Publications by authors named "Maria Bykhovskaia"

33 Publications

The Accessory Helix of Complexin Stabilizes a Partially Unzippered State of the SNARE Complex and Mediates the Complexin Clamping Function .

eNeuro 2021 Mar-Apr;8(2). Epub 2021 Apr 7.

Ophthalmology, Visual and Anatomical Sciences Department, Wayne State University School of Medicine, Detroit, MI 48202

Spontaneous synaptic transmission is regulated by the protein complexin (Cpx). Cpx binds the SNARE complex, a coil-coiled four-helical bundle that mediates the attachment of a synaptic vesicle (SV) to the presynaptic membrane (PM). Cpx is thought to clamp spontaneous fusion events by stabilizing a partially unraveled state of the SNARE bundle; however, the molecular detail of this mechanism is still debated. We combined electrophysiology, molecular modeling, and site-directed mutagenesis in to develop and validate the atomic model of the Cpx-mediated clamped state of the SNARE complex. We took advantage of botulinum neurotoxins (BoNTs) B and G, which cleave the SNARE protein synaptobrevin (Syb) at different sites. Monitoring synaptic depression on BoNT loading revealed that the clamped state of the SNARE complex has two or three unraveled helical turns of Syb. Site-directed mutagenesis showed that the Cpx clamping function is predominantly maintained by its accessory helix (AH), while molecular modeling suggested that the Cpx AH interacts with the unraveled C terminus of Syb and the SV lipid bilayer. The developed molecular model was employed to design new Cpx poor-clamp and super-clamp mutations and to tested the predictions employing molecular dynamics simulations. Subsequently, we generated lines harboring these mutations and confirmed the poor-clamp and super-clamp phenotypes Altogether, these results validate the atomic model of the Cpx-mediated fusion clamp, wherein the Cpx AH inserts between the SNARE bundle and the SV lipid bilayer, simultaneously binding the unraveled C terminus of Syb and preventing full SNARE assembly.
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http://dx.doi.org/10.1523/ENEURO.0526-20.2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026252PMC
June 2021

SNARE complex alters the interactions of the Ca sensor synaptotagmin 1 with lipid bilayers.

Biophys J 2021 02 14;120(4):642-661. Epub 2021 Jan 14.

Department of Neurology, Wayne State University, Detroit, Michigan. Electronic address:

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca sensor synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca binding. Before fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which is thought to bind the C2B domain in vivo. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. We investigated how the isolated C2 modules and the C2AB tandem of Syt1 interact with membranes mimicking either SV or PM. The simulations showed that the C2AB tandem can either bridge SV and PM or insert into PM with its Ca-bound tips and that the latter configuration is more favorable. Surprisingly, C2 domains did not cooperate in penetrating into PM but instead mutually hindered their insertion into the bilayer. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the C2AB-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains and promotes their insertion into PM. We performed the MD simulations of the prefusion protein complex positioned between the lipid bilayers mimicking PM and SV, and our results demonstrated in silico that the presence of the Ca bound C2AB tandem promotes lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.
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http://dx.doi.org/10.1016/j.bpj.2020.12.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896035PMC
February 2021

Two Pathways for the Activity-Dependent Growth and Differentiation of Synaptic Boutons in .

eNeuro 2019 Jul/Aug;6(4). Epub 2019 Aug 22.

Neurology Department, Wayne State University, Detroit, Michigan 48201

Synapse formation can be promoted by intense activity. At the larval neuromuscular junction (NMJ), new synaptic boutons can grow acutely in response to patterned stimulation. We combined confocal imaging with electron microscopy and tomography to investigate the initial stages of growth and differentiation of new presynaptic boutons at the NMJ. We found that the new boutons can form rapidly in intact larva in response to intense crawling activity, and we observed two different patterns of bouton formation and maturation. The first pathway involves the growth of filopodia followed by a formation of boutons that are initially devoid of synaptic vesicles (SVs) but filled with filamentous matrix. The second pathway involves rapid budding of synaptic boutons packed with SVs, and these more mature boutons are sometimes capable of exocytosis/endocytosis. We demonstrated that intense activity predominantly promotes the second pathway, i.e., budding of more mature boutons filled with SVs. We also showed that this pathway depends on synapsin (Syn), a neuronal protein which reversibly associates with SVs and mediates their clustering via a protein kinase A (PKA)-dependent mechanism. Finally, we took advantage of the temperature-sensitive mutant to demonstrate that seizure activity can promote very rapid budding of new boutons filled with SVs, and this process occurs at scale of minutes. Altogether, these results demonstrate that intense activity acutely and selectively promotes rapid budding of new relatively mature presynaptic boutons filled with SVs, and that this process is regulated via a PKA/Syn-dependent pathway.
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http://dx.doi.org/10.1523/ENEURO.0060-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709223PMC
March 2020

Growth and excitability at synapsin II deficient hippocampal neurons.

Mol Cell Neurosci 2019 04 9;96:25-34. Epub 2019 Mar 9.

