A"messenger zone hypothesis"based on the visual three-dimensional spatial distribution of motoneurons innervating deep limb muscles

Coordinated contraction of skeletal muscles relies on selective connections between the muscles and multiple classes of the spinal motoneuro ns.Howeve r,current research on the spatial location of the spinal motoneurons innervating differe nt muscles is limited.In this study,we investigated the spatial distribution and relative position of different motoneurons that control the deep muscles of the mouse hindlimbs,which were innervated by the obturator nerve,femoral nerve,inferior gluteal nerve,deep pe roneal nerve,and tibial nerve.Locations were visualized by combining a multiplex retrograde tracking technique compatible with three-dimensional imaging of solvent-cleared o rgans(3DISCO)and 3-D imaging technology based on lightsheet fluorescence microscopy(LSFM).Additionally,we propose the hypothesis that"messenger zones"exist as interlaced areas between the motoneuron pools that dominate the synergistic or antagonist muscle groups.We hypothesize that these interlaced neurons may participate in muscle coordination as messenger neurons.Analysis revealed the precise mutual positional relationships among the many motoneurons that innervate different deep muscles of the mouse.Not only do these findings update and supplement our knowledge regarding the overall spatial layout of spinal motoneurons that control mouse limb muscles,but they also provide insights into the mechanisms through which muscle activity is coordinated and the architecture of motor circuits.

Early electrical field stimulation prevents the loss of spinal cord anterior horn motoneurons and muscle atrophy following spinal cord injury

Our previous study revealed that early application of electrical field stimulation（EFS） with the anode at the lesion and the cathode distal to the lesion reduced injury potential, inhibited secondary injury and was neuroprotective in the dorsal corticospinal tract after spinal cord injury（SCI）. The objective of this study was to further evaluate the effect of EFS on protection of anterior horn motoneurons and their target musculature after SCI and its mechanism. Rats were randomized into three equal groups. The EFS group received EFS for 30 minutes immediately after injury at T_（10）. SCI group rats were only subjected to SCI and sham group rats were only subjected to laminectomy. Luxol fast blue staining demonstrated that spinal cord tissue in the injury center was better protected; cross-sectional area and perimeter of injured tissue were significantly smaller in the EFS group than in the SCI group. Immunofluorescence and transmission electron microscopy showed that the number of spinal cord anterior horn motoneurons was greater and the number of abnormal neurons reduced in the EFS group compared with the SCI group. Wet weight and cross-sectional area of vastus lateralis muscles were smaller in the SCI group to in the sham group. However, EFS improved muscle atrophy and behavioral examination showed that EFS significantly increased the angle in the inclined plane test and Tarlov＇s motor grading score. The above results confirm that early EFS can effectively impede spinal cord anterior horn motoneuron loss, promote motor function recovery and reduce muscle atrophy in rats after SCI.

Peripheral nerve fibroblasts secrete neurotrophic factors to promote axon growth of motoneurons

Peripheral nerve fibroblasts play a critical role in nerve development and regeneration.Our previous study found that peripheral nerve fibroblasts have different sensory and motor phenotypes.Fibroblasts of different phenotypes can guide the migration of Schwann cells to the same sensory or motor phenotype.In this study,we analyzed the different effects of peripheral nerve-derived fibroblasts and cardiac fibroblasts on motoneurons.Compared with cardiac fibroblasts,peripheral nerve fibroblasts greatly promoted motoneuron neurite outgrowth.Transcriptome analysis results identified 491 genes that were differentially expressed in peripheral nerve fibroblasts and cardiac fibroblasts.Among these,130 were significantly upregulated in peripheral nerve fibroblasts compared with cardiac fibroblasts.These genes may be involved in axon guidance and neuron projection.Three days after sciatic nerve transection in rats,peripheral nerve fibroblasts accumulated in the proximal and distal nerve stumps,and most expressed brain-derived neurotrophic factor.In vitro,brain-derived neurotrophic factor secreted from peripheral nerve fibroblasts increased the expression ofβ-actin and F-actin through the extracellular regulated protein kinase and serine/threonine kinase pathways,and enhanced motoneuron neurite outgrowth.These findings suggest that peripheral nerve fibroblasts and cardiac fibroblasts exhibit different patterns of gene expression.Peripheral nerve fibroblasts can promote motoneuron neurite outgrowth.

