Spinal Neurons Research Articles

Effects of MDA-19 on Zebrafish Larval Behavior: Perspectives From Neurodevelopment, Oxidative Stress, and Metabolomics.

As global regulations on synthetic cannabinoids tighten, illicit vendors increasingly turn to new structures of synthetic cannabinoids to evade legal scrutiny. MDA-19, a novel synthetic cannabinoid, exhibited significant agonistic effects on type 2 cannabinoid receptors invivo and showed emerging trends of abuse in illicit markets. However, research on the toxicological effects of MDA-19 remains scarce. In this study, we examined the effects of MDA-19 on neurodevelopment, behavior, oxidative stress, and metabolomics by exposing zebrafish embryos to MDA-19 solutions with concentrations of 1, 10, and 20 mg/L over 5 days. Results revealed that exposure to 10 and 20 mg/L of MDA-19 accelerated hatching in zebrafish embryos but led to reduced body length without affecting mortality or malformation. Furthermore, exposure to all concentrations of MDA-19 resulted in diminished swimming ability and reduced activity time in zebrafish. Transgenic zebrafish (hb9-GFP) exposed to MDA-19 exhibited impaired development of spinal motor neurons. Notably, exposure to 20 mg/L MDA-19 increased the levels of reactive oxygen species (ROS) in zebrafish and elevated the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), while the levels of the lipid oxidation product malondialdehyde (MDA) remained unaffected. Nontargeted metabolomics analyses showed that MDA-19 interfered with multiple metabolic pathways affecting energy metabolism, such as alanine, aspartate, and glutamate metabolism; the citric acid cycle (TCA cycle), pantothenate, and coenzyme A biosynthesis; and purine metabolism. In conclusion, the present study provided the essential evidence for the neurotoxic effects of MDA-19, which was associated with impaired neurodevelopment, dysregulation of oxidative stress homeostasis, and altered energy metabolism.

Supraspinal contributions to defective antagonistic inhibition and freezing of gait in Parkinson's disease.

The neuromuscular circuit mechanisms of freezing of gait in Parkinson's disease have received little study. Technological progress enables researchers chronically to sense local field potential activity of the basal ganglia in patients while walking. To study subthalamic activity and the circuit processes of supraspinal contributions to spinal motor integration, we recorded local field potentials, surface EMG of antagonistic leg muscles and gait kinematics in patients while walking and freezing. To evaluate the specificity of our findings, we controlled our findings to internally generated volitional stops. We found specific activation-deactivation abnormalities of oscillatory activity of the subthalamic nucleus both before and during a freeze. Furthermore, we were able to show with synchronization analyses that subthalamo-spinal circuits entrain the spinal motor neurons to a defective timing and activation pattern. The main neuromuscular correlates when turning into freezing were as follows: (i) disturbed reciprocity between antagonistic muscles; (ii) increased co-contraction of the antagonists; (iii) defective activation and time pattern of the gastrocnemius muscle; and (iv) increased subthalamo-muscular coherence with the gastrocnemius muscles before the freeze. Beyond the pathophysiological insights into the supraspinal mechanisms contributing to freezing of gait, our findings have potential to inform the conceptualization of future neurorestorative therapies.

Mechanisms of Differential Sensitivity to Ethanol-Induced Apoptosis in Mouse Spinal Cord at Different Developmental Stages-Akt/GSK Signaling and BAX.

