Making quantal analysis more convenient, fast, and accurate: User-friendly software QUANTAN

2010

Neuroscience

Synapsin is a phosphoprotein reversibly associated with synaptic vesicles. We investigated synapsin function in mediating synaptic activity during intense stimulation at Drosophila motor boutons. Electron microscopy analysis of synapsin(−) boutons demonstrated that synapsin maintains vesicle clustering over the periphery of the bouton. Cyclosporin A pretreatment disrupted peripheral vesicle clustering, presumably due to increasing synapsin phosphorylated state. Labeling recycling vesicles with a fluorescent dye FM1-43 followed by photoconversion of the dye into electron dense product demonstrated that synapsin deficiency does not affect mixing of the reserve and recycling vesicle pools but selectively reduces the size of the reserve pool. Intense stimulation produced a significant increase in vesicle abundance and vesicle redistribution toward the central core of synapsin (+) boutons, while in synapsin (−) boutons the area occupied by vesicles did not change and the increase in vesicle numbers was not as prominent. However, intense stimulation produced an increase in basal release at synapsin(−) but not in synapsin(+) boutons, suggesting that synapsin may direct vesicles to the reserve pool. Finally, synapsin deficiency inhibited an increase in quantal size and formation of endosome-like cisternae, which was activated either by intense electrical stimulation or by high K+ application. Taken together, these results elucidate a novel synapsin function, specifically, promoting vesicle reuptake and reserve pool formation upon intense stimulation.

Section: Methodsmentioning

confidence: 99%

Synapsin regulates vesicle organization and activity-dependent recycling at Drosophila motor boutons

Akbergenova

2010

Neuroscience

“…The method of direct counts was used either to detect spontaneous quanta or to detect synchronous quanta at 0.2 and 0.5 mM [Ca 2+ ] o when synaptic transmission was low, and quantal counts were reliable. Quanta were counted as inflections or peaks of EPSP traces using in-house software (Bykhovskaia 2008). At high-output synapses or at physiological Ca 2+ concentration, quantal content was calculated as the ratio between EPSP amplitude and quantal amplitude.…”

Section: Methodsmentioning

confidence: 99%

Cooperative regulation of neurotransmitter release by Rab3a and synapsin II

Coleman

Molecular and Cellular Neuroscience

2010

Self Cite

To understand how the presynaptic proteins synapsin and Rab3a may interact in the regulation of the synaptic vesicle cycle and the release process, we derived a double knockout (DKO) mouse lacking both synapsin II and Rab3a. We found that Rab3a deletion rescued epileptic-like seizures typical for synapsin II gene deleted animals (SynII(−)). Furthermore, action potential evoked release was drastically reduced in DKO synapses, although spontaneous release remained normal. At low Ca2+ conditions, quantal content was equally reduced in Rab3a(−) and DKO synapses, but as Ca2+ concentration increased, the increase in quantal content was more prominent in Rab3a(−). Electron microscopy analysis revealed that DKO synapses have a combined phenotype, with docked vesicles being reduced similar to Rab3a(−), and intraterminal vesicles being depleted similar to SynII(−). Consistently, both SynII(−) and DKO terminals had increased synaptic depression and incomplete recovery. Taken together, our results suggest that synapsin II and Rab3a have separate roles in maintaining the total store of synaptic vesicles and cooperate in promoting the latest steps of neuronal secretion.

“…Recordings were digitized with Digidata A/D board and Axoscope software (Axon Instruments) and analyzed off-line. Area and amplitude of spontaneous synaptic potentials were measured using in-house software (Bykhovskaia, 2008). Spontaneous activity was recorded for 5 min at each recording site.…”

Section: Experimental Methodsmentioning

confidence: 99%

Enhancement of the endosomal endocytic pathway increases quantal size

Akbergenova

Molecular and Cellular Neuroscience

2009

Self Cite

We combined recordings of spontaneous quantal events with electron microscopy analysis of synaptic ultrastructure to demonstrate that the size of a neurosecretory quantum increases following an activation of the endosomal endocytic pathway. We reversibly activated the endosomal endocytic pathway in Drosophila motor boutons by application of high K+ solution. This treatment produced the formation of numerous cisternae, vacuoles and enlarged vesicles. Spontaneous quantal events recorded immediately after the cessation of high K+ application were significantly enlarged, and this increase in quantal size was reversed after a 10 minute resting period. Actin depolymerization produced by latrunculin B pretreatment inhibited both the formation of endosome-like structures and the increase in quantal size. Loading the preparations with the dye FM1–43 followed by photoconversion of the dye combined with electron microscopy analysis revealed that the observed cisternae are likely to be the product of both bulk membrane retrieval and vesicle fusion.