Synphilin-1 Is Developmentally Localized to Synaptic Terminals, and Its Association with Synaptic Vesicles Is Modulated by α-Synuclein

Abstract Most vesicles in the interior of synaptic terminals are clustered in clouds close to active zone regions of the plasma membrane where exocytosis occurs. Electron-dense structures, termed bridges, have been reported between a small minority of pairs of neighboring vesicles within the clouds. Synapsin proteins have been implicated previously, but the existence of the bridges as stable structures in vivo has been questioned. Here we use electron tomography to show that the bridges are present but less frequent in synapsin knockouts compared to wildtype. An analysis of distances between neighbors in wildtype tomograms indicated that the bridges are strong enough to resist centrifugal forces likely induced by fixation with aldehydes. The results confirm that the bridges are stable structures and that synapsin proteins are involved in formation or stabilization.

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Surface Charge Manipulation and Electrostatic Immobilization of Synaptosomes for Super-resolution Imaging: A Study on Tau Compartmentalization

10.21203/rs.3.rs-402907/v1 ◽

2021 ◽

Author(s):

Ushashi Bhattacharya ◽

Jia-Fong Jhou ◽

Yi-Fong Zou ◽

Gerald Abrigo ◽

Shu-Wei Lin ◽

...

Keyword(s):

Synaptic Vesicles ◽

Protein Localization ◽

Super Resolution ◽

Synaptic Terminals ◽

Stochastic Optical Reconstruction Microscopy ◽

Neural Transmission ◽

Resolution Imaging ◽

Optical Reconstruction ◽

Cortical Synapses ◽

Induced Aggregation

Abstract Synaptosomes are subcellular fractions prepared from brain tissues that are enriched in synaptic terminals, widely used for the study of neural transmission and synaptic dysfunction. Immunofluorescence imaging is increasingly applied to synaptosomes to investigate protein localization. However, conventional methods for imaging synaptosomes over glass coverslips suffer from formaldehyde-induced aggregation. Here, we developed a simple and facile strategy to capture and image synaptosomes without aggregation artefacts. First, ethylene glycol bis(succinimidyl succinate) (EGS) is chosen as the chemical fixative to replace formaldehyde. EGS/glycine treatment makes the zeta potential of synaptosomes more negative. Second, we modified glass coverslips with 3-aminopropyltriethoxysilane (APTES) to impart positive charges. EGS-fixed synaptosomes spontaneously attach to modified glasses via electrostatic attraction while maintaining good dispersion. Individual synaptic terminals are imaged by conventional fluorescence microscopy or by super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM). We examined tau protein by two-color and three-color dSTORM to understand its spatial distribution within mouse cortical synapses, observing tau colocalization with synaptic vesicles as well postsynaptic densities.

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Roles of ATP in Depletion and Replenishment of the Releasable Pool of Synaptic Vesicles

Journal of Neurophysiology ◽

10.1152/jn.2002.88.1.98 ◽

2002 ◽

Vol 88 (1) ◽

pp. 98-106 ◽

Cited By ~ 52

Author(s):

Ruth Heidelberger ◽

Peter Sterling ◽

Gary Matthews

Keyword(s):

Synaptic Vesicles ◽

Atp Hydrolysis ◽

Synaptic Ribbons ◽

Calcium Dependent ◽

Releasable Pool ◽

Synaptic Terminals ◽

Active Zones ◽

Vesicle Movement ◽

Vesicle Pool ◽

Secretory Apparatus

Synaptic terminals of retinal bipolar neurons contain a pool of readily releasable synaptic vesicles that undergo rapid calcium-dependent release. ATP hydrolysis is required for the functional refilling of this vesicle pool. However, it was unclear which steps required ATP hydrolysis: delivery of vesicles to their anatomical release sites or preparation of synaptic vesicles and/or the secretory apparatus for fusion. To address this, we dialyzed single synaptic terminals with ATP or the poorly hydrolyzable analogue ATP-γS and examined the size of the releasable pool, refilling of the releasable pool, and the number of vesicles at anatomical active zones. After minutes of dialysis with ATP-γS, vesicles already in the releasable pool could still be discharged. This pool was not functionally refilled despite the fact that its anatomical correlate, the number of synaptic vesicles tethered to active zone synaptic ribbons, was completely normal. We conclude 1) because the existing releasable pool is stable during prolonged inhibition of ATP hydrolysis, whereas entry into the functional pool is blocked, a vesicle on entering the pool will tend to remain there until it fuses; 2) because the anatomical pool is unaffected by inhibition of ATP hydrolysis, failure to refill the functional pool is not caused by failure of vesicle movement; 3) local vesicle movements important for pool refilling and fusion are independent of conventional ATP-dependent motor proteins; and 4) ATP hydrolysis is required for the biochemical transition of vesicles and/or release sites to fusion-competent status.

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Adenosine Triphosphate and the Late Steps in Calcium-dependent Exocytosis at a Ribbon Synapse

The Journal of General Physiology ◽

10.1085/jgp.111.2.225 ◽

1998 ◽

Vol 111 (2) ◽

pp. 225-241 ◽

Cited By ~ 54

Author(s):

Ruth Heidelberger

Keyword(s):

