Histochemical and biochemical plasticity of muscle fibers in the little brown bat (Myotis lucifugus)

Summary Fiber composition, and glycolytic and oxidative capacities of the pectoralis, gastrocnemius, and cardiac muscles from active and hibernating little brown bats (Myotis lucifugus) was studied. The data were used to test two hypotheses: First, since hibernating bats maintain the capability of flight and make use of leg muscles to maintain a roosting position all winter, the fiber composition of the pectoralis and gastrocnemius muscles should not change with season. Second, we tested the hypothesis of Ianuzzo et al. (in press), who propose that the oxidative potential of mammalian cardiac muscle should increase with increasing heart rate while glycolytic potential should not. Our results indicate that the fiber composition of the pectoralis muscle was uniformly fast-twitch oxidative (FO)_ regardless of the time of year, as predicted. However, the gastrocnemius muscle exhibited a change in FO composition from 83% in active to 61% in hibernating animals. Contrary to the variable change in histochemical properties with metabolic state, a trend of reduced maximal oxidative (CS) and glycolytic (PFK) potential during hibernation in both flight and leg muscles was apparent. The oxidative potential of flight and leg muscles decreased by 15.2% and 56.5%, respectively, while the glycolytic potential of the same muscles decreased by 23.5% and 60.5%, respectively. As predicted, the glycolytic potential of cardiac muscle remained constant between active and hibernating bats, although there was a significant decrease (22.0%) in oxidative potential during hibernation.Abbreviations FO fast-twitch oxidative - FG fast-twitch glycolytic - SO slow-twitch oxidative - Vmax maximal enzyme activity - PFK phosphofructokinase - CS citrate synthase     

The determination of myogenic and cartilage cells in the early chick embryo and the modifying effect of retinoic acid

Summary The time of determination of cartilage and skeletal muscle was studied by making chimeric grafts or explants of small tissue pieces from several stages of early chick or quail embryos. Chondrogenesis was assessed by histology or with antibodies directed against type II collagen or cartilage proteoglycan, while myogenesis was detected immunohistochemically with antibodies directed against 3 different muscle markers, including muscle myosin. Grafts from Hensen's node, primitive streak and segmental plate of donor embryos of Stage 3–5 (Hamburger and Hamilton) were transplanted under the ectoderm in the extraembryonic area of Stage 12 host embryos. In addition, explants and mesodermal cells were cultured on glass in DMEM+F12 medium supplemented with 10% FCS. The results showed that determined myogenic cells could first be detected in Hensen's node and the primitive streak at Stage 3⁺–4 and that they developed from mesodermal cells located between the epiblast and hypoblast. Myogenic cells also appeared in grafted and explanted segmental plate with or without notochord from Stage 5 embryos. On the other hand, cartilage cells only formed in grafted and explanted segmental plate that also contained notochord. RA (1 ng/ml) could induce the formation of cartilage cells in the explanted primitive streak without Hensen's node or notochord taken from Stage 3–5 embryos and could also promote the differentiation of myogenic cells in primitive streak from Stage 3 embryo. Thus RA can substitute for Hensen's node or the notochord in the induction of cartilage cells and has some stimulatory effects on the differentiation of myogenic cells. Additional evidence indicates that the hypoblast might play an inductive role in the formation of the notochord which may subsequently promote the differentiation of cartilage cells. Offprint requests to: M. Solursh     

Brain tissue transplanted to the anterior chamber of the eye: 2. Fluorescence histochemistry of immature catecholamine and 5-hydroxytryptamine neurons innervating the rat vas deferens

Summary Small pieces of the wall of the rat vas deferens were homologously transplanted to the anterior chamber of the eye together with small pieces of embryonic brain stem containing either developing noradrenaline (NA) cells of the locus coeruleus or 5-hydroxytryptamine (5-HT) neurons of the developing raphe system. The eyes of the recipients were sympathetically denervated. The double transplants became rapidly vascularized from the host iris. After 31/2 months the irides, together with their two transplants were analyzed by Falck-Hillarp fluorescence microscopy. Both the NA and the 5-HT neurons had survived and matured in the eye. Fluorescent varicose nerve terminals of the NA and 5-HT type respectively were found in all three potential receptor areas, i.e. within the CNS transplants, in the host irides and in the vas deferens transplants. In the latter, the newly formed monoamine nerve terminals arborized mainly within a well developed smooth muscle layer. The density of such new fibres was higher than or similar to that of the normally present sympathetic plexus in areas of the transplant close to the CNS transplant and lower in areas at a distance from the CNS transplant. It is concluded that immature central NA and 5-HT fibres are able to grow simultaneously into different types of sympathetically denervated smooth muscle tissues to form networks of fibres in the receptor organs resembling the normal sympathetic innervation.Supported by the Swedish Medical Research Council (04X-3185), and by grants from Magnus Bergvalls Stiftelse, Tidningen Expressens Prenatalforskningsfond and Karolinska Institutets fonder. We thank miss Ingrid Strömberg for skilful technical assistance. Nialamide^® was generously donated by Swedish Pfizer AB.     

