Innervation of the limb accessory flexor muscle in several decapod crustaceans. I. Anatomy

The innervation of the accessory flexor muscle of the limbs of several decapod crustaceans was studied by means of vital staining, with methylene blue and electron microscopy. Three patterns of innervation were found. In the first pattern, the distal (DAFM) and proximal (PAFM) heads of the accessory flexor muscle were supplied by two axons (a thick and a thin) which travel in a private nerve along the length of the merus. This pattern was found in the crab (Cancer) and the lobster (Homarus), and conforms to the classical pattern established in the literature. In the second pattern, the nerve to the DAFM is made up of conjoined branches of the flexor and accessory flexor nerves. Consequently, the DAFM receives at least five axons in the portunid crabs, Carcinus, Callinectes, and Ovalipes, and occasionally six axons in Ovalipes. The PAFM in those portunids receives the usual two axons. In the third pattern, based on preliminary observations on the grapsid crab, Pachygrapsus, “super-innervation” of the accessory flexor muscle appears to include not only five axons to the DAFM but also at least three to the PAFM. In all species, methylene blue staining of the axon terminations revealed a regular pattern of blebs which are thought to correspond to synaptic terminals as revealed by electron microscopy.     

Fiber composition of the distal accessory flexor muscle in several decapod crustaceans

The fiber composition of the distal accessory flexor muscle (DAFM) and the branching pattern of its excitor axon were compared in several species of crabs, in the lobster and the crayfish. The muscle is composed exclusively of long sarcomere (> 6 μm) fibers and therefore of the slow type. In all the crab species, except one, there is a distal to proximal gradient of fibers with increasing sarcomere lengths; this gradient is reverse in lobsters and crayfish. A proximal to distal gradient of increasing fiber diameters occurs in the DAFM of all crab species but not in the lobster and crayfish, in which all the fibers are approximately equal in diameter. The single excitatory axon traverses the width of the DAFM and gives off primary branches on either side in the lobster and crayfish but on only one side in crabs. The hypothesis that the axonal branching pattern may govern the regional distribution of fibers with differing sarcomere lengths in proposed.     

Proliferation and relocation of developing lobster neuromuscular synapses

The development of multiterminal innervation from a single identifiable excitatory motoneuron to the lobster distal accessory flexor muscle (DAFM) was studied by serial section electron microscopy. The number, size, and location of neuromuscular synapses and presynaptic dense bars within the peripheral branching pattern of the axon was determined in cross sections of the DAFM in 1st (24-hr-old)-, 4th (2-week-old)-, and 12th (1-year-old)-stage lobsters. The mean size of synapses remains fairly constant in these three stages but synaptic density, i.e., the number of synapses per unit length of fiber, increased more than 20-fold between the 1st and 4th stages and more than 5-fold between the 4th and 12th stages. Synaptic surface area per fiber length showed a parallel increase. Consequently there is a proliferation of synapses along the length of individual muscle fibers during primary development. Furthermore from the 1st stage where only a few fibers are innervated, synapses proliferate to many more fibers in the 4th and to all fibers in the 12th stage. The neuromuscular synapses are distributed in different proportions within the axonal branching pattern in the three stages. Based on the number and size of synapses and presynaptic dense bars, the main axon and primary branches provide almost equal amounts of innervation in the 1st stage. With further branching in the 4th stage, the main axon accounts for only 20–25% of the innervation; the primary branches for 45% and other finer branches the remainder. By the 12th-stage synapses are found only on branches other than the main axon and its primary offshoots. There is therefore a shift in innervation from the main axon to the primary branches and then to the finer branches during primary development. This shift in innervation involves the formation of new synaptic terminals and the restructuring of existing ones into axonal areas. In this way the multiterminal innervation arising from an identifiable motoneuron is remodeled.     

A comparative study on the innervation and the vascularization of the bulbus arteriosus in teleost fish

Summary The structure of the bulbus arteriosus of a wide range of teleost fish is described with particular reference to the vascularization and innervation. The adventitia of the organ consists of blood vessels and large nerve bundles in a collagen matrix. The nerve bundles contain monoamines, and fluorescence studies show small terminal bundles penetrating the muscular media; this is confirmed by electron microscopy. The media consists of an extensive elastic tissue matrix with a spiral arrangement of smooth muscle cells joined end to end by desmosomes and presumed electrotonic junctions. The muscle cells are innervated only at the adventitia/media boundary and the significance of this innervation is discussed. It is proposed that there is a correlation between the degree of vascularization and innervation and the activity of a particular species offish.     

