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 Ca(2+) 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 Ca(2+) 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.

Leptin regulates hypothalamic POMC+ (pro-opiomelanocortin) neurons by inducing TRPC (Transient Receptor Potential Cation) channel-mediate membrane depolarization. The role of TRPC channels in POMC neuron excitability is clearly established; however, it remains unknown whether their activity alone is sufficient to trigger excitability. Here we show that the right-shift voltage induced by the leptin-induced TRPC channel-mediated depolarization of the resting membrane potential brings T-type channels into the active window current range, resulting in an increase of the steady state T-type calcium current from 40 to 70% resulting in increased intrinsic excitability of POMC neurons. We assessed the role and timing of T-type channels on excitability and leptin-induced depolarization in vitro in cultured mouse POMC neurons. The involvement of TRPC channels in the leptin-induced excitability of POMC neurons was corroborated by using the TRPC channel inhibitor 2APB, which precluded the effect of leptin. We demonstrate T-type currents are indispensable for both processes, as treatment with NNC-55-0396 prevented the membrane depolarization and rheobase changes induced by leptin. Furthermore, co-immunoprecipitation experiments suggest that TRPC1/5 channels and CaV3.1 and CaV3.2 channels co-exist in complex. The functional relevance of this complex was corroborated using intracellular Ca2+ chelators; intracellular BAPTA (but not EGTA) application was sufficient to preclude POMC neuron excitability. However, leptin-induced depolarization still occurred in the presence of either BAPTA or EGTA suggesting that the calcium entry necessary to self-activate the TRPC1/5 complex is not blocked by the presence of BAPTA in hypothalamic neurons. Our study establishes T-type channels as integral part of the signaling cascade induced by leptin, modulating POMC neuron excitability. Leptin activation of TRPC channels existing in a macromolecular complex with T-type channels recruits the latter by locally induced membrane depolarization, further depolarizing POMC neurons, triggering action potentials and excitability.

No abstract

Leptin regulates hypothalamic POMC+ (pro-opiomelanocortin) neurons by inducing TRPC (Transient Receptor Potential Cation) channel-mediate membrane depolarization. Here we assessed the role of T-type channels on POMC neuron excitability and leptin-induced depolarization in vitro. We demonstrate T-type currents are indispensable for both processes, as treatment with NNC-55-0396 prevented the membrane depolarization and rheobase changes induced by leptin in cultured mouse POMC neurons. Furthermore, we demonstrate TRPC1/C5 channels and CaV3.1 and CaV3.2 channels co-exist in complex. The functional relevance of this complex was corroborated using intracellular Ca2+ chelators; intracellular BAPTA (but not EGTA) application was sufficient to preclude POMC neuron excitability by preventing leptin-induced calcium influx through TRPC channels and T-type channel function.We conclude T-type channels are integral in POMC neuron excitability. Leptin activation of TRPC channels existing in a macromolecular complex with T-type channels recruits the latter by locally-induced membrane depolarization, further depolarizing POMC neurons, triggering action potentials and excitability.