Intermediates in synaptic vesicle recycling revealed by optical imaging of Drosophila neuromuscular junctions

Ramaswami, Mani; Krishnan, K. S.; Kelly, Regis B.

doi:10.1016/0896-6273(94)90353-0

Cited by 179 publications

(144 citation statements)

References 41 publications

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“…Indeed, various markers have been used, such as sulforhodamine (Lichtman et al 1985), fluorescently labeled antibodies (Kraszewski et al 1996;Westphal et al 2008), styryl dyes (i.e., FM dyes; Betz and Bewick 1992), and fluorescent dextrans (Holt et al 2003). Among these markers, FM dyes have proved to be valuable in a number of different preparations, including neuromuscular junctions (NMJ) from the frog (Betz and Bewick 1992), snake (Teng et al 1999), lizard (Lindgren et al 1997), and larval Drosophila (Ramaswami et al 1994); hippocampal neurons in culture (Ryan et al 1993) and acute slice (Zakharenko et al 2001); goldfish retinal bipolar cells (Zenisek et al 2000); Caenorhabditis elegans neurons (Kay et al 1999); rat calyx of Held (de Lange et al 2003); inner and outer hair cells (Griesinger et al 2002;Kaneko et al 2006); and a variety of nonneuronal preparations (for a review, see Cochilla et al 1999). …”

Section: Discussionmentioning

confidence: 99%

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

Hoopmann¹,

Rizzoli²,

Betz³

2011

Cold Spring Harb Protoc

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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)

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Section: Discussionmentioning

confidence: 99%

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

Hoopmann¹,

Rizzoli²,

Betz³

2011

Cold Spring Harb Protoc

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“…Optical measurements of endocytosis in cultured neurons suggest mild effects on endocytosis as a consequence of intersectin 1 KO (20). These results indicate that intersectin might not be mandatory for SV membrane retrieval or might regulate other steps in endocytosis that are not rate-limiting for the process per se (21).…”

mentioning

confidence: 83%

Fast neurotransmitter release regulated by the endocytic scaffold intersectin

Sakaba

Kononenko

Bacetic

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndromeassociated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain.endocytosis | synaptic transmission | synaptic vesicle recruitment N eurotransmission depends on the exocytosis of synaptic vesicles (SVs) at active zones (AZs) and the subsequent retrieval of SV membranes by endocytosis. Sustained fast neurotransmission requires rapid replenishment of release-ready SVs composing the readily releasable pool (RRP) (1-3). Whereas the machinery for SV fusion is well understood, the mechanisms controlling the number of SVs within the RRP and the rate of replenishment remain enigmatic (4, 5). It has been postulated that the rate of replenishment of release-ready SVs in addition to molecular priming reactions is regulated by the availability of SV release sites (6-8). The reuse of release sites appears to depend on components of the endocytic machinery, including dynamin (9-12). However, considering that the rate of endocytosis is ∼10-100 times slower than that of replenishment of release-ready SVs, the question arises as to how endocytic proteins affect rapid neurotransmitter release.Data from dynamin mutants can be interpreted as a deficit in SV recycling (e.g., loss of the SV reserve pool; ref. 13). Alternatively, endocytic proteins may regulate exocytosis more directly, independent of membrane retrieval. If so, interference with select endocytic factors might impair replenishment of release-ready SVs without affecting the rate of SV membrane retrieval.A possible candidate for such regulation is the early-acting multidomain endocytic protein intersectin (14-18), a protein overexpressed in Down syndrome, which also associates with components of the exocytic machinery [i.e., synaptosomal-associated protein 25 (SNAP-25)] (19). Although a role for intersectin in endocytosis has been established in Drosophila (16,17), the precise function of intersectin 1 in mammalian central synapses has not yet been analyzed extensively. Optical measurements of endocytosis in cultured ...

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“…This preparation has been extensively studied for synaptic function (Jan et al, 1977;Wu et al, 1978), development (Broadie and Bate, 1993;Kidokoro and Nishikawa, 1994;Yoshihara et al, 1997Yoshihara et al, , 2005Keshishian and Kim, 2004), plasticity (Budnik et al, 1990;Zhong and Wu, 1991;Zhong et al, 1992;Davis et al, 1996;Sigrist et al, 2003), and transmitter release mechanisms (Ramaswami et al, 1994;Kuromi and Kidokoro, 2000;Song et al, 2002;Kidokoro et al, 2004), and is therefore ideal for characterization of new mutant phenotypes. We found striking abnormalities in Shab as well as Sh larvae, although their defects were most pronounced under different stimulus conditions (Fig.…”

Section: Enhanced Synaptic Transmission At the Shab And Sh Neuromuscumentioning

confidence: 99%

Distinct Frequency-Dependent Regulation of Nerve Terminal Excitability and Synaptic Transmission by IA and IK Potassium Channels Revealed by Drosophila Shaker and Shab Mutations

Ueda

2006

Journal of Neuroscience

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Regulation of synaptic efficacy by nerve terminal excitability has not been extensively studied. We performed genetic and pharmacological dissections for presynaptic actions of K ϩ channels in Drosophila neuromuscular transmission by using electrophysiological and optical imaging techniques. Current understanding of the roles of the Shab I K channel and its mammalian Kv2 counterparts is relatively poor, as compared with that for Shaker I A channels and their Kv1 homologues. Our results revealed the striking effect of Shab mutations during high-frequency synaptic activity, as well as a functional division in synaptic regulation between the Shaker and Shab channels. Shaker channels control the basal level of release, indicated by a response to single nerve stimulation, whereas Shab channels regulate repetitive synaptic activities. These observations highlight the crucial control of nerve terminal excitability by Shaker and Shab channels to confer temporal patterns of synaptic transmission and suggest the potential participation of these channels, along with the transmitter release machinery, in activity-dependent synaptic plasticity.

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Intermediates in synaptic vesicle recycling revealed by optical imaging of Drosophila neuromuscular junctions

Cited by 179 publications

References 41 publications

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

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

Fast neurotransmitter release regulated by the endocytic scaffold intersectin

Distinct Frequency-Dependent Regulation of Nerve Terminal Excitability and Synaptic Transmission by IA and IK Potassium Channels Revealed by Drosophila Shaker and Shab Mutations

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