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Bupivacaine, a local anaesthetic, is widely applied in the epidural or subarachnoid space to clinically manage acute and chronic pain. However, the underlying mechanisms are complex and unclear. Glycine transporter 1 (GlyT1) in the spinal cord plays a critical role in various pathologic pain conditions. Therefore, we sought to determine whether bupivacaine exerts its analgesic effect by regulating GlyT1 expression and to determine the underlying mechanisms of regulation. Primary astrocytes prepared from the spinal cord of rats were treated with bupivacaine. The protein levels of GlyT1, brain-derived neurotrophic factor (BDNF) and phosphorylated adenosine 5'-monophosphate (AMP)-activated protein kinase α (p-AMPKα) were measured by western blotting or immunofluorescence. In addition, 7,8-dihydroxyflavone (7,8-DHF, BDNF receptor agonist) and AMPK shRNA were applied to verify the relationship between the regulation of GlyT1 by bupivacaine and the p-AMPKα/BDNF signalling pathway. After treatment with bupivacaine, GlyT1 expression was diminished in a concentration-dependent manner, while the expression of BDNF and p-AMPK was increased. Moreover, 7,8-DHF decreased GlyT1 expression, and AMPK knockdown suppressed the upregulation of BDNF expression by bupivacaine. Finally, we concluded that bupivacaine reduced GlyT1 expression in spinal astrocytes by activating the p-AMPKα/BDNF signalling pathway. These results provide a new mechanism for the analgesic effect of intrathecal bupivacaine in the treatment of acute and chronic pain., (© 2022. The Author(s).)

Gamma-aminobutyric acid (GABA) and glycine act as inhibitory neurotransmitters. Three types of inhibitory neurons and terminals, GABAergic, GABA/glycine coreleasing, and glycinergic, are orchestrated in the spinal cord neural circuits and play critical roles in regulating pain, locomotive movement, and respiratory rhythms. In this study, we first describe GABAergic and glycinergic transmission and inhibitory networks, consisting of three types of terminals in the mature mouse spinal cord. Second, we describe the developmental formation of GABAergic and glycinergic networks, with a specific focus on the differentiation of neurons, formation of synapses, maturation of removal systems, and changes in their action. GABAergic and glycinergic neurons are derived from the same domains of the ventricular zone. Initially, GABAergic neurons are differentiated, and their axons form synapses. Some of these neurons remain GABAergic in lamina I and II. Many GABAergic neurons convert to a coreleasing state. The coreleasing neurons and terminals remain in the dorsal horn, whereas many ultimately become glycinergic in the ventral horn. During the development of terminals and the transformation from radial glia to astrocytes, GABA and glycine receptor subunit compositions markedly change, removal systems mature, and GABAergic and glycinergic action shifts from excitatory to inhibitory.

Aims: Astrocytes re-acquire stem cell potential upon inflammation, thereby becoming a promising source of cells for regenerative medicine. Nanog is an essential transcription factor to maintain the characteristics of stem cells. We aimed to investigate the role of Nanog in astrocyte dedifferentiation., Main Methods: TNF-α was used to induce the dedifferentiation of primary rat spinal cord astrocytes. The expression of immature markers CD44 and Musashi-1 was detected by qRT-PCR and immunofluorescence. The Nanog gene is knocked down by small interference RNA. Nanog expression was measured by qRT-PCR and western blotting. BAY 11-7082 was used to suppress NF-κB signals in astrocytes. NF-κB signaling was evaluated by Western blotting., Key Findings: Our results showed that TNF-α promoted the re-expression of CD44 and Musashi-1 in astrocytes. Dedifferentiated astrocytes could be induced to differentiate into oligodendrocyte lineage cells indicating that the astrocytes had pluripotency. In addition, TNF-α treatment activated NF-κB signaling pathway and up-regulated Nanog. Knockdown of Nanog reversed the increase of CD44 and Musashi-1 induced by TNF-α without affecting the activation of NF-κB signaling. Importantly, blocking NF-κB signaling by BAY 11-7082 inhibited the expression of immature markers suggesting that TNF-α induces dedifferentiation of astrocytes through the NF-κB signaling pathway. BAY 11-7082 could also inhibit the expression of Nanog, which indicated that Nanog was regulated by NF-κB signaling pathway., Significance: These findings indicate that activation of the NF-κB signaling pathway through TNF-α leads to astrocytes dedifferentiation via Nanog. These results expand our understanding of the mechanism of astrocytes dedifferentiation., (Copyright © 2021. Published by Elsevier Inc.)

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).)

Botulinum neurotoxins (BoNTs) are notorious toxins and powerful agents and can be lethal, causing botulism, but they are also widely used as therapeutics, particularly to treat neuromuscular disorders. As of today, the commercial BoNT treatments available are from native A or B serotypes. Serotype F has shown efficacy in a clinical trial but has scarcely been used, most likely due to its medium duration of effect. Previously, the uniqueness of the light chain of the F7 subtype was identified and reported, showing an extended interaction with its substrates, VAMPs 1, 2 and 3, and a superior catalytic activity compared to other BoNT/F subtypes. In order to more extensively study the properties of this neurotoxin, we engineered a modified F7 chimera, mrBoNT/F7-1, in which all the regions of the neurotoxin were identical to BoNT/F7 except the activation loop, which was the activation loop from BoNT/F1. Use of the activation loop from BoNT/F1 allowed easier post-translational proteolytic activation of the recombinant protein without otherwise affecting its properties. mrBoNT/F7-1 was expressed, purified and then tested in a suite of in vitro and in vivo assays. mrBoNT/F7-1 was active and showed enhanced potency in comparison to both native and recombinant BoNT/F1. Additionally, the safety profile remained comparable to BoNT/F1 despite the increased potency. This new modified recombinant toxin F7 could be further exploited to develop unique therapeutics to address unmet medical needs.

Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na + and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na + -permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca 2+ release and turned off by Ca 2+ reuptake through the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output., (© 2021. The Author(s).)

Neurogenesis in adult mammals occurs mainly in the subventricular and subgranular areas of the brain, but there are also reports of its occurrence in the spinal cord. In a study on rats, neural stem cells and neuroprogenitor cells could be obtained through primary spinal cord culture, but there are no studies on these cells in canine species, to date. Dogs represent an appropriate animal model for studies on neurogenesis and neurological disorders. In addition, they are animals of great affective value, and the therapeutic use of neural stem cells can represent a breakthrough in regenerative veterinary medicine. Therefore, this study aimed to determine a protocol for the isolation, culture, and characterization of neural and neuroprogenitor stem cells derived from the spinal cord of canine fetuses. The cells were isolated from spinal cord fragments and cultured in serum-free culture medium supplemented with EGF and FGF-2 growth factors. These cells were observed daily by optical microscopy to analyze their morphological characteristics. From the third day in vitro, it was possible to observe translucent cell groupings, similar to the neurospheres, which approximately ranged from 50 µm to 200 µm at seven days in vitro. Throughout the culture period, the neurospheres developed ribbons in their periphery that migrated and communicated with other neurospheres. RT-PCR revealed that the cells expressed the characteristic genes SOX2, NESTIN, and GFAP. In addition to gene expression, the cells were phenotypically marked in the immunofluorescence assay for the proteins Nestin, GFAP, and β-tubulin III, characterizing them as neurospheres. Our results suggest that the spinal cord may be a source of neural stem cells and neural progenitor cells in canine fetuses. These cells may be an interesting option for neurogenesis and neuroregenerative therapy studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Spatial analysis of spinal neurons is currently limited by a lack of tools for efficient preparation and imaging of the whole spinal cord (SC) and the absence of a 3D reference atlas. Here, we describe protocols for efficient sectioning of whole SC using SpineRacks and subsequent image registration, atlas mapping, and 3D analysis of cells and projections, using SpinalJ. Together, these tools enable high-throughput analyses of adult mouse SC and direct comparison of spatial information of neurons between animals and studies. For complete details on the use and execution of this protocol, please refer to Fiederling et al. (2021)., Competing Interests: The authors declare that they have no competing interests., (© 2021 The Author(s).)

