neurocan guides the formation of inhibitory circuits in the brain.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1 , Egfr, and Olig2 . The increased Ascl1 expression and appearance of Egfr + and Olig2 + cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2 , a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells., Competing Interests: Competing interests statement:The authors declare no competing interest.

Human glial progenitor cells (hGPCs) exhibit diminished expansion competence with age, as well as after recurrent demyelination. Using RNA-sequencing to compare the gene expression of fetal and adult hGPCs, we identify age-related changes in transcription consistent with the repression of genes enabling mitotic expansion, concurrent with the onset of aging-associated transcriptional programs. Adult hGPCs develop a repressive transcription factor network centered on MYC, and regulated by ZNF274, MAX, IKZF3, and E2F6. Individual over-expression of these factors in iPSC-derived hGPCs lead to a loss of proliferative gene expression and an induction of mitotic senescence, replicating the transcriptional changes incurred during glial aging. miRNA profiling identifies the appearance of an adult-selective miRNA signature, imposing further constraints on the expansion competence of aged GPCs. hGPC aging is thus associated with acquisition of a MYC-repressive environment, suggesting that suppression of these repressors of glial expansion may permit the rejuvenation of aged hGPCs., (© 2024. The Author(s).)

The Drosophila larval ventral nerve cord (VNC) shares many similarities with the spinal cord of vertebrates and has emerged as a major model for understanding the development and function of motor systems. Here, we use high-quality scRNA-seq, validated by anatomical identification, to create a comprehensive census of larval VNC cell types. We show that the neural lineages that comprise the adult VNC are already defined, but quiescent, at the larval stage. Using fluorescence-activated cell sorting (FACS)-enriched populations, we separate all motor neuron bundles and link individual neuron clusters to morphologically characterized known subtypes. We discovered a glutamate receptor subunit required for basal neurotransmission and homeostasis at the larval neuromuscular junction. We describe larval glia and endorse the general view that glia perform consistent activities throughout development. This census represents an extensive resource and a powerful platform for future discoveries of cellular and molecular mechanisms in repair, regeneration, plasticity, homeostasis, and behavioral coordination., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

The features of the participation of Smad3 in the functioning of neural stem cells (NSC), neuronal committed precursors (NCP), and neuroglial elements were studied in vitro. It was found that this intracellular signaling molecule enhances the clonogenic and proliferative activities of NCP and inhibits specialization of neuronal precursors. At the same time, Smad3 does not participate in the realization of the growth potential of NSC. With regard to the secretory function (production of neurotrophic growth factors) of neuroglial cells, the stimulating role of Smad3-mediated signaling was shown. These results indicate the promise of studying the possibility of using Smad3 as a fundamentally new target for neuroregenerative agents., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Competition among adult brain cells has not been extensively researched. To investigate whether healthy glia can outcompete diseased human glia in the adult forebrain, we engrafted wild-type (WT) human glial progenitor cells (hGPCs) produced from human embryonic stem cells into the striata of adult mice that had been neonatally chimerized with mutant Huntingtin (mHTT)-expressing hGPCs. The WT hGPCs outcompeted and ultimately eliminated their human Huntington's disease (HD) counterparts, repopulating the host striata with healthy glia. Single-cell RNA sequencing revealed that WT hGPCs acquired a YAP1/MYC/E2F-defined dominant competitor phenotype upon interaction with the host HD glia. WT hGPCs also outcompeted older resident isogenic WT cells that had been transplanted neonatally, suggesting that competitive success depended primarily on the relative ages of competing populations, rather than on the presence of mHTT. These data indicate that aged and diseased human glia may be broadly replaced in adult brain by younger healthy hGPCs, suggesting a therapeutic strategy for the replacement of aged and diseased human glia., (© 2023. The Author(s).)

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely., Competing Interests: Declaration of interests L.S. is a scientific cofounder and paid consultant of BlueRock Therapeutics Inc. L.S. is a scientific cofounder of DaCapo Brainscience., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies 1 , which involve human-specific mechanisms 2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors 6 . Single-cell transcriptomics and comparison to independent neural stem cells 7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids 8 . Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity., (© 2024. The Author(s).)

