Stimulation-Dependent Alterations in Peroxidase Uptake at Lobster Neuromuscular Junctions

The uptake of cytocheinically demnonstrable horseradish peroxidase into small vesicles within nerve endings in lobster stretcher muiscles can be enhanced by electrical stimulation of transmitter release by the endings. This is observed particularly if stimulation is interrupted periodically arid the nerves peron M ar ch 2 6, 2 01 6 D ow nl oa de d fr om o n M ar ch 2 6, 2 01 6 D ow nl oa de d fr om o n M ar ch 2 6, 2 01 6 D ow nl oa de d fr om o n M ar ch 2 6, 2 01 6 D ow nl oa de d fr om vesicles responds markedly to alterations in rates of transmitter release. The excitatory and inhibitory nerves to the stretcher muscle of lobster walking limbs were prepared for external stimulation as described previously (7). The muscle itself was exposed through a hole cut in the carapace and bathed in an artificial seawater (8) containing, per liter, 465 meq of Na+, 10 of K+, 50 of Ca2+, and 525 of Cl-. Horseradish peroxidase (1.5 to 6 percent) (Sigma Biochemicals, type II) was usually added to this medium. Synaptic potentials were recorded from the muscle cells by using KCl-filled micropipettes and standard electrical equipment. Figure 1 shows two successive oscilloscope sweeps demonstrating the presence of excitatory postsynaptic potentials (EPSP's) and inhibitory postsynaptic potentials (IPSP's) in one of the peroxidase preparations. Synaptic potentials were monitored throughout all stimulation experiments; we have noted no appreciable effect of peroxidase on the electrical behavior of the neuromuscular junctions. This report is based on study of several dozen axon endings in 16 walking limb preparations; six of the muscles were soaked in peroxidase-containing medium for 45 to 70 minutes but not stimulated, eight were stimulated in the presence of peroxidase, and two were stimulated in the absence of peroxidase. All experiments were carried out tat room temperature (22°C). The stimulation experiments lasted 45 to 70 minutes. Supermaximal stimuli were delivered, usually in bursts of 1.5 seconds duration separated by 0.5-second intervals, at rates of 10 to 30 per second. The total numbers of stimuli delivered to a given preparation ranged from 15,000 to 30,000. As far as can be judged from electrophysiological evidence, essentially all the neuromuscular endings in a preparation maintained good responsiveness during the experiments, and postsynaptic potential frequency faithfully followed stimulus frequency. It should be borne in mind that the postsynaptic potentials recorded often represent the summed effects of several neighboring axon endings. In some experiments, stimulation was continuous, up to the time of tissue fixation for microscopic examination. In others, stimulation was stopped 10 minutes before fixation, and in still others, three 1 0-minute periods of stimulation were alternated with 10minute rest periods. Tissue preparation was by our standard procedures (9). 734 Fig. 2. From a preparation exposed (45 minutes) to peroxidase without electrical stimulation. The thin section was not stained with heavy metals. The electron density in the region indicated by the arrows is due to peroxidase reaction product that has accumulated in the space between muscle and an axon ending. No reaction product is seen in the vesicles (V) within the ending. As in this figure, individual thin sections of axon endings often show the axon apparently surrounded completely by muscle; from our observations we would expect to find portions of Schwann cells adjacent to regions of the surface of this ending in serial sections or in a section cut at a different angle. Bar length is 0.5 gLm. In brief, tissue was fixed with a dilute modification of Karnovsky's paraformaldehyde-glutaraldehyde mixture in phosphate buffer or in 2 percent glutaraldehyde in cacodylate buffer, rinsed in fixative-free buffer, frozen on the head of a freezing microtome, incubated (50 minutes, room temperature) in the medium of Graham and Karnovsky (9) to demonstrate sites of peroxidase activity, and then postfixed in osmium tetroxide and embedded in Epon. Thin sections were examined in an RCA EMU 3F microscope, usually without heavymetal staining. Since neuromuscular endings are relatively sparsely distributed in the tissue, the sections used were very large, and each embedded block was sampled at several levels to maximize chances of encountering an ending. Muscle not exposed to peroxidase, and peroxidase-exposed material incubated in peroxide-free medium, served as incubation controls; such preparations contained no reaction