Department of Neurology, Wayne State University School of Medicine, Detroit, MI, United States of America. Electronic address:

Synapsins are neuronal phosphoproteins that fine-tune synaptic transmission and suppress seizure activity. Synapsin II (SynII) deletion produces epileptic seizures and overexcitability in neuronal networks. Early studies in primary neuronal cultures have shown that SynII deletion results in a delay in synapse formation. More recent studies at hippocampal slices have revealed increased spontaneous activity in SynII knockout (SynII(-)) mice. To reconcile these observations, we systematically re-examined synaptic transmission, synapse formation, and neurite growth in primary hippocampal neuronal cultures. We find that spontaneous glutamatergic synaptic activity was suppressed in SynII(-) neurons during the initial developmental epoch (7 days in vitro, DIV) but was enhanced at later times (12 and18 DIV). The density of synapses, transmission between connected pairs of neurons, and the number of docked synaptic vesicles were not affected by SynII deletion. However, we found that neurite outgrowth in SynII(-) neurons was suppressed during the initial developmental epoch (7 DIV) but enhanced at subsequent developmental stages (12 and18 DIV). This finding can account for the observed effect of SynII deletion on synaptic activity. To test whether the observed phenotype resulted directly from the deletion of SynII we expressed SynII in SynII(-) cultures using an adeno-associated virus (AAV). Expression of SynII at 2 DIV rescued the SynII deletion-dependent alterations in both synaptic activity and neuronal growth. To test whether the increased neurite outgrowth in SynII(-) observed at DIV 12 and18 represents an overcompensation for the initial developmental delay or results directly from SynII deletion we performed "late expression" experiments, transfecting SynII(-) cultures with AAV at 7 DIV. The late SynII expression suppressed neurite outgrowth at 12 and 18 DIV to the levels observed in control neurons, suggesting that these phenotypes directly depend on SynII. These results reveal a novel developmentally regulated role for SynII function in the control of neurite growth.
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http://dx.doi.org/10.1016/j.mcn.2019.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6720125PMC
April 2019

Coarse-Grained Model for Zippering of SNARE from Partially Assembled States.

J Phys Chem B 2018 12 19;122(48):10834-10840. Epub 2018 Nov 19.

Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the presynaptic membrane. Vesicles are attached to the membrane via the SNARE complex, comprising the vesicle associated protein synaptobrevin (Syb), the membrane associated protein syntaxin (Syx), and the cytosolic protein SNAP25, that together form a four-helical bundle. The full assembly of Syb onto the core SNARE bundle promotes vesicle fusion. We investigated SNARE assembly using a coarse-grained model of the SNARE complex that retains chemical specificity. Steered force-control simulations of SNARE unzippering were used to set up initial disassembled states of the SNARE complex. From these states, the assembly process was simulated. We find that if Syb is in helical form and proximal to the other helices, then the SNARE complex assembles rapidly, on a microsecond time-scale, which is well within in vivo synaptic vesicle fusion time scales. Assembly times grow exponentially with a separation distance between Syb and Syx C-termini. Our results indicate that for biologically relevant rapid assembly of the SNARE complex, Syb should be in helical form, and the SNARE constituent helices brought into proximity, possibly by an agent, such as a chaperone.
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http://dx.doi.org/10.1021/acs.jpcb.8b09502DOI Listing
December 2018

Molecular Dynamics Simulations of the SNARE Complex.

Methods Mol Biol 2019 ;1860:3-13

Department of Neurology, Wayne State University School of Medicine, Detroit, MI, USA.

Molecular dynamics (MD) simulations enable in silico investigations of the dynamic behavior of proteins and protein complexes. Here, we describe MD simulations of the SNARE complex and its interactions with the neuronal protein complexin. Complexin is an effector of neuronal secretion that inhibits spontaneous fusion and is thought to clamp the fusion process via the interactions with the SNARE complex. We describe MD simulations of the SNARE complex alone and bound to complexin. The MD simulations under external forces imitating the repulsion between lipid bilayers enabled us to investigate unraveling and assembly of the SNARE complex.
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http://dx.doi.org/10.1007/978-1-4939-8760-3_1DOI Listing
June 2019

FM1-43 Photoconversion and Electron Microscopy Analysis at the Neuromuscular Junction.

Bio Protoc 2017 Sep;7(17)

Department of Neurology, Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, USA.

We developed a protocol for photoconversion of endocytic marker FM1-43 followed by electron microscopy analysis of synaptic boutons at the neuromuscular junction. This protocol allows detection of stained synaptic vesicle even when release rates are very low, such as during the spontaneous release mode. The preparations are loaded with the FM1-43 dye, pre-fixed, treated and illuminated to photoconvert the dye, and then processed for conventional electron microscopy. This procedure enables clear identification of stained synaptic vesicles at electron micrographs.
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http://dx.doi.org/10.21769/BioProtoc.2523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5661859PMC
September 2017

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction.

J Vis Exp 2017 09 25(127). Epub 2017 Sep 25.

Department of Neurology, School of Medicine, Wayne State University; Department of Anatomy and Cell Biology, School of Medicine, Wayne State University;

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has a diameter of several microns, and the release sites positioned outside of the electrode rim do not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.
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http://dx.doi.org/10.3791/56493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752324PMC
September 2017

Phosphatidylinositol (4, 5)-bisphosphate targets double C2 domain protein B to the plasma membrane.

Traffic 2017 12 23;18(12):825-839. Epub 2017 Oct 23.

Department of Neurobiology, Faculty of Life Sciences, Tel-Aviv University, Tel Aviv, Israel.

