The tensor tympani muscle of cat and dog contains IIM and slow-tonic fibres: an unusual combination of fibre types

Summary Using recently developed highly specific antisera to the full range of known adult mammalian skeletal muscle myosins, an immunohistochemical and histochemical examination was made of the middle ear muscle tensor tympani in the dog and cat. Approximately half the fibres were of the IIM type and there was a substantial population of apparently slow-tonic fibres, both these types being rare in mammals. In addition, some type I but no IIA nor IIB fibres were detected. Moreover, as no multiple end-plate innervation, thought to be typical of slow-tonic fibres, could be demonstrated in this muscle by acetylcholinesterase staining or by Ruffini gold impregnation, it is suggested that in tensor tympani the slow-tonic fibres are focally innervated. The very short length of the fibres, only 1–2 mm, is probably sufficient to permit adequate depolarization of a whole fibre by a single centrally situated end-plate. The functional implications of this combination of very rare fibre types in tensor tympani are unclear at present.     

Force and Contraction Velocity of the Middle Ear Muscles in the Gat and the Rabbit

Contractions of the stapedius and the tensor tympani muscles in cats and rabbits have been elicited at controlled length by electric stimulation of their motor nerves at varying stimulus frequencies. Both muscles were stronger than found in previous investigations. The tetanic tension of the stapedius was an average of 13.9 g (cat) and 15.4 g (rabbit). The tetanic tension of the tensor tympani averaged 54.3 g (cat) and 32.3 g (rabbit). By comparing the data from cats with the findings of Wever and Bray (1937, 1942) each muscle developed sufficient tension to cause a reduction in the sound transmission of the middle ear of more than 20 dB. With a twitch contraction time between 20 and 30 ms both muscles must be classified as fast muscles. However, different degrees of submaximal stimulation gave evidence for an additional slow twitch component in the tensor tympani muscle.     

The fibre-type composition of the first branchial arch muscles in Carnivora and Primates

A combination of standard histochemical techniques and immunohistochemical staining using myosin type-specific antisera was used to determine the fibre-type composition of the muscles of first branchial arch origin (that is, masseter, temporalis, pterygoideus medialis and lateralis, tensor veli palatini, tensor tympani, anterior digastricus and mylohyoideus) in a wide range of the Carnivora and the Primates. The rare IIM fibre type was found in the first branchial arch muscles of most of the species examined, but never in the limb muscles used as controls for this study. The jaw-closer muscles (masseter, temporalis and pterygoideus medialis) were found to contain IIM fibres in all the Carnivora except the lesser panda and in all the Primates except man. When present, the IIM fibres were usually the predominant fibre type, and the only other fibre types present were types I, II or IIC. The presence of IIM fibres in the jaw-closer muscles of most of the Carnivora and the Primates seems to be associated with an aggressive bite which is required for predation by the former and defence by the latter. In both groups of species there was the member which does not have an aggressive bite, the lesser panda and man, respectively, and these (like all other orders of mammals such as Lagomorpha, Rodentia, etc.) were found to have no IIM fibres in the jaw-closer muscles. The two muscles of the first branchial arch group which are derived from the ventral constrictor muscles of the (phylogenetically) original mandibular arch never contained IIM fibres, and were composed of type I and II fibres similar to those found in the control muscles of the limb. Tensor veli palatini and tensor tympani showed species-dependent variations in fibre-type composition and did not always reflect the composition of the jaw-closer muscles. Thus their common origin with the jaw-closers cannot be responsible for the occurrence of IIM fibres in tensor veli palatini and tensor tympani in some species. Furthermore, in tensor tympani but not in tensor veli palatini, the presence of IIM fibres was always accompanied by immunohistochemically slow-tonic fibres. Finally, the regard to the association of oxidative activity with the fibre type as defined by the myofibrillar ATPase method and by the isoform of myosin present, we suggest that in the first branchial arch muscles this is probably not directly comparable to the situation in the typical limb muscle.     

