Advertisement

Confocal and Electron Microscopic Tracking of Internalized Neuropeptides and/or Their Receptors

  • Alain Beaudet
  • Alexander C. Jackson
  • Franck Vandenbulcke

Abstract

This chapter describes confocal and electron microscopic methods for tracking peptide ligands and/or their receptors following their internalization in cell cultures or brain slices. The confocal microscopic techniques are based upon the use of high affinity fluorescent ligands that were originally developed in our laboratory to study the fate of internalized neurotensin (NT), somatostatin (SRIF), and opioid peptides (9,13,17). The electron microscopic techniques are adapted from the pre-embedding immunogold method developed by Virginia Pickel and her team (5) as applied by us to study the effect of ligand exposure on the subcellular distribution of various subtypes of neuropeptide receptors.

Keywords

Brain Slice Ringer Buffer Phenylarsine Oxide Fluorescent Ligand Antibody Dilution Buffer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Barak, L.S., S.S.G. Ferguson, J.I.E. Zhang, C. Martenson, T. Meyer, and M.G. Caron. 1997. Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate. Mol. Pharmacol. 51:177–184.PubMedGoogle Scholar
  2. 2.
    Beaudet, A., D. Nouel, T. Stroh, F. Vandenbulcke, C. Dal Farra, and J.-P. Vincent. 1998. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy. Braz. J. Med. Biol. Res. 31:1479–1489.PubMedGoogle Scholar
  3. 3.
    Boudin, H., P. Sarret, J. Mazella, A. Schonbrunn, and A. Beaudet. 2000. Somatostatin-induced regulation of sst2A receptor expression and cell surface availability in central neurons: role of receptor internalization. J. Neurosci. 20:5932–5939.PubMedGoogle Scholar
  4. 4.
    Castel, M.-N., J. Woulfe, X. Wang, P.M. Laduron, and A. Beaudet. 1992. Light and electron microscopic localization of retrogradely transported neurotensin in rat nigrostriatal dopaminergic neurons. Neuroscience 50:269–282.PubMedCrossRefGoogle Scholar
  5. 5.
    Chan, J., C. Aoki, and V.M. Pickel. 1990. Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultra-structure in brain sections before plastic embedding. J. Neurosci. 15:113–127.Google Scholar
  6. 6.
    Daaka, Y., L.M. Luttrell, S. Ahn, G.J. Delia Rocca, S.S.G. Ferguson, M.G. Caron, and R.J. Lefkowitz. 1998. Essential role for G protein-coupled receptor endocytosis in the activation of mitogen-activated protein kinase. J. Biol. Chem. 273:685–688.PubMedCrossRefGoogle Scholar
  7. 7.
    Dautry-Varsat, A., A. Ciechanover, and H.F. Lodish. 1983. pH and the recycling of transferrin during receptor-mediated endocytosis. Proc. Natl. Acad. Sci. USA 80:2258–2262.PubMedCrossRefGoogle Scholar
  8. 8.
    Dumartin, B., I. Caillé, F. Gonon, and B. Bloch. 1998. Internalization of Dl dopamine receptor in striatal neurons in vivo as evidence of action by dopamine agonists. J. Neurosci. 18:1650–1661.PubMedGoogle Scholar
  9. 9.
    Faure, M.-P., P. Gaudreau, I. Shaw, N.R. Cashman, and A. Beaudet. 1994. Synthesis of a biologically active fluorescent probe for labeling neurotensin receptors. J. Histochem. Cytochem. 42:755–763.PubMedGoogle Scholar
  10. 10.
    Faure, M.-P., A. Alonso, D. Nouel, G. Gaudriault, M. Dennis, J.-P. Vincent, and A. Beaudet. 1995. Somatodendritic internalization and perinuclear targeting of neurotensin in the mammalian brain. J. Neurosci. 15:4140–4147.PubMedGoogle Scholar
  11. 11.
