Intrafusal Research Articles

A physiologically enhanced muscle spindle model: using a Hill-type model for extrafusal fibers as template for intrafusal fibers

The muscle spindle is an essential proprioceptor, significantly involved in sensing limb position and movement. Although biological spindle models exist for years, the gold-standard for motor control in biomechanics are still sensors built of homogenized spindle output models due to their simpler combination with neuro-musculoskeletal models. Aiming to improve biomechanical simulations, this work establishes a more physiological model of the muscle spindle, aligned to the advantage of easy integration into large-scale musculoskeletal models. We implemented four variations of a spindle model in Matlab/Simulink®: the Mileusnic et al. (2006) model, Mileusnic model without mass, our enhanced Hill-type model, and our enhanced Hill-type model with parallel damping element (PDE). Different stretches in the intrafusal fibers were simulated in all model variations following the spindle afferent recorded in previous experiments in feline soleus muscle. Additionally, the enhanced Hill-type models had their parameters extensively optimized to match the experimental conditions, and the resulting model was validated against data from rats’ triceps surae muscle. As result, the Mileusnic models present a better overall performance generating the afferent firings compared to the common data evaluated. However, the enhanced Hill-type model with PDE exhibits a more stable performance than the original Mileusnic model, at the same time that presents a well-tuned Hill-type model as muscle spindle fibers, and also accounts for real sarcomere force-length and force-velocity aspects. Finally, our activation dynamics is similar to the one applied to Hill-type model for extrafusal fibers, making our proposed model more easily integrated in multi-body simulations.

Hamstrings vibration reduces tibiofemoral compressive force following anterior cruciate ligament reconstruction.

Individuals who have undergone anterior cruciate ligament reconstruction (ACLR)are at greater risk of developing knee osteoarthritis (OA).This elevated risk of knee OA is associated with high tibiofemoral (TF) compressive force, due to a combination of low knee flexion angles and increased co-contraction of the hamstrings and quadriceps during limb loading. Prolonged vibration of the hamstrings fatigues the intrafusal muscle fibers, which reduces autonomic reflexive excitation of the hamstrings and alleviates reciprocal inhibition to the quadriceps. The aim of this study was to examine the effect of prolonged hamstrings vibration on TF compressive force in individuals who have undergone ACL reconstruction. Fourteen participants with unilateral ACLR and 14 participants without knee injury performed a single-leg drop-land task before and after prolonged (20 min) vibration of the hamstrings. Peak TF compressive force, knee flexion angle, and hamstrings/quadriceps co-contraction were calculated during the deceleration phase of the drop-land task before and after vibration. The ACLR group experienced an 18% decrease in TF compressive force, a 32% increase in knee flexion angle, and a 38% decrease in hamstrings/quadriceps co-contraction after hamstrings vibration. There was no difference in any of the parameters in the noninjured group after vibration. These data suggest that acute prolonged hamstrings vibration has the potential to mitigate TF compressive force, which may protect the knee joint in the long term.Clinical significance: The results of this research are expected to lead to improved clinical care for ACLR patients because it holds promise for mitigating altered joint mechanics and perhaps slowing down the onset of posttraumatic knee osteoarthritis.

Intrafusal cross-bridge dynamics shape history-dependent muscle spindle responses to stretch.

Computational models can be critical to linking complex properties of muscle spindle organs to the sensory information that they encode during behaviours such as postural sway and locomotion where few muscle spindle recordings exist. Here, we augment a biophysical muscle spindle model to predict the muscle spindle sensory signal. Muscle spindles comprise several intrafusal muscle fibres with varied myosin expression and are innervated by sensory neurons that fire during muscle stretch. We demonstrate how cross-bridge dynamics from thick and thin filament interactions affect the sensory receptor potential at the spike initiating region. Equivalent to the Ia afferent's instantaneous firing rate, the receptor potential is modelled as a linear sum of the force and rate change of force (yank) of a dynamic bag1 fibre and the force of a static bag2/chain fibre. We show the importance of inter-filament interactions in (i) generating large changes in force at stretch onset that drive initial bursts and (ii) faster recovery of bag fibre force and receptor potential following a shortening. We show how myosin attachment and detachment rates qualitatively alter the receptor potential. Finally, we show the effect of faster recovery of receptor potential on cyclic stretch-shorten cycles. Specifically, the model predicts history-dependence in muscle spindle receptor potentials as a function of inter-stretch interval (ISI), pre-stretch amplitude and the amplitude of sinusoidal stretches. This model provides a computational platform for predicting muscle spindle response in behaviourally relevant stretches and can link myosin expression seen in healthy and diseased intrafusal muscle fibres to muscle spindle function.

