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Muscle spindles are stretch-sensitive mechanoreceptors found in the skeletal muscles of most four-limbed vertebrates. They are unique amongst sensory receptors in the ability to regulate their sensitivity by contraction of the intrafusal muscle fibres on which the sensory endings lie. Muscle spindles have revealed a remarkable diversity of functions, including reflex action in posture and locomotion, contributing to bodily self awareness, and influencing wound healing. What were the circumstances which gave rise to the evolution of such complex end-organs? We argue that spindles first appeared in early amniotes and only later in frogs and toads. This was considered an example of convergent evolution. Spindles in amphibians and reptiles are characterised by their simple structure, pointing to key features essential for spindle function. Spindle sensitivity in amphibians and reptiles is controlled by intrafusal fibre contractions evoked by branches of motor axons supplying extrafusal muscle. Modern phylogenetic evidence has revised our views on the origin of birds, placing them closer to the dinosaurs than had previously been thought. Birds are the only group, other than mammals, which has a dedicated fusimotor innervation of spindles, another example of convergent evolution, given the widely different origins of the two groups. One factor that may have played a role here was that both groups are endotherms, allowing motor control to develop further in an optimal internal environment. This, as well as other changes within the spindle, has led to the astonishing sophistication of motor control observed especially in many modern mammals., (© 2024 The Author(s). Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

A metabotropic glutamate receptor coupled to phospholipase D (PLD-mGluR) was discovered in the hippocampus over three decades ago. Its pharmacology and direct linkage to PLD activation are well established and indicate it is a highly atypical glutamate receptor. A receptor with the same pharmacology is present in spindle primary sensory terminals where its blockade can totally abolish, and its activation can double, the normal stretch-evoked firing. We report here the first identification of this PLD-mGluR protein, by capitalizing on its expression in primary mechanosensory terminals, developing an enriched source, pharmacological profiling to identify an optimal ligand, and then functionalizing it as a molecular tool. Evidence from immunofluorescence, western and far-western blotting indicates PLD-mGluR is homomeric GluK2, since GluK2 is the only glutamate receptor protein/receptor subunit present in spindle mechanosensory terminals. Its expression was also found in the lanceolate palisade ending of hair follicle, also known to contain the PLD-mGluR. Finally, in a mouse model with ionotropic function ablated in the GluK2 subunit, spindle glutamatergic responses were still present, confirming it acts purely metabotropically. We conclude the PLD-mGluR is a homomeric GluK2 kainate receptor signalling purely metabotropically and it is common to other, perhaps all, primary mechanosensory endings., (© 2023 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

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., (© 2023 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Muscle spindles encode mechanosensory information by mechanisms that remain only partially understood. Their complexity is expressed in mounting evidence of various molecular mechanisms that play essential roles in muscle mechanics, mechanotransduction and intrinsic modulation of muscle spindle firing behaviour. Biophysical modelling provides a tractable approach to achieve more comprehensive mechanistic understanding of such complex systems that would be difficult/impossible by more traditional, reductionist means. Our objective here was to construct the first integrative biophysical model of muscle spindle firing. We leveraged current knowledge of muscle spindle neuroanatomy and in vivo electrophysiology to develop and validate a biophysical model that reproduces key in vivo muscle spindle encoding characteristics. Crucially, to our knowledge, this is the first computational model of mammalian muscle spindle that integrates the asymmetric distribution of known voltage-gated ion channels (VGCs) with neuronal architecture to generate realistic firing profiles, both of which seem likely to be of great biophysical importance. Results predict that particular features of neuronal architecture regulate specific characteristics of Ia encoding. Computational simulations also predict that the asymmetric distribution and ratios of VGCs is a complementary and, in some instances, orthogonal means to regulate Ia encoding. These results generate testable hypotheses and highlight the integral role of peripheral neuronal structure and ion channel composition and distribution in somatosensory signalling., (© 2023 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Introduction: The ultimate deficit in amyotrophic lateral sclerosis (ALS) is neuromuscular junction (NMJ) loss, producing permanent paralysis, ultimately in respiratory muscles. However, understanding the functional and structural deficits at NMJs prior to this loss is crucial for therapeutic strategy design. Should early interventions focus on reversing denervation, or supporting largely intact NMJs that are functionally impaired? We therefore determined when functional and structural deficits appeared in diaphragmatic NMJs relative to the onset of hindlimb tremor (the first overt motor symptoms) in vivo in the SOD1-G93A mouse model of ALS., Materials and Methods: We employed electrophysiological recording of NMJ postsynaptic potentials for spontaneous and nerve stimulation-evoked responses. This was correlated with fluorescent imaging microscopy of the postsynaptic acetylcholine receptor (AChR) distribution throughout the postnatal developmental timecourse from 2 weeks to early symptomatic ages., Results: Significant reduction in the amplitudes of spontaneous miniature endplate potentials (mEPPs) and evoked EPPs emerged only at early symptomatic ages (in our colony, 18-22 weeks). Reductions in mEPP frequency, number of vesicles per EPP, and EPP rise time were seen earlier, at 16weeks, but this reversed by early symptomatic ages. However, the earliest and most striking impairment was an inability to maintain EPP amplitude during a 20 Hz stimulus train, which appeared 6 weeks before overt in vivo motor symptoms. Despite this, fluorescent α -bungarotoxin labelling revealed no systematic, progressive changes in 11 comprehensive NMJ morphological parameters (area, shape, compactness, number of acetylcholine receptor, AChR, regions, etc.) with disease progression. Rather, while NMJs were largely normally-shaped, from 16 weeks there was a progressive and substantial disruption in AChR concentration and distribution within the NMJ footprint., Discussion: Thus, NMJ functional deficits appear at least 6 weeks before motor symptoms in vivo , while structural deficits occur 4 weeks later, and predominantly within NMJs. These data suggest initial therapies focused on rectifying suboptimal NMJ function could produce effective relief of symptoms of weakness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 McIntosh, Mekrouda, Dashti, Giuraniuc, Banks, Miles and Bewick.)

