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.

An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.

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.

The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletalneuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.

Using light and serial electron microscopy, we show profound refinements in motor axonal branching and synaptic connectivity before and after birth. Embryonic axons become maximally connected just before birth when they innervate ∼10-fold more muscle fibers than in maturity. In some developing muscles, axons innervate almost every muscle fiber. At birth, each neuromuscular junction is coinnervated by approximately ten highly intermingled axons (versus one in adults). Extensive die off of terminal branches occurs during the first several postnatal days, leading to much sparser arbors that still span the same territory. Despite the extensive pruning, total axoplasm per neuron increases as axons elongate, thicken, and add more synaptic release sites on their remaining targets. Motor axons therefore initially establish weak connections with nearly all available postsynaptic targets but, beginning at birth, massively redistribute synaptic resources, concentrating many more synaptic sites on many fewer muscle fibers. Analogous changes in connectivity may occur in the CNS.