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Related Concept Videos

Fusion of Secretory Vesicles with the Plasma Membrane01:26

Fusion of Secretory Vesicles with the Plasma Membrane

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Proteins and neurotransmitters in secretory vesicles can be released from a cell upon vesicle docking, priming, and fusion with the plasma membrane. Vesicles are docked and primed in preparation for the quick exocytosis of their contents in response to a stimulus. The fusion process is mainly carried out by a SNAP Receptor or SNARE complex, consisting of synaptobrevin, syntaxin-1, and SNAP-25.
In 1993, Jim Rothman proposed that the antiparallel pairing of vesicular and transmembrane SNAREs, or...
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Related Experiment Video

Updated: May 3, 2026

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient
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Cryoloading: introducing large molecules into live synaptosomes.

Arup R Nath1, Robert H C Chen1, Elise F Stanley1

  • 1Laboratory of Synaptic Transmission, Toronto Western Research Institute Toronto, ON, Canada.

Frontiers in Cellular Neuroscience
|January 31, 2014
PubMed
Summary
This summary is machine-generated.

Researchers developed a cryoloading method to introduce large molecules into synaptosomes, enabling functional studies of presynaptic nerve terminals. This technique preserves synaptosome function and allows for long-term storage and identification of specific nerve terminal types.

Keywords:
intracellularpeptide loadingpresynapticrecyclingstyryl dyesynaptic vesiclesynaptosome

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Area of Science:

  • Neuroscience
  • Cell Biology
  • Biochemistry

Background:

  • Presynaptic nerve terminals, isolated as synaptosomes, are crucial for neuronal communication.
  • Their small size limits the study of large molecules like peptides and drugs within them.
  • Existing methods struggle to introduce non-membrane permeable substances into functional synaptosomes.

Purpose of the Study:

  • To develop a novel method for introducing large molecules (>150 kDa) into functional synaptosomes.
  • To enable the study of presynaptic function using previously inaccessible compounds.
  • To allow for long-term storage and identification of synaptosomes for detailed analysis.

Main Methods:

  • Developed a cryopreservation and freeze-thaw technique to load synaptosomes with large compounds.
  • Assessed synaptosome functionality using styryl dye uptake assays to monitor synaptic vesicle recycling.
  • Confirmed intracellular access and biological activity of cryoloaded compounds using BAPTA and botulinum A light chain.

Main Results:

  • Successfully introduced large molecules (>150 kDa) into synaptosomes via a freeze-thaw cryoloading method.
  • ~80% of cryoloaded synaptosomes remained functional, exhibiting normal synaptic vesicle recycling.
  • Demonstrated intracellular delivery and biological effects of cryoloaded substances, including BAPTA and botulinum A.
  • Showcased the ability to identify specific synaptosome types using immunostaining of attached postsynaptic membrane components.

Conclusions:

  • Cryoloading provides a robust method for introducing large molecules into functional synaptosomes.
  • This technique overcomes previous limitations in studying presynaptic nerve terminals.
  • Cryoloading combined with scab-staining allows for functional analysis of identified individual CNS presynaptic nerve terminals.