Across the human body, skeletal muscles have a broad range of biomechanical roles that employ complex proprioceptive control strategies to successfully execute a desired movement. This information is derived from peripherally located sensory apparatus, the muscle spindle and Golgi tendon organs. The abundance of these sensory organs, particularly muscle spindles, is known to differ considerably across individual muscles. Here we present a comprehensive data set of 119 muscles across the human body including architectural properties (muscle fibre length, mass, pennation angle and physiological cross-sectional area) and statistically test their relationships with absolute spindle number and relative spindle abundance (the residual value of the linear regression of the log-transformed spindle number and muscle mass). These data highlight a significant positive relationship between muscle spindle number and fibre length, emphasising the importance of fibre length as an input into the central nervous system. However, there appears to be no relationship between muscles architecturally optimised to function as displacement specialists and their provision of muscle spindles. Additionally, while there appears to be regional differences in muscle spindle abundance, independent of muscle mass and fibre length, our data provide no support for the hypothesis that muscle spindle abundance is related to anatomical specialisation., (© 2023. The Author(s).)

The proprioceptive system is essential for the control of coordinated movement, posture, and skeletal integrity. The sense of proprioception is produced in the brain using peripheral sensory input from receptors such as the muscle spindle, which detects changes in the length of skeletal muscles. Despite its importance, the molecular composition of the muscle spindle is largely unknown. In this study, we generated comprehensive transcriptomic and proteomic datasets of the entire muscle spindle isolated from the murine deep masseter muscle. We then associated differentially expressed genes with the various tissues composing the spindle using bioinformatic analysis. Immunostaining verified these predictions, thus establishing new markers for the different spindle tissues. Utilizing these markers, we identified the differentiation stages the spindle capsule cells undergo during development. Together, these findings provide comprehensive molecular characterization of the intact spindle as well as new tools to study its development and function in health and disease., Competing Interests: BB, LH, SK, RA, BD, DL, MK, GB, RB, EZ No competing interests declared, (© 2023, Bornstein, Heinemann-Yerushalmi et al.)

Muscle spindle abundance is highly variable within and across species, but we currently lack any clear picture of the mechanistic causes or consequences of this variation. Previous use of spindle abundance as a correlate for muscle function implies a mechanical underpinning to this variation, but these ideas have not been tested. Herein, we use integrated medical imaging and subject-specific musculoskeletal models to investigate the relationship between spindle abundance, muscle architecture and in vivo muscle behaviour in the human locomotor system. These analyses indicate that muscle spindle number is tightly correlated with muscle fascicle length, absolute fascicle length change, velocity of fibre lengthening and active muscle forces during walking. Novel correlations between functional indices and spindle abundance are also recovered, where muscles with a high abundance predominantly function as springs, compared to those with a lower abundance mostly functioning as brakes during walking. These data demonstrate that muscle fibre length, lengthening velocity and fibre force are key physiological signals to the central nervous system and its modulation of locomotion, and that muscle spindle abundance may be tightly correlated to how a muscle generates work. These insights may be combined with neuromechanics and robotic studies of motor control to help further tease apart the functional drivers of muscle spindle composition.

New Findings: What is the topic of this review? We describe the structure and function of secondary sensory endings of muscle spindles, their reflex action and role in motor control and proprioception. What advances does it highlight? In most mammalian skeletal muscles, secondary endings of spindles are more or much more numerous than primary endings but are much less well studied. By focusing on secondary endings in this review, we aim to redress the balance, draw attention to what is not known and stimulate future research., Abstract: Kinaesthesia and the control of bodily movement rely heavily on the sensory input from muscle spindles. Hundreds of these sensory structures are embedded in mammalian muscles. Each spindle has one or more sensory endings and its own complement of small muscle fibres that are activated by the CNS via fusimotor neurons, providing efferent control of sensory responses. Exactly how the CNS wields this influence remains the subject of much fascination and debate. There are two types of sensory endings, primary and secondary, with differing development, morphology, distribution and responsiveness. Spindle primary endings have received more attention than secondaries, although the latter usually outnumber them. This review focuses on the secondary endings. Their location within the spindle, their response properties, the projection of their afferents within the CNS and their reflex actions all suggest that secondaries have certain separate roles from the primaries in proprioception and motor control. Specifically, spindle secondaries seem more adapted than primaries to signalling slow and maintained changes in the relative position of bodily segments, thereby contributing to position sense, postural control and static limb positioning. By highlighting, in this way, the roles of secondary endings, a final aim of the review is to broaden understanding of muscle spindles more generally and of the important contributions they make to both sensory and motor mechanisms., (© 2021 The Authors. Experimental Physiology © 2021 The Physiological Society.)

