Histochemical analysis of glycoconjugate secretion in the alimentary canal of Anguilla anguilla L.

Conventional histochemical methods as well as lectin-binding techniques were used to study glycoconjugates that are present in the alimentary canal of the European eel (Anguilla anguilla). Specimens from pharynx, oesophagus, stomach and intestine were collected from adult ("silver eel" stage) females. Alcian Blue pH 2.5/PAS and High Iron Diamine/Alcian Blue pH 2.5 reactions were performed to stain neutral and acidic glycoconjugates. In addition, lectin histochemistry was applied to identify acidic glycoconjugates containing O-acylated sialic acids. Finally, the presence of sugar residues in the oligosaccharide side chains of glycoconjugates were investigated by using biotinylated lectins. Acidic and neutral glycoconjugates were found to be secreted throughout the alimentary canal, the acidic glycoconjugates appeared to be either sialylated or sulphated. Sialylated glycoconjugates were identified to contain sialic acid substituted at carbon in position 7 (C7). Sulphated glycoconjugates were particularly abundant in the distal intestine and were not present in the secretory products of the gastric mucosa, which contained a variety of sugar residues (D-N-acetyl-galactosamine, beta-D-galactose, alpha-D-mannose, alpha-L-fucose, D-N-acetyl-glucosamine). Lectin binding was observed in mucous cells of pharynx, oesophagus and intestine, and particularly some monosaccharides (D-N-acetyl-galactosamine and beta-D-galactose) were abundantly present.

Neurotransmitters and putative neuromodulators in the gut of Anguilla anguilla (L.). Localizations in the enteric nervous and endocrine systems.

The gut of silver eels (Anguilla anguilla L.) was investigated in order to describe both the cholinergic and adrenergic intramural innervations, and the localization of possible accessory neuromediators. Histochemical reactions for the demonstration of nicotinamide adenine dinucleotide phosphate, reduced form-(NADPH-)diaphorase and acetylcholinesterase (AChEase) were performed, as well as the immunohistochemical testing of tyrosine hydroxylase, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP), bombesin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), somatostatin, cholecystokinin-octapeptide (CCK-8), serotonin, cholineacetyl transferase. The results evidenced a different pattern in comparison with other vertebrates, namely mammals, and with other fish. Both NADPH-diaphorase and AChEase activities were histochemically detected all along the gut in the myenteric plexus, the inner musculature and the propria-submucosa. Tyrosine hydroxylase immunoreactivity was observed in the intestinal tract only, both in the myenteric plexus and in the inner musculature. Several neuropeptides (metenkephalin, CGRP, bombesin, substance P, VIP, NPY, somatostatin) were, in addition, detected in the intramural innervation; some of them also in epithelial cells of the diffuse endocrine system (met-enkephalin, substance P, NPY, somatostatin). Serotonin was only present in endocrine cells. Tyrosine hydroxylase immunoreactivity was present in localizations similar to those of NADPH-diaphorase-reactivity, and in the same nerve bundles in which substance P- and CGRP-like-immunoreactivities were detectable in the intestinal tract. In addition, NADPH-diaphorase-reactive neurons showed an anatomical relationship with AChEase-reactive nerve terminals, and a similar relationship existed between the latter and substance P-like immunoreactivity.

Different putative neuromodulators are present in the nerves which distribute to the teleost skeletal muscle.

The presence of putative neuromodulators in the nerve fibres was investigated in white skeletal muscle of two teleost fish not taxonomically correlated and showing different patterns of innervation (multiple versus focal innervation). Cryostat sections of epaxial, hypaxial and adductor mandibulae (AM) muscles of Sparus aurata and Anguilla anguilla were stained histochemically for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Other sections were used for indirect immunohistochemistry (streptavidin-biotin and rhodamine immunofluorescence methods), employing antibodies specific for putative excitatory or inhibitory peptides, including CGRP, substance P, met-enkephalin, bombesin, and VIP. In addition, ultrastructural observations were performed in order to describe the morphology of the motor endplates. A strong immunoreactivity for CGRP and substance P was found in many nerve terminals. Met-enkephalin, bombesin and VIP immunoreactivities were less frequently observed. No immunoreactivity was observed to CCK, NPY or 5-HT. NADPH-diaphorase was identified in nerve fibres of the AM complex only of A. anguilla. Electron microscopy observations evidenced more than one type of synaptic vesicle in motor endplates. Some differences in putative neuromodulator distributions were observed in the two species and muscle complexes, which may be related to the different taxonomical position as well as the different pattern of innervation of white muscle fibres.