Quantitative studies on the distribution of myofilaments in intrafusal muscle fibres

Summary Muscle spindles contain two types of intrafusal muscle fibre, nuclear bag fibres and nuclear chain fibres. The intrafusal fibres of rabbit and guinea pig spindles have been studied using quantitative stereological techniques at the ultrastructural level. The crosssectional areas occupied by myofilaments have been measured in the polar and equatorial regions of both types of intrafusal fibre. There are considerably fewer myofilaments in the equatorial regions of both types of fibre compared with their polar regions.This work was carried out with the aid of grants from the Medical Research Council and the Science Research Council of Great Britain.     

Intra- and extraganglionic nerve endings formed by neurosecretory cells of the cerebral ganglion of the earthworm (Lumbricus terrestris L.)

Summary In the cerebral (= supraesophageal, suprapharyngeal) ganglion of the earthworm, a number of neurosecretory Gomori-positive perikarya are bipolar; others are unipolar, or multipolar. Some of the neurosecretory cell processes project centrally into a fibrous zone; peripheral processes enter small nerves which leave the dorsocaudal aspect of the ganglion.In the central fibrous zone, the neurosecretory fibers form varicose Gomoripositive terminals. Here, also zinc-iodine-osmium (ZIO)-positive fibers and monoamine fluorescent fibers are found. With the electron microscope, nerve terminals containing synaptic vesicles and either large neurosecretory peptidergic granular vesicles (diameter more than 1500 Å), or smaller granular vesicles (diameter about 1300 Å, or 900 Å) are observed. These axon endings mainly form axo-dendritic synapses. Peptidergic profiles are both pre- and postsynaptic. Some of the extraganglionic peptidergic fibers appear to terminate around vessels, but most of them form terminals on the visceral muscle cells which surround the ganglion.We think that the central neurosecretory processes communicate with the fibers of the synaptic zone of the ganglion. The peripheral neurosecretory peptidergic fibers are supposed to form a primitive neurohemal area and/or to function as vasomotor nerves. The fibers innervating the visceral muscle cells may represent vegetative nerves.     

Correlations between ultrastructural features and contraction rates in Rotiferan muscle

Summary The Rotifer Trichocerca rattus has striated longitudinal retractor muscles. These muscles can be divided into two categories: 1. The central and ventral retractor muscles which, after fixation, are found in a supercontracted state: they probably contract very quickly. 2. The lateral retractor muscles which are in a relaxed state after fixation. However, if the animal is mechanically stimulated before fixation, these are also fixed in a contracted state: so, normally, these muscles probably contract more slowly than the first category.In the relaxed state, thin myofilaments of the lateral retractor muscles are folded at the I band level; this is a consequence of their compression provoked by the contraction of central and ventral retractor muscles.In muscles of the first type, the thick myofilaments are shorter (<2 ) than in the second type (2.5 ).     

Cellular and Secreted Forms of Acetylcholinesterase in Mouse Muscle Cultures

When grown in primary cell culture in the absence of neurons, muscle cells from a variety of species synthesize several forms of acetylcholinesterase (AChE), including the collagen-tailed A12 form. A12 AChE has been the subject of much study because it is thought to be a major functional enzyme form normally found in the basal lamina at the neuromuscular junction. In this paper, we show that muscle fibers derived from mouse embryos and neonates are also able to synthesize substantial percentages of their AChE as the A12 form when grown in vitro. This synthesis is modulated by a process associated with spontaneous muscle contractile activity since both total enzyme levels and the proportion of A12 AChE expressed on the cell surface are decreased when the cells are grown in the sodium channel blocker tetrodotoxin, which blocks muscle contraction. On the other hand, when the cells are treated with veratridine, which opens sodium channels, thereby mimicking one aspect of muscle contraction, their AChE levels are comparable to those of untreated cells. Although smaller in magnitude, these changes are similar to those seen in rat muscle cultures. A novel feature of mouse muscle cultures, not seen in those from rat and chick, is the presence of a secreted enzyme form that sediments in the same position as the cellular A12 form (when separated on sucrose density gradients containing high salt) and is also collagenase sensitive.     

