The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
R Llinas - One of the best experts on this subject based on the ideXlab platform.
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intraterminal injection of synapsin dependent protein kinase ii alters at the Squid Giant Synapse synaptic transmission synaptic vesicles protein phosphorylation
2016Co-Authors: R Llinas, T L Mcguinnesst, Christopher S LeonardAbstract:Synapsin I and calcium/calmodulin-depen- dent protein kinase II were pressure-injected into the preter- minal digit of the Squid Giant Synapse to test directly the possi- ble regulation of neurotransmitter release by these substances. Neurotransmitter release was determined by measuring the amplitude, rate of rise, and latency of the postsynaptic poten- tial generated in response to presynaptic depolarizing steps un- der voltage clamp conditions. Injection of dephosphosynapsin I decreased the amplitude and rate of rise of the postsynaptic potential, whereas injection of either phosphosynapsin I or heat-treated dephosphosynapsin I was without effect. Con- versely, injection of calcium/calmodulin-dependent protein ki- nase II, which phosphorylates synapsin I on site II, increased the rate of rise and amplitude and decreased the latency of the postsynaptic potential. The effects of these proteins were ob- served without any detectable change in the initial phase of the presynaptic calcium current. A synapsin I-like protein and cal- cium/calmodulin-dependent protein kinase II were demon- strated by biochemical and immunochemical techniques to be present in Squid nervous tissue. The data support the hypothe- sis that synapsin I regulates the availability of synaptic vesicles for release; we propose that calcium entry into the nerve ter- minal activates calcium/calmodulin-dependent protein kinase II, Which phosphorylates synapsin I on site II, dissociating it from the vesicles and thereby removing a constraint in the re- lease process.
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calcium role in depolarization secretion in Squid Giant Synapse tetraethylammonium tetrodotoxin suppression potenti
2016Co-Authors: R Llinas, C NicholsonAbstract:Aequorin, a protein that emits light in the presence of calicium, was injected in the presynaptic terminal of the Squid Giant Synapse. This injection was preceded by intracellular tetraethlylaminonium admini- stration, which prolonged the duration of the presynaptic action potential. After this procedure light emission was evoked by single presynaptic spikes capable of releasing synaptic transmitter. In a second set of experiments, presynaptic tetraethylammonium injection was followed by the administration of tetrodotoxin extracellularly, which abolished the pPesynaptic action potential. Under these conditions artificial depolarizatior of the presynaptic terminal triggered the release of synaptic trainsmitter, in a graded manner. However, as previously reported by other authors, memnbrane potential steps to an internal positive value of approximately + 90 mV (the suppression potential) produced a blockage of transmitter release for the dura- tion of the imposed potential. Synaptic transmission re- curred, nevertheless, as the current injection Was termi- nated. A similar set of experiments, performed after the intracelluliar injection of aequorin in the presynaptic fiber, demonstrated that the aequorin light response was evoked by membrane potential steps capable of releasing synaptic transmitter. If the membrane potential was made positive to the "suppression" level, no light response was evoked butt the light emission appeared, as did transmitter re- lease, at the end of the current pulse. These experiments demonstrate that release of transmitter is directly eor- related with intracellular calcium concentration and that the suppression potential is compatible with the existence of a calcium equilibrium potential at the presynaptic terminal.
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role of rab27 in synaptic transmission at the Squid Giant Synapse
2008Co-Authors: Eiko Kanno, Jorge E. Moreira, Mutsuyuki Sugimori, R Llinas, Soonwook Choi, Mitsunori FukudaAbstract:Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against Squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on Squid synaptic vesicles. Electrophysiological study of injected Synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca(2+) current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca(2+)-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected Giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the Squid Giant Synapse.
