Skip to main content
SpringerLink
Log in

Immunocytochemical applications in neuroanatomy

Demonstration of connections, transmitters and receptors

  • Reviews
  • Published:
Histochemistry Aims and scope Submit manuscript

Summary

In the present paper we review immunocytochemical methods for anterograde tracing with the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L), combined PHA-L tracing — neurotransmitter immunocytochemistry, and the immunocytochemical localization of receptor proteins. These methods will be mainly illustrated by examples from tracing- and neurotransmitter studies on the cholinergic basal forebrain system. The morphology of PHA-L labeled neurons strongly resembles that of Golgi impregnated neurons. The complete axonal trajectories and patterns of presynaptic endings of PHA-L labeled neurons are visualized, both for light- and electron microscopic application.

PHA-L-tracing can very well be combined with second immunocytochemical labeling procedures. In this way, traced pathways can be studied in their relation to chemically identified fiber systems or target neurons. Application of immunocytochemistry for the localization of the muscarinic acetylcholine receptor, albeit in its early stages, holds great promise for the near future.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • André C, Marullo S, Guillet JG, Convents A, Lauwereys M, Kaveri S, Hoebeke J, Strosberg AD (1987) Immunochemical studies of the muscarinic acetylcholine receptor. J Recept Res 7:89–103

    Google Scholar 

  • Araki T, Yamano M, Murakami T, Wanaka A, Betz H, Tohyama M (1988) Localization of glycine receptors in the rat central nervous system: an immunocytochemical analysis using monoclonal antibody. Neuroscience (in press)

  • Buijs RM, Van Vulpen EHS, Geffard M (1987) Ultrastructural localization of GABA in the supraoptic nucleus and neural lobe. Neuroscience 20:347–355

    Google Scholar 

  • Cordell JL, Falini B, Erber WN, Gosh AK, Abdulaziz Z, McDonald S, Pulford KAF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32:219–229

    Google Scholar 

  • Edwards SB, Hendrickson A (1981) The autoradiographic tracing of axonal connections in the central nervous system. In: Heimer L, Roberts MJ (eds) Neuroanatomical tract-tracing methods. Plenum Press, New York, pp 171–202

    Google Scholar 

  • Fink RP, Heimer L (1967) Two methods for selective silver impregnation of degnerating axons and their synaptic endings in the central nervous system. Brain Res 4:369–374

    Google Scholar 

  • Frotscher M, Léránth C (1985) Cholinergic innervation of the rat hippocampus as revealed by choline acetyltransferase immunocytochemistry: a combined light and electron microscopic study. J Comp Neurol 239:237–246

    Google Scholar 

  • Gerfen CR, Sawchenko PE (1984) An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: immunohistochemical localization of an axonally transported plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L). Brain Res 290:219–238

    Google Scholar 

  • Gerfen CR, Sawchenko PE (1985) A method for anterograde axonal tracing of chemically specified circuits in the central nervous system: combined Phaseolus vulgaris leucogglutinin (PHA-L) tract tracing and immunocytochemistry. Brain Res 343:144–150

    Google Scholar 

  • Gonatas NK, Sim SU, Stieber A, Avrameas S (1977) Internalization of lectins in neuronal GERL. J Cell Biol 73:1–13

    Google Scholar 

  • Gonatas NK, Harper C, Mizutani T, Gonatas JO (1979) Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport. J Histochem Cytochem 27:728–734

    Google Scholar 

  • Hamel E, Beaudet A (1987) Ultrastructural distribution of mu opioid receptors in rat neostriatum. In: Sandler M (ed) Neurotransmitter interactions in the basal ganglia. Raven Press, New York, pp 59–69

    Google Scholar 

  • Hancock MB (1982) DAB-Nickel substrate for the differential immunoperoxidase staining of nerve fibers and fiber terminals. J Histochem Cytochem 30:578

    Google Scholar 

  • Heimer L, RoBards MJ (eds) (1981) Neuroanatomical tract-tracing methods. Plenum Press, New York

    Google Scholar 

  • Hendrickson AE (1969) Electron microscopic autoradiography: Identification of origin of synaptic terminals in normal nervous tissue. Science 165:194–196

    Google Scholar 

  • Herkenham M (1987) Mismatches between neurotransmitter and receptor localizations in brain: observations and implications. Neuroscience 23:1–38

