Skip to main content
SpringerLink
Log in

Nonradioactive In Situ Hybridization in Combination with Tract-Tracing

  • Chapter
Neuroanatomical Tract-Tracing 3

Abstract

The use of in situ hybridization (ISH) for the detection of mRNAs in cell bodies has greatly expanded our ability to detect cellular phenotypes in the central nervous system. Riboprobes have been used in the past to identify neuropeptide precursors, distribution of receptors, ion channels, and enzymes. More recently, the discovery of unambiguous markers for the major ionotropic transmitters has made possible the definitive identification of neurons involved in fast transmission. The advantages and disadvantages of different types of probes, including DNA probes, oligonucleotides, and RNA probes for the detection of mRNAs are described. Although in situ hybridization was pioneered with the use of radioactive probes, nonradioactive alternatives are now readily available. The relative merits of nonradioactive probes, specifically for combination with tract-tracing, are discussed. This chapter focuses on in situ hybridization methods based on nonradioactive riboprobes and their use in combination with tract-tracing and immunocytochemistry.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
eBook
USD 16.99
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  • Baldwin, T. J., Tsaur, M. L., Lopez, G. A., Jan, Y. N., and Jan, L. Y., 1991, Characterization of a mammalian cDNA for an inactivating voltage-sensitive K+ channel, Neuron 7:471–483.

    Article  PubMed  CAS  Google Scholar 

  • Bellocchio, E. E., Hu, H., Pohorille, A., Chan, J., Pickel, V. M., and Edwards, R. H., 1998, The localization of the brain-specific inorganic phosphate transporter suggests a specific presynaptic role in glutamatergic transmission, J. Neurosci. 18:8648–8659.

    PubMed  CAS  Google Scholar 

  • Bloch, B., Popovici, T., Chouham, S., and Kowalski, C., 1986a, Detection of the mRNA coding for enkephalin precursor in the rat brain and adrenal by using an “in situ” hybridization procedure, Neurosci. Lett. 64:29–34.

    Article  PubMed  CAS  Google Scholar 

  • Bloch, B., Popovici, T., Le Guellec, D., Normand, E., Chouham, S., Guitteny, A. F., and Bohlen, P., 1986b, In situ hybridization histochemistry for the analysis of gene expression in the endocrine and central nervous system tissues: a 3-year experience, J. Neurosci. Res. 16:183–200.

    Article  PubMed  CAS  Google Scholar 

  • Brysch, W., Creutzfeldt, O. D., Luno, K., Schlingensiepen, R., and Schlingensiepen, K. H., 1991, Regional and temporal expression of sodium channel messenger RNAs in the rat brain during development, Exp. Brain Res. 86:562–567.

    Article  PubMed  CAS  Google Scholar 

  • Chesselet, M. F., 1990, In Situ Hybridization Histochemistry, Boston: CRC Press.

    Google Scholar 

  • Chesselet, M. F., Weiss, L., Wuenschell, C., Tobin, A. J., and Affolter, H. U., 1987, Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain, J. Comp. Neurol. 262:125–140.

    Article  PubMed  CAS  Google Scholar 

  • Clavel, C., Binninger, I., Boutterin, M. C., Polette, M., and Birembaut, P., 1991, Comparison of four non-radioactive and 35S-based methods for the detection of human papillomavirus DNA by in situ hybridization, J. Virol. Methods 33:253–266.

    Article  PubMed  CAS  Google Scholar 

  • Darby, I. A., 2000, In Situ Hybridization Protocols, 2nd ed., Totowa, NJ: Humana Press.

    Google Scholar 

  • Esclapez, M., Tillakaratne, N. J., Kaufman, D. L., Tobin, A. J., and Houser, C. R., 1994, Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms, J. Neurosci. 14:1834–1855.

    PubMed  CAS  Google Scholar 

  • Fremeau, R. T., Voglmaier, S., Seal, R. P., and Edwards, R. H., 2004, VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate, Trends Neurosci. 27:98–103.

