Frequency-Dependent Kinetics and Prevalence of Kiss-and-Run and Reuse at Hippocampal Synapses Studied with Novel Quenching Methods

Harata, Nobutoshi; Choi, Sukwoo; Pyle, Jason L.; Aravanis, Alex; Tsien, Richard W.

doi:10.1016/j.neuron.2005.12.018

Cited by 152 publications

(182 citation statements)

References 47 publications

Supporting

Mentioning

167

Contrasting

Unclassified

Order By: Relevance

“…Secondly, neuroendocrine cells utilize regulation of the fusion pore to control differential transmitter release [3,8,53], forming a critical point of control between 'breed and feed' and 'fight or flight' metabolic states. While neuronal synapses do exhibit an activity-mediated shift between 'kiss and run' and 'full collapse' vesicle cycling [21,35,54], it is not clear if a parallel physiological basis would exist in synaptic systems where vesicles do not contain a proteinacious core. Thus it may be that the divergent dependence of endocytosis on dynamin I in neuronal synapses reflects a different biological function for the transition between 'kiss and run' and 'full collapse' exo-and endocytosis [55].…”

Section: Discussionmentioning

confidence: 99%

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Fulop

Doreian

Smith

2008

Archives of Biochemistry and Biophysics

View full text Add to dashboard Cite

Under low stimulation, adrenal chromaffin cells release freely-soluble catecholamines through a restricted granule fusion pore while retaining the large neuropeptide-containing proteinacious granule core. Elevated activity causes dilation of the pore and release of all granule contents. Thus, physiological differential transmitter release is achieved through regulation of fusion pore dilation. We examined the mechanism for pore dilation utilizing a combined approach of peptide transfection, electrophysiology, electrochemistry and quantitative imaging techniques. We report that disruption of dynamin I function alters both fusion modes. Under low stimulation, interference with dynamin I does not affect granule fusion but blocks its re-internalization. In full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. These data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore and thus contributes to activity-dependent differential transmitter release from the adrenal medulla.

show abstract

Section: Discussionmentioning

confidence: 99%

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Fulop

Doreian

Smith

2008

Archives of Biochemistry and Biophysics

View full text Add to dashboard Cite

show abstract

“…Another elegant way of testing kiss-and-run fusion by use of FM dyes was devised by Harata et al (2006). They labeled hippocampal cultures with FM1-43 and destained in either the presence or the absence of BPB (a small hydrophilic quencher; Fig.…”

Section: Using Fm Dyes To Image the Fine Dynamics Of Exocytosismentioning

confidence: 99%

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

Hoopmann¹,

Rizzoli²,

Betz³

2011

Cold Spring Harb Protoc

View full text Add to dashboard Cite

The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. FM dyes reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. This protocol describes the imaging of synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Nerve terminals are stimulated (electrically or by depolarization with high K + ) in the presence of dye, their vesicles are then allowed to recycle, and finally dye is washed from the chamber. In neuromuscular junction (NMJ) preparations, movements of the muscle must be inhibited if imaging during stimulation is desired (e.g., by application of curare, a potent acetylcholine receptor inhibitor). The main characteristics of FM dyes are also reviewed here, as are recent FM dye monitoring techniques that have been used to investigate the kinetics of synaptic vesicle fusion. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. Reagents FM dye(s)Dye concentrations are usually 2-10 µM for FM1-43 or FM4-64, and 25-40 µM for FM2-10. The dyes readily dissolve in water; stocks of 1-5 mM concentration should be prepared and can be stored at 4˚C. Neuronal tissue or cell sample of interest Equipment Imaging setupAn upright epifluorescence microscope equipped with a 40×-60× water-immersion objective is optimal. When working with cell cultures, the advantages of high-resolution oil-immersion objectives (e.g., 100×/1.4 numerical aperture [NA]) can be exploited by using an inverted setup. For FM1-43, maximal fluorescence is at 465-nm excitation/560-nm emission (Henkel et al. 1996); a conventional fluorescein filter set (435-nm excitation) can also be used. Because the major concern in FM imaging is phototoxicity (which appears usually before any clear signs of photobleaching and alters the ability of the preparations to recycle vesicles), keep the illumination to a minimum by including neutral-density filters. For example, when using a 40×/0.75-NA water-immersion objective with a 100-W mercury lamp, 5%-15% transmission is sufficient. On confocal setups, an argon 488-nm laser line is typically used for excitation, but special care has to be taken to avoid phototoxicity. Images are typically acquired by charge-coupled device (CCD) cameras or photomultiplier tubes, which allows for fast acquisition rates and software-based quantitative image analysis.Neuronal stimulator (see Step 3)