Gut glycoconjugates in Sparus aurata L. (Pisces, Teleostei). A comparative histochemical study in larval and adult ages.

This study examined the gut of the euryaline fish Sparus aurata, from the pharynx to the rectum. The specimens were collected from adult animals, both sexes, and several larval and juvenile stages, from 4 to 135 days of age. Histochemical methods of distinguish neutral and acidic glycoconjugates, as well as specific techniques to identify acidic glycoconjugates which contained O-acylated sialic acids were used. The presence and distribution of sugar residues in the oligosaccharide side chain of glycoconjugates were investigated with the use of biotinylated lectins. The pharynx and oesophagus of adult fishes showed the presence of abundant secretory cells which synthesized a large quantity of neutral, as well as sulphated and sialylated glycoconjugates, with different cellular combinations of them in the proximal and distal tract. This may be related to the complex functions carried out by this end of the gut in a marine euryaline fish. Epithelial secretory cells were found in the developing oesophagus during larval life (14 days) earlier than in the stomach and intestine (34 days). The simple columnar epithelium that lined the gastric mucosa of adult fish synthesized a mixture of neutral and acidic glycoconjugates, whereas during larval life it was shown to contain neutral glycoconjugates only. The intestinal goblet cells were shown to secrete both neutral and acidic glycoconjugates, especially sulphated forms. The adherent mucus gel of the gastric and intestinal mucosa contained many sugar residues, as revealed by lectin histochemistry. This work clearly demonstrates that the quality of gut mucosubstances varies in different ages and in regions of the fish alimentary canal. This is possibly caused by changes in environmental conditions and may in turn sustain functional alterations of the digestive apparatus.

Regeneration of skeletal muscle in two teleost fish: Sparus aurata and Brachydanio rerio.

Regeneration of skeletal muscle was studied in the sea bream Sparus aurata, in which extensive post-larval muscle hyperplasia contributes to its large adult size, and in the zebrafish Brachydanio rerio, which shows little post-larval hyperplasia and reaches only a small adult size. Small mechanical lesions of body wall muscle were made under general anaesthesia, and the progress of subsequent regeneration was assessed at various intervals by histology and electron microscopy (for general morphology), by immunostaining for desmin and myosin isoforms (to identify the phenotype of new fibres), and by 5'-bromo-2'-deoxyuridine (BrdU) incorporation (to identify proliferating cells). Despite the difference in normal growth-related hyperplasia in these fish, a vigorous regeneration occurred in both species, giving rise to new fibres with an initial myosin composition that differed from that in mature fast-white fibres. However, species differences in myosin expression in these fibres suggest that they may have derived from different myoblast populations. In sea bream, myosin expression in regenerating fibres resembled that seen in new fibres produced in post-larval white muscle, whereas in the zebrafish it resembled that of the primitive monolayer fibres formed during embryonic development. Subsequently, most regenerating fibres gradually transformed into the mature fast-white phenotype in both species.

Differentiation and growth of muscle in the fish Sparus aurata (L): I. Myosin expression and organization of fibre types in lateral muscle from hatching to adult.