Expression of MyHC-15 and -2x in human muscle spindles: An immunohistochemical study.

To build on the existing data on the pattern of myosin heavy chain (MyHC) isoforms expression in the human muscle spindles, we aimed to verify whether the 'novel' MyHC-15, -2x and -2b isoforms are co-expressed with the other known isoforms in the human intrafusal fibres. Using a set of antibodies, we attempted to demonstrate nine isoforms (15, slow-tonic, 1, α, 2a, 2x, 2b, embryonic, neonatal) in different regions of intrafusal fibres in the biceps brachii and flexor digitorum profundus muscles. The reactivity of some antibodies with the extrafusal fibres was also tested in the masseter and laryngeal cricothyreoid muscles. In both upper limb muscles, the expression of slow-tonic isoform was a reliable marker for differentiating positive bag fibres from negative chain fibres. Generally, bag1 and bag2 fibres were distinguished in isoform 1 expression; the latter consistently expressed this isoform over their entire length. Although isoform 15 was not abundantly expressed in intrafusal fibres, its expression was pronounced in the extracapsular region of bag fibres. Using a 2x isoform-specific antibody, this isoform was demonstrated in the intracapsular regions of some intrafusal fibres, particularly chain fibres. To the best of our knowledge, this study is the first to demonstrate 15 and 2x isoforms in human intrafusal fibres. However, whether the labelling with an antibody specific for rat 2b isoform reflects the expression of this isoform in bag fibres and some extrafusal ones in the specialised cranial muscles requires further evaluation. The revealed pattern of isoform co-expression only partially agrees with the results of previous, more extensive studies. Nevertheless, it may be inferred that MyHC isoform expression in intrafusal fibres varies along their length, across different muscle spindles and muscles. Furthermore, the estimation of expression may also depend on the antibodies utilised, which may also react differently with intrafusal and extrafusal fibres.

What if muscle spindles were also involved in the sense of effort?

Effort perception is widely acknowledged to originate from central processes within the brain, mediated by the integration of an efference copy of motor commands in sensory areas. However, in this topical review, we aim to challenge this perspective by presenting evidence from neural mechanisms and empirical studies that suggest that reafferent signals from muscle spindles also play a significant role in effort perception. It is now imperative for future research (a) to investigate the precise mechanisms underlying the interactions between the efference copy and reafferent spindle signals in the generation of effort perception, and (b) to explore the potential for altering spindle sensitivity to affect perceived effort during ecological physical exercise and, subsequently, influence physical activity behaviours.

Degeneration of muscle spindles in a murine model of Pompe disease

Pompe disease is a debilitating medical condition caused by a functional deficiency of lysosomal acid alpha-glucosidase (GAA). In addition to muscle weakness, people living with Pompe disease experience motor coordination deficits including an instable gait and posture. We reasoned that an impaired muscle spindle function might contribute to these deficiencies and therefore analyzed proprioception as well as muscle spindle structure and function in 4- and 8-month-old Gaa−/− mice. Gait analyses showed a reduced inter-limb and inter-paw coordination in Gaa−/− mice. Electrophysiological analyses of single-unit muscle spindle proprioceptive afferents revealed an impaired sensitivity of the dynamic and static component of the stretch response. Finally, a progressive degeneration of the sensory neuron and of the intrafusal fibers was detectable in Gaa−/− mice. We observed an increased abundance and size of lysosomes, a fragmentation of the inner and outer connective tissue capsule and a buildup of autophagic vacuoles in muscle spindles from 8-month-old Gaa−/− mice, indicating lysosomal defects and an impaired autophagocytosis. These results demonstrate a structural and functional degeneration of muscle spindles and an altered motor coordination in Gaa−/− mice. Similar changes could contribute to the impaired motor coordination in patients living with Pompe disease.

There and back again: 50 years of wandering through terra incognita fusorum.

