From previous work, it appears that synaptic transmission is well preserved at aging mouse neuromuscular junctions despite profound ultrastructural changes. Scanning and light microscopy have been used to determine whether expansion or sprouting of nerve terminals or postsynaptic reorganization play a role in this apparent compensatory mechanism. The number and length of nerve terminal branches in the extensor digitorum longus of young (7 months) and old (29 months) mice were studied with a combined silver-cholinesterase method. In aged animals, there were increases in nerve terminal length and number of intrasynaptic branches, with no change in muscle fibre diameter or numbers of axons entering the junction. Neither collateral sprouting nor collateral innervation, hallmarks of partial denervation, were present. Motor endplates visualized by scanning electron microscopy appeared as slightly elevated, elliptical plateaux ('raised areas') with smooth surfaces into which the synaptic clefts were etched. In the aged endplates more than in young endplates, the primary clefts were often interrupted by narrow short outpouchings approximately perpendicular to the long axis of the primary cleft. In addition, oval primary cleft islets were more frequent and there was increased randomness and branching of secondary clefts. Both light and scanning microscopy gave concordant quantitative evidence that nerve terminals and the underlying postsynaptic cleft are longer and more branched in aged mice. The increased length of synaptic nerve terminal approximately balances the loss of girth previously reported leaving nerve terminal volume unchanged. The observed expansion of the synaptic area in the aged neuromuscular junction may be compensatory, preserving neuromuscular function. The data also point to plasticity of adult neuromuscular synaptic structure.
The early development of the circumferential axonal pathway in the brachial and lumbar spinal cord of mouse and chick embryos was studied by scanning and transmission electron microscopy. The cellular processes which comprise this pathway grow in the transverse plane and along the lateral margin of the marginal zone (i.e., circumferentially oriented), as typified by the early embryonic commissural axons. The first formative event observed was in the ventrolateral margin of the primitive spinal cord ventricular zone. Cellular processes were found near the external limiting membrane that appeared to grow a variable distance either dorsally or ventrally. Later in development, presumptive motor column neurons migrated into the ventrolateral region, distal to these early circumferentially oriented processes. Concurrently, other circumferentially oriented perikarya and processes appeared along the dorsolateral margin. Due to their aligned sites of origin and parallel growth, the circumferential processes formed a more or less continuous line or pathway, which in about 10% of the scanned specimens could be followed along the entire lateral margin of the embryonic spinal cord. Several specimens later in development had two sets of aligned circumferential processes in the ventral region. Large numbers of circumferential axons were then found to follow the preformed pathway by fasciculation, after the primitive motor column had become established. Since the earliest circumferential processes appeared to differentiate into axons and were found nearly 24 hours prior to growth of most circumferential axons, their role in guidance as pioneering axons was suggested.
The monoclonal antibody HNK-1 recognizes a carbohydrate epitope present on a host of glycoconjugates which include the glycoproteins neural cell adhesion molecules (N-CAM) myelin-associated glycoprotein and ependymins, and two glycolipids. Other antibodies, including NC-1, L2 and IgM paraproteins from neuropathy patients share similar binding specificity to the same or related sulfated glucuronyl-containing antigen (SGA). To further investigate its possible significance in early development, the distribution of SGA was studied in the head region of early developing chick (S13-S18) and mouse (E8.5-E11.5) embryos by immunocytochemistry. A striking species difference was found in the apparent distribution of immunodetectable SGA. In chick, migrating neural crest cells and their related cell types were heavily stained by HNK-1; whereas no stain was detectable on mouse neural crest cells at comparable stages, except perhaps in a restricted area adjacent to the otic placode and immediately adjacent to the neural tube. Within the developing CNS, the distribution of SGA was similar in both species. It was first expressed on neuroepithelial cells prior to axonal outgrowth, and was distributed in a continuous zone along the entire lateral walls of the early neural tube. Little or no SGA was detectable along most of the floor and roof plates. SGA appeared during this same period in the lateral basal lamina and within the adjacent mesenchyme and nearby cells. SGA was particularly evident on neuroepithelial endfeet at this stage. Early developing longitudinal axons were subsequently found to grow in association with the endfeet of SGA-positive neuroepithelial cells. These findings, in conjunction with previous studies, suggest that SGA is associated as a marker, and perhaps functionally, with the organization of early neuronal settling and axonal growth patterns within the developing vertebrate CNS.
The surface morphology of the microvasculature from mouse skeletal muscle was studied by scanning electron microscopy. Cell surfaces were exposed by digesting away extracellular collagen and other matrix by a simple HCl treatment. Four distinct subdivisions of the microvasculature (arterioles, precapillary arterioles, capillaries, and venules) were identified based on marked differences in surface features. Arterioles of 20-10 micrometers diameter had a discontinuous, single layer of smooth muscle cells encircling the vessel. These smooth muscle cells had an uneven surface with shallow grooves and depressions that were often oriented parallel to the longitudinal cell body axis. The underlying arteriolar endothelial surface was also rough with long ridges separating shallow furrows that were oriented parallel to the vessel length. As the arteriolar size decreased, the perivascular cell were found further apart, they became smooth surfaced, and were oriented preferentially parallel to the vessel. The endothelium of the precapillary arterioles, as well as, capillaries and venules had smooth surfaces. Venules had a discontinuous layer of flat, smooth surfaced pericytes. Morphologically distinct groups of smooth muscle cells (i.e., precapillary sphincters) were not found. Although pericytes normally associated with capillaries and other vessels were often removed during tissue processing, most cells and their surface feature were generally well preserved.
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