    Article  PubMed  CAS  Google Scholar 

  • Gall, J. G., and Pardue, M. L., 1969, Formation and detection of RNA-DNA hybrid molecules in cytological preparations, Proc. Natl. Acad. Sci. U. S. A. 63:378–383.

    Article  PubMed  CAS  Google Scholar 

  • Goldman, D., Simmons, D., Swanson, L. W., Patrick, J., and Heinemann, S., 1986, Mapping of brain areas expressing RNA homologous to two different acetylcholine receptor alphasubunit cDNAs, Proc. Natl. Acad. Sci. U.S.A. 83:4076–4080.

    Article  PubMed  CAS  Google Scholar 

  • Guyenet, P. G., Stornetta, R. L., Weston, M. C., McQuiston, T., and Simmons, J. R., 2004, Detection of amino acid and peptide transmitters in physiologically identified brainstem cardiorespiratory neurons, Auton. Neurosci. 114:1–10.

    Article  PubMed  CAS  Google Scholar 

  • Hisano, S., Sawada, K., Kawano, M., Kanemoto, M., Xiong, G. X., Mogi, K., Sakata-Haga, H., Takeda, J., Fukui, Y., and Nogami, H., 2002, Expression of inorganic phosphate/vesicular glutamate transporters (BNPI/VGLUT1 and DNPI/VGLUT2) in the cerebellum and precerebellar nuclei of the rat, Mol. Brain. Res. 107:23–31.

    Article  PubMed  CAS  Google Scholar 

  • Hoefler, H., Childers, H., Montminy, M. R., Lechan, R. M., Goodman, R. H., and Wolfe, H. J., 1986, In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes, Histochem. J. 18:597–604.

    Article  PubMed  CAS  Google Scholar 

  • Hwang, P. M., Glatt, C. E., Bredt, D. S., Yellen, G., and Snyder, S. H., 1992, A novel K+ channel with unique localizations in mammalian brain: molecular cloning and characterization, Neuron 8:473–481.

    Article  PubMed  CAS  Google Scholar 

  • John, H. A., Birnstiel, M. L., and Jones, K. W., 1969, RNA-DNA hybrids at the cytological level, Nature 223:582–587.

    Article  PubMed  CAS  Google Scholar 

  • Johnson, A. D., Peoples, J., Stornetta, R. L., and Van Bockstaele, E. J., 2002, Opioid circuits originating from the nucleus paragigantocellularis and their potential role in opiate withdrawal, Brain Res. 955:72–84.

    Article  PubMed  CAS  Google Scholar 

  • Julien, J. F., Legay, F., Dumas, S., Tappaz, M., and Mallet, J., 1987, Molecular cloning, expression and in situ hybridization of rat brain glutamic acid decarboxylase messenger RNA, Neurosci. Lett. 73:173–180.

    Article  PubMed  CAS  Google Scholar 

  • Kreft, S., Zajc-Kreft, K., Zivin, M., Sket, D., and Grubic, Z., 1996, Application of the nonradioactive in situ hybridization for the localization of acetylcholinesterase mRNA in the central nervous system of the rat; comparison to the radioactive technique, Pflugers Arch. 431:R309–R310.

    Article  PubMed  CAS  Google Scholar 

  • Lanaud, P., Popovici, T., Normand, E., Lemoine, C., Bloch, B., and Roques, B. P., 1989, Distribution of CCK mRNA in particular regions (hippocampus, periaqueductal grey and thalamus) of the rat by in situ hybridization, Neurosci. Lett. 104:38–42.

    Article  PubMed  CAS  Google Scholar 

  • Lenz, S., Perney, T. M., Qin, Y., Robbins, E., and Chesselet, M. F., 1994, GABA-ergic interneurons of the striatum express the Shaw-like potassium channel Kv3.1, Synapse 18:55–66.

