Slowing of synapse elimination by alpha-bungarotoxin superfusion of the neonatal rabbit soleus muscle

To examine the role of postsynaptic activity in regulating the rate of neuromuscular synapse elimination, contractile activity of neonatal rabbit soleus muscles was decreased by chronic superfusion of alpha-bungarotoxin (alpha-BGT) over their surfaces. Superfusion was begun at 6 days postnatal and continued for a variable duration (2 to 5 days) before muscles were analyzed. The percentage of polyinnervated fibers was assessed both physiologically and anatomically for alpha-BGT-treated muscles and their contralateral muscles, in addition to normal and control muscles of the same age. Within muscles exposed to alpha-BGT, polyinnervation was significantly greater than that for muscles from each of the control groups. The anatomical assay further revealed that the retention of polyinnervation in alpha-BGT-treated muscles was most pronounced near the muscle's surface, although end plates at the center were also affected. This finding, coupled with evidence that only a small percentage of the muscle fibers were completely inactivated, suggests that the activity block was also most pronounced near the surface and relatively low at the muscle's center. The percentage of end plates at which synapse elimination was delayed was greater than the estimated percentage whose activity was completely blocked, suggesting that synapse loss was slowed even in muscle fibers retaining some postsynaptic activity. These observations indicate that the rate of synapse elimination depends on the levels of functional acetylcholine receptors. This process could be mediated in a graded fashion by changes in postsynaptic activity (subthreshold or suprathreshold) or by a nonelectrical effect of blocking postsynaptic receptors.     

Effects of evoked and spontaneous motoneuronal firing on synapse competition and elimination in skeletal muscle

Synapse elimination is a general feature of the development of neural connections, including the connections of motoneurons to skeletal muscle fibers. Our work addressed two questions: (1) how the action potentials generated in the set of motoneurons innervating an individual muscle (i.e., in a motor pool) are correlated in time during development in vivo; (2) what influence different firing patterns exert on the processes of polyneuronal innervation and synapse elimination which characterize the establishment of muscle innervation. We recorded the spontaneous electromyographic activity of the tibialis anterior and soleus muscles of late embryonic and neonatal rats, identifying the firing of at least two single motor unit signals in each record. We found that a striking switch occurs a few days after birth from a highly synchronous type of firing to an asynchronous one, the first thus characterizing embryonic while the second one adult motoneurons. We also investigated the effects of an evoked synchronous type of discharge on neuromuscular synapse formation, measuring polyneuronal innervation and synapse elimination. This was done in an adult in vivo model of de novo synapse formation, while a chronic TTX nerve conduction block, placed centrally with respect to the stimulating electrodes, eliminated the natural activity of motoneurons. We found that the imposed synchronous activity greatly inhibits synapse elimination, causing polyneuronal innervation to persist. We conclude that the early synchronous firing, favors the establishment of polyneuronal innervation while the subsequent switch to an asynchronous one promotes synapse elimination.     

Ultrastructural observations on the renal papilla of the rabbit

Summary Ultrastructural studies of renal papillae of New Zealand brown rabbits under different states of water balance indicate no morphological variation between control, antidiuretic and diuretic animals; the only exception being a decrease in the amount of glycogen in the collecting duct cells in the antidiuretic state and an increase in the diuretic.The light cells of the collecting ducts have a low electron density and show a paucity of organelles. These comprise mitochondria, Golgi apparatus, multivesicular bodies, sparse endoplasmic reticulum and free ribosomes. The centrally-placed, spherical nucleus demonstrates large numbers of nuclear pores. The lateral surfaces and bases of the cells have considerable infoldings which may have functional significance.The attenuated endothelial cells of the vasa recta are punctuated by fenestrations which are most frequently crossed by membrane. The cells contain micropinocytotic and pinocytotic vesicles.The loops of Henle in the papilla are lined by squamous cells which are extended longitudinally in the form of interdigitating processes. The bases of the cells of most loops are scalloped.The interstitial cells are embedded in an amorphous matrix containing occasional collagen fibres and strands of fibrillar material. The cells are irregular in outline and have moderately developed endoplasmic reticulum and Golgi apparatuses.Tight junctions between the cells of all collecting ducts, loops of Henle and vasa recta are a constant finding. All these tubular elements are surrounded by a prominent basement membrane; that associated with the loops of Henle tends to be multiplied, particularly at scalloped regions. The membrane associated with the vasa recta is single except at regions where it projects across the interstitium to the membranes of the collecting ducts and loops of Henle.The functional implications of these findings are discussed.     