This paper is in two parts: 'There', which is a review of some of the major advances in the study of spindle structure and function during the past 50 years, serving as an introduction to the symposium entitled 'Mechanotransduction, Muscle Spindles and Proprioception' held in Munich in July 2022; and 'And Back Again', presenting new quantitative morphological results on the equatorial nuclei of intrafusal muscle fibres and of the primary sensory ending in relationship to passive stretch of the spindle.

Botulunim Toxin Type A Followed by TAR (Transversus Abdominal Release) with Abdominoplasty as the Best Combination for Big Hernia Repair

Introduction: The current incidence of incisional hernia is up to 11%. Botulinum toxin type A shows atrophy in the extrafusal and intrafusal muscle fibers. With the addition of Muscle Paralysis and improving strength, it is possible to perform an effective posterior component separation. Objective: Describe an easy algorithm to treat complex abdominal wall reconstruction and improve the quality of adjacent tissues. Material and Methods: Fifty-nine patients were analyzed over 1 year in the General Surgery and Plastic and Reconstructive Surgery Service, patients with midline abdominal wall hernias, using the effective reconstruction algorithm, placing by ultrasound-guided botulinum toxin in the muscle transverse, 1 month later performing TAR, with Abdominoplasty for skin flap management, and comparing with the control group that did not apply botulinum toxin. Discussion: Botox is a neurotoxin derived from the bacterium Clostridium botulinum (botulinum toxin type A) that has been observed at the sensory level causing atrophy in the extrafusal and intrafusal muscle fibers. Repair of the abdominal wall after incisional hernias has been a great challenge with recurrence rates of 11%. Performing the release of the transverse muscle has a recurrence of 6%, as well as the compensation of skin flaps are ideal for proper management. Results: Within the observed analyzes, the results were divided into trans-surgical (99% of the patients closed the midline), immediate post-surgical (Pain, Seroma and Infection <1%) and late (with <3% recurrence) in the experimental group. Conclusions: Within the study, we will be able to analyze that lowering the recurrence rate <3% in patients properly protocolized, is translated as an adequate and totally reproducible method in our environment.

Muscle spindles of the multifidus muscle undergo structural change after intervertebral disc degeneration

PurposeProprioceptive deficits are common in low back pain. The multifidus muscle undergoes substantial structural change after back injury, but whether muscle spindles are affected is unclear. This study investigated whether muscle spindles of the multifidus muscle are changed by intervertebral disc (IVD) degeneration in a large animal model.MethodsIVD degeneration was induced by partial thickness annulus fibrosus lesion to the L3-4 IVD in nine sheep. Multifidus muscle tissue at L4 was harvested at six months after lesion, and from six age-/sex-matched naïve control animals. Muscle spindles were identified in Van Gieson’s-stained sections by morphology. The number, location and cross-sectional area (CSA) of spindles, the number, type and CSA of intrafusal fibers, and thickness of the spindle capsule were measured. Immunofluorescence assays examined Collagen I and III expression.ResultsMultifidus muscle spindles were located centrally in the muscle and generally near connective tissue. There were no differences in the number or location of muscle spindles after IVD degeneration and only changes in the CSA of nuclear chain fibers. The thickness of connective tissue surrounding the muscle spindle was increased as was the expression of Collagen I and III.ConclusionChanges to the connective tissue and collagen expression of the muscle spindle capsule are likely to impact their mechanical properties. Changes in capsule stiffness may impact the transmission of length change to muscle spindles and thus transduction of sensory information. This change in muscle spindle structure may explain some of the proprioceptive deficits identified with low back pain.

A model for self-organization of sensorimotor function: the spinal monosynaptic loop.

