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.

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.

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.

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.

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.

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.

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.

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.

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 contusion 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 neurotrophic 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 findings 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.

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 FMNs, 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.

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)1, 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.

EFFECT OF 22kD,35kD PROTEIN MOLECULES FROM EXTRACT OF SKELETAL MUSCLE ON CULTURED ANTERIOR HORN MOTONEURON OF LUMBAR SPINE IN RAT

Ⅰ. INTRODUCTION The survival of motoneurons in vertebrates during their embryonic period depends on certain neurotrophic substances provided from their skeletal muscles. Skeletal muscles have been shown, in in vitro and in vivo experiments, to contain substances which are capable of enhancing the survival and development of motoneurons but preventing the

CGRP对离体脊髓运动神经元突触传递的影响

目的:观察降钙素基因相关肽(CGRP)对新生大鼠离体脊髓运动神经元(MN)突触传递的影响。方法:选取8~12日龄的新生SD大鼠制备离体脊髓横切片,运用细胞内记录技术进行MN生物电记录,在同侧腹外侧索(iVLF)和同侧背根(iDR)施加电刺激(单脉冲,0.1~0.2 ms,0.1 Hz,10~100 V)以诱发兴奋性突触后电位(EPSP),即iVLF-EPSP和iDR-EPSP。给予CGRP灌流,观察其对脊髓MN突触传递的影响。结果:①在7个稳定记录的MN,诱导出iVLF-EPSP并灌流1μmol/L CGRP 15 min,在其中的5个MN上观察到iVLF-EPSP的时程被缩短(P<0.05)。②在5个稳定记录的MN,诱导出iDR-EPSP并灌流1μmol/L CGRP 15 min,在其中的3个MN上观察到iDR-EPSP的曲线下面积被抑制(P<0.05)、上升时间被缩短(P<0.01)。③在2个稳定记录的MN中,诱导出iDR-EPSP并灌流1、5μmol/L CGRP各15 min,观察到iDR-EPSP幅度的平均抑制率分别为6%、36%。结论:CGRP对部分脊髓MN的下行激活和外周传入突触传递可能存在抑制作用。

高精度经颅直流电刺激对踝关节跖背屈疲劳任务中脊髓运动神经元兴奋性的影响

目的探究踝关节跖背屈疲劳任务中高精度经颅直流电刺激(high-definition transcranial direct current stimulation,HD-tDCS)对H-反射和M-波的调控效果,为HD-tDCS减轻神经肌肉疲劳的实际应用提供方向。方法招募20名健康青年男性受试者,随机分为真刺激组和假刺激组各10名。对受试者采取连续5 d的单盲HD-tDCS干预(时间20 min;强度2 mA;靶点Cz),干预前1天采集受试者安静条件下的H-反射和M-波,跖屈肌最大自主收缩(maximal voluntary isometric contraction,MVIC)时的M-波,跖屈肌和背屈肌MVIC力矩,并进行一次踝关节跖背屈运动性疲劳任务,以确定受试者达到该任务疲劳的时间。干预后1天进行与第1次疲劳任务相同的运动时间,并进行后测的数据采集。采用重复测量双因素(刺激方案×疲劳前后)方差分析其自变量对受试者肌肉力学特性、α运动神经元传导特性各指标的影响。结果相较于疲劳前,两组疲劳后的自主激活值(voluntary activation,VA)、H-反射最大值(maximal H-reflex,H_(max))、M-波最大值(maximal M-wave,Mmax)、跖屈肌和背屈肌MVIC力矩均显著降低(P<0.05),但相比于真刺激组,假刺激组的VA和背屈肌MVIC力矩下降更为显著(P<0.05)。结论连续5 d的HD-tDCS干预有助于提高脊髓节段α运动神经元的活性,且能抑制跖背屈疲劳诱发的外周“神经-肌肉”接头处信息传递能力的下降。