Lysophospholipase activity in rabbit skeletal muscle

Skeletal muscle contains appreciable lysophospholipase activity which is differentiated by muscle type with red muscle subcellular fractions having greater activity than the corresponding white ones.     

Asynchronous synapse elimination in neonatal motor units: studies using GFP transgenic mice

In developing muscle, synapse elimination reduces the number of motor axons that innervate each postsynaptic cell. This loss of connections is thought to be a consequence of axon branch trimming. However, branch retraction has not been observed directly, and many questions remain, such as: do all motor axons retract branches, are eliminated branches withdrawn synchronously, and are withdrawing branches localized to particular regions? To address these questions, we used transgenic mice that express fluorescent proteins in small subsets of motor axons, providing a unique opportunity to reconstruct complete axonal arbors and identify all the postsynaptic targets. We found that, during early postnatal development, each motor axon loses terminal branches, but retracting branches withdraw asynchronously and without obvious spatial bias, suggesting that local interactions at each neuromuscular junction regulate synapse elimination.     

Competitive mechanisms underlying synapse elimination in the lumbrical muscle of the rat

The process of neuromuscular synapse elimination has been studied in the fourth deep lumbrical (4DL) muscle of the rat, a preparation which offers technical advantages for some types of experimental work. Studies have been performed both during development and in adult denervated muscles undergoing reinnervation. Results indicate that synapse elimination is dependent upon competition between motoneurons. Cellular mechanisms underlying this competition have also been explored. Both neuromuscular activity and muscle fiber type recognition appear to play a role, but positional cues appear unimportant in this small muscle.     

Ultrastructural alterations in skeletal muscle fibers of streptozotocin-diabetic rats

Summary The ultrastructure of fast-twitch-oxidative-glycolytic (FOG), fasttwitch-glycolytic (FG) and slow-twitch-oxidative (SO) fibers in plantaris and soleus muscles of normal and streptozotocin-diabetic rats was studied. In the diabetic animals, the mitochondria of FOG and SO fibers showed a loss of cristae and an increase in electron-dense granules. There was also an increased number of lipid droplets in close proximity to the mitochondria and the nuclei, and a separation of individual muscle nuclei to form satellite cells. Higher incidences of surface projections and sarcoplasmic splittings at the nuclear region were noticed in SO fibers. The FG fibers showed some disorientation of the T-tubular system. It is concluded that streptozotocin-diabetes has differential effects on the fine structure of the three fiber types of rat skeletal muscle.Supported by USPHS Grant AM 18280-04, Boston University Grant GRS-405-BI, and a grant-in-aid award from Sigma Xi Society     

Ultrastructural changes in skeletal muscle after fatiguing exercise.

Thoroughbred horses were exercised to fatigue at 40, 85, and 100% of their maximal oxygen consumption (VO2max) on a treadmill and completed a 1,600-m gallop on a track to identify the effect of exercise of various durations and intensities on the ultrastructure of mitochondria and sarcoplasmic reticulum (SR) from the middle gluteal muscle. The percentage of the total area occupied by mitochondria and SR increased in electron micrographs of muscle samples collected at the termination of exercise and at 30 and 60 min of recovery compared with those collected before exercise. Mitochondrial area increased 3- to 4-fold and SR area approximately 1.6-fold after exercise at the intensities greater than 40% of the VO2max. Smaller increases occurred in response to exercise at 40% of the VO2max. Areas were not different from rest in samples collected after 60 min of recovery. The reversal of ultrastructural alterations paralleled the trend toward normalization of muscle temperature, muscle pH, and the concentrations of selected muscle metabolites.     

Ultrastructural observations on rabbit blastocysts after maternal exposure to cadmium chloride

Cadmium chloride (CdCl2) (0.1 or 1.0%) was given in the drinking water to pregnant rabbits on Days 0 to 5 of gestation. Electron microscopy of the blastocysts revealed significant changes in the lysosomes (e.g. autophagic vacuoles and residual bodies) of endodermal cells as well as those of the inner cell mass. No such changes were evident in the trophoblast.     

Ultrastructural observations on FUdR-induced cell death and subsequent elimination of cell debris.