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1 methyl 4 phenylpyridinium induces synaptic dysfunction through a pathway involving caspase and pkcδ enzymatic activities
2007Co-Authors: Yafell Serulle, Jorge E. Moreira, Mutsuyuki Sugimori, R Llinas, Scott T Brady, Gerardo Morfini, Gustavo PiginoAbstract:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration has been used, in various mammalian species, as an experimental model of Parkinson's disease. The pathogenesis for such pharmacologically induced Parkinson's disease involves 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. This metabolite produces rapid degeneration of nigrostriatal dopaminergic neurons, which causes the parkinsonian syndrome. In this work, we show that injection of MPP+ into the presynaptic terminal of the Squid Giant Synapse blocks synaptic transmission without affecting the presynaptic action potential or the presynaptic calcium currents. These effects of MPP+ were mimicked by the injection of an active form of caspase-3 and prevented by inhibitors of caspase-3 and protein kinase C δ. Ultrastructurally, MPP+-injected Synapses showed a dramatic reduction in the number of neurotransmitter vesicles at the presynaptic active zone, as compared with control Synapses. Otherwise, normal docking and clathrin-coated vesicles were observed, albeit at much reduced numbers. These results indicate that MPP+ acutely reduces presynaptic vesicular availability, not release, and that MPP+-induced pathogenesis results from presynaptic dysfunction that leads, secondarily, to dying-back neuropathy in affected neurons.
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vesicular reuptake inhibition by a synaptotagmin i c2b domain antibody at the Squid Giant Synapse
2004Co-Authors: R Llinas, Mutsuyuki Sugimori, Kimberly A Moran, Jorge Eduardo Moreira, Mitsunori FukudaAbstract:Synaptotagmin (Syt) I, a ubiquitous synaptic vesicle protein, comprises a transmembrane region and two C2 domains. The C2 domains, which have been shown to be essential for both synaptic vesicle exocytosis and endocytosis, are also seen as the Ca2+ sensors in synaptic vesicular release. In a previous study, we reported that a polyclonal antibody raised against the Squid (Loligo pealei) Syt I C2B domain, while inhibiting vesicular endocytosis, was synaptic release neutral at the Squid Giant Synapse. Recent reports concerning the C2B requirements for synaptic release prompted us to readdress the role of C2B in Squid Giant Synapse function. Presynaptic injection of another anti-Syt I-C2B antibody (using recombinant whole C2B domain expressed in mammalian cell culture as an antigen) into the presynaptic terminal reproduced our previous results, i.e., reduction of vesicular endocytosis without affecting synaptic release. This set of results addresses the issue of the geometrical arrangement of the Ca2+ sensor, allowing the C2B domain antibody to restrict Ca2+-dependent C2B self-oligomerization without modifying the Ca2+-dependent release process.
George J Augustine - One of the best experts on this subject based on the ideXlab platform.
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under review
2016Co-Authors: Lu-yang Wang, George J Augustine, Samuel Young, Max Planck Florida, Lee KongAbstract:ntu.edu.sg Here we summarize the evidence from two “Giant ” presynaptic terminals—the Squid Giant Synapse and the mammalian calyx of Held—supporting the involvement of nanodomain calcium signals in triggering of neurotransmitter release. At the Squid Synapse, there are three main lines of experimental evidence for nanodomain signaling. First, changing the size of the unitary calcium channel current by altering external calcium concentration causes a non-linear change in transmitter release, while changing the number of open channels by broadening the presynaptic action potential causes a linear change in release. Second, low-affinity calcium indicators, calcium chelators, and uncaging of calcium all suggest that presynaptic calcium concentrations are as high as hundreds of micromolar, which is more compatible with a nanodomain type of calcium signal. Finally, neurotransmitter release is much less affected by the slow calcium chelator, ethylene glycol tetraacetic acid (EGTA), in comparison to the rapid chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Similarly, as the calyx of Held Synapse matures, EGTA becomes less effective in attenuating transmitter release whil
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Presynaptic nanodomains: a tale of two Synapses
2015Co-Authors: George J Augustine, Lu-yang EwangAbstract:Here we summarize the evidence from two Giant presynaptic terminals - the Squid Giant Synapse and the mammalian calyx of Held - supporting the involvement of nanodomain calcium signals in triggering of neurotransmitter release. At the Squid Synapse, there are three main lines of experimental evidence for nanodomain signaling. First, changing the size of the unitary calcium channel current by altering external calcium concentration causes a non-linear change in transmitter release, while changing the number of open channels by broadening the presynaptic action potential causes a linear change in release. Second, low-affinity calcium indicators, calcium chelators, and uncaging of calcium all suggest that presynaptic calcium concentrations are as high