Transmission Electron Microscopic Imaging of Synaptic Vesicle Endocytosis in Mouse Hippocampal Neurons

Abstract

Source: Villarreal, S., et. al., Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons. J. Vis. Exp. (2017)

This video demonstrates the analysis of synaptic vesicle endocytosis in cultured hippocampal neurons through horseradish peroxidase labeling and electron microscopy.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

Electron Microscopy

1. Prepare a high K⁺ stimulation solution with HRP (horseradish peroxidase) as 31.5 mM NaCl, 2 mM CaCl₂, 90 mM KCl, 25 mM HEPES (pH 7.4), 30 mM glucose, 2 mM MgCl₂, 0.01 mM CNQX, 0.05 mM AP5, and 5 mg/mL HRP, then adjust to pH 7.4 with 5 M NaOH.
   1. Stimulate the hippocampal neuron culture with 1.5 mL high K⁺ stimulation solution at room temperature (referred to as K⁺) by addition of 1.5 mL to each well for 90 s. In the resting condition (ref....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Neurobasal medium	Thermo Fisher	21103-049	Growth medium for neuron, Warm up to 37°C before use
B27	Thermo Fisher	17504-044	Gradient for neuronal differentiation
Glutamax	Thermo Fisher	35050-061	Gradient for neuronal culture
Trypsin XI from bovine pancrease	Sigma	T1005	Neuronal culture-digest hippocampal tissues
Deoxyribonuclease I from bovine pancreas	Sigma	D5025	Neuronal culture-inhibits viscous cell suspension
Poly-l-lysine	Sigma	P4832	Electron microscopy, substrate for neuronal growth, apply on multiwell plate for 1 h at room temperature then wash with sterilized water 3 times
Horseradish peroxidase(HRP)	Sigma	P6782	Electron microscopy, labeling of endocytosed synaptic vesicles by catalyzing DAB in presence hydrogen peroxide, final concentration is 5 mg/mL in normal saline, make fresh before use
Sodium cacodylate	Electron Microscopy Sciences	12300	Electron microscopy, buffer for fixatives and washing, final concentration is 0.1 N
3,3′-Diaminobenzidine(DAB)	Sigma	D8001	Electron microscopy, labeling of endocytosed synaptic vesicles, substrate for HRP, final concentration is 0.5 mg/mL in DDW and filtered, make fresh before use
Hydrogen peroxide solution	Sigma	H1009	Electron microscopy, labeling of endocytosed synaptic vesicles by inducing HRP-DAB reaction, final concentration is 0.3% in DDW, make fresh before use
Glutaraldehyde	Electron Microscopy Sciences	16365	Electron microscopy, fixatives, final concentration is 4% in Na-cacodylate buffer, make fresh before use, shake well before to use
TEM	JEOL	200CX	Electron microscopy, imaging of endocytosed vesicles and ultrastructural changes
CCD digital camera	AMT	XR-100	Electron microscopy, capturing images
Lead citrate	Leica microsystems	16707235	Electron microscopy, grid staining