Introduction: Ocular diseases pose a significant health concern for donkeys. However, studies examining the microanatomy and cell populations of the donkey retina are scarce. The current study aimed to describe the vascular pattern of the donkey retina and document its cellular components., Methods: The donkey retina specimens were obtained from different retinal regions and prepared for semithin sectioning and immunohistochemistry., Results: The donkey has a paurangiotic retina in which retinal vessels are confined to a narrow area around the optic disc. Glial cells coexist with the blood vessels being very numerous in the vascular region and become scanty in the avascular ones. S-100-positive astrocytes could be observed in these avascular areas. Ganglion cells are organized in a single layer with the least population existing in the peripheral retina. Acidic fibroblast growth factor (AFGF) is immunoreactive in amacrine and ganglion cells. A subpopulation of amacrine cells reacted strongly to tyrosine hydroxylase (TH), and others reacted positively to S-100 protein. Ganglion cell nuclei exhibited a strong immunoreactivity to S-100 protein as well. Furthermore, glial fibrillary acidic protein (GFAP) is used to identify Müller cells that extend their processes across the retina from the inner to the outer limiting membrane., Conclusions: In conclusion, our findings provide novel insights into the normal retinal organization. The donkey retina shows the characteristic expression of immunohistochemical markers for the major cell types. In addition, the distribution of glial cells is comparable between the vascular and avascular regions., (© 2024 S. Karger AG, Basel.)

Mosaic Analysis with Double Markers (MADM) is a powerful genetic method typically used for lineage tracing and to disentangle cell autonomous and tissue-wide roles of candidate genes with single cell resolution. Given the relatively sparse labeling, depending on which of the 19 MADM chromosomes one chooses, the MADM approach represents the perfect opportunity for cell morphology analysis. Various MADM studies include reports of morphological anomalies and phenotypes in the central nervous system (CNS). MADM for any candidate gene can easily incorporate morphological analysis within the experimental workflow. Here, we describe the methods of morphological cell analysis which we developed in the course of diverse recent MADM studies. This chapter will specifically focus on methods to quantify aspects of the morphology of neurons and astrocytes within the CNS, but these methods can broadly be applied to any MADM-labeled cells throughout the entire organism. We will cover two analyses-soma volume and dendrite characterization-of physical characteristics of pyramidal neurons in the somatosensory cortex, and two analyses-volume and Sholl analysis-of astrocyte morphology., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The proliferative and neurogenic potential of retinal Müller glia after injury varies widely across species. To identify the endogenous mechanisms regulating the proliferative response of mammalian Müller glia, we comparatively analyzed the expression and function of nestin, an intermediate filament protein established as a neural stem cell marker, in the mouse and rat retinas after injury., Methods: Nestin expression in the retinas of C57BL/6 mice and Wistar rats after methyl methanesulfonate (MMS)-induced photoreceptor injury was examined by immunofluorescence and Western blotting. Adeno-associated virus (AAV)-delivered control and nestin short hairpin RNA (shRNA) were intravitreally injected to rats and Müller glia proliferation after MMS-induced injury was analyzed by BrdU incorporation and immunofluorescence. Photoreceptor removal and microglia/macrophage infiltration were also analyzed by immunofluorescence., Results: Rat Müller glia re-entered the cell cycle and robustly upregulated nestin after injury whereas Müller glia proliferation and nestin upregulation were not observed in mice. In vivo knockdown of nestin in the rat retinas inhibited Müller glia proliferation while transiently stimulating microglia/macrophage infiltration and phagocytic removal of dead photoreceptors., Conclusions: Our findings suggest a critical role for nestin in the regulation of Müller glia proliferation after retinal injury and highlight the importance of cross species analysis to identify the molecular mechanisms regulating the injury responses of the mammalian retina.