The molecular mechanisms that produce the full array of neuronal subtypes in the vertebrate nervous system are incompletely understood. Here, we provide evidence of a global temporal patterning program comprising sets of transcription factors that stratifies neurons based on the developmental time at which they are generated. This transcriptional code acts throughout the central nervous system, in parallel to spatial patterning, thereby increasing the diversity of neurons generated along the neuraxis. We further demonstrate that this temporal program operates in stem cell-derived neurons and is under the control of the TGFβ signaling pathway. Targeted perturbation of components of the temporal program, Nfia and Nfib, reveals their functional requirement for the generation of late-born neuronal subtypes. Together, our results provide evidence for the existence of a previously unappreciated global temporal transcriptional program of neuronal subtype identity and suggest that the integration of spatial and temporal patterning mechanisms diversifies and organizes neuronal subtypes in the vertebrate nervous system., Competing Interests: The authors have declared that no competing interests exist.

Interferons (IFNs) are cytokines that possess antiviral, antiproliferative, and immunomodulatory actions. IFN-α and IFN-β are two major family members of type-I IFNs and are used to treat diseases, including hepatitis and multiple sclerosis. Emerging evidence suggests that type-I IFN receptors (IFNARs) are also expressed by microglia, astrocytes, and neurons in the central and peripheral nervous systems. Apart from canonical transcriptional regulations, IFN-α and IFN-β can rapidly suppress neuronal activity and synaptic transmission via non-genomic regulation, leading to potent analgesia. IFN-γ is the only member of the type-II IFN family and induces central sensitization and microglia activation in persistent pain. We discuss how type-I and type-II IFNs regulate pain and infection via neuro-immune modulations, with special focus on neuroinflammation and neuro-glial interactions. We also highlight distinct roles of type-I IFNs in the peripheral and central nervous system. Insights into IFN signaling in nociceptors and their distinct actions in physiological vs. pathological and acute vs. chronic conditions will improve our treatments of pain after surgeries, traumas, and infections., Competing Interests: R-RJ is a consultant of Boston Scientific and received a research grant from the company. This activity is not related to this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tan, Ji, Yeh and Ji.)

Background: Spinal cord injury (SCI) is characterized by autonomic dysreflexia, chronic pain, sensory and motor deficits. Resveratrol has shown potential neuroprotective function in several neurodegenerative diseases' models. However, if resveratrol could improve the function recovery after SCI and the further mechanism have not been investigated., Methods: SCI rat model was established through laminectomy at lamina T9-10 aseptically. Basso, beattie and bresnahan (BBB) and inclined plane score, sensory recovery, spinal cord content, and inflammatory factors were measured. The levels of GAP43, NF421, GFAP, Bax, Bcl-2 and caspase-3 were measured using immunohistochemical staining. Tunel staining was applied to detect apoptosis level., Results: Resveratrol significantly improved the function recovery, promoted axonal regeneration, suppressed apoptosis after SCI. The activation of Wnt/β-catenin signaling pathway was achieved by resveratrol. XAV939 significantly reversed the influence of resveratrol on function recovery, axonal regeneration, apoptosis after SCI., Conclusions: Resveratrol could promote the function recovery and axonal regeneration, improve histological damage, inhibit apoptosis level after SCI through regulating Wnt/β-catenin signaling pathway. This research expanded the regulatory mechanism of resveratrol in SCI injury.

The effects of stem cells from human exfoliated deciduous teeth (SHED) on mechanical allodynia were examined in mice. A single intravenous injection of SHED and conditioned medium from SHED (SHED-CM) through the left external jugular vein significantly reversed the established mechanical allodynia induced by spinal nerve transection at 6 days after injection. SHED or SHED-CM significantly decreased the mean numbers of activating transcription factor 3-positive neurons and macrophages in the ipsilateral side of the dorsal root ganglion (DRG) at 20 days after spinal nerve transection. SHED or SHED-CM also suppressed activation of microglia and astrocytes in the ipsilateral side of the dorsal spinal cord. A single intravenous injection of secreted ectodomain of sialic acid-binding Ig-like lectin-9 and monocyte chemoattractant protein-1 had no effect on the established mechanical allodynia, whereas a single intravenous injection of protein component(s) contained in SHED-CM with molecular weight of between 30 and 50 kDa reversed the pain. Therefore, it may be concluded that protein component(s) with molecular mass of 30-50 kDa secreted by SHED could protect and/or repair DRG neurons damaged by nerve transection, thereby ameliorating mechanical allodynia., (© 2021. The Author(s).)

Studies on the development of central nervous system (CNS) primarily rely on the use of specific molecular markers for different types of neural cells. S100B is widely being used as a specific marker for astrocytes in the CNS. However, the specificity of its expression in astrocyte lineage has not been systematically investigated and thus has remained a lingering issue. In this study, we provide several lines of molecular and genetic evidences that S100B is expressed in both protoplasmic astrocytes and myelinating oligodendrocytes. In the developing spinal cord, S100B is first expressed in the ventral neuroepithelial cells, and later in ALDH1L1+/GS+ astrocytes in the gray matter. Meanwhile, nearly all the S100B+ cells in the white matter are SOX10+/MYRF+ oligodendrocytes. Consistent with this observation, S100B expression is selectively lost in the white matter in Olig2-null mutants in which oligodendrocyte progenitor cells (OPCs) are not produced, and dramatically reduced in Myrf-conditional knockout mutants in which OPCs fail to differentiate. Similar expression patterns of S100B are observed in the developing forebrain. Based on these molecular and genetic studies, we conclude that S100B is not a specific marker for astrocyte lineage; instead, it marks protoplasmic astrocytes in the gray matter and differentiating oligodendrocytes., (© 2021. The Author(s).)

