Introduction of impermeant molecules into synaptosomes using freeze/thaw permeabilization

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• Synaptosomes are crucial for understanding neurotransmission.
• Investigating protein phosphorylation in nerve terminals requires access to the interior.

Purpose of the Study:

• To explore brief freezing as a method for transiently permeabilizing synaptosomes.
• To assess the impact of freeze-thaw on synaptosome function and molecular interactions.

Main Methods:

• Rat brain synaptosomes were subjected to brief freezing and thawing with dimethyl sulfoxide.
• Neurotransmitter release ([3H]norepinephrine, [14C]acetylcholine) was measured in response to elevated potassium and calcium.
• Protein phosphorylation was assessed using [gamma-32P]ATP and specific protein kinases (cAMP-dependent, Ca2+/calmodulin-dependent).

Main Results:

• Freeze-thawed synaptosomes released neurotransmitters in a calcium-dependent manner.
• Synaptosomes retained resting and stimulated protein phosphorylation capabilities post-freeze/thaw.
• The synaptosomal interior became accessible to protein kinases and inhibitors, demonstrated by substrate phosphorylation (e.g., synapsin I).
• Rapid resealing of synaptosomes occurred post-freezing, preventing further agent entry.

Conclusions:

• Brief freezing provides transient permeabilization of synaptosomes.
• This technique allows for the study of intracellular processes like protein phosphorylation within nerve terminals.
• The method facilitates research into the functional roles of phosphoproteins, kinases, and phosphatases in synaptic function.