Muscle spindles and Golgi tendon organs (GTOs) are two types of sensory receptors that respond to changes in length or tension of skeletal muscles. These mechanosensors have long been known to participate in both proprioception and stretch reflex. Here, we present recent findings implicating these organs in maintenance of spine alignment as well as in realignment of fractured bones. These discoveries have been made in several mouse lines lacking functional mechanosensors in part or completely. In both studies, the absence of functional spindles and GTOs produced a more severe phenotype than that of spindles alone. Interestingly, the spinal curve phenotype, which appeared during peripubertal development, bears resemblance to the human condition adolescent idiopathic scoliosis. This similarity may contribute to the study of the disease by offering both an animal model and a clue as to its aetiology. Moreover, it raises the possibility that impaired proprioceptive signalling may be involved in the aetiology of other conditions. Overall, these new findings expand considerably the scope of involvement of proprioception in musculoskeletal development and function.This article is part of the Theo Murphy meeting issue 'Mechanics of development'., (© 2018 The Author(s).)

Acid-sensing ion channel 3 (ASIC3) is involved in acid nociception, but its possible role in neurosensory mechanotransduction is disputed. We report here the generation of Asic3-knockout/eGFPf-knockin mice and subsequent characterization of heterogeneous expression of ASIC3 in the dorsal root ganglion (DRG). ASIC3 is expressed in parvalbumin (Pv+) proprioceptor axons innervating muscle spindles. We further generate a floxed allele of Asic3 (Asic3(f/f)) and probe the role of ASIC3 in mechanotransduction in neurite-bearing Pv+ DRG neurons through localized elastic matrix movements and electrophysiology. Targeted knockout of Asic3 disrupts spindle afferent sensitivity to dynamic stimuli and impairs mechanotransduction in Pv+ DRG neurons because of substrate deformation-induced neurite stretching, but not to direct neurite indentation. In behavioural tasks, global knockout (Asic3(-/-)) and Pv-Cre::Asic3(f/f) mice produce similar deficits in grid and balance beam walking tasks. We conclude that, at least in mouse, ASIC3 is a molecular determinant contributing to dynamic mechanosensitivity in proprioceptors.

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

All sensory receptors adapt, i.e. they constantly adjust their sensitivity to external stimuli to match the current demands of the natural environment. Electrophysiological responses of sensory receptors from widely different modalities seem to exhibit common features related to adaptation, and these features can be used to examine the underlying sensory transduction mechanisms. Among the principal senses, mechanosensation remains the least understood at the cellular level. To gain greater insights into mechanosensory signalling, we investigated if mechanosensation displayed adaptive dynamics that could be explained by similar biophysical mechanisms in other sensory modalities. To do this, we adapted a fly photoreceptor model to describe the primary transduction process for a stretch-sensitive mechanoreceptor, taking into account the viscoelastic properties of the accessory muscle fibres and the biophysical properties of known mechanosensitive channels (MSCs). The model's output is in remarkable agreement with the electrical properties of a primary ending in an isolated decapsulated spindle; ramp-and-hold stretch evokes a characteristic pattern of potential change, consisting of a large dynamic depolarization during the ramp phase and a smaller static depolarization during the hold phase. The initial dynamic component is likely to be caused by a combination of the mechanical properties of the muscle fibres and a refractory state in the MSCs. Consistent with the literature, the current model predicts that the dynamic component is due to a rapid stress increase during the ramp. More novel predictions from the model are the mechanisms to explain the initial peak in the dynamic component. At the onset of the ramp, all MSCs are sensitive to external stimuli, but as they become refractory (inactivated), fewer MSCs are able to respond to the continuous stretch, causing a sharp decrease after the peak response. The same mechanism could contribute a faster component in the 'sensory habituation' of mechanoreceptors, in which a receptor responds more strongly to the first stimulus episode during repetitive stimulation., (© 2015 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

I present a brief review of current understanding of the innervation of the mammalian muscle spindle, from a personal historical perspective. The review begins with comparative studies on the numbers of spindle afferents and considers how their relative abundance may best be assessed. This is followed by an examination of the distribution and some functional properties of the motor innervation. The primary ending is the subject of the final section, in particular, I look at what can be learned from serial sectioning and volumetric reconstruction, and present new results on a model and simulations concerning sensory terminal deformation during stretch., (© 2015 Anatomical Society.)

Mechanoreception is an essential feature of many sensory modalities. Nevertheless, the mechanisms that govern the conversion of a mechanical force to distinct patterns of action potentials remain poorly understood. Proprioceptive mechanoreceptors reside in skeletal muscle and inform the nervous system of the position of body and limbs in space. We show here that Whirlin/Deafness autosomal recessive 31 (DFNB31), a PDZ-scaffold protein involved in vestibular and auditory hair cell transduction, is also expressed by proprioceptive sensory neurons (pSNs) in dorsal root ganglia in mice. Whirlin localizes to the peripheral sensory endings of pSNs and facilitates pSN afferent firing in response to muscle stretch. The requirement of Whirlin in both proprioceptors and hair cells suggests that accessory mechanosensory signaling molecules define common features of mechanoreceptive processing across sensory systems., (Copyright © 2015 de Nooij et al.)

