Effects of wortmannin and latrunculin A on slow endocytosis at the frog neuromuscular junction.

Phosphoinositides are key regulators of synaptic vesicle cycling and endocytic traffic; the actin cytoskeleton also seems to be involved in modulating these processes. We investigated the effects of perturbing phosphoinositide signalling and actin dynamics on vesicle cycling in frog motor nerve terminals, using fluorescence and electron microscopy, and electrophysiology. Antibody staining for beta-actin revealed that actin surrounds but does not overlap with synaptic vesicle clusters. Latrunculin A, which disrupts actin filaments by binding actin monomers, and wortmannin, an inhibitor of phosphatidyl inositol-3-kinase (PI3-kinase), each disrupted the pattern of presynaptic actin staining, but not vesicle clusters in resting terminals. Latrunculin A, but not wortmannin, also reduced vesicle mobilization and exocytosis. Both drugs inhibited the stimulation-induced uptake of the styryl dye FM1-43 and blocked vesicle reformation from internalized membrane objects after tetanic stimulation. These results are consistent with a role of PI3-kinase and the actin cytoskeleton in the slow pathway of vesicle endocytosis, used primarily by reserve pool vesicles.

Sustained stimulation of exocytosis triggers continuous membrane retrieval in rat pituitary somatotrophs.

We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch-clamp capacitance, FM1-43 fluorescence imaging and amperometry. Stimulation of exocytosis through voltage-dependent Ca2+ channels by depolarizations (1-5 s) increased the capacitance by 4.3 +/- 0.9 % and the fluorescence by 6.6 +/- 1.1 % (10 cells). The correlation between the capacitance and fluorescence changes indicated that the cell membrane and granule membrane added via exocytosis were stained with the membrane-bound fluorescent dye FM1-43 in a quantitatively similar manner. Intracellular dialysis (0.5-4.5 min) with elevated Ca2+ (1.5-100 microM) evoked continuous exocytosis that was detected with a carbon fibre electrode from dopamine-loaded cells (10 cells) or as an increase in FM1-43 fluorescence (56 +/- 10 %; 21 cells). Interestingly during Ca2+ dialysis the capacitance did not significantly change (2 +/- 1 %; 31 cells), indicating that endocytosis efficiently retrieved increased cell membrane. Sustained endocytosis was not blocked when the intracellular GTP (300 microM) was replaced with GTP[gamma]S. Replacing intracellular Ca2+ (100 microM) with Ba2+ (300 microM) or Sr2+ (200 microM), or reducing the pH of the intracellular solution from 7.2 to 6.2 did not block sustained endocytosis. Our results suggest that pituitary somatotrophs have the ability to undergo continuous exocytosis and membrane retrieval that persist in whole-cell recordings.

Intraterminal Ca(2+) and spontaneous transmitter release at the frog neuromuscular junction.

We investigated the relationship between intraterminal Ca(2+) concentration ([Ca(2+)](i)) and the frequency of miniature end plate potentials (MEPPs) at the frog neuromuscular junction by use of ratiometric imaging of fura-2-loaded nerve terminals and intracellular recording of MEPPs. Elevation of extracellular [KCl] over the range of 2-20 mM resulted in increases in [Ca(2+)](i) and MEPP frequency. Loading terminals with the fast and slow Ca(2+)-buffers bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl (BAPTA-AM) and EGTA-AM resulted in equivalent reductions in the KCl-dependent increases in MEPP frequency. The [Ca(2+)](i) dependence of MEPP frequency determined by elevation of [Ca(2+)](i) due to application of 0.1-10 microM ionomycin was similar to that determined when [Ca(2+)](i) was raised by increasing extracellular KCl. Measurements in 10 mM extracellular KCl revealed that application of the phorbol ester phorbol 12 myristate 13-acetetate (PMA) caused an increase in MEPP frequency while the inactive analogue, 4 alpha-PMA, did not. PMA application also caused an increase in [Ca(2+)](i). The relationship between [Ca(2+)](i) and MEPP frequency in PMA was the same as was determined by the other methods of raising [Ca(2+)](i). Under all conditions tested, our data revealed a low [Ca(2+)](i) threshold for activation of transmitter release and are consistent with a K(d) for [Ca(2+)](i) on the order of 1 microM.

Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals.

