- Short report
- Open Access
Common strength and localization of spontaneous and evoked synaptic vesicle release sites
https://doi.org/10.1186/1756-6606-7-23
© Loy et al.; licensee BioMed Central Ltd. 2014
- Received: 8 July 2013
- Accepted: 29 March 2014
- Published: 2 April 2014
Abstract
Background
Different pools and functions have recently been attributed to spontaneous and evoked vesicle release. Despite the well-established function of evoked release, the neuronal information transmission, the origin as well as the function of spontaneously fusing synaptic vesicles have remained elusive. Recently spontaneous release was found to e.g. regulate postsynaptic protein synthesis or has been linked to depressive disorder. Nevertheless the strength and cellular localization of this release form was neglected so far, which are both essential parameters in neuronal information processing.
Findings
Here we show that the complete recycling pool can be turned over by spontaneous trafficking and that spontaneous fusion rates critically depend on the neuronal localization of the releasing synapse. Thereby, the distribution equals that of evoked release so that both findings demonstrate a uniform regulation of these fusion modes.
Conclusions
In contrast to recent works, our results strengthen the assumption that identical vesicles are used for evoked and spontaneous release and extended the knowledge about spontaneous fusion with respect to its amount and cellular localization. Therefore our data supported the hypothesis of a regulatory role of spontaneous release in neuronal outgrowth and plasticity as neurites secrete neurotransmitters to initiate process outgrowth of a possible postsynaptic neuron to form a new synaptic connection.
Keywords
- Synaptic Vesicle
- Spontaneous Release
- Vesicle Release
- Reserve Pool
- Calcium Dependent Manner
Findings
Background
Central neurons display two different forms of vesicle release: stimulation dependent, i.e. evoked release and stimulation independent spontaneous release, which occurs at resting membrane potential. Spontaneous neurotransmitter release is thought to play a crucial role in synaptic plasticity, memory and learning [1] as well as in pathophysiology [2]. Furthermore spontaneous neurotransmitter release was found to regulate postsynaptic dendritic protein synthesis [3, 4]. The origin of spontaneous release remains controversial, with some evidence arguing for the recycling pool [5, 6] of vesicles and some for the reserve pool [7]. Here we seek to analyze the amount of spontaneous release regarding its turnover kinetics and cellular location. While a pool can be defined for each form of release, our results indicate a common regulation of both vesicle populations and thus a common origin of both pools from the recycling pool of vesicles.
Results
The recycling pool of hippocampal synapses is turned over completely by spontaneous vesicle recycling. A Scheme of experimental setup: Boutons were first labeled with FM1-43 by spontaneous uptake for different time periods. At the end of the first part of the experiment, boutons were completely destained using twice 900 pulses to determine the amount of spontaneous turnover. After a 10 minute recovery synaptic boutons were labeled a second time using 1200 electrically evoked action potentials and again completely destained to determine the recycling pool size. B Difference images for 15 and 120 minutes spontaneous FM1-43 uptake and evoked staining, respectively. Scale bar, 10 μm. C Corresponding mean fluorescence profiles to determine ∆Fspontaneous and ∆Fevoked for each time period. D Time course of ∆Fspontaneous to ∆Fevoked ratio depending on extracellular calcium concentration (t ½ in minutes: 0 mM Ca2+ = 43.96, 2.5 mM Ca2+ = 20.58, 5 mM Ca2+ = 15.73). Arrows mark the exemplary time points. E Correlation of spontaneous and evoked release at individual synaptic boutons (time point at 60 min).
At soma-near synapses the number of spontaneously fused vesicles as well the recycling pool vesicles is higher than in distal cellular compartments. A Analysis of spontaneous and evoked release differentiated between process and soma using dual color experiments of spH transfected hippocampal neurons spontaneously labeled with αGFP-CypHer5E™. The spontaneous release (SR), recycling pool (RP), reserve pool (resP) and the total pool (TP) were determined. Scheme of experimental setup added with the mean values of spH and CypHer5E™ fluorescence used for further analysis. Boutons of spH transfected hippocampal neurons were first labeled with αGFP-CypHer5E™ by spontaneous uptake during a 120 minute period. Then a stitched image was captured to encompass the whole neuron with its processes at high resolution in both fluorescence channels (120 min). After stimulation with 40 mM K+ solution for 2 minutes in the presence of Bafilomycin an image of the same region was recorded (135 min), followed by an image after NH4+ application (140 min). B Images depicting whole neuron. Insets: Synaptic boutons at higher magnification. Scale bar, 50 μm. C Scheme of image analysis used for distinction between process and soma. D Quantification of pool sizes: RP (two-sample t-test: p = 0.001), SR (two-sample t-test: p = 0.042) and SR to RP ratio (SR/RP; two-sample t-test: p = 0.005; 11 experiments). Correlations of distinct vesicle pools differentiated for synapses located at the process or the soma: SR and RP (E; Pearson’s r process = 0.96 ± 0.01, r soma = 0.84 ± 0.04), SR and ResP (F; Pearson’s r process = 0.19 ± 0.14, r soma = 0.51 ± 0.11) and SR and TP (G; Pearson’s r process = 0.64 ± 0.09, r soma = 0.78 ± 0.05). Dashed line in E represents the linear fit of a subset of data putative resembling the processes running above or beneath the soma (slopeprocess = 0.55 ± 0.02, fit: y = mx + t).