The cytoskeleton of ependymal cells is fundamental to organize and maintain the normal architecture of the central canal (CC). However, little is known about the plasticity of cytoskeletal components after spinal cord injury. Here, we focus on the structural organization of the cytoskeleton of ependymal cells in the normal and injured spinal cord of mice (both females and males) using immunohistochemical and electron microscopy techniques. We found that in uninjured animals, the actin cytoskeleton (as revealed by phalloidin staining) was arranged following the typical pattern of polarized epithelial cells with conspicuous actin pools located in the apical domain of ependymal cells. Transmission electron microscopy images showed microvilli tufts, long cilia, and characteristic intercellular membrane specializations. After spinal cord injury, F-actin rearrangements paralleled by fine structural modifications of the apical domain of ependymal cells were observed. These changes involved disruptions of the apical actin pools as well as fine structural modifications of the microvilli tufts. When comparing the control and injured spinal cords, we also found modifications in the expression of vimentin and glial fibrillary acidic protein (GFAP). After injury, vimentin expression disappeared from the most apical domains of ependymal cells but the number of GFAP-expressing cells within the CC increased. As in other polarized epithelia, the plastic changes in the cytoskeleton may be critically involved in the reaction of ependymal cells following a traumatic injury of the spinal cord., (© 2021 Wiley Periodicals LLC.)

There are several therapeutic options for spinal cord injury (SCI), among these strategies stem cell therapy is a potential treatment. The stem cells based therapies have been investigating in acute phase of clinical trials for promoting spinal repair in humans through replacement of functional neuronal and glial cells. The aim of this study was to evaluate the differentiation of Human Dental Pulp Stem Cells (hDPSCs) into functional motor neuron like cells (MNLCs) and promote neuroregeneration by stimulating local neurogenesis in the adult spinal cord slice culture. The immunocytochemistry analysis demonstrated that hDPSCs were positive for mesenchymal stem cell markers (CD73, CD90 and CD105) and negative for the hematopoietic markers (CD34 and CD45). hDPSCs were induced to neurospheres (via implementing B27, EGF, and bFGF) and then neural stem cells (NSC). The NSC differentiated into MNLCs in two steps: first by Shh and RA and ; then with GDNF and BDNF administration. The NS and the NSC were assessed for Oct4, nestin, Nanog, Sox2 expression while the MNLCs were evaluated by ISLET1, Olig2, and HB9 genes. Our results showed that hDPSC can be differentiated into motor neuron phenotype with expression of the motor neuron genes. The functionality of MNLCs was demonstrated by FM1-43, intracellular calcium ion shift and co- culture with C2C12. We co-cultivated hDPSCs with adult rat spinal slices in vitro. Immunostaining and hoechst assay showed that hDPSCs were able to migrate, proliferate and integrate in both the anterolateral zone and the edges of the spinal slices., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Location and distribution of spinal sympathetic preganglionic neurons projecting to the superior cervical ganglion were investigated in a rodent model organism for photoperiodic regulation, the Djungarian hamster ( Phodopus sungorus ). Upon unilateral injection of Fluoro-Gold into the superior cervical ganglia, retrograde neuronal tracing demonstrated labeled neurons ipsilateral to the injection site. They were seen in spinal segments C8 to Th5 of which the segments Th1 to Th3 contained about 98% of the labeled cells. Neurons were found in the spinal cord predominantly in the intermediolateral nucleus pars principalis and pars funicularis. At the same time, the central autonomic area and the intercalated region contained only very few labeled cells. In the intermediolateral nucleus, cells often were arranged in clusters, of which several were seen in each spinal segment. Selected sections were exposed to antibodies directed against arginine-vasopressin, neuronal nitric oxide synthase, neuropeptide Y, neurotensin, oxytocin or substance P. It was found that about two-thirds of sympathetic preganglionic neurons produced the gaseous neuroactive substance nitric oxide and that few contained small amounts of neuropeptide Y. Fibers of putative supraspinal origin immunopositive for either arginine-vasopressin, neuronal nitric oxide synthase, neuropeptide Y, neurotensin, oxytocin or, in particular, substance P were found in the vicinity of labeled sympathetic preganglionic neurons. These results demonstrate the location of relay neurons for autonomic control of cranial and cardial structures and provide further knowledge on neurochemical properties of sympathetic preganglionic neurons and related structures., Competing Interests: The author declares no conflict of interest., (© 2021 The Author(s). Published by IMR Press.)

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework. We report a hierarchical structure of postnatal cell type relationships, with location providing the highest level of organization, then neurotransmitter status, family, and finally, dozens of refined populations. We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord. We also show complex lineage relationships among postnatal cell types. Additionally, we develop an open-source cell type classifier, SeqSeek, to facilitate the standardization of cell type identification. This work provides an integrated view of spinal cell types, their gene expression signatures, and their molecular organization., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Clinically, posttraumatic stress disorder (PTSD) and chronic pain are highly comorbid conditions, but the underlying mechanisms of and therapeutic strategies against PTSD-related pain remain unclear. Our previous studies suggested that dysregulation of neuroinflammation contributes to the development of stress-induced hyperalgesia. Recent studies reported that angiotensin II was a 'stress-related hormone', and could induce glial activation by stimulating the type 1 receptor (AT1R). In the present study, we aimed to investigate whether AT1R blockade could attenuate mechanical allodynia induced by PTSD-like stress. Adult male rats were exposed to single prolonged stress (SPS) to establish a model of PTSD-pain comorbidity. Our results showed that SPS exposure increased the levels of angiotensin II in the hippocampus, prefrontal cortex (PFC) and spinal cord; intraperitoneal injection of losartan attenuated SPS-induced mechanical allodynia, and suppressed SPS-induced glial activation (both microglia and astrocytes) and proinflammatory cytokine expression in the PFC and spinal cord, but not in the hippocampus. We further showed that intrathecal injection of losartan also exerted anti-hyperalgesic effect and suppressed SPS-induced glial activation and proinflammatory cytokine expression in the spinal cord. These results indicated that AT1R blockade by losartan attenuated mechanical allodynia induced by PTSD-like stress, and this may be attributed to the suppression of glial activation and proinflammatory cytokine expression in the spinal cord. Although further research is warranted to verify our findings in female rodents and to assess pharmacological effects of AT1R blockade in PFC and hippocampus, our study suggested the therapeutic potential of targeting AT1R in the treatment of PTSD-related chronic pain., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In species that regenerate the injured spinal cord, the ependymal region is a source of new cells and a prominent coordinator of regeneration. In mammals, cells at the ependymal region proliferate in normal conditions and react after injury, but in humans, the central canal is lost in the majority of individuals from early childhood. It is replaced by a structure that does not proliferate after damage and is formed by large accumulations of ependymal cells, strong astrogliosis and perivascular pseudo-rosettes. We inform here of two additional mammals that lose the central canal during their lifetime: the Naked Mole-Rat (NMR, Heterocephalus glaber ) and the mutant hyh ( hydrocephalus with hop gait ) mice. The morphological study of their spinal cords shows that the tissue substituting the central canal is not similar to that found in humans. In both NMR and hyh mice, the central canal is replaced by tissue reminiscent of normal lamina X and may include small groups of ependymal cells in the midline, partially resembling specific domains of the former canal. However, no features of the adult human ependymal remnant are found, suggesting that this structure is a specific human trait. In order to shed some more light on the mechanism of human central canal closure, we provide new data suggesting that canal patency is lost by delamination of the ependymal epithelium, in a process that includes apical polarity loss and the expression of signaling mediators involved in epithelial to mesenchymal transitions.

