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 smooth muscle tumors

The ultrastructure of a series of primary and metastatic smooth muscle tumors is reviewed. Myofilaments and other smooth muscle features were present in all primary leiomyosarcomas of the soft tissues and uterus. They were also present but were less plentiful in most of the metastatic leiomyosarcomas. Electron microscopy is therefore a useful method to establish the diagnosis of a suspected leiomyosarcoma. Stromal tumors of the gastro-intestinal tract may require correlated immunocytochemical and ultrastructural studies for their identification: 9 of the 50 cases examined were positive with immunostaining for S-100 protein, and 16 tumors with epithelioid transformation did not show evidence of smooth muscle differentiation by electron microscopy.     

Ultrastructural observations on muscle spindles in extraocular muscles of pig

Human and some mammals such as the sheep, goat and domestic and wild pigs have more or less muscle spindles in the extrinsic eye muscles, especially the domestic pigs having abundant muscle spindles (Matsuyama, 1987). The muscle spindles play a large role in maintaining the stable visual posture of the eyeballs. To define the morphological properties of the muscle spindles relative to the eye movement, the ultrastructure of the spindles was investigated in 6 extraocular muscles of the pigs by electron microscopy. The muscle spindles in the pig extraocular muscles consist of 4 to 5 intrafusal muscle fibers, one of which is nuclear bag fiber and 3 to 4 are nuclear chain fibers. The outer capsule is thin, composing of few layers, and the inner capsule ramifying to enwrap the individual fiber, accompanied by the medullated and unmedullated nerve fibers and blood capillaries. The nuclear bag fiber, 14 micron in diameter, is innervated by the atypical annulospiral sensory terminals and the chain fiber by the typical annulospiral terminal packed with mitochondria and microvesicles. The intrafusal fibers are innervated by the flower-spray sensory terminals anchoring deeply into the sarcoplasma, having abundant neurotubules and few mitochondria. The gamma motor end-plates have a relatively smooth synaptic cleft with a width of 70 nm and synaptic boutons containing few synaptic vesicles, sometimes, revealing a shallow fold of postsynaptic sarcolemma and abundant synaptic vesicles. The alpha motor end-plates reveal a relatively smooth synaptic cleft with a width of 80 nm, sometimes with a rough postsynaptic infolding, and boutons containing few synaptic vesicles and small-sized mitochondria. The satellite cells are innervated by the sensory terminals in various ways. The muscle spindles in the pig extraocular muscles are found to be much simpler in structure than those in the other antigravity muscles of the body. Their ultrastructure seems to reflect the morphological adaptation relative to the eyebal movement.     

Development of neuromuscular synapse in the skeletal muscle after chemical desympathization

The purpose of this investigation was the study of cholinesterase (ChE)-positive zones of neuromuscular synapse (NMS) in gastrocnemius and plantaris muscles of albino rats after guanethidine sympathectomy. Qualitative and morphometric characteristics of ChE-positive zones were studied, which were demonstrated using a modified thioacetic acid method in 72 control and experimental animals aged from 5 days to 3 months. It was found that in both gastrocnemius and plantaris muscles the growth of muscle fiber diameter and section area in the region of NMS was delayed after desympathization. In gastrocnemius muscle, smaller fiber diameter persisited up to the age of 3 months, while in plantaris muscle it was larger than in control animals of the same age. The differences in dimensional parameters of NMS sections were leveled off by the age of 3 months because of their accelerated growth. Desympathization resulted in the premature appearance of dystrophic changes in MNS region as compared to those developing in control animals, in which they are the features of involution processes.     

A note on the elimination of polyneuronal innervation of skeletal muscles in neonatal rats

The elimination of the polyneuronal innervation was reinvestigated in normal rats from the tenth day after birth. The extent of polyneuronal innervation was measured by intracellular recordings in cut muscle fibre preparations (Barstad 1962). This preparation has the advantage that it favours the recording of small signals. With this increased sensitivity we detected a class of small end-plate potentials which may represent nerve terminal function in the course of elimination. These small potentials were always observed in fibres with an additional large end-plate potential and were occassionally present even in one-month old rats.     