Double C2 domain protein B (DOC2B) is a high-affinity Ca sensor that translocates from the cytosol to the plasma membrane (PM) and promotes vesicle priming and fusion. However, the molecular mechanism underlying its translocation and targeting to the PM in living cells is not completely understood. DOC2B interacts in vitro with the PM components phosphatidylserine, phosphatidylinositol (4, 5)-bisphosphate [PI(4, 5)P ] and target SNAREs (t-SNAREs). Here, we show that PI(4, 5)P hydrolysis at the PM of living cells abolishes DOC2B translocation, whereas manipulations of t-SNAREs and other phosphoinositides have no effect. Moreover, we were able to redirect DOC2B to intracellular membranes by synthesizing PI(4, 5)P in those membranes. Molecular dynamics simulations and mutagenesis in the calcium and PI(4, 5)P -binding sites strengthened our findings, demonstrating that both calcium and PI(4, 5)P are required for the DOC2B-PM association and revealing multiple PI(4, 5)P -C2B interactions. In addition, we show that DOC2B translocation to the PM is ATP-independent and occurs in a diffusion-like manner. Our data suggest that the Ca -triggered translocation of DOC2B is diffusion-driven and aimed at PI(4, 5)P -containing membranes.
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http://dx.doi.org/10.1111/tra.12528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967617PMC
December 2017

A synaptotagmin suppressor screen indicates SNARE binding controls the timing and Ca cooperativity of vesicle fusion.

Elife 2017 09 12;6. Epub 2017 Sep 12.

Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, United States.

The synaptic vesicle Ca sensor Synaptotagmin binds Ca through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, we took advantage of observations that mutations in the C2B domain Ca-binding pocket dominantly disrupt release from invertebrates to humans. We performed an intragenic screen for suppressors of lethality induced by expression of Synaptotagmin C2B Ca-binding mutants in . This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, we propose a sequence of molecular interactions mediated by Synaptotagmin that promote Ca activation of the synaptic vesicle fusion machinery.
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http://dx.doi.org/10.7554/eLife.28409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617632PMC
September 2017

Electrophysiological analysis of synaptic transmission in Drosophila.

Wiley Interdiscip Rev Dev Biol 2017 09 24;6(5). Epub 2017 May 24.

Department of Neurology, Wayne State University, Detroit, MI, USA.

Synaptic transmission is dynamic, plastic, and highly regulated. Drosophila is an advantageous model system for genetic and molecular studies of presynaptic and postsynaptic mechanisms and plasticity. Electrical recordings of synaptic responses represent a wide-spread approach to study neuronal signaling and synaptic transmission. We discuss experimental techniques that allow monitoring synaptic transmission in Drosophila neuromuscular and central systems. Recordings of synaptic potentials or currents at the larval neuromuscular junction (NMJ) are most common and provide numerous technical advantages due to robustness of the preparation, large and identifiable muscles, and synaptic boutons which can be readily visualized. In particular, focal macropatch recordings combined with the analysis of neurosecretory quanta enable rigorous quantification of the magnitude and kinetics of transmitter release. Patch-clamp recordings of synaptic transmission from the embryonic NMJ enable overcoming the problem of lethality in mutant lines. Recordings from the adult NMJ proved instrumental in the studies of temperature-sensitive paralytic mutants. Genetic studies of behavioral learning in Drosophila compel an investigation of synaptic transmission in the central nervous system (CNS), including primary cultured neurons and an intact brain. Cholinergic and GABAergic synaptic transmission has been recorded from the Drosophila CNS both in vitro and in vivo. In vivo patch-clamp recordings of synaptic transmission from the neurons in the olfactory pathway is a very powerful approach, which has a potential to elucidate how synaptic transmission is associated with behavioral learning. WIREs Dev Biol 2017, 6:e277. doi: 10.1002/wdev.277 For further resources related to this article, please visit the WIREs website.
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http://dx.doi.org/10.1002/wdev.277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980642PMC
September 2017

Synapsin II Regulation of GABAergic Synaptic Transmission Is Dependent on Interneuron Subtype.

J Neurosci 2017 02 13;37(7):1757-1771. Epub 2017 Jan 13.

Department of Neurology,

Synapsins are epilepsy susceptibility genes that encode phosphoproteins reversibly associated with synaptic vesicles. Synapsin II (SynII) gene deletion produces a deficit in inhibitory synaptic transmission, and this defect is thought to cause epileptic activity. We systematically investigated how SynII affects synchronous and asynchronous release components of inhibitory transmission in the CA1 region of the mouse hippocampus. We found that the asynchronous GABAergic release component is diminished in SynII-deleted (SynII(-)) slices. To investigate this defect at different interneuron subtypes, we selectively blocked either N-type or P/Q-type Ca channels. SynII deletion suppressed the asynchronous release component at synapses dependent on N-type Ca channels but not at synapses dependent on P/Q-type Ca channels. We then performed paired double-patch recordings from inhibitory basket interneurons connected to pyramidal neurons and used cluster analysis to classify interneurons according to their spiking and synaptic parameters. We identified two cell subtypes, presumably parvalbumin (PV) and cholecystokinin (CCK) expressing basket interneurons. To validate our interneuron classification, we took advantage of transgenic animals with fluorescently labeled PV interneurons and confirmed that their spiking and synaptic parameters matched the parameters of presumed PV cells identified by the cluster analysis. The analysis of the release time course at the two interneuron subtypes demonstrated that the asynchronous release component was selectively reduced at SynII(-) CCK interneurons. In contrast, the transmission was desynchronized at SynII(-) PV interneurons. Together, our results demonstrate that SynII regulates the time course of GABAergic release, and that this SynII function is dependent on the interneuron subtype. Deletion of the neuronal protein synapsin II (SynII) leads to the development of epilepsy, probably due to impairments in inhibitory synaptic transmission. We systematically investigated SynII function at different subtypes of inhibitory neurons in the hippocampus. We discovered that SynII affects the time course of GABA release, and that this effect is interneuron subtype specific. Within one of the subtypes, SynII deficiency synchronizes the release and suppresses the asynchronous release component, while at the other subtype SynII deficiency suppresses the synchronous release component. These results reveal a new SynII function in the regulation of the time course of GABA release and demonstrate that this function is dependent on the interneuron subtype.
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http://dx.doi.org/10.1523/JNEUROSCI.0844-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320607PMC
February 2017