Dual end-plate potentials at the single neuromuscular junction of the adult frog

Summary Electrophysiological evidence is presented that at least 30 percent of sartorius muscle fibres of adult frogs are innervated by two or more axons at a single end-plate zone. In these fibres, increasing stimulation of the common sartorius nerve led to the appearance of two or more distinct levels of end-plate potentials (e.p.p.) (or currents, measured by the voltage clamp technique). They had an identical time course, reversal potential and delay to nerve stimulation. When the recording microelectrode was moved along the same fibre and reinserted, both components of e.p.p. decreased proportionally. This indicated that both components of e.p.p. originated very closely to each other on the muscle fibre, presumably in one end-plate zone. Many fibres of the sartorius muscle of adult frogs therefore possess polyneural innervation of a single end-plate zone, which is otherwise typical for early stages of ontogenesis.     

Slowing of the discharge of secondary endings of cat muscle spindles during fusimotor stimulation

Summary Responses of secondary endings of muscle spindles of the peroneus tertius muscle of the anaesthetized cat have been recorded during repetitive stimulation of functionally single fusimotor fibres that produced slowing of the discharge. In a sample of 125 pairs of single fusimotor fibres and secondary spindle afferents 5 examples of slowing were seen. The amount of slowing became less at longer muscle lengths. Conditioning the spindle by stimulating the muscle nerve at fusimotor strength, at a length 2.5 mm longer than the test length, and then returning to the test length 3 seconds later led to a greater degree of slowing of the discharge than after conditioning stimulation at the test length. With one exception, responses to muscle stretch were reduced during stimulation of a fusimotor fibre that produced slowing. On two occasions stimulating a fusimotor fibre that produced slowing of the response of one secondary ending, led to excitation of two other endings. Two possible explanations for the generation of slowing responses have been considered. The first is that the slowing is the result of contraction of the region of intrafusal fibre directly underlying the secondary sensory ending. The second, which we favour since it accounts for the facts more adequately, is that slowing is the result of shortening of the region of nuclear chain fibres on which the sensory ending lies, produced by movement in an adjacent nuclear bag fibre.     

The complex-type oligosaccharide binding lectin Datura stramonium agglutinin detects type II A muscle fibres in the brachial biceps from man and cat

Complex-type oligosaccharides were detected in the sarcoplasm of muscle fibres from cat and human biceps using lectins and anticarbohydrate antibodies. The lectin Datura stramonium agglutinin strongly stained type II A fibres as identified by myosin ATPase activity after alkaline and acid preincubation. In contrast, all muscle fibres showed a moderate coarse granular staining after incubation with Tetracarpidum conophorum agglutinin and Telfairia occidentalis agglutinin which recognize tri-antennary complex glycans poorly bound by D. stramonium agglutinin. Strong sarcoplasmic staining in all muscle fibres was obtained after incubation with an antibody against branched N-acetyllactosamine structure while an antibody against binary 2←3 sialyllactosamine glycans failed to detect the muscle fibres. Treatment of the muscle sections with sialidase prior to incubation with D. stramonium agglutinin did not influence the lectin staining pattern. Staining of blots from electrophoretically separated muscle proteins obtained byhomogenization, solubilization and centrifugation of small muscle pieces showed D. stramonium agglutinin binding to a number of bands ranging from 200 kDa to 30 kDa. No D. stramonium agglutinin positive bands were observed in blots from separated mitochondrial proteins while blots from sarcoplasmic reticulum separated by electrophoresis stained many bands in the range from 200 kDa to 30 kDA. It may be concluded that all muscle fibres inhuman and cat biceps hold intracellular non-sialylated complex-type oligosaccharides and further, that a specific tri-antennary complex-type glycoform is strongly expressed in type II A fibres as recognized by D. stramonium agglutinin. These results indicate a differential glycosylation of certain myofibrillar-associated proteins in muscle fibre types This revised version was published online in July 2006 with corrections to the Cover Date.     

