Synaptic repression at crayfish neuromuscular junctions. II. Evidence for calcium involvement

The inability of synaptic junctions to generate normalsized postsynaptic potentials under normal physiological conditions was studied at crayfish neuromuscular synapses. Synaptic repression in the superficial flexor muscle system of the crayfish was induced by surgery: the nerve was cut in the middle of the target field, and the lateral muscle fibers were removed. After this surgery, the remaining medial synapses were unable to generate normal-sized junction potentials (jp) over the medial muscle population. In an attempt to study the mechanism underlying this response, we varied the extracellular calcium concentration of the Ringers solution bathing the preparation, in both repressed and control animals, while monitoring the size of the same junction potential. The junction potential generated by the spontaneous activity of the nerve increased in size with increasing calcium concentrations in control animals, but failed to do so in repressed animals, that is, changes in external calcium concentrations did not affect repressed synapses. However, in the presence of the calcium ionophore A23187, control and repressed synapses both show an increase in the junction potential sizes they generate. Our data suggest that calcium is involved in the mechanisms that underlie synaptic repression in this crustacean neuromuscular system. © 1993 John Wiley & Sons, Inc.     

Depressing Effects of Caffeine at Crayfish Neuromuscular Synapses I. Dosage Response and Ca<Superscript>++</Superscript> Gradient Effects

The response of crayfish synaptic terminals to drugs began to be studied to characterize the terminal’s physiological characteristics. Caffeine, the first drug to be studied, was selected to enhance synaptic transmission because of its ability to increase calcium release from internal stores. 1. The largest excitor neuron to the superficial flexor muscle system of Procambarus clarkii was stimulated at 10 Hz while recording junction potentials from several lateral muscle fibers. 2. Caffeine unexpectedly decreased synaptic transmission in this system in a dosage-dependent manner. The depressing effect of caffeine was observed at 5 mM caffeine and junction potentials disappeared completely at 50 mM. Washing the preparation in fresh control Ringers did not restore the amplitudes of the junction potentials. 3. Changes in extracellular calcium concentrations delayed or depressed the caffeine effect depending on the calcium gradient across the membrane or the caffeine dosage. The data suggest that calcium is involved in caffeine’s response in this system in a way yet to be determined.     

Agrin and acetylcholine receptors are removed from abandoned synaptic sites at reinnervated frog neuromuscular junctions

Changes in the distribution of agrin and acetylcholine receptors (AChRs) were examined during reinnervation and following permanent denervation as a means of understanding mechanisms controlling the distribution of these molecules. Following nerve damage in the peripheral nervous system, regenerating nerve terminals preferentially return to previous synaptic sites leading to the restoration of synaptic activity. However, not all portions of original synaptic sites are reoccupied: Some of the synaptic sites are abandoned by both the nerve terminal and the Schwann cell. Abandoned synaptic sites contain agrin, AChRs, and acetylcholinesterase (AChE) without an overlying nerve terminal or Schwann cell providing a unique location to observe changes in the distribution of these synapse-specific molecules. The distribution of anti-agrin and AChR staining at abandoned synaptic sites was altered during the process of reinnervation, changing from a dense, wide distribution to a punctate, pale pattern, and finally becoming entirely absent. Agrin and AChRs were removed from abandoned synaptic sites in reinnervated frog neuromuscular junctions, while in contralateral muscles which were permanently denervated, anti-agrin and AChR staining remained at abandoned synaptic sites. Decreasing synaptic activity during reinnervation delayed the removal of agrin and AChRs from abandoned synaptic sites. Altogether, these results support the hypothesis that synaptic activity controls a cellular mechanism that directs the removal of agrin from synaptic basal lamina and the loss of agrin leads to the dispersal of AChRs. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 999–1018, 1997     

The area composita of adhering junctions connecting heart muscle cells of vertebrates. I. Molecular definition in intercalated disks of cardiomyocytes by immunoelectron microscopy of desmosomal proteins

Among sarcomeric muscles the cardiac muscle cells are unique by, inter alia, a systemic and extended cell-cell contact structure, the intercalated disk (ID), comprising frequent and closely spaced arrays of plaque-coated cell-cell adhering junctions (AJs). As some of these junctions may look somewhat like desmosomes and others like fasciae adhaerentes, the dogma has emerged in the literature that IDs contain - like epithelial cells - both kinds of AJs formed by - for the most - mutually exclusive molecular ensembles. This, however, is not the case. In comprehensive immunoelectron microscopic studies of mammalian (human, bovine, rat, mouse) and non-mammalian (chicken, amphibia, fishes) heart muscle tissues, we have localized major constituents of the desmosomal plaques of polar epithelia, desmoplakin, plakophilin-2 and plakoglobin, as well as the desmosomal cadherins, desmoglein Dsg2 and desmocollin Dsc2, in both kinds of ID AJs, independent of the specific morphological appearance. The desmosomal molecules are not restricted to the desmosome-like-looking junctions but can also be detected in junctions appearing similar to the zonula or fascia adhaerens structures. These AJs of cardiac ID are therefore subsumed under the collective term area composita. We discuss our results with respect to the importance of ID junction molecules for the formation, maintenance and function of the heart, particularly in relation to recent findings that deletions of - or mutations in - genes encoding such proteins can cause severe, sometimes lethal damages.