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Simultaneous ultrastructural demonstration of retrogradely transported horseradish peroxidase and choline acetyltransferase immunoreactivity

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Summary

In order to study the synaptic connections of neurons identified by their projection target and neurotransmitter content, we have adapted a method of combining retrograde tracing of horseradish peroxidase (HRP) and immunocytochemistry at the electron microscopic level. HRP was injected into the rat amygdala. Sections from the rostral forebrain were processed according to the 3,3′-diaminobenzidine/glucose oxidase reaction followed by choline acetyltransferase (ChAT) localization. Neurons in the ventral pallidum which contained both the diffuse immunoperoxidase reaction product (ChAT) and large electron dense bodies characteristic of retrogradely transported HRP were defined as double labeled, i.e. cholinergic neurons that project to the amygdaloid body.

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References

  • Albanese A, Bentivoglio M (1982) Retrograde fluorescent neuronal tracing combined with acetylcholinesterase histochemistry. J Neurosci Methods 6:121–127

    Google Scholar 

  • Armstrong DM, Saper CB, Levey AI, Wainer BH, Terry RD (1983) Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase. J Comp Neurol 216:53–68

    Google Scholar 

  • Barber RP, Phelps PE, Houser CR, Crawford GD, Salvatera PM, Vaughn JE (1984) The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: An immunocytochemical study. J Comp Neurol 229:329–346

    Google Scholar 

  • Berod A, Hartman BK, Pujol JF (1981) Importance of fixation in immunohistochemistry: use of flormaldehyde at variable pH for the localization of tyrosine hydroxylase. J Histochem Cytochem 29:844–850

    Google Scholar 

  • Bolam JP, Wainer BH, Smith AD (1984) Characterization of cholinergic neurons in the rat neostriatum. A combination of choline acetyltransferase immunocytochemistry, Golgi-impregnation and electron microscopy. Neuroscience 12:711–718

    Google Scholar 

  • Bowker RM, Westlund KN, Sullivan MC, Coulter JD (1982) A combined retrograde transport and immunocytochemical staining method for demonstrating the origins of serotonergic projections. J Histochemn Cytochem 30:805–810

    Google Scholar 

  • Butcher LL (1983) Acetylcholinesterase histochemistry. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy, vol 1: Methods in chemical neuroanatomy. Elsevier, Amsterdam, pp 1–49

    Google Scholar 

  • Carlsen J, Záborszky L, Heimer L (1984) Cholinergic innervation of rat amygdala as determined by combined retrograde fluorescent tracing and neurotransmitter histochemistry. Soc Neurosci (Abst) 10:8

    Google Scholar 

  • Carson KA, Mesulam MM (1982) Electron microscopic demonstration of neural connections using horseradish peroxidase: a comparison of the tetramethylbenzidine procedure with seven other histochemical methods. J Histochem Cytochem 30:425–435

    Google Scholar 

  • Eckenstein F, Thoenen H (1982) Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons. EMBO J 1:363–368

    Google Scholar 

  • Hardy H, Heimer L, Switzer R, Watkins D (1976) Simultaneous demonstration of horseradish peroxidase and acetylcholinesterase. Neurosci Lett 3:1–5

    Google Scholar 

  • Hökfelt T, Skagerberg G, Skirboll L, Björklund (1983) Combination of retrograde tracing and neurotransmitter histochemistry. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatomy, vol 1: Methods in chemical neuroanatomy, Elsevier, Amsterdam, pp 228–285

    Google Scholar 

  • Hsu S, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 29:577–580

    Google Scholar 

  • Itoh Z, Akiva K, Namura S, Miguno N, Nakamura Y, Sugimoto T (1979) Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method. Brain Res 175:341–346

    Google Scholar 

  • Keefer DA (1978) Horseradish peroxidase as a retrogradely transported, detailed dendritic marker. Brain Res 140:15–32

