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Confocal Imaging of Nerve Cells and Their Connections

  • Andrew J. Todd
Chapter

Abstract

This chapter outlines the use of confocal microscopy combined with immunofluorescence staining for examining nerve cells in the central nervous system (CNS) and studying the synaptic connections between them. A method for carrying out immunolabeling with two or three different fluorescent dyes is described, together with modifications that allow this to be combined with tract tracing,intracellular injection, or lectin labeling, and also a new technique for combining confocal and electron microscopy (EM) on sections which have been processed for immunocytochemistry. With these approaches, it is possible to search for and quantify contacts between neurons at the light microscopic level and then examine representative contacts at the ultrastructural level to determine whether synapses are present.

Keywords

Dorsal Horn Confocal Image Dendritic Tree Primary Afferents Immunoreactive Neuron 
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.

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References

  1. 1.
    Belichenko, P.V. and A. Dahlström. 1995. Confocal laser scanning microscopy and 3-D reconstructions of neuronal structures in human brain cortex. Neuroimage 2:201–207.PubMedCrossRefGoogle Scholar
  2. 2.
    Bleazard, L., R.G. Hill, and R. Morris. 1994. The correlation between the distribution of NK1 receptor and the actions of tachykinin agonists in the dorsal horn of the rat indicates that substance P does not have a functional role on substantia gelatinosa (lamina II) neurons. J. Neurosci. 14:7655–7664.PubMedGoogle Scholar
  3. 3.
    Brelje, T.C., M.W. Wessendorf, and R.L. Sorenson. 1993. Multicolor laser scanning confocal immunofluorescence microscopy: practical applications and limitations, p. 97–181. In B. Matsumoto (Ed.), Cell Biological Applications of Confocal Microscopy. Acadamic Press, San Diego.Google Scholar
  4. 4.
    Brown, J.L., H. Liu, J.E. Maggio, S.R. Vigna, P.W. Mantyh, and A.I. Basbaum. 1995. Morphological characterization of substance P receptor-immunoreactive neurons in rat spinal cord and trigeminal nucleus caudalis J. Comp. Neurol. 356:327–344.PubMedCrossRefGoogle Scholar
  5. 5.
    Jankowska, E., D.J. Maxwell, S. Dolk, and A. Dahlström. 1997. A confocal and electron microscopic study of contacts between 5-HT fibres and feline dorsal horn interneurons in pathways from muscle afferents. J. Comp. Neurol. 387:430–438.PubMedCrossRefGoogle Scholar
  6. 6.
    Ju, G., T. Hökfelt, E. Brodin, J. Fahrenkrug, J.A. Fischer, P. Frey, R.P. Elde, and J.C. Brown. 1987. Primary sensory neurons of the rat showing calcitonin generelated peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide-and cholecystokinin-immunoreactive ganglion cells. Cell Tissue Res. 247:417–431.PubMedCrossRefGoogle Scholar
  7. 7.
    Kosaka, T., I. Nagatsu, J.-Y. Wu, and K. Hama. 1986. Use of high concentrations of glutaraldehyde for immunocytochemistry of transmitter-synthesizing enzymes in the central nervous system. Neuroscience 18:975–990.PubMedCrossRefGoogle Scholar
  8. 8.
    Lawson, S.N. 1992. Morphological and biochemical cell types of sensory neurons, p. 27–59. In S.A. Scott (Ed.), Sensory Neurones: Diversity, Development and Plasticity. Oxford University Press, New York.Google Scholar
  9. 9.
    Li, J.-L., T. Kaneko, S. Nomura, Y.-Q. Li, and N. Mizuno. 1997. Association of serotonin-like immuno-reactive axons with nociceptive projection neurons in the caudal spinal trigeminal nucleus of the rat. J. Comp. Neurol. 384:127–141.PubMedCrossRefGoogle Scholar
