Basal lamina components are concentrated in premuscle masses and at early acetylcholine receptor clusters in chick embryo hindlimb muscles.

As an initial step in characterizing the function of basal lamina components during muscle cell differentiation and innervation in vivo, we have determined immunohistochemically the pattern of expression of three components--laminin, proteins related to agrin (an acetylcholine receptor (AChR)-aggregating protein), and a heparan sulfate proteoglycan--during the development of chick embryo hindlimb muscles. Monoclonal antibodies against agrin were used to purify the protein from the Torpedo ray and to characterize agrin-like proteins from embryonic and adult chicken. In early hindlimb buds (stage 19), antibodies against laminin and agrin stained the ectodermal basement membrane and bound to limb mesenchyme with a generalized, punctate distribution. However, as dorsal and ventral premuscle masses condensed (stage 22-23), mesenchymal immunoreactivity for laminin and agrin-like proteins, but not the proteoglycan, became concentrated in these myogenic regions. Significantly, the preferential accumulation of these molecules in myogenic regions of the limb preceded by 1-2 days the appearance of muscle-specific proteins, myoblast fusion, and muscle innervation. All three basal lamina components were preferentially associated with all AChR clusters from the time we first observed them on newly formed myotubes at stage 26. Localization of these antigens in three-dimensional collagen gel cultures of limb mesenchyme, explanted prior to innervation of the limb, paralleled the staining patterns seen during limb development in the embryo. These results indicate that basal lamina molecules intrinsic to limb mesenchyme are early markers for myogenic and synaptic differentiation, and suggest that these components play important roles during the initial phases of myogenesis and synaptogenesis.

The regulation of intramuscular nerve branching during normal development and following activity blockade.

In vertebrates, approximately 50% of the lumbosacral motoneurons die during a short period of development that coincides with synaptogenesis in the limb. Although it has been postulated that these motoneurons die because they fail to obtain adequate trophic support from the muscles, it is not clear how this factor is supplied. The mechanism by which activity blockade prevents motoneurons cell death is also unknown. In order to begin to understand the nature of these proposed trophic interactions, we have examined the temporal sequence of axonal invasion and ramification within two muscles of the chick hindlimb, the predominantly slow iliofibularis and the fast posterior iliotibialis, during the cell death period. We found striking differences in intramuscular nerve ingrowth and branching between fast and slow muscle. We also observed differences in the molecular composition of fast and slow myotubes that may contribute to the nerve pattern differences. In addition, we observed a progressive increase in the degree of intramuscular nerve fasciculation as well as a precise temporal sequence of nerve branching. The earliest detectable response to chronic curarization was a dramatic decrease in the degree of intramuscular nerve fasciculation. Activity blockade also greatly enhanced nerve branching within the muscles from the time that nerve branches normally formed, and, additionally, interfered with the normal cessation of axon growth. Our results support the idea that nerve endings are the sites of trophic uptake. Furthermore, although our results do not allow us to exclude other activity-dependent influences on motoneuron survival, they suggest the following testable hypotheses: (1) the normal regulation of motoneuron survival may result from the precise control of intramuscular nerve branching, (2) activity blockade may increase motoneuron survival by enhancing intramuscular nerve branching, and (3) anything which affects this complex process of nerve branching may also alter motoneuron survival.

[Synaptogenesis and axon collaterals coming from the white matter in the upper cervical cord of the 10 day (stage 36) chick embryo--Golgi and electron microscopic studies].

Our previous study with 3H-thymidine autoradiography showed that neurons of the zona spongiosa, the nucleus proprius of the dorsal horn, the zona intermedia and the ventralhorn differentiated earlier than those of the substantia gelatinosa and the neck and the base of the dorsal horn, and that neurons of the substantia gelatinosa which were the last to differentiate reached their final position at stage 36 (Fig. 1). In the upper cervical cord of chick embryos at stage 36 when all spinal neurons finished cell migration and the cytoarchitecture similar to that of the cat spinal cord (Rexed, 1952) could be recognized (cf. Figs. 1, 3B), we studied the distribution of synapses by the electron microphotomontage (Fig. 3 A) and the morphology of axon collaterals coming from the white matter by the Golgi method (Fig. 4), in order to examine i) which spinal neurons have synaptic contacts at this stage and ii) what part of the axon collateral makes synaptic contacts. In the white matter, synapses were numerous around the gray matter and they were few in the peripheral part along the external surface of the cord. The paucity of synapses in the peripheral part was explained by a finding that dendrites reaching the external surface of the cord were few in number at this stage (cf. Fig. 3 C). In the gray matter, synapses were more numerous and denser in the zona intermedia and the ventral horn than in the dorsal horn.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased motor neuron projection during development does not increase the number of neuromuscular synapses.

We investigated in developing embryos whether the total number of neuromuscular synapses is determined by the muscle or by the number of innervating motor neurons. The superior oblique muscle of duck embryos was hyperinnervated by preventing the naturally occurring death of trochlear motor neurons using immunoglobulin G from patients with acquired myasthenia gravis. In spite of a significant increase in the number of motor neurons innervating the muscle, a corresponding increase in the number of neuromuscular synapses did not occur. These results suggest that the total number of synapses in a muscle is independent of the number of innervating motor neurons and that it is determined intrinsically by the muscle itself.

Aggregating factor from Torpedo electric organ induces patches containing acetylcholine receptors, acetylcholinesterase, and butyrylcholinesterase on cultured myotubes.