We have identified and characterized two vesicle recycling pathways in frog motor nerve terminals. We exploited the differential staining properties of FM dyes of varying hydrophobicity to label selectively two different vesicle pools, using optical imaging and electron microscopy of photoconverted dyes. During a 1 min tetanus, a rapidly recycling route places vesicles selectively into a small readily releasable pool comprising about 20% of vesicles. After the tetanus, a much slower pathway (from which FM2-10 but not FM1-43 can be rinsed) delivers vesicles via infoldings and cisternae selectively to a reserve pool with a halftime of about 8 min. Mixing between the two pools is slow. During stimulation at 30 Hz, 10-15 s is required to mobilize and release dye from the reserve pool.

Differential regulation of granule-to-granule and granule-to-plasma membrane fusion during secretion from rat pituitary lactotrophs.

We used fluorescence imaging of individual exocytic events together with electron microscopy to study the regulation of dense core granule-to-plasma membrane fusion and granule-to-granule fusion events that occur during secretion from rat pituitary lactotrophs. Stimulating secretion with elevated extracellular potassium, with the calcium ionophore ionomycin, or with thyrotropin releasing hormone or vasoactive intestinal polypeptide resulted in abundant exocytic structures. Approximately 67% of these structures consisted of multiple granules fused together sharing a single exocytic opening with the plasma membrane, i.e., compound exocytosis. For all of these stimulation conditions there appeared to be a finite number of plasma membrane fusion sites, approximately 11 sites around each cellular equator. However, a granule could fuse directly with another granule that had already fused with the plasma membrane even before all plasma membrane sites were occupied. Granule-to-plasma membrane and granule-to-granule fusion events were subject to different regulations. Forskolin, which can elevate cAMP, increased the number of granule-to-granule fusion events without altering the number of granule-to-plasma membrane fusion events. In contrast, the phorbol ester PMA, which activates protein kinase C increased both granule-to-granule and granule-to-plasma membrane fusion events. These results provide a cellular mechanism that can account for the previously demonstrated potentiation of secretion from lactotrophs by cAMP- and PKC-dependent pathways.

Spatial variability in release at the frog neuromuscular junction measured with FM1-43.

We quantified the spatial variability in release properties at different synaptic vesicle clusters in frog motor nerve terminals, using a combination of fluorescence and electron microscopy. Individual synaptic vesicle clusters labeled with FM1-43 varied more than 10-fold in initial intensity (integrated FM1-43 fluorescence) and in absolute rate of dye loss during tetanic electrical nerve stimulation. Most of this variability arose because large vesicle clusters spanned more than one presynaptic active zone (inferred from postsynaptic acetylcholine receptor stripes labeled with rhodamine-conjugated alpha-bungarotoxin); when the rate of dye loss was normalized to the length of receptor stripe covered, variability from spot to spot was greatly reduced. In addition, electron microscopic measurements showed that large vesicle clusters (i.e., those spanning multiple active zones) were also thicker, and the increased depth of vesicles led to increased total spot fluorescence without a corresponding increase in the rate of dye loss during stimulation. These results did not reveal the presence of "hot zones" of secretory activity.

Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events.

Using FM1-43 fluorescence, we have optically detected single exocytic and endocytic events in rat pituitary lactotrophs. About fifty discrete fluorescent spots abruptly appear around the entire surface of a cell bathed in FM1-43 and high-potassium saline. The spots, which also immunostain for prolactin, reflect the labeling of dense cores as well as membranes of exocytosed secretory granules. Stained cores are not released, but remain attached to the cell and are eventually endocytosed. However, in cells exposed to dopamine (or an analog, bromocriptine), the cores dissolve and are secreted after several seconds. Solubilization of dense cores is mediated through a reduction in cytoplasmic cyclic AMP. Thus, the composition of secretions from individual secretory granules is regulated.

Monitoring secretory membrane with FM1-43 fluorescence.

FM1-43 and similar styryl dyes have proven useful as probes for membrane trafficking because they reversibly stain membranes, are impermeable to membranes, and are more fluorescent when bound to membranes than when in solution. Because these dyes stain membranes in an activity-dependent manner, they are ideal for studies of neurotransmitter release mechanisms such as synaptic vesicle recycling, exocytosis, and endocytosis. FM dyes have been used in conjunction with other techniques such as fluorescent calcium indicator dyes and electrophysiological techniques to elucidate mechanisms of presynaptic calcium homeostasis and modulation of neurotransmitter release. Presynaptic membranes have been marked by FM dyes in studies of synaptogenesis and reinnervation. As a probe for endocytosed membranes, these dyes have been used to examine vacuole formation in yeast. These versatile membrane dyes are useful in a variety of applications.

