Summary
In order to associate specific fiber projections in the central nervous system with specific target neurons, procedures were developed in which the anterograde neuroanatomical tracing technique utilizing Phaseolus vulgaris-leucoagglutinin (PHA-L) is combined with immunocytochemistry of three (different) neuronal markers: gammaamino butyric acid, choline acetyltransferase, and serotonin. A double, indirect, peroxidase-antiperoxidase staining method is used on free-floating brain sections. The primary antiserum against the PHA-L (first primary antiserum) is mixed with the primary antiserum against the neuronal marker (second primary antiserum). These primary antisera are raised in different animal species. Following the incubation in the cocktail of primary antisera, the sections are incubated in a cocktail of two secondary antisera. The transported PHA-L is then visualized by incubation in a peroxidase-antiperoxidase complex and subsequent reaction with nickel-enhanced diaminobenzidine/H2O2 (blue reaction product in PHA-L-labeled neurons and fibers). Incubation is continued with peroxidase-antiperoxidase antibodies raised in the animal species in which the second primary antiserum is developed, and the staining is completed by treatment with diaminobenzidine/H2O2 (brown reaction product in the target neurons). The present results suggest that PHA-L-tracing can be combined with immunocytochemistry of a variety of target neuron-related antigens.
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References
Amaral DG, Kurz J (1985) An analysis of the origins of the cholinergic and noncholinergic septal projections to the hippocampal formation of the rat. J. Comp Neurol 240:37–59
Berendse HW, Voorn P, Te Kortschot A, Groenewegen HJ (1988) Nuclear origin of thalamic afferents of the ventral striatum determines their relation to patch/matrix configurations in enkephalin immunoreactivity of the rat. J. Chem. Neuroanat 1:3–10
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
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. Brain Res 290:219–238
Gerfen CR, Sawchenko PE (1985) A method of anterograde axonal tracing of chemically specified circuits in the central nervous system: Combined Phaseolus vulgaris-leucoagglutinin (PHA-L) tract tracing and immunohistochemistry. Brain Res 343:144–150
Groenewegen HJ, van Dijk CA (1984) Efferent connections of the dorsal tegmental region in the rat, studied by means of anterograde transport of the lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L). Brain Res 304:367–371
Groenewegen HJ, Ahlenius S, Haber, SN, Kowall NW, Nauta WJH (1986) Cytoarchitecture, fiber connections, and some histochemical aspects of the interpeduncular nucleus in the rat. J Comp Neurol 249:65–102
Grove EA, Domesick VB, Nauta WJH (1986) Light microscopic evidence of striatal input to intrapallidal neurons of cholinergic cell group Ch4 in the rat: a study employing the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Brain Res 367:379–384
Hancock MB (1982) DAB-Nickel substrate for the differential immunoperoxidase staining of nerve fibers and fiber terminals. J. Histochem Cytochem 30:578P
Hancock MB (1987) Projections from the nucleus of the solitary tract (NTS) onto PNMT-immunoreactive cells in the ventral medulla of the rat. Abstr Soc Neurosci 13:278
Hodgson AJ, Penke B, Erdei A, Chubb IW, Somogyi P (1985) Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system. J. Histochem Cytochem 33:229–239
Jonkers BW, Sterk JC, Wouterlood FG (1984) Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device. J. Neurosci Methods 12:141–149
Kita H, Kitai ST (1987) Large PHA-L labeled pallidal terminals form symmetrical synapses on the somata and dendrites of subthalamic and substantia nigral neurons. Abstr Soc Neurosci 13:1567
Mason DY, Woolston R-E (1982) Double immunoenzymatic labelling. In: Bullock GR, Petrusz P (eds) Techniques in immunocytochemistry. Academic Press, London, pp 135–154
