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IntroductionThe 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 methodsWe 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.ResultsSignificant 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.DiscussionThus, 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.

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.

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.

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. 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.

Muscle spindle afferents are slowly adapting low threshold mechanoreceptors which have both dynamic and static sensitivity to muscle stretch. The exact mechanism by which these neurons translate muscle movement into action potentials is not well understood, although the PIEZO2 mechanically sensitive cation channel is essential for stretch sensitivity. PIEZO2 is rapidly adapting, suggesting the requirement for additional molecular elements to maintain firing during stretch. Spindle afferent sensory endings contain glutamate-filled synaptic-like vesicles which are released in a stretch and calcium dependent manner. Previous work has shown that glutamate can increase and a phospholipase-D coupled metabotropic glutamate antagonist can abolish firing during static stretch. Here we test the hypothesis that vesicle-released glutamate is necessary for maintaining muscle spindle afferent excitability during static but not dynamic stretch. To test this hypothesis, we used a mouse muscle-nerve ex vivo preparation to measure identified muscle spindle afferent responses to stretch and vibration. In C57BL/6 adult mice, bath applied glutamate significantly increased the firing rate during the plateau phase of stretch, but not during the dynamic phase of stretch. Blocking the packaging of glutamate into vesicles by the sole vesicular glutamate transporter, VGLUT1, either with xanthurenic acid or by using a transgenic mouse with only one copy of the VGLUT1 gene (VGLUT1(+/−)) decreased muscle spindle afferent firing during sustained stretch, but not during vibration. Our results suggest a model of mechanotransduction where calcium entering the PIEZO2 channel can cause the release of glutamate from synaptic-like vesicles which then helps to maintain afferent depolarization and firing.

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.

Mechanosensory transduction (MST) in proprioceptors, and other low threshold mechanosensory nerve terminals (LTMT), has been debated intensely for decades. MST in muscle spindles produces a receptor potential that encodes stimulus speed and duration, is predominantly due to Na+, a little Ca2+, plus some transient, non-mechanically-gated K+ ion fluxes. The abundant, multiple Na+-selective DEG/ENaC channel isoforms present in all LTMTs seemed obvious Na+ sources, perhaps supplemented with Ca2+-selective TRPs, and Ca2+-activated K+ channels. However, genetic deletions of even multiple DEG/ENaC genes produces only mild functional perturbation. Conversely, deleting the more recently discovered Piezo2 mechanosensory protein profoundly impairs LTMT responses, including in muscle spindles. Yet its transient opening, non-Na+-selectivity and its pharmacology do not reflect known receptor potential and response properties. A Ca2+-dependent recycling vesicle pool that we have shown is essential for mechanosensitivity, plus other recent DEG/ENaC discoveries, may reconcile these conflicting observations. We propose the abundance of axolemmal MST complexes, comprising untested DEG/ENaC combinations, is controlled by Piezo2-gated Ca2+ influx that regulates their vesicular insertion and retrieval.

Baroreceptors are a promising new drug target for treating high blood pressure (hypertension). Hypertension is the world’s commonest killer disease because it is the biggest underlying risk factor for cardiovascular disease, stroke and kidney failure (WHO). These account for over 7 m deaths per year. The WHO regards current treatments as inadequate. Even in the developed world most patients need multiple drugs, both for the initial condition and combatting the side-effects the primary medication produces. Even with multi-drug therapies,>10% of patients are unresponsive. No more effective drugs have been introduced for over 20 years. We hypothesise this is because of the focus on old targets. Therefore, the WHO has asked urgently for new therapeutic targets. Baroreceptors are emerging as such a novel, and highly effective target. Electrical stimulation of baroreceptor nerves reduces long-term blood pressure by >20 mmHg even in the most drug-resistant patients. In some cases, it can even replace medication. However, only a small number of patients get this treatment, as it is highly invasive, expensive and requires rare surgical expertise. Thus, only resistant hypertensives are treated. Even then, this is a high-risk patient group, and only 50% are deemed suitable due to the anatomical condition of the stimulation site, and the difficulties of post-operative stimulation optimisation, gagging reflex activation, equipment maintenance, etc. We propose a pharmaceutical targeting of baroreceptors could be just as effective, and would be suitable for all patients, plus avoid the side-effects associated with current medications. We have discovered a modulatory system controlling mechanosensory terminal sensitivity that involves an unusual glutamate receptor with highly atypical pharmacology. We are therefore pharmacologically characterising the receptor in the aorta-aortic depressor nerve (ADN) system, as a translational step towards the clinic. Isolated aorta-ADN or working-heart brainstem (WHBS) preparations were humanely prepared (ASPA, 1986) from adult Sprague-Dawley rats. Baroreceptor immunolabelling for the atypical glutamate receptor (PLD-GluR) showed it to be present. In physiological experiments, pressure pulses were delivered periodically to the isolated or WHBS aorta. The effects of glutamate receptor or transducer channel ligands on ADN firing was recorded, as well as effects on sympathetic output and heart rate. PLD-GluR agonists robustly increased baroreceptor firing. Concanavalin A increased kainate-mediated enhancement by blocking receptor desensitisation. Blocking TRP or DEG/ENaC channels with 2-APB or amiloride inhibited pressure-evoked firing. Glutamatergic stimulation of baroreceptors also inhibited sympathetic output, indicating decreased sympathetic tone. Selective PLD-GluR antagonist PCCG-13 inhibited pressure-evoked firing and changes in sympathetic tone. These data show baroreceptor firing can be pharmacologically controlled. And, this influences baroreflex, inhibiting sympathetic output. Thus, glutamate receptor activation on baroreceptors offer a novel target, increasing baroreceptor stretch-sensitivity and the baroreflex response which may have important clinical implications. Further experiments are planned to fully characterise this response and the GluR involved.

