Synaptic Vesicle Recycling in Cultured Cerebellar Granule Cells: Role of Vesicular Acidification and Refilling

Abstract

Abstract:The role of the transvesicular protonmotive force in synaptic vesicle recycling was investigated in cultured cerebellar granule cells. The vesicular V-ATPase was inhibited by 1 ,uM bafilomycin Al; as an alternative, the pH component of the gradient was selectively collapsed by equilibration of the cells with 10 mM methylamine and monitored with the fluorescent probe Lysosensor Green. Electrical field-evoked exocytosis of D-[ 3H]aspartate was inhibited by bafilomycin Al but not by methylamine, indicating that a transvesicular membrane potential rather than pH gradient is required for transmitter retention within vesicles. In contrast, neither compound affected the field-evoked uptake, recycling, or destaining of the vesicle-specific dye FM2-10; thus, vesicles whose lumens were neutral and/or depleted of transmitter could still recycle in the nerve terminal. No exhaustion of o-[3H]aspartate exocytosis was observed when cells were subjected to six consecutive trains of field stimuli (40 Hz/l0 s separated by 10 s). In contrast, the release of preloaded FM2-10 was reduced by -~50%, with each stimulus indicating that unlabeled vesicles with accumulated D-[3H]aspartate were competing with labeled vesides for exocytosis. As D-[3HI aspartate was accumulated rapidly across the vesicle membrane from the large cytoplasmic pool, the transmitter-loaded but unlabelled vesides may represent refilled recycling vesicles. FM2-l 0 destaining and D-[3H]aspartate exocytosis were reduced in parallel at low frequencies, challenging a role for transient vesicle fusion. Key Words: Exocytosis-Vesicle-Refilling-D-[3H]Aspartate-FM1 -43-Granule cell.

Vesicular Release of L- and D-Aspartate from Hippocampal Nerve Terminals: Immunogold Evidence~!2008-09-01~!2008-10-15~!2008-12-05~!

“…Thus, the present results and those of Fleck and co-workers [14] strongly suggest that also D-aspartate is transported into synaptic vesicles, probably by the same transporter as Laspartate. This is in line with the large body of evidence showing exocytosis of both L-aspartate [1-4, 6-10, 14] and D-aspartate [11,12,[40][41][42][43][44][45].…”

“…Strongly in favour of an exocytotic release mechanism are our previous immunocytochemical results showing that K + induced depolarisation elicited depletion of L-aspartate from nerve endings, which could be inhibited by low extracellular Ca 2+ -concentrations and tetanus toxin [8][9][10]. In addition, Daspartate has been shown to be subject to exocytotic release both from cultured neurons [11] There is ample evidence that excitatory nerve endings in the brain contain functional plasma membrane excitatory amino acid transporters (EAATs) [16][17][18][19][20]. The previous aspartate release data do not clearly distinguish between Laspartate release via exocytosis and via these EAATs, which could release aspartate either through reversal of the transporter or through heteroexchange with glutamate released from synaptic vesicles.…”

“…Strongly in favour of an exocytotic release mechanism are our previous immunocytochemical results showing that K + induced depolarisation elicited depletion of L-aspartate from nerve endings, which could be inhibited by low extracellular Ca 2+ -concentrations and tetanus toxin [8][9][10]. In addition, Daspartate has been shown to be subject to exocytotic release both from cultured neurons [11] synaptosomes [12] and intact brain tissue [13]. Taken together, these results indicate that synaptic vesicles harbour a transporter for L-and/or Daspartate (see [14]).…”

Prolonged Reciprocal Signaling via NMDA and GABA Receptors at a Retinal Ribbon Synapse

“…Pipettes were coated with regular dental wax (Cavex, West Chester, PA) to reduce pipette capacitance and electrical noise and filled with solution comprising the following (in mM): 95 Cs-gluconate, 25 HEPES, 10 TEACl, 3 Mg-ATP, 0.5 Na-GTP, and 0.5 EGTA, adjusted to pH 7.2 with CsOH. Methylamine HCl (10 mM) was also routinely included to buffer vesicular pH (Cousin and Nicholls, 1997;Vigh et al, 2005). The osmolarity was adjusted to 257 Ϯ 2 mOsm.…”

Glucose is Necessary to Maintain Neurotransmitter Homeostasis during Synaptic Activity in Cultured Glutamatergic Neurons

“…Conceivably, glucose and lactate may contribute differently to maintain these processes. The release of glutamate was monitored by the use of the nonmetabolizable glutamate analog D-[ 3 H]aspartate, which labels the vesicular as well as the cytoplasmic glutamate pools (Belhage et al, 1992;Cousin and Nicholls, 1997;Fleck et al, 2001). To examine a possible contribution of the transporters to the release of D-[ 3 H]aspartate, the nontransportable glutamate transport inhibitor DL-threo-b-benzyloxyaspartate (TBOA) was employed (Shimamoto et al, 1998;Waagepetersen et al, 2001;Bak et al, 2003Bak et al, , 2004.…”