Uel J. McMahan

The architecture of active zone material at the frog's neuromuscular junction

Active zone material at the nervous systems synapses is situated next to synaptic vesicles that are docked at the presynaptic plasma membrane, and calcium channels that are anchored in the membrane. Here we use electron microscope tomography to show the arrangement and associations of structural components of this compact organelle at a model synapse, the frogs neuromuscular junction. Our findings indicate that the active zone material helps to dock the vesicles and anchor the channels, and that its architecture provides both a particular spatial relationship and a structural linkage between them. The structural linkage may include proteins that mediate the calcium-triggered exocytosis of neurotransmitter by the synaptic vesicles during synaptic transmission.

The agrin gene codes for a family of basal lamina proteins that differ in function and distribution.

We isolated two cDNAs that encode isoforms of agrin, the basal lamina protein that mediates the motor neuron-induced aggregation of acetylcholine receptors on muscle fibers at the neuromuscular junction. Both proteins are the result of alternative splicing of the product of the agrin gene, but unlike agrin, they are inactive in standard acetylcholine receptor aggregation assays. They lack one (agrin-related protein 1) or two (agrin-related protein 2) regions in agrin that are required for its activity. Expression studies provide evidence that both proteins are present in the nervous system and muscle and that, in muscle, myofibers and Schwann cells synthesize the agrin-related proteins while the axon terminals of motor neurons are the sole source of agrin.

The Shapes of Sensory and Motor Neurones and the Distribution of their Synapses in Ganglia of the Leech: A Study Using Intracellular Injection of Horseradish Peroxidase

Three types of sensory neurones and two kinds of motor neurones in the segmental ganglion of the leech were examined with the light and electron microscope after intracellular injection of horseradish peroxidase (HRP) for a histological marker. The aim was to develop a method for identifying the synapses of specific cells in the ganglion’s complex neuropil and to form a picture of their distribution and structure. Reaction of HRP with different benzidine derivatives produces opaque and electron dense deposits. For light microscopy a blue stain is formed that makes processes visible in whole mounts millimeters away from the injection site at the soma. The reaction product for electron microscopy is distributed throughout the cytoplasm, yet ultrastructural details are preserved. The sensory neurones that respond specifically to touch or pressure or noxious mechanical stimuli to the skin share in their branching pattern a number of common features. A single process arising from each cell body forms large primary branches that pass through the neuropil and leave the ganglion by the ipsilateral connectives and roots. Within the neuropil these branches give rise to numerous smaller secondary processes. In contrast, the annulus erector and large longitudinal motoneurones send their main process across the ganglion to bifurcate and enter the contralateral roots. Secondary processes of the motoneurones are highly branched and more numerous than those of the sensory cells. Each type of sensory and motor cell is distinguished by the shape, length and distribution of its secondary processes. Secondary processes of sensory neurones exhibit numerous swellings and irregularly shaped fingers. Electron micrographs show that the sensory neurones make synapses at these specializations, each of which contacts several postsynaptic processes. The sensory neurones receive inputs at the same fingers and swellings, an arrangement suggesting that regions within a cell’s arborization may function semi-autonomously. The main process and large branches of the two motor neurones are studded with spines a few micrometres long and a fraction of a micrometre in diameter. Vesicle-containing varicosities from other cells make synaptic contact primarily with the spines, which themselves have few vesicles. These two motor neurones are largely, if not entirely, postsynaptic to other neurones within the leech nervous system.

Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions

To test the hypothesis that agrin mediates motor neuron-induced aggregation of acetylcholine receptors (AChRs) in skeletal muscle fibers and to determine whether the agrin active in this process is released by motor neurons, we raised polyclonal antibodies to purified ray agrin that blocked its receptor aggregating activity. When the antibodies were applied to chick motor neuron--chick myotube cocultures, they inhibited the formation of AChR aggregates at and near neuromuscular contacts, demonstrating that agrin plays a role in the induction of the aggregates. Rat motor neurons, like chick motor neurons, induce AChR aggregates on chick myotubes. This effect was not inhibited by our antibodies, indicating that, although the antibodies inhibited the activity of chick agrin, they did not have a similar effect on rat agrin. We conclude that agrin released by rat motor neurons induced the chick myotubes to aggregate AChRs.

cDNA that encodes active agrin

Agrin is thought to mediate the motor neuron-induced aggregation of AChRs and AChE on the surface of muscle fibers at neuromuscular junctions. We have isolated a cDNA from a chick brain library that, based on sequence homology and expression experiments, codes for active agrin. Examination of the sequence reveals considerable similarity to homologous cDNAs previously isolated from ray and rat libraries. A conspicuous difference is an insertion of 33 bp in chick agrin cDNA, which endows the encoded protein with AChR/AChE aggregating activity. Homologous transcripts having the 33 bp insertion were detected in the ray CNS, which indicates that an insertion of similar size is conserved in agrin in many, if not all, vertebrate species. Results of in situ hybridization studies and PCR experiments on mRNA isolated from motor neuron-enriched fractions of the spinal cord indicate that, consistent with the agrin hypothesis, motor neurons contain transcripts that code for active agrin.