Complexin Mutants Reveal Partial Segregation between Recycling Pathways That Drive Evoked and Spontaneous Neurotransmission.

J Neurosci 2017 01;37(2):383-396

Department of Neurology and

Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. We examined synaptic vesicle recycling pathways at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. We combined loading of the lipophilic dye FM1-43 with photoconversion, electron microscopy, and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. We found that the total number of recycling vesicles was equal to those retrieved through spontaneous and evoked pools, suggesting that retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1-43 destaining and synaptic depression measured in the presence of the vesicle-refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1-43 photoconversion combined with electron microscopy analysis indicated that spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, whereas vesicles recycled following evoked release involve a larger intraterminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion.

Significance Statement: Neurotransmitter release involves fusion of synaptic vesicles with the plasma membrane in response to an action potential, or spontaneously in the absence of stimulation. Upon fusion, vesicles are retrieved and recycled, and it is unclear whether recycling pathways for evoked and spontaneous vesicles are segregated after fusion. We addressed this question by taking advantage of preparations lacking the synaptic protein complexin, which have elevated spontaneous release that enables reliable tracking of the spontaneous recycling pool. Our results suggest that spontaneous and evoked recycling pathways are segregated during the retrieval process but can partially intermix during stimulation.
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http://dx.doi.org/10.1523/JNEUROSCI.1854-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242395PMC
January 2017

Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion.

Biophys J 2016 Nov;111(9):1954-1964

Department of Neurology, Wayne State University, Detroit, Michigan; Department of Anatomy and Cell Biology, Wayne State University, Detroit, Michigan. Electronic address:

Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the plasma membrane. Vesicles attach to membranes via a specialized protein machinery composed of membrane-attached (t-SNARE) and vesicle-attached (v-SNARE) proteins that zipper together to form a coiled-coil SNARE bundle that brings the two fusing membranes into close proximity. Neurotransmitter release may occur either in response to an action potential or through spontaneous fusion. A cytosolic protein, Complexin (Cpx), binds the SNARE complex and restricts spontaneous exocytosis by acting as a fusion clamp. We previously proposed a model in which the interaction between Cpx and the v-SNARE serves as a spring to prevent premature zippering of the SNARE complex, thereby reducing the likelihood of fusion. To test this model, we combined molecular-dynamics (MD) simulations and site-directed mutagenesis of Cpx and SNAREs in Drosophila. MD simulations of the Drosophila Cpx-SNARE complex demonstrated that Cpx's interaction with the v-SNARE promotes unraveling of the v-SNARE off the core SNARE bundle. We investigated clamping properties in the syx paralytic mutant, which has a single-point mutation in the t-SNARE and displays enhanced spontaneous release. MD simulations demonstrated an altered interaction of Cpx with the SNARE bundle that hindered v-SNARE unraveling by Cpx, thus compromising clamping. We used our model to predict mutations that should enhance the ability of Cpx to prevent full assembly of the SNARE complex. MD simulations predicted that a weakened interaction between the Cpx accessory helix and the v-SNARE would enhance Cpx flexibility and thus promote separation of SNAREs, reducing spontaneous fusion. We generated transgenic Drosophila with mutations in Cpx and the v-SNARE that disrupted a salt bridge between these two proteins. As predicted, both lines demonstrated a selective inhibition in spontaneous release, suggesting that Cpx acts as a fusion clamp that restricts full SNARE zippering.
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http://dx.doi.org/10.1016/j.bpj.2016.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5102999PMC
November 2016

Calcium binding promotes conformational flexibility of the neuronal Ca(2+) sensor synaptotagmin.

Biophys J 2015 May;108(10):2507-2520

Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico; Department of Neurology, Wayne State University, Detroit, Michigan. Electronic address:

Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that serves as a calcium sensor of neuronal secretion. It is established that calcium binding to Syt1 triggers vesicle fusion and release of neuronal transmitters, however, the dynamics of this process is not fully understood. To investigate how Ca(2+) binding affects Syt1 conformational dynamics, we performed prolonged molecular dynamics (MD) simulations of Ca(2+)-unbound and Ca(2+)-bound forms of Syt1. MD simulations were performed at a microsecond scale and combined with Monte Carlo sampling. We found that in the absence of Ca(2+) Syt1 structure in the solution is represented by an ensemble of conformational states with tightly coupled domains. To investigate the effect of Ca(2+) binding, we used two different strategies to generate a molecular model of a Ca(2+)-bound form of Syt1. First, we employed subsequent replacements of monovalent cations transiently captured within Syt1 Ca(2+)-binding pockets by Ca(2+) ions. Second, we performed MD simulations of Syt1 at elevated Ca(2+) levels. All the simulations produced Syt1 structures bound to four Ca(2+) ions, two ions chelated at the binding pocket of each domain. MD simulations of the Ca(2+)-bound form of Syt1 revealed that Syt1 conformational flexibility drastically increased upon Ca(2+) binding. In the presence of Ca(2+), the separation between domains increased, and interdomain rotations became more frequent. These findings suggest that Ca(2+) binding to Syt1 may induce major changes in the Syt1 conformational state, which in turn may initiate the fusion process.
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http://dx.doi.org/10.1016/j.bpj.2015.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457270PMC
May 2015

Coarse-Grained Model of SNARE-Mediated Docking.

