No abstract

A study was conducted on the influence of 4‐aminopyridine (4‐AP) on the radiocalcium uptake and membrane potential of rat cortical synaptosomes previously depolarized by biochemical procedures. The initial calcium entry into isolated nerve terminals was substantally enhanced in the presence of 10‐−4M 4‐AP in potassium‐rich media (60 mM). The fast initial phase of potassium‐stimulated calcium entry involves different kinetic characteristics in the presence and the absence of 4‐AP. In the presence of 4‐AP, the fast component of calcium entry reached a peak during the first 15 s immediately following depolarization. The potassium‐stimulated synaptosomal calcium influx at 15 s was 3.5 ± 0.17 times higher in the presence of 4‐AP (10‐−4M) and 2.6 ± 0.11 times higher in the absence of 4‐AP as compared with the respective unstimulated uptake values. In the absence of 4‐AP, however, the calcium entry did not show a peak until 45 s after depolarization. The total amount of calcium accumulated in the synaptosome treated and untreated by 4‐AP is equal at the end of this initial uptake period. The effect of 4‐AP on membrane potential during depolarization of synaptosomes evoked by potassium‐rich medium was also determined by means of a potential sensitive fluorescent dye. It was found that 4‐AP had no effect on the final level of membrane potential, induced by high [K+]o; however, the kinetics showed significant differences. The initial phase of synaptosomal membrane depolarization was slower in the presence of 4‐AP: τ‐4‐AP: 42 ± 4.0 s, τ‐control: 18 ± 2.5 s. The initial calcium entry into the nerve terminals was increased during the 4‐AP treatment in potassium‐rich media—as resulted from our calcium uptake studies—and thus, during this higher calcium influx, the degree of membrane depolarization was decreased. This faster initial calcium influx constitutes a feasible explanation for the unique effect of 4‐AP termed “chemical potentiation” on transmitter release at chemical synapses.

Two of the key enzymes involved in glutamate metabolism, glutaminase and glutamine synthetase, were quantitatively localized to axons and glia of the crayfish giant nerve fiber by immunocytochemistry and electron microscopy of antibody-linked gold microspheres. In Western blots, rabbit antisera for glutamine synthetase and glutaminase specifically recognized crayfish polypeptides corresponding approximately in size to subunits of purified mammalian brain enzymes. Glutamine synthetase immunoreactivity was found to be 11 times greater in the adaxonal glial cells than in the axon. Glutaminase immunoreactivity was found in somewhat greater concentration (2.5:1) in glia as compared to axoplasm. Glutamate immunoreactivity also was evaluated and found to be present in high concentration in both glia and axons, as might be expected for an important substrate of cellular metabolism. Using radiolabeled substrates it was demonstrated that glutamine and glutamate were interconverted by the native enzymes in the intact crayfish giant nerve fiber and that the formation of glutamine from glutamate occurred in the axoplasm-free nerve fiber, the cellular component of which is primarily periaxonal glia. The results of this investigation provide immunocytochemical and metabolic evidence consistent with an intercellular glutamine cycle that modulates the concentration of periaxonal glutamate and glutamine in a manner similar to that described for perisynaptic regions of the vertebrate central nervous system. These findings further corroborate previous electrophysiological evidence that glutamate serves as the axon-to-glial cell neurochemical signal that activates glial cell mechanisms responsible for periaxonal ion homeostasis.

Oxygen consumption (QO2) of single isolated axons and their associated glial cell sheath was investigated under a variety of conditions to determine the contribution of each cell type to whole tissue QO2. It was found that the QO2 of the sheath, in the absence of a functional axon, represented approximately 30% of the total tissue QO2. When the axon was injected with carboxyatractyloside, an inhibitor of mitochondrial oxidative phosphorylation that is membrane impermeant, electrophysiological properties of the axon were not affected and glial sheath respiratory activity was stimulated by 1.7 to 2.7 times the untreated control level. These results suggest that glial cell metabolic activity is regulated by the metabolic activity of the axon. Depending on the experimental conditions the glial sheath accounts for 30% to nearly 100% of the QO2 of axon-glial cell tissue. On the basis of these and morphometric measurements we estimate that in a normally functioning axon-glial cell system the glial sheath accounts for 90% of the tissue QO2.