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PubMed1990

The expression of myosin genes in developing skeletal muscle in the mouse embryo.

Using in situ hybridization, we have investigated the temporal sequence of myosin gene expression in the developing skeletal muscle masses of mouse embryos. The probes used were isoform-specific, 35S-labeled antisense cRNAs to the known sarcomeric myosin heavy chain and myosin alkali light chain gene transcripts. Results showed that both cardiac and skeletal myosin heavy chain and myosin light chain mRNAs were first detected between 9 and 10 d post coitum (p.c.) in the myotomes of the most rostral somites. Myosin transcripts appeared in more caudal somites at later stages in a developmental gradient. The earliest myosin heavy chain transcripts detected code for the embryonic skeletal (MHCemb) and beta-cardiac (MHC beta) isoforms. Perinatal myosin heavy chain (MHCpn) transcripts begin to accumulate at 10.5 d p.c., which is much earlier than previously reported. At this stage, MHCemb is the major MHC transcript. By 12.5 d p.c., MHCpn and MHCemb mRNAs are present to an equal extent, and by 15.5 d p.c. the MHCpn transcript is the major MHC mRNA detected. Cardiac MHC beta transcripts are always present as a minor component. In contrast, the cardiac MLC1A mRNA is initially more abundant than that encoding the skeletal MLC1F isoform. By 12.5 d p.c. the two MLC mRNAs are present at similar levels, and by 15.5 d p.c., MLC1F is the predominant MLC transcript detected. Transcripts for the ventricular/slow (MLC1V) and another fast skeletal myosin light chain (MLC3F) are not detected in skeletal muscle before 15 d p.c., which marks the beginning of the fetal stage of muscle development. This is the first stage at which we can detect differences in expression of myosin genes between developing muscle fibers. We conclude that, during the development of the myotome and body wall muscles, different myosin genes follow independent patterns of activation and accumulation. The data presented are the first detailed study of myosin gene expression at these early stages of skeletal muscle development.

Animals↗
PubMed1993

Muscle contraction and inward current induced by silver and effect of Ca2+ channel blockers.

Single fibers from toe or anterior tibialis muscle contracted transiently and then tonically in the presence of 1.8 mM Ca2+ on addition of 10 microM Ag+. Exposure of fibers to Cd2+ completely inhibited tonic contraction and modified phasic contraction to some extent. Nifedipine at 10 microM initially potentiated and then completely inhibited twitch tension; subsequently, fibers no longer contracted phasically in response to 20 microM Ag+, whereas slight tonic contraction still occurred. Fibers with membrane potential clamped at -90 mV produced maintained inward current on application of Ag+. Simultaneous administration of 1 mM Cd2+ and 10 microM Ag+ to fibers voltage clamped with the double mannitol gap technique almost completely blocked the inward current. Removal of Cd2+ elicited a rapid and large inward current. Ag(+)-induced inward current was inhibited when 1 mM Cd2+ was applied to fibers during development of the inward current. In fibers paralyzed with 10 microM nifedipine, the inward current induced by 10 microM Ag+ was partially inhibited. These results suggest that phasic contraction induced by Ag+ is controlled by L-type Ca2+ channels (probably voltage sensors) located in the T-tubular membrane, whereas tonic contraction involves Ca2+ channels sensitive and/or insensitive to dihydropyridine in the surface and T-tubular membranes.

Animals↗
PubMed1999

Classification of teased nerve fibers for multicenter clinical trials.

Teased nerve fibers are used widely in both clinical and experimental neuropathology, but anecdotal evidence indicates that even experienced readers find little agreement on categories for teased fiber classification. To develop a classification scheme that could be used and understood by both experienced and naive readers, specific criteria were developed for normal fibers and those exhibiting Wallerian degeneration, demyelination, hypomyelination, remyelination, and abnormal paranodal myelination. Twenty fibers teased from human sural nerve biopsies were selected as examples of one or more of these categories. Ten readers, including seven having no previous experience with teased fibers, were given a set of instructions and asked to score each fiber for all matching categories. These readers averaged high rates of true positive (56-85%) classifications, while average false positive (3-18%) rates were much lower. Among the three experienced readers, true positive agreements averaged between 75 and 100% across the fiber classifications. False positives were correspondingly low, ranging between 0 and 8%. These results suggest that it is possible to design an easily learned, meaningful scheme for classifying teased nerve fibers.