The current study investigated differences in ethanol-induced apoptosis of spinal cord dorsal horn neurons at different developmental stages and the molecular mechanisms involved. A mouse ethanol intervention model was established on postnatal days 4, 7, and 12. Primary cells were derived from the spinal cord at postnatal day 4. Western blotting, immunofluorescence, and flow cytometry were used to detect apoptosis-related proteins in the spinal cord and primary cells. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes originating from the Gene Expression Omnibus dataset GSE184615 was conducted. Effects on Akt/GSK3β pathway proteins were investigated using the GSK3β inhibitor AR-A014418, and the Akt inhibitor DHA. Lentiviral knockdown and overexpression of intervening GSK3β were used in HT22 cell lines to investigate the effects of alcohol on GSK 3β and caspase proteins. J-aggregates, reactive oxygen species assays, and calcein-AM assays were used to investigate mitochondrial function and cell viability. Ethanol caused downregulation of Akt activity and upregulation of GSK3β activity and apoptosis. DHA, AR-A014418, and knockdown of GSK3β effectively counteracted ethanol-induced apoptosis, whereas overexpression of GSK3β enhanced the injury process. PI3K activity was unchanged during these processes. Fluorescence colocalization analysis indicated that BAX was translocated to mitochondria during the apoptotic process. BAX was downregulated as the spinal cord developed, consistent with a reduced susceptibility to ethanol-induced apoptosis. Akt/GSK3β signaling and BAX together determine the direction of alcohol-induced apoptosis and its susceptibility to change during developmental stages in the spinal cord.

Exploring P2X7 receptor antagonism as a therapeutic target for neuroprotection in an hiPSC motor neuron model.

ATP is present in negligible concentrations in the interstitium of healthy tissues but accumulates to significantly higher concentrations in an inflammatory microenvironment. ATP binds to 2 categories of purine receptors on the surface of cells, the ionotropic P2X receptors and metabotropic P2Y receptors. Included in the family of ionotropic purine receptors is P2X7 (P2X7R), a non-specific cation channel with unique functional and structural properties that suggest it has distinct roles in pathological conditions marked by increased extracellular ATP. The role of P2X7R has previously been explored in microglia and astrocytes within the context of neuroinflammation, however the presence of P2X7R on human motor neurons and its potential role in neurodegenerative diseases has not been the focus of the current literature. We leveraged the use of human iPSC-derived spinal motor neurons (hiPSC-MN) as well as human and rodent tissue to demonstrate the expression of P2X7R on motor neurons. We extend this observation to demonstrate that these receptors are functionally active on hiPSC-MN and that ATP can directly induce death via P2X7R activation in a dose dependent manner. Finally, using a highly specific P2X7R blocker, we demonstrate how modulation of P2X7R activation on motor neurons is neuroprotective and could provide a unique pharmacologic target for ATP-induced MN death that is distinct from the role of ATP as a modulator of neuroinflammation.

Metal Ruthenium Complexes Treat Spinal Cord Injury By Alleviating Oxidative Stress Through Interaction With Antioxidant 1 Copper Chaperone Protein.

Oxidative stress is a major factor affecting spinal cord injury (SCI) prognosis. A ruthenium metal complex can aid in treating SCI by scavenging reactive oxygen species via a protein-regulated mechanism to alleviate oxidative stress. This study aimed to introduce a pioneering strategy for SCI treatment by designing two novel half-sandwich ruthenium (II) complexes containing diverse N^N-chelating ligands. The general formula is [(η6-Arene)Ru(N^N)Cl]PF6, where arene is either 2-phenylethanol-1-ol (bz-EA) or 3-phenylpropanol-1-ol (bz-PA), and the N^N-chelating ligands are fluorine-based imino-pyridyl ligands. This study shows that these ruthenium metal complexes protect neurons by scavenging reactive oxygen species. Notably, η6-Arene substitution from bz-PA to bz-EA significantly enhances reactive oxygen species scavenging ability and neuroprotective effect. Additionally, molecular dynamics simulations indicate that the ruthenium metal complex increases Antioxidant 1 Copper Chaperone protein expression, reduces oxidative stress, and protects neurons during SCI treatment. Furthermore, ruthenium metal complex protected spinal cord neurons and stimulated their regeneration, which improves electrical signals and motor functions in mice with SCI. Thus, this treatment strategy using ruthenium metal complexes can be a new therapeutic approach for the efficient treatment of SCI.

Spinal lumbar Urocortin 3-expressing neurons are associated with both scratching and Compound 48/80-induced sensations.