Fusimotor Effects on Position and Velocity Sensitivity of Spindle Endings in the External Intercostal Muscle of the Gat

Position and velocity responses of spindle endings in the external intercostal muscle of the cat have been determined in the absence and in the presence of fusimotor fibre stimulation at a constant rate. The length input signal had triangular wave form. A close functional resemblance was found between intercostal and leg muscle spindles: primary and secondary endings could be separated among intercostal afferents with the same methods that have been used in leg muscles; dynamic and static fibres were discerned and the fusimotor effects on position and velocity sensitivity were the same as in the hind limb; the ratio between dynamic and static fibres was about 1: 3, i.e. the same as in the hind leg. Some dissimilarities existed: the number of ‘intermediate’ endings was larger in the intercostal than in the leg muscles; position and velocity sensitivities were generally larger in intercostal endings. These discrepancies are discussed. As a general conclusion it is stated that a spindle model elaborated from studies on leg spindles can easily be modified to account also for intercostal spindle behaviour.     

Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog

1. End-plate potentials were recorded intracellularly at the frog neuromuscular junction bathed in a solution containing a low concentration of calcium and a high concentration of magnesium.2. The muscle was subsequently subjected to ;cholinesterase staining', and the area of the individual end-plates, studied with intracellular electrodes, was measured.3. A positive correlation was found between the end-plate area and the diameter of muscle fibres.4. The mean quantum content (m) showed a positive correlation with the size of end-plates.5. The frequency of spontaneous miniature end-plate potentials was positively correlated with m as well as with end-plate area.6. It is concluded that the amount of transmitter released following nerve stimulation is related to the size of nerve endings.     

An ultrastructural study of nerve terminal degeneration in muscle spindles of the tenuissimus muscle of the cat

Summary Muscle spindles in the tenuissimus muscle of the cat were studied between 12 and 168 h after cutting or freezing the nerve to this muscle. Degenerative changes in sensory and motor nerve terminals on intrafusal muscle fibres were observed using the electron microscope. Comparisons were made with spindles from unoperated or sham-operated cats.The earliest degenerative changes were seen in sensory and motor terminals at 20–24 h after the lesion. No nerve endings were seen by 114 h after denervation. The most consistent initial signs of degeneration were: (1) the presence of abnormal mitochondria and dense bodies in sensory terminals, and (2) a decrease in the number and clumping of synaptic vesicles combined with an increase in glycogen and neurofilaments in motor endings. Intrafusal fibres participate in the removal of degenerating sensory endings. Schwann cells phagocytose degenerating motor terminals. The disappearance of nerve terminals precedes the complete degeneration of preterminal myelinated fibres within the muscle spindle.     

Where in the muscle spindle is the resting discharge generated?

This is a report of experiments on muscle spindles of the soleus muscle of the anaesthetized cat. Following a step shortening of the muscle, muscle spindles fall silent. At suitable muscle lengths their discharge may restart several seconds later to gradually recover a maintained rate of discharge. These experiments examine the question of where within the spindle the resumption of a resting discharge may originate. It was found that stimulation of some static fusimotor fibres immediately after the shortening led to early recovery of the resting discharge. Stimulation of dynamic and other static gamma motoneurones had much less effect. Since the dynamic gamma axons innervate almost exclusively the bag1 intrafusal fibre, contraction of this fibre appears to have little influence on the mechanisms responsible for restarting the resting discharge. Bag2 and chain fibres do seem to be involved. For primary endings, the bag2 fibre contraction was especially effective since static axons, which did not evoke 'driving' of the afferent response, and which are thought to predominantly innervate bag2 fibres, did restart the resting discharge. For secondary endings, stimulation of nearly all gamma axons led to an early restart of the resting discharge suggesting that here the nuclear chain fibres were responsible.     

Palisade endings are present in canine extraocular muscles and have a cholinergic phenotype

