TECHNICAL NOTE |
Correspondence to: Michael Kressel, Inst. of Anatomy, University of Erlangen, Krankenhausstr. 9, D-91054 Erlangen, Germany.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Current protocols for a combined approach of anterograde tracing with carbocyanine dyes or horseradish peroxidase (HRP) conjugates and immunohistochemistry represent a compromise between sensitive detection of the tracer and the immunohistochemical procedure. Therefore, it was investigated whether the use of tyramide amplification allows sensitive anterograde tracing with wheat-germ agglutinin conjugated to horseradish peroxidase (WGAHRP) in conjunction with simultaneous immunohistochemistry. Vagal afferents were anterogradely labeled by injection of WGAHRP into the nodose ganglion of rats. By use of tyramidebiotin amplification, a dense fiber plexus of vagal afferents was visualized centrally in the nucleus of the solitary tract and in retrogradely labeled neurons in the dorsal vagal nucleus. In the esophagus and duodenum, large- and small-caliber vagal fibers and terminals could be demonstrated comparably to conventional tracing techniques using carbocyanine dyes or WGAHRP and TMB histochemistry. Combination with immunohistochemistry could easily be done, requiring only one more incubation step, and did not result in loss of sensitivity of the tracing. With this method and confocal microscopy, the presence of Ca binding proteins in vagal afferent terminals could be demonstrated. Tyramide amplification allows sensitive anterograde tracing with low background staining in conjunction with immunohistochemistry of intra-axonal markers. (J Histochem Cytochem 46:527533, 1998)
Key Words: tyramides, carbocyanine dyes, anterograde tracing, calretinin, calbindin, wheat-germ agglutinin, confocal microscopy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The development of powerful anterograde and retrograde tract tracing methods has led to our current understanding of the complex organization of the central and peripheral nervous systems. By tracing techniques, the route taken, the area, and the terminal structures of a projection originating from a defined population of neurons either within the central nervous system or in the skin or visceral organs can be determined precisely. Elucidation of the underlying neural connections of the nervous system per se provides only restricted insight into its mechanisms. One alternative to gain insight into the nature of neural networks is a combination of tracing methods with immunohistochemistry (
The projections originating from ganglia of the peripheral nervous system distribute over wide distances in the body and are heterogeneous with respect to their postsynaptic targets, neurotransmitter content, and functional role. In this part of the nervous system, elucidation of the chemical composition of fibers with a known origin and projection to a specific organ or tissue component is possible only by a combination of anterograde tracing and immunohistochemistry. The tracers, which have been used successfully in the peripheral nervous system, are the carbocyanine dye DiI (1,1'-dioleyl-3,3,3',3'tetramethylindocarbocyanine) or DiA (4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide) and horseradish peroxidase coupled to wheat-germ agglutinin and cholera toxin subunit B (
It is shown here that anterograde tracing with WGAHRP can reproducibly and easily be combined with immunohistochemistry of traced axons by use of peroxidase-mediated deposition of tyramidebiotin as the detection system for the tracer. Moreover, because the endproduct of this reaction can be visualized with fluorescent dyes, this method is compatible with multicolor detection of more antigens within the same section and subsequent use of confocal laser scanning microscopy for image analysis and three-dimensional reconstruction.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anterograde Tracing and Tissue Preparation
Adult male Wistar rats were used. For all procedures performed on animals, the federal animal welfare legislation rules were followed. The rats were anesthetized with Hypnorm (Janssen; Neuss, Germany) (0.4 ml/kg) and Dormicum (Roche; GrenzachWyhlen, Germany) (0.2 mg/kg). When the animals were fully unresponsive, the left or both vagal nerves were dissected free by a midline incision in the neck and traced cranially to the nodose ganglion. The capsule of the nodose ganglion was slit with the tip of a 26-gauge hypodermic needle. Four µl of WGAHRP or WGA conjugated to biotin (2% in PBS) (SigmaAldrich Chemie; Deisenhofen, Germany) was pressure-injected into the ganglion by a glass micropipette with a tip diameter of 4060 µm. Six animals received a WGAHRP injection into the left (n = 4) or both (n = 2) nodose ganglia. Two rats were injected with WGA conjugated to biotin into the left nodose ganglion. After the injections the animals were allowed to survive for 24 hr. Then they were perfused under deep thiopental anesthesia (250 mg/kg) (Nycomed; München, Germany) through the ascending aorta, initially with 300 ml of saline containing 10 IE heparin/ml followed by 500 ml of 3% paraformaldehyde in 0.1 M PO4 buffer, pH 7.4. The brainstem, esophagus, and duodenum were removed and stored overnight at 4C in 0.1 M PO4 buffer containing 15% sucrose. The tissue was then mounted on Tissue-Tek, rapidly frozen in isopentane at -75C, and stored at -20C until cryosectioning. Ten- or 15-µm-thick transverse sections from the brainstem or longitudinal sections from the esophagus and duodenum were cut in a cryostat and mounted on poly-L-lysine-coated glass slides.
