Drosophila MICAL regulates myofilament organization and synaptic structure

The overall size and structure of a synaptic terminal is an important determinant of its function. In a large-scale mutagenesis screen, designed to identify Drosophila mutants with abnormally structured neuromuscular junctions (NMJs), we discovered mutations in Drosophila mical, a conserved gene encoding a multi-domain protein with a N-terminal monooxygenase domain. In mical mutants, synaptic boutons do not sprout normally over the muscle surface and tend to form clusters along synaptic branches and at nerve entry sites. Consistent with high expression of MICAL in somatic muscles, immunohistochemical stainings reveal that the subcellular localization and architecture of contractile muscle filaments are dramatically disturbed in mical mutants. Instead of being integrated into a regular sarcomeric pattern, actin and myosin filaments are disorganized and accumulate beneath the plasmamembrane. Whereas contractile elements are strongly deranged, the proposed organizer of sarcomeric structure, D-Titin, is much less affected. Transgenic expression of interfering RNA molecules demonstrates that MICAL is required in muscles for the higher order arrangement of myofilaments. Ultrastructural analysis confirms that myosin-rich thick filaments enter submembranous regions and interfere with synaptic development, indicating that the disorganized myofilaments may cause the synaptic growth phenotype. As a model, we suggest that the filamentous network around synaptic boutons restrains the spreading of synaptic branches.     

Infradiagnóstico de enfermedades neuromusculares en ancianos de 80 y más años

Myasthenia gravis (MG), amyotrophic lateral sclerosis and Guillain-Barre syndrome (GBS) have been classically considered as exceptional or unusual diseases in people with a geriatric profile. Over the past 25 years, several population-based studies have been conducted in the Osona area (Barcelona), which, for the first time, has led to describing the high global incidences in the elderly, especially those over 80 years-old. The results suggest the possibility of underdiagnosis of these neuromuscular diseases in the elderly, a fact that could be especially relevant in the case of MG and GBS, since they are 2 potentially reversible entities with high mortality in the event of underdiagnosis and absence of treatment.     

Neuromuscular adaptations predict functional disability independently of clinical pain and psychological factors in patients with chronic non-specific low back pain

Patients with chronic low back pain exhibit characteristics such as clinical pain, psychological symptoms and neuromuscular adaptations. The purpose of this study was to determine the independent contribution of clinical pain, psychological factors and neuromuscular adaptations to disability in patients with chronic low back pain. Clinical pain intensity, pain catastrophizing, fear-avoidance beliefs, anxiety, neuromuscular adaptations to chronic pain and neuromuscular responses to experimental pain were assessed in 52 patients with chronic low back pain. Lumbar muscle electromyographic activity was assessed during a flexion–extension task (flexion relaxation phenomenon) to assess both chronic neuromuscular adaptations and neuromuscular responses to experimental pain during the task. Multiple regressions showed that independent predictors of disability included neuromuscular adaptations to chronic pain (β = 0.25, p = 0.006, sr² = 0.06), neuromuscular responses to experimental pain (β = −0.24, p = 0.011, sr² = 0.05), clinical pain intensity (β = 0.28, p = 0.002, sr² = 0.08) and psychological factors (β = 0.58, p < 0.001, sr² = 0.32). Together, these predictors accounted for 65% of variance in disability (R² = 0.65 p < 0.001). The current investigation revealed that neuromuscular adaptations are independent from clinical pain intensity and psychological factors, and contribute to inter-individual differences in patients’ disability. This suggests that disability, in chronic low back pain patients, is determined by a combination of factors, including clinical pain, psychological factors and neuromuscular adaptations.     

Transmembrane mechanisms in the assembly of the postsynaptic apparatus at the neuromuscular junction