Classical proprioceptors, like Golgi tendon organs and muscle spindles are absent in the extraocular muscles (EOMs) of most mammals. Instead, a nerve end organ was detected in the EOMs of each species including sheep, cat, rabbit, rat, monkey, and human examined so far: the palisade ending. Until now no clear evidence appeared that palisade endings are also present in canine EOMs. Here, we analyzed dog EOMs by confocal laser scanning microscopy, 3D reconstruction, and transmission electron microscopy. In EOM wholemount preparations stained with antibodies against neurofilament and synaptophysin we could demonstrate typical palisade endings. Nerve fibers coming from the muscle extend into the tendon. There, the nerve fibers turn 180° and return to branch into preterminal axons which establish nerve terminals around a single muscle fiber tip. Fine structural analysis revealed that each palisade ending in dog EOMs establish nerve terminals on the tendon. In some palisade endings we found nerve terminals contacting the muscle fiber as well. Such neuromuscular contacts have a basal lamina in the synaptic cleft. By using an antibody against choline acetyltransferase (ChAT) we proved that canine palisade endings are ChAT-immunoreactive. This study shows that palisade endings are present in canine EOMs. In line with prior findings in cat and monkey, palisade endings in dog have a cholinergic phenotype.     

Morphological identification and intrafusal distribution of the endings of static fusimotor axons in the cat

1. Tenuissimus muscles of the cat were prepared in which the motor innervation was reduced to a single gamma axon by cutting all the other motor axons and allowing them to degenerate during a period of 7-12 days. The function of the surviving gamma axon was then determined, and the distribution of its endings ascertained in teased, silver preparations.2. In the ten muscles successfully prepared the function of the surviving gamma axon was static and the motor innervation distributed to the spindles consisted of trail endings. The conduction velocities of the axons ranged from 33 to 48 m/sec.3. A detailed histological analysis was made of thirty spindles innervated by six of the surviving static axons.4. The six static axons distributed trail endings to both bag and chain muscle fibres in the poles of thirty spindles with about twice the frequency of supplying them to poles in which the distribution was restricted exclusively to one type of muscle fibre or the other.5. The density of trail innervation supplied to the bag fibres, in terms of the mean number of terminals per fibre, was typically from one and a half to twice that supplied to the chain fibres. On the other hand, whereas the number of bag fibres supplied with trail endings in a spindle pole was seldom more than one, the number of chain fibres innervated was usually two in a range of one to four.6. The possible effects that partial denervation might have had on the spindles are discussed, but it is concluded that they are unlikely to have affected the results.     

The effects of suxamethonium and acetylcholine on the behaviour of cat muscle spindles during dynamics stretching, and during fusimotor stimulation

1. The responses of primary and secondary afferent fibres from muscle spindles in cat soleus were studied during constant velocity stretching.2. Intravenous Suxamethonium (SCh) caused a large increase in the response of the primary afferents to the dynamic phase of stretching, and a smaller increase in their response to static extension. The effects of SCh were similar to the effects of dynamic fusimotor stimulation.3. Increasing doses of SCh increased the response of primary afferents to dynamic stretching up to a point, but a peak discharge frequency was encountered beyond which the afferent fibre could not be induced to discharge.4. Suxamethonium increased the response of the secondary afferent fibres by a smaller amount than the primaries, and in particular caused a smaller increase in the response to dynamic extension.5. Acetylcholine given by close arterial injection had an effect similar to the effect of SCh.6. The effects of static and dynamic fusimotor stimulation on the response of primary afferents summed with the effects of small doses of SCh. When large amounts of SCh were used fusimotor stimulation sometimes had no further effect on the afferent discharge. It was not possible to say whether the fusimotor activity was then inhibited, or submerged in the SCh activity.7. The actions of SCh and of acetylcholine emphasized the differences in response of primary and secondary afferent endings to dynamic stretching. The use of these drugs enabled us to classify fibres of intermediate conduction velocity.8. Suxamethonium is known to activate slow muscle fibre systems with distributed nerve endings. The similarity between dynamic fusimotor activity and the effect of SCh suggests that the dyanimic fusimotor fibres act on slow intrafusal muscle fibres through multiple distributed endings.     