    Article  PubMed  CAS  Google Scholar 

  • Lewis, M. E., and Baldino, F., 1990, Probes for in situ hybridization histochemistry, In: Chesselet, M. F. (ed.), In Situ Hybridization Histochemistry, Boca Raton, FL: CRC Press, pp. 1–21.

    Google Scholar 

  • Lewis, M. E., Sherman, T. G., and Watson, S. J., 1985, In situ hybridization histochemistry with synthetic oligonucleotides: strategies and methods, Peptides 6(Suppl. 2):75–87.

    Article  PubMed  CAS  Google Scholar 

  • Malherbe, P., Sigel, E., Baur, R., Persohn, E., Richards, J. G., and Mohler, H., 1990, Functional expression and sites of gene transcription of a novel alpha subunit of the GABAA receptor in rat brain, FEBS Lett. 260:261–265.

    Article  PubMed  CAS  Google Scholar 

  • McKinnon, D., 1989, Isolation of a cDNA clone coding for a putative second potassium channel indicates the existence of a gene family, J. Biol. Chem. 264:8230–8236.

    PubMed  CAS  Google Scholar 

  • Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R., 1984, Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter, Nucleic Acids Res. 12:7035–7056.

    PubMed  CAS  Google Scholar 

  • Mezey, E., 1989, Phenylethanolamine N-methyltransferase-containing neurons in the limbic system of the young rat, Proc. Natl. Acad. Sci. U. S. A. 86:347–351.

    Article  PubMed  CAS  Google Scholar 

  • Mitchell, V., Gambiez, A., and Beauvillain, J. C., 1993, Fine-structural localization of proenkephalin mRNAs in the hypothalamic magnocellular dorsal nucleus of the guinea pig: a comparison of radioisotopic and enzymatic in situ hybridization methods at the light-and electron-microscopic levels, Cell Tissue Res. 274:219–228.

    Article  PubMed  CAS  Google Scholar 

  • Park, J. S., Kurman, R. J., Kessis, T. D., and Shah, K. V., 1991, Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections, Mod. Pathol. 4:81–85.

    PubMed  CAS  Google Scholar 

  • Pelletier, G., Liao, N., Follea, N., and Govindan, M. V., 1988, Distribution of estrogen receptors in the rat pituitary as studied by in situ hybridization, Mol. Cell. Endocrinol. 56:29–33.

    Article  PubMed  CAS  Google Scholar 

  • Perney, T. M., Marshall, J., Martin, K. A., Hockfield, S., and Kaczmarek, L. K., 1992, Expression of the mRNAs for the Kv3.1 potassium channel gene in the adult and developing rat brain, J. Neurophysiol. 68:756–766.

    PubMed  CAS  Google Scholar 

  • Pochet, R., Brocas, H., Vassart, G., Toubeau, G., Seo, H., Refetoff, S., Dumont, J. E., and Pasteels, J. L., 1981, Radioautographic localization of prolactin messenger RNA on histological sections by in situ hybridization, Brain Res. 211:433–438.

    Article  PubMed  CAS  Google Scholar 

  • Poyatos, I., Ponce, J., Aragon, C., Gimenez, C., and Zafra, F., 1997, The glycine transporter GLYT2 is a reliable marker for glycine-immunoreactive neurons, Mol. Brain Res. 49:63–70.

    Article  PubMed  CAS  Google Scholar 

  • Rogers, S. W., Hughes, T. E., Hollmann, M., Gasic, G. P., Deneris, E. S., and Heinemann, S., 1991, The characterization and localization of the glutamate receptor subunit GluR1 in the rat brain, J. Neurosci. 11:2713–2724.

    PubMed  CAS  Google Scholar 

  • Rudy, B., Kentros, C., Weiser, M., Fruhling, D., Serodio, P., Vega-Saenz, D. M., Ellisman, M. H., Pollock, J. A., and Baker, H., 1992, Region-specific expression of a K+ channel gene in brain, Proc. Natl. Acad. Sci. U. S. A. 89:4603–4607.