Urocortin 3 is a neuropeptide that belongs to the corticotropin-releasing hormone family and is involved in mechanosensation and stress regulation. In this study, we show that Urocortin 3 marks a population of excitatory neurons in the mouse spinal cord, divided into 2 nonoverlapping subpopulations expressing protein kinase C gamma or calretinin/calbindin 2, populations previously associated with mechanosensation. Electrophysiological experiments demonstrated that lumbar spinal Urocortin 3 neurons receive both glycinergic and GABAergic local tonic inhibition, and monosynaptic inputs from both Aβ and C fibers, which could be confirmed by retrograde trans-synaptic rabies tracing. Furthermore, fos analyses showed that subpopulations of lumbar Urocortin 3 neurons are activated by artificial scratching or Compound 48/80-induced sensations. Chemogenetic activation of lumbar Urocortin 3-Cre neurons evoked a targeted biting/licking behavior towards the corresponding dermatome and chemogenetic inhibition decreased Compound 48/80-induced behavior. Hence, spinal lumbar Urocortin 3 neurons represent a mechanically associated population with roles in both scratching and Compound 48/80-induced sensations.

Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.

Effect of hypoxia in the post-hatching development of the salmon (Salmo salar L.) spinal cord

IntroductionHypoxia has a teratogenic effect on the fish during embryonic development. Nevertheless, the effects on the larval stage are not yet known. Therefore, the aim of this study was to assess the effects of hypoxia on the number of neurons and their apoptotic rate in the spinal cord of Salmo salar alevins after hatching.MethodsWe used a total of 400 alevins, establishing both hypoxia and control (normoxia) groups (n = 8), considering post-hatching days 1, 3, 5, and 7, each with 50 individuals. Transversal sections of 50 μm thickness were cut from the alevin body. We performed cresyl-violet staining and counted the spinal cord neurons. Also, immunohistochemistry for HIF-1α and caspase-3 were used. For statistical analysis ANOVA one-way and Tukey's Test were used.ResultsHIF-1α was expressed in spinal neurons in both the hypoxic and normoxic groups, with the former being significantly higher. Both the hypoxic and normoxic groups evidenced the process of neuronal apoptosis, with the hypoxic groups demonstrating a higher significance. The number of neurons in the spinal cord was significantly lower in the hypoxic group.DiscussionWe found that when oxygen levels in the aquatic environment were low in Salmo salar farming alevins post-hatch, the number of spinal neurons dropped by half. These results contribute to increasing our knowledge of the biological development of salmon, in particular the genesis of the spinal cord, and the effects of hypoxic conditions on the development of this structure of the nervous system.

Immune cells promote paralytic disease in mice infected with enterovirus D68.

Enterovirus D68 (EV-D68) is associated with acute flaccid myelitis (AFM), a poliomyelitis-like illness causing paralysis in young children. However, mechanisms of paralysis are unclear, and antiviral therapies are lacking. To better understand EV-D68 disease, we inoculated newborn mice intracranially to assess viral tropism, virulence, and immune responses. Wild-type (WT) mice inoculated intracranially with a neurovirulent strain of EV-D68 showed infection of spinal cord neurons and developed paralysis. Spinal tissue from infected mice revealed increased levels of chemokines, inflammatory monocytes, macrophages, and T cells relative to controls, suggesting that immune cell infiltration influences pathogenesis. To define the contribution of cytokine-mediated immune cell recruitment to disease, we inoculated mice lacking CCR2, a receptor for several EV-D68-upregulated cytokines, or RAG1, which is required for lymphocyte maturation. WT, Ccr2 -/- , and Rag1 -/- mice had comparable viral titers in spinal tissue. However, Ccr2 -/- and Rag1 -/- mice had significantly less paralysis relative to WT mice. Consistent with impaired T cell recruitment to sites of infection in Ccr2 -/- and Rag1 -/- mice, antibody-mediated depletion of CD4 + or CD8 + T cells from WT mice diminished paralysis. These results indicate that immune cell recruitment to the spinal cord promotes EV-D68-associated paralysis and illuminate new targets for therapeutic intervention.