Previous research advocates that exercise is a non-pharmacological therapy for Parkinson's disease (PD). However, few studies have investigated the effects of exercise on central nervous system structures other than the nigrostriatal pathway by using PD animal models. This study investigated the effects of exercise on tyrosine hydroxylase (TH)- and cerebral dopamine neurotrophic factor (CDNF)-containing spinal-cord neurons. Male Swiss mice were divided into 4 groups: sedentary control (SEDCONT), exercise control (EXERCONT), sedentary Parkinson (SEDPD), and exercise Parkinson (EXERPD). The PD groups were submitted to a surgical procedure for stereotaxic bilateral injection of 6-hydroxydopamine into the striatum. TH- and CDNF-containing spinal-cord neurons were evaluated in all groups, using immunohistochemistry and western-blotting. TH content in the ventral horn differed notably between the SEDPD and EXERPD groups. CDNF content was highest in the EXERPD group. SEDPD and EXERPD groups differed the most, as shown by immunohistochemistry and western-blotting. The EXERPD group showed the most intense labeling in immunohistochemistry compared to the SEDCONT and EXERCONT groups. Therefore, we showed here that exercise increased the content of both TH and CDNF in the spinal-cord neurons of a bilateral PD mouse model. We may assume that the spinal cord is affected in a PD model, and therefore this central nervous system region deserves more attention from researchers dealing with PD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The microglial reaction is a hallmark of neurodegenerative conditions, and elements thereof may exert differential effects on disease progression, either worsening or ameliorating severity. In amyotrophic lateral sclerosis (ALS), a syndrome characterized by cytoplasmic aggregation of TDP-43 protein and atrophy of motor neurons in the cortex and spinal cord, the transcriptomic signatures of microglia during disease progression are incompletely understood. Here, we performed longitudinal RNAseq analysis of cortical and spinal cord microglia from rNLS8 mice, in which doxycycline-regulatable expression of human TDP-43 (hTDP-43) in the cytoplasm of neurons recapitulates many features of ALS. Transgene suppression in rNLS8 mice leads to functional, anatomical and electrophysiological resolution that is dependent on a microglial reaction that is concurrent with recovery rather than disease onset. We identified basal differences between the gene expression profiles of microglia dependent on localization in spinal cord or cortex. Microglia subjected to chronic hTDP-43 overexpression demonstrated transcriptomic changes in both locations. We noted strong upregulation of Apoe, Axl, Cd63, Clec7a, Csf1, Cst7, Igf1, Itgax, Lgals3, Lilrb4, Lpl and Spp1 during late disease and recovery. Importantly, we identified a distinct suite of differentially expressed genes associated with each phase of disease progression and recovery. Differentially expressed genes were associated with chemotaxis, phagocytosis, inflammation, and production of neuroprotective factors. These data provide new insights into the microglial reaction in TDP-43 proteinopathy. Genes differentially expressed during progression and recovery may provide insight into a unique instance in which the microglial reaction promotes functional recovery after neuronal insult., (© 2021. The Author(s).)

Thyroid hormones are messengers that bind to specific nuclear receptors and regulate a wide range of physiological processes in the early stages of vertebrate embryonic development, including neurodevelopment and myelogenesis. We here tested the effects of reduced T3 availability upon the myelination process by treating zebrafish embryos with low concentrations of iopanoic acid (IOP) to block T4 to T3 conversion. Black Gold II staining showed that T3 deficiency reduced the myelin density in the forebrain, midbrain, hindbrain and the spinal cord at 3 and 7 dpf. These observations were confirmed in 3 dpf mbp:egfp transgenic zebrafish, showing that the administration of IOP reduced the fluorescent signal in the brain. T3 rescue treatment restored brain myelination and reversed the changes in myelin-related gene expression induced by IOP exposure. NG2 immunostaining revealed that T3 deficiency reduced the amount of oligodendrocyte precursor cells in 3 dpf IOP-treated larvae. Altogether, the present results show that inhibition of T4 to T3 conversion results in hypomyelination, suggesting that THs are part of the key signaling molecules that control the timing of oligodendrocyte differentiation and myelin synthesis from very early stages of brain development., Competing Interests: The authors have declared that no competing interests exist.

Peripheral and intraspinal feedback is required to shape and update the output of spinal networks that execute motor behavior. We report that lumbar dI2 spinal interneurons in chicks receive synaptic input from afferents and premotor neurons. These interneurons innervate contralateral premotor networks in the lumbar and brachial spinal cord, and their ascending projections innervate the cerebellum. These findings suggest that dI2 neurons function as interneurons in local lumbar circuits, are involved in lumbo-brachial coupling, and that part of them deliver peripheral and intraspinal feedback to the cerebellum. Silencing of dI2 neurons leads to destabilized stepping in posthatching day 8 hatchlings, with occasional collapses, variable step profiles, and a wide-base walking gait, suggesting that dI2 neurons may contribute to the stabilization of the bipedal gait., Competing Interests: BH, YH, NB, AK, MD, YC, AL, AK none, (© 2021, Haimson et al.)

Physical exercise stimulates adult neurogenesis, yet the underlying mechanisms remain poorly understood. A fundamental component of the innate neuroregenerative capacity of zebrafish is the proliferative and neurogenic ability of the neural stem/progenitor cells. Here, we show that in the intact spinal cord, this plasticity response can be activated by physical exercise by demonstrating that the cholinergic neurotransmission from spinal locomotor neurons activates spinal neural stem/progenitor cells, leading to neurogenesis in the adult zebrafish. We also show that GABA acts in a non-synaptic fashion to maintain neural stem/progenitor cell quiescence in the spinal cord and that training-induced activation of neurogenesis requires a reduction of GABA A receptors. Furthermore, both pharmacological stimulation of cholinergic receptors, as well as interference with GABAergic signaling, promote functional recovery after spinal cord injury. Our findings provide a model for locomotor networks' activity-dependent neurogenesis during homeostasis and regeneration in the adult zebrafish spinal cord., (© 2021. The Author(s).)