Inhibitory innervation of a lobster muscle

Summary Inhibitory neuromuscular synapses formed by the common inhibitor (CI) neuron on the distal accessory flexor muscle (DAFM) in the lobster, Homarus americanus, were studied with electrophysiological and electron-microscopic (thin-section and freeze-fracture) techniques. Postsynaptic inhibition as indicated by inhibitory junctional potentials was several-fold stronger on distal compared to proximal muscle fibers. This difference correlated with the results of serial thin-section studies, which showed more inhibitory synapses on distal fibers than on their proximal counterparts. Effects of postsynaptic inhibition on excitatory junctional potentials via current shunting had a morphological correlate in the spatial relationship between inhibitory and excitatory synapses on the distal fibers. Inhibitory synapses were larger than their excitatory counterparts and had fewer glial processes. In freeze-fracture views, inhibitory synapses did not appear as raised plateaus in the P-face as do excitatory synapses, and their active zones were more widely scattered. The intramembrane particles in the inhibitory postsynaptic membrane-representing neurotransmitter receptors-are arranged in parallel rows in the sarcolemmal P-face and have complementary furrows in the sarcolemmal E-face. Altogether, our findings help to describe a population of inhibitory neuromuscular synapses formed by the CI neuron in lobster muscle.     

Retinal vascularization in the grass goby,Zosterisessor ophiocephalus: a scanning electron-microscopic study of vascular corrosion casts

Synopsis The retinal vascularization ofZosterisessor ophiocephalus is described by scanning electron microscopy of vascular corrosion casts. The retina is vascularized sclerad by the choroidal body and the choriocapillaris, and vitread by the lentiform body and by a dense network of hyaloid vessels. In the bodies there is a parallel arrangement of capillaries indicating a counter current exchange mechanism. The well developed vascularization and its correlation with the hypoxic conditions of the muddy habitat of the species are discussed.     

Pineal gland of a nocturnal bird, Indian spotted owlet, Athene brama: morphological and endocrine observations

It has been reported that owls (Strigiformes) do not have a pineal gland. However, our light microscopy study revealed an intermediate form of tubulofollicular and solid-type large pineal gland in a tropical owlet, Athene brama. The epithelial cells forming follicles (6-8) in the distal region and the solid cluster of parenchymal cells of different diameters in the proximal region anteriorly tapered with a long cylindrical stalk and continued into commissural organs and choroid plexus. The intrapineal localization of perivascular nerve fibers and blood vessels clearly explained the sympathetic innervation as well as vascularization of this neuroendocrine gland. Further, electron microscopy revealed a developed intracellular structure of the pinealocytes with a large number of mitochondria, Golgi bodies, and granular as well as clear vesicles in the process terminals. The evidence of intrapinealocyte lipid droplets and dense bodies and a moderate amount of melatonin in plasma (ranging from 100-365 pg/mL) during different reproductive phases finally proved a defined secretory activity of the gland in this tropical, nocturnal bird.     

Adaptive morphology of the heart of Southern‐Fur‐Seal (Arctocephalus australis – Zimmermamm, 1783)

The Southern‐fur‐seal belongs to the order Carnivora, suborder Pinnipedia, and Otariidae family. This species inhabits aquatic and terrestrial environments, thus presenting important morphophysiological adaptive changes, especially in the cardiac system. For this purpose, Southern‐fur‐seal (Arctocephalus australis) hearts were used from animals that died from natural causes. Gross morphology observations were supported by light, scanning and transmission electron microscopy. The heart was long and flat; it was lined by pericardium and partly covered by lungs. Structurally, atrium and ventricle muscle fibers exhibit typical features of cardiac fibers revealing myofibrils bundles, mitochondria, plate‐shaped junctions, anastomosis between myofibrils bundles, and electron‐dense granule natriuretic around the nucleus and mitochondria of atrium muscle cells. The Southern‐fur‐seal heart was structurally similar to other mammals; however, it presented morphological changes that assist in their adaptation to their environment.     

