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

Published: April 30, 2024

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​ 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 p…

Representative Results

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 populations reveals the expected increase in surface expression of AMPAR. (A) Single plane and (B) Z-stacked (maximum…

Divulgaciones

The authors have nothing to disclose.

Materials

18 mm dia. #1.5 thick coverglasses Neuvitro GG181.5
Alexa 555-conjugated goat anti-mouse secondary Life Technologies A21424
Alexa 555-conjugated goat anti-rabbit secondary Life Technologies A21429
Alexa 647-conjugated goat anti-mouse secondary Life Technologies A21236
Alexa 647-conjugated goat anti-rabbit secondary Life Technologies A21245
B27 Gibco 17504044
CaCl2 Sigma C7902
Corning Costar Flat Bottom Cell Culture Plates Corning 3513
Dynasore Tocris 2897
Glucose Sigma G8270
Glycine Tocris 219
Goat anti-rabbit Fab fragments Sigma SAB3700970
HEPES Sigma H7006
KCl Sigma P9541
L-Glutamine Sigma G7513
Lipofectamine 2000 Invitrogen 11668019
Mouse anti-GluA1 antibody Millipore MAB2263
NaCl Sigma S6546
Neurobasal Media Gibco 21103049
NGS Abcam Ab7481
Parafilm Bemis PM999
PBS Gibco 10010023
Pelco BioWave Ted Pella 36500
PFA Alfa Aesar 43368
Picrotoxin Tocris 1128
Poly-D-lysine hydrobromide Sigma P7280
ProLong Gold Antifade Mountant Life Technologies P36934
Rabbit anti-GFP antibody Invitrogen A11122
Rabbit anti-PSD-95 antibody Cell Signaling 2507
Strychnine Tocris 2785
Sucrose Sigma S0389
Superfrost plus microscope slides Fisher 12-550-15
Triton X-100 Sigma X100
TTX Tocris 1078

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An In Vitro Technique to Study Glutamate Receptor Trafficking in Hippocampal Neurons. J. Vis. Exp. (Pending Publication), e22129, doi: (2024).

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