Biophys J 2015 May;108(9):2258-69

Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, Pennsylvania; Bioengineering Program, Lehigh University, Bethlehem, Pennsylvania. Electronic address:

Synaptic transmission requires that vesicles filled with neurotransmitter molecules be docked to the plasma membrane by the SNARE protein complex. The SNARE complex applies attractive forces to overcome the long-range repulsion between the vesicle and membrane. To understand how the balance between the attractive and repulsive forces defines the equilibrium docked state we have developed a model that combines the mechanics of vesicle/membrane deformation with an apparently new coarse-grained model of the SNARE complex. The coarse-grained model of the SNARE complex is calibrated by comparison with all-atom molecular dynamics simulations as well as by force measurements in laser tweezer experiments. The model for vesicle/membrane interactions includes the forces produced by membrane deformation and hydration or electrostatic repulsion. Combining these two parts, the coarse-grained model of the SNARE complex with membrane mechanics, we study how the equilibrium docked state varies with the number of SNARE complexes. We find that a single SNARE complex is able to bring a typical synaptic vesicle to within a distance of ∼ 3 nm from the membrane. Further addition of SNARE complexes shortens this distance, but an overdocked state of >4-6 SNAREs actually increases the equilibrium distance.
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http://dx.doi.org/10.1016/j.bpj.2015.03.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423046PMC
May 2015

A continuum model of docking of synaptic vesicle to plasma membrane.

J R Soc Interface 2015 Jan;12(102):20141119

Neurotransmitter release from neuronal terminals is governed by synaptic vesicle fusion. Vesicles filled with transmitters are docked at the neuronal membrane by means of the SNARE machinery. After a series of events leading up to the fusion pore formation, neurotransmitters are released into the synaptic cleft. In this paper, we study the mechanics of the docking process. A continuum model is used to determine the deformation of a spherical vesicle and a plasma membrane, under the influence of SNARE-machinery forces and electrostatic repulsion. Our analysis provides information on the variation of in-plane stress in the membranes, which is known to affect fusion. Also, a simple model is proposed to study hemifusion.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277110PMC
http://dx.doi.org/10.1098/rsif.2014.1119DOI Listing
January 2015

Synapsin regulates activity-dependent outgrowth of synaptic boutons at the Drosophila neuromuscular junction.

J Neurosci 2014 Aug;34(32):10554-63

Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico 00960-6032, and

Patterned depolarization of Drosophila motor neurons can rapidly induce the outgrowth of new synaptic boutons at the larval neuromuscular junction (NMJ), providing a model system to investigate mechanisms underlying acute structural plasticity. Correlative light and electron microscopy analysis revealed that new boutons typically form near the edge of postsynaptic reticulums of presynaptic boutons. Unlike mature boutons, new varicosities have synaptic vesicles which are distributed uniformly throughout the bouton and undeveloped postsynaptic specializations. To characterize the presynaptic mechanisms mediating new synaptic growth induced by patterned activity, we investigated the formation of new boutons in NMJs lacking synapsin [Syn(-)], a synaptic protein important for vesicle clustering, neurodevelopment, and plasticity. We found that budding of new boutons at Syn(-) NMJs was significantly diminished, and that new boutons in Syn(-) preparations were smaller and had reduced synaptic vesicle density. Since synapsin is a target of protein kinase A (PKA), we assayed whether activity-dependent synaptic growth is regulated via a cAMP/PKA/synapsin pathway. We pretreated preparations with forskolin to raise cAMP levels and found this manipulation significantly enhanced activity-dependent synaptic growth in control but not Syn(-) preparations. To examine the trafficking of synapsin during synaptic growth, we generated transgenic animals expressing fluorescently tagged synapsin. Fluorescence recovery after photobleaching analysis revealed that patterned depolarization promoted synapsin movement between boutons. During new synaptic bouton formation, synapsin redistributed upon stimulation toward the sites of varicosity outgrowth. These findings support a model whereby synapsin accumulates at sites of synaptic growth and facilitates budding of new boutons via a cAMP/PKA-dependent pathway.
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http://dx.doi.org/10.1523/JNEUROSCI.5074-13.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200108PMC
August 2014

Synapsin II and Rab3a cooperate in the regulation of epileptic and synaptic activity in the CA1 region of the hippocampus.

J Neurosci 2013 Nov;33(46):18319-30

Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico 00956, and Department of Pharmacology Wayne State University School of Medicine, Detroit, Michigan 48001.