Recent spinal cord literature abounds with descriptions of genetic preprogramming and the molecular control of circuit formation. In this paper, we explore to what extent circuit formation based on learning rather than preprogramming could explain the selective formation of the monosynaptic projections between muscle spindle primary afferents and homonymous motoneurons. We adjusted the initially randomized gains in the neural network according to a Hebbian plasticity rule while exercising the model system with spontaneous muscle activity patterns similar to those observed during early fetal development. Normal connectivity patterns developed only when we modeled β motoneurons, which are known to innervate both intrafusal and extrafusal muscle fibers in vertebrate muscles but were not considered in previous literature regarding selective formation of these synapses in animals with paralyzed muscles. It was also helpful to correctly model the greatly reduced contractility of extrafusal muscle fibers during early development. Stronger and more coordinated muscle activity patterns such as observed later during neonatal locomotion impaired projection selectivity. These findings imply a generic functionality of a musculoskeletal system to imprint important aspects of its mechanical dynamics onto a neural network, without specific preprogramming other than setting a critical period for the formation and maturation of this general pattern of connectivity. Such functionality would facilitate the successful evolution of new species with altered musculoskeletal anatomy, and it may help to explain patterns of connectivity and associated reflexes that appear during abnormal development.NEW & NOTEWORTHY A novel model of self-organization of early spinal circuitry based on a biologically realistic plant, sensors, and neuronal plasticity in conjunction with empirical observations of fetal development. Without explicit need for guiding genetic rules, connection matrices emerge that support functional self-organization of the mature pattern of Ia to motoneuron connectivity in the spinal circuitry.

Neuregulin 1 Drives Morphological and Phenotypical Changes in C2C12 Myotubes: Towards De Novo Formation of Intrafusal Fibres In Vitro.

Muscle spindles are sensory organs that detect and mediate both static and dynamic muscle stretch and monitor muscle position, through a specialised cell population, termed intrafusal fibres. It is these fibres that provide a key contribution to proprioception and muscle spindle dysfunction is associated with multiple neuromuscular diseases, aging and nerve injuries. To date, there are few publications focussed on de novo generation and characterisation of intrafusal muscle fibres in vitro. To this end, current models of skeletal muscle focus on extrafusal fibres and lack an appreciation for the afferent functions of the muscle spindle. The goal of this study was to produce and define intrafusal bag and chain myotubes from differentiated C2C12 myoblasts, utilising the addition of the developmentally associated protein, Neuregulin 1 (Nrg-1). Intrafusal bag myotubes have a fusiform shape and were assigned using statistical morphological parameters. The model was further validated using immunofluorescent microscopy and western blot analysis, directed against an extensive list of putative intrafusal specific markers, as identified in vivo. The addition of Nrg-1 treatment resulted in a 5-fold increase in intrafusal bag myotubes (as assessed by morphology) and increased protein and gene expression of the intrafusal specific transcription factor, Egr3. Surprisingly, Nrg-1 treated myotubes had significantly reduced gene and protein expression of many intrafusal specific markers and showed no specificity towards intrafusal bag morphology. Another novel finding highlights a proliferative effect for Nrg-1 during the serum starvation-initiated differentiation phase, leading to increased nuclei counts, paired with less myotube area per myonuclei. Therefore, despite no clear collective evidence for specific intrafusal development, Nrg-1 treated myotubes share two inherent characteristics of intrafusal fibres, which contain increased satellite cell numbers and smaller myonuclear domains compared with their extrafusal neighbours. This research represents a minimalistic, monocellular C2C12 model for progression towards de novo intrafusal skeletal muscle generation, with the most extensive characterisation to date. Integration of intrafusal myotubes, characteristic of native, in vivo intrafusal skeletal muscle into future biomimetic tissue engineered models could provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications.

Diversity of Mammalian Motoneurons and Motor Units.

Although they share the common function of controlling muscle fiber contraction, spinal motoneurons display a remarkable diversity. Alpha-motoneurons are the "final common pathway", which relay all the information from spinal and supraspinal centers and allow the organism to interact with the outside world by controlling the contraction of muscle fibers in the muscles. On the other hand, gamma-motoneurons are specialized motoneurons that do not generate force and instead specifically innervate muscle fibers inside muscle spindles, which are proprioceptive organs embedded in the muscles. Beta-motoneurons are hybrid motoneurons that innervate both extrafusal and intrafusal muscle fibers. Even among alpha-motoneurons, there exists an exquisite diversity in terms of motoneuron electrical and molecular properties, physiological and structural properties of their neuromuscular junctions, and molecular and contractile properties of the innervated muscle fibers. This diversity, across species, across muscles, and across muscle fibers in a given muscle, underlie the vast repertoire of movements that one individual can perform.

Evaluating Sexual Dimorphism of the Muscle Spindles and Intrafusal Muscle Fibers in the Medial Gastrocnemius of Male and Female Rats.