show abstract

“…We visualized vesicle populations using a quenching approach: spH-expressing neurons were stimulated in presence of folimycin, to inhibit re-acidification, which allowed us to measure the size of the vesicle pool released actively. After allowing for complete compensatory endocytosis (90 s) we applied bromophenol blue (BPB, 2mM) to quench the remaining surface spH fluorescence 14 . The non-acidified vesicles that had been endocytosed remained fluorescent.…”

mentioning

confidence: 99%

The same synaptic vesicles drive active and spontaneous release

Groemer²,

2010

View full text Add to dashboard Cite

Synaptic vesicles release neurotransmitter both actively (upon stimulation) and spontaneously (at rest). It has been long assumed that identical vesicles use both modes of release; however, recent evidence has challenged this view. Using several assays (FM dye imaging, pHluorin imaging and antibody-labeling of synaptotagmin), in neuromuscular preparations from Drosophila, frog and mouse as well as rat cultured neurons, we suggest that the same vesicles participate in active and spontaneous release.1 Synaptic vesicles fuse with the neuronal plasma membrane to release their neurotransmitter contents both upon the arrival of action potentials (active release) and at rest (spontaneous release). It has been assumed for almost six decades that identical vesicles exocytose in both cases, an assumption which has been used to generate the quantal (vesicular) release theory 1 . This assumption is in agreement with the fact that prolonged active 2 or spontaneous release 3 can both release essentially all vesicles (see also 4,5 ). The issue of whether the same vesicles can be released both spontaneously and actively has been tested recently by investigating the loading and unloading of styryl (FM) dyes during vesicle recycling 6 . In cultured hippocampal neurons, the vesicles loaded with FM dyes during active or spontaneous recycling were unloaded efficiently only by the same release paradigm. Also, inhibiting synaptic vesicle re-acidification (and therefore refilling with neurotransmitter) by use of folimycin during spontaneous release specifically depleted the spontaneous pool 6 . Surprisingly, both of these findings have been later contested in similar experiments -FM dyes taken up either at rest or during stimulation could be released with identical kinetics, and the inhibition of reacidification during depolarization led to a cessation of spontaneous release 7 (see also 5 ). However, later studies have again found different FM dye release kinetics when using different loading paradigms 8,9 , and labeling experiments using the expression of a biotinylated variant of the synaptic vesicle marker VAMP2 (synaptobrevin) also supported the hypothesis that spontaneously and actively recycling vesicles are different 10 .We tested here this controversial issue by combining several fluorescence imaging assays. We sought to reproduce the FM dye loading/unloading paradigms used in the past. A number of FM dyes have been used, including FM 1-43 and FM 2-10 6-9 ; both dyes have provided evidence for a difference between the spontaneously and actively recycling vesicles 8,9 . It has been recently suggested that FM 2-10 reports this difference better than FM 1-43 9 . However, as FM 2-10 is less bright when compared to FM 1-43, it is typically used at very high concentrations (400 µM; ~4-fold higher than its membrane dissociation constant 11 ). Since the FM dyes have a detergent-like structure, they inhibit vesicle recycling at high concentrations 11 . We therefore decided to employ FM 1-43 in our work (the concentration typically used,...

show abstract

Frequency-Dependent Kinetics and Prevalence of Kiss-and-Run and Reuse at Hippocampal Synapses Studied with Novel Quenching Methods

Cited by 152 publications

References 47 publications

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

Imaging Synaptic Vesicle Recycling by Staining and Destaining Vesicles with FM Dyes

The same synaptic vesicles drive active and spontaneous release

Contact Info

Product

Resources

About