Precision of reinnervation of original postsynaptic sites in frog muscle after a nerve crush

SummaryRegenerating neuromuscular junctions in the cutaneous pectoris muscle of the frog were examined by light and electron microscopy up to three months after crushing the motor nerve. The aim was to determine the precision of reinnervation of the original synaptic sites. More than 95% of the original postsynaptic membrane is recovered by nerve terminals and little, if any, synaptic contact is made on other portions of the muscle fibre surface. Even after prolonged denervation when the Schwann cells have retracted from 70–80% of the postsynaptic membrane, regenerating terminals return to and cover a large fraction of it. Although synapses are confined to the original synaptic sites, the pattern of innervation of muscle fibres is altered in several ways: (a) regenerating axon terminals can fail to branch leaving small stretches of postsynaptic membrane uncovered; (b) two terminal branches can lie side by side over a stretch of postsynaptic membrane normally occupied by one terminal; and (c) after growing along a stretch of postsynaptic membrane on one muscle fibre, terminals can leave it to end either in extracellular space or on the postsynaptic membrane of another fibre. Altogether the results demonstrate a strong and specific affinity between the original synaptic sites and regenerating nerve terminals.

Agrin-Induced Postsynaptic-like Apparatus in Skeletal Muscle Fibersin Vivo

We find that when extrajunctional regions of denervated soleus muscles in adult rats are transfected with cDNA encoding rat agrin isoform Y4Z8, which is normally secreted by motor neurons at adult neuromuscular junctions, the myofibers express and secrete the neural agrin. Muscle fibers in the vicinity of transfection form at their surface specialized areas having extracellular, plasma membrane, and cytoplasmic protein aggregates, narrow and deep plasma membrane infoldings, and an accumulation of myonuclei, all of which are characteristic of the postsynaptic apparatus at neuromuscular junctions. We conclude that at ectopic neuromuscular junctions that form in the extrajunctional region of denervated adult soleus muscles after implantation of a foreign nerve, a single neural-derived factor, agrin, is sufficient not only to cause protein aggregation in the early stages of postsynaptic apparatus formation, as predicted by the agrin hypothesis, but also to bring about changes in conformation of the muscle fiber surface and distribution of organelles which appear as the apparatus reaches maturity.

Agrin isoforms and their role in synaptogenesis

Agrin is thought to mediate the motor neuron-induced aggregation of synaptic proteins on the surface of muscle fibers at neuromuscular junctions. Recent experiments provide direct evidence in support of this hypothesis, reveal the nature of agrin immunoreactivity at sites other than neuromuscular junctions, and have resulted in findings that are consistent with the possibility that agrin plays a role in synaptogenesis throughout the nervous system.

Fine structure of synapses in the dorsal nucleus of the lateral geniculate body of normal and blinded rats

SummaryDegenerating boutons, observed from 2 to 60 days after eye enucleation, displayed decreased plasma membrane density, increased axoplasmic density, and enlarged mitochondria with deformed cristae when compared with boutons from normal animals. There was also a loss of synaptic plasma membrane specialization and the boutons abnormally indented contiguous dendrites. The number and appearance of synaptic vesicles in some degenerating boutons were notably altered. Phagocytosis of boutons in most instances appeared to be accomplished by astrocytes. When degeneration was first apparent in some boutons, the subsynaptic organelle in the adjacent dendritic cytoplasm was enlarged, somewhat less dense and was associated with small granular and circular profiles. Subsynaptic organelles in experimental animals were absent from contiguities between dendrites and other cell processes, except in a few instances when only small portions of boutons remained at their synaptic sites, suggesting that the organelles disappeared when boutons had been completely phagocytized.Degenerating myelinated axons, observed from 2 to 300 days after enucleation, exhibited the same triad of features as degenerating boutons. They appeared to be phagocytized in most instances by dense glial processes, presumably oligodendrocytic, which were normally situated between the axon and its myelin sheath and were related to the inner mesaxon.

Visual Identification of Nerve Terminals in Living Isolated Skeletal Muscle

1. The unmyelinated terminal branches of motor nerve fibres were clearly resolved in live, unstained skeletal muscles of the frog and of the mudpuppy (Necturus maculosus), using Nomarski optics. The observations were supplemented by several histological procedures, including electron microscopy, and by extracellular recordings from the nerve terminals. 2. In live motor nerve terminals of the mudpuppy one can see a series of varicosities, which in the electron microscope are shown to contain accumulations of synaptic vesicles. Junctional folds in the muscle fibres are confined to the areas opposite the varicosities. Terminal branches of the frog’s motor axon are also varicose, but the swellings are so closely spaced that they can be seen only after staining or by electron microscopy. 3. Nuclei of Schwann cells are recognized along living nerve terminals. Electrophoretic injection of a fluorescent dye, Procionyellow, into the cell bodies of Schwann cells enables one to see the distribution of their processes with the light microscope. 4. Visibility of terminal arborizations was improved by bathing nerve-muscle preparations in solutions of collagenase for 15 to 30 min, thereby removing much of the connective tissue. After longer collagenase treatment nerve terminals could be lifted off muscle fibres with a micropipette, thus exposing the postsynaptic membrane.