Post-hatching development of lateral muscle in a teleost fish, Sparus aurata (L) was examined. At hatching only two fibre types were present, several layers of mitochondria-poor, myofibril-rich deep muscle fibres surrounded the notochord and were covered by a superficial monolayer of mitochondria-rich, myofibril-poor A third ultrastructurally distinct fibre type first appeared as one or two fibres located just under the lateral line at 6 days post-hatching. This type, which gradually increased in number during larval life, contained a slow isoform of myosin, identified by mATPase staining and immunostaining with myosin isoform-specific antibodies. Deep muscle fibres--the presumptive fast-white type--contained a fast myosin, and superficial monolayer fibres an isoform similar but not identical to that in adult pink muscle fibres. The only fibres present during larval life which showed a clear change in myosin expression were the superficial monolayer fibres, which gradually transformed into the slow type post-larvally. Pink muscle fibres first appeared near the end of larval life. Both slow and pink muscle fibres remained concentrated around the horizontal septum under the lateral line during larval life, expanding outwards towards the apices of the myotomes only after metamorphosis. Between 60 and 90 days very small diameter fibres with a distinct mATPase profile appeared scattered throughout the deep, fast-white muscle layer, giving it a 'mosaic' appearance, which persisted into adult life. A marked expansion in the slow muscle layer began at the same time, partly by transformation of superficial monolayer fibres, but mainly by addition of new fibres both on the deep surface of the superficial monolayer and close to the lateral line. The order of appearance of these fibre types, their myosin composition, and the significance of the superficial monolayer layer are discussed and compared to muscle fibre type development in higher vertebrates.

Differentiation and growth of muscle in the fish Sparus aurata (L): II. Hyperplastic and hypertrophic growth of lateral muscle from hatching to adult.

Post-hatching growth of lateral muscle in a teleost fish, Sparus aurata (L) was studied morphometrically to identify and quantify muscle fibre hyperplasia and hypertrophy, and by in vivo nuclear labelling with 5-bromo-deoxyuridine to identify areas of myoblast proliferation. Muscle fibre types were identified principally by myosin ATPase histochemistry and immunostaining, and labelled nuclei were identified at light and electronmicroscope level by immunostaining with a specific monoclonal antibody. Hyperplastic growth was slow at hatching, but then increased to a maximum at the mid-point of larval life. Larval hyperplastic growth occurred by apposition of new fibres along proliferation zones, principally just under the lateral line and in the apical regions of the myotome, but also just under the superficial monolayer at intermediate positions. The first of these zones gave rise to slow and pink muscle fibres, in a process which continued through into postlarval life. The other zones added new fibres to the fast-white muscle layer in a process which was exhausted by the end of larval life. Post-larvally, between 60 and 90 days posthatching, a new hyperplastic process started in the fast-white muscle as nuclei proliferated and new muscle fibres were formed throughout the whole layer. This process resulted in a several-fold increase in the number of fast-white fibres over a few weeks, and then waned to very low levels in juveniles. Hyperplasia by apposition continued for some time postlarvally on the deep surface of the superficial monolayer, but at this stage gave rise to slow fibres only. Hypertrophic growth occurred at all ages, but was the dominant mechanism of muscle growth only in the juvenile and adult stages. Mechanisms giving rise to these different growth processes in fish muscle are discussed, and compared with muscle development in higher vertebrates.

Muscle growth and myosin isoform transitions during development of a small teleost fish, Poecilia reticulata (Peters) (Atheriniformes, Poeciliidae): a histochemical, immunohistochemical, ultrastructural and morphometric study.

The myosin composition of lateral muscle in Poecilia reticulata from birth to adult was studied by ATPase histochemistry and immunostaining with myosin isoform-specific antibodies. At birth the muscle consists of two layers containing developmental isoforms of myosin. In deep layer fibres the developmental myosin is replaced by the adult fast-white isoform soon after birth. In the epaxial and hypaxial monolayer fibres the myosin composition present at birth (J1) is replaced within 3 days by another (J2). In some fibres, this J2 composition is retained in the adult, but in others it is slowly replaced by the adult slow-red muscle isoform. Close to the lateral line, all monolayer fibres are already in transition between the J2 myosin and the adult slow-red form at birth, and rapidly complete the transition to slow-red form. These fibres, together with others generated de novo in an underlying hyperplastic zone, form the red muscle layer of the adult. The pink muscle develops during the first month after birth, and by 31 days it consists of an outer, middle and inner layer. A few middle layer fibres are already present at birth, while the outer layer fibres first appear 3 days after birth. The thin inner layer is probably a transitional form between the middle pink and adult white types, and appears at about 31 days. A morphometric analysis showed that growth of the white muscle occurs principally by hypertrophy. Even at the magnification level of the electron microscope, no satellite cells or myoblasts which could give rise to new fibres were found in the white muscle, except in the far epaxial and hypaxial regions and only in the first 10 days. A zone of hyperplastic growth was also found lying just under the superficial monolayer close to the lateral line, and this presumably contributes fibres to the red and pink muscle layers.

