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

Active zone material at the nervous system's 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 frog's 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.

Automatic acquisition of fiducial markers and alignment of images in tilt series for electron tomography.

Three-dimensional reconstruction of a section of biological tissue by electron tomography requires precise alignment of a series of two-dimensional images of the section made at numerous successive tilt angles. Gold beads on or in the section serve as fiducial markers. A scheme is described that automatically detects the position of these markers and indexes them from image to image. The resulting set of position vectors are arranged in a matrix representation of the tilt geometry and, by inversion, alignment information is obtained. The scheme is convenient, requires little operator time and provides an accuracy of < 2 pixels RMS. A tilt series of 60-70 images can be aligned in approximately 30 min on any modern desktop computer.

Regulation of the size and distribution of agrin-induced postsynaptic-like apparatus in adult skeletal muscle by electrical muscle activity.

We compared actylcholine receptor (AChR) aggregates induced by neural agrin released from transfected muscle fibers with AChR aggregates induced by transplanted axons in extrajunctional regions of denervated rat soleus muscles. Both neural agrin and transplanted axons induced multiple, irregularly distributed AChR aggregates on muscle fibers. Direct electrical muscle stimulation of transfected muscles for up to 10 weeks removed all agrin-induced AChR aggregates (the losers) except one (the winner) on many fibers. Axon-induced AChR aggregates underwent comparable selection of winners and losers. The results suggest that agrin and acetylcholine-driven muscle activity provided by transplanted axons are sufficient to elicit in a denervated adult muscle fiber processes that regulate the size and distribution of ectopic neuromuscular junctions.

Neuregulins and erbB receptors at neuromuscular junctions and at agrin-induced postsynaptic-like apparatus in skeletal muscle.

We demonstrate by immunohistochemistry that at least two isoforms of neuregulin (NRG) are concentrated at neuromuscular junctions in adult rat muscles. One is NRGbeta3, a secreted protein which is bound to basal lamina that occupies the synaptic cleft. The other(s), NRG-a, is in the muscle fibers' plasma membrane. We show further that muscle NRG, including NRG-a, is concentrated at postsynaptic-like apparatus induced to form in the extrajunctional region of the soleus muscle by exposure to neural agrin. The agrin-induced postsynaptic-like apparatus also includes aggregates of the NRG receptors erbB2 and erbB3 as does postsynaptic apparatus at neuromuscular junctions. These findings together with those of others suggest a mechanism by which neural agrin induces the expression of epsilon-AChR subunits in postsynaptic-like apparatus, and they support the hypothesis that agrin has a similar function at neuromuscular junctions.

Dissection of active zones at the neuromuscular junction by EM tomography.

We used EM tomography to examine the fine structure of the apparently amorphous electron dense material that is seen at active zones of axon terminals when viewed by conventional 2D electron microscopy. Serial 1-nm optical slices from 3D reconstructions of individual thin tissue sections reveal that the material is composed of an interconnecting network of elongate components directly linked to synaptic vesicles and the presynaptic membrane. Each vesicle at the active zone that lies adjacent to the presynaptic plasma membrane has several such connections. Information provided by reconstruction data may be useful in generating experiments aimed at understanding the mechanisms involved in the docking of synaptic vesicles and their exocytosis during synaptic transmission.

Ligands for ErbB-family receptors encoded by a neuregulin-like gene.

Neuregulins (also called ARIA, GGF, heregulin or NDF) are a group of polypeptide factors that arise from alternative RNA splicing of a single gene. Through their interaction with the ErbB family of receptors (ErbB2, ErbB3 and ErbB4), neuregulins help to regulate cell growth and differentiation in many tissues. Here we report the cloning of a second neuregulin-like gene, neuregulin-2. The encoded product of the neuregulin-2 gene has a motif structure similar to that of neuregulins and an alternative splicing site in the epidermal growth factor(EGF)-like domain gives rise to two isoforms (alpha and beta). Northern blot and in situ hybridization analysis of adult rat tissues indicate that expression of neuregulin-2 is highest in the cerebellum, and the expression pattern is different from that of neuregulins. Recombinant neuregulin-2beta induces the tyrosine-phosphorylation of ErbB2, ErbB3 and ErbB4 in cell lines expressing all of these ErbB-family receptors. However, in cell lines with defined combinations of ErbBs, neuregulin-2beta only activates those with ErbB3 and/or ErbB4, suggesting that signalling by neuregulin-2 is mediated by ErbB3 and/or ErbB4 receptors.