In a control experiment, the vagal nerve was cut below the nodose ganglion and WGAHRP was injected into the cervical vagal nerve.
Antisera and Reagents
Rabbit antisera raised against calbindin and calretinin were obtained from SWant (Bellinzona, Switzerland) and have been described in previous work (-calcitonin gene-related peptide (CGRP) was used from Affiniti (Biotrend; Köln, Germany). Tyramide amplification reagents, including streptavidin-coupled horseradish peroxidase (streptavidinHRP), were purchased from DuPont NEN (Brussels, Belgium).
Tyramide Amplification and Immunohistochemistry
Slides were allowed to dry for 60 min. After rehydration in 0.1 M Tris-HCl, 0.15 M NaCl, 0.05% Tween 20, pH 7.5 (TNT), the slides were quenched for 15 min in freshly prepared NaBH4 (0.5 mg/ml) (Fluka; Buchs, Switzerland) in 0.1 M Tris-HCl, 0.15 M NaCl, pH 7.5 (TN). After washing in TNT, they were permeabilized for 15 min in 0.1% Triton X-100 in TN buffer. After 60-min incubation in TNT plus 2% bovine serum albumin (TNTBSA), the slides were washed in TNT and then incubated for 15 min in either tyramide conjugated to fluorescein (tyramideFITC) (direct method) or biotinylated tyramide (indirect method) diluted 1:50 in amplification diluent (DuPont). After a washing step in TNT the primary antibodies were applied at a dilution of 1:1000 for anti-calbindin/calretinin and 1:250 for anti-CGRP in TNTBSA overnight at 4C. The primary antibodies were detected by incubation with donkey anti-rabbit IgG conjugated to lissamine rhodamine (LRSC) or indocarbocyanine (Cy3) diluted at 1:200 and donkey anti-sheep indodicarbocyanine (Cy5) at 1:200 (Jackson Laboratories; Dianova, Hamburg, Germany). Incorporated biotintyramide was visualized by adding streptavidinFITC 1:200 (Molecular Probes; Eugene, OR) to the last incubation solution. After a final washing step the slides were mounted in Vectashield (Vector Laboratories; Camon, Wiesbaden, Germany). For detection of anterogradely transported WGAbiotin tracer, the slides were incubated for 4 hr in streptavidinHRP diluted 1:500 in TNTBSA before the tyramide amplification step. Further processing of the bound streptavidinHRP was done by tyramidebiotin amplification and detection by streptavidinFITC as described for the indirect method.
Confocal Microscopy
Slides were examined with a Bio-Rad MRC-1000 confocal laser scanning microscope. Serial optical sections were taken typically at 1-µm depth increments using the x 60 objective of a Nikon 300 inverted microscope. For detection of FITC, LRSC, Cy3, and Cy5, the 488-, 568- and 630-nm laser lines of the krypton/argon laser were used. It was confirmed that there was no fluorescence bleed-through between the red and green fluorescence channels. In dual and triple label studies, the fluorescence of each channel was recorded separately and an extended depth-of-focus image of all the fluorescence within the slide was taken. Subsequently the images from the two or three channels were digitally superimposed in pseudocolors to obtain all information in one picture.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Central Projections of Nodose Ganglion Cells
By tyramide amplification alone or in combination with immunohistochemistry, HRP activity could be demonstrated in the central projections of the nodose ganglion cells. Tyramide amplification for detection of WGAHRP was done by two protocols. A direct approach used tyramideFITC as substrate for the peroxidase reaction, which produced a fluorescent reaction product seen in the FITC filter. The indirect approach consisted of amplification of tyramidebiotin, which was subsequently detected by incubation with streptavidinFITC. By both procedures a dense plexus of anterogradely labeled fibers and terminals was revealed in the nucleus of the solitary tract of the ipsilateral brainstem (Figure 1). Single fibers were also seen on the contralateral side and in the area postrema, as described in the literature (
|
Tyramide amplification of brainstem sections was combined with immunohistochemistry (Figure 2). Slides were always incubated with the primary and secondary antibodies after the tyramide reaction because the enzymatic activity of HRP was rapidly lost after cryosectioning. Overnight storage of slides at 4C in buffer, as done for incubations with the primary antibodies, completely abolished all HRP activity. HRP activity was also lost by addition of normal serum containing NaN3 as a preservative to the incubation solution before the amplification step. However, no loss in intensity of the tyramide reaction product was observed when the immunohistochemical procedure was performed after the tyramide incubation. With the combined approach it became evident that the central projections of the nodose ganglion cells were negative for calretinin immunoreactivity (Figure 2).
