Sumner I. Zacks

UPTAKE OF EXOGENOUS HORSERADISH PEROXIDASE BY COATED VESICLES IN MOUSE NEUROMUSCULAR JUNCTIONS

Following intramuscular injection of horseradish peroxidase into mouse skeletal muscle adjacent to areas of innervation, rapid uptake of the label was observed by coated but not synaptic vesicles. The tracer was also found in Schwann cells, external mesaxons and within peripheral myelin lamellae, but never in axoplasm of intramuscular nerves proximal to the injection site. The data suggest that the tracer is taken up by the coated vesicles and may be rapidly discharged into the synaptic clefts from which it is cleared by a combination of phagocytic activity and absorption via pinocytotic vesicles and intercellular clefts of adjacent capillaries or, more probably, enzyme activity is lost within the coated vesicles. The significance of these observations as related to neuromuscular function is discussed.

Fine structure of neuromuscular junctions after nerve section and implantation of nerve in denervated muscle

Abstract The first evidence of spontaneous reinnervation of neuromuscular junctions occurred 4 weeks after nerve section. In these experiments, the regenerating axons returned to old subneural apparatuses. There was little tendency to form new junctions as judged by light and electron microscopic studies. When the peroneal nerve was cut and reimplanted in the m. tibialis anterior at a new site distant from the original innervation band, a new innervation band developed near the point of entry of the transplanted nerve 6–8 weeks after operation. Electron microscopic study revealed atrophic subneural apparatuses with characteristic features of previous denervation and other junctions with fine structure resembling that observed in the embryonic morphogenesis of neuromuscular junctions. Similar patterns of neuromuscular junction reinnervation were observed in experiments in which after denervation, a foreign nerve was implanted into the muscle. Under these circumstances, an additional observation was the formation of abnormal tubular structures probably related to the regeneration of the transverse tubular system in atrophic muscle.

STRUCTURE AND STAINING CHARACTERISTICS OF MYOFIBER EXTERNAL LAMINA

A variety of cytochemical methods were used to investigate the composition of mouse sarcolemma. We found that the sarcolemma is a complex composed of the myofiber cell membrane, a thin zone external to the cell membrane that contains groups that bind colloidal iron and thorium at low pH and a thicker amorphous layer that fails to stain with colloidal iron and thorium at low pH. The entire complex is periodic acid-Schiff-positive and stains with ruthenium red and strongly acid solutions of phosphotungstic acid. Although the specificity of these cytochemical stains is controversial, data obtained with them and from preliminary analyses of myofiber external lamina (EL) indicate that the myofiber cell coat is chiefly composed of glycoprotein containing a large number of carboxyl groups. The EL within the subneural apparatus of the neuromuscular junction differs from noninnervated areas in the fusion of Schwann cell and myofiber EL, the absence of collagen microfibrils, the more intense binding of divalent cations and the less intense stain with phosphotungstic acid in strongly acid solutions.

Fine structure observations of denervation and reinnervation of neuromuscular junctions in mouse foot muscle.

The fine structure changes which occurred during denervation and reinnervation of neuromuscular junctions for a period of thirty weeks after nerve section were studied in muscles of the mouse foot. Axons were absent by the end of the first week after nerve section and there was retraction of Schwann cells and perineural epithelial cells. The primary synaptic cleft became shallow, the secondary clefts, shorter and wider, but the basement lamina filling the subneural apparatus was unaltered. The characteristic thickening of the muscle surface membrane in the primary synaptic cleft persisted, and there were increased numbers of clustered ribosomes. A loss of coated vesicles was observed in the sole-plate sarcoplasm. The earliest returning axon sprouts were first observed at three weeks and became numerous by four weeks. These originally contained few and variably sized synaptic vesicles but no coated vesicles. In the succeeding weeks the primary clefts deepened, the secondary clefts became elongated and narrower, and, by fifteen to thirty weeks, all neuromuscular junctions studied had normal fine structure. No fine-structure evidence of de novo formation of neuromuscular junctions was obtained by electron microscopy, although occasional small neuromuscular junctions formed by escaped fibers were observed in dual-stained sections studied in the light microscope. Persistence of tenuous processes of both Schwann and perineural epithelial cells in the immediate vicinity of denervated neuromuscular junctions appeared to play a major role in guiding returning axons to old sites of innervation.

CYTOCHEMICAL AND PHYSICAL PROPERTIES OF MYOFIBER EXTERNAL LAMINA

The structure and staining characteristics of isolated rat muscle sarcolemmal tubes were examined in the optical and electron microscopes with and without prior staining with ruthenium red. The sarcolemmal tubes, which chiefly consisted of a layer of external lamina with a few collagen microfilaments embedded in the surface and segments of cell membrane, were little affected by incubation with neuraminidase, hyaluronidase and collagenase. However, approximately 50% of the external lamina (EL) was extracted by 0.1-0.3 M lithium diiodosalicylate. Dialysis of the lithium diiodosalicylate extract yielded a fluffy, poorly soluble, thixotropic, acidic (pK 9.9) gel (external lamina substance) which had staining properties similar to the EL in tissue sections. External lamina substance has properties consistent with an acidic glycoprotein or proteoglycan.

Chemical composition of isolated rat skeletal sarcolemma

SummaryIsolated rat skeletal muscle sarcolemmal tubes contained 78.1% protein, 13.1% lipid and 3.3% neutral carbohydrate. Gas-liquid chromatography of the total sarcolemmal carbohydrates showed the presence of fucose (0.28%), mannose (0.44%), galactose (0.95%), neuraminic acid (0.31%) and larger quantities of glucose (1.58%), glucosamine (2.59%) and galactosamine (2.03%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed 30 to 35 protein bands including three major bands at 170,000, 140,000 and 44,000 mol wt. Amino acid analysis showed the sarcolemma to contain small amounts of proteins characteristic of collagen and relatively large amounts of acidic amino acids (21.5% glutamic and aspartic acids). Lithium 3,5-diiodo salicylate (LIS) extracted a fraction of the external lamina of the sarcolemma (designated ELS). The ELS was rich in glucuronic acid and hexosamines as well as having an increased proportion of acidic amino acids. The ELS contained no hydroxyproline and had less lipid than whole sarcolemma.

Studies on Tetanus ToxinI. Formation of Intramitochondrial Dense Granules in Mice Acutely Poisoned with Tetanus Toxin

When mice are acutely poisoned by either crude or purified tetanus toxin, a marked increase in number and a moderate increase in size of intramitochondrial dense granules occurs. This is most pronounced in skeletal muscle but also may be observed in brain and spinal cord. The formation of abnormal dense granules is prevented by prior protection of the experimental mice with tetanus antitoxin. Evidence, suggesting mitochondria as an important site of tetanus toxin action, is discussed.

The localization of botulinum A toxin in the mouse.

Abstract Crystalline botulinum A toxin was labelled with fluorescein or rhodamine labels under conditions of high and low ionic strength buffers without loss of toxicity. In vitro binding to various mouse tissues was studied by darkfield fluorescence microscopy. Binding was almost entirely confined to structures in skeletal muscle consistent in size, shape and location with motor endplates. This localization was confirmed by localization of unlabelled botulinum toxin by means of fluorescein-labelled antitoxin. Appropriate control preparations confirmed the selectivity of toxin binding.