This study sought to investigate the sexual dimorphism of muscle spindles in rat medial gastrocnemius muscle. The muscles were cut transversely into 5–10 and 20 μm thick serial sections and the number, density, and morphometric properties of the muscle spindles were determined. There was no significant difference (p > 0.05) in the number of muscle spindles of male (14.45 ± 2.77) and female (15.00 ± 3.13) rats. Muscle mass was 38.89% higher in males (1.08 vs. 0.66 g in females), making the density of these receptors significantly higher (p < 0.01) in females (approximately one spindle per 51.14 mg muscle mass vs. one per 79.91 mg in males). There were no significant differences between the morphometric properties of intrafusal muscle fibers or muscle spindles in male and female rats (p > 0.05): 5.16 ± 2.43 and 5.37 ± 2.27 μm for male and female intrafusal muscle fiber diameter, respectively; 5.57 ± 2.20 and 5.60 ± 2.16 μm for male and female intrafusal muscle fiber number, respectively; 25.85 ± 10.04 and 25.30 ± 9.96 μm for male and female shorter muscle spindle diameter, respectively; and 48.99 ± 20.73 and 43.97 ± 16.96 μm for male and female longer muscle spindle diameter, respectively. These findings suggest that sexual dimorphism in the muscle spindles of rat medial gastrocnemius is limited to density, which contrasts previous findings reporting differences in extrafusal fibers diameter.

Localization of PIP5Kγ selectively in proprioceptive peripheral fields and also in sensory ganglionic satellite cells as well as neuronal cell membranes and their central terminals.

Based on a previous study by others reporting that PIP5Kγ (phosphatidylinositol 4-phosphate 5-kinase γ) and its product, phosphatidylinositol 4,5 bisphosphate (PIP2 ), are involved in the regulation of nociception, the present immunohistochemical study examined the localization of PIP5Kγ-immunoreactivity in dorsal root ganglia (DRG) and their peripheral and central terminal fields. PIP5Kγ-immunoreactivity was localized for the first time in the muscle spindles, in which it was found in I-bands of polar regions of intrafusal muscle fibers and also in sensory nerve terminals abutting on equatorial regions of the muscle fibers. This finding indicates the involvement of PIP5Kγ in the proprioception and suggests somehow complicated mechanisms of its involvement because of its heterogeneous localization in intra-I-band structures. In DRG, on the other hand, PIP5Kγ-immunoreactivity was shown to be localized heterogeneously, but not evenly, over apposed plasma membranes of both neurons and ganglionic satellite cells in immune electron microscopy. In addition, no peripheral nerve terminals of DRG showing its distinct immunoreactivity were found in most peripheral fields of nociception and any other sensory perception except for the proprioception through muscle spindles. In contrast, numerous central terminals of DRG in the spinal posterior horn were immunoreactive for it. This finding leads us to consider the possibility that the regulation by PIP5Kγ of nociception is dominantly exerted in DRG and sensory neural tracts central, rather than peripheral, to DRG.

Development of an Optogenetic Method to Stimulate Gamma Motor Neurons In Vitro

The muscle spindle is a sensory organ located in the skeletal muscle that is critical for motor control and proprioception. The muscle spindle is innervated by stretch sensitive muscle spindle afferents and gamma motor neurons that control the length of the intrafusal muscle fibers and therefore the sensitivity of the stretch sensitive afferents. However, it has been challenging to study the gamma motor neurons since it is hard to specifically stimulate the gamma but not the alpha motor neurons that control the force generating extrafusal fibers. A previous study showed that alpha motor neurons were recruited from small diameter to large diameter with increasing optical intensities in mice expressing the blue light gated Channelrhodopsin2 (ChR2) in motor neurons (Llewellyn, et al., 2010). This is the reverse recruitment pattern of electrical stimulation. We hypothesized that gamma motor neurons, which are smaller than even the smallest alpha motor neurons, will be recruited first using low optical stimuli. The extensor digitorum longus muscle and sciatic nerve were dissected and placed in a tissue bath with oxygenated synthetic interstitial fluid. The sciatic nerve was attached to an extracellular suction electrode that recorded muscle spindle afferent activity. We used a light guide to deliver blue LED light (470nm; 0.5mW-5 mW) to the end of the nerve. We found that at lower levels of optical stimulation we recruited the gamma motor neurons as evidenced by an increased muscle spindle afferent firing rate and a pause after stimulation, which is typically observed following the release of stretch. Higher optical intensities were required to recruit alpha motor neurons to produce a twitch muscle contraction. Higher frequencies of optical stimulation led to greater increases in muscle spindle afferent firing rates as expected. We are currently testing additional stimulation frequencies and increasing our sample size to understand individual variability. We are also verifying that ChR2 is found in motor neurons only using immunohistochemistry. We will use this technique to study gamma motor neuron function and to produce a more physiological relevant system to study muscle spindle afferents. We can also use this method to screen for intrafusal fiber dysfunction in disease models.

