B Ceccarelli

Coated vesicles and pits during enhanced quantal release of acetylcholine at the neuromuscular junction

SummaryFrog neuromuscular junctions were stimulated by different methods to secrete quanta of ACh, and the attendant changes in the ultrastructure of the nerve terminal were assessed by morphometric analysis of electron micrographs. Secretion was stimulated by electrical stimulation at 2 Hz or by application of the secretagogues, lanthanum, ouabain or black widow spider venom, either in the presence or in the absence of extracellular Ca2+. The numbers of synaptic vesicles, coated vesicles and coated pits, and the length of axolemma and area of axoplasm were measured on the micrographs. There was a significant increase (about threefold) in the total number of coated structures (vesicles plus pits) per μm2 of axoplasm, but the fractional increase in the number of coated pits exceeded the fractional increase in the number of coated vesicles. These increases were positively correlated with the increase in the length of axolemma per unit area and negatively correlated with the changes in concentration of synaptic vesicles, suggesting that they were due to the increases in the surface area of the terminal that accompany a loss of vesicles. However, the increase in the concentration of coated structures was not related to the number of quanta secreted or to the estimated number of vesicles recycled. The lack of correspondence between the fractional increases in the coated pits and coated vesicles and the poor correlation between the numbers of these structures and the overall parameters of the secretory process suggest that, in contrast to the situation in other secretory systems, coated pits and coated vesicles may not play a crucial role in maintaining the functional population of synaptic vesicles at rapidly secreting neuromuscular junctions.

The effect of potassium on exocytosis of transmitter at the frog neuromuscular junction.

1. Electrophysiology and morphology have been combined to investigate the time course of the exocytosis of quanta of neurotransmitter induced by elevated concentrations of K+ at the frog neuromuscular junction. 2. Replicas of freeze‐fractured resting nerve terminals fixed in the presence of 20 mM‐K+ showed images of fusion of synaptic vesicles with the presynaptic axolemma which were closely associated with the active zones. After 1 min in 20 nM‐K+ fusions appeared also outside the active zones, and by 5 min they became uniformly distributed over the presynaptic membrane. 3. The average total density of fusions was not significantly different at the various times examined since it decreased at the active zones while it increased over the rest of the membrane. 4. Resting terminals fixed in 20 mM‐K+ released 33,000‐45,000 quanta after the addition of fixative; terminals stimulated by 20 mM‐K+ for 1‐5 min released 50,000‐100,000 quanta during fixation. The fixative potentiated K+‐induced transmitter release. 5. Fusions were uniformly distributed in terminals pre‐incubated for 5 min in 20 mM‐K+ without added Ca2+, stimulated by adding Ca2+ for 30 s, and then fixed. Conversely, after 5 min stimulation in hypertonic Ringer solution fusions remained predominantly located near the active zones. A similar distribution was observed after 15 min stimulation by a lower concentration of K+ (15 mM). 6. At all concentrations of K+ tested (10, 15, 20, 25 mM) miniature end‐plate potential (MEPP) rate attained a steady‐state value within 10‐15 min. Values from a single junction were generally lower at higher concentrations of K+, which indicates partial inactivation of the secretion‐recycling process. 7. The data indicate that K+ initially activates exocytosis at the active zones. Subsequently, ectopic exocytosis is activated while sites at the active zones appear to undergo partial inactivation. These phenomena are not related to the intensity or to the amount of previous secretion.

Correlation between quantal secretion and vesicle loss at the frog neuromuscular junction.