    Google Scholar 

  • Kristensson K, Olsson Y (1976) Retrograde transport of horseradish peroxidase in transected axons. 3. Entry into injured axons and subsequent localization in perikaryon. Brain Res 115:201–213

    Google Scholar 

  • La Vail JH, La Vail MM (1974) The retrograde intraaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic study. J Comp Neurol 157:303–358

    Google Scholar 

  • Lechan DM, Nestler J, Jacobson SJ (1981) Immunohistochemical localization of retrogradely and anterogradely transported wheat germ agglutinin (WGA) within the central nervous system of the rat: application to immunostaining of a second antigen within the same neuron. J Histochem Cytochem 29:1255–1262

    Google Scholar 

  • Léránth C, Fehér E (1983) Synaptology and sources of vasoactive intestinal polypeptide and substance P containing axons of the celiac ganglion. An experimental electron microscopic immunohistochemical study. Neuroscience 10:947–958

    Google Scholar 

  • Léránth C, Sakamoto H, MacLusky N, Shanabrough M, Naftolin F (1985) ACTH-containing neurons of the rat hypothalamus establish synaptic connections with LH-RH neurons of the medial preoptic area. (In preparation)

  • Levey AI, Wainer BH, Mufson EJ, Mesulam MM (1983) Co-localization of acetylcholinesterase and choline acetyltransferase in the rat cerebrum. Neuroscience 9:9–22

    Google Scholar 

  • Lewis PR, Henderson Z (1980) Tracing putative cholinergic pathways by a dual cytochemical technique. Brain Res 196:489–493

    Google Scholar 

  • Mesulam M-M (1982) Principles of horseradish peroxidase neurohistochemistry and their applications for tracing neural pathways-axonal transport, enzyme histochemistry and light microscopic analysis. In: Mesulam M-M (ed) Tracing neural connections with horseradish peroxidase. John Wiley and Sons, Chichester, pp 1–152

    Google Scholar 

  • Mesulam MM, Mufson EJ, Wainer BH, Levey AI (1983) Central cholinergic pathways in rat: an overview based on an alternate nomenclature (Chl-Ch6). Neuroscience 10:1185–1201

    Google Scholar 

  • Morell JI, Grenberger LM, Pfaff DW (1981) Comparison of horseradish peroxidase visualization methods: quantitative results and further technical specifics. J Histochem Cytochem 29:903–916

    Google Scholar 

  • Oldfield BJ, Hou-Yu A, Silverman AJ (1983) Technique for the simultaneous ultrastructural demonstration of anterogradely transported horseradish peroxidase and an immunocytochemically identified neuropeptide. J Histochem Cytochem 31:1145–1150

    Google Scholar 

  • Paxinos G, Watson C (1982) The rat brain in stereotaxic coordinates. Academic Press, New York

    Google Scholar 

  • Priestly JV, Somogyi P, Cuello AC (1981) Neurotransmitter-specific projection neurons revealed by combining PAP immunohistochemistry with retrograde transport of HRP. Brain Res 220: 231–240

    Google Scholar 

  • Reaves TA, Jr, Cumming R, Libber MT, Hayward JN (1982) A technique combining intracellular dye-marking, immunocytochemical identification and ultrastructural analysis of physiologically identified single neurons. Neurosci Lett 29:195–199

    Google Scholar 

  • Rosene DL, Mesulam MM (1978) Fixation variables in horseradish peroxidase neurochemistry. I. The effects of fixation time and perfusion procedures upon enzyme activity. J Histochem Cytochem 26:28–39

    Google Scholar 

  • Ruda MA, Coffield J, Dubner R (1984) Demonstration of postsynaptic opioid modulation of thalamic projection by the combined techniques of retrograde horseradish peroxidase and enkephalin immunocytochemistry. J Neurosci 4:2117–2132

    Google Scholar 

  • Sawchenko P, Swanson LW (1981) A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques. Brain Res 210:31–51