as hundreds of micromolar, which is more compatible with a nanodomain type of calcium signal. Finally, neurotransmitter release is much less affected by the slow calcium chelator, EGTA, in comparison to the rapid chelator BAPTA. Similarly, as the calyx of Held Synapse matures, EGTA becomes less effective in attenuating transmitter release while the number of calcium channels required to trigger a single fusion event declines. This suggests a developmental transformation of microdomain to nanodomain coupling between calcium channels and transmitter release. Calcium imaging and uncaging experiments, in combination with simulations of calcium diffusion, indicate the peak calcium concentration seen by presynaptic calcium sensors reaches at least tens of micromolar. Taken together, data from these provide a compelling argument that nanodomain calcium signaling gates very rapid transmitter release
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tonically active protein kinase a regulates neurotransmitter release at the Squid Giant Synapse
2001Co-Authors: George J Augustine, Andrew J Czernik, Sabine Hilfiker, Paul GreengardAbstract:1. Electrophysiological and microinjection methods were used to examine the role of cyclic AMP-dependent protein kinase A (PKA) in regulating transmitter release at the Squid Giant Synapse. 2. Excitatory postsynaptic potentials (EPSPs) evoked by presynaptic action potentials were not affected by presynaptic injection of an exogenous active catalytic subunit of mammalian PKA. 3. In contrast, presynaptic injection of PKI-amide, a peptide that inhibits PKA with high potency and specificity, led to a reversible inhibition of EPSPs. 4. Injection of several other peptides that serve as substrates for PKA also reversibly inhibited neurotransmitter release. The ability of these peptides to inhibit release was correlated with their ability to serve as PKA substrates, suggesting that these peptides act by competing with endogenous substrates for phosphorylation by active endogenous PKA. 5. We suggest that the phosphorylation of PKA substrates is maintained at a relatively high state under basal conditions and that this tonic activity of PKA is to a large degree required for evoked neurotransmitter release at the Squid Giant presynaptic terminal.
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Two sites of action for synapsin domain E in regulating neurotransmitter release.
1998Co-Authors: Sabine Hilfiker, Andrew J Czernik, Felix E. Schweizer, Hung-teh Kao, George J AugustineAbstract:Synapsins, a family of synaptic vesicle proteins, have been shown to regulate neurotransmitter release; the mechanism(s) by which they act are not fully understood. Here we have studied the role of domain E of synapsins in neurotransmitter release at the Squid Giant Synapse. Two Squid synapsin isoforms were cloned and found to contain a carboxy (C)-terminal domain homologous to domain E of the vertebrate a-type synapsin isoforms. Presynaptic injection of a peptide fragment of domain E greatly reduced the number of synaptic vesicles in the periphery of the active zone, and increased the rate and extent of synaptic depression, suggesting that domain E is essential for synapsins to regulate a reserve pool of synaptic vesicles. Domain E peptide had no effect on the number of docked synaptic vesicles, yet reversibly inhibited and slowed the kinetics of neurotransmitter release, indicating a second role for synapsins that is more intimately associated with the release process itself. Thus, synapsin domain E is involved in at least two distinct reactions that are crucial for exocytosis in presynaptic terminals.
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a neuronal sec1 homolog regulates neurotransmitter release at the Squid Giant Synapse
1998Co-Authors: George J Augustine, Thomas Dresbach, Marie E Burns, Vincent Oconnor, William M Debello, Heinrich BetzAbstract:Sec1-related proteins are essential for membrane fusion at distinct stages of the constitutive and regulated secretory pathways in eukaryotic cells. Studies of neuronal isoforms of the Sec1 protein family have yielded evidence for both positive and negative regulatory functions of these proteins in neurotransmitter release. Here, we have identified a Squid neuronal homolog (s-Sec1) of Sec1 proteins and examined its function in neurotransmitter release at the Squid Giant Synapse. Microinjection of s-Sec1 into the presynaptic terminal of the Giant Synapse inhibited evoked neurotransmitter release, but this effect was prevented by coinjecting the cytoplasmic domain of Squid syntaxin (s-syntaxin), one of the binding partners of s-Sec1. A 24 amino acid peptide fragment of s-Sec1, which inhibited the binding of s-Sec1 to s-syntaxin in vitro , completely blocked release, suggesting an essential function of the s-Sec1/s-syntaxin interaction in transmitter release. Electron microscopy showed that injection of s-Sec1 did not change the spatial distribution of synaptic vesicles at presynaptic release sites (“active zones”), whereas the inhibitory peptide increased the number of docked vesicles. These distinct morphological effects lead us to conclude that Sec1 proteins function at different stages of synaptic vesicle exocytosis, and that an interaction of s-Sec1 with syntaxin—at a stage blocked by the peptide—is necessary for docked vesicles to fuse.