Kinetics of synaptic depression and vesicle recycling after tetanic stimulation of frog motor nerve terminals.

We measured the time courses of two key components of the synaptic vesicle cycle during recovery from synaptic depression under different conditions, and used this and other information to create a kinetic model of the vesicle cycle. End plate potential (EPP) amplitudes were used to follow recovery from synaptic depression after different amounts of tetanic stimulation. This provided an estimate of the time course of vesicle mobilization from the reserve pool to the docked (readily releasable) pool. In addition, FM1-43 was used to measure the rate of membrane retrieval after tetanic stimulation, and the amount of membrane transferred to the surface membrane. This provided a measure of the rate of refilling of the reserve pool with recycled vesicles. The time courses of both synaptic depression and endocytosis were slowed by prolonged tetanic stimulation. This behavior could be fitted by a simple model, assuming a first-order kinetics for both vesicle endocytosis and mobilization. The results show that a nearly 20-fold decrease in the rate constant of endocytosis greatly delays refilling of the depleted reserve pool. However, to fully account for the slower recovery of depression, a decrease in the rate constant of vesicle mobilization from the reserve pool of about sixfold is also required.

The synaptic vesicle cycle.

The ins and outs of the synaptic vesicle cycle are being examined in increasing detail with diverse investigative tools in a variety of cell types, particularly those with large granules. The cycle begins with the opening of a fusion pore that connects the vesicle lumen to the extracellular fluid. Sensitive electrophysiological techniques reveal the often-stuttering behavior of single pores in non-neuronal cells, through which small molecules trickle until the fusion pore expands and the remaining contents erupt from the vesicle. The granule membranes are then retrieved by multiple processes that appear to act in parallel and that are distinguished from each other kinetically and ultrastructurally. Following endocytosis, synaptic vesicles are then shuttled back into the vesicle pool, where they briefly mix with other vesicles, become immobilized, and remain gelled with their neighbors, even while moving en masse again to the presynaptic membrane as a prelude for another round of exocytosis.

Monitoring secretion in real time: capacitance, amperometry and fluorescence compared.

Techniques for measuring exocytosis, endocytosis and vesicle cycling in living cells in real time have resulted in a rapid expansion in the knowledge of these processes in neurons and other secretory cells. Several experimental approaches, developed during the past decade, have played key roles in this expansion. In this review we focus on three techniques: electrophysiological methods for monitoring membrane capacitance, electrochemical methods for detecting released secretory contents and optical methods for imaging membranes of endosomes and recycled vesicles that are stained with fluorescent dyes. Each technique has contributed unique and complementary information about the vesicle cycle, advancing our knowledge of the kinetics of membrane fusion and retrieval, the identity of the secretory contents and the spatial patterns and directional pathways involved in secretory membrane recycling. Naturally, each technique has inherent limitations; some of these shortcomings have recently been resolved by using more than one method simultaneously.

Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction.

We used FM1-43 imaging and intracellular recordings of synaptic potentials to measure the time course of endocytosis in frog motor nerve terminals following tetanic nerve stimulation, and we used fura-2 imaging of intraterminal Ca2+ concentration to compare endocytic rate and [Ca2+]i. Following a 30 Hz tetanus, endocytosis declined exponentially with a time constant that depended on the duration of stimulation. The level of [Ca2+]i rose from a resting value of about 100 nM to more than 500 nM during 30 Hz stimulation, and rapidly declined to 200-250 nM after stimulation. [Ca2+]i returned to resting level with a time course that, like endocytosis, depended on the duration of tetanic stimulation. However, the rate of [Ca2+]i recovery was much slower than the rate of endocytosis, leading to the conclusion that endocytic rate is not determined solely by the instantaneous level of [Ca2+]i.

Imaging exocytosis and endocytosis.

