Neuron-fibroblast coculture assay to demonstrate presynaptic differentiation induced by the NGL family of cell adhesion molecules
Related Journal & Article Information
Journal: Nature Neuroscience
Article Title: NGL family PSD-95–interacting adhesion molecules regulate excitatory synapse formation
Introduction
Expression of synaptic cell adhesion molecules, such as neuroligin, neurexin and SynCAM, in nonneural cells can induce pre- or postsynaptic differentiation in contacting neurites of cocultured neurons [1-3]. We used this assay to demonstrate that NGL, a family of cell adhesion molecules that associates with the netrin-G family of cell adhesion molecules and the postsynaptic scaffolding protein PSD-95, can induce presynaptic differentiation in contacting axons of cocultured neurons. Presynaptic differentiation was visualized by immunostaining for the presynaptic marker synapsin I. In addition, functional presynaptic differentiation was demonstrated by vesicle turnover experiments, which monitor the uptake of synaptotagmin luminal domain antibodies [4].
Materials
Reagents
Equipment
Procedure
Preparation of HEK293T cells
1. Grow HEK293T cells on 60-mm culture dishes to around 50% confluency.
2. Transfect HEK293T cells with Myc-NGL-2 or Myc-NGL-2 ECD (a negative control that lacks the extracellular domain) by the calcium phosphate method.
3. After 24–48 h, trypsinize the cells and resuspend 10% of the cells from a single 60-mm dish in 1 mL of phosphate-buffered saline (PBS).
4. Harvest the cells by centrifugation and then resuspend them in 1 mL of PBS.
Coculture of HEK293T cells and neurons
5. Add 10% (100 μl) of the resuspended cells onto cultured hippocampal neurons (DIV8) grown on a 18-mm coverslip (medium density).
6. Add cytosine arabinoside to the culture medium to a final concentration of 0.5 μM to inhibit overgrowth of HEK293T cells.
7. Incubate the coculture in the CO2 incubator for 3 days.
Immunostaining
8. Fix the coculture in 4% paraformaldehyde and 4% sucrose in phosphate buffered saline at room temperature for 5 min.
9. Incubate the cells with primary antibodies (synapsin I and Myc; 1 μg/ml) followed by Cy3- or FITC-conjugated secondary antibodies.
Vesicle turnover experiment
10. Perform vesicle turnover experiments in parallel with the immunostaining. Briefly wash the cells with the extracellular solution (in mM): 150 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, 10 HEPES (pH 7.4).
11. Incubate the cells in high-potassium solution (in mM; 97 NaCl, 57 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, 10 HEPES, pH 7.4) containing synaptotagmin luminal domain antibodies (Synaptic Systems, 1:10) at room temperature for 5 min.
12. Wash the cells twice with the extracellular solution, then subject them to fixation and immunostaining with appropriate antibodies.
Image acquisition and quantitation
13. Capture Z-stacked images of randomly chosen HEK293T cells by confocal microscopy (63 x objective), maintaining the same acquisition parameters throughout the acquisition.
14. For quantitation, manually trace the boundaries of the transfected cells. Normalize the integrated synapsin I immunofluorescence from a transfected HEK293T cell by the cell area.
Troubleshooting
Critical Steps
Anticipated Results
References
1. Scheiffele, P., Fan, J., Choih, J., Fetter, R. & Serafini, T. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons. Cell 101, 657-69 (2000).
2. Biederer, T. et al. SynCAM, a synaptic adhesion molecule that drives synapse assembly. Science 297, 1525-31 (2002).
3. Graf, E. R., Zhang, X., Jin, S. X., Linhoff, M. W. & Craig, A. M. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins. Cell 119, 1013-26 (2004).
4. Kraszewski, K. et al. Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin. J Neurosci 15, 4328-42 (1995).
Acknowledgements
Keywords