Biopsy↗
PubMed2003

[Unmyelinated nerve fibers].

The paper presents a critical review of various current concepts of the structure and kinetics of unmyelinated nerve fiber. A classification of nerve fibers, different from the earlier ones, is proposed, that demonstrates not only the morphological fiber types, but also the kinetics of their reversible transition stages from non-glial to myelinated fiber. Evidence is presented to show the erroneousness of conceptions, still appearing in many publications, that consider the unmyelinated nerve fiber as "the Remak's cable type fiber". According to the current data, "Remak's fiber" is a glial-neurite complex, i.e. a bundle of unmyelinated nerve fibers covered with a single glial cell. Using the electron microscope, it was demonstrated that comparable glial-neurite complexes of myelinated nerve fibers, formed in CNS in a similar way by a single oligodendrocyte, cannot be named a single fiber. Cutting the nerves makes visible that the single fibers forming "the Remak's fiber" stem from different cells, therefore they cannot be a single "fiber". It has been shown for the first time experimentally, that in extreme situations, as a result of contraction of gliocyte processes, unmyelinated fibers may "leave" the glial-neurite complexes and become the nonglial fibers. Some data are presented that may serve as criteria for differentiation of unmyelinated fiber with a stratified sheath from developing myelinated fiber.

Animals↗
PubMed1976

Temperature-dependence of tension development by glycerinated muscle fibers of rabbit psoas in Mn (II)-ATP and Mg-ATP solutions.

The tension of single fibers isolated from glycerinated rabbit psoas muscle was measured at various temperatures using Mg-ATP or Mn-ATP as a substrate. The tension developed in Mn-ATP was 80-89% of that in Mg-ATP at 4 degrees-16 degrees, and both tensions decreased as the temperature was reduced. Myosin forms the myosin-product complex predominantly generated on admixture with ADP (and Pi) at the burst site during Mn(II)-ATP hydrolysis below 10 degrees, while it forms the myosin-product complex predominantly formed via ATP hydrolysis upon hydrolysis above 10 degrees, as it does during Mg-ATP hydrolysis (Hozumi & Tawada (1975) Biochim. Biophys. Acta 376, 1; Tawada & Yoshida (1975) J. Biochem. 78, 293; Yazawa & Morita (1973) J. Biochem. 74, 1107). Since the cycle time of cross-bridge attachment to and detachment from actin in muscle is about 1-10 sec and because the spontaneous decay time of the myosin-product complex into the myosin-product complex in the absence of actin is less than 1 sec, then if most of the cross-bridges are detached from actin as suggested by X-ray data, it can be inferred that most of the cross-bridges detached from actin may form the predominant myosin-product complex in Mn-ATP below 10 degrees. If this is so, the tension development in Mn-ATP below 10 degrees cannot be compatible with some muscle models which assume the formation of the myosin-product complex by the cross-bridges prior to combination with actin during contraction.

Adenosine Triphosphate↗
PubMed2003

Cellular aspects of elastogenesis in the elastic tendon of the chicken wing.

Morphological, immunocytochemical and ultrastructural methods were used to investigate the role of cells during elastogenesis in the elastic tendon of the chicken wing. Intimate contact of the cell processes with elastic fibers was observed in adult birds. During development there was a sequential appearance of microfibril bundles that became progressively impregnated with amorphous elastin, which eventually predominated in fully developed elastic fibers. The growing elastic fibers were usually enveloped by recesses of the cell surface. The tendon cells were polarized in their association with fibrous components of the extracellular matrix. This arrangement suggests that these cells secrete and organize elastic and collagen fibers to different extracellular compartments. These results show that cells are intimately involved in producing components of different extracellular matrix fibers, in controlling their assembly, and in defining their borders and associations during development.

Animals↗
PubMed1989

Transient direct connection of vestibular mossy fibers to the vestibulocerebellar Purkinje cells in early postnatal development of kittens.

