William J. Betz

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.

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.

Depression of transmitter release at the neuromuscular junction of the frog

1. Depression of transmitter release produced by preceding conditioning stimulation was studied at the frogs neuromuscular junction.

Optical monitoring of transmitter release and synaptic vesicle recycling at the frog neuromuscular junction.

1. Frog cutaneous pectoris motor nerve terminals were loaded with the fluorescent dye FM1‐43, which produced a series of discrete spots along the length of terminals, each spot evidently marking a cluster of synaptic vesicles. Terminals were imaged for 2‐10 min as they destained during repetitive nerve stimulation. Endplate potentials (EPPs) were recorded simultaneously from the muscle fibres innervated by these terminals; their summed amplitudes provided a measure of cumulative transmitter release. 2. Individual fluorescent spots in any one terminal varied in initial brightness but destained at similar fractional rates. 3. The rates of cumulative transmitter release and destaining increased with stimulus frequency in the range 2‐30 Hz. At 40 Hz, however, both transmitter release and destaining were slower than at 30 Hz. 4. In twenty‐six experiments, rates of dye loss and transmitter release were compared quantitatively. When the time course of summed EPPs was scaled to fit the time course of dye loss during the first 30‐60 s of destaining, the two curves usually diverged at later times, the dye loss curve falling below the summed EPP curve. Thus, assuming that dye loss and transmitter release are proportional at early times, at later times the rate of dye loss decreases relative to the rate of transmitter release. 5. At stimulus frequencies from 2 to 30 Hz, the results could be fitted by a simple model in which vesicles lose their dye during exocytosis and, after a fixed recycle ‘dead time’, they re‐enter the vesicle pool, mixing randomly with other vesicles. 6. Unlike stimulation at lower frequencies, at 40 Hz dye loss and summed EPP amplitude curves did not significantly diverge. Stimulation periods lasted up to about 2 min. Interpreted according to the model of vesicle recycling, this suggests that vesicle recycling is inhibited at 40 Hz. 7. The model led to predictions about the relative number, N, of vesicles (labelled and unlabelled) in the terminal at any time during stimulation. The calculated value of N decreased at times less than the recycle ‘dead time’, and then increased, reflecting the appearance of recycled vesicles in the vesicle pool. 8. From estimates of N and recorded EPP amplitudes, the fraction of vesicles released per shock, F, could be calculated during the entire stimulation period. At low stimulus frequencies (2‐5 Hz), after an initial rapid fall, F decreased slowly and monotonically by about 50% in 6 min. At higher stimulus frequencies, a different process was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions

1. Frog cutaneous pectoris nerve‐muscle preparations were incubated with collagenase and protease and examined with electrophysiological and electron microscopic techniques.

Synaptic vesicle pools at the frog neuromuscular junction

We have characterized the morphological and functional properties of the readily releasable pool (RRP) and the reserve pool of synaptic vesicles in frog motor nerve terminals using fluorescence microscopy, electron microscopy, and electrophysiology. At rest, about 20% of vesicles reside in the RRP, which is depleted in about 10 s by high-frequency nerve stimulation (30 Hz); the RRP refills in about 1 min, and surprisingly, refilling occurs almost entirely by recycling, not mobilization from the reserve pool. The reserve pool is depleted during 30 Hz stimulation with a time constant of about 40 s, and it refills slowly (half-time about 8 min) as nascent vesicles bud from randomly distributed cisternae and surface membrane infoldings and enter vesicle clusters spaced at regular intervals along the terminal. Transmitter output during low-frequency stimulation (2-5 Hz) is maintained entirely by RRP recycling; few if any vesicles are mobilized from the reserve pool.

Imaging synaptic vesicle exocytosis and endocytosis with FM dyes

FM dyes have been used to label and then monitor synaptic vesicles, secretory granules and other endocytic structures in a variety of preparations. Here, we describe the general procedure for using FM dyes to study endosomal trafficking in general, and synaptic vesicle recycling in particular. The dye, dissolved in normal saline solution, is added to a chamber containing the preparation to be labeled. Stimulation evokes exocytosis, and compensatory endocytosis that follows traps FM dye inside the retrieved vesicles. The extracellular dye is then washed from the chamber, and labeled endocytic structures are examined with a fluorescence microscope. Fluorescence intensity provides a direct measure of the labeled vesicle number, a good measure of the amount of exocytosis. If the preparation is stimulated again, without dye in the chamber, dimming of the preparation provides a measure of exocytosis of labeled vesicles. With a synaptic preparation on hand, this protocol requires 1 day.

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.

The effects of partial denervation at birth on the development of muscle fibres and motor units in rat lumbrical muscle.

1. Two aspects of nerve‐muscle development were studied in neonatal rats, the role of competition between motor neurones during the elimination of polyneuronal innervation, and the dependence of muscle fibre production upon the number of motor neurones innervating the muscle. 2. Rat lumbrical muscles were partially denervated at birth by cutting the lateral plantar nerve. Many muscles remained innervated by a single motor axon from the sural nerve. These motor units developed in the complete absence of competition from other motor units. In the adult muscles the number of innervated muscle fibres was approximately the same as at birth (about 120 muscle fibres). 3. In muscles that were totally denervated at birth, the normal post‐natal production of muscle fibres was arrested. In partially denervated muscles, the production of new muscle fibres depended on the number of remaining motor units. The relationship between the total number of muscle fibres and the number of remaining motor units was fitted by a simple model. 4. The results suggest that in the lumbrical muscle, the decrease in motor unit size that occurs during normal development can be accounted for entirely by competition between motor nerve terminals. 5. The results also suggest that the normal post‐natal increase in the total number of muscle fibres depends on a trophic interaction between the muscle and its innervation.