Neonatal myosin in bovine and pig tensor tympani muscle fibres.

In previous studies of middle ear muscles, the classification of fibre types by histochemical methods was particularly difficult in the bovine and porcine tensor tympani muscle, suggesting the presence of immature fibres. We therefore reexamined the tensor tympani from pigs and cattle of various ages immunohistochemically, using a panel of antimyosin antibodies, including one (anti-NE) specific for neonatal and embryonic myosins. Fibres positive to anti-NE were found in tensor tympani in both species in all ages examined; only a few of these fibres reacted exclusively with this antibody; some also contained slow myosin and the majority also contained adult fast (type IIA) myosin. Furthermore, although the remaining fibres included some of the classical types I and IIA, the majority of them showed a mismatch between their histochemical and immunohistochemical profiles. The morphological appearance of the muscle, the widespread presence of neonatal myosin (often together with another myosin in the same fibre) and the persistence of this composition from birth to adulthood, could be explained by an incomplete development of the muscle fibres, resulting in a 'muscle' much better suited to the role of a ligament.

Hyperplastic and hypertrophic growth of lateral muscle in Dicentrarchus labrax (L.). An ultrastructural and morphometric study.

In this EM study of lateral muscle in Dicentrarchus labrax, we observed that during the larval period, growth of the presumptive red and white muscle layers occurs both by hypertrophy (as fibres already present at hatching complete their maturation) and by production of new fibres in germinal zones specific to the two muscle layers. In the first half of larval life the presumptive white muscle increases in thickness by the addition, superficially, of new fibres derived from a germinal zone of presumptive myoblasts lying beneath the red muscle layer. In the second half of larval life new fibres produced in this same zone form the intermediate (or pink) muscle layer. Dorsoventrally the myotome grows throughout larval life, largely by addition of new fibres from germinal zones at the hypo- and epi-axial extremities. Towards the end of larval life all these germinal zones are becoming exhausted, but another source of fibres arises as satellite cells, associated with large-diameter presumptive white muscle fibres, are activated to produce new fibres. The addition of small, new fibres gives the white muscle its mosaic appearance. Morphometric analysis of fibre diameters in the white muscle confirms that whereas these hyperplastic processes are important during the larval and juvenile periods, when growth is very rapid, they have ceased by the time the adult stage is attained. By contrast, fibre hypertrophy continues through into adult life. The presumptive red muscle consists initially of a monolayer of fibres present only near the lateral line, and during larval life it grows hypo- and epi-axially by addition of fibres derived from myoblasts already present in these areas at hatching. Lying superficially to the presumptive red muscle monolayer there is a near-continuous layer of external cells with a "flattened" profile. During the second half of larval life, differentiation of these external cells into myoblasts provides the source of new fibres which are added to the red muscle layer. This process, which occurs initially in the region around the lateral line and later spreads outwards, is responsible for the increase in thickness of the red muscle.

The adductor mandibulae muscle in teleost fish with protrusible or non protrusible jaws: a histochemical and immunohistochemical study.