The focus of this review is on the principal sensory ending of the mammalian muscle spindle, known as the primary ending. The process of mechanosensory transduction in the primary ending is examined under five headings: (i) action potential responses to defined mechanical stimuli-representing the ending's input-output properties; (ii) the receptor potential-including the currents giving rise to it; (iii) sensory-terminal deformation-measurable changes in the shape of the primary-ending terminals correlated with intrafusal sarcomere length, and what may cause them; (iv) putative stretch-sensitive channels-pharmacological and immunocytochemical clues to their identity; and (v) synaptic-like vesicles-the physiology and pharmacology of an intrinsic glutamatergic system in the primary and other mechanosensory endings, with some thoughts on the possible role of the system. Thus, the review highlights spindle stretch-evoked output is the product of multi-ionic receptor currents plus complex and sophisticated regulatory gain controls, both positive and negative in nature, as befits its status as the most complex sensory organ after the special senses.

Processes underlying mechanotransduction and its regulation are poorly understood. Inhibitors of Ca2+-activated K+ channels cause a dramatic increase in afferent output from stretched muscle spindles. We used immunocytochemistry to test for the presence and location of small conductance Ca2+-activated K+ channels (SK1-3) in primary endings of muscle spindles and lanceolate endings of hair follicles in the rat. Tissue sections were double immunolabelled with antibodies to one of the SK channel isoforms and to either synaptophysin (SYN, as a marker of synaptic like vesicles (SLV), present in many mechanosensitive endings) or S100 (a Ca2+-binding protein present in glial cells). SK channel immunoreactivity was also compared to immunolabelling for the Na+ ion channel ASIC2, previously reported in both spindle primary and lanceolate endings. SK1 was not detected in sensory terminals of either muscle spindles or lanceolate endings. SK2 was found in the terminals of both muscle spindles and lanceolate endings, where it colocalised with the SLV marker SYN (spindles and lanceolates) and the satellite glial cell (SGC) marker S100 (lanceolates). SK3 was not detected in muscle spindles; by contrast it was present in hair follicle endings, expressed predominantly in SGCs but perhaps also in the SGC: terminal interface, as judged by colocalisation statistical analysis of SYN and S100 immunoreactivity. The possibility that all three isoforms might be expressed in pre-terminal axons, especially at heminodes, cannot be ruled out. Differential distribution of SK channels is likely to be important in their function of responding to changes in intracellular [Ca2+] thereby modulating mechanosensory transduction by regulating the excitability of the sensory terminals. In particular, the presence of SK2 throughout the sensory terminals of both kinds of mechanoreceptor indicates an important role for an outward Ca2+-activated K+ current in the formation of the receptor potential in both types of ending.

Adult muscle size and fibre-type composition are heritable traits that vary substantially between individuals. We used inbred mouse strains in which soleus muscle mass varied by an order of magnitude to explore whether properties of muscle spindles can also be influenced by genetic factors. Skip-serial cross-sections of soleus muscles dissected from 15 male mice of BEH, BEL, C57BL/6J, DUH, LG/J and SM/J strains were analysed for number of muscle spindles and characteristics of intrafusal and extrafusal fibres following ATPase staining. The BEL and DUH strains determined the range of: soleus mean size, a 10-fold difference from 2.1 to 22.3 mg, respectively; the mean number of extrafusal fibres, a 2.5-fold difference from 497 to 1249; and mean fibre-cross-sectional area, three-fold difference, e.g. for type 1 fibres, from 678 to 1948 μm². The range of mean proportion of type 1 fibres was determined by C57BL/6J (31%) and DUH (64%) strains. The mean number of spindles per muscle ranged between nine (LG/J) and 13 (BEL) (strain effect P < 0.02). Genetic correlations between spindle count and muscle weight or properties of extrafusal fibres were weak and not statistically significant. However, there was a strong correlation between the proportion of spindles with more than one bag2 fibre and the proportion of extrafusal fibres that were of type 1, and strain-dependent variation in the numbers of such spindles was statistically significant. The numbers of intrafusal fibres per spindle ranged from 2 to 8, with the most common complement of four found in 75.6% of spindles. There were no significant differences between the strains in the mean numbers of intrafusal fibres; however, the variance of the number was significantly less for the C57BL/6J strain than for any of the others. We conclude that abundance of muscle spindles and their intrafusal-fibre composition are substantially determined by genetic factors, which are different from those affecting muscle size and properties of the extrafusal fibres., (© 2013 Anatomical Society.)

Our aim in the present study was to determine whether a glutamatergic modulatory system involving synaptic-like vesicles (SLVs) is present in the lanceolate ending of the mouse and rat hair follicle and, if so, to assess its similarity to that of the rat muscle spindle annulospiral ending we have described previously. Both types of endings are formed by the peripheral sensory terminals of primary mechanosensory dorsal root ganglion cells, so the presence of such a system in the lanceolate ending would provide support for our hypothesis that it is a general property of fundamental importance to the regulation of the responsiveness of the broad class of primary mechanosensory endings. We show not only that an SLV-based system is present in lanceolate endings, but also that there are clear parallels between its operation in the two types of mechanosensory endings. In particular, we demonstrate that, as in the muscle spindle: (i) FM1-43 labels the sensory terminals of the lanceolate ending, rather than the closely associated accessory (glial) cells; (ii) the dye enters and leaves the terminals primarily by SLV recycling; (iii) the dye does not block the electrical response to mechanical stimulation, in contrast to its effect on the hair cell and dorsal root ganglion cells in culture; (iv) SLV recycling is Ca(2+) sensitive; and (v) the sensory terminals are enriched in glutamate. Thus, in the lanceolate sensory ending SLV recycling is itself regulated, at least in part, by glutamate acting through a phospholipase D-coupled metabotropic glutamate receptor.