Matching of facilitation at the neuromuscular junction of the lobster: a possible case for influence of muscle on nerve

1. The facilitation of neuromuscular transmission, which occurs during repetitive activation, was examined in the proximal accessory flexor muscle in walking legs of the lobster using electrophysiological techniques.2. Post-synaptic potentials (p.s.p.s) in different muscle fibres facilitated to markedly different degrees. P.s.p.s in some fibres did not facilitate at all, while in others they increased in size by 20-30 times during stimulation at 20 Hz even though all the excitatory neuromuscular synapses are made by a single axon.3. Stimulation of widely separated groups of synapses on any single muscle fibre evoked p.s.p.s with closely matched facilitation properties. Extracellular p.s.p.s recorded from single synaptic spots showed the same characteristics of facilitation as those of intracellular p.s.p.s in the same muscle fibre, suggesting that individual synaptic contacts on any single fibre are similar to each other.4. Facilitation can be accounted for by an increase in the number of quanta released from the nerve terminals. There is no evidence for an increase in post-synaptic membrane sensitivity.5. Low Ca solutions reduce transmitter release with comparatively little change in facilitation, while Cs solutions increase the size of p.s.p.s without increasing the amplitude of spontaneous miniature potentials. Thus, at poorly facilitating synapses it is unlikely that the absence of facilitation is caused by the saturation of some post-synaptic process.6. It is concluded that the excitatory presynaptic nerve terminals on a single muscle fibre have matching facilitation characteristics. Some interaction between individual muscle fibres and their associated nerve endings may be required to establish or maintain this matching.     

Effects ofAnemonia sulcata toxin II on presynaptic currents and evoked transmitter release at neuromuscular junctions of the mouse

The effect ofAnemonia sulcata toxin II (ATX-II) on the amount of transmitter released by nerve impulses was investigated in motor end-plates of the mouse. ATX-II (80 nM) caused repetitive end-plate potentials in response to a single nerve stimulus and a 3- to 4-fold increase in the quantal content of the phasic end-plate potential. This increase is less than what would be expected if ATX-II induced plateau action potentials at the motor endings. To solve this discrepancy presynaptic currents were recorded by focal extracellular electrodes. It was found that the K current present at the endings is strong enough to prevent the development of presynaptic plateau action potentials, in contrast to what has been observed in other excitable membranes (unmyelinated axons, nodes of Ranvier and skeletal muscle fibres). By using tetraethylammonium and 3,4-diaminopyridine to block K channels and Co²⁺ to block Ca channels, ATX-II allowed the development of prolonged plateau responses at the endings upon motor nerve stimulation. These results suggest that the mouse motor endings are endowed with a relatively powerful K channel system, which effectively controls the amount of presynaptic depolarization.     

The formation of synapses in reinnervated mammalian striated muscle

1. The hemidiaphragm of the adult rabbit has a single band of end-plates running around the middle of the muscle. A study has been made of the formation of synapses during spontaneous reinnervation of this muscle, using histological, ultrastructural and electrophysiological techniques.2. Following spontaneous reinnervation, silver-stained nerve terminals were found in association with cholinesterase-stained end-plates only in the region of the muscle corresponding to the original innervation band.3. The regenerated nerve terminals were observed with the electronmicroscope in positions overlying or adjacent to the old synaptic folds.4. Spontaneous miniature end-plate potentials and evoked synaptic potentials were recorded only in the middle of the muscle fibres after reinnervation.5. The growth of the regenerating axons was not oriented towards the end-plate zone but followed muscle fibres and blood vessels in random directions.6. It is concluded that, in adult mammalian striated muscle, the old end-plate region is preferentially reinnervated as a consequence of some special property of the muscle fibre at this site.     

The fine structure of innervated myotendinous cylinders in extraocular muscles of rhesus monkeys

Summary Many of the myelinated nerve fibres of the distal myotendinous region of rectus muscles terminate on muscle fibre tips. The terminal expansions contain aggregated, small clear vesicles and mitochondria. Neuromuscular clefts at the contacts measure 20–40 nm and are uninterrupted by a basal lamina; the sarcoplasm opposite the contacts is unmodified. Some terminals invaginate the muscle fibre tips and others contact the sides of processes formed by splitting of the tips. The muscle fibre termination, its tendon and the nerve fibre branches are encapsulated to form an end-organ averaging 125 m in length and described as a myotendinous cylinder.Approximately 350 innervated myotendinous cylinders were estimated to be present in the horizontal recti with rather fewer in the vertical rectus muscles. Many of them occur shortly before the main myotendinous junction. All muscle fibres contributing to myotendinous cylinders were identified as the compact, felderstruktur, multi-innervated variety with directly apposed myofibrils that are known to be non-twitch fibres. All felderstruktur fibre terminations examined were encapsulated but 19% of them were not innervated.The nerve terminals of myotendinous cylinders are similar to those described by Dogiel (1906) as palisade endings and it is argued that they meet the morphological criteria of sensory neuromuscular endings. Their disposition suggests a capacity to monitor felderstruktur muscle fibre contraction.     