Agrin-induced postsynaptic-like apparatus in skeletal muscle fibers in 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.

gamma-AChR/epsilon-AChR switch at agrin-induced postsynaptic-like apparatus in skeletal muscle.

We transfected the extrajunctional region of denervated soleus muscles in adult rats with neural agrin cDNA to induce myofibers to form postsynaptic-like apparatus containing acetylcholine receptor (AChR) aggregates. By 1 week approximately 30% of the AChR aggregates contained a mixture of epsilon-AChRs and gamma-AChRs while approximately 70% had only gamma-AChRs. If the transfected muscles were reinnervated in the original junctional region, the postsynaptic-like apparatus, despite the absence of apposed axon terminals, gradually came to have only epsilon-AChRs. We conclude that at the postsynaptic apparatus of ectopic neuromuscular junctions formed by a foreign nerve implanted into the extra-junctional region of denervated muscles, agrin secreted by the axon terminal plays a direct role in the gamma-AChR/epsilon-AChR switch that occurs as the apparatus reaches maturity. Our findings, together with results from other studies, indicate further that agrin and acetylcholine are the only nerve-derived factors required for this switch.

Globular and asymmetric acetylcholinesterase in the synaptic basal lamina of skeletal muscle.

The aim of this study was to characterize the molecular forms of acetylcholinesterase (AChE) associated with the synaptic basal lamina at the neuromuscular junction. The observations were made on the neuromuscular junctions of cutaneous pectoris muscles of frog, Rana pipiens, which are similar to junctions of most other vertebrates including mammals, but are especially convenient for experimentation. By measuring relative AChE activity in junctional and extrajunctional regions of muscles after selective inactivation of extracellular AChE with echothiophate, or of intracellular AChE with DFP and 2-PAM, we found that > 66% of the total AChE activity in the muscle was junction-specific, and that > 50% of the junction-specific AChE was on the cell surface. More than 80% of the cell surface AChE was solubilized in high ionic strength detergent-free buffer, indicating that most, if not all, was a component of the synaptic basal lamina. Sedimentation analysis of that fraction indicated that while asymmetric forms (A12, A8) were abundant, globular forms sedimenting at 4-6 S (G1 and G2), composed > 50% of the AChE. It was also found that when muscles were damaged in various ways that caused degeneration of axons and muscle fibers but left intact the basal lamina sheaths, the small globular forms persisted at the synaptic site for weeks after phagocytosis of cellular components; under certain damage conditions, the proportion of globular to asymmetric forms in the vacated basal lamina sheaths was as in normal junctions. While the asymmetric forms required high ionic strength for solubilization, the extracellular globular AChE could be extracted from the junctional regions of normal and damaged muscles by isotonic buffer. Some of the globular AChE appeared to be amphiphilic when examined in detergents, suggesting that it may form hydrophobic interactions, but most was non-amphiphilic consistent with the possibility that it forms weak electrostatic interactions. We conclude that the major form of AChE in frog synaptic basal lamina is globular and that its mode of association with the basal lamina differs from that of the asymmetric forms.

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.

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.

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.

Isolation and characterization of a cDNA that encodes an agrin homolog in the marine ray.