Slack potassium channels in spinal dorsal horn neurons control neuropathic pain and acute itch.

The sodium-activated potassium channel Slack (KNa1.1, Kcnt1) plays a critical role in tuning neuronal excitability. Previous studies have revealed that Slack is expressed in neurons of the superficial dorsal horn of the spinal cord. However, the precise role of Slack in spinal dorsal horn neurons is unclear. In this study, we used mice in which Slack is conditionally ablated in spinal dorsal horn neurons (Lbx1-Slack-/- mice) and analyzed their behaviors in various models of pain and itch. Lbx1-Slack-/- mice exhibited increased neuropathic pain behavior after peripheral nerve injury but normal responses in a model of inflammatory pain. Unexpectedly, Lbx1-Slack-/- mice demonstrated increased scratching after intradermal injection of chloroquine, LY344864, and histamine. Moreover, neuromedin B receptors are coexpressed with Slack in the dorsal horn, and scratching after intrathecal delivery of neuromedin B was increased in Lbx1-Slack-/- mice. Our study provides in vivo evidence that Slack expressed in spinal dorsal horn neurons inhibits nerve injury-induced allodynia and acute itch induced by various pruritogens.

Targeting exosomal double-stranded RNA-TLR3 signaling pathway attenuates morphine tolerance and hyperalgesia

Long-term morphine use leads to tolerance and hyperalgesia in patients with chronic pain, with neuroinflammation playing a key role, but its underlying mechanisms remain elusive. This study determines that repeated intrathecal morphine injections increase double-stranded RNA (dsRNA) production in spinal neurons, due to downregulated adenosine deaminase RNA specific 1 (ADAR1) expression. Lentivirus-induced ADAR1 elevation decreases the high levels of intracellular dsRNA and attenuates morphine tolerance and hyperalgesia. dsRNA is released into cerebrospinal fluid via exosomes (Exos) after repeated morphine injections and is taken up by microglia for TLR3-TRIF-IL-6 signaling activation. Blocking Exos release with GW4869 or inhibition of TLR3 signaling mitigates neuroinflammation, preventing the development of morphine tolerance and hyperalgesia. Intrathecal injection of TLR3 inhibitor alone shows analgesic effects in neuropathic pain, and co-administration with morphine amplifies the analgesic efficacy of morphine. These findings demonstrate that targeting dsRNA-TLR3 signaling to mitigate neuroinflammation could be a promising treatment for morphine tolerance.

A human-specific progenitor sub-domain extends neurogenesis and increases motor neuron production.

Neurogenesis lasts ~10 times longer in developing humans compared to mice, resulting in a >1,000-fold increase in the number of neurons in the CNS. To identify molecular and cellular mechanisms contributing to this difference, we studied human and mouse motor neurogenesis using a stem cell differentiation system that recapitulates species-specific scales of development. Comparison of human and mouse single-cell gene expression data identified human-specific progenitors characterized by coexpression of NKX2-2 and OLIG2 that give rise to spinal motor neurons. Unlike classical OLIG2+ motor neuron progenitors that give rise to two motor neurons each, OLIG2+/NKX2-2+ ventral motor neuron progenitors remain cycling longer, yielding ~5 times more motor neurons that are biased toward later-born, FOXP1-expressing subtypes. Knockout of NKX2-2 converts ventral motor neuron progenitors into classical motor neuron progenitors. Such new progenitors may contribute to the increased production of human motor neurons required for the generation of larger, more complex nervous systems.

Remote limb ischemic preconditioning alleviated spinal cord injury through inhibiting proinflammatory immune response and promoting the survival of spinal neurons.