Attempt to produce a chick/mouse heteroclass musculature

Summary Heteroclass chick/mouse chimaeras were prepared by transplanting somitic presumptive myogenic cells or limb bud myoblasts from donor mouse embryos into chick hosts, to replace (1) previously extirpated brachial somitic mesoderm or (2) experimentally deleted limb premuscular masses. Since mouse and chick cells can be distinguished by differential staining affinities, this parameter was used to verify the viability of the implant and to assess its fate. Our analyses showed that transplanted mouse somitic myogenic stem cells or limb bud myoblasts did not participate in the host brachial musculature, whatever the experimental conditions.     

Use of cryostat sections for measurement of Ca2+ uptake by sarcoplasmic reticulum.

Calcium uptake of cardiac muscle and fast-twitch and slow-twitch skeletal muscle of rabbit was measured in 10-μm thin sections. Ca²⁺ uptake showed K⁺, Mg²⁺, Ca²⁺, and oxalate dependence and required ATP. The contribution of mitochondria to the Ca²⁺ uptake could be ruled out, since inclusion of ruthenium red or sodium azide in the medium did not show inhibition. The method, which avoids unphysiological fragmentation of the sarcoplasmic reticulum, has the added advantage of requiring only 0.1 to 0.3 g of muscle and permitting simultaneous histochemical studies from the same muscle block.     

A quantitative analysis of elastic,entropic, electrostatic,and osmotic forces within relaxed skinned muscle fibers

The elastic behavior of mechanically skinned skeletal muscle fibers in relaxing solution is modelled using equations developed by Flory (1953) for the elasticity of non-biological polymers. Mechanically, the relaxed skinned fiber is considered to be a semi-crystalline network of inextensible polymer chains, which are periodically cross-linked and which are bathed in an aqueous medium. We consider (1) configurational elastic forces in the network, (2) entropic forces due to mixing of polymer and water, (3) electrostatic forces due to fixed charges on the muscle proteins and mobile charges in the bathing solution, and (4) compressive forces due to large colloids in the bathing solution. Van der Waals forces are not considered since calculations show that they are probably negligible under our conditions. We derive an expression which relates known quantities (ionic strength, osmotic compressive pressure, and fiber width), experimentally estimated quantities (fixed charge density and volume fraction of muscle proteins), and derived quantities (concentration of cross-links and a parameter reflecting the interaction energy between protein and water).The model was tested by comparison with observed changes in skinned fiber width under a variety of experimental conditions which included changes in osmotic compressive pressure, pH, sarcomere length, and ionic strength. Over a wide range of compressive pressure (0–36 atm) the theory predicted the nonlinear relation between fiber width and logarithm of pressure. The direction and magnitude of the decrease in width when pH was decreased to 4 could be modelled asssuming the fixed charge density on the protein network was 0.34 moles of electrons per liter protein, a value in accordance with the estimates of others. The relation between width and sarcomere length over the complete range of compressive pressures could be modelled with the assumption that the number of cross-links increases somewhat with sarcomere length. Changes of width with ionic strength were modelled assuming that increasing salt concentration both increased the electrostatic shielding of fixed charges and decreased the number of cross-links. The decrease of fiber width in 1% glutaraldehyde was modelled by assuming that the concentration of crosslinks increased by some 10%. The theory predicted the order of magnitude but not the detailed shape of the passive tension-length relation which may indicate that, as with non-biological polymers, the theory does not adequately describe the behavior of semi-crystalline networks at high degrees of deformation.In summary, the theory provides a semiquantitative approach to an understanding of the nature and relative magnitudes of the forces underlying the mechanical behavior of relaxed skinned fibers. It indicates, for instance, that when fibers are returned to near their in vivo size with 3% PVP, the forces in order of their importance are: ¦ elastic forces ¦ ¦ entropic forces > ¦ electrostatic forces ¦ ¦ osmotic compressive forces ¦.