1. We measured the rate of occurrence of miniature endplate potentials (MEPPs) at identified endplates in frog cutaneous pectoris muscles treated with crude black widow spider venom (BWSV) or purified alpha‐latrotoxin (alpha‐LTX) in calcium‐free solutions, and we examined the relationship between the length of the nerve terminal and the total number of quanta secreted, and the relationship between the number of quanta secreted and the number of vesicles remaining at different times. 2. The venom, or toxin, was applied in a modified Ringer solution with tetrodotoxin, 1 mM‐EGTA and no divalent cations, and quantal secretion was started by applying Ca2(+)‐free solutions with Mg2+. This was done to synchronize the quantal discharge at the various junctions in a muscle. Ringer solution was applied after the MEPP rate had declined to low levels, and then the muscle fibre was injected with Lucifer Yellow, the endplate stained for acetylcholinesterase and the length of the nerve terminal and the length of a sarcomere were measured on the fluorescent fibre. 3. The total number of quanta secreted by a terminal was measured under a wide variety of experimental conditions: the weights of the frogs ranged from 13 to 68 g, the temperature from 9 to 28 degrees C, and the concentration of Mg2+ from 2 to 10 mM. In one series of experiments the Mg2+ was withdrawn after 3‐4 min and reapplied 35‐40 min later in order to divide the total output of quanta into two approximately equal bouts of secretion that were well separated in time. 4. The total number of MEPPs recorded at a junction was loosely correlated with the length of its nerve terminal, but it was not affected by the temperature, the concentration of Mg2+ or the division of secretion into well‐separated bouts of quantal release. The average total secretion per unit length was about 3700 quanta/sarcomere or about 1200 quanta/microns. 5. The average time course of quantal secretion per micrometre of terminal was determined at single junctions in muscles held at 22‐23 degrees C or at 9‐10 degrees C. Other muscles were fixed at various times during the course of secretion at each temperature and the number of synaptic vesicles remaining in cross‐sections of the terminals were counted on electron micrographs. The number of vesicles remaining per micrometre of terminal was determined from the number per cross‐section and the section thickness.(ABSTRACT TRUNCATED AT 400 WORDS)

The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, enhances the evoked quanta release of acetylcholine at the frog neuromuscular junction

The possible role of protein kinase C in the regulation of quantal transmitter release was studied at the frog neuromuscular junction by using the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a compound known to mimic the effects of the physiological activator of the enzyme, endogenous diacylglycerol. The main effect of the phorbol ester was to increase the quantal content,m, of the endplate potential. The initial values ofm were adjusted over a wide range by changing the Ca2+ concentration of the extracellular medium, and the TPA-induced fractional increase inm was significantly greater at junctions with a lower initial quantal content. On the other hand, the absolute increases inm induced by the phorbol ester were positively correlated with the square root of the initial quantal content. The possible physiological significance of this correlation is discussed in view of the well known relationship between extracellular Ca2+ concentration and the quantal content of the end plate potential.

New Evidence Supporting the Vesicle Hypothesis for Quantal Secretion at the Neuromuscular Junction

Acetylcholine (ACh) is stored in two compartments within vertebrate motor terminals: the cytoplasm and the synaptic vesicles (Whittaker et al., 1964; Dunant et al., 1972); it is released from terminals in two ways: continuously in a relatively steady molecular stream (Katz and Miledi, 1977; Vyskocil and Illes, 1979), and intermittantly in pulses, or quanta, (Fatt and Katz, 1952, del Castillo and Katz, 1954) which contain about 104 molecules (Kuffler and Yoshikami, 1975). The function of the continuous leak is not understood (Edwards et al., 1985), but the spontaneous or neurally evoked release of quanta generates the discrete, transient miniature endplate potentials (mepps) or endplate potentials (epps) that mediate neuromuscular transmission. Two hypotheses have been proposed for the origin of the quanta: a) the cytoplasmic hypothesis which postulates that quanta are comprised of cytoplasmic ACh which diffuses in pulses through channels in the axolemma which become intermittantly permeable to it (Israel and Manaranche, 1985), and b) the vesicle hypothesis which postulates that quanta are comprised of ACh which is released by exocytosis from the interiors of synaptic vesicles whose membranes have fused with the axolemma (del Castillo and Katz, 1956). In our opinion, the vast preponderance of evidence supports the vesicle hypothesis of quantal secretion, though its proof is not yet absolute and most of the mechanistic details of the fusion and recovery processes have yet to be worked out.

Chapter 5 Exocytosis and Membrane Recycling

Publisher Summary This chapter deals with the problem of how the membranes of intracellular vesicles that fuse with the plasma membrane are retrieved, rather than lost to the economy of the cell. Exocytosis is the process developed by cells to carry out this essential function and has some distinct advantages over simple transmembrane transport processes. Three in particular can be listed: (1) exocytosis permits cells to store large quantities of secretory products and to release them as required without the need to expend large amounts of energy, (2) release can take place at specific, strategically located portions of the plasmalemma (polarized release), so that high concentrations of secretory products can be achieved in the extracellular space immediately adjacent to the release sites, and (3) exocytosis permits rapid and efficient regulation of release. The chapter summarizes the many important advances in knowledge of exo- and endocytosis that have occurred in the field. In particular, the attempts that have been made to reconstruct the process in vitro, starting from completely disassembled components, have not been successful.