Depletion of Vesicles From Frog Neuromuscular Junctions by Prolonged Tetanic Stimulation

Abstract

A B S T R A C TCurarized cutaneous pectoris nerve muscle preparations from frogs were subjected to prolonged indirect stimulation at 2/see while recording" from end plate regions. At the ends of the periods of stimulation, the curare was removed and the preparations were fixed for electron microscopy or treated with black widow spider venom to determine the degree to which their stores of transmitter had been depleted. After 6--8 hr of stimulation the nerve terminals were almost completely depleted of their stores of transmitter and of their population of vesicles. Most of the transmitter release occurred during the first 4 hr of stimulation, and after this time most (about 80%) of the fibers were depleted of about 80% of tbeir transmitter. The organization of the nerve terminals in 4-hr preparations appeared normal and the terminals still contained many vesicles. When peroxidase was present in the bathing medium, terminals from stimulated preparations showed many vesicles that contained peroxidase, whereas the rested control p;eparations showed few such vesicles The fact that after 4 hr the total number of vesicles is not markedly changed while a large fraction (up to 45%) contained peroxidase suggests that in our experiments vesicles were continuously fusing with and reforming from the axolemma. I . N T R O D U C T I O NSeveral workers have tried to deplete neuromuscular junctions of their stores of transmitter and of their synaptic vesicles by tetanic stimulation of the nerve (1, 2, 3, 4). Depiction of transmitter has been obtained only when synthesis was inhibited by hemicholinium (2, 3) and, under this condition, a reduction in the number of vesicles occurred only in the regions of the axoplasm immediately adjacent to the axolemma (4). In these previous works the preparations were stimulated for from several minutes to a few hours at frequencies of 10/see or more. We have stimulated a neuromuscular preparation from the frog for 6-9 hr at a rate of 2/see in the absence of hemicholinium and have successfully depleted the terminals of their store of transmitter and of their population of vesicles. M A T E R I A L S A N D M E T H O D SThe cutaneous pectorls muscle of the frog, Rana p¢zens, was used. The muscles were mounted in the chamber described previously (5) and maintained at about 22°C in a Ringer's soIution that contained 116 rn~i NaC1, 2.0 mu KC1, 1.8 m:~ CaC12, 1 rn~ NaH2PO4, and 2 m~ Na2HPO4 (pH 7.0). End plate regions were impaled with mieropipettes filled with 3 • KC1. Conventional recording equipment was used and photographic records of the end plate potentials @.p.p.s) and miniature end plate potenrials (m.e.p.p.s) were obtained. The nerve was stimulated with square pulses 0.1 msec in duration and amplitude three to four times threshold. The muscle twitch was blocked by adding curare to the bath at a concentration of 3 X 10 -6 g/ml, an 30THE JOV~,tL O~" CELL BIOLOGr -VoLtnuE 54~ 197g • pages 30-38 on

Acetylcholine compartments in mouse diaphragm. Comparison of the effects of black widow spider venom, electrical stimulation, and high concentrations of potassium.

“…Hence, ACh synthesis at rest may not be inhibited by HC-3 either because HC-3 does not have access to the sites of synthesis or because the synthesis is not dependent on extracellular choline. In stimulated diaphragms, however, newly synthesized ACh is preferentially released (9), perhaps because it is used to reload recycled vesicles (6,7,18,37). This synthesis may occur only in highly restricted regions of the terminal immediately adjacent to the sites of ACh release (10) and may be critically dependent on choline supplied by a high-affinity transport system (15).…”

Membrane flow during pinocytosis. A stereologic analysis.

“…For example, in neurohypophysis (13), anterior pituitary (20), adrenal medulla (1), peripheral nerve terminals (5,27), and islet cells (36), it has been suggested that membrane components delivered to the cell surface during the exocytosis of the secretory granule are retrieved by the cell through a burst of pinocytic activity. This hypothesis has not been tested quantitatively, but a stereologic analysis with HRP migh be a useful means of doing so.…”

Synaptic Vesicle Mobility in Mouse Motor Nerve Terminals with and without Synapsin

“…Recruitment comes from synaptic vesicles that can reside several vesicle diameters away from fusion sites (Ceccarelli et al, 1972). Directed transport via an actin-myosin mechanism has been proposed to facilitate movement, based primarily on evidence of myosin involvement in vesicle recruitment (Prekeris and Terrian, 1997;Ryan, 1999;Verstreken et al, 2005) (but see Tokuoka and Goda, 2006).…”