Peripheral Projections of the Nodose Afferents
HRP activity developed by tyramide amplification also stained fibers and terminal endings of vagal afferents in the esophagus and duodenum brilliantly fluorescent. Axons in the vagal trunk or in single nerve fibers were outlined by small fluorescent granules. The two types of vagal afferent endings in the wall of the rat gastrointestinal tract, which have been described using anterograde tracing by DiI or WGAHRP and TMB histochemistry, could be visualized by tyramide amplification. The small-caliber intramuscular fibers were observed running parallel to the bundles of the smooth musculature and ramifying in different focal planes, as described in the pylorus and stomach (
For combined tracing and immunohistochemistry antisera raised against calretinin and calbindin were used. Calretinin and calbindin are Ca-binding proteins frequently used for neuronal identification which, because they occur throughout the entire cytoplasm, delineate neurons and axons in a Golgi-like staining manner (
In the control animal, no fluorescence at all could be detected after tyramidebiotin amplification, apart from unspecific staining of single erythrocytes or macrophages.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many procedures have been developed to circumvent the problems associated with the combination of DiI tracers or HRP conjugates and immunohistochemistry. One way is a sequential two-step procedure on single sections. During the first step the tracer is developed, and thereafter its localization is fully documented. In a second incubation step, the slides are processed for immunohistochemistry with sacrifice of the tracer (
Previous studies using the highly sensitive tracers DiI or WGAHRP visualized by TMB histochemistry described two types of vagal afferent endings in the musculature of the gastrointestinal tract: intramuscular afferents and IGLEs (
Combination with immunohistochemistry could be readily accomplished, requiring only one more incubation step. By anterograde tracing it could be shown that vagal afferents originating from neurons in the nodose ganglion terminated in countless leaf-like structures on myenteric ganglia, which were immunoreactive for the Ca binding proteins calretinin and calbindin. Calbindin-positive nerve endings in the rat esophagus have already been described by
![]() |
Acknowledgments |
---|
Supported by the Deutsche Forschungsgemeinschaft (KR 1665/2-1).
I thank Ms Anita Hecht for diligently preparing the cryocuts.
Received for publication May 9, 1997; accepted October 21, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bentivoglio M, Chen S (1993) Retrograde neuronal tracing combined with immunocytochemistry. In Cuello AC, ed. Immunohistochemistry II. Chichester, Wiley, 301-328
Berthoud HR (1995) Anatomical demonstration of vagal input to nicotinamide acetamide dinucleotide phosphate diaphorase-positive (nitrergic) neurons in the rat fundic stomach. J Comp Neurol 358:428-439[Medline]
Berthoud HR (1996) Morphological analysis of vagal input to gastrin releasing peptide and vasoactive intestinal peptide containing neurons in the rat glandular stomach. J Comp Neurol 370:61-70[Medline]
Berthoud HR, Kressel M, Neuhuber WL (1992a) An anterograde tracing study of the vagal innervation of rat liver, portal vein and biliary system. Anat Embryol 186:431-442[Medline]
Berthoud HR, Kressel M, Neuhuber WL (1995) Vagal afferent innervation of rat abdominal paraganglia as revealed by anterograde DiI-tracing and confocal microscopy. Acta Anat 152:127-132[Medline]
Berthoud HR, Neuhuber WL (1994) Distribution and morphology of vagal afferents and efferents supplying the digestive system. In Taché Y, Wingate DL, Burks TF, eds. Innervation of the Gut. Boca Raton, FL, CRC Press, 43-66
Berthoud HR, Patterson LM, Neumann F, Neuhuber WL (1997) Distribution and structure of vagal afferent intraganglionic laminar endings (IGLEs) in the rat gastrointestinal tract. Anat Embryol 195:183-191[Medline]
Berthoud HR, Powley TL (1992b) Vagal afferent innervation of the rat fundic stomach: morphological characterization of the gastric tension receptor. J Comp Neurol 319:261-276[Medline]
Clerc N, Condamin M (1987) Selective labeling of vagal sensory nerve fibers in the lower esophageal sphincter with anterogradely transported WGA-HRP. Brain Res 424:216-224[Medline]