靶肌肉注射胶质细胞源性神经营养因子对面神经损伤修复的作用

目的 通过靶肌肉注射胶质细胞源性神经营养因子(GDNF),观察其对面神经压榨损伤后大鼠功能恢复、神经形态学及GDNF在中枢面神经元中表达的影响,探讨经靶肌肉注射GDNF治疗周围性面瘫的可行性及作用机理。方法 SD大鼠随机分为假手术组(只暴露右侧面神经主干)、模型组(面神经主干压榨)、实验对照组(面神经主干压榨+颊肌注射生理盐水)和实验组(面神经主干压榨+颊肌注射GDNF),通过颊肌电生理、面瘫症状评分观察大鼠的神经功能恢复,Masson染色观察颊肌纤维形态学变化、甲苯胺蓝染色观察面神经形态学变化、Western Blot检测面神经元中GDNF的表达。结果 (1)大鼠面瘫症状评分:假手术组无面瘫表现,评分为0分,造模后各组大鼠均出现周围性面瘫表现,评分均为5分;术后第28天,实验组大鼠面瘫症状已基本完全恢复,模型组及实验对照组较前有不同程度改善但未完全恢复,评分均>3分;(2)颊肌电生理:各组造模后与假手术组相比,峰潜伏期有不同程度延长、最大振幅有不同程度下降;随时间的推移,实验组峰潜伏期较模型组及实验对照组明显缩短(P<0.05),最大振幅较其明显上升(P<0.05);(3)甲苯胺蓝染色:模型组、实验对照组、实验组术后均出现神经纤维形态不规则,外膜不连续不清晰,轴突数量减少。术后第28天,实验组面神经形态与假手术无明显差异,较模型组及实验对照组有明显恢复;(4)Masson染色:各组造模后肌纤维数量减少,肌肉组织占比面积减少;实验组肌纤维形态较模型组及实验对照组恢复快,至术后第28天,肌纤维形态接近正常(P<0.05);(5)Western Bolt检测:各组造模后中枢面神经元组织GDNF的蛋白表达下降,观察期内逐渐增强;与模型组及实验对照组相比,术后各时间点,实验组中枢面神经元组织GDNF的蛋白表达显著增强(P<0.01)。结论 靶肌肉注射GDNF可作为改善周围神经损伤的有效给药方式之一,促进神经纤维修复、靶肌肉功能的恢复及中枢神经系统对周围损伤神经的修复。

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

Relationship between the partitioning properties of motoneuron recruitment and the needle feeling propagation

Histological and physiological studies indicated that most skeletal muscles can be divided into a series of relatively independent sub-volumes: "neuromuscular compartments" and the partitioning property of the muscle result in the localization of muscle reflex. In the present experiment we studied the recruitment properties of medial gastracnemius (MG) muscle motoneuron pool of decerebrate cat with two kinds of local mechanical stimulation: local stretch and acupuncture-like stimuli. The results indicate that: (ⅰ) there is an obvious property of localization of recruitment activity, only the MNs which innervate the stimulated compartment were recruited by weaker and shorter stimuli; (ⅱ) recruitment activity spread to those MNs which supply the adjacent and distal compartments during the strength of stimulation or duration of the stimulation was increased; and (ⅲ) the recruitment property of muscle activity elicited by the local mechanical stimulation is thought similar to that of "needle feeling" along the meridian pathway during stimulation of acupuncture point.

NE对脊髓运动神经元外周传入突触传递及其表观受体动力学的影响

目的:探讨去甲肾上腺素(NE)对新生大鼠离体脊髓运动神经元(MN)外周传入所诱发的兴奋性突触后电位(EPSP)及其表观受体动力学的影响。方法:应用新生(8~14 d)SD大鼠脊髓横切片(400~500μm)MN细胞内记录技术,观察NE对MN细胞电生理特性及同侧背根(iDR)电刺激诱发的兴奋性突触后电位(iDR-EPSP)的影响,并通过表观受体动力学方法分析iDR-EPSPs的受体动力学。结果:在7个稳定记录的MN,累积灌流1、5和25μmol/L的NE各15 min,不仅均浓度依赖性诱导去极化反应,增大膜电阻和升高动作电位发放频率(I-F曲线上移)(均P<0.01),还降低iDR-EPSP幅度、曲线下面积、时程和衰减时间(P<0.05);在5μmol/L NE抑制iDR-EPSP的10个MN,其iDR-EPSPs的表观受体动力学分析显示,NE降低表观最大反应(V max)和表观结合速率常数(K 1)(均P<0.05),而增加表观平衡解离常数(K T)(P<0.05),及表观解离速率常数(K 2)可能存在增大趋势(P>0.05)。结论:在NE通过兴奋MN而抑制外周传入兴奋性突触传递的调控中可能涉及降低突触后受体亲和力的机制。