A comparative analysis of the encapsulated end-organs of mammalian skeletal muscles and of their sensory nerve 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.

Comment on "Two enigmas in proprioception: abundance and location of muscle spindles" by T. Kokkorogiannis.

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].

Sensory reinnervation of muscles following nerve section and suture in cats.

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.

An allometric analysis of the number of muscle spindles in mammalian skeletal muscles.

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.

The distribution of static gamma-axons in the tenuissimus muscle of the cat.

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.

A histological study of the motor innervation of the cat's muscle spindle.

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.

Observations on the primary sensory ending of tenuissimus muscle spindles in the cat.

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.

Intrafusal motor innervation: a quantitative histological analysis of tenuissimus muscle spindles in the cat.

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.

Characteristic properties of superficial lumbrical spindles in the cat hind limb, related to their bag1 fibres.

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.

Specificities of afferents reinnervating cat muscle spindles after nerve section.

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)

Correlated histological and physiological observations on a case of common sensory output and motor input of the bag1 fibre and a chain fibre in a cat tenuissimus spindle.

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.

Testing the classification of static gamma 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 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.

A study of mammalian intrafusal muscle fibres using a combined histochemical and ultrastructural technique.

A direct correlation of the histochemical and ultrastructural properties of intrafusal muscle fibres has been achieved by cutting frozen serial sections for histochemical applications (15 micron thick sections) and for electron microscopy (60 micron thick sections) in a repeating sequence. Three types of intrafusal fibre were recognized, including two types of nuclear-bag fibre, designated bag1 and bag2. In addition to histochemical and ultrastructural differences, the three types of fibre differed in length and diameter. Regional variations of histochemical and ultrastructural properties were found. The results are compared with previous attempts to correlate histochemical and ultrastructural properties of intrafusal muscle fibres based on indirect methods.

Form and classification of motor endings in mammalian muscle spindles.

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.

Pacemaker activity in a sensory ending with multiple encoding sites: the cat muscle spindle primary ending.

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.

Histological analysis of cat muscle spindles following direct observation of the effects of stimulating dynamic and static motor axons.

1. Eleven cat tenuissimus spindles have been analysed mainly by cutting serial, transverse, 1 micrometer thick sections following direct observation of the effects of dynamic motor (gamma or beta) stimulation. 2. Histological results from these spindles were also used to interpret the effects of static fusimotor stimulation of other spindles. 3. Dynamic motor stimulation usually produced contractions seen as convergent movements of sarcomeres in single bag fibres, identified as bag1 fibres for reasons given in the text. 4. In one spindle a single dynamic axon produced a translational movement in one pole of a bag1 fibre and a convergent movement in each pole of a bag2 fibre, together with movements in other unidentified (presumably chain) fibres. Subsequent analysis showed that besides innervating both bag fibres the axon also supplied two chain fibres. 5. Contrary to expectation, motor endings on the bag1 fibres seldom occurred at the sites of convergent movement. Only two cases of coincidence occurred among sixteen foci and twenty-one motor endings; otherwise focus and nearest ending were separated by distances of 0.85--2.5 mm. 6. Most of the convergent movements of sarcomeres observed in bag1 fibres occurred in a region of the pole that is ultrastructurally distinct from the region where most of the motor endings were located. The possible relevance of this to the production of contractions in the bag1 fibre is discussed. 7. Convergent movement foci in bag2 fibres produced by the stimulation of static axons occurred largely within the same regions of the pole as the motor endings were located, though, whereas foci were observed in both intra- and extracapsular regions, most of the endings were intracapsular.

A possible role for placental lysosomes in the formation of villous syncytiotrophoblast.

An ultrastructural and ultrahistochemical study of first trimester human placentae confirms previous reports that the cytotrophoblastic cells show a spectrum of differentiation, that dissolution of the limiting membrane of the cytotrophoblastic cells occurs and that fragments of free membrane can be found in the syncytiotrophoblast. There is an aggregation of primary lysosomes in the region of approximation of the cytotrophoblast to the syncytiotrophoblast, free lysosomal enzymes are found in the space between the two trophoblastic components, secondary lysosomes have been noted in the vicinity of fragmenting cytotrophoblastic cell membrane and the incorporation of a segment of free membrane into a vesicular structure has been noted. It is suggested that placental lysosomes mediate the dissolution of the cytotrophoblastic cell membranes that is a necessary prerequisite for their full differentiation into syncytiotrophoblast and it is further suggested that one of the principal roles of placental lysosomes is in the structural refashioning of the organ that occurs during the first trimester.