Synaptic Vesicles ◽

Flash Photolysis ◽

Average Rate ◽

Small Sample Size ◽

Small Sample ◽

Calcium Dependent ◽

Synaptic Terminals ◽

First Response ◽

Synaptic Vesicle Fusion ◽

Kinetics Of

The ATP dependence of the kinetics of Ca2+-dependent exocytosis after flash photolysis of caged Ca2+ was studied by capacitance measurements with submillisecond resolution in single synaptic terminals of retinal bipolar neurons. After control experiments verified that this combination of techniques is valid for the study of exocytosis in synaptic terminals, a comparison was made between the Ca2+ dependence of the rate of exocytosis in synaptic terminals internally dialyzed with MgATP, MgATP-γ-S, or no added Mg2+ or nucleotide. The Ca2+ threshold for release, the maximum rate of release, and the overall relationship between the rate of synaptic vesicle fusion and [Ca2+]i were found to be independent of MgATP. A decrease in the average rate at near-threshold [Ca2+]i was observed in terminals with MgATP-γ-S, but due to the small sample size is of unclear significance. The Ca2+ dependence of the delay between the elevation of [Ca2+]i and the beginning of the capacitance rise was also found to be independent of MgATP. In contrast, MgATP had a marked effect on the ability of terminals to respond to multiple stimuli. Terminals with MgATP typically exhibited a capacitance increase to a second stimulus that was >70% of the amplitude of the first response and to a third stimulus with a response amplitude that was >50% of the first, whereas terminals without MgATP responded to a second stimulus with a response <35% of the first and rarely responded to a third flash. These results suggest a major role for MgATP in preparing synaptic vesicles for fusion, but indicate that cytosolic MgATP may have little role in events downstream of calcium entry, provided that [Ca2+]i near release sites is elevated above ≈30 μM.

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A marginal band of microtubules transports and organizes mitochondria in retinal bipolar synaptic terminals

The Journal of General Physiology ◽

10.1085/jgp.201511396 ◽

2015 ◽

Vol 146 (1) ◽

pp. 109-117 ◽

Cited By ~ 10

Author(s):

Malkolm Graffe ◽

David Zenisek ◽

Justin W. Taraska

Keyword(s):

Electron Microscopy ◽

Transmitter Release ◽

Synaptic Vesicles ◽

Bipolar Cells ◽

Synaptic Activity ◽

Synaptic Terminal ◽

Synaptic Terminals ◽

Marginal Band ◽

Microtubule Structure

A set of bipolar cells in the retina of goldfish contains giant synaptic terminals that can be over 10 µm in diameter. Hundreds of thousands of synaptic vesicles fill these terminals and engage in continuous rounds of exocytosis. How the cytoskeleton and other organelles in these neurons are organized to control synaptic activity is unknown. Here, we used 3-D fluorescence and 3-D electron microscopy to visualize the complex subcellular architecture of these terminals. We discovered a thick band of microtubules that emerged from the axon to loop around the terminal periphery throughout the presynaptic space. This previously unknown microtubule structure associated with a substantial population of mitochondria in the synaptic terminal. Drugs that inhibit microtubule-based kinesin motors led to accumulation of mitochondria in the axon. We conclude that this prominent microtubule band is crucial to the transport and localization of mitochondria into the presynaptic space to provide the sustained energy necessary for continuous transmitter release in these giant synaptic terminals.

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Surface charge manipulation and electrostatic immobilization of synaptosomes for super-resolution imaging: a study on tau compartmentalization

Scientific Reports ◽

10.1038/s41598-021-98142-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ushashi Bhattacharya ◽

Jia-Fong Jhou ◽

Yi-Fong Zou ◽

Gerald Abrigo ◽

Shu-Wei Lin ◽

...

Keyword(s):

Synaptic Vesicles ◽

Protein Localization ◽

Super Resolution ◽

Synaptic Terminals ◽

Stochastic Optical Reconstruction Microscopy ◽

Neural Transmission ◽

Resolution Imaging ◽

Optical Reconstruction ◽

Cortical Synapses ◽

Induced Aggregation

AbstractSynaptosomes are subcellular fractions prepared from brain tissues that are enriched in synaptic terminals, widely used for the study of neural transmission and synaptic dysfunction. Immunofluorescence imaging is increasingly applied to synaptosomes to investigate protein localization. However, conventional methods for imaging synaptosomes over glass coverslips suffer from formaldehyde-induced aggregation. Here, we developed a facile strategy to capture and image synaptosomes without aggregation artefacts. First, ethylene glycol bis(succinimidyl succinate) (EGS) is chosen as the chemical fixative to replace formaldehyde. EGS/glycine treatment makes the zeta potential of synaptosomes more negative. Second, we modified glass coverslips with 3-aminopropyltriethoxysilane (APTES) to impart positive charges. EGS-fixed synaptosomes spontaneously attach to modified glasses via electrostatic attraction while maintaining good dispersion. Individual synaptic terminals are imaged by conventional fluorescence microscopy or by super-resolution techniques such as direct stochastic optical reconstruction microscopy (dSTORM). We examined tau protein by two-color and three-color dSTORM to understand its spatial distribution within mouse cortical synapses, observing tau colocalization with synaptic vesicles as well postsynaptic densities.

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The Lateral Giant Fiber to Motor Giant Fiber Synapse in Crayfish

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094103 ◽

1972 ◽

Vol 30 ◽

pp. 170-171

Author(s):

Charles A. Stirling

Keyword(s):

Synaptic Vesicles ◽

Procambarus Clarkii ◽

Synaptic Contact ◽

Giant Fiber ◽

Synaptic Junctions

The lateral giant (LG) to motor giant (MoG) synapses in crayfish (Procambarus clarkii) abdominal ganglia are the classic electrotonic synapses. They have previously been described as having synaptic vesicles and as having them on both the pre- and postsynaptic sides of symmetrical synaptic junctions. This positioning of vesicles would make these very atypical synapses, but in the present work on the crayfish Astacus pallipes the motor giant has never been found to contain any type of vesicle at its synapses with the lateral giant fiber.The lateral to motor giant fiber synapses all occur on short branches off the main giant fibers. Closely associated with these giant fiber synapses are two small presynaptic nerves which make synaptic contact with both of the giant fibers and with their small branches.

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