Frataxin gene editing rescues Friedreich\u2019s ataxia pathology in dorsal root ganglia organoid-derived sensory neurons

Friedreich’s ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.

Differentiation of Intrafusal Fibers from Human Induced Pluripotent Stem Cells

Human-based "body-on-a-chip" technology provides powerful platforms in developing models for drug evaluation and disease evaluations in phenotypic models. Induced pluripotent stem cells (iPSCs) are ideal cell sources for generating different cell types for these in vitro functional systems and recapitulation of the neuromuscular reflex arc would allow for the study of patient specific neuromuscular diseases. Regarding relevant afferent (intrafusal fibers, sensory neurons) and efferent (extrafusal fibers, motoneurons) cells, in vitro differentiation of intrafusal fiber from human iPSCs has not been established. This work demonstrates a protocol for inducing an enrichment of intrafusal bag fibers from iPSCs using morphological analysis and immunocytochemistry. Phosphorylation of the ErbB2 receptors and S46 staining indicated a 3-fold increase of total intrafusal fibers further confirming the efficiency of the protocol. Integration of induced intrafusal fibers would enable more accurate reflex arc models and application of this protocol on patient iPSCs would allow for patient-specific disease modeling.

Impaired muscle spindle function in murine models of muscular dystrophy.

Muscular dystrophy patients suffer from progressive degeneration of skeletal muscle fibres, sudden spontaneous falls, balance problems, as well as gait and posture abnormalities. Dystrophin- and dysferlin-deficient mice, models for different types of muscular dystrophy with different aetiology and molecular basis, were characterized to investigate if muscle spindle structure and function are impaired. The number and morphology of muscle spindles were unaltered in both dystrophic mouse lines but muscle spindle resting discharge and their responses to stretch were altered. In dystrophin-deficient muscle spindles, the expression of the paralogue utrophin was substantially upregulated, potentially compensating for the dystrophin deficiency. The results suggest that muscle spindles might contribute to the motor problems observed in patients with muscular dystrophy. Muscular dystrophies comprise a heterogeneous group of hereditary diseases characterized by progressive degeneration of extrafusal muscle fibres as well as unstable gait and frequent falls. To investigate if muscle spindle function is impaired, we analysed their number, morphology and function in wildtype mice and in murine model systems for two distinct types of muscular dystrophy with very different disease aetiology, i.e. dystrophin- and dysferlin-deficient mice. The total number and the overall structure of muscle spindles in soleus muscles of both dystrophic mouse mutants appeared unchanged. Immunohistochemical analyses of wildtype muscle spindles revealed a concentration of dystrophin and β-dystroglycan in intrafusal fibres outside the region of contact with the sensory neuron. While utrophin was absent from the central part of intrafusal fibres of wildtype mice, it was substantially upregulated in dystrophin-deficient mice. Single-unit extracellular recordings of sensory afferents from muscle spindles of the extensor digitorum longus muscle revealed that muscle spindles from both dystrophic mouse strains have an increased resting discharge and a higher action potential firing rate during sinusoidal vibrations, particularly at low frequencies. The response to ramp-and-hold stretches appeared unaltered compared to the respective wildtype mice. We observed no exacerbated functional changes in dystrophin and dysferlin double mutant mice compared to the single mutant animals. These results show alterations in muscle spindle afferent responses in both dystrophic mouse lines, which might cause an increased muscle tone, and might contribute to the unstable gait and frequent falls observed in patients with muscular dystrophy.

Evidence for central antispastic effect of botulinum toxin type A.