The study of psychiatric and neurological diseases requires the substrate in which the disorders occur, that is, the nervous tissue. Currently, several types of human bio-specimens are being used for research, including postmortem brains, cerebrospinal fluid, induced pluripotent stem (iPS) cells, and induced neuronal (iN) cells. However, these samples are far from providing a useful predictive, diagnostic, or prognostic biomarker. The olfactory epithelium is a region close to the brain that has received increased interest as a research tool for the study of brain mechanisms in complex neuropsychiatric and neurological diseases. The olfactory sensory neurons are replaced by neurogenesis throughout adult life from stem cells on the basement membrane. These stem cells are multipotent and can be propagated in neurospheres, proliferated in vitro and differentiated into multiple cell types including neurons and glia. For all these reasons, olfactory epithelium provides a unique resource for investigating neuronal molecular markers of neuropsychiatric and neurological diseases. Here, we describe the isolation and culture of human differentiated neurons and glial cells from olfactory epithelium of living subjects by an easy and non-invasive exfoliation method that may serve as a useful tool for the research in brain diseases., (© 2023. The Author(s).)

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system 1 . The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG + and IgA + plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs., (© 2023. The Author(s).)

Photoreceptors in the vertebrate eye are dependent on the retinal pigmented epithelium for a variety of functions including retinal re-isomerization and waste disposal. The light-sensitive pineal gland of fish, birds, and amphibians is evolutionarily related to the eye but lacks a pigmented epithelium. Thus, it is unclear how these functions are performed. Here, we ask whether a subpopulation of zebrafish pineal cells, which express glial markers and visual cycle genes, is involved in maintaining photoreceptors. Selective ablation of these cells leads to a loss of pineal photoreceptors. Moreover, these cells internalize exorhodopsin that is secreted by pineal rod-like photoreceptors, and in turn release CD63-positive extracellular vesicles (EVs) that are taken up by pdgfrb-positive phagocytic cells in the forebrain meninges. These results identify a subpopulation of glial cells that is critical for pineal photoreceptor survival and indicate the existence of cells in the forebrain meninges that receive EVs released by these pineal cells and potentially function in waste disposal., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to simultaneously assess transcription and chromatin accessibility in EGCs undergoing spontaneous neurogenesis in culture, as well as small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. A subset of in vivo EGCs, highly enriched within the myenteric ganglia and that persist into adulthood, have a gene expression program and chromatin state consistent with neurogenic potential. These results clarify the mechanisms underlying EGC potential for neuronal fate transition., Competing Interests: Declaration of interests A.M.G. receives research funds from Takeda Pharmaceutical Company., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ability of Timm's sulphide silver method to stain zincergic terminal fields has made it a useful neuromorphological marker. Beyond its roles in zinc-signalling and neuromodulation, zinc is involved in the pathophysiology of ischemic stroke, epilepsy, degenerative diseases and neuropsychiatric conditions. In addition to visualising zincergic terminal fields, the method also labels transition metals in neuronal perikarya and glial cells. To provide a benchmark reference for planning and interpretation of experimental investigations of zinc-related phenomena in rat brains, we have established a comprehensive repository of serial microscopic images from a historical collection of coronally, horizontally and sagittally oriented rat brain sections stained with Timm's method. Adjacent Nissl-stained sections showing cytoarchitecture, and customised atlas overlays from a three-dimensional rat brain reference atlas registered to each section image are included for spatial reference and guiding identification of anatomical boundaries. The Timm-Nissl atlas, available from EBRAINS, enables experimental researchers to navigate normal rat brain material in three planes and investigate the spatial distribution and density of zincergic terminal fields across the entire brain., (© 2023. The Author(s).)

Lipids comprise an extremely heterogeneous group of compounds that perform a wide variety of biological functions. Traditional view of lipids as important structural components of the cell and compounds playing a trophic role is currently being supplemented by information on the possible participation of lipids in signaling, not only intracellular, but also intercellular. The review article discusses current data on the role of lipids and their metabolites formed in glial cells (astrocytes, oligodendrocytes, microglia) in communication of these cells with neurons. In addition to metabolic transformations of lipids in each type of glial cells, special attention is paid to the lipid signal molecules (phosphatidic acid, arachidonic acid and its metabolites, cholesterol, etc.) and the possibility of their participation in realization of synaptic plasticity, as well as in other possible mechanisms associated with neuroplasticity. All these new data can significantly expand our knowledge about the regulatory functions of lipids in neuroglial relationships.