From the secretion of neurotransmitters via synaptic vesicles to the expulsion of cellular waste via contractile vacuoles, exocytosis and its sequel, endocytosis, are being explored with a variety of new optical tools. Fluorescent markers, especially styryl dyes such as FM1-43 (which reversibly labels endosomal membranes), have been used to follow exo- and endocytic events in many cell types. Even though the development of new dyes is still largely empirical, some theoretical principles have emerged to guide future dye chemistry. Moreover, advances in optical imaging technology that augment conventional fluorescence microscopy are appearing. For example, interference reflection microscopy (which requires no flurophore) and total internal reflection microscopy have recently been used to observe single exocytic events at the contact point between a glass coverslip and the plasma membrane.

Synaptic vesicle movements monitored by fluorescence recovery after photobleaching in nerve terminals stained with FM1-43.

We used the fluorescence recovery after photobleaching technique to monitor movements of synaptic vesicles in top views of living frog motor nerve terminals that had been prestained with the fluorescent dye FM1-43. In each experiment, a small portion of a single stained vesicle cluster was bleached with a laser and monitored subsequently for signs of recovery as neighboring, unbleached vesicles moved into the bleached region. In resting terminals, little or no recovery from photobleaching occurred. Repetitive nerve stimulation, which caused all fluorescent spots to grow dim as dye was released from exocytosing vesicles, did not promote recovery from photobleaching. Pretreatment with botulinum toxin (type A, C, or D) blocked exocytosis and destaining, but intense nerve stimulation still did not cause significant recovery in bleached regions. These results suggest that lateral movements of synaptic vesicles are restricted severely in both resting and stimulated nerve terminals. We tested for laser-induced photodamage in several ways. Bleached regions could be restained fully with FM1-43, and these restained regions could be destained fully by nerve stimulation. Partially bleached regions could be destained, although the rate of destaining was lower than normal. Brisk recovery from photobleaching occurred after treatment with okadaic acid, which disrupts synaptic vesicle clusters and causes vesicles to spread throughout the nerve terminal. These results suggest that vesicle translocation and recycling machinery was intact in photobleached regions.

Simultaneous independent measurement of endocytosis and exocytosis.

Studies of membrane traffic between the cytoplasm and surface of a cell suggest that membrane internalization is tightly coupled to secretion. Studies using the capacitance technique show that endocytosis can follow evoked exocytosis within a second or less. The capacitance technique, however, measures only the net change in cell surface area, and thus separating exocytosis from endocytosis requires that the two events do not overlap in time. This condition is probably met with small, brief stimuli, but during prolonged stimulation it is more likely that exocytosis and endocytosis occur simultaneously, We used FM1-43 fluorescence, which provides a cumulative measure of exocytosis, independent of endocytosis, in combination with capacitance monitoring to track unidirectional movements of membrane simultaneously and in real time in bovine adrenal chromaffin cells. We confirm that, with relatively small stimuli, exocytosis ceases before endocytosis begins (no overlap). In contrast, during prolonged stimulation, the onset of endocytosis is delayed by 2-3 min, but then the rate of endocytosis quickly grows to equal that of exocytosis. The delayed onset of endocytosis may be an emergency defence against catastrophic cell swelling.

FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.

Previous work has shown that the fluorescent styryl dye FM1-43 stains nerve terminals in an activity-dependent fashion. This dye appears to label the membranes of recycled synaptic vesicles by being trapped during endocytosis. Stained terminals can subsequently be destained by repeating nerve stimulation in the absence of dye; the destaining evidently reflects escape of dye into the bathing medium from membranes of exocytosing synaptic vesicles. In the present study we tested two key aspects of this interpretation of FM1-43 behavior, namely: (i) that the dye is localized in synaptic vesicles, and (ii) that it is actually released into the bathing medium during destaining. To accomplish this, we first photolyzed the internalized dye in the presence of diaminobenzidine. This created an electron-dense reaction product that could be visualized in the electron microscope. Reaction product was confined to synaptic vesicles, as predicted. Second, using spectrofluorometry, we quantified the release of dye liberated into the medium from tubocurarine-treated nerve-muscle preparations. Nerve stimulation increased the amount of FM1-43 released, and we estimate that normally a stained synaptic vesicle contains a few hundred molecules of the dye. The key to the successful detection of released FM1-43 was to add the micelle-forming detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which increased FM1-43 quantum yield by more than two orders of magnitude.