Exocytosis and Endocytosis of Synaptic Vesicles and Functional Roles of Vesicle Pools: Lessons from the Drosophila Neuromuscular Junction

Institute for Behavioral Sciences, Gunma University School of Medicine, 3-39-22 Showamachi, Maebashi 371-8511, Japan.
The Neuroscientist (Impact Factor: 6.84). 05/2005; 11(2):138-47. DOI: 10.1177/1073858404271679
Source: PubMed


To maintain synaptic transmission during intense neuronal activities, the synaptic vesicle (SV) pool at release sites is effectively replenished by recruitment of SVs from the reserve pool and/or by endocytosis. The authors have studied dynamics of SVs using a fluorescence dye, FM1-43, which is incorporated into SVs during endocytosis and released by exocytosis. Drosophila is one of the most suitable preparations for genetic and pharmacological analyses, and this provides a useful model system. The authors found at the neuromuscular junctions of Drosophila that exocytosis and endocytosis of SVs are triggered by Ca(2+) influx through distinct routes and that selective inhibition of exocytosis or endocytosis resulted in depression of synaptic transmission with a distinct time course. They identified two SV pools in a single presynaptic bouton. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low-frequency stimulation and locates close to release sites in the periphery of boutons, whereas the reserve pool (RP) is loaded and unloaded only during high-frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the quantal content of evoked release, suggesting that SVs in the ECP are primarily involved in synaptic transmission. SVs in the RP are recruited to synaptic transmission by a process involving the cAMP/PKA cascade during high-frequency stimulation. Cytochalasin D blocked this recruitment process, suggesting involvement of filamentous actin. Endocytosed SVs replenish the ECP during stimulation and the RP after tetanic stimulation. Replenishment of the ECP depends on Ca(2+) influx from external solutions, and that of the RP is initiated by Ca(2+) release from internal stores. Thus, SV dynamics is closely involved in modulation of synaptic efficacy and influences synaptic plasticity.