The regularity of primary and secondary muscle spindle afferent discharges

1. The patterns of nerve impulses in the afferent fibres from muscle spindles have been studied using the soleus muscle of the decerebrate cat. Impulses from up to five single units were recorded simultaneously on magnetic tape, while the muscle was stretched to a series of different lengths. Various statistics were later determined by computer analysis.2. After the ventral roots were cut to eliminate any motor outflow to the muscle spindles, both primary and secondary spindle endings discharged very regularly. At frequencies around 30 impulses/sec the coefficient of variation of the interspike interval distributions had a mean value of only 0.02 for the secondary endings and 0.058 for the primary endings. The values obtained for the two kinds of ending did not overlap.3. When the ventral roots were intact, the ;spontaneous' fusimotor activity considerably increased the variability of both kinds of endings. Secondary endings still discharged much more regularly than primary endings, even when the fusimotor activity increased the frequency of firing equally for the two kinds of endings. At frequencies around 30/sec the average coefficient of variation of the interval distributions was then 0.064 for the secondary endings and 0.25 for the primary endings.4. When the ventral roots were intact there was usually an inverse relation between the values of successive interspike intervals. The first serial correlation coefficient often had values down to - 0.6 for both kinds of ending. Higher order serial correlation coefficients were also computed.5. Approximate calculations, based on the variability observed when the ventral roots were intact, suggested that when the length of the muscle was constant an observer analysing a 1 sec period of discharge from a single primary ending would only be able to distinguish about six different lengths of the muscle. The corresponding figure for a secondary ending was twenty-five lengths.6. The increase in variability with fusimotor activity, and the pattern of serial correlations, were probably caused by static fusimotor fibres firing at rates below the fusion frequency of the intrafusal muscle fibres that they supply.     

Junctions between subsynaptic folds and rough sarcoplasmic reticulum of muscle fibres

Serial sections through motor end plate regions of mouse muscle fibres demonstrated junctions between the subsynaptic folds and the rough sarcoplasmic reticulum of the sole plate nuclei. The shape of these structures resembles that of the well-known peripheral couplings, diads and triads of muscle fibres. However, the location of the new junctions between the surface membrane and the sole plate nuclei at a large distance from myofibrils, indicates a different function. The connection with the rough sarcoplasmic reticulum possibly influence the regulation of fibre protein metabolism, for example, gene expression for acetylcholine receptor synthesis.     

The interaction between foreign and original motor nerves innervating the soleus muscle of rats.

1. The fibular nerve was transplanted on to the soleus muscle of the rats. Interruption of the original soleus nerve then permitted cross-innervation, and subsequently, over a period of weeks, re-innervation by the original nerve. 2. Individual muscle fibres were often innervated by both the original and the foreign nerve. The original and foreign end-plates were located in separate regions of the muscle. There were no indications that the original nerve could displace or repress the foreign innervation. 3. The extent of re-innervation by the original nerve depended upon the method of denervation. A single crush of the nerve was followed by virtually complete re-innervation, even of muscle fibres already innervated by the foreign nerve. When re-innervation was delayed by resection of a segment of the nerve only muscle fibres without foreign nerve innervation were re-innervated. Denervation by a simple nerve cut gave an intermediate result. 4. Re-innervation by the original nerve can take place without measurable extrajunctional sensitivity to ACh. 5. The original end-plate region could retain high and localized sensitivity to ACh for several months despite degeneration of its motor nerve terminal and activity of the muscle fibre. 6. Established foreign end-plates were re-innervated by the foreign nerve on muscle fibres with intact original innervation. 7. The factors controlling synapse formation in skeletal muscles are discussed.