We investigated whether channels of the epithelial sodium/amiloride-sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch-evoked afferent discharge was reduced in a dose-dependent manner by amiloride and three analogues - benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching > or = 85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC delta subunit agonists (icilin and capsazepine). HMA's profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P < 0.01 each) at 1 microm, while 10 microm HMA was required for highly significant inhibition (P < 0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low-affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC alpha, beta and gamma subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC alpha, beta or gamma relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P < 0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1alpha) were also highly significantly greater (P < 0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic-like vesicles which regulate afferent excitability in these mechanosensitive endings.

The encapsulated sensory endings of mammalian skeletal muscles are all mechanoreceptors. At the most basic functional level they serve as length sensors (muscle spindle primary and secondary endings), tension sensors (tendon organs), and pressure or vibration sensors (lamellated corpuscles). At a higher functional level, the differing roles of individual muscles in, for example, postural adjustment and locomotion might be expected to be reflected in characteristic complements of the various end-organs, their sensory endings and afferent nerve fibres. This has previously been demonstrated with regard to the number of muscle-spindle capsules; however, information on the other types of end-organ, as well as the complements of primary and secondary endings of the spindles themselves, is sporadic and inconclusive regarding their comparative provision in different muscles. Our general conclusion that muscle-specific variability in the provision of encapsulated sensory endings does exist demonstrates the necessity for the acquisition of more data of this type if we are to understand the underlying adaptive relationships between motor control and the structure and function of skeletal muscle. The present quantitative and comparative analysis of encapsulated muscle afferents is based on teased, silver-impregnated preparations. We begin with a statistical analysis of the number and distribution of muscle-spindle afferents in hind-limb muscles of the cat, particularly tenuissimus. We show that: (i) taking account of the necessity for at least one primary ending to be present, muscles differ significantly in the mean number of additional afferents per spindle capsule; (ii) the frequency of occurrence of spindles with different sensory complements is consistent with a stochastic, rather than deterministic, developmental process; and (iii) notwithstanding the previous finding, there is a differential distribution of spindles intramuscularly such that the more complex ones tend to be located closer to the main divisions of the nerve. Next, based on a sample of tendon organs from several hind-foot muscles of the cat, we demonstrate the existence in at least a large proportion of tendon organs of a structural substrate to account for multiple spike-initiation sites and pacemaker switching, namely the distribution of sensory terminals supplied by the different first-order branches of the Ib afferent to separate, parallel, tendinous compartments of individual tendon organs. We then show that the numbers of spindles, tendon organs and paciniform corpuscles vary independently in a sample of (mainly) hind-foot muscles of the cat. Grouping muscles by anatomical region in the cat indicated the existence of a gradual proximo-distal decline in the overall average size of the afferent complement of muscle spindles from axial through hind limb to intrinsic foot muscles, but with considerable muscle-specific variability. Finally, we present some comparative data on muscle-spindle afferent complements of rat, rabbit and guinea pig, one particularly notable feature being the high incidence of multiple primary endings in the rat.

We describe a new method of investigation of the integrative action of fusimotor inputs in mammalian muscle spindles by stimulation of multiple fusimotor axons using independent pseudorandom pulse trains, each of low mean rate with pseudorandomly distributed stimulus intervals. Technically it was feasible only because of the development of (1) a novel, highly efficient approach to functional isolation of fusimotor efferents in ventral-root filaments, which we have called the isodyne strategy; (2) a real-time, microprocessor-based stimulus artefact cancellation device (SACAD); and (3) a highly adjustable, multi-branch stimulation electrode array. The general approach of using multiple, independent, pseudorandom stimulation of several input channels has wider applications in controlled-activation paradigms.

I present observations on the numbers and distributions of muscle spindles indicating that spindle number is an important controlled variable of muscle design, but the distribution of spindles within a muscle is not. Although our understanding of the functional correlates of spindle number is far from complete, I argue that nothing that is known about their number or distribution is inconsistent with them acting as length sensors, in contradiction to Kokkorogiannis's comment [Brain Res. Bull., this issue] on Windorst's review [U. Windhorst, Muscle proprioceptive feedback and spinal networks, Brain Res. Bull. 73 (2007) 155-202].

An allometric analysis of the number of muscle spindles in relation to muscle mass in mammalian (mouse, rat, guinea-pig, cat, human) skeletal muscles is presented. It is shown that the trend to increasing number as muscle mass increases follows an isometric (length) relationship between species, whereas within a species, at least for the only essentially complete sample (human), the number of spindles scales, on average, with the square root rather than the cube root of muscle mass. An attempt is made to reconcile these apparently discrepant relationships. Use of the widely accepted spindle density (number of spindles g(-1) of muscle) as a measure of relative abundance of spindles in different muscles is shown to be grossly misleading. It is replaced with the residuals of the linear regression of ln spindle number against ln muscle mass. Significant differences in relative spindle abundance as measured by residuals were found between regional groups of muscles: the greatest abundance is in axial muscles, including those concerned with head position, whereas the least is in muscles of the shoulder girdle. No differences were found between large and small muscles operating in parallel, or between antigravity and non-antigravity muscles. For proximal vs. distal muscles, spindles were significantly less abundant in the hand than the arm, but there was no difference between the foot and the leg.