Neuroprotective effects of recombinant human erythropoietin on facial motoneurons after facial nerve injury in rats

BACKGROUND： Erythropoietin and recombinant human erythropoietin （rhEPO） inhibit apoptosis of motor neurons caused by spinal cord injury and brain damage in rats. However, it still remains to be shown whether rhEPO can protect facial motoneurons （FMNs） as Well. OBJECTIVE： To test the neuroprotective effects of rhEPO on injured VMNs, as well as the influence on Caspase-3 expression. DESIGN, TIME AND SETTING： Randomized, controlled, animal experiment. This study was performed at the Central Laboratory of Basic Medical College, Chongqing Medical University from January to October 2007. MATERIALS： Seventy-five female SD rats, weighing 210-230 g. rhEPO injection was provided by Sansheng pharmaceuticals company, Shenyang City, Liaoning Province, China, and the License number was HMLN S20010001. METHODS： A total of 75 female rats were randomly divided into rhEPO treatment, control, and sham operation groups, with 25 rats in each group. Rat models of facial nerve injury were established in the rhEPO treatment group and the control group by crushing the main trunk of the left facial nerve. Surgical microscopic observation of the facial nerve damage displayed perineurial disruption. The left stylomastoid foramen of the sham operation group were only exposed, but without nerve injury. The rhEPO treatment group was treated with rhEPO （5 000 U/kg, i.p.） once following injury and once a day for two weeks. The control and sham operation groups were treated with the same dose of normal saline （i.p.）, once following injury and once a day for two weeks. MAIN OUTCOME MEASURES： Rats were sacrificed 3, 7, 14, 21, and 28 days after injury, FMN survival after facial nerve injury was analyzed by Toluidine blue staining, and then survival ratios （L/R） were calculated. The number of apoptotic profiles in the injured FMNs were evaluated by TUNEL staining. Expression of Caspase-3 in the facial nucleus was detected by immunohistochemistry methods. RESULTS： A total of 75 rats were included in the final analysis. FMN survival ratios, both in rhEPO treatment group and control group, decreased gradually between seven and 28 days; however, FMN survival ratios were significantly greater in the rhEPO treatment group compared to the control group （P 〈 0.05）. No TUNEL-positive cells were observed three days after injury in the rhEPO treatment and control groups; however, by seven days after injury, apoptotic cells were observed and peaked by 14 days in the control group. Between seven and 21 days, apoptotic cell numbers were significantly lower in the rhEPO treatment group compared to the control group （P 〈 0.05）. The expression of Caspase-3 increased three days after injury and peaked at 14 days in the control group. Nevertheless, Caspase-3 expression was significantly lower in the rhEPO treatment group compared to the control group at each time point （P 〈 0.05）. CONCLUSION： Treatment with rhEPO can effectively protect facial motoneurons by reducing expression of Caspase-3 and inhibiting apoptosis.

Vascular endothelial growth factor：an essential neurotrophic factor for motoneurons？

Vascular endothelial growth factor（VEGF）,an angiogenic factor with neuroprotective effects：The VEGF was initially characterized by its vasculogenic and angiogenic activities and its capacity to promote vascular permeability（Yancopoulos et al.,2000）.VEGF is also known as VEGF-A and is the prototype member of a related group of five trophic factors,VEGF-B,VEGF-C,VEGF-D and placental growth factor（Pl GF;Lange et al.,2016）.

Time windows for postnatal changes in morphology and membrane excitability of genioglossal and oculomotor motoneurons