    Article  PubMed  CAS  Google Scholar 

  • Schmued, L. C., and Fallon, J. H., 1986, Fluoro-gold: a new fluorescent retrograde axonal tracer with numerous unique properties, Brain Res. 377:147–154.

    Article  PubMed  CAS  Google Scholar 

  • Schreihofer, A.M., Stornetta, R.L., and Guyenet, P.G., 1999, Evidence for glycinergic respiratory neurons: Bötzinger neurons express mRNA for glycenergic transporter 2, J. Comp. Neurol. 407:583–597.

    Article  PubMed  CAS  Google Scholar 

  • Seroogy, K., Schalling, M., Brene, S., Dagerlind, A., Chai, S. Y., Hokfelt, T., Persson, H., Brownstein, M., Huan, R., Dixon, J., Filer, D., Schlessinger, D., and Goldstein, M., 1989, Cholecystokinin and tyrosine hydroxylase messenger RNAs in neurons of rat mesencephalon: peptide/monoamine coexistence studies using in situ hybridization combined with immunocytochemistry, Exp. Brain Res. 74:149–162.

    Article  PubMed  CAS  Google Scholar 

  • Shivers, B. D., Schachter, B. S., and Pfaff, D. W., 1986, In Situ hybridization for the study of gene expression in the brain, Meth. Enzymol. 124: 497–510.

    Article  PubMed  CAS  Google Scholar 

  • Siegel, R. E., and Young III, W. S., 1985, Detection of preprocholecystokinin and preproenkephalin A mRNAs in rat brain by hybridization histochemistry using complementary RNA probes, Neuropeptides 6:573–580.

    Article  PubMed  CAS  Google Scholar 

  • Stornetta, R. L., Akey, P. J., and Guyenet, P. G., 1999, Location and electrophysiological characterization of rostral medullary adrenergic neurons that contain neuropeptide Y mRNA in rat medulla, J. Comp. Neurol. 415:482–500.

    Article  PubMed  CAS  Google Scholar 

  • Stornetta, R. L., and Guyenet, P. G., 1999, Distribution of glutamic acid decarboxylase mRNA containing neurons in rat medulla projecting to thoracic spinal cord in relation to monoaminergic brainstem neurons, J. Comp. Neurol. 407:367–380.

    Article  PubMed  CAS  Google Scholar 

  • Stornetta, R. L., McQuiston, T. J., and Guyenet, P. G., 2004, GABAergic and glycinergic presympathetic neurons of rat medulla oblongata identified by retrograde transport of pseudorabies virus and in situ hybridization, J. Comp. Neurol. 479:257–270.

    Article  PubMed  CAS  Google Scholar 

  • Stornetta, R. L., Sevigny, C. P., and Guyenet, P. G., 2003, Inspiratory augmenting bulbospinal neurons express both glutamatergic and enkephalinergic phenotypes, J. Comp. Neurol. 455:113–124.

    Article  PubMed  CAS  Google Scholar 

  • Stornetta, R. L., Sevigny, C. P., Schreihofer, A. M., Rosin, D. L., and Guyenet, P. G., 2002, Vesicular glutamate transporter DNPI/GLUT2 is expressed by both C1 adrenergic and nonaminergic presympathetic vasomotor neurons of the rat medulla, J. Comp. Neurol. 444:207–220.

    Article  PubMed  CAS  Google Scholar 

  • Stornetta, R.L., Schreihofer, A.M., Pelaez, N.M., Sevigny, C.P., and Guyenet, P.G., 2001, Preproenkephalin mRNA is expressed by C1 and non-C1 barosensitive bulbospinal neurons in the rostral ventrolateral medulla of the rat, J. Comp. Neurol. 435:111–126.