    Google Scholar 

  • Houser CR, Crawford GD, Barber RP, Salvaterra PM, Vaughn JE (1983) Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase. Brain Res 266:97–119

    Google Scholar 

  • Jacob MH, Berg DK, Lindstrom JM (1984) Shared antigenic determinant between the Electrophorus acetylcholine receptor and a synaptic component on chicken ciliary ganglion neurons. Proc Natl Acad Sci USA 81:3223–3227

    Google Scholar 

  • Kaveri SV, Cervantes-Olivier P, Delavier-Klutchko C, Strosberg AD (1987) Monoclonal antibodies directed against the human A431 β2-adrenergic receptor recognize two major polypeptide chains. Eur J Biochem 167:449–456

    Google Scholar 

  • Kiss A, Palkovits M, Skirboll LR (1988) Light microscopic triplecolored immunohistochemical staining on the same vibratome section using the avidin-biotin-peroxidase complex technique. Histochemistry 88:353–356

    Google Scholar 

  • Kita H, Kitai ST (1987) Efferent projections of the subthalamic nucleus in the rat: light and electron microscopic analysis with the PHA-L method. J Comp Neurol 260:435–452

    Google Scholar 

  • Kristensson K, Olsson Y (1971) Retrograde axonal transport of protein. Brain Res 29:363–365

    Google Scholar 

  • Kubota Y, Inagaki S, Shimada S, Kito S, Eckenstein F, Tohyama M (1987) Neostriatal cholinergic neurons receive direct synaptic inputs from dopaminergic axons. Brain Res 413:179–184

    Google Scholar 

  • Kuhar MJ (1985) The mismatch problem in receptor mapping studies. Trends Neurosci 8:190–191

    Google Scholar 

  • Kuhar MJ, Taylor N, Wamsley JK, Hulme EC, Birdsall NJM (1981) Muscarinic cholinergic receptor localization in brain by electron microscopic autoradiography. Brain Res 216:1–9

    Google Scholar 

  • LaVail JH, LaVail MM (1972) Retrograde axonal transport in the central nervous system. Science 176:1416–1417

    Google Scholar 

  • Leiber D, Harbon S, Guillet JG, André C, Strosberg AD (1984) Monoclonal antibodies to purified muscarinic receptor display agonist-like activity. Proc Natl Acad Sci USA 81:4331–4334

    Google Scholar 

  • Léránth C, Frotscher M (1987) Cholinergic innervation of hippocampal GAD- and somatostatin-immunoreactive commissural neurons. J Comp Neurol 261:33–47

    Google Scholar 

  • Levey AI, Bolam JP, Rye DB, Hallanger AE, Demuth RM, Mesulam MM, Wainer BH (1986) A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride. J Histochem Cytochem 34:1449–1457

    Google Scholar 

  • Lin C-S, Lu SM, Yawamaki RM (1987) Laminar and synaptic organization of terminals from the ventrobasal and posterior thalamic nuclei in ratg barrel cortex. Abstr Soc Neurosci 13:248

    Google Scholar 

  • Luiten PGM, Spencer DG Jr, Traber J, Gaykema RPA (1985a) The pattern of cortical projections from the intermediate parts of the magnocellular nucleus basalis in the rat demonstrated by tracing with Phaseolus vulgaris leucoagglutinin. Neurosci Lett 57:137–142

    Google Scholar 

  • Luiten PGM, Ter Horst GJ, Karst H, Steffens AB (1985b) The course of pareventricular hypothalamic efferents to autonomic structures in medulla and spinal cord. Brain Res 329:374–378

    Google Scholar 

  • Luiten PGM, Gaykema RPA, Traber J, Spencer DG Jr (1987) Cortical projection patterns of magnocellular basal nucleus subdivisions as revealed by anteriogradely transported Phaseolus vulgaris leucoagglutinin. Brain Res 413:229–250

    Google Scholar 

  • Matsuyama T, Luiten PGM, Spencer DG Jr, Strosberg AD (1988) Ultrastructural localization of immunoreactive sites for muscarinic acetylcholine receptor proteins in the rat cerebral cortex. Neurosci Res Commun 2:64–76

    Google Scholar 

  • Nauta WJH (1950) Über die sogenannte terminale Degeneration im Zentralnervensystem und ihre Darstellung durch Silberimpregnation. Arch Neurol Psychiatry 66:353–376