Arrangement and structure of sinus hair muscles in the big-clawed shrew,Sorex unguiculatus

The arrangement and structure of sinus hair muscles in the snout of the shrew, Sorex unguiculatus, were studied by electron microscopy and serial section light microscopy. Both striated and smooth muscles are directly associated with sinus hair follicles. The striated muscle fibers originate from the base of a follicle and insert onto the superficial portion of adjoining caudally positioned follicles. Some fibers insert into the corium instead of inserting into a follicle. The fibers show a fine structure typical of red fibers. Smooth muscle cells form a network with elastic fibers beneath the corium. Some cells are directly attached to the capsule of the sinus, thus forming a type of M. arrector pili. Striated muscle fibers that appear to end in the corium are connected with the smooth muscle network through the elastic fibers which appear to function as the tendon of these two types of muscle cell.     

Microvascularization of the interhyoid muscle in larval Xenopus laevis (Daudin): Scanning electron microscopy of vascular corrosion casts and correlative light microscopy

The interhyoid muscle in tadpoles of Xenopus laevis (Daudin) is an important part of the buccal pump, a functional unit that provides unidirectional flow of water through mouth and pharynx. In anuran tadpoles, this flow is crucial in both respiration (gas exchange) and food intake (ingestion). The microvascular anatomy of the interhyoid muscles of 43 tadpoles of X. laevis from developmental stages 49–60 was examined by scanning electron microscopy of vascular corrosion casts and correlative light microscopy of paraplast embedded Goldner stained serial tissue sections. Analysis of vascular corrosion casts of the interhyoid muscle showed that several descending branches of external carotid arteries supplied the interhyoid muscle. Arteries splitted into many arterioles at the dorsal surface of the interhyoid muscle and formed sheaths of longitudinally orientated capillaries around muscle fibers. Postcapillary vessels formed perpendicularly orientated arrays of collecting venules (mean diameter: 15.6 μm), which drained the interhyoid muscle from the ventral surface into external jugular veins. Cast analyses revealed sprouting angiogenesis at the capillary level and nonsprouting angiogenesis at distal domains of the venous system. Both means of angiogenesis that persisted throughout the developmental periods examined are thought to represent a superposition of concurrent developmental and physiological processes. The dense microvascular bed of the interhyoid muscle reflects its high demand for supply with oxygen and nutrients. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Histochemical and ultrastructural studies of the innervation of the lymph-vessel and blood-vessel wall. 1. Adrenergic innervation

The adrenergic innervation of the lymph vascular wall was studied by means of the Falck fluorescence histochemical tecnique and electron microscopy with Tranzer and Richards' histochemical tecnique. The lymph vessels wall, compared with that of blood vessels, shows very few adrenergic nerve fibers located in the adventitia outside the smooth muscle cells. The possible role of the nervous system in the motor control of the lymph vessels is discussed.     

Ring bands in fish skeletal muscle: Reorienting the myofibrils and microtubule cytoskeleton within a single cell

Skeletal muscle cells (fibers) contract by shortening their parallel subunits, the myofibrils. Here we show a novel pattern of myofibril orientation in white muscle fibers of large black sea bass, Centropristis striata. Up to 48% of the white fibers in fish >1168 g had peripheral myofibrils undergoing an ～90^o shift in orientation. The resultant ring band wrapped the middle of the muscle fibers and was easily detected with polarized light microscopy. Transmission electron microscopy showed that the reoriented myofibrils shared the cytoplasm with the central longitudinal myofibrils. A microtubule network seen throughout the fibers surrounded nuclei but was mostly parallel to the long‐axis of the myofibrils. In the ring band portion of the fibers the microtubule cytoskeleton also shifted orientation. Sarcolemmal staining with anti‐synapsin was the same in fibers with or without ring bands, suggesting that fibers with ring bands have normal innervation and contractile function. The ring bands appear to be related to body‐mass or age, not fiber size, and also vary along the body, being more frequent at the midpoint of the anteroposterior axis. Similar structures have been reported in different taxa and appear to be associated with hypercontraction of fibers not attached to a rigid structure (bone) or with fibers with unusually weak links between the sarcolemma and cytoskeleton, as in muscular dystrophy. Fish muscle fibers are attached to myosepta, which are flexible and may allow for fibers to hypercontract and thus form ring bands. The consequences of such a ring band pattern might be to restrict the further expansion of the sarcolemma and protect it from further mechanical stress. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Biomechanical properties of native basement membranes

Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.     