A study was made of the morphology and fibre type composition of the adductor mandibulae (AM) muscle in Teleosts with very protrusible (carp), moderately protrusible (cod) and non-protrusible (trout and cat-fish) jaws. In contrast to the trout and cat-fish, in which the AM is formed by only 2 components (mandibular and mental), in the carp and cod there is a third portion (maxillary) which is more or less developed in relation to the extent of jaw protrusion. Fibre types were identified by means of histochemical staining for succinate dehydrogenase and myosin ATPase activities, and by immunohistochemistry with anti-sera specific for fish fast and slow myosins. In all the species AM is composed principally of white (fast) fibres, with a smaller proportion of red (slow) fibres. The red fibres, which appear in the deep layers only of the muscle are not found in all of the components, and in the different species are not always present in the same parts. In those parts of the AM which are mixed, a transition zone lies between the red and white areas, and is usually composed of a third, or intermediate, type of fibre with histochemical and immunohistochemical properties similar to those of the pink zone of lateral muscle. The anatomical characteristics and different fibre type compositions of the various components forming the AM are discussed in relation to the extent of jaw protrusion and the relevant physiological data concerning other movements in which this muscle participants.

Histochemical, immunohistochemical, and ultrastructural observations on the iris muscles of Gallus gallus.

The distribution and typology of fibers in the two muscular systems (sphincter and dilator) of the iris in Gallus gallus were determined histochemically, immunohistochemically, and ultrastructurally. The sphincter muscle in proximity to the ciliary margin was composed predominantly of slow fibers. In the intermediate tract, a large group of fast oxidative fibers were evident and the pupillary margin was exclusively composed of slow fibers. The fast fibers had histochemical and immunohistochemical patterns similar to the alpha fibers in the skeletal control muscle (biventer cervicis). In contrast, the slow fibers were composed of at least three slow types, which were comparable to the isoforms of the different myosins in beta 1 and beta 2 skeletal fibers. In the dilator muscle, the oblique system was uniquely composed of fast oxidative fibers. The radial system was predominantly composed of slow fibers with isoforms of myosins different from the slow fibers of the sphincter and control muscles. Ultrastructural features (width of Z bands, extension of the sarcoplasmic reticulum and SR-T tubule junctions, and number of mitochondria) confirm the histochemical and immunohistochemical assessments of fiber types, even if some peculiar aspects in several fibers were observed. Smooth muscle cells separated from striated fibers were evident at the pupillary margin. The hypothesis of a mesenchymal origin for all irideal striated muscles is discussed.

Developmental transitions of myosin isoforms and organisation of the lateral muscle in the teleost Dicentrarchus labrax (L.).

In Dicentrarchus labrax (the sea bass) the differentiation of lateral muscle fibres occurs at different stages and in different ways in the superficial (red), intermediate (pink) and deep (white) regions of the myotome. At hatching the myotomes are composed of presumptive white and red fibres, the latter forming a superficial monolayer present only near the transverse septum. At this stage, differences between the fibre types are mainly ultrastructural. From their different reactions with isoform-specific antibodies to mullet myosin, and the appearance of histochemical mATPase activity, it appears that in both red and white muscle fibres there is a transition in myosin composition from an early larval form (L1R and L1W respectively) to a late larval form (L2R and L2W) and then to the isoforms typical of adult red and white muscle. The transition from L1W to L2W in the deep muscle occurs very rapidly and early in larval life (between 10 and 28 days), whereas the equivalent transition in the superficial muscle (from L1R to L2R) is a gradual process beginning in fibres near the transverse septum and spreading hypo- and epi-axially as this layer grows around the deep muscle. The definitive adult forms (AR and AW), distinguishable by the appearance of characteristic histochemical myosin ATPase activity, are present in the superficial red muscle by 80 days, but later in the deep white muscle (by 20 months), respectively. Compared to the superficial red and deep white muscle, the intermediate (pink) muscle layer first appears relatively late (80 days), but then acquires the histo- and immunohistochemical profile characteristic of the adult form much more rapidly.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of myosins present in the lateral muscle of some fish: species variations in myosin isoforms and their distribution in red, pink and white muscle.