    Google Scholar 

  • Nauta WJH, Gygax PA (1954) Silver impregnation of degencrating axons in the central nervous system: a modified technic. Stain Technol 29:91–93

    Google Scholar 

  • Nyakas C, Luiten PGM, Traber J, Spencer DG Jr (1987) Detailed projection patterns of septal and diagonal band efferents to the hippocampus in the rat with emphasis on innervation of CAA1 and dentate gyrus. Brain Res Bull 18:533–545

    Google Scholar 

  • Nyakas C, Luiten PGM, Balkan B, Spencer DG Jr (1988) Changes in septo-hippocampal and entorhino-hippocampal projections after lateral entorhinal, septal or septal-raphé lesions as studied by anterograde tracing methods. Brain Res Bull (in press)

  • Oertel WH, Tappaz ML, Berod A, Mugnaini E (1982) Two-color immunocytochemistry for dopamine and GABA neurons in rat substantia nigra and zona incerta. Brain Res Bull 9:463–474

    Google Scholar 

  • Pfeiffer F, Simlir R, Grenningloh G, Betz H (1984) Monoclonal antibodies and peptides mapping reveal structural similarities between the subunits of the glycine receptors of rat spinal cord. Proc Natl Acad Sci USA 81:7224–7227

    Google Scholar 

  • Raposo G, Dunia J, Marullo S, André C, Guillet JG, Strosberg AAD, Benedetti EL, Hoebeke J (1987) Redistribution of muscarinic acetylcholine receptors on human fibroblasts induced by regulatory ligands. Biol Cell 60:117–124

    Google Scholar 

  • Room P, Groenewegen HJ (1986) Connections of the parahippocampal cortex. I. Cortical afferents. J Comp Neurol 251:415–450

    Google Scholar 

  • Rye DB, Wainer BH, Mesulam MM, Mufson EJ, Saper CB (1984) Cortical projections arising from the basal forebrain: a study of cholinergic and noncholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase. Neuroscience 13:627–643

    Google Scholar 

  • Sawchenko PE, Gerfen CR (1985) Plant lectins and bacterial toxins as tools for tracing neuronal connections. Trends Neurosci 8:378–384

    Google Scholar 

  • Scott Young III W, Wamsley K, Zarbin MA, Kuhar MJ (1980) Opioid receptors undergo axonal flow. Science 210:76–77

    Google Scholar 

  • Spencer DG Jr, Horvath E, Traber J (1986) Direct autoradiographic determination of M1 and M2 muscarinic acetylcholine receptor distribution in the rat brain: relation to cholinergic nuclei and projections. Brain Res 380:59–68

    Google Scholar 

  • Sternberger LA (1979) Immunocytochemistry, 2nd edn. John Wiley, New York

    Google Scholar 

  • Swanson LW, Lindstrom J, Tzartos S, Schmued LC, O'Leary DDM, Cowan WM (1983) Immunohistochemical localization of monoclonal antibodies to the nicotinic acetylcholine receptor in chick midbrain. Proc Natl Acad Sci USA 80:4532–4536

    Google Scholar 

  • Swanson LW, Simmons DM, Whiting PJ, Lindstrom J (1987) Immunohistochemical localization of neuronal nicotinic receptors in the rodent central nervous system. J Neurosci 7:3334–3342

    Google Scholar 

  • Takeda N, Inagaki S, Shiosaka S, Taguchi Y, Oertel WH, Tohyama M, Watanabe T, Wada H (1984) Immunohistochemical evidence for the coexistence of histidine decarboxylase-like and glutamate decarboxylase-like immunoreactivities in nerve cells of the magnocellular nucleus of the posterior hypothalamus of rats. Proc Natl Acad Sci USA 81:7647

    Google Scholar 

  • Ter Horst GJ, Luiten PGM (1987) Phaseolus vulgaris leuco-agglutinin tracing of intrahypothalamic connections of the lateral, ventromedial, dorsomedial and paraventricular hypothalamic nuclei in the rat. Brain Res Bull 18:191–203

    Google Scholar 

  • Ter Horst GJ, Groenewegen HJ, Karst H, Luiten PGM (1984) Phaseolus vulgaris leuco-agglutinin immunohistochemistry. A comparison between autoradiographic and lectin tracing of neuronal efferents. Brain Res 307:379–383