Some forms of idiopathic epilepsy in animals and humans are associated with deficiency of synapsin, a phosphoprotein that reversibly associates with synaptic vesicles. We have previously shown that the epileptic phenotype seen in synapsin II knock-out mice (SynII(-)) can be rescued by the genetic deletion of the Rab3a protein. Here we have examined the cellular basis for this rescue using whole-cell recordings from CA1 hippocampal pyramidal cells in brain slices. We find that SynII(-) neurons have increased spontaneous activity and a reduced threshold for the induction of epileptiform activity by 4-aminopyridine (4-AP). Using selective recordings of glutamatergic and GABAergic activity we show that in wild-type neurons low concentrations of 4-AP facilitate glutamatergic and GABAergic transmission in a balanced way, whereas in SynII(-) neurons this balance is shifted toward excitation. This imbalance reflects a deficit in inhibitory synaptic transmission that appears to be secondary to reduced Ca(2+) sensitivity in SynII(-) neurons. This suggestion is supported by our finding that synaptic and epileptiform activity at SynII(-) and wild-type synapses is similar when GABAergic transmission is blocked. Deletion of Rab3a results in glutamatergic synapses that have a compromised responsiveness to either low 4-AP concentrations or elevated extracellular Ca(2+). These changes mitigate the overexcitable phenotype observed in SynII(-) neurons. Thus, Rab3a deletion appears to restore the excitatory/inhibitory imbalance observed in SynII(-) hippocampal slices indirectly, not by correcting the deficit in GABAergic synaptic transmission but rather by impairing excitatory glutamatergic synaptic transmission.
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http://dx.doi.org/10.1523/JNEUROSCI.5293-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3893359PMC
November 2013

Interaction of the complexin accessory helix with the C-terminus of the SNARE complex: molecular-dynamics model of the fusion clamp.

Biophys J 2013 Aug;105(3):679-90

Neuroscience Department, Universidad Central del Caribe, Bayamon, Puerto Rico.

SNARE complexes form between the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 to drive membrane fusion. A cytosolic protein, complexin (Cpx), binds to the SNARE bundle, and its accessory helix (AH) functions to clamp synaptic vesicle fusion. We performed molecular-dynamics simulations of the SNARE/Cpx complex and discovered that at equilibrium the Cpx AH forms tight links with both synaptobrevin and SNAP25. To simulate the effect of electrostatic repulsion between vesicle and membrane on the SNARE complex, we calculated the electrostatic force and performed simulations with an external force applied to synaptobrevin. We found that the partially unzipped state of the SNARE bundle can be stabilized by interactions with the Cpx AH, suggesting a simple mechanistic explanation for the role of Cpx in fusion clamping. To test this model, we performed experimental and computational characterizations of the syx(3-69)Drosophila mutant, which has a point mutation in syntaxin that causes increased spontaneous fusion. We found that this mutation disrupts the interaction of the Cpx AH with synaptobrevin, partially imitating the cpx null phenotype. Our results support a model in which the Cpx AH clamps fusion by binding to the synaptobrevin C-terminus, thus preventing full SNARE zippering.
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http://dx.doi.org/10.1016/j.bpj.2013.06.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736676PMC
August 2013

Adar is essential for optimal presynaptic function.

Mol Cell Neurosci 2013 Jan 3;52:173-80. Epub 2012 Nov 3.

Institute of Neurobiology, Medical Sciences Campus, University of Puerto Rico, PR.

RNA editing is a powerful way to recode genetic information. Because it potentially affects RNA targets that are predominantly present in neurons, it is widely hypothesized to affect neuronal structure and physiology. Across phyla, loss of the enzyme responsible for RNA editing, Adar, leads to behavioral changes, impaired locomotion, neurodegeneration and death. However, the consequences of a loss of Adar activity on neuronal structure and function have not been studied in detail. In particular, the role of RNA editing on synaptic development and physiology has not been investigated. Here we test the physiological and morphological consequences of the lack of Adar activity on the Drosophila neuromuscular junction (NMJ). Our detailed examination of synaptic transmission showed that loss of Adar increases quantal size, reduces the number of quanta of neurotransmitter released and perturbs the calcium dependence of synaptic release. In addition, we find that staining for several synaptic vesicle proteins is abnormally intense at Adar deficient synapses. Consistent with this finding, Adar mutants showed a major alteration in synaptic ultrastructure. Finally, we present evidence of compensatory changes in muscle membrane properties in response to the changes in presynaptic activity within the Adar mutant NMJs.
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http://dx.doi.org/10.1016/j.mcn.2012.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3613243PMC
January 2013

Adhesion energy can regulate vesicle fusion and stabilize partially fused states.

J R Soc Interface 2012 Jul 18;9(72):1555-67. Epub 2012 Jan 18.

Field of Theoretical and Applied Mechanics, Cornell University, Ithaca, NY, USA.

Release of neurotransmitters from nerve terminals occurs by fusion of synaptic vesicles with the plasma membrane, and this process is highly regulated. Although major molecular components that control docking and fusion of vesicles to the synaptic membrane have been identified, the detailed mechanics of this process is not yet understood. We have developed a mathematical model that predicts how adhesion forces imposed by docking and fusion molecular machinery would affect the fusion process. We have computed the membrane stress that is produced by adhesion-driven vesicle bending and find that it is compressive. Further, our computations of the membrane curvature predict that strong adhesion can create a metastable state with a partially opened pore that would correspond to the 'kiss and run' release mode. Our model predicts that the larger the vesicle size, the more likely the metastable state with a transiently opened pore. These results contribute to understanding the mechanics of the fusion process, including possible clamping of the fusion by increasing molecular adhesion, and a balance between 'kiss and run' and full collapse fusion modes.
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http://dx.doi.org/10.1098/rsif.2011.0827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367824PMC
July 2012

Synapsin regulation of vesicle organization and functional pools.