Time windows for postnatal changes in morphology and membrane excitability of genioglossal(GG) and oculomotor(OCM) motoneurons(MNs) are yet to be fully described. Analysis of data on brain slices in vitro of the 2 populations of MNs point to a well-defined developmental program that progresses with common age-related changes characterized by:(1) increase of dendritic surface along with length and reshaping of dendritic tree complexity;(2) disappearance of gap junctions early in development;(3) decrease of membrane passive properties, such as input resistance and time constant, together with an increase in the number of cells displaying sag, and modifications in rheobase;(4) action potential shortening and afterhyperpolarization; and(5) an increase in gain and maximum firing frequency. These modifications take place at different time windows for each motoneuronal population. In GG MNs, active membrane properties change mainly during the first postnatal week, passive membrane properties in the second week, and dendritic increasing length and size in the third week of development. In OCM MNs, changes in passive membrane properties and growth of dendritic size take place during the first postnatal week, while active membrane properties and rheobase change during the second and third weeks of development. The sequential order of changes is inverted between active and passive membrane properties, and growth in size does not temporally coincide for both motoneuron populations. These findings are discussed on the basis of environmental cues related to maturation of the respiratory and OCM systems.

EXCITATORY CONNECTIONS BETWEEN SPINAL MOTONEURONS IN THE ADULT RAT

BZ]Dendro dendritic and dendro somatic projections are common between spinal motoneurons. We attempted to clarify whether there are functional connections through these projections. Methods. Motoneurons were antidromically stimulated by the muscle nerve and recorded intracellularly to examine the direct interaction between them, after the related dorsal roots had been cut. Results. Excitatory connections, demonstrated by depolarizing potentials in response to muscle nerve stimulation, were found between motoneurons innervating the same muscle or synergistic muscles, but never between motoneurons innervating antagonistic muscles. These potentials were finely graded in response to a series of increasing stimuli and resistant to high frequency (50Hz) stimulation. Conclusions.These results indicate that excitatory connections, with certain specificity of spatial and temporal distribution, occur in the spinal motoneurons. It is also suggested that electrical coupling should be involved in these connections and this mechanism should improve the excitability of the motoneurons in the same column.

P75 and phosphorylated c-Jun are differentially regulated in spinal motoneurons following axotomy in rats

The neurotrophin receptor （p75） activates the c-Jun N-terminal kinase （JNK） pathway. Activation of JNK and its substrate c-Jun can cause apoptosis. Here we evaluate the role of p75 in spinal motoneurons by comparing immunoreactivity for p75 and phosphorylated c-Jun （p-c-Jun）, the production of JNK activation in axotomized motoneurons in postnatal day （PN）I, PN7, PN14 and adult rats. Intensive p-c-Jun was induced in axotomized motoneurons in PN1 and PN7. In PN14, p-c-Jun expression was sharply reduced after the same injury. The decreased expression of p-c-Jun at this age coincided with a developmental switch of re-expression of p75 in axotomized cells. In adult animals, no p-c-Jun but intensive p75 was detected in axotomized motoneurons. These results indicate differential expression or turnover of phosphorylation of c-Jun and p75 in immature versus mature spinal motoneurons in response to axonal injury. The non-co-occurrence of p75 and p-c-Jun in injured motoneurons indicated that p75 may not activate JNK pathway, suggesting that the p75 may not be involved in cell death in axotomized motoneurons.

BDNF secreted from nerve fibroblasts promotes neurite outgrowth of motoneurons by activating ERK and AKT pathway

Peripheral nerve fibroblasts play an important role in the process of nerve development and regeneration.The present study revealed that fibroblasts with different tissue sources,such as peripheral nerve fibroblasts(N-Fbs)and cardiac Fbs(C-Fbs),exerted distinct effects on motoneurons.Compared to C-Fbs,N-Fbs significantly promoted neurite outgrowth of motoneurons in vitro.mRNA sequencing identified a total of 491 differentially expressed genes between N-Fbs and C-Fbs.Out of them,130 genes were significantly upregulated in N-Fbs than in C-Fbs,and these genes might be involved in axon guidance and neuron projection.