Conclusion and discussion
We provide new evidence that spontaneous vesicle turnover can reach the level of the recycling pool of vesicles in a time and calcium dependent manner (Figure 1D, Additional file 1: Figure S1C). Considering these results together with the fact that the amount of recycling pool vesicles correlates robustly with the spontaneous release at each bouton (Figure 1E), suggest that spontaneous vesicles originate from the recycling pool of vesicles rather than from the reserve pool. This underlines previous findings [5, 6, 19], but is contrary to studies that pointed at the reserve pool as origin of spontaneous vesicles [7]. These previous studies used a sequential labeling paradigm instead of a simultaneous labeling that we used in our study. We cannot exclude with our experimental approach that, after total recycling pool turnover, additional vesicles recycle spontaneously from within the reserve pool of vesicles, due to a lack of discrimination without sequential labeling after saturation. However such vesicles would account for a minority of spontaneously fusing vesicles due to the lack of correlation with the reserve pool (Figure 2F). Besides we found that the soma has absolutely the larger synapses with the larger recycling pool and the higher spontaneous release, but if spontaneous and evoked turnover is normalized on the size of the synapse, the relative release is higher at the processes. Recent publications found a distance from soma dependency of synapse size and evoked release at the processes [20, 21]. In accordance with evoked release [21], spontaneous release declined along the processes with increasing distance to the soma (Additional file 1: Figure S5A), but remained constant, if spontaneous release was normalized on synapse size (Additional file 1: Figure S5B). Our functional measurements indicated, that both forms of release exhibit the same relationship regarding distance from the soma with smaller, but more effective synapses at the process [11]. These results therefore point to a common developmental origin of these release modes with the vesicle populations stemming both from the recycling pool of vesicles. We also found differences between soma and processes regarding synapse size, relative and absolute release and confirmed, that smaller synapses release more efficiently. In conclusion we found a multitude of commonalities of spontaneous release and evoked release, e.g. correlation and identical size with recycling pool, vesicles were immediately re-releasable, same cellular localization with respect to release characteristics which together suggests the recycling pool as the common source of spontaneous and evoked released vesicles. Nevertheless a definition of a spontaneous vesicle pool is valid as vesicles differ with respect to their neuronal function and more future research is needed to substantiate the origin of spontaneous vesicles and to further address the function of these vesicles.
Notes
Declarations
Acknowledgments
We thank D. Gilbert and O. Friedrich (Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nuremberg, Paul-Gordan-Strasse 3, Erlangen, Germany) for providing their imaging setup.