McMullen NT, Prescott AM (1987) Light microscopic evidence for thalamocortical input to GABAergic neurons in auditory neocortex: combination of PHA-L and target neuron immunocytochemistry. Abstr Soc Neurosci 13:326
Meredith GE, Wouterlood FG, Groenewegen HJ (1987) Hippocampal innervation of cholinergic neurons in the ventral striatum (nucleus accumbens) of the rat. A double-label study combining PHA-L-tracing and immunocytochemistry of choline acetyltransferase. Abstr Soc Neurosci 13:446
Mugnaini E, Oertel WH (1985) An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunocytochemistry. In: Björklund A, Hökfelt T (eds) Handbook of chemical neuroanatamy, vol 4. Elsevier, Amsterdam, pp 436–608
Nieuwenhuys R (1985) The chemoarchitecture of the brain. Springer, Berlin Heidelberg New York, pp 1–246
Nyakas C, Luiten PGM, Spencer DG, Traber J (1987) Detailed projection pattern of septal and diagonal band efferents to the hippocampus in the rat with emphasis on innervation of CA1 and dentate gyrus. Brain Res Bull 18:533–545
Paxinos G, Watson C (1982) The rat brain in stereotaxic coordinates. Academic Press, New York
Seguela P, Geffard M, Buijs RM, Le Moal M (1984) Antibodies against γ-amino butyric acid. Specificity studies and immunocytochemical results. Proc Natl Acad Sci USA 81:3888–3892
Sloviter RS, Nilaver G (1987) Immunocytochemical localization of GABA-,cholecystokinin-,vasoactive intestinal polypeptide-,and somatostatin-like immunoreactivity in the area dentata and hippocampus of the rat. J Comp Neurol 256:42–60
Somogyi P, Hodgson AJ, Chubb IW, Penke B, Erdei A (1985) Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system. J. Histochem Cytochem 33:240–248
Steinbusch HWM (1980) Distribution of serotonin-immunoreactivity in the central nervous system of the rat. Cell bodies and terminals. Neuroscience 6:557–618
Sternberger LA (1979) Immunocytochemistry. 2nd edn. John Wiley, New York, pp 1–354
Swanson LW, Cowan WM (1979) The connections of the septal region in the rat. J. Comp Neurol 186:621–656
Wouterlood FG, Groenewegen HJ (1985) Neuroanatomical tracing by use of Phaseolus vulgaris-leucoagglutinin (PHA-L): Electron microscopy of PHA-L-filled neuronal somata, dendrites, axons and axon terminals. Brain Res 326:188–191
Wouterlood FG, Mugnaini E, Nederlof J (1985) Projection of olfactory bulb efferents to layer I GABAergic neurons in the entorhinal area. Combination of anterograde degeneration and immunoelectron microscopy in rat. Brain Res 343:283–296
Wouterlood FG, Bol JGJM, Steinbusch HWM (1987a) Doublelabel immunocytochemistry: Combination of anterograde tracing with Phaseolus vulgaris-leucoagglutinin and enzyme immunocytochemistry of the target neurons. J. Histochem Cytochem 35:817–823
Wouterlood FG, Steinbusch HWM, Luiten PGM, Bol JGJM (1987b) Projection of the prefronatal 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
Wouterlood FG, Steinbusch HWM, Watanabe T (1987c) Neurons in the medial septal nucleus innervate cholinergic neurons in the diagonal band, histaminergic neurons in the mammillary body, and serotonergic neurons in the nucleus raphe dorsalis. A double-label study combining PHA-L tracing and enzyme immunocytochemistry. Abstr Soc Neurosci 13:444
Wouterlood FG, Sauren YMHF, Pattiselanno A (1988a) Compromises between penetration of antisera and preservation of ultrastructure in preembedding electron microscopic immunocytochemistry. J. Chem. Neuroanat 1 (in press)
Wouterlood FG, Gaykema RPA, Steinbusch HWM, Watanabe T, Wada H (1988b) 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 24 (in press)
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Wouterlood, F.G. Anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin combined with immunocytochemistry of gamma-amino butyric acid, choline acetyltransferase or serotonin. Histochemistry 89, 421–428 (1988). https://doi.org/10.1007/BF00492597
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DOI: https://doi.org/10.1007/BF00492597