The axis of the vertebrate neural tube is patterned, in part, by a ventral to dorsal gradient of Shh signaling. In the ventral spinal cord, Shh induces concentration-dependent expression of transcription factors, subdividing neural progenitors into distinct domains that subsequently produce distinct neuronal and glial subtypes. In particular, progenitors of the pMN domain express the bHLH transcription factor Olig2 and produce motor neurons followed by oligodendrocytes, the myelinating glial cell type of the central nervous system. In addition to its role in patterning ventral progenitors, Shh signaling must be maintained through development to specify pMN progenitors for oligodendrocyte fate. Using a forward genetic screen in zebrafish for mutations that disrupt the development of oligodendrocytes, we identified a new mutant allele of boc, which encodes a type I transmembrane protein that functions as a coreceptor for Shh. Embryos homozygous for the bocco25 allele, which creates a missense mutation in a Fibronectin type III domain that binds Shh, have normally patterned spinal cords but fail to maintain pMN progenitors, resulting in a deficit of oligodendrocytes. Using a sensitive fluorescent detection method for in situ RNA hybridization, we found that spinal cord cells express boc in a graded fashion that is inverse to the gradient of Shh signaling activity and that boc function is necessary to maintain pMN progenitors by shaping the Shh signaling gradient., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In the spinal cord, cerebrospinal fluid-contacting neurons (CSF-cNs) are GABAergic interoceptive sensory neurons that detect spinal curvature via a functional coupling with the Reissner fiber. This mechanosensory system has recently been found to be involved in spine morphogenesis and postural control but the underlying mechanisms are not fully understood. In zebrafish, CSF-cNs project an ascending and ipsilateral axon reaching two to six segments away. Rostralmost CSF-cNs send their axons ipsilaterally into the hindbrain, a brain region containing motor nuclei and reticulospinal neurons (RSNs), which send descending motor commands to spinal circuits. Until now, the synaptic connectivity of CSF-cNs has only been investigated in the spinal cord, where they synapse onto motor neurons and premotor excitatory interneurons. The identity of CSF-cN targets in the hindbrain and the behavioral relevance of these sensory projections from the spinal cord to the hindbrain are unknown. Here, we provide anatomical and molecular evidence that rostralmost CSF-cNs synapse onto the axons of large RSNs including Mauthner cells and V2a neurons. Functional anatomy and optogenetically assisted mapping reveal that rostral CSF-cNs also synapse onto the soma and dendrites of cranial motor neurons innervating hypobranchial muscles. During acousto-vestibular evoked escape responses, ablation of rostralmost CSF-cNs results in a weaker escape response with a decreased C-bend amplitude, lower speed, and deficient postural control. Our study demonstrates that spinal sensory feedback enhances speed and stabilizes posture, and reveals a novel spinal gating mechanism acting on the output of descending commands sent from the hindbrain to the spinal cord., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In the spinal cord, ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities including locomotion. Interneurons arise during embryonic development from distinct progenitor domains orderly distributed along the dorso-ventral axis of the neural tube. The p2 progenitor domain generates at least five V2 interneuron populations. However, identification and characterization of all V2 populations remain currently incomplete and the mechanisms that control their development remain only partly understood. In this study, we report the generation of a Vsx1-CreER T2 BAC transgenic mouse line that drives CreER T2 recombinase expression mimicking endogenous Vsx1 expression pattern in the developing spinal cord. We showed that the Vsx1-CreER T2 transgene can mediate recombination in V2 precursors with a high efficacy and specificity. Lineage tracing demonstrated that all the V2 interneurons in the mouse developing spinal cord derive from cells expressing Vsx1. Finally, we confirmed that V2 precursors generate additional V2 populations that are not characterized yet. Thus, the Vsx1-CreER T2 line described here is a useful genetic tool for lineage tracing and for functional studies of the mouse spinal V2 interneurons., (© 2021 Wiley Periodicals LLC.)

Metformin, the first medication that is often prescribed for the treatment of type 2 diabetes mellitus, was recently found to be neuroprotective. To study the mechanism underlying the neuroprotective effect of metformin, we pretreated primary spinal cord neurons with 50 µM or 100 µM metformin for 2 h prior to treatment with hydrogen peroxide (H 2 O 2 ) for up to 48 h. Our results showed that H 2 O 2 increased the expression of purinergic receptor P2X7 (P2X7R) in spinal cord neurons, which promoted the downstream pro-inflammatory cytokines release and oxidative stress. We found that metformin could reverse these pro-inflammatory and pro-oxidative effects of H 2 O 2 . Besides, P2X7R knockdown by siRNA suppressed H 2 O 2 -induced pro-inflammatory cytokine release and oxidative stress response. In conclusion, our results show that metformin can alleviate H 2 O 2 -induced inflammation and oxidative stress via modulating the P2X7R signaling pathway.

Reportedly, TWIK-related spinal cord K + (TRESK) deficiency in spinal cord neurons positively correlates with the mechanism underlying neuropathic pain (NP). However, the precise effects of TRESK on neurons of the spinal cord remain elusive. In the present study, we investigated the impact of TRESK silencing on spinal cord neurons to further elucidate the downstream mechanisms of TRESK. Herein, neurons of the dorsal spinal cord were cultured as a cell model for investigations. Apoptosis, oxidative stress, and DNA damage-related proteins were evaluated. Additionally, flow cytometry, microarray profiling, real-time polymerase chain reaction (PCR), western blotting, fluorescence in situ hybridization (FISH), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were performed. In cultured neurons, the downregulation of TRESK mRNA expression induced apoptosis of dorsal spinal cord neurons. Using real-time PCR and western blotting, the upregulation of LncRNA Gm11874 (Gm11874) and ATP5i, screened from the gene chip, was confirmed. On silencing TRESK, expression levels of γ-H2AX, poly [ADP-ribose] polymerase 1 (PARP-1), FoxO1, FoxO3, MitoSOX, malondialdehyde (MDA), and 8-hydroxy-2' -deoxyguanosine (8-OHdG), which are known indices of oxidative stress and DNA damage, were significantly elevated. Moreover, ATP induced oxidative stress, DNA damage, and apoptosis were reduced by ATP5i siRNA. Finally, Gm11874 and ATP5i were co-expressed in spinal cord neurons in a FISH experiment, and the expression of ATP5i was positively regulated by Gm11874. These results implied that ATP5i induced oxidative stress and DNA damage, resulting in neuronal apoptosis, and Gm11874 was confirmed to act upstream of ATP5i. Our study revealed that TRESK silencing upregulated Gm11874 to induce apoptosis of spinal cord neurons, which resulted in ATP5i promoting oxidative stress and DNA damage. These findings could highlight the TRESK-mediated NP mechanism.

Spinal cord injury (SCI) is a traumatic disease that can cause severe nervous system dysfunction. SCI often causes spinal cord mitochondrial dysfunction and produces glucose metabolism disorders, which affect neuronal survival. Zinc is an essential trace element in the human body and plays multiple roles in the nervous system. This experiment is intended to evaluate whether zinc can regulate the spinal cord and neuronal glucose metabolism and promote motor functional recovery after SCI. Then we explore its molecular mechanism. We evaluated the function of zinc from the aspects of glucose uptake and the protection of the mitochondria in vivo and in vitro. The results showed that zinc elevated the expression level of GLUT4 and promoted glucose uptake. Zinc enhanced the expression of proteins such as PGC-1 α and NRF2, reduced oxidative stress, and promoted mitochondrial production. In addition, zinc decreased neuronal apoptosis and promoted the recovery of motor function in SCI mice. After administration of AMPK inhibitor, the therapeutic effect of zinc was reversed. Therefore, we concluded that zinc regulated the glucose metabolism of the spinal cord and neurons and promoted functional recovery after SCI through the AMPK pathway, which is expected to become a potential treatment strategy for SCI., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Hengshuo Hu et al.)

Neurogenesis timing is an essential developmental mechanism for neuronal diversity and organization throughout the central nervous system. In the mouse spinal cord, growing evidence is beginning to reveal that neurogenesis timing acts in tandem with spatial molecular controls to diversify molecularly and functionally distinct post-mitotic interneuron subpopulations. Particularly, in some cases, this temporal ordering of interneuron differentiation has been shown to instruct specific sensorimotor circuit wirings. In zebrafish, in vivo preparations have revealed that sequential neurogenesis waves of interneurons and motor neurons form speed-dependent locomotor circuits throughout the spinal cord and brainstem. In the present review, we discuss temporal principals of interneuron diversity taken from both mouse and zebrafish systems highlighting how each can lend illuminating insights to the other. Moving forward, it is important to combine the collective knowledge from different systems to eventually understand how temporally regulated subpopulation function differentially across speed- and/or state-dependent sensorimotor movement tasks.