Experimental animal study. To investigate the protective effect of remote limb ischemia preconditioning (RLPreC) on traumatic spinal cord injury (SCI) and explore the underlying biological mechanisms using RNA sequencing. China Rehabilitation Science Institute; Beijing; China. spinal cord injury was induced in mice using a force of 0.7 N. RLPreC treatment was administered. Motor function, pain behavior, and gene expression were assessed. RLPreC treatment significantly improved motor function and reduced pain-like behavior in SCI mice. RNA-Seq analysis identified 5247 differentially expressed genes (DEGs). GO analysis revealed enrichment of immune response, inflammatory signaling, and synaptic transmission pathways among these DEGs. KEGG analysis indicated suppression of inflammation and promotion of synapse-related pathways. RLPreC is a promising therapeutic strategy for improving motor function and alleviating pain after traumatic SCI. RNA-Seq analysis provides insights into potential therapeutic targets and warrants further investigation.

STM2457 Inhibits METTL3-Mediated m6A Modification of miR-30c to Alleviate Spinal Cord Injury by Inducing the ATG5-Mediated Autophagy.

The study aimed to investigate the role of N6-methyladenosine (m6A) modification in spinal cord injury (SCI) and its underlying mechanism, focusing on the interplay between m6A methyltransferase-like 3 (METTL3), miR-30c, and autophagy-related proteins. An SCI model was established in rats, and changes in autophagy-related proteins, m6A methylation levels, and miR-30c levels were analyzed. Hydrogen peroxide (H2O2)-stimulated spinal cord neuron cells (SCNCs) were used to assess the impact of METTL3 overexpression. The effects of STM2457, an antagonist of METTL3, were evaluated on cell viability, apoptosis, and autophagy markers in H2O2-stimulated SCNCs. In the SCI model, decreased levels of autophagy markers and increased m6A methylation, miR-30c levels, and METTL3 were observed. Overexpression of METTL3 in SCNCs led to reduced cell viability, increased apoptosis, and suppressed autophagy. Conversely, co-overexpression of autophagy-related protein 5 (ATG5) or miR-30c inhibition reversed these effects. Knocking out METTL3 yielded opposite results. STM2457 treatment improved cell viability, reduced apoptosis, and upregulated autophagy markers in SCNCs, which also enhanced functional recovery in rats as measured by the Basso-Beattie-Bresnahan score and inclined plate test. STM2457 alleviated SCI by suppressing METTL3-mediated m6A modification of miR-30c, which in turn induces ATG5-mediated autophagy. This study provides insights into the role of m6A modification in SCI and suggests a potential therapeutic approach through targeting METTL3.

Expression of Neurotrophic Factors and Their Receptors in the Rat Spinal Trigeminal Nucleus

The spinal trigeminal nucleus as part of the trigeminal sensory nuclear complex is associated with the transmission of discriminative tactile sensations, primarily pain, and temperature, from the orofacial region. It is divided into three parts – caudal, interpolar, and oral subnucleus which process pain and temperature stimulate. Our previous experiments have revealed the presence of certain neurotrophic factors such as the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) in this nucleus. These neurotrophins facilitate neuronal differentiation, survival, and plasticity by signalling through high-affinity transmembrane receptors. By using primary antibodies against NGF, BDNF, NT-3, and glial-derived neurotrophic factor (GDNF), and their corresponding receptors of the tyrosine receptor kinase (Trk) proto-oncogene family and GFRα1, we found immunoreactive cells scattered along the whole length of the nucleus in rats in all three subnuclei. In particular, we observed that the majority of spinal trigeminal neurons were intensely immunostained for all the neurotrophic factors examined and that they were richly endowed with their Trk receptors. Our results also showed that NGF, NT-3, TrkC and GFRα1 expression did not differ between different topographic regions of the nucleus, while BDNF, GDNF, and TrkA expression was highest in the SpVo, and TrkB expression was highest in the SpVi and SpVc. Given that these neurotrophic factors are involved in mechanisms of central sensitization in trigeminal nociceptive pathways, it can be inferred that neurotrophins may contribute to a better understanding of the fundamental mechanisms of orofacial pain.