Mutsuyuki Sugimori - One of the best experts on this subject based on the ideXlab platform.
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blocking effects of human tau on Squid Giant Synapse transmission and its prevention by t 817 ma
2011Co-Authors: Jorge E. Moreira, Soonwook Choi, Herman Moreno, Janaina Brusco, Jesus Avila, Mutsuyuki SugimoriAbstract:Filamentous tau inclusions are hallmarks of Alzheimer's disease and related neurodegenerative tauopathies, but the molecular mechanisms involved in tau-mediated changes in neuronal function and their possible effects on synaptic transmission are unknown. We have evaluated the effects of human tau protein injected directly into the presynaptic terminal axon of the Squid Giant Synapse, which affords functional, structural, and biochemical analysis of its action on the synaptic release process. Indeed, we have found that at physiological concentration recombinant human tau (h-tau42) becomes phosphorylated, produces a rapid synaptic transmission block, and induces the formation of clusters of aggregated synaptic vesicles in the vicinity of the active zone. Presynaptic voltage clamp recordings demonstrate that h-tau42 does not modify the presynaptic calcium current amplitude or kinetics. Analysis of synaptic noise at the post-synaptic axon following presynaptic h-tau42 microinjection revealed an initial phase of increase spontaneous transmitter release followed by a marked reduction in noise. Finally, systemic administration of T-817MA, a proposed neuro-protective agent, rescued tau-induced synaptic abnormalities. Our results show novel mechanisms of h-tau42 mediated synaptic transmission failure and identify a potential therapeutic agent to treat tau-related neurotoxicity.
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role of rab27 in synaptic transmission at the Squid Giant Synapse
2008Co-Authors: Eiko Kanno, Jorge E. Moreira, Mutsuyuki Sugimori, R Llinas, Soonwook Choi, Mitsunori FukudaAbstract:Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against Squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on Squid synaptic vesicles. Electrophysiological study of injected Synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca(2+) current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca(2+)-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected Giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the Squid Giant Synapse.
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1 methyl 4 phenylpyridinium induces synaptic dysfunction through a pathway involving caspase and pkcδ enzymatic activities
2007Co-Authors: Yafell Serulle, Jorge E. Moreira, Mutsuyuki Sugimori, R Llinas, Scott T Brady, Gerardo Morfini, Gustavo PiginoAbstract:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration has been used, in various mammalian species, as an experimental model of Parkinson's disease. The pathogenesis for such pharmacologically induced Parkinson's disease involves 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. This metabolite produces rapid degeneration of nigrostriatal dopaminergic neurons, which causes the parkinsonian syndrome. In this work, we show that injection of MPP+ into the presynaptic terminal of the Squid Giant Synapse blocks synaptic transmission without affecting the presynaptic action potential or the presynaptic calcium currents. These effects of MPP+ were mimicked by the injection of an active form of caspase-3 and prevented by inhibitors of caspase-3 and protein kinase C δ. Ultrastructurally, MPP+-injected Synapses showed a dramatic reduction in the number of neurotransmitter vesicles at the presynaptic active zone, as compared with control Synapses. Otherwise, normal docking and clathrin-coated vesicles were observed, albeit at much reduced numbers. These results indicate that MPP+ acutely reduces presynaptic vesicular availability, not release, and that MPP+-induced pathogenesis results from presynaptic dysfunction that leads, secondarily, to dying-back neuropathy in affected neurons.