The meninges are a membranous structure enveloping the central nervous system (CNS) that host a rich repertoire of immune cells mediating CNS immune surveillance. Here, we report that the mouse meninges contain a pool of monocytes and neutrophils supplied not from the blood but by adjacent skull and vertebral bone marrow. Under pathological conditions, including spinal cord injury and neuroinflammation, CNS-infiltrating myeloid cells can originate from brain borders and display transcriptional signatures distinct from their blood-derived counterparts. Thus, CNS borders are populated by myeloid cells from adjacent bone marrow niches, strategically placed to supply innate immune cells under homeostatic and pathological conditions. These findings call for a reinterpretation of immune-cell infiltration into the CNS during injury and autoimmunity and may inform future therapeutic approaches that harness meningeal immune cells., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Astrocytes are a viable source for generating new neurons via direct conversion. However, little is known about the neurogenic cascades triggered in astrocytes from different regions of the CNS. Here, we examine the transcriptome induced by the proneural factors Ascl1 and Neurog2 in spinal cord-derived astrocytes in vitro. Each factor initially elicits different neurogenic programs that later converge to a V2 interneuron-like state. Intriguingly, patch sequencing (patch-seq) shows no overall correlation between functional properties and the transcriptome of the heterogenous induced neurons, except for K-channels. For example, some neurons with fully mature electrophysiological properties still express astrocyte genes, thus calling for careful molecular and functional analysis. Comparing the transcriptomes of spinal cord- and cerebral-cortex-derived astrocytes reveals profound differences, including developmental patterning cues maintained in vitro. These relate to the distinct neuronal identity elicited by Ascl1 and Neurog2 reflecting their developmental functions in subtype specification of the respective CNS region., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.

Central nervous system injury and neurodegenerative diseases cause irreversible loss of neurons. Overexpression of exogenous specific transcription factors can reprogram somatic cells into functional neurons for regeneration and functional reconstruction. However, these practices are potentially problematic due to the integration of vectors into the host genome. Here, we showed that the activation of endogenous genes Ngn2 and Isl1 by CRISPRa enabled reprogramming of mouse spinal astrocytes and embryonic fibroblasts to motor neurons. These induced neurons showed motor neuronal morphology and exhibited electrophysiological activities. Furthermore, astrocytes in the spinal cord of the adult mouse can be converted into motor neurons by this approach with high efficiency. These results demonstrate that the activation of endogenous genes is sufficient to induce astrocytes into functional motor neurons in vitro and in vivo. This direct neuronal reprogramming approach may provide a novel potential therapeutic strategy for treating neurodegenerative diseases and spinal cord injury., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic spinal cord injury (SCI) is a catastrophic condition associated with significant neurological deficit and social and financial burdens. It is currently being managed symptomatically with no real therapeutic strategies available. In recent years, a number of innovative regenerative strategies have emerged and have been continuously investigated in clinical trials. In addition, several more are coming down the translational pipeline. Among ongoing and completed trials are those reporting the use of mesenchymal stem cells, neural stem/progenitor cells, induced pluripotent stem cells, olfactory ensheathing cells, and Schwann cells. The advancements in stem cell technology, combined with the powerful neuroimaging modalities, can now accelerate the pathway of promising novel therapeutic strategies from bench to bedside. Various combinations of different molecular therapies have been combined with supportive scaffolds to facilitate favorable cell-material interactions. In this review, we summarized some of the most recent insights into the preclinical and clinical studies using stem cells and other supportive drugs to unlock the microenvironment in chronic SCI to treat patients with this condition. Successful future therapies will require these stem cells and other synergistic approaches to address the persistent barriers to regeneration, including glial scarring, loss of structural framework, and immunorejection.

Microglia are important immune cells in the central nervous system (CNS). Mutations in microglia may cause CNS disorders. Replacement of dysfunctional microglia with allogeneic wild-type microglia can correct the gene deficiency, thus treating the neurogenic diseases. However, traditional approaches cannot efficiently replace microglia at the adulthood. Here, we introduce a potentially clinical-feasible strategy named microglia replacement by bone marrow transplantation that achieves efficient microglia replacement at the whole CNS scale, including the brain, spinal cord, and retina in adult mice. For complete details on the use and execution of this protocol, please refer to Xu et al. (2020). The original abbreviation of this microglia replacement strategy is mrBMT. We hereby change the name to Mr BMT., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Scaffolds of recombinant spider silk protein (spidroin) and hyaluronic acid (HA) hydrogel hold promise in combination with cell therapy for spinal cord injury. However, little is known concerning the human immune response to these biomaterials and grafted human neural stem/progenitor cells (hNPCs). Here, we analyzed short- and long-term in vitro activation of immune cells in human peripheral blood mononuclear cells (hPBMCs) cultured with/without recombinant spidroins, HA hydrogels, and/or allogeneic hNPCs to assess potential host-donor interactions. Viability, proliferation and phenotype of hPBMCs were analyzed using NucleoCounter and flow cytometry. hPBMC viability was confirmed after exposure to the different biomaterials. Short-term (15 h) co-cultures of hPBMCs with spidroins, but not with HA hydrogel, resulted in a significant increase in the proportion of activated CD69 + CD4 + T cells, CD8 + T cells, B cells and NK cells, which likely was caused by residual endotoxins from the Escherichia coli expression system. The observed spidroin-induced hPBMC activation was not altered by hNPCs. It is resource-effective to evaluate human compatibility of novel biomaterials early in development of the production process to, when necessary, make alterations to minimize rejection risk. Here, we present a method to evaluate biomaterials and hPBMC compatibility in conjunction with allogeneic human cells.

Nucleocytosolic transport, a membrane process, is impaired in motor neurons in amyotrophic lateral sclerosis (ALS). This study analyzes the nuclear lipidome in motor neurons in ALS and examines molecular pathways linked to the major lipid alterations. Nuclei were obtained from the frozen anterior horn of the lumbar spinal cord of ALS patients and age-matched controls. Lipidomic profiles of this subcellular fraction were obtained using liquid chromatography and mass spectrometry. We validated the mechanisms behind presumable lipidomic changes by exploring ALS surrogate models including human motor neurons (derived from ALS lines and controls) subjected to oxidative stress, the hSOD-G93A transgenic mice, and samples from an independent cohort of ALS patients. Among the differential lipid species, we noted 41 potential identities, mostly belonging to phospholipids (particularly ether phospholipids, as plasmalogens), as well as diacylglycerols and triacylglycerides. Decreased expression of alkyldihydroxyacetonephosphate synthase (AGPS)-a critical peroxisomal enzyme in plasmalogen synthesis-is found in motor neuron disease models; this occurs in parallel with an increase in the expression of sterol carrier protein 2 (SCP2) mRNA in ALS and Scp2 levels in G93A transgenic mice. Further, we identified diminished expression of diacylglycerol-related enzymes, such as phospholipase C βI (PLCβI) and protein kinase CβII (PKCβII), linked to diacylglycerol metabolism. Finally, lipid droplets were recognized in the nuclei, supporting the identification of triacylglycerides as differential lipids. Our results point to the potentially pathogenic role of altered composition of nuclear membrane lipids and lipids in the nucleoplasm in the anterior horn of the spinal cord in ALS. Overall, these data support the usefulness of subcellular lipidomics applied to neurodegenerative diseases., (© 2021 International Society for Neurochemistry.)