    Article  PubMed  CAS  Google Scholar 

  • Surmeier, D. J., Eberwine, J., Wilson, C. J., Cao, Y., Stefani, A., and Kitai, S. T., 1992, Dopamine receptor subtypes colocalize in rat striatonigral neurons, Proc. Natl. Acad. Sci. U. S. A. 89:10178–10182.

    Article  PubMed  CAS  Google Scholar 

  • Terenghi, G., Polak, J. M., Hamid, Q., O’Brien, E., Denny, P., Legon, S., Dixon, J., Minth, C. D., Palay, S. L., Yasargil, G., and Chan-Palay, V., 1987, Localization of neuropeptide Y mRNA in neurons of human cerebral cortex by means of in situ hybridization with a complementary RNA probe, Proc. Natl. Acad. Sci. U. S. A. 84:7315–7318.

    Article  PubMed  CAS  Google Scholar 

  • Valentino, K. L., Eberwine, J. H., and Barchas, J. D., 1987, In Situ Hybridization: Applications to Neurobiology, New York: Oxford University Press.

    Google Scholar 

  • Wada, K., Ballivet, M., Boulter, J., Connolly, J., Wada, E., Deneris, E. S., Swanson, L. W., Heinemann, S., and Patrick, J., 1988, Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor, Science 240:330–334.

    Article  PubMed  CAS  Google Scholar 

  • Wang, H., Kunkel, D. D., Schwartzkroin, P. A., and Tempel, B. L., 1994, Localization of Kv1.1 and Kv1.2, two K channel proteins, to synaptic terminals, somata, and dendrites in the mouse brain, J. Neurosci. 14:4588–4599.

    PubMed  CAS  Google Scholar 

  • Wilkinson, D. G., 1998, In Situ Hybridization: A Practical Approach, 2nd ed., Oxford: Oxford University Press.

    Google Scholar 

  • Wisden, W., and Morris, B. J., 1994, In Situ Hybridization Protocols for the Brain, London: Academic Press, Harcourt Brace & Co.

    Google Scholar 

  • Wisden, W., Morris, B. J., Darlison, M. G., Hunt, S. P., and Barnard, E. A., 1988, Distinct GABAA receptor alpha subunit mRNAs show differential patterns of expression in bovine brain, Neuron 1:937–947.

    Article  PubMed  CAS  Google Scholar 

  • Wuenschell, C. W., Fisher, R. S., Kaufman, D. L., and Tobin, A. J., 1986, In situ hybridization to localize mRNA encoding the neurotransmitter synthetic enzyme glutamate decarboxylase in mouse cerebellum, Proc. Natl. Acad. Sci. U. S. A. 83:6193–6197.

    Article  PubMed  CAS  Google Scholar 

  • Young III, W. S., 1990, In situ hybridization histochemistry, In: Björklund, A., Hökfelt, T., Wouterlood, F. G., and Van den Pol, A. N. (eds.), Analysis of Neuronal Microcircuits and Synaptic Interactions, Amsterdam: Elsevier.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacology, University of Virginia, Charlottesville, VA, 22908

    Ruth L. Stornetta & Patrice G. Guyenet

Authors
  1. Ruth L. Stornetta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Patrice G. Guyenet
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Center for Molecular and Behavioral Neuroscience, Rutgers University, Newark, NJ, USA

    Laszlo Zaborszky

  2. Department of Anatomy, Vrije University Medical Center, Amsterdam, The Netherlands

    Floris G. Wouterlood

  3. Neurosciences Division, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

    José Luis Lanciego

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Springer Science+Business Media, Inc.

About this chapter

Cite this chapter

Stornetta, R.L., Guyenet, P.G. (2006). Nonradioactive In Situ Hybridization in Combination with Tract-Tracing. In: Zaborszky, L., Wouterlood, F.G., Lanciego, J.L. (eds) Neuroanatomical Tract-Tracing 3. Springer, Boston, MA . https://doi.org/10.1007/0-387-28942-9_8

Download citation

Publish with us

Policies and ethics

Search

Navigation