An analysis of the interactions between sympathetic nerve fibers and smooth muscle cells in tissue culture

The interactions between sympathetic nerve fibers and smooth muscle cells and fibroblasts from the newborn guinea pig vas deferens were studied in tissue culture with phase contrast microscopy, time-lapse microcinematography, catecholamine fluorescence histochemistry and scanning and transmission electron microscopy. The amount of sympathetic nerve fiber growth, its catecholamine fluorescence reaction and the size of the nerve cell bodies and their nuclei all increased in the presence of vas deferens tissue. Specific growth of nerve fibers to large clumps of vas deferens tissue was seen from distances of up to 2 mm. In contrast, no specific growth from a distance occurred to single cells or small groups of cells. However, random contact with a muscle cell often led to close, extensive, and long-lasting associations. Contact with fibroblasts was always transitory.The rate of sympathetic nerve fiber growth over individual muscle cells was faster than over fibroblasts, which, in turn, was faster than over the collagen-coated surface of the coverslip. Palpation of a muscle cell by a nerve fiber growth cone increased the rate of spontaneous contraction of the muscle cell, the extent of the increase being dependent on the number of nerve fibers involved. Multiple innervation of a smooth muscle cell occurred if nerve fibers reached the cell at about the same time, but not if there was a close association already established. These results are discussed in relation to possible interactions of sympathetic nerve fibers with smooth muscle cells in vivo.     

免疫荧光法显示贝居腹盾吸虫肌肉神经组织的实验研究

从背角无齿蚌(Anodonta woodiana)围心腔内检获自然感染的贝居腹盾吸虫(Aspidogaster conchicola),根据虫体内睾丸、卵巢、子宫及卵黄腺4种生殖器官的发育程度,将所获虫体分为幼虫组和成虫组,所有虫体用4%甲醛固定后,分别采用鬼笔环肽染色法和乙酰化微管蛋白免疫组织化学法显示贝居腹盾吸虫成虫和幼虫的肌肉纤维和神经纤维,使用激光共聚焦显微镜观察。结果显示,鬼笔环肽荧光染色的贝居腹盾吸虫成虫较幼虫肌肉组织更为发达,口周围、盾盘附着器、子宫末段及阴茎囊肌肉纤维分布较为密集。乙酰化微管蛋白荧光染色的贝居腹盾吸虫成虫较幼虫神经网络更为复杂,在部分生殖器官如子宫末段、阴茎囊和睾丸部位存在神经纤维。双重染色结果显示,该吸虫不同器官上的肌肉纤维分布与神经纤维的分布相互吻合。该结果提示,贝居腹盾吸虫可能通过神经系统对其肌肉活动进行调控。     

Scanning electron microscopy of giemsa-stained chromosomes and surface-spread chromosomes

Giemsa-stained chromosomes as prepared for light microscopy, and including G-banded, C-banded, and FPG-stained chromosomes, were examined by scanning electron microscopy. Although suitable for light microscopy, these chromosomes were too flat for a close examination of their fine structure by scanning electron microscopy. The surface of Giemsa-positive regions was rough and bright, whereas that of unstained or poorly stained regions was smoother and less bright. Giemsa-staining, therefore, seems to produce the bulkiness of the chromosomes. On topographical examination by scanning electron microscopy, the transparent chromosomes as observed with the light microscope proved to be footprints. Stereographical examinations of surface-spread chromosomes showed that minimally stretched chromosomes were composed of a mass of nodular and twisted looping fibers with an average diameter of about 300 Å. The substructure of these chromosome fibers was not determined. The kinetochore region was discernible as a constriction in the mass of the chromosome fibers, and was distinguishable from gaps by the presence of several chromosome fibers parallel to the axis of the chromatid. The organization of the chromosome fibers, however, was disordered rather than regular.     

Pathological steps of cancer-related lymphedema: histological changes in the collecting lymphatic vessels after lymphadenectomy

Introduction

To date, an electron microscopy study of the collecting lymphatic vessels has not been conducted to examine the early stages of lymphedema. However, such histological studies could be useful for elucidating the mechanism of lymphedema onset. The aim of this study was to clarify the changes occurring in collecting lymphatic vessels after lymphadenectomy.

Methods

The study was conducted on 114 specimens from 37 patients who developed lymphedema of the lower limbs after receiving surgical treatment for gynecologic cancers and who consulted the University of Tokyo Hospital and affiliated hospitals from April 2009 to March 2011. Lymphatic vessels that were not needed for lymphatico venous anastomosis surgery were trimmed and subsequently examined using electron microscopy and light microscopy.