The resumption of blood supply in spinal cord (SC) after injury is a prerequisite of its recovery. To expose the mechanisms of damaged SC revascularization we have used an organotypic SC/aortic fragments (AF) co-culture where, as we showed previously, damaged SC tissue induces AF cell sprouting but repels them away. Supplementation of culture medium with exogenous VEGF-A 165 redirects the migrating aortic endothelial cells towards SC tissue. This effect and the pattern of sFlt1 expression (a soluble form of VEGFR1) suggest that the low level of SC-secreted VEGF and the presence of sFlt1 in SC slices together prevent the migration of aortic CD31 + cells to the SC in the absence of exogenous VEGF. VEGF-A 165 supplementation sequesters this inhibitory activity of sFlt1 by direct binding thus allowing CD31 + cell migration in to SC tissue. Proteome analysis has shown that migration/proliferation of CD31 + and αSMA + aortic cells in neuronal culture medium used in our SC/AF model (which obstruct sprouting by itself) was resumed by combined action of several pro- (aFGF, bFGF, Osteopontin, TF, IGFBP2, SDF1) and anti-angiogenic (Endostatin/Collagen18) factors. The mutual influence of AF and SC tissues is a key factor balancing these factors and thus driving endothelial sprouting in SC injury zone., (© 2021 John Wiley & Sons Ltd.)

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose pathophysiology is largely unknown. Despite the fact that motor neuron (MN) death is recognized as the key event in ALS, astrocytes dysfunctionalities and neuroinflammation were demonstrated to accompany and probably even drive MN loss. Nevertheless, the mechanisms priming astrocyte failure and hyperactivation are still obscure. In this work, altered pathways and molecules in ALS astrocytes were unveiled by investigating the proteomic profile and the secreted metabolome of primary spinal cord astrocytes derived from transgenic ALS mouse model overexpressing the human (h)SOD1(G93A) protein in comparison with the transgenic counterpart expressing hSOD1(WT) protein. Here we show that ALS primary astrocytes are depleted of proteins-and of secreted metabolites-involved in glutathione metabolism and signaling. The observed increased activation of Nf-kB, Ebf1, and Plag1 transcription factors may account for the augmented expression of proteins involved in the proteolytic routes mediated by proteasome or endosome-lysosome systems. Moreover, hSOD1(G93A) primary astrocytes also display altered lipid metabolism. Our results provide novel insights into the altered molecular pathways that may underlie astrocyte dysfunctionalities and altered astrocyte-MN crosstalk in ALS, representing potential therapeutic targets to abrogate or slow down MN demise in disease pathogenesis.

Platinum-based chemotherapeutics still play an essential role in cancer treatment. Despite their high effectiveness, severe side effects such as chemotherapy-induced neuropathy (CIPN) occur frequently. The pathophysiology of CIPN by platinum-based chemotherapeutics is not fully understood yet, but primarily the disturbance of dorsal root ganglion cells is discussed. However, there is increasing evidence of central nervous system involvement with activation of spinal cord astrocytes after treatment with chemotherapeutics. We investigated the influence of cis- or oxaliplatin on the functionality of cultured rat spinal cord astrocytes by using immunocytochemistry and patch-clamp electrophysiology. Cis- or oxaliplatin activated spinal astrocytes and led to downregulation of the excitatory amino acid transporter 1 (EAAT1) expression. Furthermore, the expression and function of potassium channel K ir4.1 were modulated. Pre-exposure to a specific Kir4.1 blocker in control astrocytes led to a reduced immune reactivity (IR) of EAAT1 and a nearly complete block of the current density. When spinal astrocytes were pre-exposed to antibiotic minocycline, all effects of cis- or oxaliplatin were abolished. Taken together, the modulation of K ir4.1 and EAAT1 proteins in astrocytes could be linked to the direct impact of cis- or oxaliplatin, identifying spinal astrocytes as a potential target in the prevention and treatment of chemotherapy-induced neuropathy.

NK cells are innate immune cells implicated in ALS; whether NK cells impact ALS in a sex- and age-specific manner was investigated. Herein, NK cells were depleted in male and female SOD1G93A ALS mice, survival and neuroinflammation were assessed, and data were stratified by sex. NK cell depletion extended survival in female but not male ALS mice with sex-specific effects on spinal cord microglia. In humans, NK cell numbers, NK cell subpopulations, and NK cell surface markers were examined in prospectively blood collected from subjects with ALS and control subjects; longitudinal changes in these metrics were correlated to revised ALS functional rating scale (ALSFRS-R) slope and stratified by sex and age. Expression of NK cell trafficking and cytotoxicity markers was elevated in subjects with ALS, and changes in CXCR3+ NK cells and 7 trafficking and cytotoxicity markers (CD11a, CD11b, CD38, CX3CR1, NKG2D, NKp30, NKp46) correlated with disease progression. Age affected the associations between ALSFRS-R and markers NKG2D and NKp46, whereas sex impacted the NKp30 association. Collectively, these findings suggest that NK cells contribute to ALS progression in a sex- and age-specific manner and demonstrate that age and sex are critical variables when designing and assessing ALS immunotherapy.

In brain astrocytes, noradrenaline (NA) has been shown to up-regulate IL-6 production via β-adrenoceptors (ARs). However, the underlying intracellular mechanisms for this regulation are not clear, and it remains unknown whether α-ARs are involved. In this study, we investigated the AR-mediated regulation of IL-6 mRNA levels in the cultured astrocytes from rat spinal cord. NA, the α 1 -agonist phenylephrine, and the β-agonist isoproterenol increased IL-6 mRNA levels. The phenylephrine-induced IL-6 increase was accompanied by an increase in ERK phosphorylation, and these effects were blocked by inhibitors of PKC and ERK. The isoproterenol-induced IL-6 increase was accompanied by an increase in CREB phosphorylation, and these effects were blocked by a PKA inhibitor. Our results indicate that IL-6 increases by α 1 - and β-ARs are mediated via the PKC/ERK and cAMP/PKA/CREB pathways, respectively. Moreover, conditioned medium collected from astrocytes treated with the α 2 -AR agonist dexmedetomidine, increased IL-6 mRNA in other astrocytes. In this study, we elucidate that α 1 - and α 2 -ARs, in addition to β-ARs, promote IL-6 transcription through different pathways in spinal cord astrocytes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Nervous systems are designed to become extra sensitive to afferent nociceptive stimuli under certain circumstances such as inflammation and nerve injury. How pain hypersensitivity comes about is key issue in the field since it ultimately results in chronic pain. Central sensitization represents enhanced pain sensitivity due to increased neural signaling within the central nervous system (CNS). Particularly, much evidence indicates that underlying mechanism of central sensitization is associated with the change of spinal neurons. Extracellular signal-regulated kinases have received attention as key molecules in central sensitization. Previously, we revealed the isoform-specific function of extracellular signal-regulated kinase 2 (Erk2) in spinal neurons for central sensitization using mice with Cre-loxP-mediated deletion of Erk2 in the CNS. Still, how extracellular signal-regulated kinase 5 (Erk5) in spinal neurons contributes to central sensitization has not been directly tested, nor is the functional relevance of Erk5 and Erk2 known. Here, we show that Erk5 and Erk2 in the CNS play redundant and/or distinct roles in central sensitization, depending on the plasticity context (cell types, pain types, time, etc.). We used male mice with Erk5 deletion specifically in the CNS and found that Erk5 plays important roles in central sensitization in a formalin-induced inflammatory pain model. Deletion of both Erk2 and Erk5 leads to greater attenuation of central sensitization in this model, compared to deletion of either isoform alone. Conversely, Erk2 but not Erk5 plays important roles in central sensitization in neuropathic pain, a type of chronic pain caused by nerve damage. Our results suggest the elaborate mechanisms of Erk signaling in central sensitization., (© 2021 Wiley Periodicals LLC.)