Semin Cell Dev Biol 2011 Jun 31;22(4):387-92. Epub 2011 Jul 31.

Universidad Central del Caribe, Neuroscience Department, 2U6 Ave Laurel, Bayamon, PR 00956, USA.

Synaptic vesicles are organized in clusters, and synapsin maintains vesicle organization and abundance in nerve terminals. At the functional level, vesicles can be subdivided into three pools: the releasable pool, the recycling pool, and the reserve pool, and synapsin mediates transitions between these pools. Synapsin directs vesicles into the reserve pool, and synapsin II isoform has a primary role in this function. In addition, synapsin actively delivers vesicles to active zones. Finally, synapsin I isoform mediates coupling release events to action potentials at the latest stages of exocytosis. Thus, synapsin is involved in multiple stages of the vesicle cycle, including vesicle clustering, maintaining the reserve pool, vesicle delivery to active zones, and synchronizing release events. These processes are regulated via a dynamic synapsin phosphorylation/dephosphorylation cycle which involves multiple phosphorylation sites and several pathways. Different synapsin isoforms have unique and non-redundant roles in the multifaceted synapsin function.
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http://dx.doi.org/10.1016/j.semcdb.2011.07.003DOI Listing
June 2011

Cooperative regulation of neurotransmitter release by Rab3a and synapsin II.

Mol Cell Neurosci 2010 Jun 23;44(2):190-200. Epub 2010 Mar 23.

Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA.

To understand how the presynaptic proteins synapsin and Rab3a may interact in the regulation of the synaptic vesicle cycle and the release process, we derived a double knockout (DKO) mouse lacking both synapsin II and Rab3a. We found that Rab3a deletion rescued epileptic-like seizures typical for synapsin II gene deleted animals (Syn II(-)). Furthermore, action potential evoked release was drastically reduced in DKO synapses, although spontaneous release remained normal. At low Ca2+ conditions, quantal content was equally reduced in Rab3a(-) and DKO synapses, but as Ca2+ concentration increased, the increase in quantal content was more prominent in Rab3a(-). Electron microscopy analysis revealed that DKO synapses have a combined phenotype, with docked vesicles being reduced similar to Rab3a(-), and intraterminal vesicles being depleted similar to Syn II(-). Consistently, both Syn II(-) and DKO terminals had increased synaptic depression and incomplete recovery. Taken together, our results suggest that synapsin II and Rab3a have separate roles in maintaining the total store of synaptic vesicles and cooperate in promoting the latest steps of neuronal secretion.
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http://dx.doi.org/10.1016/j.mcn.2010.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522281PMC
June 2010

Rab3a-mediated vesicle recruitment regulates short-term plasticity at the mouse diaphragm synapse.

Mol Cell Neurosci 2009 Jun 5;41(2):286-96. Epub 2009 Apr 5.

Department of Biological Sciences, Lehigh University, 111 Research Dr., Bethlehem, PA 18015, USA.

Rab3a is a small GTP-binding protein associated with presynaptic vesicles. We have measured the releasable pool in the neuromuscular junction of Rab3a(-) mice by recordings of the asynchronous release activity produced by local applications of hypertonic solutions and demonstrated that the releasable pool is significantly reduced in Rab3a(-) synapses. We found that the activity-dependent vesicle recruitment, as well as the synaptic enhancement associated with it, is disrupted in Rab3a(-) synapses. We employed Ca2+ chelators and disruption of Ca2+ sensitivity of fusion machinery by botulinum neurotoxin type-A microinjections, and demonstrated that local Ca2+ elevation may overcome the Rab3a deficiency in maintaining the releasable pool. Rab3a(-) terminals demonstrated a small but significant low-frequency depression, probably due to insufficient refilling of the releasable pool. Our results, taken together, support the hypothesis that Rab3a maintains the pool of fusion competent vesicles tightly coupled to Ca2+ channels.
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http://dx.doi.org/10.1016/j.mcn.2009.03.008DOI Listing
June 2009

Stimulation-induced formation of the reserve pool of vesicles in Drosophila motor boutons.

J Neurophysiol 2009 May 11;101(5):2423-33. Epub 2009 Mar 11.

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

We combined electron microscopy (EM), synaptic vesicle staining by fluorescent marker FM1-43, photoconversion of the dye into an electron dense product, and electrical recordings of synaptic responses to study the distribution of reserve and recycling vesicles and its dependence on stimulation in Drosophila motor boutons. We showed that, at rest, vesicles are distributed over the periphery of the bouton, with the recycling and reserve pools being intermixed and the central core of the bouton being devoid of vesicles. Continuous high-frequency stimulation followed by a resting period mobilized the reserve vesicles into the recycling pool and, most notably, produced an increase in vesicle abundance. Recordings of synaptic activity from the temperature-sensitive endocytosis mutant shibire during continuous stimulation until complete depression provided an independent estimate of the increase in vesicle abundance on intense stimulation. EM analysis demonstrated that continuous stimulation produced an increase in the vesicle density, whereas during a subsequent resting period, vesicles filled empty areas of the bouton, spreading toward its central core. Although the observed structural potentiation did not alter basal transmitter release, it produced an increased synaptic enhancement during high-frequency stimulation. The latter effect was not observed when the boutons were potentiated using high-frequency stimulation without a subsequent resting period. We concluded therefore that the newly formed vesicles replenish the reserve pool during a resting period following intense stimulation.
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http://dx.doi.org/10.1152/jn.91122.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2681433PMC
May 2009

Synapsin I accelerates the kinetics of neurotransmitter release in mouse motor terminals.