Botulinum toxin type A (BoNT/A) injections into hyperactive muscles provide effective treatment for spasticity and dystonias, presumably due to its local effects on extrafusal and intrafusal motor fibres. A recent discovery of toxin's retrograde axonal transport to CNS might suggest additional action sites. However, in comparison to cholinergic peripheral terminals, functional consequences of BoNT/A direct central action on abnormally increased muscle tone are presently unknown. To address this question, the central effects of BoNT/A were assessed in experimental local spastic paralysis. Local spastic paralysis was induced by injection of tetanus toxin (1.5ng) into rat gastrocnemius. Subsequently, BoNT/A (5U·kg-1 ) was applied i.m. into the spastic muscle or intraneurally (i.n.) into the sciatic nerve to mimic the action of axonally transported toxin. Functional role of BoNT/A transcytosis in spinal cord was evaluated by lumbar i.t. application of BoNT/A-neutralizing antitoxin. BoNT/A effects were studied by behavioural motor assessment and cleaved synaptosomal-associated protein 25 (SNAP-25) immunohistochemistry. Tetanus toxin evoked muscular spasm (sustained rigid hind paw extension and resistance to passive ankle flexion). Subsequent injections of BoNT/A, i.m. or i.n, reduced tetanus toxin-evoked spastic paralysis. Beneficial effects of i.n. BoNT/A and occurrence of cleaved SNAP-25 in ventral horn were prevented by i.t. antitoxin. Axonally transported BoNT/A relieves muscle hypertonia induced by tetanus toxin, following the trans-synaptic movement of BoNT/A in the CNS. These results suggest that such direct, centrally mediated reduction of abnormal muscle tone might contribute to the effectiveness of BoNT/A in spasticity and hyperkinetic movement disorders.

The loss of slow skeletal muscle isoform of troponin T in spindle intrafusal fibres explains the pathophysiology of Amish nemaline myopathy.

The pathogenic mechanism and the neuromuscular reflex-related phenotype (e.g. tremors accompanied by clonus) of Amish nemaline myopathy, as well as of other recessively inherited TNNT1 myopathies, remain to be clarified. The truncated slow skeletal muscle isoform of troponin T (ssTnT) encoded by the mutant TNNT1 gene is unable to incorporate into myofilaments and is degraded in muscle cells. By contrast to extrafusal muscle fibres, spindle intrafusal fibres of normal mice contain a significant level of cardiac TnT and a low molecular weight splice form of ssTnT. Intrafusal fibres of ssTnT-knockout mice have significantly increased cardiac TnT. Rotarod and balance beam tests have revealed abnormal neuromuscular co-ordination in ssTnT-knockout mice and a blunted response to a spindle sensitizer, succinylcholine. The loss of ssTnT and a compensatory increase of cardiac TnT in intrafusal nuclear bag fibres may increase myofilament Ca2+ -sensitivity and tension, impairing spindle function, thus identifying a novel mechanism for the development of targeted treatment. A nonsense mutation at codon Glu180 of TNNT1 gene causes Amish nemaline myopathy (ANM), a recessively inherited disease with infantile lethality. TNNT1 encodes the slow skeletal muscle isoform of troponin T (ssTnT). The truncated ssTnT is unable to incorporate into myofilament and is degraded in muscle cells. The symptoms of ANM include muscle weakness, atrophy, contracture and tremors accompanied by clonus. An ssTnT-knockout (KO) mouse model recapitulates key features of ANM such as atrophy of extrafusal slow muscle fibres and increased fatigability. However, the neuromuscular reflex-related symptoms of ANM have not been explained. By isolating muscle spindles from ssTnT-KO and control mice aiming to examine the composition of myofilament proteins, we found that, in contrast to extrafusal fibres, intrafusal fibres contain a significant level of cardiac TnT and the low molecular weight splice form of ssTnT. Intrafusal fibres from ssTnT-KO mice have significantly increased cardiac TnT. Rotarod and balance beam tests revealed impaired neuromuscular co-ordination in ssTnT-KO mice, indicating abnormality in spindle functions. Unlike the wild-type control, the beam running ability of ssTnT-KO mice had a blunted response to a spindle sensitizer, succinylcholine. Immunohistochemistry detected ssTnT and cardiac TnT in nuclear bag fibres, whereas fast skeletal muscle TnT was detected in nuclear chain fibres, and cardiac α-myosin was present in one of the two nuclear bag fibres. The loss of ssTnT and a compensatory increase of cardiac TnT in nuclear bag fibres would increase myofilament Ca2+ -sensitivity and tension, thus affecting spindle activities. This mechanism provides an explanation for the pathophysiology of ANM, as well as a novel target for treatment.