Agrin, the protein thought to trigger motor neuron-induced aggregation of postsynaptic molecules at the developing neuromuscular junction, has been purified from the synapse-rich electric organ of the marine ray. In order to study agrin's role in synaptogenesis and to examine its relationship to antigenically similar proteins, we isolated from a marine ray library a partial cDNA, OL4, which codes for a member of the agrin protein family. Sequence analysis shows that agrin and agrin-related proteins contain regions similar to basal lamina proteins and other secreted molecules including laminin, epidermal growth factor, and pancreatic secretory trypsin inhibitors. Northern blot analysis revealed transcripts in several different tissues, but the highest levels of expression are in brain and spinal cord. In situ hybridization studies demonstrate that agrin/agrin-related mRNAs are present in motor neurons that innervate the electric organ and skeletal muscle. They also reveal that agrin/agrin-related transcripts have a broad distribution in neurons and nonneural cells in the CNS, raising the possibility that agrin and/or agrin-related proteins mediate formation of the postsynaptic apparatus at neuron-to-neuron synapses.

Synthesis and transport of agrin-like molecules in motor neurons.

Several lines of evidence indicate that agrin, or a protein very similar to it, directs the formation and maintenance of the postsynaptic apparatus at the neuromuscular junction. We discuss the results of studies involving immunohistochemical, biochemical and in situ hybridization techniques that support the hypothesis that agrin or agrin-like molecules active at the junction are produced by motor neurons.

Agrin-like molecules in motor neurons.

According to the agrin hypothesis molecules that mediate the nerve-induced aggregation of acetylcholine receptors and acetylcholinesterase on developing and regenerating skeletal muscle fibers are similar or identical to agrin, a protein extracted from the electric organ of marine rays. Here we present evidence that agrin is highly concentrated in the cell bodies of motor neurons and is transported to axon terminals which is consistent with the agrin hypothesis.

Motor neurons contain agrin-like molecules.

Molecules antigenically similar to agrin, a protein extracted from the electric organ of Torpedo californica, are highly concentrated in the synaptic basal lamina of neuromuscular junctions in vertebrate skeletal muscle. On the basis of several lines of evidence it has been proposed that agrin-like molecules mediate the nerve-induced formation of acetylcholine receptor (AChR) and acetylcholinesterase (AChE) aggregates on the surface of muscle fibers at developing and regenerating neuromuscular junctions and that they help maintain these postsynaptic specializations in the adult. Here we show that anti-agrin monoclonal antibodies selectively stain the cell bodies of motor neurons in embryos and adults, and that the stain is concentrated in the Golgi apparatus. We also present evidence that motor neurons in both embryos and adults contain molecules that cause the formation of AChR and AChE aggregates on cultured myotubes and that these AChR/AChE-aggregating molecules are antigenically similar to agrin. These findings are consistent with the hypothesis that agrin-like molecules are synthesized by motor neurons, and are released from their axon terminals to become incorporated into the synaptic basal lamina where they direct the formation of synapses during development and regeneration.

Agrin-like molecules at synaptic sites in normal, denervated, and damaged skeletal muscles.

Several lines of evidence have led to the hypothesis that agrin, a protein extracted from the electric organ of Torpedo, is similar to the molecules in the synaptic cleft basal lamina at the neuromuscular junction that direct the formation of acetylcholine receptor and acetylcholinesterase aggregates on regenerating myofibers. One such finding is that monoclonal antibodies against agrin stain molecules concentrated in the synaptic cleft of neuromuscular junctions in rays. In the studies described here we made additional monoclonal antibodies against agrin and used them to extend our knowledge of agrin-like molecules at the neuromuscular junction. We found that anti-agrin antibodies intensely stained the synaptic cleft of frog and chicken as well as that of rays, that denervation of frog muscle resulted in a reduction in staining at the neuromuscular junction, and that the synaptic basal lamina in frog could be stained weeks after degeneration of all cellular components of the neuromuscular junction. We also describe anti-agrin staining in nonjunctional regions of muscle. We conclude the following: (a) agrin-like molecules are likely to be common to all vertebrate neuromuscular junctions; (b) the long-term maintenance of such molecules at the junction is nerve dependent; (c) the molecules are, indeed, a component of the synaptic basal lamina; and (d) they, like the molecules that direct the formation of receptor and esterase aggregates on regenerating myofibers, remain associated with the synaptic basal lamina after muscle damage.