Ultrastructural observations on rabbit,hamster and mouse eggs following electrical stimulation in vitro

The fine structural changes were studied in the plasma membrane, cortical granules (CGs) and meiotic spindle of rabbit, hamster and mouse eggs in response to electrical stimulation. Eggs were collected 16 to 18 hours after HCG injection, and freed from the cumulus oophorus. They were stimulated in vitro by delivering a single monophasic square wave pulse of 150 V for 1 msec. Stimulated and unstimulated eggs were fixed for fine structural observations 1, 30 and 60 minutes after stimulation. Within one minute of stimulation the microvilli of rabbit eggs were long, branching and had bulbous ends. Umbonate protrusions were also present on their surface. By 30 minutes after stimulation the rabbit eggs lacked microvilli and the perivitelline space was filled with detached vesicles. Degenerating changes were readily noticeable in the microvilli of hamster eggs by 60 minutes. Changes were not noted in the microvilli of stimulated mouse eggs. There were markedly fewer CGs in the hamster and mouse, but not rabbit, eggs by 30 minutes after stimulation, and nearly all of them disappeared in the mouse and hamster eggs by 60 minutes. The density of the CGs in the rabbit was not altered. The meiotic spindle of hamster eggs dissipated within one minute of stimulation, however, the microtubules reappeared by 60 minutes after stimulation. Rotation of the meiotic spindle occurred in the mouse and hamster eggs by 30 minutes after stimulation. In some of the mouse eggs the spindle migrated to the center and the chromosomes were in telophase.     

Ultrastructural observations on gynecomastia

Ten cases of gynecomastia were studied by electron microscopy. The ducts showed proliferation of both epithelial and myoepithelial cells. Intracytoplasmic lumina, previously thought to be a feature of malignant breast lesions, were seen. Squamous metaplasia was observed in some cases. The stroma showed fibroblasts, myofibroblasts, and, occasionally, pericytes. The general morphology of gynecomastia is similar to that of benign lesions of the female breast at the ultrastructural level. The features of the stromal cells reflect the effects of estrogenic stimulation.     

Ultrastructural alterations in skeletal muscle of pigs with acute monensin myotoxicosis

Large doses of monensin, a Na+-selective carboxylic ionophore, produce polyfocal, monophasic necrosis of skeletal muscle, with Type I fiber selectivity, in swine. For a study of the sequential ultrastructural alterations in affected skeletal muscles, 14 weanling pigs were given 40 mg monensin/kg body weight and were euthanatized 1, 2, 4, 8, and 16 days later. Myotoxicosis and myoglobinuria were apparent clinically. At necropsy, white, dry areas of necrosis were present in the muscle masses of the anterior and posterior thigh, shoulder, and loin. Two patterns of skeletal muscle necrosis were observed on Day 1, especially in Type I fibers. In fibers exhibiting the first of these patterns, the contractile material was disrupted, forming dense amorphous and filamentous clumps scattered within the persistent sheaths of external lamina (sarcolemmal tubes); the mitochondria were swollen and contained flocculent matrix densities, and the nuclei were pyknotic. Fibers showing the second pattern were uniformly dense, but their sarcoplasm was not disrupted. Sublethally injured fibers were also observed and showed focal myofibrillar lysis. On Days 2 and 4, the necrotic muscle had marked infiltration of macrophages in the interstitium and within sarcolemmal tubes. Rapid resolution of the fiber necrosis occurred by phagocytosis of the sarcoplasmic debris. Regeneration of affected muscles developed early following injury and progressed rapidly to complete restoration of the necrotic muscles without residual fibrosis. Regeneration was initiated on Day 1 by activation of satellite cells to form presumptive myoblasts; on Days 4 and 8 these cells showed evidence of fusion, forming myotubes to restore the necrotic fibers.     