Zika virus (ZIKV) is an arbovirus member of the Flaviviridae family that causes severe congenital brain anomalies in infected fetuses. The key target cells of ZIKV infection, human neural progenitor cells (hNPCs), are highly permissive to infection that causes the inhibition of cell proliferation and induces cell death. We have previously shown that pharmaceutical-grade heparin inhibits virus-induced cell death with negligible effects on in vitro virus replication in ZIKV-infected hNPCs at the "high" multiplicity of infection (MOI) of 1. Here, we show that heparin inhibits formation of ZIKV-induced intracellular vacuoles, a signature of paraptosis, and inhibits necrosis and apoptosis of hNPCs grown as neurospheres (NS). To test whether heparin preserved the differentiation of ZIKV-infected hNPCs into neuroglial cells, hNPCs were infected at the MOI of 0.001. In this experimental condition, heparin inhibited ZIKV replication by ca. 2 log 10 , mostly interfering with virion attachment, while maintaining its protective effect against ZIKV-induced cytopathicity. Heparin preserved differentiation into neuroglial cells of hNPCs that were obtained from either human-induced pluripotent stem cells (hiPSC) or by fetal tissue. Quite surprisingly, multiple additions of heparin to hNPCs enabled prolonged virus replication while preventing virus-induced cytopathicity. Collectively, these results highlight the potential neuroprotective effect of heparin that could serve as a lead compound to develop novel agents for preventing the damage of ZIKV infection on the developing brain. IMPORTANCE ZIKV is a neurotropic virus that invades neural progenitor cells (NPCs), causing inhibition of their proliferation and maturation into neurons and glial cells. We have shown previously that heparin, an anticoagulant also used widely during pregnancy, prevents ZIKV-induced cell death with negligible inhibition of virus replication. Here, we demonstrate that heparin also exerts antiviral activity against ZIKV replication using a much lower infectious inoculum. Moreover, heparin interferes with different modalities of virus-induced cell death. Finally, heparin-induced prevention of virus-induced NPC death allows their differentiation into neuroglial cells despite the intracellular accumulation of virions. These results highlight the potential use of heparin, or pharmacological agents derived from it, in pregnant women to prevent the devastating effects of ZIKV infection on the developing brain of their fetuses.

During cortical development, the balance between progenitor self-renewal and neurogenesis is critical for determining the size/morphology of the cortex. A fundamental feature of the developing cortex is an increase in the length of G1 phase in RGCs over the course of neurogenesis, which is a key determinant of progenitor fate choice. How the G1 length is temporally regulated remains unclear. Here, Pdk1, a member of the AGC kinase family, was conditionally disrupted by crossing an Emx1-Cre mouse line with a Pdk1fl/fl line. The loss of Pdk1 led to a shorter cell cycle accompanied by increased RGC proliferation specifically at late rather than early/middle neurogenic stages, which was attributed to impaired lengthening of G1 phase. Coincidently, apical-to-basal interkinetic nuclear migration was accelerated in Pdk1 cKO cortices. Consequently, we detected an increased neuronal output at P0. We further showed the significant upregulation of the cell cycle regulator cyclin D1 and its activator Myc in the cKO cortices relative to those of control animals. Overall, we have identified a novel role for PDK1 in cortical neurogenesis. PDK1 functions as an upstream regulator of the Myc-cyclin D1 pathway to control the lengthening of G1 phase and the balance between RGC proliferation and differentiation., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The purpose of this study was to investigate Müller cells during the fetal development of the human eye. Müller cells in eyes of 39 human fetuses (11-38 weeks of gestation, WOG) and 6 infants (5 died of abusive head trauma, AHT, aged 1-9 months) were immunohistochemically stained and investigated for spatial and temporal immunoreaction of nestin, CD44, collagen IX and GFAP, which are stem cell markers or markers of intermediate filaments, respectively, in one of the hitherto largest cohorts of fetal eyes. Müller cells could be detected immunohistochemically as early as 12 WOG by immunohistochemical staining with nestin. Nestin was more strongly expressed in Müller cells of the peripheral retina and a centroperipheral gradient of immunoreaction over time was observed. CD44 was predominantly expressed in fetal eyes of the late second and early third trimester between (23 and 27 WOG) and significantly stronger in the infant eyes. Collagen IX labeling in the central retina was significantly stronger than in more peripheral sectors and increased with fetal age. GFAP staining in Müller cells was seen in the eye of a fetus of 38 WOG who died postnatally and in the infant eyes with and without history of AHT. Additionally, GFAP staining was present in the astrocytes of fetal and infant eyes. All examined markers were expressed by Müller cells at different developmental stages highlighting the plasticity of Müller cells during the development of the human eye. GFAP should be cautiously used as a marker for AHT as it was also expressed in fetal astrocytes and Müller cells in eyes without history of AHT., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuronal disorder characterized by neuronal degeneration and currently no effective cure is available to stop or delay the disease from progression. Transplantation of murine glial-restricted precursors (mGRPs) is an attractive strategy to modulate ALS development and advancements such as the use of immune modulators could potentially extend graft survival and function. Using a well-established ALS transgenic mouse model (SOD1 G93A ), we tested mGRPs in combination with the immune modulators synthetic PreImplantation Factor (sPIF), Tacrolimus (Tac), and Costimulatory Blockade (CB). We report that transplantation of mGRPs into the cisterna magna did not result in increased mice survival. The addition of immunomodulatory regimes again did not increase mice lifespan but improved motor functions and sPIF was superior compared to other immune modulators. Immune modulators did not affect mGRPs engraftment significantly but reduced pro-inflammatory cytokine production. Finally, sPIF and CB reduced the number of microglial cells and prevented neuronal number loss. Given the safety profile and a neuroprotective potential of sPIF, we envision its clinical application in near future., (© 2022. The Author(s).)

