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Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons

Nature Protocols volume 1, pages 1887–1897 (2006)Cite this article

Abstract

Elucidating the molecular organization of synapses is essential for understanding brain function and plasticity. Immunofluorescence, combined with various fluorescent probes, is a sensitive and versatile method for morphological studies. However, analysis of synaptic proteins in situ is limited by epitope-masking after tissue fixation. Furthermore, postsynaptic proteins (such as ionotropic receptors and scaffolding proteins) often require weaker fixation for optimal detection than most intracellular markers, thereby hindering simultaneous visualization of these molecules. We present three protocols, which are alternatives to perfusion fixation, to overcome these restrictions. Brief tissue fixation shortly after interruption of vital functions preserves morphology and antigenicity. Combined with specific neuronal markers, selective detection of γ-aminobutyric acid A (GABAA) receptors and the scaffolding protein gephyrin in relation to identified inhibitory presynaptic terminals in the rodent brain is feasible by confocal laser scanning microscopy. The most sophisticated of these protocols can be associated with electrophysiology for correlative studies of synapse structure and function. These protocols require 2–3 consecutive days for completion.

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Figure 1: A flow chart of the experimental procedure for the three options for tissue preparation.
Figure 2: Examples of double immunofluorescence staining of mouse and rat brain tissue with options A and B compared with perfusion-fixed tissue.
Figure 3: Examples of double and triple immunofluorescence staining of mouse brain tissue with option C.

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References

  1. Racz, B., Blanpied, T.A., Ehlers, M.D. & Weinberg, R.J. Lateral organization of endocytic machinery in dendritic spines. Nat. Neurosci. 7, 917–918 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Triller, A., Cluzeaud, F., Pfeiffer, F., Betz, H. & Korn, H. Distribution of glycine receptors at central synapses: an immunoelectron microscopy study. J. Cell Biol. 101, 683–688 (1985).

    Article  CAS  PubMed  Google Scholar 

  3. Somogyi, P. in Neural Mechanisms of Visual Perception (eds. Lam, D.K.T. & Gilbert, C.D.) 35–62 (Portfolio Publishing Co, Houston, 1989).

    Google Scholar 

  4. Lujan, R., Nusser, Z., Roberts, J.D., Shigemoto, R. & Somogyi, P. Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus. Eur. J. Neurosci. 8, 1488–1500 (1996).

    Article  CAS  PubMed  Google Scholar 

  5. Landis, D.M. Membrane and cytoplasmic structure at synaptic junctions in the mammalian central nervous system. J. Electr. Microsc. Tech. 10, 129–151 (1988).

    Article  CAS  Google Scholar 

  6. Harlow, M.L., Ress, D., Stoschek, A., Marshall, R.M. & McMahan, U.J. The architecture of active zone material at the frog's neuromuscular junction. Nature 409, 479–484 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Dick, O. et al. The presynaptic active zone protein bassoon is essential for photoreceptor ribbon synapse formation in the retina. Neuron 37, 775–786 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Baron, M.K. et al. An architectural framework that may lie at the core of the postsynaptic density. Science 311, 531–535 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Ottersen, O.P. & Landsend, A.S. Organization of glutamate receptors at the synapse. Eur. J. Neurosci. 9, 2219–2224 (1997).

    Article  CAS  PubMed  Google Scholar 

  10. Cowan, W.M., Sudhof, T.C. & Stevens, C.F. Synapses (Johns Hopkins Univ. Press, Baltimore, 2001).

    Google Scholar 

  11. Phillips, G.W. & Bridgman, P.C. Immunoelectron microscopy of acetylcholine receptors and 43 kD protein after rapid freezing, freeze-substitution, and low-temperature embedding in Lowicryl K11M. J. Histochem. Cytochem. 39, 625–634 (1991).

    Article  CAS  PubMed  Google Scholar 

  12. Nusser, Z. et al. Immunocytochemical localization of the α1 and β2/3 subunits of the GABAA receptor in relation to specific GABAergic synapses in the dentate gyrus. Eur. J. Neurosci. 7, 630–646 (1995).