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vesicular reuptake inhibition by a synaptotagmin i c2b domain antibody at the Squid Giant Synapse
2004Co-Authors: R Llinas, Mutsuyuki Sugimori, Kimberly A Moran, Jorge Eduardo Moreira, Mitsunori FukudaAbstract:Synaptotagmin (Syt) I, a ubiquitous synaptic vesicle protein, comprises a transmembrane region and two C2 domains. The C2 domains, which have been shown to be essential for both synaptic vesicle exocytosis and endocytosis, are also seen as the Ca2+ sensors in synaptic vesicular release. In a previous study, we reported that a polyclonal antibody raised against the Squid (Loligo pealei) Syt I C2B domain, while inhibiting vesicular endocytosis, was synaptic release neutral at the Squid Giant Synapse. Recent reports concerning the C2B requirements for synaptic release prompted us to readdress the role of C2B in Squid Giant Synapse function. Presynaptic injection of another anti-Syt I-C2B antibody (using recombinant whole C2B domain expressed in mammalian cell culture as an antigen) into the presynaptic terminal reproduced our previous results, i.e., reduction of vesicular endocytosis without affecting synaptic release. This set of results addresses the issue of the geometrical arrangement of the Ca2+ sensor, allowing the C2B domain antibody to restrict Ca2+-dependent C2B self-oligomerization without modifying the Ca2+-dependent release process.
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Role of the conserved WHXL motif in the C terminus of synaptotagmin in synaptic vesicle docking.
2000Co-Authors: Mitsunori Fukuda, Jorge E. Moreira, Mutsuyuki Sugimori, Katsuhiko Mikoshiba, Vincent Hok Liu, Rodolfo R. LlinásAbstract:Synaptotagmin (Syt) I , an abundant synaptic vesicle protein, consists of one transmembrane region, two C2 domains, and a short C terminus. This protein is essential for both synaptic vesicle exocytosis and endocytosis via its C2 domains. Although the short C terminus is highly conserved among the Syt family and across species, little is known about the exact role of the conserved C terminus of Syt I. In this paper, we report a function of the Syt I C terminus in synaptic vesicle docking at the active zones. Presynaptic injection of a peptide corresponding to the C-terminal 21 amino acids of Syt I (named Syt-C) into the Squid Giant Synapse blocked synaptic transmission without affecting the presynaptic action potential or the presynaptic Ca2+ currents. The same procedure repeated with a mutant C-terminal peptide (Syt-CM) had no effect on synaptic transmission. Repetitive presynaptic stimulation with Syt-C produced a rapid decrease in the amplitude of the postsynaptic potentials as the synaptic block progressed, indicating that the peptide interferes with the docking step rather than the fusion step of synaptic vesicles. Electron microscopy of the Synapses injected with the Syt-C peptide showed a marked decrease in the number of docked synaptic vesicles at the active zones, as compared with controls. These results indicate that Syt I is a multifunctional protein that is involved in at least three steps of synaptic vesicle cycle: docking, fusion, and reuptake of synaptic vesicles.
Jorge E. Moreira - One of the best experts on this subject based on the ideXlab platform.
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blocking effects of human tau on Squid Giant Synapse transmission and its prevention by t 817 ma
2011Co-Authors: Jorge E. Moreira, Soonwook Choi, Herman Moreno, Janaina Brusco, Jesus Avila, Mutsuyuki SugimoriAbstract:Filamentous tau inclusions are hallmarks of Alzheimer's disease and related neurodegenerative tauopathies, but the molecular mechanisms involved in tau-mediated changes in neuronal function and their possible effects on synaptic transmission are unknown. We have evaluated the effects of human tau protein injected directly into the presynaptic terminal axon of the Squid Giant Synapse, which affords functional, structural, and biochemical analysis of its action on the synaptic release process. Indeed, we have found that at physiological concentration recombinant human tau (h-tau42) becomes phosphorylated, produces a rapid synaptic transmission block, and induces the formation of clusters of aggregated synaptic vesicles in the vicinity of the active zone. Presynaptic voltage clamp recordings demonstrate that h-tau42 does not modify the presynaptic calcium current amplitude or kinetics. Analysis of synaptic noise at the post-synaptic axon following presynaptic h-tau42 microinjection revealed an initial phase of increase spontaneous transmitter release followed by a marked reduction in noise. Finally, systemic administration of T-817MA, a proposed neuro-protective agent, rescued tau-induced synaptic abnormalities. Our results show novel mechanisms of h-tau42 mediated synaptic transmission failure and identify a potential therapeutic agent to treat tau-related neurotoxicity.