Amyotrophic lateral sclerosis (ALS) is an incurable disease characterized by proteinaceous aggregate accumulation and neuroinflammation culminating in rapidly progressive lower and upper motor neuron death. To interrogate cell-intrinsic and inter-cell type perturbations in ALS, single-nucleus RNA sequencing was performed on the lumbar spinal cord in the murine ALS model SOD1 G93A transgenic and littermate control mice at peri-symptomatic onset stage of disease, age 90 days. This work uncovered perturbed tripartite synapse functions, complement activation and metabolic stress in the affected spinal cord; processes evidenced by cell death and proteolytic stress-associated gene sets. Concomitantly, these pro-damage events in the spinal cord co-existed with dysregulated reparative mechanisms. This work provides a resource of cell-specific niches in the ALS spinal cord and asserts that interwoven dysfunctional neuronal-glial communications mediating neurodegeneration are underway prior to overt disease manifestation and are recapitulated, in part, in the human post-mortem ALS spinal cord., (© 2022. The Author(s).)

Cellular identity is established through complex layers of genetic regulation, forged over a developmental lifetime. An expanding molecular toolbox is allowing us to manipulate these gene regulatory networks in specific cell types in vivo. In principle, if we found the right molecular tricks, we could rewrite cell identity and harness the rich repertoire of possible cellular functions and attributes. Recent work suggests that this rewriting of cell identity is not only possible, but that newly induced cells can mitigate disease phenotypes in animal models of major human diseases. So, is the sky the limit, or do we need to keep our feet on the ground? This Spotlight synthesises key concepts emerging from recent efforts to reprogramme cellular identity in vivo. We provide our perspectives on recent controversies in the field of glia-to-neuron reprogramming and identify important gaps in our understanding that present barriers to progress., (© 2022. Published by The Company of Biologists Ltd.)

