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.
1. Preparation Before Labeling
2. Live Labeling of Surface-expressed Receptors
3. Surface Expression (Figure 1)
4. Internalization (Figure 2)
5. Recycling (Figure 3)
6. Mounting and Imaging of Samples
7. Time Considerations
8. Image Analysis
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 intensity projection) confocal pictures. Scale bars = 50 µm (whole cell) or 5 µm (dendrite). Graph shows the increased surface expression of GluA1 after cLTP protocol. Surface expression index: surface/intracellular receptors (n = 3; number of cells: con = 7; cLTP = 7; values represent mean ± SEM; ****p < 0.0001 using Mann-Whitney U test). (C) Control experiment in which the permeabilization step was skipped. In addition to surface and internal GluA1, the intracellular excitatory synaptic marker PSD-95 was evaluated. Scale bars = 50 µm (whole cell) or 5 µm (dendrite).
Figure 2: Phosphorylation of GluN2B at S1480 promotes NMDAR internalization. Primary hippocampal neurons were transfected with either GFP-GluN2B WT or the phospho-mimetic mutant GFP-GluN2B S1480E on DIV11-12. Following 3-4 days of protein expression, surface GFP was labeled on live cells with a rabbit anti-GFP antibody and cells were then returned to 37 °C to allow for receptor internalization by endocytosis. Surface-expressed exogenous receptors were visualized with Alexa 555-conjugated secondary antibody, and the internalized population identified after permeabilization using Alexa 647-conjugated antibody. For clarity, surface GFP-GluN2B is pseudocolored in green and internalized GFP-GluN2B is pseudocolored in white. (A) Single plane and (B) Z-stacked (maximum intensity projection) confocal pictures. Scale bars = 50 µm (whole cell) or 5 µm (dendrite). Graph shows the elevated internalization displayed by the phospho-mimetic mutant GluN2B S1480E. Internalization index: internalized receptors/surface-expressed receptors (n = 6; number of cells: WT = 34; S1480E = 28; values represent mean ± SEM; ***p < 0.001 using Mann-Whitney U test). (C) Control experiment in which the internalization step (Intenaliz.) was performed at 4 °C. Scale bars = 50 µm (whole cell) or 5 µm (dendrite).
Figure 3: Phosphorylation of GluN2B at S1480 does not modify NMDAR recycling. Primary hippocampal neurons were transfected with either GFP-GluN2B WT or the phospho-mimetic mutant GFP-GluN2B S1480E on DIV11-12 as shown in Figure 3. Following 3-4 days of protein expression, surface GFP was labeled on live cells with a rabbit anti-GFP antibody and cells were then returned to 37 °C to allow for receptor internalization by endocytosis. Remaining surface-expressed receptors were blocked by Fab incubation and recycling was allowed for 45 min. Available surface expressed exogenous receptors (recycled) were visualized with Alexa 555-conjugated secondary antibody, and the internalized population identified after permeabilization using Alexa 647-conjugated antibody. For clarity, surface GFP-GluN2B is pseudocolored in white and internalized GFP-GluN2B is pseudocolored in green. (A) Single plane and (B) Z-stacked (maximum intensity projection) confocal pictures. Scale bars = 50 µm (whole cell) or 5 µm (dendrite). Graph shows the lack of effect the GluN2B S1480 phosphorylation has on recycling. Recycling index: recycled receptors/internalized receptors (n = 5; number of cells: WT = 27; S1480E = 24; values represent mean ± SEM; n.s. = non-significant using Mann-Whitney U test). (C) Control experiment in which the Fab incubation step to block surface-expressed epitopes was skipped. Scale bars = 50 µm (whole cell) or 5 µm (dendrite).
The authors have nothing to disclose.
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 |