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