Estimation of the Readily Releasable Synaptic Vesicle Pool at the Drosophila Larval Neuromuscular Junction (original) (raw)
Related papers
Neuron, 2000
Valdivia terminal during sustained neuronal activity, we asked the following questions: what is the size of vesicle pools? Chile † Escuela de Postgrado What is the rate of vesicle mobilization from these pools? And to what extent vesicle recycling is contributing in Fac. de Ciencias Universidad de Chile maintaining synaptic transmission during prolonged tetanic stimulation? Combining measurements of nerve- ‡ Gunma University School of Medicine 3-39-22 Showa-machi evoked synaptic currents and imaging of FM1-43 in synaptic boutons, these questions can be answered by Maebashi 371-8511 Japan taking advantage of a temperature-sensitive paralytic mutant, shibire ts (shi ts ). In shi ts endocytosis ceases and synaptic vesicles are completely depleted after synaptic activation at nonpermissive temperatures, while synap-Summary tic transmission at room temperature is normal (Koenig and Ikeda, 1989, 1996; Koenig et al., 1989; Estes et al., Two vesicle pools, readily releasable (RRP) and re -1996). This mutant provides a means to estimate the serve (RP) pools, are present at Drosophila neuromussize of vesicle pools and their rates of mobilization at cular junctions. Using a temperature-sensitive mutant, the larval neuromuscular junction. The contribution of shibire ts , we studied pool sizes and vesicle mobilizarecycling for synaptic transmission can be examined in tion rates. In shibire ts , due to lack of endocytosis at wild-type larvae at steady state during repetitive nerve nonpermissive temperatures, synaptic currents constimulation.
—The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals, at crayfish and larval Drosophila neuromuscular junctions (NMJs), were examined for physiological differentiation into distinctly separate functional groups. These NMJs are glutamatergic and produce graded excitatory postsynap-tic potentials (EPSPs). The packaging of glutamate was perturbed by blocking the vesicular glutamate transporter (VGlut) with bafilomycin A1. Various frequencies of motor nerve stimulation, exposure time, and concentration of bafilomycin A1 were examined. The low-output tonic opener NMJs in crayfish exposed to 4 lM bafilomycin A1 and 20-Hz continuous stimulation decreased the EPSP amplitude to 50% in 30minwithcontrolslasting3h.AfteractivityandbafilomycinA1−inducedsynapticdepression,theEPSPswererapidlyrevitalizedbyserotonin(5−HT,1lM)inthecrayfishpreparations.The5−HTactioncanbeblockedwithaPLCinhibitor.Wepostulate5−HTrecruitsunusedvesiclesfromtheRP.TheperceptionistheRRPisselectivelyactivatedduringrapidelectricalstimulation(20Hz)sparingtheRP.Whenstimulationfrequencyishigh(40Hz)theRPisrecruitedtotheRRPanddampenssubsequentrecruitmentwith5−HT.ThehigheroutputsynapsesofthelarvalDrosophilaNMJwhenstimulatedat1Hzor5Hzandexposedto4lMofbafilo−mycinA1showedadepressionrateof5030 min with controls lasting 3 h. After activity and bafilomycin A1-induced synaptic depression, the EPSPs were rapidly revitalized by serotonin (5-HT, 1 lM) in the crayfish preparations. The 5-HT action can be blocked with a PLC inhibitor. We postulate 5-HT recruits unused vesicles from the RP. The perception is the RRP is selectively activated during rapid electrical stimulation (20 Hz) sparing the RP. When stimulation frequency is high (40 Hz) the RP is recruited to the RRP and dampens subsequent recruitment with 5-HT. The higher output synapses of the larval Drosophila NMJ when stimulated at 1 Hz or 5 Hz and exposed to 4 lM of bafilo-mycin A1 showed a depression rate of 50% within 30minwithcontrolslasting3h.AfteractivityandbafilomycinA1−inducedsynapticdepression,theEPSPswererapidlyrevitalizedbyserotonin(5−HT,1lM)inthecrayfishpreparations.The5−HTactioncanbeblockedwithaPLCinhibitor.Wepostulate5−HTrecruitsunusedvesiclesfromtheRP.TheperceptionistheRRPisselectivelyactivatedduringrapidelectricalstimulation(20Hz)sparingtheRP.Whenstimulationfrequencyishigh(40Hz)theRPisrecruitedtotheRRPanddampenssubsequentrecruitmentwith5−HT.ThehigheroutputsynapsesofthelarvalDrosophilaNMJwhenstimulatedat1Hzor5Hzandexposedto4lMofbafilo−mycinA1showedadepressionrateof5010 min with controls lasting $40 min. After low frequency depression and/or exposure to bafilomycin A1 a burst of higher frequency (10 Hz) can recruit vesicles from the RP to the RRP.
FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction
2008
To maintain transmitter release during intense stimulation, neurons need to efficiently recycle vesicles at the synapse. Following membrane fusion, vesicles are reshaped and formed from the plasma membrane by bulk or clathrin-mediated endocytosis. Most synapses, including the Drosophila neuromuscular junction (NMJ), can also recycle synaptic vesicles directly by closing the fusion pore, a process referred to as "kiss and run." While the process of clathrin-mediated vesicle retrieval is under intense investigation, the kiss-and-run phenomenon remains much less accepted. To gain better insight into the mechanisms of synaptic vesicle recycling, it is therefore critical not only to identify and characterize novel players involved in the process, but also to develop novel methods to study vesicle recycling. Although in recent years numerous techniques to study vesicle traffic have been developed (see also this volume), in this chapter we outline established procedures that use the fluorescent dye FM 1-43 or related compounds to study vesicle cycling. We describe how FM 1-43 can be used to study and visualize clathrin-mediated or bulk endocytosis from the presynaptic membrane as well as exocytosis of labeled vesicles at the Drosophila NMJ, one of the best-characterized model synapses to study synaptic function in a genetic model system.
A New Kinetic Framework for Synaptic Vesicle Trafficking Tested in Synapsin Knock-Outs
The Journal of …, 2011
At least two rate-limiting mechanisms in vesicle trafficking operate at mouse Schaffer collateral synapses, but their molecular/physical identities are unknown. The first mechanism determines the baseline rate at which reserve vesicles are supplied to a readily releasable pool. The second causes the supply rate to depress threefold when synaptic transmission is driven hard for extended periods. Previous models invoked depletion of a reserve vesicle pool to explain the reductions in the supply rate, but the mass-action assumption at their core is not compatible with kinetic measurements of neurotransmission under maximal-use conditions. Here we develop a new theoretical model of rate-limiting steps in vesicle trafficking that is compatible with previous and new measurements. A physical interpretation is proposed where local reserve pools consisting of four vesicles are tethered to individual release sites and are replenished stochastically in an all-or-none fashion. We then show that the supply rate depresses more rapidly in synapsin knock-outs and that the phenotype can be fully explained by changing the value of the single parameter in the model that would specify the size of the local reserve pools. Vesicle-trafficking rates between pools were not affected. Finally, optical imaging experiments argue against alternative interpretations of the theoretical model where vesicle trafficking is inhibited without reserve pool depletion. This new conceptual framework will be useful for distinguishing which of the multiple molecular and cell biological mechanisms involved in vesicle trafficking are rate limiting at different levels of synaptic throughput and are thus candidates for physiological and pharmacological modulation.
Neuroscience, 2003
The formation of chemical synapses in the mammalian brain involves complex pre-and postsynaptic differentiation processes. Presynaptically, the progressive accumulation of synaptic vesicles is a hallmark of synapse maturation in the neocortex [J Neurocytol 12 (1983b) 697]. In this study, we analyzed the functional consequences of presynaptic vesicle-pool maturation at central glutamatergic and GABAergic synapses. Using (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43) staining of recycling synaptic vesicles, we demonstrate a pronounced developmental increase in presynaptic vesicle accumulation during differentiation of neocortical neurons in culture. Using electrophysiological methods to study functional synaptic maturation, we found an improved recovery from hypertonic solution-induced depletion. As supported by the FM1-43 staining results, this change is most likely caused by a developmental increase in the number of reserve-pool vesicles. In addition, assuming a rapid reuse of freshly recycled vesicles, a developmental maturation of the endocytosis process may also contribute. The observed presynaptic maturation process occurred selectively at glutamatergic synapses, while GABAergic synapses did not show similar developmental alterations. Furthermore, we used high-frequency stimulation (HFS) of glutamatergic and GABAergic synapses to reveal the physiological consequences of reserve-pool maturation. As expected, recovery from HFS-induced depletion was incomplete at immature glutamatergic synapses and strongly improved during synapse maturation. Again, GABAergic synapses did not show similar developmental changes. Taken together, our study characterizes the functional consequences of a pronounced accumulation of reserve-pool vesicles occurring selectively at glutamatergic synapses.
Cycling of Synaptic Vesicles: How Far? How Fast!
Science Signaling, 2001
Synaptic transmission is based on the regulated exocytotic fusion of synaptic vesicles filled with neurotransmitter. In order to sustain neurotransmitter release, these vesicles need to be recycled locally. Recent data suggest that two tracks for the cycling of synaptic vesicles coexist: a slow track in which vesicles fuse completely with the presynaptic plasma membrane, followed by clathrin-mediated recycling of the vesicular components, and a fast track that may correspond to the transient opening and closing of a fusion pore. In this review, we attempt to provide an overview of the components involved in both tracks of vesicle cycling, as well as to identify possible mechanistic links between these two pathways.