Clerc N, Mazzia C (1994) Morphological relationships of choleragenoid horseradish peroxidase-labeled spinal primary afferents with myenteric ganglia and mucosal associated lymphoid tissue in the cat esophagogastric junction. J Comp Neurol 347:171-186[Medline]
Elberger AJ, Honig MG (1990) Double labeling of tissue containing the carbocyanine dye DiI for immunocytochemistry. J Histochem Cytochem 38:735-739[Abstract]
Elfvin LG, Aldskogius H, Johansson J (1992) Splenic primary sensory afferents in the guinea pig demonstrated with anterogradely transported wheat-germ agglutinin conjugated to horseradish peroxidase. Cell Tissue Res 269:229-234[Medline]
Elfvin LG, Aldskogius H, Johansson J (1993) Primary sensory afferents in the thymus of the guinea pig demonstrated with anterogradely transported horseradish peroxidase conjugates. Neurosci Lett 150:35-38[Medline]
Freund TF (1993) Anterograde PHA-L tracing combined with pre- and post-embedding immunocytochemistry. In Cuello AC, ed. Immunohistochemistry II. Chichester, Wiley, 329-348
Fundin BT, Rice FL, Pfaller K, Arvidsson J (1994) The innervation of the mystacial pad in adult rat studied by anterograde transport of HRP conjugates. Exp Brain Res 99:233-246[Medline]
Groenewegen HJ, Wouterlood FG (1990) Light and electron microscopic tracing of neuronal connections with Phaseolus vulgaris leucoagglutinin (PHA-L), and combinations with other neuroanatomical techniques. In Björklund A, Hökfelt T, Wouterlood FG, van den Pol AN, eds. Handbook of Chemical Neuroanatomy. Vol 8. Analysis of Neuronal Microcircuits and Synaptic Interactions. New York, Elsevier Science, 47-124
Härtig W, Brückner G, Brauer K, Seeger G, Bigl V (1996) Triple immunofluorescence labelling of parvalbumin, calbindin-D28k and calretinin in rat and monkey brain. J Neurosci Methods 67:89-95[Medline]
Kressel M, Berthoud HR, Neuhuber WL (1994) Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy. Cell Tissue Res 275:109-123[Medline]
Kuramoto H, Kuwano R (1995) Location of sensory nerve cells that provide calbindin-containing laminar nerve endings in myenteric ganglia of the rat esophagus. J Autonom Nerv Syst 54:126-136[Medline]
Lechan RM, Nestler JL, Jacobson S (1981) Immunohistochemical localization of retrogradely and anterogradely transported wheat germ agglutinin within the central nervous system of the rat. J Histochem Cytochem 29:1255-1262[Abstract]
Leslie RA, Gwyn DG, Hopkins DA (1982) The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat. Brain Res Bull 8:37-43[Medline]
Lindh B, Aldskogius H, Hökfelt T (1989) Simultaneous immunohistochemical demonstration of intra-axonally transported markers and neuropeptides in the peripheral nervous system of guinea pig. Histochemistry 92:367-376[Medline]
Mesulam MM (1978) Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents. J Histochem Cytochem 26:106-117[Abstract]
Miller RJ (1995) Regulation of calcium homeostasis in neurons: the role of calcium-binding proteins. Biochem Soc Trans 23:629-636[Medline]
Neuhuber WL (1987) Sensory vagal innervation of the rat esophagus and cardia: a light and electron microscopic anterograde tracing study. J Autonom Nerv Syst 20:243-255[Medline]
Neuhuber WL, Clerc N (1990) Afferent innervation of the esophagus in cat and rat. In Zenker W, Neuhuber WL, eds. The Primary Afferent Neuron. New York, Plenum Press, 93-107
Rogers JH (1992) Immunohistochemical markers in rat cortex: co-localization of calretinin and calbindin-D28k with neuropeptides and GABA. Brain Res 587:147-157[Medline]
Rodrigo J, Hernandez CJ, Vidal MA, Pedrosa JA (1975) Vegetative innervation of the esophagus. II. Intraganglionic laminar endings. Acta Anat 92:79-100[Medline]
Rosene DL, Mesulam MM (1978) Fixation variables in horseradish peroxidase neurohistochemistry. I. The effects of fixation time and perfusion procedures upon enzyme activity. J Histochem Cytochem 26:28-39[Abstract]
Schwaller B, Buchwald P, Blümcke I, Celio MR, Hunziker W (1993) Characterization of a polyclonal antiserum against the purified human recombinant calcium binding protein calretinin. Cell Calcium 14:639-648[Medline]
Smith Y, Bolam JP (1992) Combined approaches to experimental neuroanatomy: combined tracing and immunocytochemical techniques for the study of neuronal microcircuits. In Bolam JP, ed. Experimental Neuroanatomy. New York, Oxford University Press, 239-266
Tanaka Y, Yoshida Y, Hirano M (1993) CGRP-immunoreactive cells supplying laryngeal sensory nerve fibers in the cat's nodose ganglion. J Laryngol Otol 107:916-919[Medline]