    Article  CAS  PubMed  Google Scholar 

  13. Kulik, A. et al. Compartment-dependent colocalization of Kir3.2-containing K+ channels and GABAB receptors in hippocampal pyramidal cells. J. Neurosci. 26, 4289–4297 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hagiwara, A., Fukazawa, Y., Deguchi-Tawarada, M., Ohtsuka, T. & Shigemoto, R. Differential distribution of release-related proteins in the hippocampal CA3 area as revealed by freeze-fracture replica labeling. J. Comp. Neurol. 489, 195–216 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Rash, J.E., Yasumura, T. & Dudek, F.E. Ultrastructure, histological distribution, and freeze-fracture immunocytochemistry of gap junctions in rat brain and spinal cord. Cell Biol. Int. 22, 731–749 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Rash, J.E. et al. Ultrastructural localization of connexins (Cx36, Cx43, Cx45), glutamate receptors and aquaporin-4 in rodent olfactory mucosa, olfactory nerve and olfactory bulb. J. Neurocytol. 34, 307–341 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Wouterlood, F.G., Bockers, T.M. & Witter, M.P. Synaptic contacts between identified neurons visualized in the confocal laser scanning microscope. Neuroanatomical tracing combined with immunofluorescence detection of post-synaptic density proteins and target neuron-markers. J. Neurosci. Meth. 128, 129–142 (2003).

    Article  CAS  Google Scholar 

  18. Koulen, P., Sassoè-Pognetto, M., Grünert, U. & Wässle, H. Selective clustering of GABAA and glycine receptors in the mammalian retina. J. Neurosci. 16, 2127–2140 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Fritschy, J.M., Weinmann, O., Wenzel, A. & Benke, D. Synapse-specific localization of NMDA- and GABAA-receptor subunits revealed by antigen-retrieval immunohistochemistry. J. Comp. Neurol. 390, 194–210 (1998).

    Article  CAS  PubMed  Google Scholar 

  20. Geiman, E.J., Zheng, W., Fritschy, J.M. & Alvarez, F.J. Glycine and GABAA receptor subunits on Renshaw cells: relationship with presynaptic neurotransmitters and postsynaptic gephyrin clusters. J. Comp. Neurol. 444, 275–289 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Sassoe-Pognetto, M., Wassle, H. & Grunert, U. Glycinergic synapses in the rod pathway of the rat retina: cone bipolar cells express the α1 subunit of the glycine receptor 14, 5131–5146 (1994).

  22. Melone, M., Burette, A. & Weinberg, R.J. Light microscopic identification and immunocytochemical characterization of glutamatergic synapses in brain sections. J. Comp. Neurol. 492, 495–509 (2005).

    Article  PubMed  Google Scholar 

  23. Watanabe, M. et al. Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre-recipient layer) of the mouse hippocampal CA3 subfield. Eur. J. Neurosci. 10, 478–487 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Nagy, G.G., Watanabe, M., Fukaya, M. & Todd, A.J. Synaptic distribution of the NR1, NR2A and NR2B subunits of the N-methyl-D-aspartate receptor in the rat lumbar spinal cord revealed with an antigen-unmasking technique. Eur. J. Neurosci. 20, 3301–3312 (2004).

    Article  PubMed  Google Scholar 

  25. Barnard, E.A. et al. International Union of Pharmacology. XV. Subtypes of γ-aminobutyric acidA receptors: classification on the basis of subunit structure and function. Pharmacol. Rev. 50, 291–313 (1998).

    CAS  PubMed  Google Scholar 

  26. Sieghart, W. & Ernst, M. Heterogeneity of GABAA receptors: revived interest in the development of subtype-selective drugs. Curr. Med. Chem. 5, 217–242 (2005).

    CAS  Google Scholar 

  27. Colquhoun, D. & Sivilotti, L.G. Function and structure in glycine receptors and some of their relatives. Trends Neurosci. 27, 337–344 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Lynch, J.W. Molecular structure and function of the glycine receptor chloride channel. Physiol. Rev. 84, 1051–1095 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Kneussel, M. & Betz, H. Receptors, gephyrin and gephyrin-associated proteins: novel insights into the assembly of inhibitory postsynaptic membrane specializations. J. Physiol. 525, 1–9 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sassoè-Pognetto, M. & Fritschy, J.M. Gephyrin, a major postsynaptic protein of GABAergic synapses. Eur. J. Neurosci. 7, 2205–2210 (2000).