    Google Scholar 

  • Silver MA, Jacobowitz DM (1975) Specific uptake and retrograde flow of antibody to dopamine-β-hydroxlase by central nervous system noradrenergic neurons in vivo. Brain Res 167:65–75

    Google Scholar 

  • Sofroniew MV, Schrell U (1981) Evidence for a direct projection from oxytocin and vasopressin neurons in the hypothalamic paraventricular nucleus to the medulla oblongata: immunohistochemical visualization of both the horseradish peroxidase transported and the peptide produced by the same neurons. Neurosci Lett 22:211–217

    Google Scholar 

  • Sofroniew MV, Eckenstein F, Thoenen H, Cuello AC (1982) Topography of choline acetyltransferase-containing neurons in the forebrain of the rat. Neurosci Lett 33:7–12

    Google Scholar 

  • Somogyi P, Takagi H (1982) A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry. Neuroscience 7:1779–1784

    Google Scholar 

  • Streit P (1980) Selective retrograde labeling indicating the transmitter of neuronal pathways. J Comp Neurol 191:429–463

    Google Scholar 

  • Streit P, Reubi JC (1977) A new and sensitive staining method for axonally transported horseradish peroxidase (HRP) in the pigeon visual system. Brain Res 126:530–537

    Google Scholar 

  • van der Kooy D, Sawchenko PE (1982) Characterization of serotoninergic neurons using concurrent fluorescent retrograde axonal tracing and immunohistochemistry. J Histochem Cytochem 30:794–798

    Google Scholar 

  • Wahle P, Sanides-Buchholtz C, Eckenstein F, Albus K (1984) Concurrent visualization of choline acetyltransferase-like immunoreactivity and retrograde transport of neocortically injected markers in basal forebrain neurons of cat and rat. Neurosci Lett 44:223–228

    Google Scholar 

  • Wainer BH, Rye DB (1984) Retrograde horseradish tracing combined with localization of choline acetyltransferase immunoreactivity. J Histochem Cytochem 32:439–443

    Google Scholar 

  • Winfield DA, Gatter KC, Powell TPS (1975) An electron microscopic study of retrograde and orthograde transport of horseradish peroxidase to the lateral geniculate nucleus of the monkey. Brain Res 92:462–467

    Google Scholar 

  • Záborszky L, Eckenstein F, Léránth C, Oertel W, Schmechel D, Alones V, Heimer L (1984a) Cholinergic cells of the ventral pallidum: a combined electron microscopic immunocytochemical, degeneration and HRP study. Soc. Neurosci (Abst) 10:8

    Google Scholar 

  • Záborszky L, Léránth C, Heimer L (1984b) Ultrastructural evidence of amygdalofugal axons terminating on cholinergic cells of the rostral forebrain. Neurosci Lett 52:219–225

    Google Scholar 

  • Záborszky L, Carlsen J, Brashear HR, Heimer L (1985a) Cholinergic and GABA-ergic projections to the olfactory bulb in the rat. (Submitted)

  • Záborszky L, Zahm SD, Oertel WH, Heimer L (1985b) Afferents to the GAD-containing cells in the ventral pallidum. Anat Rec 211:221A

    Google Scholar 

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  1. L. Záborszky & C. Léránth

    Present address: Semmelweis University, Hungary

Authors and Affiliations

  1. Department of Neurology, University of Virginia Medical Center, Box 394, 22908, Charlottesville, Virginia, USA

    L. Záborszky

  2. Department of Obstetrics and Gynecology and Section of Neuroanatomy, Yale University School of Medicine, New Haven, USA

    C. Léránth

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  1. L. Záborszky
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  2. C. Léránth
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Záborszky, L., Léránth, C. Simultaneous ultrastructural demonstration of retrogradely transported horseradish peroxidase and choline acetyltransferase immunoreactivity. Histochemistry 82, 529–537 (1985). https://doi.org/10.1007/BF00489974

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  • DOI: https://doi.org/10.1007/BF00489974

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