Postnatal development of mossy fiber afferents from the vestibular and the visual system to the vestibulocerebellum was studied electrophysiologically and morphologically. In kittens anesthetized with pentobarbital sodium and N2O plus halothane, extracellular simple and complex spikes of Purkinje cells were recorded in the flocculus, nodulus and uvula. In the flocculus, stimulation of the VIIIth, but not the optic nerve, evoked simple spike responses with a latency of 16 ms at the day of birth which decreased to 5 ms by day 15 (short latency group). On the other hand, another group of simple spike responses with much longer latencies (50-80 ms) began to be elicited on day 7 via both the optic and VIIIth nerves. The latency decreased to 24 ms by day 15 and 10 ms on day 30. These latencies further shortened with development to the adult latency value (3-5 ms). Simple spike responses of the short latency group were also evoked in the nodulus and uvula from the VIIIth nerve with a slightly longer latency than that in the flocculus (23 ms on day 3 and 12 ms on day 17). Because of the immaturity of granule cells in early postnatal days, short latency simple spike responses from the VIIIth nerve suggested the direct synaptic connection of vestibular mossy fibers with Purkinje cells. Horseradish peroxidase was injected into the white matter of the flocculus, nodulus and uvula in slice preparations. Mossy fibers labeled with horseradish peroxidase showed fine branches extending to reach Purkinje cell somata from mossy swellings in the internal granular layer during days 2-20. Electron microscopy showed that the labeled mossy fibers made intimate contacts with Purkinje cell somata and the terminals contained many round synaptic vesicles. Pre and postsynaptic densities were occasionally found. After day 20, direct mossy fiber connections with Purkinje cells could not be observed. During days 7-20, these direct connections, as well as mossy fiber-granule cell connections could be observed. It was demonstrated that during early postnatal development, vestibular mossy fibers temporarily make direct contact with Purkinje cells, through which impulses could be transmitted to elicit simple spikes in Purkinje cells.

Action Potentials↗
PubMed2000

Involvement of gap junctional communication in myogenesis.

Cell-to-cell communication plays important roles in development and in tissue morphogenesis. Gap junctional intercellular communication (GJIC) has been implicated in embryonic development of various tissues and provides a pathway to exchange ions, secondary messengers, and metabolites through the intercellular gap junction channels. Although GJIC is absent in adult skeletal muscles, the formation of skeletal muscles involves a sequence of complex events including cell-cell interaction processes where myogenic cells closely adhere to each other. Much experimental evidence has shown that myogenic precursors and developing muscle fibers can directly communicate through junctional channels. This review summarizes current knowledge on the GJIC and developmental events involved in the formation of skeletal muscle fibers and describes recent progress in the investigation of the role of GJIC in myogenesis: evidence of gap junctions in somitic and myotomal tissue as well as in developing muscle fibers in situ, GJIC between perfusion myoblasts in culture, and involvement of GJIC in cytodifferentiation of skeletal muscle cells and in myoblast fusion. A model of intercellular signaling is proposed where GJIC participates to coordinate a multicellular population of interacting myogenic precursors to allow commitment to the skeletal muscle fate.

Animals↗
PubMed1999

Mossy fiber sprouting in the dentate gyrus in a newly developed epileptic mutant, Ihara epileptic rat.

We examined the correlation between seizure activity and development of mossy fiber sprouting in the hippocampal formation using Timm staining in a newly developed Ihara epileptic rat (IER). The sprouting of mossy fibers were clearly shown in the inner molecular portion of the dentate gyrus and in the stratum oriens of CA3 pyramidal cell layer with repeated seizures. A positive correlation between the frequency of generalized tonic and clonic convulsions and the Timm staining score in molecular layer of dentate gyrus was revealed. Sprouting of mossy fiber in IER seems to be linked with seizure activities resulting from epileptic bursts, not to the genetic mutation.

Animals↗
PubMed2001

Pax6 regulates granule cell polarization during parallel fiber formation in the developing cerebellum.

The molecular mechanisms that govern the coordinated programs of axonogenesis and cell body migration of the cerebellar granule cell are not well understood. In Pax6 mutant rats (rSey2/rSey2), granule cells in the external germinal layer (EGL) fail to form parallel fiber axons and to migrate tangentially along these fibers despite normal expression of differentiation markers. In culture, mutant cells sprout multiple neurites with enlarged growth cones, suggesting that the absence of Pax6 function perturbs cytoskeletal organization. Some of these alterations are cell-autonomous and rescuable by ectopic expression of Pax6 but not by co-culture with wild-type EGL cells. Cell-autonomous control of cytoskeletal dynamics by Pax6 is independent of the ROCK-mediated Rho small GTPase pathway. We propose that in addition to its roles during early patterning of the CNS, Pax6 is involved in a novel regulatory step of cytoskeletal organization during polarization and migration of CNS neurons.