The vertebrate neuromuscular junction (NMJ) is marked by molecular specializations that include postsynaptic clusters of acetylcholine receptor (AChR) and acetylcholinesterase (AChE). Whereas AChRs are aggregated in the postsynaptic muscle membrane to a density of 10,000/mum(2), AChE is concentrated, also to a high density, in the synaptic basement membrane (BM). In recent years considerable progress has been made in understanding the cellular and molecular mechanisms of AChR clustering. It is known that during the early stages of motoneuron-muscle interaction, the nerve-secreted proteoglycan agrin activates the muscle-specific kinase MuSK, which leads to the formation of a postsynaptic cytoskeletal scaffold that immobilizes and concentrates AChRs through a process generally accepted to involve diffusion-mediated trapping of the receptors. We have recently tested this diffusion-trap model at the single molecule level for the first time by using quantum-dot labeling to track individual AChRs during NMJ development. Our results showed that single AChRs exhibit Brownian-type movement, with diffusion coefficients of 10(-11) to 10(-9)cm(2)/s, until they become immobilized at "traps" assembled in response to synaptogenic stimuli. Thus, free diffusion of AChRs is an integral part of their clustering mechanism. What is the mechanism for AChE clustering? We previously showed that the A(12) asymmetric form of AChE binds to perlecan, a heparan-sulfate proteoglycan which in turn interacts with the transmembrane dystroglycan complex. Through this linkage AChE becomes bound to the muscle membrane and, like AChRs, may exhibit lateral mobility along the membrane. Consistent with this idea, pre-existent AChE at the cell surface becomes clustered together with AChRs following synaptogenic stimulation. Future studies testing diffusion-mediated trapping of AChE should provide insights into the synaptic localization of BM-bound molecules at the NMJ.     

Quaternary derivatives of granatanol diesters: potent, ultrashort acting non-depolarizing neuromuscular relaxants

The purpose of this study was to explore the feasibility of utilizing the granatanol: N-methyl [9-azabicyclo (3.3.1) nonane] 3-alpha-ol as the terminal group in a series of new bisquaternary azabicycyclic diester-type neuromuscular blocking agents. Fifty two bisquaternary ammonium derivatives of several dicarboxylic acid esters of granatanol and three similar derivatives of pseudo granatanol have been investigated for neuromuscular blocking (NMB) potency (ED(50) s), onset and recovery of action and for cardiovascular side effects. All agents were studied first in anesthetized rats, and selected agents were subjected to further pharmacodynamic testing in rabbits, juvenile pigs, cats, dogs and monkeys. One agent was tested in continuous i.v. infusion mode in comparison with its corresponding tropine diester and the aminosteroid muscle relaxant, rocuronium. Several new and highly potent NMB granatanol derivatives are described, which are largely similar in NMB potency to the previously described tropine: N-methyl [8-azabicyclo (3.2.1)] 3-alpha-ol diester derivatives. The majority of the presently described granatanol derivatives displayed ultrashort onset and duration of actions. In that respect some of these agents proved to be the fastest and shortest acting non-depolarizing muscle relaxants described so far. On the negative side, many, but not all, granatanol derivatives produced cardiovascular side effects: e.g. changes in heart rate and blood pressure. Like with the similar tropinyl diester derivatives, cardiac vagal block was present with the majority of these agents as assessed in the rat, pig and cat. Few glutaryl, fumaryl and cyclobutane (trans) 1,2-dicarboxylyl granatanol diesters quaternized with disubstituted benzyl halides, bearing p-acyloxy radicals, showed excellent NMB profile. In these derivatives, however, the rapid decomposition of the p-acyloxy groups leads to formation of toxic quinone methene metabolites which precludes their further pharmaceutical development. The pseudo granatanol derivatives were less potent in the rat than the corresponding granatanols and were not further investigated. We conclude that the 9-azabicyclo (3.3.1) nonane (granatane) ring system can successfully replace the similar 8-azabicyclo (3.2.1) octane (tropane) ring system in building potent, utrashort acting NMB agents.     

神经肌肉电刺激联合吞咽康复训练对脑卒中后吞咽障碍患者吞咽功能、表面肌电图及舌骨喉复合体动度的影响

摘要 目的：观察吞咽康复训练、神经肌肉电刺激（NMES）联合治疗对脑卒中后吞咽障碍患者吞咽功能、表面肌电图及舌骨喉复合体动度的影响。方法：选取2018 年9月~2020年10 月期间在我院治疗的70例脑卒中后吞咽障碍患者,根据住院号尾数的奇偶将患者分为对照组和实验组,各35例。对照组仅进行吞咽康复训练,实验组在此基础上接受NMES治疗,两组均治疗2个疗程,观察治疗前后两组患者吞咽功能、表面肌电图、舌骨喉复合体动度及生活质量变化。结果：治疗2个疗程后,实验组患者的吞咽功能改善总有效率高于对照组,组间比较有显著性差异（P<0.05）。治疗2个疗程后,两组吞咽时程缩短、最大波幅值升高,且实验组的吞咽时程短于对照组,最大波幅值高于对照组（P<0.05）。治疗2个疗程后,两组舌骨上移距离、舌骨前移距离、甲状软骨上移距离、甲状软骨前移距离增大,且实验组的舌骨上移距离、舌骨前移距离、甲状软骨上移距离、甲状软骨前移距离大于对照组（P<0.05）。治疗2个疗程后,两组SWAL-QOL总分均升高,且实验组的SWAL-QOL总分高于对照组（P<0.05）。结论：NMES联合吞咽康复训练应用于脑卒中后吞咽障碍患者,可有效促进其吞咽功能改善,提高舌骨肌肌群肌力及生活质量。     