A factor in extracts of the electric organ of Torpedo californica causes the formation of clusters of acetylcholine receptors (AChRs) and aggregates of acetylcholinesterase (AChE) on myotubes in culture. In vivo, AChRs and AChE accumulate at the same locations on myofibers, as components of the postsynaptic apparatus at neuromuscular junctions. The aim of this study was to compare the distribution of AChRs, AChE, and butyrylcholinesterase (BuChE), a third component of the postsynaptic apparatus, on control and extract-treated myotubes. Electric organ extracts induced the formation of patches that contained high concentrations of all three molecules. The extract-induced aggregation of AChRs, AChE, and BuChE occurred in defined medium, and these components accumulated in patches simultaneously. Three lines of evidence indicate that a single factor in the extracts induced the aggregation of all three components: the dose dependence for the formation of patches of AChRs was the same as that for patches of AChE and BuChE; the AChE- and BuChE-aggregating activities co-purified with the AChR-aggregating activity; and all three aggregating activities were immunoprecipitated at the same titer by a monoclonal antibody against the AChR-aggregating factor. We have shown previously that this monoclonal antibody binds to molecules concentrated in the synaptic cleft at neuromuscular junctions. Taken together, these results suggest that during development and regeneration of myofibers in vivo, the accumulation at synaptic sites of at least three components of the postsynaptic apparatus, AChRs, AChE, and BuChE, are all triggered by the same molecule, a molecule similar if not identical to the electric organ aggregating factor.

Synaptic development in the human fetus: a morphometric analysis of normal and Down's syndrome neocortex.

Postmortem tissue was obtained from six normal and four Down's syndrome brains ranging in age from 12 to 40 weeks postconception. Tissue was processed for electron microscopy using routine osmium and EPTA staining procedures, and to examine synaptic development, photomicrographs were systematically taken throughout the molecular layer of the sensorimotor neocortex. The number of EPTA-stained synapses were consistently greater than the number of osmium-stained synaptic contacts. A progressive increase in synaptic density throughout the range of ages examined was observed for both normal and Down's syndrome tissue. There was also an increase with developmental age in apparent measures of synaptic maturity, e.g., an increased ratio of mature to primitive contacts and asymmetrical to symmetrical contacts. In normal tissue, pre- and postsynaptic membrane lengths were observed to increase with the ages studied, whereas synaptic membrane widths appeared to attain mature values by 29 weeks postconception. Cleft width remained fairly constant to 28 weeks postconception. Although direct statistical comparisons could not be made, in Down's tissue synaptic parameter development was generally less consistent and the parameters appeared to be reduced during the later stages of development studied.

Ontogenesis of the myenteric plexus in the cat gastrointestinal sphincters. III. Synaptogenesis.

Synaptogenesis in the cat myenteric ganglia of the lower esophageal, pyloric and ileocecal sphincters in the fetal life and at different postnatal stages of development was examined. The synaptic formation began in the early fetal period. The first immature synapses were of axosomatic and axodendritic types. The new formation and maturation of the synaptic contacts was an active process in the fetal period and in the first postnatal weeks, but it continued to the adult level. It was expressed quantitatively in the increase of the surface area of the synaptic contact zones, the number of the synapses, the length of the synaptic contact zones and the number of the synaptic vesicles. Fine structural features of the synaptic maturation were the increased pleomorphism of the synaptic vesicles, widing and lengthening of the thickening of the opposed membranes, the appearance of the coated evaginations and vesicles. Synaptic morphology became more complex because of the including of different in size and structure pre- and postsynaptic components. The frequency of the synapses on dendritic spines and synapses "en passant" increased. The synaptogenesis in the three sphincters studied exhibited a different dynamics. The parallel advance in the neuronal and synaptic differentiation and the relationship between the desmosomal junctions and synapses were discussed.

On the development of the cerebellum of the trout, Salmo gairdneri. IV. Development of the pattern of connectivity.

Synaptogenesis has been studied in the corpus cerebelli of the trout Salmo gairdneri, Richardson, 1836. The first synapses are observed in hatchlings and occur between parallel fibres and the shafts of Purkinje dendrites. Subsequently the axosomatic synapses of Purkinje axon collaterals on the neurons of the ganglionic layer appear, and finally the synapses made by climbing fibres and mossy fibres, and by stellate cell axons develop. Young synapses in the cerebellum of the trout resemble the mature structures so closely that the criteria for the identification of the latter can also be applied to the former. The number of parallel fibre synapses and of Purkinje axon collateral synapses increases considerably during development. Eurydendroid cells, the axons of which leave the cerebellum, receive an abundance of Purkinje axon collaterals on their somata and main dendritic trunks. Mossy fibre synapses are numerous in the granular layer. Climbing fibre contacts and synapses of stellate cell axons, both with Purkinje cells, are found occasionally. The following pattern of connectivity is proposed. The main input-output system is formed by the mossy fibres, the granule cells, the Purkinje cells and the eurydendroid cells. Additional pathways are formed by (1) the mossy fibres, granule cells and eurydendroid cells, and (2) the climbing fibres, Purkinje cells and eurydendroid cells. The afferent-efferent systems, mentioned above, are influenced by a number of internuncial elements: (1) The Golgi cells receive their input from the parallel fibres and contact with their axon collaterals the dendrites of granule cells. (2) Axon collaterals of Purkinje cells are in synaptic relation with Golgi cells. (3) Axon collaterals of Purkinje cells impinge upon the somata and main dendrites of other Purkinje cells. (4) Stellate cells, which derive their input from the parallel fibres, synapse with dendrites and somata of Purkinje cells. The possible functional roles of all of these neuronal elements are discussed.