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’.

Muscle spindles and tendon organs are types of encapsulated proprioceptive sense organs found in skeletal muscle, and important for motor control. Tendon organs typically have single sensory endings of group Ib nerve fibers and are located at the myo-tendinous junction. They respond best to actively generated muscle force. Muscle spindles are more complex. They may have one or more sensory endings as well as their own motor innervation. They are located between fascicles of extrafusal muscle fibers and respond to muscle length and its rate of change. Each muscle has its own characteristic complement of muscle spindles and tendon organs.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00424-014-1536-9) contains supplementary material, which is available to authorized users.

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.

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 (Asic3f/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::Asic3f/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., Acid-sensing ion channel 3 (ASIC3) is known to play a role in nociception, but its role in low threshold neurosensory mechanotransduction is unclear. Here, the authors target ASIC3 expression in dorsal root ganglion parvalbumin positive neurons and find ASIC3 contributes to dynamic proprioception responses.

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.

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 ≥85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC δ 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 μm, while 10 μm 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 α, β and γ subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC α, β or γ 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 (BNaC1α) 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.

All sensory receptors adapt, i.e., they constantly adjust their sensitivity to external stimuli to match the current natural environment [1]. 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 [1,2]. Among the principal senses, mechanosensation remains the least understood at the cellular level [3]. 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 [4] to describe the primary transduction process for a stretch-sensitive mechanoreceptor, taking into account the viscoelastic properties of the accessory muscle fibres [5] 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 of 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 [6]. The initial dynamic component is likely to be caused by both the mechanical properties of the muscle fibres and a refractory state of MSCs. Consistent with literature, the current model predicts that the dynamical component is due to a rapid stress increase during the ramp [7]. More novel predictions from the model are the mechanisms to explain the initial peak in the dynamical component. At the onset of the ramp, all MSCs are sensitive to external stimuli, but as they become refractory (clipped inactivated state), 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 'sensory habituation' of a mechanoreceptor, in which a receptor responds more strongly to the first stimulus episode during repetitive stimulation [8].

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.

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.

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].

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 bag(1) fibres and static axons to the bag(2) (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 bag, fibre and to the bag(2) 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 bag(1) fibre, including one terminal that was shared directly with the bag, (sensory cross-terminal). The other first-order branch supplied the bag(2) 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 bag(1), 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 bag, 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.

Item does not contain fulltext Abstract 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.

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.

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.

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 FM 143 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 la 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.

Testing the classification of static γ axons using different patterns of random stimulation. 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 γ 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 γ 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 γ 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 γ axons cannot be used for unequivocally identifying the type(s) of intrafusal muscle fiber these axons supply.

In this chapter we shall examine the development and application of histological methods at the tissue and system level of neural organisation. The principal techniques — fixation, dyeing, metal impregnation and histochemistry — have already been encountered in Chapter 1, where the emphasis was at the cytological level. The two chapters are intended to be considered together, thus the specific techniques covered in each one are applicable at both cellular and tissue levels. Once again, the methods to be described here will, whenever possible, be placed in the context of particular functional problems. There are, however, no hard and fast rules about which is the most appropriate method to apply to a given problem; this will depend on many factors not all under the investigator’s control, including one’s experience, material resources and availability of imaging and data-recording facilities. Of course, it should be borne in mind that different methods will almost certainly generate somewhat different data while addressing the same problem. This point will be illustrated in example 4 below with a case study from my own work on the innervation of muscle spindles.