Animals↗
PubMed1994

Effects of Aramid, a high strength synthetic fiber, on respiratory cells in vitro.

Industry continues to develop synthetic fibers for new technologies and as replacements for asbestos, a toxic and carcinogenic fiber. To determine whether the in vitro effects of the aromatic polyamide fiber, Aramid (Kevlar, Twaron), resembled those induced by asbestos, fibers were surveyed for (1) cytotoxicity as measured by total cell protein, and (2) proliferative capacity as measured by [3H]thymidine incorporation, colony forming efficiency (CFE), and ornithine decarboxylase (ODC) activity in two target cells of mineral dust-induced lung damage, hamster tracheal epithelial (HTE) cells and rat lung (RL90) fibroblasts. Results of cytotoxicity tests indicated that Aramid was as toxic to HTE and RL90 cells as were crocidolite and chrysotile asbestos when expressed on both an equal mass and equal fiber number basis. In HTE cells, Aramid caused a statistically significant increase in [3H]thymidine incorporation and CFE and produced a dose-dependent induction of ODC enzyme activity. Proliferative effects by asbestos or Aramid were not observed in RL90 fibroblasts. Thus, when tested over a respirable size range, Aramid exhibited many of the same effects on epithelial cells in vitro as did asbestos, including increased radiolabeled nucleotide incorporation into DNA and induction of ODC enzyme activity.

Animals↗
PubMed1993

Development of dopamine-beta-hydroxylase-positive fiber innervation of the rat hippocampus.

Development of the noradrenergic fiber innervation of the rat hippocampus by the locus coeruleus was examined immunohistochemically in fixed tissue from animals aged 4 days through 55 days postnatal. The presence of tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) immunoreactive cells and fibers was evaluated in sections of hippocampus and locus coeruleus. Large, multipolar TH- and DBH-positive cells with long beaded fibers were visible within locus coeruleus at all ages; no immunopositive cell bodies were found in hippocampus. In hippocampal sections from mature animals (PN55), the highest density of DBH-stained fibers was found in stratum lucidum of CA3 and in the hilus and inner molecular layer of the dentate gyrus. Whereas similar patterns of fiber positivity were found at PN21 and PN10 (although with somewhat reduced density of immunopositive fibers), the pattern was quite different at PN4. Although fiber staining was relatively sparse at PN4, relative density of DBH fibers was highest in stratum radiatum of CA1 and subiculum. This change in staining pattern suggests that noradrenergic function in hippocampus may change as the rat matures. Double immunofluorescence techniques showed an overlap of DBH and TH positive fibers in all hippocampal regions at all ages. DBH immunostaining appeared to be somewhat more sensitive than the TH staining. These data made it impossible to confirm the presence of significant numbers of nonnoradrenergic, catecholamine-containing fibers in hippocampus.

Animals↗
PubMed1985

The development of the dentate area and the hippocampal mossy fiber projection of the rat.

The development of the dentate area and the hippocampal mossy fiber system of the rat has been investigated at the light microscopic level by using fluorescent tracing, Nissl, and Timm's histochemical methods. Although the cytoarchitectonic development of the dentate granular layer is mainly a postnatal phenomenon, the initial events take place before birth. The aggregation and maturation of the cells in the granular layer proceed in a graded fashion from the lateral to the medial and from the superficial to the deep aspects of the layer. The earliest-formed granule cells are probably derived directly from the cells of the ventricular zone. They start to form mossy fibers prenatally, either during the relatively long period of migration to the granular layer or soon after their arrival. However, most of the granule cells are derived from a secondary proliferative center in the hilus. They start to produce mossy fibers postnatally a while after arriving at the granular layer. The total complement of granule cells starts to grow mossy fibers in a sequence that is related to the final position of the cells of origin within the granular layer. This sequence also proceeds in a graded fashion from the lateral to the medial and from the superficial to the deep aspects of the layer. In the beginning the mossy fibers elongate relatively rapidly. Already at birth the Timm-stained mossy fiber zone occupies the anterolateral part of the hilus and the adjacent suprapyramidal parts of the regio inferior. Once the mossy fibers have reached the distal end of the regio inferior they elongate along the longitudinal axis of the hippocampus more slowly. At the same time the Timm-stainability of the mossy fiber zone, which, during the first postnatal week, is weaker toward the regio superior, develops a mature pattern in which the distal part of the zone stains most intensely. Throughout development, fibers from the granule cells that form first are longer and diverge more in the septotemporal dimension than fibers from later-forming granule cells. In contrast to other axonal systems which appear to be sculptured from a diffuse set of connections the results presented here provide evidence that the topographic relationships of the mossy fiber system develop in a stepwise fashion.

