Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potassium.

Abstract

Frog cutaneous pectoris nerve muscle preparations were studied by the freezefracture technique under the following conditions: (a) during repetitive indirect stimulation for 20 min, 10/s; (b) during recovery from this stimulation; and (c) during treatment with 20 mM K +.Indirect stimulation causes numerous dimples or protuberances to appear on the presynaptic membrane of the nerve terminal, and most are located near the active zones. Deep infoldings of the axolemma often develop between the active zones. Neither the number nor the distribution of dimples, protuberances, or infoldings changes markedly during the first minute of recovery. The number of dimples, protuberances, and infoldings is greatly reduced after l0 min of recovery. Since endocytosis proceeds vigorously during the recovery periods, we conclude that endocytosis occurs mostly at the active zones, close to the sites of exocytosis.20 mM K § also causes many dimples or protuberances to appear on the axolemma of the nerve terminal but they are distributed almost uniformly along the presynaptic membrane. Experiments with horseradish peroxidase (HRP) show that recycling of synaptic vesicles occurs in 20 mM K § This recycling is not accompanied by changes in the number of coated vesicles. Since both exocytosis and endocytosis occur in 20 mM K § it is difficult to account for this unique distribution. However, we suggest that K § causes dimples or protuberances to appear between the active zones because it activates latent sites of exocytosis specified by small numbers of large intramembrane particles located between active zones. The activation of latent release sites may be related to the complex effects that K § has on the quantal release of neurotransmitter. KEY WORDS membrane fusionOur previous paper showed that black widow active zones potassium endocytosis spider venom (BWSV) induces the appearance of vesicle retrieval many dimples (P face) or protuberances (E face) 178J. CELL BIOLOGY t~) The Rockefeller University Press 9

The influence of 2‐chloroadenosine on potassium‐evoked and neurally‐evoked acetylcholine secretion from normal or from latent active zones in the frog

“…frequency (Figure 2a) is first elevated to immeasurable levels (b) and then, as depletion of ACh quanta ensues (Ceccarelli et al, 1979b), decays over time course 0.5-2 h to lower frequencies which remain relatively stable (c). Figure 2(c-i) illustrates that 2-chloroadenosine is an effective inhibitor of ACh release during the steady-state period of ACh release evoked by 20mmK.…”

“…Figure 2). This stable period, which is also associated with the frequent appearance of a small mode m.e.p.ps, delineates the time over which most of the ACh release occurs between the original active zones (Ceccarelli et al, 1979b;Haimann et al, 1980;Ceccarelli & Hurlbut, 1980). For the studies in which the active zone is disorganized by Ca-free Ringer containing Mg, a reduction in m.e.p.p.…”

“…Specifi-cally, it has been found that a large number of latent release sites appear after exposure to 20 mM K whereby the original active zone of ACh secretion now contributes only minimally to the overall quantal output (Ceccarelli et al 1979b;Ceccarelli & Hurlbut, 1980). It has thus been suggested that the absence of an effect of adenosine derivatives on Kevoked release, if true, could be due to such an alteration of the active zone structure (see Silinsky 1985b, for discussion).…”

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

“…Whatever the cause of the difference between the two sets of results, it appears that in those regions of the muscles where an adequate degree of tissue preservation is achieved the plane of fracture is quite different from the one normally obtained in chemically fixed and cryoprotected specimens (6,7,27). This is not surprising since there are no convincing a priori reasons to believe that in a quasi-vitrified portion of a cell the inner membrane domain should be more prone to be freeze-cleaved (22).…”

“…Although these observations indicate that vesicles fuse with and are recovered from the axolemma when quanta are actively secreted, the slowness of chemical fixation precludes demonstrating that vesicle fusion and transmitter release are coincident. The images of fusion seen after chemical fixation represent various states of association of vesicles with the axolemma, trapped during the several seconds required for complete fixation, and depict an integration of the events that occur over the fixation time (7). The rapid-freezing technique (18,(47)(48)(49), has been used in the attempt to correlate more precisely in time the appearance of images of fusion with the enhanced release of quanta of acetylcholine evoked by a single action potential in frog nerve-muscle preparations treated with 4-aminopyridine (4-AP) ~ (26,28).…”