Fifty-nanometre diameter, clear, synaptic-like vesicles (SLVs) are found in primary mechanosensory nerve terminals of vertebrate and invertebrate animals. We have investigated their role in mechanosensory function using the muscle spindle primary endings of rat Ia afferents as a model. Uptake and release of the synaptic vesicle marker FM1-43 indicated that SLVs recycle like synaptic vesicles and do so in a Ca(2+)-sensitive manner. Mechanical stimulation increased SLV recycling, increasing both dye uptake and release. Immunogold/electronmicroscopy showed that, like the central synaptic endings, Ia peripheral endings are enriched with glutamate. Moreover, exogenous glutamate enhanced stretch-induced Ia excitability. Enhanced excitability persisted in the presence of antagonists to the commonest ionotropic and metabotropic glutamate receptors (kynurenate, MCPG, CPPG and MAP4). However, excitation by glutamate was abolished by (R,S)-3,5-dihydroxyphenylglycine (DHPG), and rather more effectively by (2R,1'-S,2'-R,3'-S)-2-(2'-carboxy-3'-phenylcyclopropyl) glycine (PCCG-13). PCCG-13 also significantly reduced stretch-activated excitability in the absence of exogenous glutamate. These data indicate that SLVs recycle at rest, releasing glutamate, and that mechanical activity increases this process. The blockade with DHPG and PCCG-13 suggests that endogenous glutamate release acts, at least in part, through the recently described phospholipase D-linked metabotropic Glu receptor to maintain the excitability of the sensory endings.

Sensory terminals of muscle spindles and similar mechanosensory neurons contain large numbers of 50 nm, "synaptic-like" vesicles (SLVs), about whose role very little is known. Using fluorescence microscopy, immunocytochemistry and electrophysiological recording, we present evidence that SLVs undergo a recycling process, and that they release glutamate that has an autogenic excitatory effect on mechanosensory transduction, probably involving a metabotropic receptor linked to phospholipase D. The rate of recycling of SLVs is activity dependent, at least in part, as shown by an increased rate of destaining of preparations labelled with FMI-43 during high-frequency, small-amplitude vibration. Immunogold labelling showed levels of glutamate-like reactivity in the sensory terminals at least as great as in probable Ia presynaptic terminals in the spinal cord. Exogenously applied glutamate has an excitatory effect on the spindle's response to stretch, which is blocked by 3,5-dihydroxyphenylglycine.

The possibility of using randomly generated stimulus intervals (with a Poisson distribution) to identify the type(s) of intrafusal fiber activated by the stimulation of single static gamma axons was tested in Peroneus tertius muscle spindles of anesthetized cats. Three patterns of random stimulation with different values of mean intervals [20 +/- 4. 47, 30 +/- 8.94, and 40 +/- 8.94 (SD) ms] were used. Single static gamma axons activating, in single spindles, either the bag2 fiber alone or the chain fibers alone or both types of intrafusal fiber were prepared. Responses of spindle primary endings elicited by the stimulation of gamma axons were recorded from Ia fibers in cut dorsal root filaments. Cross-correlograms between stimuli and spikes of the primary ending responses, autocorrelograms, interval histograms of responses, and stimulations were built. The characteristics of cross-correlograms were found to be related not only to the type of intrafusal muscle fibers activated but also to the parameters of the stimulation. Moreover some cross-correlograms with similar characteristics were produced by the activation of different intrafusal muscle fibers. It also was observed that, whatever the type of intrafusal muscle fiber activated, cross-correlograms could exhibit oscillations after an initial peak, provided the extent in frequency of the primary ending response was small; these oscillations arise in part from the autocorrelation of the primary ending responses. Therefore, cross-correlograms obtained during random stimulation of static gamma axons cannot be used for unequivocally identifying the type(s) of intrafusal muscle fiber these axons supply.

In muscle spindles of the cat, independent control of dynamic and static components of the response of the primary sensory ending to stretch is provided by separate motor inputs to the various kinds of intrafusal muscle fibre: dynamic axons (gamma or beta) to the bag1 fibres and static axons to the bag2 (typically gamma only) and chain (gamma or beta) fibres. Nonlinear summation of separately evoked effects during combined stimulation of dynamic and static motor axons appears to be due to mutual resetting by antidromic invasion of separate encoding sites, leading to partial occlusion of the momentarily lesser response by the greater. The encoding sites are thought to be located within the primary ending's preterminal branches which from first-order level are normally segregated to the bag1 fibre and to the bag2 and chain fibres. Here we describe the analysis of a special case that arose in a histophysiological study which had shown that the degree of occlusion was related to the minimum number of nodes between the putative encoding sites. Three-dimensional reconstruction of the primary ending revealed that the terminals of one chain fibre were derived entirely from the first-order branch that supplied the bag1 fibre, including one terminal that was shared directly with the bag1 (sensory cross-terminal). The other first-order branch supplied the bag2 and remaining chain fibres as normal. The degree of occlusion seen during simultaneous stimulation of a dynamic beta axon and a static gamma axon indicated that the encoding sites were separated by both first-order branches. Schematic reconstruction of the motor innervation revealed that the static gamma axon was most unlikely to have supplied the chain fibre which shared sensory terminals with the bag1, but that these fibres also shared a motor input with histological characteristics of beta type. Ramp-frequency stimulation of the dynamic beta axon at constant length evoked a driving effect which persisted after fatiguing the extrafusal component and was therefore explicable on the basis of the observed pattern of motor innervation, though the identity of the axon could not be conclusively proved. Individually, instances of shared sensory terminals and motor input of bag1 and chain fibres are rare in the cat; their combination in a single spindle with correlated physiology is described here for the first time. The observation is considered in relation to the importance of dynamic and static segregation in motor control, since it may imply that there is a lower limit to the degree of segregation that the developmental programme can provide.