    Google Scholar 

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

    Google Scholar 

  • Van den Pol AN (1985) Silver-intensified gold and peroxidase as dual ultrastructural immunolabels for pre- and postsynaptic neurotransmitters. Science 228:332–335

    Google Scholar 

  • Van den Pol AN, Smith AD, Powell JF (1985) GABA axons in synaptic contact with dopamine neurons in the substantia nigra: double immunocytochemistry with biotin-peroxidase and protein A-colloidal gold. Brain Res 348:146–154

    Google Scholar 

  • Van Hoesen GW, Hyman BT, Damasio AR (1986) Cell-specific pathology in neural systems of the temporal lobe in Alzheimer's disease. Prog Brain Res 10:321–335

    Google Scholar 

  • Watts AG, Swanson LW, Sanchez-Watts G (1987) Efferent projections of the suprachiasmatic nucleus: I. Studies using anterograde transport of Phaseolus vulgaris leucogglutinin in the rat. J Comp Neurol 258:204–229

    Google Scholar 

  • Witter MP, Groenewegen HJ (1984) Laminar origin and septotemporal distribution of entorhinal and perirhinal projections to the hippocampus in the cat. J Comp Neurol 224:371–385

    Google Scholar 

  • Woolf NJ, Hernit MC, Butcher LL (1986) Cholinergic and noncholinergic projections from the rat basal borebrain revealed by combined choline acetyltransferase and Phaseolus vulgaris leucoagglutinin immunohistochemistry. Neurosci Lett 66:281–286

    Google Scholar 

  • Wouterlood FG (1987) Combined light and electron microscopy of central nervous system neurons and their afferent and efferent synaptic connections. In: Hayat MA (ed) Correlative microscopy in biology: instrumentation and methods. Academic Press, New York, pp 83–119

    Google Scholar 

  • Wouterlood FG (1988) Anterograde neuroanatomical tracing with Phaseolus vulgaris leucoagglutinin combined with immunocytochemistry of gamma-amino butyric acid, choline acetyltransferase or serotonin. Histochemistry 89:421–428

    Google Scholar 

  • Wouterlood FG, Groenewegen HJ (1985) Neuroanatomical tracing by use of Phaseolus vulgaris leucoagglutinin (PHA-L): electron microscopy of PHA-L filled neuronal somata, dendrites, and axon terminals. Brain Res 326:188–191

    Google Scholar 

  • Wouterlood FG, Bol JGJM, Steinbusch HWM (1987a) Doublelabel immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris leucoagglutinin and enzyme immunocytochemistry of target neurons. J Histochem Cytochem 35:817–823

    Google Scholar 

  • Wouterlood FG, Steinbusch HWM, Luiten PGM, Bol JGJM (1987b) Projection from the prefrontal cortex to histaminergic cell groups in the posterior hypothalamic region of the rat. Anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with immunocytochemistry of histidine decarboxylase. Brain Res 406:330–336

    Google Scholar 

  • Wouterlood FG, Gaykema RPA, Steinbusch HWM, Watanabe T, Wada H (1988) The connections between the septum-diagonal band complex and histaminergic neurons in the posterior hypothalamus of the rat. Anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with immunocytochemistry of histidine decarboxylase. Neuroscience (in press)

  • Zarbin MA, Wamsley JK, Kuhar MJ (1982) Axonal transport of muscarinic cholinergic receptors in rat vagus nerve: high and low affinity agonist receptors move in opposite directions and differ in nucleotide sensitivity. J Neurosci 2:934–941

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Animal Physiology, University of Groningen, P.O. Box 14, 9750 AA, Haren, The Netherlands

    P. G. M. Luiten, T. Matsuyama, B. Buwalda & R. P. A. Gaykema

  2. Department of Anatomy, Vrije Universiteit, Van der Boechorststraat 7, 1007 MC, Amsterdam, The Netherlands

    F. G. Wouterlood

  3. Institut Pasteur, 28 Rue du Docteur Roux, F-75724, Paris Cedex 15, France

    A. D. Strosberg

Authors
  1. P. G. M. Luiten
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. F. G. Wouterlood
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T. Matsuyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. D. Strosberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. B. Buwalda
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. R. P. A. Gaykema
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Luiten, P.G.M., Wouterlood, F.G., Matsuyama, T. et al. Immunocytochemical applications in neuroanatomy. Histochemistry 90, 85–97 (1988). https://doi.org/10.1007/BF00500973

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00500973

Keywords

Search

Navigation