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role of rab27 in synaptic transmission at the Squid Giant Synapse
2008Co-Authors: Eiko Kanno, Jorge E. Moreira, Mutsuyuki Sugimori, R Llinas, Soonwook Choi, Mitsunori FukudaAbstract:Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against Squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on Squid synaptic vesicles. Electrophysiological study of injected Synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca(2+) current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca(2+)-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected Giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the Squid Giant Synapse.
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1 methyl 4 phenylpyridinium induces synaptic dysfunction through a pathway involving caspase and pkcδ enzymatic activities
2007Co-Authors: Yafell Serulle, Jorge E. Moreira, Mutsuyuki Sugimori, R Llinas, Scott T Brady, Gerardo Morfini, Gustavo PiginoAbstract:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration has been used, in various mammalian species, as an experimental model of Parkinson's disease. The pathogenesis for such pharmacologically induced Parkinson's disease involves 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. This metabolite produces rapid degeneration of nigrostriatal dopaminergic neurons, which causes the parkinsonian syndrome. In this work, we show that injection of MPP+ into the presynaptic terminal of the Squid Giant Synapse blocks synaptic transmission without affecting the presynaptic action potential or the presynaptic calcium currents. These effects of MPP+ were mimicked by the injection of an active form of caspase-3 and prevented by inhibitors of caspase-3 and protein kinase C δ. Ultrastructurally, MPP+-injected Synapses showed a dramatic reduction in the number of neurotransmitter vesicles at the presynaptic active zone, as compared with control Synapses. Otherwise, normal docking and clathrin-coated vesicles were observed, albeit at much reduced numbers. These results indicate that MPP+ acutely reduces presynaptic vesicular availability, not release, and that MPP+-induced pathogenesis results from presynaptic dysfunction that leads, secondarily, to dying-back neuropathy in affected neurons.
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Role of the conserved WHXL motif in the C terminus of synaptotagmin in synaptic vesicle docking.
2000Co-Authors: Mitsunori Fukuda, Jorge E. Moreira, Mutsuyuki Sugimori, Katsuhiko Mikoshiba, Vincent Hok Liu, Rodolfo R. LlinásAbstract:Synaptotagmin (Syt) I , an abundant synaptic vesicle protein, consists of one transmembrane region, two C2 domains, and a short C terminus. This protein is essential for both synaptic vesicle exocytosis and endocytosis via its C2 domains. Although the short C terminus is highly conserved among the Syt family and across species, little is known about the exact role of the conserved C terminus of Syt I. In this paper, we report a function of the Syt I C terminus in synaptic vesicle docking at the active zones. Presynaptic injection of a peptide corresponding to the C-terminal 21 amino acids of Syt I (named Syt-C) into the Squid Giant Synapse blocked synaptic transmission without affecting the presynaptic action potential or the presynaptic Ca2+ currents. The same procedure repeated with a mutant C-terminal peptide (Syt-CM) had no effect on synaptic transmission. Repetitive presynaptic stimulation with Syt-C produced a rapid decrease in the amplitude of the postsynaptic potentials as the synaptic block progressed, indicating that the peptide interferes with the docking step rather than the fusion step of synaptic vesicles. Electron microscopy of the Synapses injected with the Syt-C peptide showed a marked decrease in the number of docked synaptic vesicles at the active zones, as compared with controls. These results indicate that Syt I is a multifunctional protein that is involved in at least three steps of synaptic vesicle cycle: docking, fusion, and reuptake of synaptic vesicles.