The acetylcholine receptor gamma-to-epsilon switch occurs in individual endplates

Maturation of the neuromuscular junction is accompanied by molecular switching of acetylcholine receptor (AChR) channels from embryonic types with gamma-subunits to adult ones with epsilon-subunits after birth. As a step toward understanding the molecular mechanisms of the gamma-to-epsilon switch, we addressed the question of whether embryonic- and adult-type AChRs constitute different endplates during the transitional period. From analyses with double- or triple-staining with anti-gamma- and/or anti-epsilon-antibodies together with alpha-bungarotoxin, which binds to alpha-subunits, we demonstrated that during neonatal stages in mice, adult-type AChRs are incorporated into individual endplates expressing embryonic-AChRs and replace these embryonic-AChRs gradually. The main period of AChR transition in the mouse diaphragm was between postnatal days 5 (P5) and P7, similar to the period described previously in which endplates shift from multi-axon to single-axon innervation. This finding will help our understanding of the mechanisms of the gamma-to-epsilon switch during establishment of the neuromuscular junction.     

Semaphorin 6C expression in innervated and denervated skeletal muscle

Semaphorins are secreted or transmembrane proteins important for axonal guidance and for the structuring of neuronal systems. Semaphorin 6C, a transmembrane Semaphorin, has growth cone collapsing activity and is expressed in adult skeletal muscle. In the present study the expression of Semaphorin 6C mRNA and immunoreactivity has been compared in innervated and denervated mouse hind-limb and hemidiaphragm muscles. Microscopic localization of immunoreactivity was studied in innervated and denervated rat skeletal muscle. The results show that Semaphorin 6C mRNA expression and immunoreactivity on Western blots are down-regulated following denervation. The mRNA of Semaphorin 6C as well as immunoreactivity determined by Western blots are expressed in extrasynaptic as well as perisynaptic regions of muscle. Immunohistochemical studies, however, show Semaphorin 6C-like immunoreactivity to be concentrated at neuromuscular junctions. The results suggest a role for Semaphorin 6C in neuromuscular communication.     

Time-dependent effects of neuromuscular electrical stimulation on changes in spinal excitability are dependent on stimulation frequency: A preliminary study in healthy adults

Neuromuscular electrical stimulation (NMES) can be used as treatment for spasticity. The present study examined differences in time-dependent effects of NMES depending on stimulation frequency. Forty healthy subjects were separated into four groups (no-stim, NMES of 50, 100, and 200?Hz). The un-conditioned H-reflex amplitude and the H-reflex conditioning-test paradigm were used to measure the effectiveness on monosynaptic Ia excitation of motoneurons in the soleus (SOL) muscle, disynaptic reciprocal Ia inhibition from tibialis anterior (TA) to SOL, and presynaptic inhibition of SOL Ia afferents. Each trial consisted of a 30-min period of NMES applied to the deep peroneal nerve followed by a 30-min period with no stimulation to measure prolonged effects. Measurements were performed periodically. Stimulation applied at all frequencies produced a significant reduction in monosynaptic Ia excitation of motoneurons in the SOL muscle, however, only stimulation with 50?Hz showed prolonged reduction after NMES. NMES frequency did not affect the amount of disynaptic reciprocal Ia inhibition and presynaptic inhibition of Ia afferents. The results show a frequency-dependent effect of NMES on the monosynaptic Ia excitation of motoneurons. This result has implications for selecting the optimal NMES frequency for treatment in patients with spasticity.     

Acetylcholine Translocating Protein: Mediatophore at Rat Neuromuscular Synapses

The neuromuscular synapses of the rat sternomastoid muscles contain a membrane protein, mediatophore, that endows artificial membranes with a calcium-dependent acetylcholine release mechanism. Mediatophore and choline acetylase had similar distributions along the muscle. Sciatic nerve membranes contain mediatophore, and a purified preparation was obtained from the nerve.