1. A combined physiological, histological and computer modelling study was carried out on muscle spindles of the cat tenuissimus muscle to examine whether there was any correlation between the functional interaction of putative encoding sites, operated separately by static and dynamic fusimotor neurones, and the topological structure of the preterminal branches of the primary sensory ending. 2. Spindles, whose I a responses to stretch and separate and combined static and dynamic fusimotor stimulation were recorded in physiological experiments, were located in situ. Subsequently the ramifications of the sensory ending were reconstructed histologically, and the topology of the branch tree was used in computer simulations of I a responses to examine the effect of the electronic separation of encoding sites on the static-dynamic interaction pattern. 3. Interactions between separate static and dynamic inputs, manifest in responses to combineed stimulation, were quantified by a coefficient of interaction (Ci) which, by definition, was 1 for strictly linear summation of separate inputs and zero for maximum occlusion between inputs. 4. For the majority of spindles static-dynamic interactions were characterized by pronounced occlusion (C1 < 0.35). In these spindles putative encoding sites (the peripheral heminodes of the branches supplying the intrafusal fibres activated by individual fusimotor efferents) were separated by a minimum conduction path of between three and ten myelinated segments (2-9 nodes of Ranvier). In contrast, significant summation (C1, approximately 0.7) was found in only one spindle. In this case putative encoding sites were separated by a single node. 5. Occlusion was not due to encoder saturation and it could not be accounted for by any other known physiological mechanisms (intrafusal fatigue or unloading). It is therefore attributed to competitive pacemaker interaction between encoding sites which are largely selectively operated by static and dynamic fusimotor efferents. 6. Model simulations of real preterminal-branch tree structures confirmed that short conduction paths between encoding sites were associated with manifest summation, whereas longer minimum conduction paths favoured pronounced occlusion. 7. In the extreme, occlusion could be so pronounced as to give rise to negative values of C1 during critical segments of response cycles. This was associated with lower discharge rates during combined static and dynamic stimulation than the higher of the individual stimulation effects. This phenomenon is referred to as hyperocclusion. Computer simulations demonstrated that hyperocclusion could be accounted for by a slow ionic adaptation process. e.g. by a very slowly activating K+ conductance.

Superficial lumbrical spindles of the cat show several important differences as compared with the classic picture of the spindle, derived principally from tenuissimus. Thus long chain fibres occur in a high proportion of superficial lumbrical spindles, and there is great individual variation in the number of bag fibres present. This study is especially concerned with the morphology and relationships of the bag1, fibre, which are again highly unusual. Most notably, bag1 fibres were almost always the longest intrafusal fibres, typically exceeding bag2 fibres by about 2 mm in total length. They also commonly exhibited the greatest diameter, especially in the extracapsular region where, uniquely, they normally increased in size on leaving the capsule, often becoming similar to extrafusal fibres in this respect. In a few spindles where there was only a single bag fibre this was more often a bag1 than is the case for similar spindles in tenuissimus, and in tandem-linked spindles the continuous bag fibres were more often bag1 than bag2. These results contribute to growing evidence that different muscles exhibit characteristic features of the structure and innervation of their proprioceptors, which may reflect adaptations to local control requirements.

A quantitative analysis of the motor innervation of intrafusal muscle fibres is described, based on teased silver-impregnated spindles of the tenuissimus muscle of the cat. Included in the analysis are the number and distribution of intrafusal branches of both skeletofusimotor (beta) and purely fusimotor (gamma) axons, and the form of their endings. The number of axonal branches per spindle was found to follow binomial probability distributions, as had previously been shown for the afferent axons. There was a strong correlation between the numbers of gamma intrafusal branches and afferent axons, but none for the intrafusal branches of beta axons. The degree of segregation of gamma input to bag2 and chain fibres was assessed and was found, among other things, to be related to the presence of secondary sensory endings in the same pole. In this and other respects it did not appear to have the properties that would be expected if independent activation of the bag2 and chain fibres were to be functionally important. Morphometric analysis of the motor endings supplied to bag2 or chain fibres by gamma axons revealed some differences between those of intrafusal branches with segregated as opposed to unsegregated distributions, but this cannot be taken as evidence of more than one type of static gamma motoneuron because of the likely contribution of other influential factors such as fibre size. Finally, the relevance of studies on intrafusal motor innervation to the concept of the motor unit and its development are discussed.

1. The distribution of static gamma-axons within and among muscle spindles of the tenuissimus muscle has been studied in the anaesthetized cat, on the basis of the effects on the responses of primary endings when bag2 or chain fibres or both are activated by static gamma-stimulation. 2. Locations of spindles were marked for subsequent histological analysis using teased, silver-impregnated preparations. 3. Static effects were classified into: (i) biassing; (ii) driving; or (iii) indeterminate categories. 4. Critical correlations established that the biassing type was produced by bag2 activity, either alone or in combination with chain fibres, whereas the driving type was produced by chain fibres active alone. Indirect evidence suggested that indeterminate effects were produced by bag2 and chain fibres active together. 5. The static gamma-axons showed some differential distribution according to their conduction velocities: faster-conducting axons were likely to be more widely distributed among spindles but less likely to innervate chain fibres alone than were more slowly conducting axons. 6. The results are discussed in terms of their possible functional and developmental significance.