Results

Based on macroscopic findings, the histochemical changes in the collecting lymphatic vessels were defined as follows: normal, ectasis, contraction, and sclerosis type (NECST). In the ectasis type, an increase in endolymphatic pressure was accompanied by a flattening of the lymphatic vessel endothelial cells. In the contraction type, smooth muscle cells were transformed into synthetic cells and promoted the growth of collagen fibers. In the sclerosis type, fibrous elements accounted for the majority of the components, the lymphatic vessels lost their transport and concentrating abilities, and the lumen was either narrowed or completely obstructed.

Conclusions

The increase in pressure inside the collecting lymphatic vessels after lymphadenectomy was accompanied by histological changes that began before the onset of lymphedema.     

Autonomic innervation of receptors and muscle fibres in cat skeletal muscle

Cat hindlimb muscles, deprived of their somatic innervation, have been examined with fluorescence and electron microscopy and in teased, silver preparations; normal diaphragm muscles have been examined with electron microscopy only. An autonomic innervation was found to be supplied to both intra- and extrafusal muscle fibres. It is not present in all muscle spindles and is not supplied at all to tendon organs. Fluorescence microscopy revealed a noradrenergic innervation distributed to extrafusal muscle fibres and some spindles. On the basis of the vesicle content of varicosities the extrafusal innervation was identified as noradrenergic (32 axons traced), and the spindle innervation as involving noradrenergic, cholinergic and non-adrenergic axons (14 traced). Some of the noradrenergic axons that innervate spindles and extrafusal muscle fibres are branches of axons that also innervate blood vessels. We cannot say whether there are any noradrenergic axons that are exclusively distributed to intra- or extrafusal muscle fibres. The varicosities themselves may be in neuroeffective association with striated muscle fibres only, or with both striated fibres and the smooth muscle cells in the walls of blood vessels. The functional implications of this direct autonomic innervation of muscle spindles and skeletal muscle fibres are discussed and past work on the subject is evaluated.     

Vascularization of the pars intermedia of the hypophysis in the toad,Bufo bufo (L.) (Amphibia,Anura)

The vascularization of the pars intermedia of the hypophysis of the toad, Bufo bufo (L.) was studied by injection of a mixture of India-ink and gelatine into the circulatory system of the head via the arteria carotis communis. Further methyl-methacrylate corrosion casts of brains were made and the hypophysial region of the corrosion casts was examined with the scanning electron microscope. The results showed that the vascularization of the pars intermedia of the toad hypophysis consists of a single-layered vascular network, which is located on the ventral surface of the pars intermedia. The network is formed by capillaries, which primarily run caudally in a fan-like manner and which show only a few cross-connections. In the rostral region of the pars intermedia this network lies rather superficially, while in the caudal region it slightly penetrates the parenchyma. The vascular network originates from vessels of the neural stalk and from wide capillaries of the rostro-ventral region of the neurointermediate junction. The venous drainage of the pars intermedia is exerted by veins, which leave the caudal region and drain into the veins leaving the venous pole of the pars distalis. The flat, wide meshed vascular net on the ventral side of the pars intermedia, demonstrated in this study, fits into the concept that the pars intermedia of the anuran hypophysis is under the control of nerve fibers coming from the hypothalamus.     

An investigation of lymphatic vessels in the feline dental pulp

The existence of lymphatic vessels in the dental pulp has been a matter of continuing controversy. We have now used light microscopy to examine semithin transverse sections of perfusion-fixed incisors and canines in cats. Lymphatics were found in all the teeth studied. In most teeth they were present in the coronal, middle, and apical regions of the pulp; but in a few they were lacking coronally and in the middle. Within individual teeth, lymphatics were found in the subodontoblastic zone or more centrally in the pulp; but none were found in the odontoblast layer or in the pulp horns. Vessels located by light microscopy were subsequently examined by transmission electron microscopy. Their ultrastructural features were typical of lymphatics and included irregular, attenuated endothelium with adjacent cells joined in different ways. Occasional gaps connected the extracellular spaces with their lumens, and abluminal endothelial projections appeared to form open end bulbs. There was very little basement membrane, but anchoring filaments were found near the abluminal surface of the endothelium and near collagen fibrils. The total cross-sectional area of lymphatic vessels was measured in semithin sections and, with pulp area, increased from the coronal region to the middle. However, both areas decreased from the middle to the apical region suggesting either that lymph flows faster as it reaches the foramens of the apical delta or that some vessels leave the tooth through lateral root canals. Using the methods of light and transmission electron microscopy, therefore, we have shown that pulp lymphatic vessels exist. Questions remain, however, about their distribution within teeth, variations between teeth, and routes of exit from teeth.