Ultrastructural localization of calcium in normal and abnormal skeletal muscle.

Calcium was demonstrated ultrastructurally as a fine black reaction product with unbuffered 2% saturated potassium pyroantimonate, pH 9.4. In comparison with normal muscle, there was increased precipitate in degenerating skeletal muscle fibers and some degenerating-regenerating fibers occurring in pathologic human muscle, regardless of disease entity, and in experimentally injured rat muscle. The pathologically increased calcium was mainly within the sarcoplasmic reticulum and mitochondria. Both structures could be completely blackened. Nuclear calcium was also increased, the precipitates being localized as circular profiles within the nucleoli and heterochromatin as well as being associated with the nuclear envelope. Myofibrillar calcium was only modestly increased. When normal rat muscle was preincubated in 136 mM calcium-enhanced Hanks' medium, calcium accumulated in the muscle fibers--it was especially heavy in the mitochondria and sarcoplasmic reticulum and appeared identical with the pathologic human and rat muscle fibers. Preincubation of normal rat muscle in 0.1 M acetate buffer (pH 4.65) before calcium loading augmented myofibrillar staining, mainly in the H-zone of the A-bands excluding the M-zone and in broad irregular N1, N2, and "N3" lines of the I-bands. EMMA-4 electron probe microanalysis and EGTA (ethylene glycolbis (beta-aminoethyl ether)N,N'-tetraacetic acid) chelation prior to staining confirmed that the precipitate in the several loci was calcium antimonate. It is proposed that in skeletal muscle fibers injured by various pathologic processes, a breach of the plasmalemma barrier to calcium occurs as a very early abnormality. Extracellular calcium would then pour into the aqueous sarcoplasm of the muscle fiber, from which it would be withdrawn by and accumulated with the still active organelles normally having a great avidity for uptake of this ion, especially the mitochondria and sarcoplasmic reticulum. The resultant organellar calcification would impair function and damage the structure of proteins and phospholipids.     

Ultrastructural features of Adriamycin-induced skeletal and cardiac muscle toxicity.

In this study, the authors examined the effect of the anti-tumor agent Adriamycin, a known cardiotoxin, on mouse heart, diaphragm, and gastrocnemius muscle. Using an established model of Adriamycin cardiac toxicity, they found that 4 days after the intraperitoneal injection of 20 mg/kg of Adriamycin, characteristic heart lesions, including vacuolation of the sarcoplasmic reticulum, interstitial edema, and mitochondrial degeneration, were demonstrated in all treated animals. Furthermore, similar, but much more severe, myocyte damage was demonstrated in the diaphragm; muscle toxicity followed a decreasing gradient of injury from the peritoneal to the thoracic surface of the tissue. On the other hand, treatment with Adriamycin resulted in an increase in the size and number of lipid droplets in the red fibers of the gastrocnemius muscle without any other ultrastructural evidence of drug-induced damage to myocytes. An examination of the pharmacokinetics and metabolism of Adriamycin after intraperitoneal treatment revealed that relative drug levels in muscle (diaphragm much greater than heart much greater than gastrocnemius) paralleled the degree of ultrastructural damage observed. This study indicates that treatment with Adriamycin can produce significant injury to non-cardiac muscle in a fashion that strongly resembles the characteristic pattern of Adriamycin-related damage to the heart, and that the degree of myocyte damage is apparently dependent upon the Adriamycin concentration in the tissue.     

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1–50 M, ryanodine slightly but significantly increased the submaximal Ca²⁺-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine 1 M decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and activity-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 M and 5 M for SL and AM, respectively, following ryanodine administration in the release phase, and 100 M and 30 M, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca²⁺ underlies the contracture caused by this agent in intact skeletal muscles. The delayed decreased Ca²⁺ efflux by caffeine, as evidenced by depression of tension transient with no change in the calcium content may be responsible for the decreased twitch tension caused by this agent.