L-type calcium channels are involved in fast endocytosis at the mouse neuromuscular junction

• Published in: The European journal of neuroscience 2008-03, Vol.27 (6), p.1333-1344
• Main Authors: Perissinotti, Paula P., Tropper, Bárbara Giugovaz, Uchitel, Osvaldo D.
• Format: Article
• Language: English
• Subjects: Animals; Calcium Channel Blockers - pharmacology; Calcium Channels, L-Type - physiology; Electric Stimulation - methods; Endocytosis - drug effects; Endocytosis - physiology; FM2-10; levator auris longus; Male; Mice; Neuromuscular Junction - drug effects; Neuromuscular Junction - physiology; Presynaptic Terminals - drug effects; Presynaptic Terminals - physiology; Synaptic Vesicles - drug effects; Synaptic Vesicles - physiology; Time Factors; vesicle pools; vesicle recycling
• ISSN: 0953-816X; 1460-9568
• DOI: 10.1111/j.1460-9568.2008.06113.x

We used fluorescence microscopy of FM dyes‐labeled synaptic vesicles and electrophysiological recordings to examine the functional characteristics of vesicle recycling and study how different types of voltage‐dependent Ca2+ channels (VDCCs) regulate the coupling of exocytosis and endocytosis at mouse neuromuscular junction. Our results demonstrate the presence of at least two different pools of recycling vesicles: a high‐probability release pool (i.e. a fast destaining vesicle pool), which is preferentially loaded during the first 5 s (250 action potentials) at 50 Hz; and a low‐probability release pool (i.e. a slow destaining vesicle pool), which is loaded during prolonged stimulation and keeps on refilling after end of stimulation. Our results suggest that a fast recycling pool mediates neurotransmitter release when vesicle use is minimal (i.e. during brief high‐frequency stimulation), while vesicle mobilization from a reserve pool is the prevailing mechanism when the level of synaptic activity increases. We observed that specific N‐ and L‐type VDCC blockers had no effect on evoked transmitter release upon low‐frequency stimulation (5 Hz). However, at high‐frequency stimulation (50 Hz), L‐type Ca2+ channel blocker increased FM2‐10 destaining and at the same time diminished quantal release. Furthermore, when L‐type channels were blocked, FM2‐10 loading during stimulation was diminished, while the amount of endocytosis after stimulation was increased. Our experiments suggest that L‐type VDCCs promote endocytosis of synaptic vesicles, directing the newly formed vesicles to a high‐probability release pool where they compete against unused vesicles.