Kappa opioids inhibit spinal output neurons to suppress itch.

Itch is a protective sensation that drives scratching. Although specific cell types have been proposed to underlie itch, the neural basis for itch remains unclear. Here, we used two-photon Ca2+ imaging of the dorsal horn to visualize neuronal populations that are activated by itch-inducing agents. We identify a convergent population of spinal interneurons recruited by diverse itch-causing stimuli that represents a subset of neurons that express the gastrin-releasing peptide receptor (GRPR). Moreover, we find that itch is conveyed to the brain via GRPR-expressing spinal output neurons that target the lateral parabrachial nuclei. We then show that the kappa opioid receptor agonist nalfurafine relieves itch by selectively inhibiting GRPR spinoparabrachial neurons. These experiments provide a population-level view of the spinal neurons that respond to pruritic stimuli, pinpoint the output neurons that convey itch to the brain, and identify the cellular target of kappa opioid receptor agonists for the inhibition of itch.

Spastin accumulation and motor neuron defects caused by a novel SPAST splice site mutation

BackgroundHereditary spastic paraplegia (HSP) is a rare genetically heterogeneous neurodegenerative disorder. The most common type of HSP is caused by pathogenic variants in the SPAST gene. Various hypotheses regarding the pathogenic mechanisms of HSP-SPAST have been proposed. However, a single hypothesis may not be sufficient to explain HSP-SPAST.ObjectiveTo determine the causative gene of autosomal dominant HSP-SPAST in a pure pedigree and to study its underlying pathogenic mechanism.MethodsA four-generation Chinese family was investigated. Genetic testing was performed for the causative gene, and a splice site variant was identified. In vivo and in vitro experiments were conducted separately. Western blotting and immunofluorescence were performed after transient transfection of cells with the wild-type (WT) or mutated plasmid. The developmental expression pattern of zebrafish spasts was assessed via whole-mount in situ hybridization. The designed guide RNA (gRNA) and an antisense oligo spast-MO were microinjected into Tg(hb9:GFP) zebrafish embryos, spinal cord motor neurons were observed, and a swimming behavioral analysis was conducted.ResultsA novel heterozygous intron variant, c.1004 + 5G > A, was identified in a pure HSP-SPAST pedigree and shown to cosegregate with the disease phenotypes. This intron splice site variant skipped exon 6, causing a frameshift mutation that resulted in a premature termination codon. In vitro, the truncated protein was evenly distributed throughout the cytoplasm, formed filamentous accumulations around the nucleus, and colocalized with microtubules. Truncated proteins diffusing in the cytoplasm appeared denser. No abnormal microtubule structures were observed, and the expression levels of α-tubulin remained unchanged. In vivo, zebrafish larvae with this mutation displayed axon pathfinding defects, impaired outgrowth, and axon loss. Furthermore, spast-MO larvae exhibited unusual behavioral preferences and increased acceleration.ConclusionThe adverse effects of premature stop codon mutations in SPAST result in insufficient levels of functional protein, and the potential toxicity arising from the intracellular accumulation of spastin serves as a contributing factor to HSP-SPAST.

Differential susceptibility of human motor neurons to infection with Usutu and West Nile virus

West Nile virus (WNV) and Usutu virus (USUV) are closely related flaviviruses with differing capacities to cause neurological disease in humans. WNV is thought to use a transneural route of neuroinvasion along motor neurons and causes severe motor deficits. The potential for use of transneural routes of neuroinvasion by USUV has not been investigated experimentally, and evidence from the few clinical case reports of USUV-associated neuroinvasive disease is lacking. We hypothesised that, compared with WNV, USUV is less able to infect motor neurons, and therefore determined the susceptibility of human induced pluripotent stem cell (iPSC)-derived spinal cord motor neurons to infection. Both viruses could grow to high titres in iPSC-derived neural cultures. However, USUV could not productively infect motor neurons due to restriction by the antiviral response, which was not induced upon WNV infection. Inhibition of the antiviral response allowed for widespread infection and transportation of USUV along motor neurons within a compartmented culture system. These results show a stark difference in the ability of these two viruses to evade initiation of intrinsic antiviral immunity. Our data suggests that USUV cannot infect motor neurons in healthy individuals but in case of immunodeficiency may pose a risk for motor-related neurological disease and transneural invasion.

