An In Vitro Technique to Study Glutamate Receptor Trafficking in Hippocampal Neurons

In This Article

Abstract

Source: Chiu, A. M., et al. An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures. J. Vis. Exp. (2019)

This video demonstrates an antibody-feeding approach to study the trafficking of glutamate receptors in primary hippocampal neuronal cultures. This approach allows the observation of receptor populations at both the plasma and internal membranes. Discrimination between these subsets is achieved by labeling the receptors before and after membrane permeabilization, using the same primary antibody but secondary antibodies conjugated to different fluorophores.

Protocol

1. Preparation Before Labeling

Preparation and maintenance of primary hippocampal cultures
1. Prepare primary hippocampal cultures at a density of 150,000 cells plated on poly-D-lysine-coated (0.1 mg/mL) 18 mm cover glasses. Excellent guides for dissociated neuronal culture preparation are available.
  NOTE: If required, the cultures may be treated with cytosine arabinoside (Ara-C, 10 μM from days in vitro 1 [DIV1]) to avoid glial proliferation in the preparation.
  NOTE: Alternative coating reagents such as fibronectin (1 mg/mL) or laminin (5 μg/mL) m....

Access restricted. Please log in or start a trial to view this content.

Results

Immunofluorescence microscopy showing GluA1 surface/intracellular distribution; cLTP-induced changes.
Figure 1: cLTP increases surface expression of GluA1. Primary hippocampal neurons at DIV21 were subjected to chemical LTP (cLTP) by incubating with glycine-containing ECS. Distinct labeling of surface-expressed (red) vs. intracellular (blue) GluA1 popu.......