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We have investigated whether rat motor nerve terminals with different in vivo activity patterns also have different vesicle trafficking characteristics. To do this, we monitored, using combined optical and electrical techniques, the rate of exocytosis (during different frequencies and patterns of activity), the releasable pool size, and the recycle time of synaptic vesicles in terminals on soleus (slow-twitch) and extensor digitorum longus [(EDL); fast-twitch] muscle fibers. EDL terminals had a higher initial quantal content (QC) than soleus, but during tonic or phasic stimulation at 20-80 Hz, EDL QC ran down to a greater extent than soleus QC. By recording loss of fluorescence from exocytosing vesicles labeled with the dye FM1-43, EDL terminals were found to destain faster than those in soleus. Simultaneous intracellular recording of end plate potentials, to count the number of vesicles released, permitted estimation of the total vesicle pool (VP) size and the recycle time by combining the optical and electrophysiological data. Soleus vesicle pool was larger than EDL, but recycle time was not significantly different. These terminals, therefore, are adapted to their in vivo activity patterns by alterations in QC and VP size but not recycle time.

Congenital myasthenic syndromes are a heterogeneous group of conditions in which muscle weakness resulting from impaired neuromuscular transmission is often present from infancy. One form of congenital myasthenic syndrome is due to a reduction of the number of acetylcholine receptors (AChRs) at the neuromuscular junction. We describe four new cases of AChR deficiency, characterized by a reduction in both miniature endplate potential amplitude and AChR abundance accompanied by elongation of the neuromuscular junction and some decrease in postsynaptic folding. A number of cytoplasmic proteins are normally associated with the postsynaptic membrane and may contribute to the clustering of AChRs at the neuromuscular junction. We therefore investigated the expression of several of these proteins in these AChR-deficiency patients. In each patient, immunolabelling of the neuromuscular junction for rapsyn, dystrophin, beta-dystroglycan and a form of beta-spectrin was strong but that for utrophin was markedly reduced or absent. This suggested that a defect in utrophin expression might underlie the congenital AChR deficiency. However, a reduction in utrophin labelling was also seen in three patients with adult acquired autoimmune myasthenia gravis in whom AChR loss results directly from the extracellular binding of autoantibodies. We conclude that the loss of AChRs in AChR deficiency does not result from the absence of rapsyn or beta-dystroglycan and that reduction of utrophin is probably secondary to the loss of AChRs. The possible role of AChRs and/or utrophin in determining the extent of postsynaptic folding is discussed.

The styryl pyridinium dye RH414, which we have used recently for optical monitoring of synaptic vesicle membrane trafficking in frog motor nerve terminals, reversibly inhibited contractions in the frog nerve-muscle preparations used for those studies. We report here the results of experiments into the basis of this inhibition and how the blocking effects can be modulated by incident light, by using conventional intracellular recording and iontophoretic techniques. Bath application of 5-42.5 microM RH414 blocked nerve-evoked twitches in frog cutaneus pectoris muscles, although subthreshold endplate potentials and miniature endplate potentials could still be recorded. In the presence of the dye, illumination of the recording area with light from a mercury arc lamp over a wide range of wavelengths (340-560 nm) potentiated the amplitude of endplate potentials, miniature endplate potentials, and depolarizations resulting from iontophoretic application of acetylcholine. The magnitude of both the light-induced disinhibition of endplate and iontophoretic potentials increased with the intensity of the illumination and developed exponentially with a time constant of several hundred milliseconds. Both the dye-induced inhibition and the light-induced disinhibition disappeared after washing in dye-free physiological saline. Muscle fibre resting membrane potential, input conductance and miniature endplate potential frequency, however, were not affected by these manipulations. These data are consistent with a specific, curare-like interaction of the dye with acetylcholine receptors which can be modulated by light.

Living motor nerve terminals from several species can be stained in an activity-dependent fashion by certain styryl dyes, such as RH414, RH795, and a new dye, FM1-43, which can be imaged independently of the others. The dyes evidently become trapped within recycled synaptic vesicles. In frog cutaneus pectoris muscle, bright fluorescent spots spaced regularly along the length of the nerve terminals appear after stimulation in the presence of the dye. The spots align well with postsynaptic ACh receptors and are persistent for many hours, unless further stimulation is given, in which case the spots disappear. Destaining, like staining, requires transmitter release and proceeds gradually over several minutes at high stimulus frequencies (e.g., 30 Hz), and fluorescent spots in the same terminal disappear at about the same rate. We suggest that each spot is a cluster of hundred of synaptic vesicles and that the mechanism of staining involves the ability of the dyes to partition reversibly into the outer leaflet of surface membranes, without being able to penetrate the entire membrane thickness. Then, during endocytosis following transmitter release, dye molecules become trapped in recycled synaptic vesicle membranes. The dyes therefore make it possible optically to study vesicle exocytosis and recycling in living nerve terminals in real time, and should be useful for marking terminals in a variety of preparations according to their level of activity.

The fluorescent dyes FM1-43 and RH414 label motor nerve terminals in an activity-dependent fashion that involves dye uptake by synaptic vesicles that are recycling. This allows optical monitoring of vesicle recycling in living nerve terminals to determine how recycled vesicles reenter the vesicle pool. The results suggest that recycled vesicles mix with the pool morphologically and functionally. One complete cycle of release of transmitter, recycling of a vesicle, and rerelease of transmitter appears to take about 1 minute.