Animals↗
PubMed2005

A dominant negative form of Rac1 affects myogenesis of adult thoracic muscles in Drosophila.

Blocking Rac1 function in precursors of the indirect flight muscle of Drosophila severely disrupts muscle formation. The DLM fibers that develop using larval scaffolds are reduced in number and fiber size, while the DVMs, which develop using founder cells, are mostly absent. These adult muscle phenotypes are in part due to a reduced myoblast pool present at the third larval instar. BrDU labeling studies indicated that this is primarily due to a reduction in proliferation. In addition, DVM myoblasts display altered morphology and are unable to segregate into primordia. This defect precedes the evident block in fusion. We also show that the recently described DVM founder cells can be labeled with 22C10 and beta-3 tubulin, and that they are present under conditions of dominant negative Rac1(N17) expression. Despite the presence of founder cells, DVM fiber formation is rarely observed. Although DLM myoblasts are able to segregate around their larval scaffolds, the pace of fusion is reduced and consequently there is a delay in DLM fiber formation. Thus, in addition to its well-established role in fusion, Rac1 is also involved in the regulation of myoblast proliferation and segregation during adult myogenesis. These are two new roles for Rac1 in Drosophila.

Animals↗
PubMed1982

Acetylcholine receptors and cholinesterase in developing chick skeletal muscle fibers.

The developmental changes in the distribution of acetylcholine receptors (AChRs) and cholinesterase (ChE) were investigated in the posterior latissimus dorsi (PLD) muscle of chick embryos by double staining with rhodamine-labeled erabutoxin b (TMR-Eb) for AChRs and Karnovsky's method for ChE. During the development, the TMR-Eb and ChE positive areas changed in their shapes and sizes. In early stages, the TMR-Eb positive areas appeared as fine fluorescent dots of about 0.3 micrometer in diameter or small aggregates of such dots. These areas then became enlarged and exhibited the following sequential changes in their configuration: spindle-, round-, ring-, C- and finally tree-shaped. The transformation in the configuration of the areas appeared to be caused by the changes of the fluorescent dots in their number and distribution. Simultaneous staining with ChE revealed that at early stages the TMR-Eb positive areas were not stained with ChE, but then they were stained later. The ChE deposits usually accumulated at the borders of the TMR-Eb positive areas and thereby outlined them. Electron microscopy using horseradish peroxidase-labeled erabutoxin b revealed that the fluorescent dots represent the discrete regions of high AChR concentration at the muscle membrane.

Animals↗
PubMed1984

Histofluorescence evaluation of sympathetic nerve fibers in hearts of developing rats exposed to ethanol during gestation.

Histofluorescence for catecholamines has been used to examine the ingrowth of the sympathetic innervation in the heart of control rats and rats exposed either pre- or perinatally to ethanol. In all groups, sympathetic innervation began in primary association with large blood vessels and, to a lesser extent, in the myocardial surface between 2 and 6 days of postnatal age. From day 9 onward, innervation progressed in density first in the atria and right ventricle. The deep muscle of the left ventricle was least innervated of all areas of the heart at all time periods examined. In pups exposed continuously to ethanol or withdrawn at birth, the innervation pattern and density were similar to controls, although slight qualitative differences may have occurred in the first two weeks of the postnatal period in the group exposed continuously to ethanol. The reported altered cardiac biochemical responses to sympathetic agents after pre- or perinatal exposure to ethanol did not appear to be associated with significant alterations in the growth of sympathetic nerve fibers into the heart. It is suggested that the alterations in the development of the cardiac sympathetic nervous system induced by ethanol probably occurs at the level of nerve terminals or adrenergic receptors.

Animals↗