    Faure, M.-R., D. Nouel, and A. Beaudet. 1995. Axonal and dendritic transport of internalized neurotensin in rat mesostriatal dopaminergic neurons. Neuroscience 68:519–529.PubMedCrossRefGoogle Scholar
  12. 12.
    Ferguson, S.S.G., J. Zhang, L.S. Barak, and M.G. Caron. 1998. Role of Β:-arrestins in the intracellular trafficking of G-protein-coupled receptors. Adv. Pharmacol. 42:420–424.Google Scholar
  13. 13.
    Gaudriault, G., D. Nouel, C. Dal Farra, A. Beaudet, and J.-P. Vincent. 1997. Receptor-induced internalization of selective peptidic µ and δ opioid ligands. J. Biol. Chem. 272:2880–2888.PubMedCrossRefGoogle Scholar
  14. 14.
    Ghinea, N., M.T. Vu Hai, M.T. Groyer-Picard, A. Houllier, D. Schoevaert, and E. Milgrom. 1992. Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells. J. Cell Biol. 118:1347–1358.PubMedCrossRefGoogle Scholar
  15. 15.
    Grady, E.F., A.M. Garland, P.D. Gamp, M. Lovett, D.G. Payan, and N.W. Bunnett. 1995. Delineation of the endocytic pathway of substance P and its seventransmembrane domain NK1 receptor. Mol. Biol. Cell 6:509–524.PubMedGoogle Scholar
  16. l6.
    Mantyh, P.W., C.J. Allen, J.R. Ghilardi, S.D. Rogers, C.R. Mantyh, H. Liu, A.I. Basbaum, S.R. Vigna, and J.E. Maggio. 1995. Rapid endocytosis of a G proteincoupled receptor: substance P-evoked internalization of its receptor in the rat striatum in vivo. Proc. Natl. Acad. USA 92:2622–2626.CrossRefGoogle Scholar
  17. 17.
    Nouel, D., G. Gaudriault, M. Houle, T. Reisine, J.-P. Vincent, J. Mazella, and A. Beaudet. 1997. Differential internalization of somatostatin in COS-7 cells transfected with sst1 and sst2 receptor subtypes: a confocal microscopic study using novel fluorescent somatostatin derivatives. Endocrinology 138:296–306.PubMedCrossRefGoogle Scholar
  18. 18.
    Nouel, D., M.-P Faure, J.-A. St. Pierre, R. Alonso, R. Quirion, and A. Beaudet. 1997b. Differential binding profile and internalization process of neurotensin via neuronal and glial receptors. J. Neurosci. 17:1795–1803.PubMedGoogle Scholar
  19. 19.
    Pearse, B.M. 1982. Coated vesicles from human placenta carry ferritin, transferrin, and immunoglobulin G. Proc. Natl. Acad. Sei. USA 79:451–455.CrossRefGoogle Scholar
  20. 20.
    Sarret, P., D. Nouel, C. Dal Farra, J.-P. Vincent, A. Beaudet, and J. Mazella. 1999. Receptor-mediated internalization is critical for the inhibition of the expression of growth-hormone by somatostatin in the pituitary cell line AtT-20. J. Biol. Chem. 274:19294–19300.PubMedCrossRefGoogle Scholar
  21. 21.
    Stroh, T., A.C. Jackson, P. Sarret, C. DalFarra, J.-P. Vincent, H.J. Kreienkamp, J. Mazella, and A. Beaudet. 2000. Intracellular dynamics of sst5 receptors in transfected COS-7 cells: maintenance of cell surface receptors during ligand-induced endocytosis. Endocrinology 141:354–365.PubMedCrossRefGoogle Scholar
  22. 22.
    Stroh, T., A.C. Jackson, C. DalFarra, A. Schonbrun, J.R Vincent, and A. Beaudet. Receptor-mediated internalization of somatostatin in rat cortical and hippocampal neurons. Synapse (In press).Google Scholar
  23. 23.
    von Zastrow, M. and B.K. Kobilka. 1992. Ligand-regulated internalization and recycling of human β2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J. Biol. Chem. 267:3530–3538.Google Scholar

Copyright information

© Springer-Verlag New York, Inc. 2002

Authors and Affiliations

  • Alain Beaudet
    • 1
  • Alexander C. Jackson
    • 1
  • Franck Vandenbulcke
    • 1
  1. 1.Montreal Neurological InstituteMcGill UniversityMontrealCanada

Personalised recommendations