  10. 10.
    Llewellyn-Smith, I.J. and J.B. Minson. 1992. Complete penetration of antibodies into Vibratome sections after glutaraldehyde fixation and ethanol treatment: light and electron microscopy for neuropeptides. J. Histochem. Cytochem. 40:1741–1749.PubMedGoogle Scholar
  11. 11.
    Mailof, L. and P.-O. Forsgren. 1993. Confocal microscopy: important considerations for accurate imaging, p. 79–95. In B. Matsumoto (Ed.), Cell Biological Applications of Confocal Microscopy. Aadamic Press, San Diego.Google Scholar
  12. 12.
    Marshall, G.E., S.A.S. Shehab, R.C. Spike, and A.J. Todd. 1996. Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat. Neuroscience 72:255–263.PubMedCrossRefGoogle Scholar
  13. 13.
    Mason, P., S.A. Back, and H.L. Fields. 1992. A confocal laser microscopic study of enkephalin-immunoreactive appositions onto physiologically identified neurons in the rostral ventromedial medulla. J. Neurosci. 12:4023–4036.PubMedGoogle Scholar
  14. 14.
    Naim, M., R.C. Spike, C. Watt, S.A.S. Shehab, and A.J. Todd. 1997. Cells in laminae III and IV of the rat spinal cord which possess the neurokinin-1 receptor and have dorsally-directed dendrites receive a major synaptic input from tachykinin-containing primary afferents. J. Neurosci. 17:5536–5548.PubMedGoogle Scholar
  15. 15.
    Naim, M., S.A.S. Shehab, and A.J. Todd. 1998. Cells in laminae III and IV of the rat spinal cord which possess the neurokinin-1 receptor receive monosynaptic input from myelinated primary afferents. Eur. J. Neurosci. 10:3012–3019.PubMedCrossRefGoogle Scholar
  16. 16.
    Pollock, R., R. Kerr, and D.J. Maxwell. 1997. An immunocytochemical investigation of the relationship between substance P and the neurokinin-1 receptor in the lateral horn of the rat thoracic spinal cord. Brain Res. 777:22–30.PubMedCrossRefGoogle Scholar
  17. 17.
    Robertson, B. and G. Grant. 1985. A comparison between wheatgerm agglutinin and choleragenoid-horseradish peroxidase as anterogradely transported markers in central branches of primary sensory neurones in the rat with some observations in the cat. Neuroscience 14:895–905.PubMedCrossRefGoogle Scholar
  18. 18.
    Sakamoto, H., R.C. Spike, and A.J. Todd. 1999. Neurons in laminae III and IV of the rat spinal cord with the neurokinin-1 receptor receive few contacts from unmyelinated primary afferents which do not contain substance P. Neuroscience 94:903–908.PubMedCrossRefGoogle Scholar
  19. 19.
    Todd, A.J. 1997. A method for combining confocal and electron microscopic examination of sections processed for double-or triple-labeling immunocyto-chemistry. J. Neurosci. Methods 73:149–157.PubMedCrossRefGoogle Scholar
  20. 20.
    Todd, A.J., C. Watt, R.C. Spike, and W Sieghart. 1996. Colocalization of GABA, glycine and their receptors at synapses in the rat spinal cord. J. Neurosci. 16:974–982.PubMedGoogle Scholar
  21. 21.
    Vulchanova, L., M.S. Riedl, S.J. Shuster, L.S. Stone, K.M. Hargreaves, G. Buell, A. Surprenant, R.A. North, and R. Elde. 1998. P2X3 is expressed by DRG neurons that terminate in inner lamina II. Eur. J. Neurosci. 10:3470–3478.PubMedCrossRefGoogle Scholar
  22. 22.
    Willis, W.D. and R.E. Coggeshall. 1991. Sensory Mechanisms of the Spinal Cord. Plenum, New York.Google Scholar

Copyright information

© Springer-Verlag New York, Inc. 2002

Authors and Affiliations

  • Andrew J. Todd
    • 1
  1. 1.Laboratory of Human Anatomy, Institute of Biomedical and Life SciencesUniversity of GlasgowGlasgowUK, EU

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