Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion.
Fast activity-driven turnover of neurotransmitter-filled vesicles at presynaptic terminals is a crucial step in information transfer in the CNS. Characterization of the relationship between the nanoscale organization of synaptic vesicles and their functional properties during transmission is currently of interest. Here we outline a procedure for ultrastructural investigation of functional vesicles in synapses from native mammalian brain tissue. FM dye is injected into the target region of a brain slice and upstream axons are electrically activated to stimulate vesicle turnover and dye uptake. In the presence of diaminobenzidine (DAB), photoactivation of dye-filled vesicles yields an osmiophilic precipitate that is visible in electron micrographs. When combined with serial-section electron microscopy, fundamental ultrastructure-function relationships of presynaptic terminals in native circuits are revealed. We outline the utility of this protocol for the 3D reconstruction of a recycling vesicle pool in CA3-CA1 synapses from an acute hippocampal slice and for the characterization of its anatomically defined docked pool. This protocol requires 6-7 d.
1] School of Life Sciences, University of Sussex, Brighton, UK. [2] Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.
Vincenzo
V
Marra
Medical Research Council Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, London, UK.
Jemima J
JJ
Burden
School of Life Sciences, University of Sussex, Brighton, UK.
Freya
F
Crawford
School of Life Sciences, University of Sussex, Brighton, UK.
Kevin
K
Staras
D015100:3,3'-Diaminobenzidine; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000166:cytology*; D008854:Microscopy, Electron; D009474:Neurons / Q000648:ultrastructure*; D017729:Presynaptic Terminals / Q000648:ultrastructure*; D013572:Synaptic Vesicles / Q000648:ultrastructure*
D005456:Fluorescent Dyes; D015100:3,3'-Diaminobenzidine