Expression of neurofilament gene in spinal motoneurons during neural regeneration

The technique in situ hybridization was used to measure the levels of light （NF-L）,medium（NF-M） and heavy （NF-H） neurofilament protein subunits mRNA in L4-6 spinal motoneurons in adult rat during regeneration following a unilateral crush of the sciatic nerve.It was found that the hybridization signals of each neurofilament subunit mRNA were dramatically decreased in spinal motoneurons postaxotomy by light microscopy. The hybridization signals of NF-L and NF-M mRNA were located in cytoplasm of neurons, whereas NF-H mRNA was found in both nucleus and cytoplasm of neurons. lmage analysis showed that the encoding levels of mRNA for each of neurofilament subunit mRNA reduced on the 3rd d and returned to control levels on the 28th d following the lesion. The relative levels of mRNA coding for each neurofilament subunit were significantly different. The lowest level of NF-L mRNA was observed at 5 d postaxotomy, and that of NF-M, NF-H mRNA on the 7th and 10th d after injury. Moreover,the levels of HF-M and

Expression of α Tubulin Gene in Motoneurons of Spinal Cord During Nerve Regeneration

We have previously reported that the injury of sciatic nerve causes not only the change of axonal ultrastructures, but also the increase of β tubulin gene expression in the neural soma. And the microtubule, as a carrier of transported materials, of axonal architecture plays an important role. It has also been found that the content and transported velocity of tubulin in the regeneration nerve increase significantly owing to the requirements of axonal reconstruction. As the NOR-Ag staining technique is specific

Intraspinal transplantation of motoneuron-like cell combined with delivery of polymer-based glial cell line-derived neurotrophic factor for repair of spinal cord contusion injury

To evaluate the effects of glial cell line-derived neurotrophic factor transplantation combined with adipose-derived stem cells-transdifferentiated motoneuron delivery on spinal cord con-tusion injury, we developed rat models of spinal cord contusion injury, 7 days later, injected adipose-derived stem cells-transdifferentiated motoneurons into the epicenter, rostral and caudal regions of the impact site and simultaneously transplanted glial cell line-derived neuro-trophic factor-gelfoam complex into the myelin sheath. Motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery reduced cavity formations and increased cell density in the transplantation site. The combined therapy exhibited superior promoting effects on recovery of motor function to transplantation of glial cell line-derived neurotrophic factor, adipose-derived stem cells or motoneurons alone. These ifndings suggest that motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery holds a great promise for repair of spinal cord injury.

Downregulation of EphB2 by RNA interference attenuates glial/fibrotic scar formation and promotes axon growth

The rapid formation of a glial/fibrotic scar is one of the main factors hampering axon growth after spinal cord injury. The bidirectional Eph B2/ephrin-B2 signaling of the fibroblast-astrocyte contact-dependent interaction is a trigger for glial/fibrotic scar formation. In the present study, a new in vitro model was produced by coculture of fibroblasts and astrocytes wounded by scratching to mimic glial/fibrotic scar-like structures using an improved slide system. After treatment with RNAi to downregulate Eph B2, changes in glial/fibrotic scar formation and the growth of VSC4.1 motoneuron axons were examined. Following RNAi treatment, fibroblasts and astrocytes dispersed without forming a glial/fibrotic scar-like structure. Furthermore, the expression levels of neurocan, NG2 and collagen I in the coculture were reduced, and the growth of VSC4.1 motoneuron axons was enhanced. These findings suggest that suppression of Eph B2 expression by RNAi attenuates the formation of a glial/fibrotic scar and promotes axon growth. This study was approved by the Laboratory Animal Ethics Committee of Jiangsu Province, China(approval No. 2019-0506-002) on May 6, 2019.

Current landscape in motoneuron regeneration and reconstruction for motor cranial nerve injuries

The intricate anatomy and physiology of cranial nerves have inspired clinicians and scientists to study their roles in the nervous system. Damage to motor cranial nerves may result from a variety of organic or iatrogenic insults and causes devastating functional impairment and disfigurement. Surgical innovations directed towards restoring function to injured motor cranial nerves and their associated organs have evolved to include nerve repair, grafting, substitution, and muscle transposition. In parallel with this progress, research on tissue-engineered constructs, development of bioelectrical interfaces, and modulation of the regenerative milieu through cellular, immunomodulatory, or neurotrophic mechanisms has proliferated to enhance the available repertoire of clinically applicable reconstructive options. Despite these advances, patients continue to suffer from functional limitations relating to inadequate cranial nerve regeneration, aberrant reinnervation, or incomplete recovery of neuromuscular function. These shortfalls have profound quality of life ramifications and provide an impetus to further elucidate mechanisms underlying cranial nerve denervation and to improve repair. In this review, we summarize the literature on reconstruction and regeneration of motor cranial nerves following various injury patterns. We focus on seven cranial nerves with predominantly efferent functions and highlight shared patterns of injuries and clinical manifestations. We also present an overview of the existing reconstructive approaches, from facial reanimation, laryngeal reinnervation, to variations of interposition nerve grafts for reconstruction. We discuss ongoing endeavors to promote nerve regeneration and to suppress aberrant reinnervation and the development of synkinesis. Insights from these studies will shed light on recent progress and new horizons in understanding the biomechanics of peripheral nerve neurobiology, with emphasis on promising strategies for optimizing neural regeneration and identifying future directions in the field of motor cranial neuron research.