Synapse 2009 Jun;63(6):531-3

Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, USA.

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http://dx.doi.org/10.1002/syn.20635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706446PMC
June 2009

Enhancement of the endosomal endocytic pathway increases quantal size.

Mol Cell Neurosci 2009 Feb 5;40(2):199-206. Epub 2008 Nov 5.

Lehigh University, Dept. of Biological Sciences, 111 Research Dr., Bethlehem, PA 18015, USA.

We combined recordings of spontaneous quantal events with electron microscopy analysis of synaptic ultrastructure to demonstrate that the size of a neurosecretory quantum increases following an activation of the endosomal endocytic pathway. We reversibly activated the endosomal endocytic pathway in Drosophila motor boutons by application of high K+ solution. This treatment produced the formation of numerous cisternae, vacuoles and enlarged vesicles. Spontaneous quantal events recorded immediately after the cessation of high K+ application were significantly enlarged, and this increase in quantal size was reversed after a 10 minute resting period. Actin depolymerization produced by latrunculin B pretreatment inhibited both the formation of endosome-like structures and the increase in quantal size. Loading the preparations with the dye FM1-43 followed by photoconversion of the dye combined with electron microscopy analysis revealed that the observed cisternae are likely to be the product of both bulk membrane retrieval and vesicle fusion.
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http://dx.doi.org/10.1016/j.mcn.2008.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522282PMC
February 2009

Synapsin II and calcium regulate vesicle docking and the cross-talk between vesicle pools at the mouse motor terminals.

J Physiol 2008 Oct 31;586(19):4649-73. Epub 2008 Jul 31.

Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA.

The synapsins, an abundant and highly conserved family of proteins that associate with synaptic vesicles, have been implicated in regulating the synaptic vesicle cycle. However, it has not been determined whether synapsin directly regulates the number of docked vesicles. Here we document that reducing Ca(2+) concentration [Ca(2+)](o) in the extracellular medium from 2 to 0.5 mm led to an approximately 40% decrease in both docked and undocked synaptic vesicles in wild-type nerve terminals of the mouse diaphragm. The same treatment reduced the number of undocked vesicles in nerve terminals derived from synapsin II gene deleted animals, but surprisingly it did not decrease vesicle docking, indicating that synapsin II inhibits docking of synaptic vesicles at reduced [Ca(2+)](o). In accordance with the morphological findings, at reduced [Ca(2+)](o) synapsin II (-) terminals had a higher rate of quantal neurotransmitter release. Microinjection of a recombinant synapsin II protein into synapsin II (-) terminals reduced vesicular docking and inhibited quantal release, indicating a direct and selective synapsin II effect for regulating vesicle docking and, in turn, quantal release. To understand why [Ca(2+)](o) has a prominent effect on synapsin function, we investigated the effect of [Ca(2+)](o) on the distribution of synaptic vesicles and on the concentration of intraterminal Ca(2+). We found that reduced [Ca(2+)](o) conditions produce a decrease in intracellular Ca(2+) and overall vesicle depletion. To explore why at these conditions the role of synapsin II in vesicle docking becomes more prominent, we developed a quantitative model of the vesicle cycle, with a two step synapsin action in stabilizing the vesicle store and regulating vesicle docking. The results of the modelling were in a good agreement with the observed dependence of vesicle distribution on synapsin II and calcium deficiency.
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http://dx.doi.org/10.1113/jphysiol.2008.154666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2614040PMC
October 2008

Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN.

J Neurosci Methods 2008 Mar 23;168(2):500-13. Epub 2007 Oct 23.

Lehigh University, Department of Biological Sciences, 111 Research Drive, Bethlehem, PA 18015, United States.

Quantal analysis of synaptic transmission is an important tool for understanding the mechanisms of synaptic plasticity and synaptic regulation. Although several custom-made and commercial algorithms have been created for the analysis of spontaneous synaptic activity, software for the analysis of action potential evoked release remains very limited. The present paper describes a user-friendly software package QUANTAN which has been created to analyze electrical recordings of postsynaptic responses. The program package is written using Borland C++ under Windows platform. QUANTAN employs and compares several algorithms to extract the average quantal content of synaptic responses, including direct quantal counts, the analysis of synaptic amplitudes, and the analysis of integrated current traces. The integration of several methods in one user-friendly program package makes quantal analysis of action potential evoked release more reliable and accurate. To evaluate the variability in quantal content, QUANTAN performs deconvolution of the distributions of amplitudes or areas of synaptic responses employing a ridge regression method. Other capabilities of QUANTAN include the analysis of the time-course and stationarity of quantal release. In summary, QUANTAN uses digital records of synaptic responses as an input and computes the distribution of quantal content and synaptic parameters. QUANTAN is freely available to other scholars over the internet.
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http://dx.doi.org/10.1016/j.jneumeth.2007.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2290970PMC
March 2008