2001
Neurotransmitter release is a highly efficient secretory process exhibiting resistance to fatigue and plasticity attributable to the existence of distinct pools of synaptic vesicles (SVs), namely a readily releasable pool and a reserve pool from which vesicles can be recruited after activity. Synaptic vesicles in the reserve pool are thought to be reversibly tethered to the actin-based cytoskeleton by the synapsins, a family of synaptic vesicleassociated phosphoproteins that have been shown to play a role in the formation, maintenance, and regulation of the reserve pool of synaptic vesicles and to operate during the postdocking step of the release process. In this paper, we have investigated the physiological effects of manipulating synapsin levels in identified cholinergic synapses of Aplysia californica. When endogenous synapsin was neutralized by the injection of specific anti-synapsin antibodies, the amount of neurotransmitter released per impulse was unaffected, but marked changes in the secretory response to high-frequency stimulation were observed, including the disappearance of post-tetanic potenti-ation (PTP) that was substituted by post-tetanic depression (PTD), and increased rate and extent of synaptic depression. Opposite changes on post-tetanic potentiation were observed when synapsin levels were increased by injecting exogenous synapsin I. Our data demonstrate that the presence of synapsin-dependent reserve vesicles allows the nerve terminal to release neurotransmitter at rates exceeding the synaptic vesicle recycling capacity and to dynamically change the efficiency of release in response to conditioning stimuli (e.g., posttetanic potentiation). Moreover, synapsin-dependent regulation of the fusion competence of synaptic vesicles appears to be crucial for sustaining neurotransmitter release during short periods at rates faster than the replenishment kinetics and maintaining synchronization of quanta in evoked release.
A readily retrievable pool of synaptic vesicles
Nature neuroscience, 2011
Although clathrin-mediated endocytosis is thought to be the predominant mechanism of synaptic vesicle recycling, it seems to be too slow for fast recycling. Therefore, it was suggested that a presorted and preassembled pool of synaptic vesicle proteins on the presynaptic membrane might support a first wave of fast clathrin-mediated endocytosis. In this study we monitored the temporal dynamics of such a 'readily retrievable pool' of synaptic vesicle proteins in rat hippocampal neurons using a new type of probe. By applying cypHer5E, a new cyanine dye-based pH-sensitive exogenous marker, coupled to antibodies to luminal domains of synaptic vesicle proteins, we could reliably monitor synaptic vesicle recycling and demonstrate the preferential recruitment of a surface pool of synaptic vesicle proteins upon stimulated endocytosis. By using fluorescence nanoscopy of surface-labeled synaptotagmin 1, we could resolve the spatial distribution of the surface pool at the periactive z...
Synaptic vesicle life cycle and synaptic turnover
Journal of Physiology-Paris, 1993
Cholinergic synaptic vesicles contain a mixture of soluble low molecular mass constituents. Besides acetylcholine these include Ca 2+, ATP, GTP, small amounts of ADP and AMP, and also the diadenosine polyphosphates AP4A and ApsA. In synaptic vesicles isolated from the electric ray these diadenosine polyphosphates occur in mmol concentrations and might represent a novel cotransmitter. The membrane proteins of cholinergic synaptic vesicles presumably are identical to those in other types of electron-lucent synaptic vesicles. A presumptive exception are the transmitter-specific carriers. The life cycle of the synaptic vesicle in intact neurons and in situ was investigated by analysis of all cytoplasmic membrane compartments that share membrane integral proteins with synaptic vesicles. The results suggest that the synaptic vesicle membrane compartment might originate from the trans-Golgi network and, after cycles of exo-and endocytosis in the nerve terminal, might fuse into an endosomal membrane compartment early on retrograde transport. Tracer experiments using membrane proteins and soluble contents suggest that the synaptic vesicle membrane compartment does not intermix with the presynaptic plasma membrane on repeated cycles of exo-and endocytosis if low frequency stimulation is applied. A cDNA has been isolated from the electric ray electric lobe that codes for o-rab3, a small GTP-binding protein highly homologous to mammalian rab3. While abundant in the nerve terminals of the electric organ and at the neuromuscular junction this protein occurs only in limited subpopulations of nerve terminals in electric ray brain. Immunocytochemical analysis using the colloidal gold technique and a monospecific antibody against o-rab3 suggests that the GTP-binding protein remains attached to recycling synaptic vesicles. No evidence was found for a major contribution of an intraterminal endosomal sorting compartment involved in synaptic vesicle recycling.