Activation of glial cells and neuroinflammation play an important role in the onset and development of Alzheimer's disease (AD). Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor in the brain that is involved in regulating neuroinflammation. However, the precise effects of TREM2 on neuroinflammatory responses and its underlying molecular mechanisms in AD have not been studied in detail. Here, we employed a lentiviral-mediated strategy to downregulation of TREM2 expression on microglia in the brain of APPswe/PS1dE9 (APP/PS1) transgenic mice and BV2 cells. Our results showed that downregulation of TREM2 significantly aggravated AD-related neuropathology including Aβ accumulation, peri-plaque microgliosis and astrocytosis, as well as neuronal and synapse-associated proteins loss, which was accompanied by a decline in cognitive ability. The further mechanistic study revealed that downregulation of TREM2 expression initiated neuroinflammatory responses through toll-like receptor 4 (TLR4)-mediated mitogen-activated protein kinase (MAPK) signaling pathway and subsequent stimulating the production of pro-inflammatory cytokines in vivo and in vitro. Moreover, blockade of p38, JNK, and ERK1/2 inhibited the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) induced by Aβ 1-42 in TREM2-knocked down BV2 cells. Taken together, these findings indicated that TREM2 might be a potential therapeutic target for AD and other neuroinflammation-related diseases., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Axolotls are an important model organism for multiple types of regeneration, including functional spinal cord regeneration. Remarkably, axolotls can repair their spinal cord after a small lesion injury and can also regenerate their entire tail following amputation. Several classical signaling pathways that are used during development are reactivated during regeneration, but how this is regulated remains a mystery. We have previously identified miR-200a as a key factor that promotes successful spinal cord regeneration. Here, using RNA-seq analysis, we discovered that the inhibition of miR-200a results in an upregulation of the classical mesodermal marker brachyury in spinal cord cells after injury. However, these cells still express the neural stem cell marker sox2. In vivo cell tracking allowed us to determine that these cells can give rise to cells of both the neural and mesoderm lineage. Additionally, we found that miR-200a can directly regulate brachyury via a seed sequence in the 3'UTR of the gene. Our data indicate that miR-200a represses mesodermal cell fate after a small lesion injury in the spinal cord when only glial cells and neurons need to be replaced., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The biological basis of male-female brain differences has been difficult to elucidate in humans. The most notable morphological difference is size, with male individuals having on average a larger brain than female individuals 1,2 , but a mechanistic understanding of how this difference arises remains unknown. Here we use brain organoids 3 to show that although sex chromosomal complement has no observable effect on neurogenesis, sex steroids-namely androgens-lead to increased proliferation of cortical progenitors and an increased neurogenic pool. Transcriptomic analysis and functional studies demonstrate downstream effects on histone deacetylase activity and the mTOR pathway. Finally, we show that androgens specifically increase the neurogenic output of excitatory neuronal progenitors, whereas inhibitory neuronal progenitors are not increased. These findings reveal a role for androgens in regulating the number of excitatory neurons and represent a step towards understanding the origin of sex-related brain differences in humans., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Diabetes is a risk factor for Alzheimer's disease (AD), a chronic neurodegenerative disease. We and others have shown prediabetes, including hyperglycemia and obesity induced by high fat and high sucrose diets, is associated with exacerbated amyloid beta (Aβ) accumulation and cognitive impairment in AD transgenic mice. However, whether hyperglycemia reduce glial clearance of oligomeric amyloid-β (oAβ), the most neurotoxic Aβ aggregate, remains unclear. Mixed glial cultures simulating the coexistence of astrocytes and microglia in the neural microenvironment were established to investigate glial clearance of oAβ under normoglycemia and chronic hyperglycemia. Ramified microglia and low IL-1β release were observed in mixed glia cultures. In contrast, amoeboid-like microglia and higher IL-1β release were observed in primary microglia cultures. APPswe/PS1dE9 transgenic mice are a commonly used AD mouse model. Microglia close to senile plaques in APPswe/PS1dE9 transgenic mice exposed to normoglycemia or chronic hyperglycemia exhibited an amoeboid-like morphology; other microglia were ramified. Therefore, mixed glia cultures reproduce the in vivo ramified microglial morphology. To investigate the impact of sustained high-glucose conditions on glial oAβ clearance, mixed glia were cultured in media containing 5.5 mM glucose (normal glucose, NG) or 25 mM glucose (high glucose, HG) for 16 days. Compared to NG, HG reduced the steady-state level of oAβ puncta internalized by microglia and astrocytes and decreased oAβ degradation kinetics. Furthermore, the lysosomal acidification and lysosomal hydrolysis activity of microglia and astrocytes were lower in HG with and without oAβ treatment than NG. Moreover, HG reduced mitochondrial membrane potential and ATP levels in mixed glia, which can lead to reduced lysosomal function. Overall, continuous high glucose reduces microglial and astrocytic ATP production and lysosome activity which may lead to decreased glial oAβ degradation. Our study reveals diabetes-induced hyperglycemia hinders glial oAβ clearance and contributes to oAβ accumulation in AD pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