Neural computational modeling reveals a major role of corticospinal gating of central oscillations in the generation of essential tremor

Essential tremor, also referred to as familial tremor, is an autosomal dominant genetic disease and the most common movement disorder. It typically involves a postural and motor tremor of the hands, head or other part of the body. Essential tremor is driven by a central oscillation signal in the brain. However, the corticospinal mechanisms involved in the generation of essential tremor are unclear. Therefore, in this study, we used a neural computational model that includes both monosynaptic and multisynaptic corticospinal pathways interacting with a propriospinal neuronal network. A virtual arm model is driven by the central oscillation signal to simulate tremor activity behavior. Cortical descending commands are classified as alpha or gamma through monosynaptic or multisynaptic corticospinal pathways, which converge respectively on alpha or gamma motoneurons in the spinal cord. Several scenarios are evaluated based on the central oscillation signal passing down to the spinal motoneurons via each descending pathway. The simulated behaviors are compared with clinical essential tremor characteristics to identify the corticospinal pathways responsible for transmitting the central oscillation signal. A propriospinal neuron with strong cortical inhibition performs a gating function in the generation of essential tremor. Our results indicate that the propriospinal neuronal network is essential for relaying the central oscillation signal and the production of essential tremor.

Network-centric medicine for peripheral nerve injury:treating the whole to boost endogenous mechanisms of neuroprotection and regeneration

Peripheral nerve injuries caused by accidents may lead to paralysis,sensory disturbances,anaesthesia,and lack of autonomic functions.Functional recovery after disconnection of the motoneuronal soma from target tissue with proximal rupture of axons is determined by several factors:motoneuronal soma viability,proper axonal sprouting across inhibitory zones and elongation toward specific muscle,effective synapse contact rebuilding,and prevention of muscle atrophy.Therapies,such as adjuvant drugs with pleiotropic effects,that promote functional recovery after peripheral nerve injury are needed.Toward this aim,we designed a drug discovery workflow based on a network-centric molecular vision using unbiased proteomic data and neural artificial computational tools.Our focus is on boosting intrinsic capabilities of neurons for neuroprotection;this is in contrast to the common approach based on suppression of a pathobiological pathway known to be associated with disease condition.Using our workflow,we discovered neuroheal,a combination of two repurposed drugs that promotes motoneuronal soma neuroprotection,is anti-inflammatory,enhances axonal regeneration after axotomy,and reduces muscle atrophy.This drug discovery workflow has thus yielded a therapy that is close to its clinical application.

Locomotor analysis identifies early compensatory changes during disease progression and subgroup classification in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a motoneuron degenerative disease that is challenging to diagnose and presents with considerable variability in survival.Early identification and enhanced understanding of symptomatic patterns could aid in diagnosis and provide an avenue for monitoring disease progression.Use of the m SOD1 G93 A mouse model provides control of the confounding environmental factors and genetic heterogeneity seen in amyotrophic lateral sclerosis patients,while investigating underlying disease-induced changes.In the present study,we performed a longitudinal behavioral assessment paradigm and identified an early hindlimb symptom,resembling the common gait abnormality foot drop,along with an accompanying forelimb compensatory mechanism in the m SOD1 G93 A mouse.Following these initial changes,m SOD1 mice displayed a temporary hindlimb compensatory mechanism resembling an exaggerated steppage gait.As the disease progressed,these compensatory mechanisms were not sufficient to sustain fundamental locomotor parameters and more severe deficits appeared.We next applied these initial findings to investigate the inherent variability in B6 SJL m SOD1 G93 A survival.We identified four behavioral variables that,when combined in a cluster analysis,identified two subpopulations with different disease progression rates：a fast progression group and a slow progression group.This behavioral assessment paradigm,with its analytical approaches,provides a method for monitoring disease progression and detecting m SOD1 subgroups with different disease severities.This affords researchers an opportunity to search for genetic modifiers or other factors that likely enhance or slow disease progression.Such factors are possible therapeutic targets with the potential to slow disease progression and provide insight into the underlying pathology and disease mechanisms.