Spinal neuron diversity scales exponentially with swim-to-limb transformation during frog metamorphosis.

Vertebrates exhibit a wide range of motor behaviors, ranging from swimming to complex limb-based movements. Here we take advantage of frog metamorphosis, which captures a swim-to-limb-based movement transformation during the development of a single organism, to explore changes in the underlying spinal circuits. We find that the tadpole spinal cord contains small and largely homogeneous populations of motor neurons (MNs) and V1 interneurons (V1s) at early escape swimming stages. These neuronal populations only modestly increase in number and subtype heterogeneity with the emergence of free swimming. In contrast, during frog metamorphosis and the emergence of limb movement, there is a dramatic expansion of MN and V1 interneuron number and transcriptional heterogeneity, culminating in cohorts of neurons that exhibit striking molecular similarity to mammalian motor circuits. CRISPR/Cas9-mediated gene disruption of the limb MN and V1 determinants FoxP1 and Engrailed-1, respectively, results in severe but selective deficits in tail and limb function. Our work thus demonstrates that neural diversity scales exponentially with increasing behavioral complexity and illustrates striking evolutionary conservation in the molecular organization and function of motor circuits across species.

Heterogeneity of synaptic NMDA receptor responses within individual lamina I pain-processing neurons across sex in rats and humans.

Excitatory glutamatergic NMDA receptors (NMDARs) are key regulators of spinal pain processing, and yet the biophysical properties of NMDARs in dorsal horn nociceptive neurons remain poorly understood. Despite the clinical implications, it is unknown whether the molecular and functional properties of synaptic NMDAR responses are conserved between males and females or translate from rodents to humans. To address these translational gaps, we systematically compared individual and averaged excitatory synaptic responses from lamina I pain-processing neurons of adult Sprague-Dawley rats and human organ donors, including both sexes. By combining patch-clamp recordings of outward miniature excitatory postsynaptic currents with non-biased data analyses, we uncovered a wide range of decay constants of excitatory synaptic events within individual lamina I neurons. Decay constants of synaptic responses were distributed in a continuum from 1-20ms to greater than 1000ms, suggesting that individual lamina I neurons contain AMPA receptor (AMPAR)-only as well as GluN2A-, GluN2B- and GluN2D-NMDAR-dominated synaptic events. This intraneuronal heterogeneity in AMPAR- and NMDAR-mediated decay kinetics was observed across sex and species. However, we discovered an increased relative contribution of GluN2A-dominated NMDAR responses at human lamina I synapses compared with rodent synapses, suggesting a species difference relevant to NMDAR subunit-targeting therapeutic approaches. The conserved heterogeneity in decay rates of excitatory synaptic events within individual lamina I pain-processing neurons may enable synapse-specific forms of plasticity and sensory integration within dorsal horn nociceptive networks. KEY POINTS: Synaptic NMDA receptors (NMDARs) in spinal dorsal horn nociceptive neurons are key regulators of pain processing, but it is unknown whether their functional properties are conserved between males and females or translate from rodents to humans. In this study, we compared individual excitatory synaptic responses from lamina I pain-processing neurons of male and female adult Sprague-Dawley rats and human organ donors. Individual lamina I neurons from male and female rats and humans contain AMPA receptor-only as well as GluN2A, GluN2B- and GluN2D-NMDAR-dominated synaptic events. This may enable synapse-specific forms of plasticity and sensory integration within dorsal horn nociceptive networks. Human lamina I synapses have an increased relative contribution of GluN2A-dominated NMDAR responses compared with rodent synapses. These results uncover a species difference relevant to NMDAR subunit-targeting therapeutic approaches.