Estrogens are well-known to regulate development of sexual dimorphism of the brain; however, their role in embryonic brain development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs from early neurogenesis specifically in a type of glia in the olfactory bulb (OB), which we name estrogen-responsive olfactory bulb (EROB) cells. In response to estrogen, EROB cells overlay the outermost layer of the OB and interact tightly with olfactory sensory neurons at the olfactory glomeruli. Inhibiting estrogen activity using an estrogen receptor antagonist, ICI182,780 (ICI), and/or EROB cell ablation impedes olfactory glomerular development, including the topological organisation of olfactory glomeruli and inhibitory synaptogenesis in the OB. Furthermore, activation of estrogen signalling inhibits both intrinsic and olfaction-dependent neuronal activity in the OB, whereas ICI or EROB cell ablation results in the opposite effect on neuronal excitability. Altering the estrogen signalling disrupts olfaction-mediated behaviour in later larval stage. We propose that estrogens act on glia to regulate development of OB circuits, thereby modulating the local excitability in the OB and olfaction-mediated behaviour., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Characterizing the molecular signature of a cell subtype leads to a better understanding of cell diversity, as this molecular data can identify new cellular markers and offer insights about cell function. Here, we describe an efficient protocol to separate a subtype of astrocytes, the Olig2-AS, from other glial cells by using a double reporter mouse approach and to determine the transcriptome profile of the Olig2-AS from the postnatal spinal cord using RNA-sequencing analysis. For complete details on the use and execution of this protocol, please refer to Ohayon et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Larvicide pyriproxyfen (PPF), used in drinking water reservoirs to control Aedes mosquitoes, has already been shown as a possible cause of congenital anomalies in the central nervous system. However, the neurotoxic effects of PPF on the development of vertebrate embryos are still underexplored. Thus, the aim of this study was to investigate the effects of PPF on the morphometric parameters of the head and brain, as well as on the cell layers of the forebrain and midbrain, using embryos of Gallus domesticus as a model. Two sublethal PPF concentrations (0.01 mg/L and 10 mg/L), as defined by a survival curve, were tested. Analysis of the biometry of embryos showed significant reduction in body and brain mass and also in measurements of the head and brain. A reduction in cell layer thickness of the forebrain and midbrain was observed, accompanied by a reduction in the numerical density of cells per area. Changes in brain and head sizes and in the thickness of the cell layers of the forebrain and midbrain were significant at 10 mg/L PPF. Notably, PPF caused DNA doublestrand breaks and induced apoptosis in embryos exposed to 10 mg/L, which were accompanied by a reduction in cell proliferation. Regarding neuronal and glial differentiation, no changes were observed in the number of neurons and glial cells on the analyzed layers. Furthermore, PPF did not impact the head ossification process. These findings reveal that PPF is a strong stressor for neurodevelopment, causing damage to the cell architecture of brain vesicles., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Glial cells are essential for functionality of the nervous system. Growing evidence underscores the importance of astrocytes; however, analogous astroglia in peripheral organs are poorly understood. Using confocal time-lapse imaging, fate mapping, and mutant genesis in a zebrafish model, we identify a neural crest-derived glial cell, termed nexus glia, which utilizes Meteorin signaling via Jak/Stat3 to drive differentiation and regulate heart rate and rhythm. Nexus glia are labeled with gfap, glast, and glutamine synthetase, markers that typically denote astroglia cells. Further, analysis of single-cell sequencing datasets of human and murine hearts across ages reveals astrocyte-like cells, which we confirm through a multispecies approach. We show that cardiac nexus glia at the outflow tract are critical regulators of both the sympathetic and parasympathetic system. These data establish the crucial role of glia on cardiac homeostasis and provide a description of nexus glia in the PNS., Competing Interests: The authors have declared that no competing interests exist.

Uncovering the architecture of white matter axons is fundamental to the study of brain networks. We developed a method for quantifying axonal orientations at a resolution of ~15 micrometers. This method is based on the common Nissl staining technique for postmortem histological slices. Nissl staining reveals the spatial organization of glial cells along axons. Using structure tensor analysis, we leveraged this patterned organization to uncover local axonal orientation. We used Nissl-based structure tensor analysis to extract fine details of axonal architecture and demonstrated its applicability in multiple datasets of humans and nonhuman primates. Nissl-based structure tensor analysis can be used to compare fine-grained features of axonal architecture across species and is widely applicable to existing datasets.