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    • "Upon stimulation, FM 1-43 dye is taken up by synaptic vesicles and labels newly endocytosed vesicles within the nerve terminal. Thus, defects in synaptic labeling with FM 1-43 dye are indicative of compromised vesicle cycling (Kuromi and Kidokoro, 2005; Verstreken et al., 2008). Stimulation with 90 mM KCl caused robust labeling of synaptic boutons in wild-type larvae (Fig. 5, A and D). "
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    ABSTRACT: Transcription factors are essential for regulating neuronal microtubules (MTs) during development and after axon damage. In this paper, we identify a novel neuronal function for Drosophila melanogaster FoxO in limiting MT stability at the neuromuscular junction (NMJ). foxO loss-of-function NMJs displayed augmented MT stability. In contrast, motor neuronal overexpression of wild-type FoxO moderately destabilized MTs, whereas overexpression of constitutively nuclear FoxO severely destabilized MTs. Thus, FoxO negatively regulates synaptic MT stability. FoxO family members are well-established components of stress-activated feedback loops. We hypothesized that FoxO might also be regulated by cytoskeletal stress because it was well situated to shape neuronal MT organization after cytoskeletal damage. Indeed, levels of neuronal FoxO were strongly reduced after acute pharmacological MT disruption as well as sustained genetic disruption of the neuronal cytoskeleton. This decrease was independent of the dual leucine zipper kinase-Wallenda pathway and required function of Akt kinase. We present a model wherein FoxO degradation is a component of a stabilizing, protective response to cytoskeletal insult.
    The Journal of Cell Biology 02/2012; 196(3):345-62. DOI:10.1083/jcb.201105154 · 9.83 Impact Factor
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    • "Calcium also plays a critical role in the endocytosis of synaptic vesicles (Sudhof, 2004; Kuromi and Kidokoro, 2005; Balaji et al., 2008; Yamashita et al., 2010). To measure Ca 2+ levels and endocytosis of synaptic vesicles, we stimulated cells coexpressing SyGCaMP3 and VGLUT1-2XmOr2 in the absence of bafilomycin (Figures 4A,B). "
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    ABSTRACT: Synaptic transmission involves the calcium dependent release of neurotransmitter from synaptic vesicles. Genetically encoded optical probes emitting different wavelengths of fluorescent light in response to neuronal activity offer a powerful approach to understand the spatial and temporal relationship of calcium dynamics to the release of neurotransmitter in defined neuronal populations. To simultaneously image synaptic vesicle recycling and changes in cytosolic calcium, we developed a red-shifted reporter of vesicle recycling based on a vesicular glutamate transporter, VGLUT1-mOrange2 (VGLUT1-mOr2), and a presynaptically localized green calcium indicator, synaptophysin-GCaMP3 (SyGCaMP3) with a large dynamic range. The fluorescence of VGLUT1-mOr2 is quenched by the low pH of synaptic vesicles. Exocytosis upon electrical stimulation exposes the luminal mOr2 to the neutral extracellular pH and relieves fluorescence quenching. Reacidification of the vesicle upon endocytosis again reduces fluorescence intensity. Changes in fluorescence intensity thus monitor synaptic vesicle exo- and endocytosis, as demonstrated previously for the green VGLUT1-pHluorin. To monitor changes in calcium, we fused the synaptic vesicle protein synaptophysin to the recently improved calcium indicator GCaMP3. SyGCaMP3 is targeted to presynaptic varicosities, and exhibits changes in fluorescence in response to electrical stimulation consistent with changes in calcium concentration. Using real time imaging of both reporters expressed in the same synapses, we determine the time course of changes in VGLUT1 recycling in relation to changes in presynaptic calcium concentration. Inhibition of P/Q- and N-type calcium channels reduces calcium levels, as well as the rate of synaptic vesicle exocytosis and the fraction of vesicles released.
    Frontiers in Molecular Neuroscience 11/2011; 4:34. DOI:10.3389/fnmol.2011.00034 · 4.08 Impact Factor
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    • "In general, SVs can be attributed to three functionally different pools, termed the readily releasable pool, the recycling pool and the reserve pool (RP) (Rizzoli and Betz, 2005). Together, the readily releasable pool and the recycling pool, sometimes referred to as the exo-endo cycling pool (ECP), typically represent about 10–20% of the SVs and are released under moderate or intense neuronal activity, whereas vesicles from the RP are recruited only upon high frequency stimulation (Figure 2) (Kuromi and Kidokoro, 2005; Rizzoli and Betz, 2005). Based on these characteristics, different stimulation protocols have been developed that allow the specific mobilization of SVs from different pools as well as their labeling with fluorescent dyes (Kuromi and Kidokoro, 2002; Verstreken et al., 2008). "
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    ABSTRACT: Cell types rich in mitochondria, including neurons, display a high energy demand and a need for calcium buffering. The importance of mitochondria for proper neuronal function is stressed by the occurrence of neurological defects in patients suffering from a great variety of diseases caused by mutations in mitochondrial genes. Genetic and pharmacological evidence also reveal a role of these organelles in various aspects of neuronal physiology and in the pathogenesis of neurodegenerative disorders. Yet the mechanisms by which mitochondria can affect neurotransmission largely remain to be elucidated. In this review we focus on experimental data that suggest a critical function of synaptic mitochondria in the function and organization of synaptic vesicle pools, and in neurotransmitter release during intense neuronal activity. We discuss how calcium handling, ATP production and other mitochondrial mechanisms may influence synaptic vesicle pool organization and synaptic function. Given the link between synaptic mitochondrial function and neuronal communication, efforts toward better understanding mitochondrial biology may lead to novel therapeutic approaches of neurological disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and psychiatric disorders that are at least in part caused by mitochondrial deficits.
    Frontiers in Synaptic Neuroscience 09/2010; 2:139. DOI:10.3389/fnsyn.2010.00139
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