Twelve-day mouse embryos were treated with fluorodeoxyuridine (FUdR) and sacrificed at various time intervals after treatment. The neuroepithelial cells were then examined to determine by electron microscopy the primary site of action of the drug. About two hours after treatment mitotic activity ceased and a number of cells were found with a normal interphase nucleus, but with a cytoplasm in which the ribosomes had lost their normal polysomal configuration and were dispersed as single ribosomes. At about the same time cells were seen with an accumulation of chromatin at the nuclear membrane and the segregation of chromatin masses within the somewhat denser karyoplasm. Concurrent with the nuclear changes was the appearance of a condensed cytosome containing monoribosomes. The condensation of the nucleus and cytoplasm was followed by fragmentation of the cell into membrane bound bodies. Since condensed cells always contained monodispersed ribosomes, it seems likely that the dispersal of the ribosomes is the first morphological sign of the action of FUdR. Since about half of the neuroepithelial population underwent cell degeneration, the second goal of this experiment was to study the fate of the dying cells. Some fragments from condensed cells were found within apparently normal neuroepithelial cells, indicating phagocytosis. In addition macrophages were seen containing phagosomes from fragmented cells. Most of the fragments, however, remained free and were not immediately phagocytosed. These unphagocytosed fragments lysed and became ghosts, thereby giving the neureopithlium a vacuolated appearance. Hence, cellular debris was eliminated partially by neuroepithelial cells, partially by macrophages and to a great extent by lysis.     

Ultrastructural observations of isolated intact and fragmented junctions of skeletal muscle by use of tannic acid mordanting

Tannic acid mordanting during fixation of isolated vesicles from skeletal muscle enhanced the resolution of the images. Isolated triadic junctions displayed two characteristic features not previously described: (a) a clear gap separated terminal cisternae from transverse tubules; (b) this gap was bridged by a separating array of structures which resembled the "feet" of intact muscle. When the triad was broken in a French press and subsequently reassembled by joining the two organelles, a similar gap was seen but the structure of the feet was less well defined. When the membrane of the triad was extracted by Triton X-100, the junctional region was retained and a similar gap between the two organelles could be discerned. The terminal cisternae characteristically displayed a thickening of the cytoplasmic leaflet of the membrane in select areas in which electron-dense material was apposed on the luminal leaflet. This thickened membrane was not observed in longitudinal reticulum or in terminal cisternae regions distal to the electron-dense matter. This thickened leaflet was not invariably associated with the junction, and some junctional regions did not display discernible thickening of the membrane. When the triad was treated with KCl, the electron-dense aggregate was dispersed and the thickened leaflet of the terminal cisternae dissipated, whereas the triadic junctional region with its feet remained unchanged. KCl treatment caused dissolution of three proteins of Mr = 77,000, 43,000, and 38,000. Treatment of Triton-resistant vesicles with KCl caused the loss of electron-dense aggregate but did not otherwise influence the appearance of the junction. A good degree of correlation both qualitatively and in quantitative parameters between the isolated vesicles and the intact muscle was observed.     

Phosphorylation of rabbit skeletal muscle myosin in situ

Myosin light chain (P light chain) is phosphorylated by Ca2+ X calmodulin-dependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow-twitch soleus and fast-twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast-twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin-activated ATPase activity, may be related to isometric twitch potentiation.     

Static and kinetic studies on rabbit skeletal muscle troponin

Fluorescence titration curves of 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled troponin (IAANS-labeled Tn) and troponin-1-anilinonaphthalene-8-sulfonic acid (Tn-ANS) complex indicated that the fluorescent moiety, IAANS or ANS, detects conformational change of troponin I (TnI) or Tn due to the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of troponin C (TnC) component. A fluorescence stopped-flow study showed that the kinetic behavior of IAANS-labeled Tn reflects a change in state of the TnI component induced by the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of TnC component. The state change of TnI induced by the Ca2+ binding was complete within the instrumental dead time. On the other hand, that induced by the Ca2+ removal had a rate constant of around 13 s-1. ANS, which is noncovalently bound to Tn, reflects the kinetic properties of both the TnI component and the low affinity Ca2+-binding region of TnC component. The fluorescence intensity change of ANS induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC was complete within the instrumental dead time, while that induced by the Ca2+ removal from the same sites was biphasic. The rate constants of the biphasic process were found to be 62 +/- 7 s-1 and 16 +/- 4 s-1. The former value corresponds to the rate constant of the Ca2+ removal reaction from the low affinity Ca2+-binding sites of TnC component, and the latter value to the rate constant observed in the case of IAANS-labeled Tn. Based on these experimental results and on the discussion in our previous paper (Iio, T. & Kondo, H. (1981) J. Biochem. 90, 163-175), we have refined the two-way information-transfer mechanism which we previously proposed in order to explain the biological function of Tn.