ALS is characterized by progressive inability to execute movements. Motor neurons innervating fast-twitch muscle-fibers preferentially degenerate. The reason for this differential vulnerability and its consequences on motor output is not known. Here, we uncover that fast motor neurons receive stronger inhibitory synaptic inputs than slow motor neurons, and disease progression in the SOD1 G93A mouse model leads to specific loss of inhibitory synapses onto fast motor neurons. Inhibitory V1 interneurons show similar innervation pattern and loss of synapses. Moreover, from postnatal day 63, there is a loss of V1 interneurons in the SOD1 G93A mouse. The V1 interneuron degeneration appears before motor neuron death and is paralleled by the development of a specific locomotor deficit affecting speed and limb coordination. This distinct ALS-induced locomotor deficit is phenocopied in wild-type mice but not in SOD1 G93A mice after appearing of the locomotor phenotype when V1 spinal interneurons are silenced. Our study identifies a potential source of non-autonomous motor neuronal vulnerability in ALS and links ALS-induced changes in locomotor phenotype to inhibitory V1-interneurons.

The neuronal networks that generate locomotion are well understood in swimming animals such as the lamprey, zebrafish and tadpole. The networks controlling locomotion in tetrapods remain, however, still enigmatic with an intricate motor pattern required for the control of the entire limb during the support, lift off, and flexion phase, and most demandingly when the limb makes contact with ground again. It is clear that the inhibition that occurs between bursts in each step cycle is produced by V2b and V1 interneurons, and that a deletion of these interneurons leads to synchronous flexor-extensor bursting. The ability to generate rhythmic bursting is distributed over all segments comprising part of the central pattern generator network (CPG). It is unclear how the rhythmic bursting is generated; however, Shox2, V2a and HB9 interneurons do contribute. To deduce a possible organization of the locomotor CPG, simulations have been elaborated. The motor pattern has been simulated in considerable detail with a network composed of unit burst generators; one for each group of close synergistic muscle groups at each joint. This unit burst generator model can reproduce the complex burst pattern with a constant flexion phase and a shortened extensor phase as the speed increases. Moreover, the unit burst generator model is versatile and can generate both forward and backward locomotion.

Background and Purpose: Ongoing neuropathic pain is one of the most challenging clinical problems which have detrimental effects on a patient's life quality. Conventional therapies for chronic neuropathic pain majorly includes centrally acting analgesics. Unfortunately, the unceasing use of these drugs results in adverse effects, such as CNS in-coordination, respiratory depression and substance use disorder. DALDA ([D-Arg 2 , Lys 4 ]-Dermorphin-(1-4)-amide), a peripherally acting opioid have been shown to possess potent analgesic activity without causing CNS toxicities in nerve-injured rats. However, the mechanism(s) underpinning DALDA induced-attenuation of ongoing neuropathic pain is yet to identify [1]., Experimental Design: In this study, we have measured the in-silico ligand-receptor binding affinity of DALDA against potential inflammatory targets by utilizing glide module of schrödinger software. Effect of DALDA on oxido-inflammatory stress was evaluated in LPS-induced C6 glial cells. In-vitro studies were followed by the behavioral assessments, where effect of DALDA was measured in chronic constriction injured rats. To examine the effect of DALDA on dopaminergic neurotransmission, cerebrospinal fluid of nerve-injured rats was assessed using LC-MS/QToF (Liquid Chromatography-Mass spectrometry/ Quadrapole time of flight Analyzer)., Results: DALDA has shown a good binding affinity with chemokine receptor type-2 (CCR2), chemokine CX3C receptor 1 (CX3CR1) and purinergic receptor (P2×4), major receptor subtypes involved in pain and inflammation. Findings from the in-vitro studies suggest that DALDA possesses potent anti-oxidant activity leading to inhibition of p38-MAPK pathway [1]. Moreover, the subcutaneous administration of DALDA leads to dose-dependent attenuation of thermal and mechanical hypersensitivity along with inhibition of neuroinflammatory mediators in serum and spinal cord of nerve-injured rats. Most importantly, DALDA treated neuropathic rats showed a preference for the DALDA-treated chamber, which was attenuated on pre-treatment with dopaminergic receptor antagonist, flupenthixol. LC-MS analysis further confirms the enhanced dopaminergic transmission in the brain of DALDA-treated neuropathic rats., Conclusion: Our findings suggest that DALDA mediated attenuation of ongoing neuropathic pain may be associated with a decrease in spinal neuroinflammatory signalling and subsequent increase in the brain dopamine level; may serve a potential therapeutic for the treatment of ongoing neuropathic pain., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Neuregulin-1 (NRG1), a family of EGF-like factors that activates ErbB receptors, can regulate the proliferation, migration, and myelinating of Schwann cells. We previously reported that NRG1/ErbB signal is responsible for organophosphate (OP)-induced delayed neuropathy (OPIDN) in hens, a susceptive animal model to neuropathic organophosphorous compounds. Our previous study discovered that a neuropathic OP, tri-o-cresyl phosphate (TOCP) activated NRG1/ErbB signaling pathway in both spinal cord and sciatic nerves of hens during the formation of OPIDN and lapatinib, a non-selective antagonist of ErbB1 and ErbB2 receptors, alleviated the toxicity. In this study, we intended to further look into the potential role of NRG1 in the pathogenesis of TOCP-induced axon damage in spinal cord and sciatic nerves and whether lapatinib could also rescue this damage in mice, an OPIDN-resistant animal model. The results revealed that no obvious toxic signs were observed after single TOCP exposure. However, slight histopathological wreck in lumbar spinal cord and sciatic nerves was found following TOCP intoxication, and the damage in sciatic nerves was characterized by axon degeneration of myelin sheath but not the loss of neural skeleton. Only histopathological damage induced by TOCP in spinal cord could be prevented by lapatinib. The translational expression of NRG1/ErbB signaling molecules was analyzed by both in vivo and in vitro studies. In general, NRG1/ErbB pathway was activated by TOCP while combined treatment with lapatinib attenuated TOCP-induced NRG1/ErbB signaling cascade. The results implied that NRG1/ErbB system may predominately play functional role in spinal cord (central nervous system) but not in sciatic nerves (peripheral nervous system) of mouse subjected to neurotoxic OP, which was confirmed by the study in vitro that lapatinib was not able to attenuate TOCP-induced neurotoxicity in rodent Schwann cell line RSC 96 cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)