Myosin isoforms and their distribution in the various fibre types of the lateral muscle of eight teleost fish (representing a wide range of taxonomic groups and lifestyles) were investigated electrophoretically, histochemically and immunohistochemically. Polyclonal antisera were raised against slow (red muscle) and fast (white muscle) myosins of the mullet, and used to stain sections of lateral muscle. Antisera specific for fast and slow myosin heavy chains only (anti-FHC and anti-SHC respectively) and for whole fast and slow myosins (anti-F and anti-S respectively) were obtained, and their specificity was confirmed by immunoblotting against electrophoretically separated myofibrillar proteins. The ATPase activity of myosin isoforms was examined histochemically using methods to demonstrate their acid- and alkali-lability and their Ca-Mg dependent actomyosin ATPase. As expected, the predominant myosin (and fibre) type in the red muscle showed an alkali-labile ATPase activity, reacted with the anti-S and anti-SHC sera (but not anti-F or anti-FHC) and contained two 'slow' light chains, whereas the predominant myosin (and fibre) type in the white muscle showed an alkali-stable ATPase activity, reacted with anti-F and anti-FHC sera (but not anti-S or anti-SHC) and contained three 'fast' light chains. However, superimposed upon this basic pattern were a number of variations, many of them species-related. On analysis by two-dimensional gel electrophoresis fish myosin light chains LC1s, LC2s and LC2f migrated like the corresponding light chains of mammalian myosins, but fish LC1f consistently had a more acidic pI value than mammalian LC1f. Fish LC3f varied markedly in Mr in a species-related manner: in some fish (e.g. eel and mullet) the Mr value of LC3f was less than that for the other two light chains (as in mammalian myosin), whereas in others it was similar to that of LC2f (e.g. cat-fish) or even greater (e.g. goldfish). Species differences were also seen in the relative intensity of LC1f and LC3f spots given by the fish fast myosins. In most of the fish examined the red muscle layer showed some micro-heterogeneity, containing (in addition to the typical slow fibres) small numbers of fibres with a histo- and immunohistochemical profile typical of white muscle (fast) fibres. However, other immunohistochemically distinct minority fibres were found in the red muscle of the goldfish. Three types of pink muscle were distinguished: a mosaic of immunohistochemically typical red and white fibres (e.g. grey mullet).(ABSTRACT TRUNCATED AT 400 WORDS)

An immunohistochemical study of the middle ear muscles of some carnivores and primates, with special reference to the IIM and slow-tonic fibre types.

The middle ear muscles of several species of carnivores (cat, dog, fox, ferret and stone-marten) and some New World monkeys (Callithrix, Saimiri) and Old World monkeys (Cercopithecus, Macaca) were examined. The fibre type compositions of these muscles were determined by a combination of the standard histochemical myofibrillar ATPase method, and immunohistochemical techniques using myosintype-specific antisera. Immunohistochemically slow-tonic fibres were found in the stapedius muscles of only two carnivores, the ferret and stone-marten. In all the carnivores and the New World monkeys, tensor tympani muscle contained IIM, slow-tonic and slow-twitch fibres, but in the Old World monkeys it resembled stapedius muscle, and contained only Type I (slow-twitch) and IIA fibres. Thus, because all the species examined had IIM fibres in the jaw-closer muscles, this means that the common embryological origin of tensor tympani muscle and the jaw-closers does not necessarily result in tensor tympani muscle containing this fibre type even though IIM fibres occur only in first branchial arch muscles. This fact, together with other species differences in the fibre type composition of these muscles, shows that there is no typical composition of middle ear muscles in general, and suggests that the differences are related to very different functional requirements.

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.

Preparation of type-specific antimyosin antibodies and determination of their specificity by biochemical and immunohistochemical methods.

This paper reports the preparation of specific anti-slow myosin antibodies (anti-I) and anti-fast myosin antibodies (anti-IIA) raised against myosins from sheep and guinea pig masseter muscles. The specificity of the antibodies has been studied by immunodiffusion in agar and by the GEDELISA test using slow-twitch (type I), fast-twitch red (type IIA) and fast-twitch white (type IIB) myofibrils isolated from guinea pig muscles. The principal specificity of the anti-I and anti-IIA antibodies was for the heavy chains of type I and IIA myosins, respectively. A smaller reaction with the corresponding light chains was also detected. Immunohistochemical staining of muscle sections using these antibodies confirmed their fibre type specificity.

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

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.