The motor innervation of four muscle spindles from the tenuissimus muscle of the cat was demonstrated using reconstructions of 1 micrometer thick, serial transverse sections. Analysis of the results clearly indicates that the bag intrafusal muscle fibre usually does not receive a static fusimotor input via trail innervation. In contrast to the highly selective innervation of bag fibres, almost half the axons supplying bag or chain fibres branched to terminate on both types of fibre. The significance of these results is discussed in relation to previous studies on fusimotor innervation and to their functional implications. The presence of autonomic innervation is a further complication that appears to have led to erroneous conclusions concerning the nature of the trail innervation of chain fibres in a recent study of the distribution of cholinesterase activity in the spindle.

The common peroneal nerve was transected and repaired by epineurial suture in nine cats. In a further nine the nerve was transected twice and similarly repaired so as to produce a short autograft. Recovery of stretch receptors in peroneus brevis was monitored histologically and physiologically from six to fifty weeks. In recovery after single neurotomy functionally identifiable muscle-spindle and tendon-organ afferents were reduced to 25% and 45% of normal, respectively; after double neurotomy (autograft) both were reduced to about 10% of normal. Muscle spindles were reinnervated with annulospiral terminals, or wholly abnormal fine axon terminals, or both. Recovery evidently entails not only a reduction in number of stretch afferents, but also the making of some incorrect reconnections that presumably result in abnormal proprioceptive feedback and reflex action. When a graft is used the sensory impairment is compounded.

1. We have made quantitative assessments of the sensory reinnervation and recovery of peroneus brevis muscle spindles following section and epineurial repair of the common peroneal nerve. After 6-50 weeks recovery, single-unit, dorsal-root recordings were made of the responses to ramp-and-hold or sinusoidal stretch of the reinnervated spindles, which were subsequently examined in teased, silver preparations. 2. Assessments of recovery used data obtained from cross-union experiments in which foreign afferents (including Ib) were given the opportunity of reinnervating spindles in the absence of their native (Ia, spindle II) afferents; and from an examination of tenuissimus spindles reinnervated by Ia and spindle II afferents in the absence of Ib afferents. These studies revealed: (i) that regenerating Ib afferents can terminate in sites originally occupied by the endings of Ia or spindle II afferents, and respond to stretch like normal Ia and spindle II afferents; (ii) that Ib and spindle II afferents reinnervating spindles are histologically identical apart from diameter range; and (iii) that some cutaneous afferents can reinnervate spindles and give highly abnormal, phasic stretch responses. 3. Recovery of afferents reinnervating spindles was marked by increases in conduction velocity and proportions firing tonically, but their firing rates at the three phases of ramp-and-hold stretch were considerably lower than normal and showed no tendency to increase. 4. Some relatively fast afferents that gave spindle II-type responses were identified as Ib afferents reinnervating secondary-ending sites; conversely, some relatively slow afferents that gave Ia-type responses were identified as spindle II afferents reinnervating primary-ending sites. 5. The estimated loss of spindle afferents from tenuissimus after nerve section (52% Ia, 49% spindle II) was considerably less than the estimated loss of these afferents from peroneus brevis after section of the common peroneal nerve (79% Ia, 86% spindle II). The proportion of spindles in tenuissimus reinnervated by free-ending afferents was also much lower (22%) than in peroneus brevis (73%). These differences are partly attributed to the greater size and degree of afferent complexity of the common peroneal nerve. 6. Similar proportions of spindles in peroneus brevis were reinnervated by Ia and Ib afferents after both partial (27% Ia, 20% Ib) and complete (21% Ia, 20% Ib) section of the common peroneal nerve.(ABSTRACT TRUNCATED AT 400 WORDS)

The arrangement of preterminal and terminal axon branches in the primary sensory endings of cat tenuissimus muscle spindles was studied using whole-mount and serial-section techniques. Although in every case one first-order preterminal branch was supplied exclusively to the bag1 type of intrafusal muscle fibre, the preterminal branching patterns differed considerably in detail. Terminals varied widely in size and location. Their precise form varied according to their position on the intrafusal muscle fibres rather than their relationship to preterminal branches. Terminals derived from separate preterminal branches remained separate and did not fuse with themselves or each other. Individually bag1 fibres had most terminals, chain fibres least. The surface of the muscle fibres were differentially indented by the terminals, least in bag1 fibres and most in chain fibres. The results are discussed in relation to mechanosensory transduction and to the factors involved in determining the form of the primary ending.

The presynaptic features of 234 motor endings supplied to cat hindlimb muscle spindles have been studied in teased, silver preparations, and the postsynaptic features of a further 27 endings have been studied in serial, 1 micron thick, transverse sections. In the presynaptic study motor endings received by the three types of intrafusal muscle fibre were compared with the endings supplied to spindles by the various functional categories of motor axon. Three forms of motor ending were found that had significantly different presynaptic features. These forms correspond closely to those previously identified in the literature as p1 (beta), p2 (dynamic gamma) and trail (static gamma). The results of the postsynaptic study showed that the degree of indentation of the intrafusal muscle fibres by motor axon terminals increases with greater distance from the primary ending, irrespective of muscle-fibre type. We conclude that the postsynaptic form of intrafusal motor endings is determined by distance from primary ending and muscle-fibre type. It is not determined by type of motor axon, and cannot be correlated with presynaptic form so as to produce a unified classification of intrafusal motor endings.