    Article  Google Scholar 

  31. Fritschy, J.M. & Mohler, H. GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J. Comp. Neurol. 359, 154–194 (1995).

    Article  CAS  PubMed  Google Scholar 

  32. Panzanelli, P., Perazzini, A.Z., Fritschy, J.M. & Sassoè-Pognetto, M. Heterogeneity of γ-aminobutyric acid type A receptors in mitral and tufted cells of the rat main olfactory bulb. J. Comp. Neurol. 484, 121–131 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Giustetto, M., Kirsch, J., Fritschy, J.M., Cantino, D. & Sassoè-Pognetto, M. Localisation of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb of the rat. J. Comp. Neurol. 395, 231–244 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Triller, A., Cluzeaud, F. & Korn, H. γ-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses. J. Cell Biol. 104, 947–956 (1987).

    Article  CAS  PubMed  Google Scholar 

  35. Sassoè-Pognetto, M. et al. Colocalization of gephyrin and GABAA-receptor subunits in the rat retina. J. Comp. Neurol. 357, 1–14 (1995).

    Article  PubMed  Google Scholar 

  36. Sassoè-Pognetto, M., Panzanelli, P., Sieghart, W. & Fritschy, J.M. Co-localization of multiple GABAA receptor subtypes with gephyrin at postsynaptic sites. J. Comp. Neurol. 420, 481–498 (2000).

    Article  PubMed  Google Scholar 

  37. Schweizer, C. et al. The γ2 subunit of GABAA receptors is required for maintenance of receptors at mature synapses. Mol. Cell. Neurosci. 24, 442–450 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Koksma, J.J., Fritschy, J.M., Mack, V., Van Kesteren, R.E. & Brussaard, A.B. Differential GABAA receptor clustering determines GABA synapse plasticity in rat oxytocin neurons around parturition and the onset of lactation. Mol. Cell. Neurosci. 28, 128–140 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Lorenzo, L.E., Barbe, A., Portalier, P., Fritschy, J.M. & Bras, H. Differential expression of GABAA and glycine receptors in ALS-resistant vs. ALS-vulnerable motoneurons: possible implications for selective vulnerability of motoneurons. Eur. J. Neurosci. 23, 3161–3170 (2006).

    Article  PubMed  Google Scholar 

  40. Fagiolini, M. et al. Specific GABAA circuits for visual cortical plasticity. Science 303, 1681–1683 (2004).

    Article  CAS  PubMed  Google Scholar 

  41. Knuesel, I., Zuellig, R.A., Schaub, M.C. & Fritschy, J.M. Alterations in dystrophin and utrophin expression parallel the reorganization of GABAergic synapses in a mouse model of temporal lobe epilepsy. Eur. J. Neurosci. 13, 1113–1124 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Knuesel, I. et al. Altered synaptic clustering of GABAA-receptors in mice lacking dystrophin (mdx mice). Eur. J. Neurosci. 11, 4457–4462 (1999).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors wish to thank C. Sidler for excellent technical assistance and L. Viltono for providing Figure 2g. The work was supported by a Swiss National Science Foundation grant (3100A0-108260) to J.-M.F. and an Italian MIUR grant (PRIN 2005059123 002) to M.S.-P.

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Author notes

  1. Edith M Schneider Gasser and Carolin J Straub: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, Zurich, CH – 8057, Switzerland

    Edith M Schneider Gasser, Carolin J Straub & Jean-Marc Fritschy

  2. Departments of Anatomy, Pharmacology, and Forensic Medicine, University of Turin, Corso Massimo d'Azeglio 52, Torino, I – 10126, Italy

    Patrizia Panzanelli & Marco Sassoè-Pognetto

  3. Brain Research Institute, University of Zurich,

    Oliver Weinmann

  4. Rita Levi Montalcini Center for Brain Repair, University of Turin,

    Marco Sassoè-Pognetto

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  1. Edith M Schneider Gasser
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Correspondence to Jean-Marc Fritschy.

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Schneider Gasser, E., Straub, C., Panzanelli, P. et al. Immunofluorescence in brain sections: simultaneous detection of presynaptic and postsynaptic proteins in identified neurons. Nat Protoc 1, 1887–1897 (2006). https://doi.org/10.1038/nprot.2006.265

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