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Synaptophysin regulates clathrin-independent endocytosis of synaptic vesicles
2000Co-Authors: Christopher Daly, Jorge E. Moreira, Mutsuyuki Sugimori, Edward B. Ziff, Rodolfo R. LlinásAbstract:The GTPase dynamin I is required for synaptic vesicle (SV) endocytosis. Our observation that dynamin binds to the SV protein synaptophysin in a Ca2+-dependent fashion suggested the possibility that a dynamin/synaptophysin complex functions in SV recycling. In this paper we show that disruption of the dynamin/synaptophysin interaction by peptide injection into the Squid Giant Synapse preterminal results in a decrease in transmitter release during high-frequency stimulation, indicating an inhibition of SV recycling. Electron microscopy of these Synapses reveals a depletion of SVs, demonstrating a block of vesicle retrieval after fusion. In addition, we observed an increase in clathrin-coated vesicles, indicating that the peptide does not block clathrin-dependent endocytosis. We conclude that the dynamin/synaptophysin complex functions in a clathrin-independent mechanism of SV endocytosis that is required for efficient synaptic transmission.
Paul Greengard - One of the best experts on this subject based on the ideXlab platform.
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tonically active protein kinase a regulates neurotransmitter release at the Squid Giant Synapse
2001Co-Authors: George J Augustine, Andrew J Czernik, Sabine Hilfiker, Paul GreengardAbstract:1. Electrophysiological and microinjection methods were used to examine the role of cyclic AMP-dependent protein kinase A (PKA) in regulating transmitter release at the Squid Giant Synapse. 2. Excitatory postsynaptic potentials (EPSPs) evoked by presynaptic action potentials were not affected by presynaptic injection of an exogenous active catalytic subunit of mammalian PKA. 3. In contrast, presynaptic injection of PKI-amide, a peptide that inhibits PKA with high potency and specificity, led to a reversible inhibition of EPSPs. 4. Injection of several other peptides that serve as substrates for PKA also reversibly inhibited neurotransmitter release. The ability of these peptides to inhibit release was correlated with their ability to serve as PKA substrates, suggesting that these peptides act by competing with endogenous substrates for phosphorylation by active endogenous PKA. 5. We suggest that the phosphorylation of PKA substrates is maintained at a relatively high state under basal conditions and that this tonic activity of PKA is to a large degree required for evoked neurotransmitter release at the Squid Giant presynaptic terminal.
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regulation by synapsin i and ca 2 calmodulin dependent protein kinase ii of the transmitter release in Squid Giant Synapse
1991Co-Authors: R Llinas, Teresa L Mcguinness, J A Gruner, M Sugimori, Paul GreengardAbstract:1. Presynaptic or simultaneous pre- and postsynaptic voltage-clamp protocols were implemented in the Squid Giant Synapse in order to determine the magnitude and time course of the presynaptic calcium current (ICa) and its relation to transmitter release before and after presynaptic injection of proteins. These included several forms of synapsin I, calcium-calmodulin-dependent protein kinase II (CaM kinase II) and avidin. 2. The quantities and location of these proteins were monitored by fluorescence video-enhanced microscopy during the electrophysiological measurements. 3. Presynaptic injection of dephosphorylated synapsin I inhibited synaptic transmission with a time course consistent with diffusion of the protein through the terminal and action at the active release zone. A mathematical model relating the diffusion of synapsin I into the terminal with transmitter release was developed to aid in the interpretation of these results. 4. Synapsin I inhibition of transmitter release was reversible. 5. The action of synapsin I was highly specific, as phosphorylation of the tail region only or head and tail regions prevented synapsin I from inhibiting release. 6. Injections of heat-treated synapsin I or of avidin, a protein with a size and isoelectric point similar to those of synapsin I, had no effect on transmitter release. 7. CaM kinase II injected presynaptically was found to facilitate transmitter release. This facilitation, which could be as large as 700% of the control response, was related to the level of penetration of the enzyme along the length of the preterminal A mathematical model of this facilitation indicates a reasonable fit between the distribution of CaM kinase II within the terminal and the degree of facilitation. 8. The overall shape of the postsynaptic response was not modified by either synapsin I or CaM kinase II injection. 9. The data suggest that, in addition to releasing transmitter, calcium also penetrates the presynaptic cytosol and activates CaM kinase II. When activated, CaM kinase II phosphorylates synapsin I, which reduces its binding to vesicles and/or cytoskeletal structures, enabling more vesicles to be released during a presynaptic depolarization. The amplitude of the postsynaptic response will then be both directly and indirectly regulated by depolarization induced Ca2+ influx. This model provides a molecular mechanism for synaptic potentiation.