Authors’ Affiliations
References
- Emptage NJ, Reid CA, Fine A: Calcium stores in hippocampal synaptic boutons mediate short-term plasticity, store-operated Ca2+ entry, and spontaneous transmitter release. Neuron. 2001, 29: 197-208. 10.1016/S0896-6273(01)00190-8.PubMedView ArticleGoogle Scholar
- Autry AE, Adachi M, Nosyreva E, Na ES, Los MF, Cheng PF, Kavalali ET, Monteggia LM: NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses. Nature. 2011, 475: 91-95. 10.1038/nature10130.PubMedPubMed CentralView ArticleGoogle Scholar
- Sutton MA, Taylor AM, Ito HT, Pham A, Schuman EM: Postsynaptic decoding of neural activity: eEF2 as a biochemical sensor coupling miniature synaptic transmission to local protein synthesis. Neuron. 2007, 55: 648-661. 10.1016/j.neuron.2007.07.030.PubMedView ArticleGoogle Scholar
- Sutton MA, Wall NR, Aakalu GN, Schuman EM: Regulation of dendritic protein synthesis by miniature synaptic events. Science. 2004, 304: 1979-1983. 10.1126/science.1096202.PubMedView ArticleGoogle Scholar
- Hua Y, Sinha R, Martineau M, Kahms M, Klingauf J: A common origin of synaptic vesicles undergoing evoked and spontaneous fusion. Nat Neurosci. 2010, 13: 1451-1453. 10.1038/nn.2695.PubMedView ArticleGoogle Scholar
- Groemer TW, Klingauf J: Synaptic vesicles recycling spontaneously and during activity belong to the same vesicle pool. Nat Neurosci. 2007, 10: 145-147. 10.1038/nn1831.PubMedView ArticleGoogle Scholar
- Fredj NB, Burrone J: A resting pool of vesicles is responsible for spontaneous vesicle fusion at the synapse. Nat Neurosci. 2009, 12: 751-758. 10.1038/nn.2317.PubMedPubMed CentralView ArticleGoogle Scholar
- Marra V, Burden JJ, Thorpe JR, Smith IT, Smith SL, Hausser M, Branco T, Staras K: A preferentially segregated recycling vesicle pool of limited size supports neurotransmission in native central synapses. Neuron. 2012, 76: 579-589. 10.1016/j.neuron.2012.08.042.PubMedPubMed CentralView ArticleGoogle Scholar
- Kavalali ET, Chung C, Khvotchev M, Leitz J, Nosyreva E, Raingo J, Ramirez DM: Spontaneous neurotransmission: an independent pathway for neuronal signaling?. Physiology (Bethesda). 2011, 26: 45-53. 10.1152/physiol.00040.2010.View ArticleGoogle Scholar
- Miesenbock G, De Angelis DA, Rothman JE: Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature. 1998, 394: 192-195. 10.1038/28190.PubMedView ArticleGoogle Scholar
- Welzel O, Henkel AW, Stroebel AM, Jung J, Tischbirek CH, Ebert K, Kornhuber J, Rizzoli SO, Groemer TW: Systematic heterogeneity of fractional vesicle pool sizes and release rates of hippocampal synapses. Biophys J. 2011, 100: 593-601. 10.1016/j.bpj.2010.12.3706.PubMedPubMed CentralView ArticleGoogle Scholar
- Branco T, Staras K, Darcy KJ, Goda Y: Local dendritic activity sets release probability at hippocampal synapses. Neuron. 2008, 59: 475-485. 10.1016/j.neuron.2008.07.006.PubMedView ArticleGoogle Scholar
- Ryan TA, Reuter H, Smith SJ: Optical detection of a quantal presynaptic membrane turnover. Nature. 1997, 388: 478-482. 10.1038/41335.PubMedView ArticleGoogle Scholar
- Pyle JL, Kavalali ET, Piedras-Renteria ES, Tsien RW: Rapid reuse of readily releasable pool vesicles at hippocampal synapses. Neuron. 2000, 28: 221-231. 10.1016/S0896-6273(00)00098-2.PubMedView ArticleGoogle Scholar
- Sholl DA: Dendritic organization in the neurons of the visual and motor cortices of the cat. J Anat. 1953, 87: 387-406.PubMedPubMed CentralGoogle Scholar
- Moulder KL, Jiang X, Taylor AA, Shin W, Gillis KD, Mennerick S: Vesicle pool heterogeneity at hippocampal glutamate and GABA synapses. J Neurosci. 2007, 27: 9846-9854. 10.1523/JNEUROSCI.2803-07.2007.PubMedView ArticleGoogle Scholar
- Branco T, Marra V, Staras K: Examining size-strength relationships at hippocampal synapses using an ultrastructural measurement of synaptic release probability. J Struct Biol. 2010, 172: 203-210. 10.1016/j.jsb.2009.10.014.PubMedPubMed CentralView ArticleGoogle Scholar
- Murthy VN, Sejnowski TJ, Stevens CF: Heterogeneous release properties of visualized individual hippocampal synapses. Neuron. 1997, 18: 599-612. 10.1016/S0896-6273(00)80301-3.PubMedView ArticleGoogle Scholar
- Wilhelm BG, Groemer TW, Rizzoli SO: The same synaptic vesicles drive active and spontaneous release. Nat Neurosci. 2010, 13: 1454-1456. 10.1038/nn.2690.PubMedView ArticleGoogle Scholar
- Peng X, Parsons TD, Balice-Gordon RJ: Determinants of synaptic strength vary across an axon arbor. J Neurophysiol. 2012, 107: 2430-2441. 10.1152/jn.00615.2011.PubMedPubMed CentralView ArticleGoogle Scholar
- de Jong AP, Schmitz SK, Toonen RF, Verhage M: Dendritic position is a major determinant of presynaptic strength. J Cell Biol. 2012, 197: 327-337. 10.1083/jcb.201112135.PubMedPubMed CentralView ArticleGoogle Scholar
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