Neurohistology is perhaps traditionally thought of as supplying information only about the spatial or structural aspects of the neuron; however, it is my intention in the present chapter and its companion (Chapter 15 in Sect.II) to present as far as possible a unified view of neurohistology as a set of related problems centred on the relationship between structure and function of nerve cells. Such problems are not unique to the subject, but in the context of neurohistology they are uniquely complex. Neurons, as cells, are unrivalled in diversity of type, and other kinds of cells rarely match any neuron in the complexity of their spatio-temporal properties or in the range of genes expressed. The status of neurohistology as a recognisable discipline is therefore dependent on these properties of nerve cells and nervous tissue, and its history is largely one of the development of methods aimed at overcoming the difficulties presented by them. Of course, recognisable disciplines need not necessarily have sharp boundaries and it is perhaps already apparent that I intend to take a fairly relaxed view as to what constitutes neurohistology. The essential criteria are whether the investigation involves the nervous system and whether it uses microscopy. Beyond those, it is a matter of taste where macroscopically neuroanatomy and neuroimaging give way to neurohistology, and microscopically neurohistology gives way to cellular and molecular biology.

Nuclear chain fibres are classically regarded as being much shorter and thinner than bag fibres, either bag1 or bag2. However, Barker et al. (1976) noticed that some spindles possessed one or more chain fibres with unusually long poles, often of similar length to the bag fibres. As an estimate of the incidence of these ‘long chain’ fibres they assessed the proportion of spindles containing at least one chain fibre pole that extended for more than 1mm beyond the end of the capsule. Kucera (1980) formalised this practical criterion as a definition of a class of long chain fibres each of which possessed at least one long pole. Since that time, long chain fibres have often been treated as though they formed a distinct type of intrafusal fibre with a specific static beta (βs) innervation (Kucera, 1982, 1984; Barker & Banks, 1986). Here we give evidence in detail of both static β and static γ innervation of long chain fibres.

The abductor digiti quinti medius (adqm) is an intrinsic muscle of the foot which abducts the fifth digit. Despite its small size (about 30mg) it contains all the elements of a complete skeletomotor and fusimotor system. It may therefore provide a practical model for studying integrated fusimotor and skeletomotor activity under a variety of conditions. Morphological investigation of the innervation of the adqm muscle of the cat hind limb was carried out as an adjunct to electrophysiological studies.

Histophysiological experiments using the superficial lumbrical muscle of the cat have provided the opportunity to analyse the structure of a large number of spindles (Decorte, Emonet-Denand, Ginape & Laporte, 1986). This muscle was initially chosen because of its small size, spindle content and known motor-unit properties (Emonet-Denand, Hunt, Petit & Pollin, 1988).

An important, if largely unrecognised, feature of the design of the mammalian skeletomotor proprioceptive system is the characteristic variation from muscle to muscle in the provision of its components. This applies not only to the relative abundance of muscle spindles, but also to quantitative differences in their sensory ending complement (Banks & Stacey, 1988). Furthermore, Banks (1994) has shown that there is a linear correlation between the numbers of static γ and afferent axons supplied to each spindle, at least in the tenuissimus. Considerably more comparative information of this type is needed for a fuller understanding of the contribution that proprioception makes to particular motor tasks. Here we describe some preliminary observations on the innervation of the superficial lumbrical muscle of the cat’s hind foot.

Mammalian Ia afferents have a tendency to fire action potentials phase-locked to periodic stimuli, when certain static gamma axons are stimulated at constant frequency. This driving is thought to be mediated by chain fibres, which, in contrast to other intrafusal fibres, have fast contraction properties, comparable to type II extrafusal fibres. Intrafusal contractions are mechanically transmitted to the sensory ending, which transduces the movement into a receptor current. The ensuing oscillatory changes in receptor potential which accompany these rapid chain fibre contractions then cause the generation of one spike during each contraction cycle. If stimulation frequencies are high, or the bag2 fibre is coactivated, the afferent may fire at subharmonic frequencies, giving 1:2 or 1:3 driving. Also, very irregular spike intervals can be observed, which gives the impression of an erratic firing response. Changes in the initial muscle length often cause transitions between subharmonic, 1:1 driving, and irregular firing (Boyd, Murphy & Mann, 1985; Banks, 1991).

Peripheral sensory endings often consist of groups of unmyelinated nerve terminals, specialised for transduction, that ultimately converge on the single afferent axon through a system of myelinated preterminal branches. At least in the mammalian muscle spindle the sensory terminals are in continuity only through the preterminal branches (Banks, 1986), so each may be supposed to have an associated spike-initiation (encoding) site, potentially able to act as a separate pacemaker. Interaction in such a system is expected to be highly competitive (Eagles & Purple, 1974) so that the final output of the afferent is in general a non-linear function of the activities of the separate encoders.

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 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.

Nuclear bag and nuclear chain intrafusal fibres are present in guinea-pig muscle spindles. Unlike muscle spindles in other species two types of nuclear chain fibre seem to be present. The electron microscopical appearance of one type of nuclear chain fibre is similar to that of nuclear bag fibres.

The distribution of motor and sensory axons to the three types of intrafusal muscle fibre have been determined using reconstructions of serially sectioned spindles and teased, silver-impregnated, whole spindles.