NEUROBIOLOGICAL EFFECTS OF NRCF DERIVED FROM DISTAL STUMPS OF MOTOR NERVE AND SENSORY NERVE AND b-FGF ON CULTURED MOTONEURON IN VITRO

Objective: To explore the mechanism of neurotropism in peripheral nerve regeneration by assessing the bioactivity of regeneration microcircumstance on motoneurons. Methods The motor branch Of femoral nerve to quadriceps was incised and the distal stump was sutured with one-end silicone chamber. The nerve regeneration chamber fluids from distal motor nerve stumps (motor branch of femoral nerve ) (MD-NRCF) was collected 7d post-operatively, and with the same method, nerve regeneration conditioned fluids from distal stumps nerve stumps (saphenous nerve ) (SD-NRCF) was collected. The dissociated rat’s motoneurons were co-cultured with MD-NRCF, SD-NRCF, basic fibroblast growth factor (b-FGF) and serum-free medium for 72h respectively and then were photographed under phase-contrast microscope. The longest neurites and cellbody areas of motoneurons from each group were measured by cell image processing computer system. MTT colorimetric assay was also used to measure cell activation. Results The cells of MD-NRCF group had significantly longer neurites than the other 3 groups, and their activation was also superior to those of the other groups. Conclusion These results indicate that MD-NRCF has more significant neurite-promoting and neurobiological effects on motoneuron than SD-NRCF and b-FGF.

神经递质释放中的有关蛋白

神经递质释放中的有关蛋白ＮＩＰＳ１９９５年２月号综述了神经递质释放过程中的许多重要蛋白质的作用。这些蛋白质分属于突触囊泡膜、胞液及神经末梢质膜。在突触囊泡膜上的蛋白有：Ｓｙｎａｐｔｏｂｒｅｖｉｎ，Ｓｙｎａｐｔｏｔａｇｍｉｎ，Ｓｙｎａｐｔｏｐｈｙ－ｓｉ...

F Wave Study in Amyotrophic Lateral Sclerosis： Assessmen of Segmental Motoneuronal Dysfunction

Background： Dysfunctional spinal circuit may play a role in the pathophysiology ofarnyotrophic lateral sclerosis （ALS）. The purpose of this study was to use F waves for assessment of segmental motoneuronal excitability following upper motor neuron （UMN） dysfunctions in ALS. Meihods： We studied the F waves of 152 ulnar nerves recorded fi＇om abductor digiti minimi in 82 patients with ALS. Two groups of hands were defined based on the presence or absence of pyramidal signs in the same upper limb. The group with pyramidal signs in tile upper limbs was designated as the P group, and the group without pyramidal signs in the upper limbs was designated as the NP group. Results： The mean （P 〈 0.001）, median （P 〈 0.001） and maximum （P = 0.035） F wave amplitudes, mean （P 〈 0.001 ）, median （P 〈 0.001） and maximum （P - 0.003） F/M amplitude ratio, index repeating neuron （P 〈 0.001 ） and index repeater F waves （P 〈 0.001 ） of the P group were significantly increased compared with the NP group. No significant differences were identified for F wave chronodispersion （P= 0.628）, mean F wave latency （P 0.151 ）, minimum F wave latency （P = 0.211 ）, maximum F wave latency （13 = 0. 199）. F wave persistence （P = 0.738）. F wave duration （P = 0. 152）, F wave conduction velocity （P = 0.813） and number of giant F waves （P = 0.072） between the two groups. Conclusions＂ In this study, increased F wave amplitude, F/M amplitude ratio and number of repeater F waves reflected enhanced segmental lnotoneuronal excitability following UMN dysfunctions in ALS.