Tyramide Amplification Allows Anterograde Tracing by Horseradish Peroxidase-conjugated Lectins in Conjunction with Simultaneous Immunohistochemistry

Abstract

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 (WGA–HRP) in conjunction with simultaneous immunohistochemistry. Vagal afferents were anterogradely labeled by injection of WGA–HRP into the nodose ganglion of rats. By use of tyramide–biotin 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 WGA–HRP 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 con-focal 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.

Keywords

tyramides carbocyanine dyes anterograde tracing calretinin calbindin wheat-germ agglutinin confocal microscopy

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 (Smith and Bolam 1992; Bentivoglio and Chen 1993; Freund 1993). Antibodies against many parts of the neuronal machinery can be used for chemical identification and functional characterization of neural projections or their postsynaptic targets, and the list of specific antibodies is increasing rapidly.

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)-iV-methylpyridinium iodide) and horseradish peroxidase coupled to wheat-germ agglutinin and cholera toxin subunit B (Elfvin et al. 1992,1993; Clerc and Mazzia 1994; Fundin et al. 1994; Berthoud et al. 1992a,1995). However, current protocols for a combined approach of anterograde tracing and immunohistochemistry represent a compromise between sensitive detection of the tracer and the immunohistochemical procedure. For optimal sensitivity, anterograde tracing with horseradish peroxidase requires inclusion of glutaraldehyde in the fixation solution and tetramethylbenzidine (TMB) as the histochemical detection system, which limit simultaneous immunohistochemical applications (Mesulam 1978; Rosene and Mesulam 1978; Lechan et al. 1981; Groenewegen and Wouterlood 1990). DiI, on the other hand, is extremely sensitive to detergents, which are necessary for good tissue penetration of the primary antibodies (Elberger and Honig 1990; Kressel et al. 1994).

It is shown here that anterograde tracing with WGA–HRP can reproducibly and easily be combined with immunohistochemistry of traced axons by use of peroxidase-mediated deposition of tyramide–biotin 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 Hyp-norm (Janssen; Neuss, Germany) (0.4 ml/kg) and Dormicum (Roche; Grenzach–Wyhlen, 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 WGA–HRP or WGA conjugated to biotin (2% in PBS) (Sigma–Aldrich Chemie; Deisenhofen, Germany) was pressure-injected into the ganglion by a glass micropipette with a tip diameter of 40–60 μm. Six animals received a WGA–HRP 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 WGA–HRP 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 (Rogers 1992; Schwaller et al. 1993). Sheep polyclonal antibody to rat α-calcitonin generelated peptide (CGRP) was used from Affiniti (Biotrend; Köln, Germany). Tyramide amplification reagents, including streptavidin-coupled horseradish peroxidase (streptavidin-HRP), 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 (TNT–BSA), the slides were washed in TNT and then incubated for 15 min in either tyramide conjugated to fluorescein (tyramide–FITC) (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 TNT–BSA 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 biotin–tyramide was visualized by adding streptavidin–FITC 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 WGA–biotin tracer, the slides were incubated for 4 hr in streptavidin–HRP diluted 1:500 in TNT–BSA before the tyramide amplification step. Further processing of the bound streptavidin-HRP was done by tyramide–biotin amplification and detection by streptavidin-FITC 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 WGA–HRP was done by two protocols. A direct approach used tyramide–FITC as substrate for the peroxidase reaction, which produced a fluorescent reaction product seen in the FITC filter. The indirect approach consisted of amplification of tyramide–bio-tin, which was subsequently detected by incubation with streptavidin–FITC. 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 (Leslie et al. 1982). At higher magnification, single nerve fibers could easily be discriminated. Intensely fluorescent granules were also observed in the cytoplasm of retrogradely labeled neurons in the dorsal motor nucleus and nucleus ambiguus of the vagus. Comparison of both methods, however, showed significantly increased fluorescence intensity by the indirect approach. The amount and extent of staining in the nucleus of the solitary tract were significantly diminished by the direct compared to the indirect method.

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 WGA–HRP 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 (Berthoud and Powley 1992b; Kressel et al. 1994) (Figure 5). The highly arborizing terminal endings at the myenteric ganglia of the esophagus, which have been called intraganglionic laminar endings (IGLEs) (Rodrigo et al. 1975; Berthoud et al. 1997), were marked by intense fluorescence (Figure 6). In addition, fibers were also observed in the submucous and mucous layer in the cervical and lower part of the esophagus, as described in previous studies (Clerc and Condamin 1987; Neuhuber 1987) (Figure 3). Unspecific staining was occasionally found in erythrocytes and macrophages. As found for the brainstem, use of the indirect method increased the fluorescence intensity of the reaction product, whereas the background fluorescence of the surrounding tissues was only slightly increased. Although the prominent staining of IGLEs in the esophagus was not diminished in number of labeled terminals using tyramide–FITC amplification (Figure 8), it was found that the fine-caliber intramuscular afferents in the duodenum (Figure 4) could be observed only after tyramide–biotin amplification. To test whether the sensitivity of anterograde tracing and tyramide amplification could be improved, WGA–biotin was used as tracer. The biotin moiety was detected by incubation with streptavidin–HRP. Further processing was done according to the indirect method by tyra-mide–biotin amplification and streptavidin–FITC as a last step. By this method, anterogradely traced fibers could be identified in the brainstem and the myenteric ganglia of the esophagus (Figure 7). However, there was enhanced background fluorescence, but no increase in the fluorescence intensity of vagal terminals was observed. Small-caliber intramuscular fibers were also absent, as found for tyramide–FITC amplification.

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 (Härtig et al. 1996). By confocal microscopy, tyramide staining and immunohistochemical labeling were recorded separately using the appropriate filter sets. Then the images of each channel were digitally superimposed in pseudocolors both on the level of single z-sections and of extended depth-of-focus images of all the fluorescence within the slide. By comparing tracing and immunohistochemistry for calretinin and calbindin, it became evident that the tracer was contained in calretinin- and calbindin-immunoreactive large-caliber nerve fibers, which terminated as IGLEs at the myenteric ganglia of the esophagus (Figures 6–8). Whereas in nerve fibers WGA–HRP was found as small granules, terminals were often completely filled by the tyramide reaction product. In addition, triple labeling was done with an antiserum raised against CGRP. The CGRP-positive fibers took an independent course in the ganglion and contained neither tracer nor calretinin or calbindin (Figure 8).

In the control animal, no fluorescence at all could be detected after tyramide–biotin amplification, apart from unspecific staining of single erythrocytes or macrophages.

Figure 1

Figures 1–8 Anterogradely traced nerve fibers visualized by tyramide–biotin amplification (Figures 1–7) and tyramide–FITC (Figure 8). Bars = 25 μm.

Figure 2

Higher magnification of fibers and terminals in the nucleus of the solitary tract. The projections of ganglion nodose vagal afferents are shown in red. Anterograde tracing was combined with immunohistochemistry against calretinin (green). Immunoreactivity for calretinin is not co-concentrated in terminals originating from the ganglion nodosum.

Figure 3

Vagal afferent fiber terminating in the esophageal epithelium. Three images have been digitally superimposed in pseudocolors: WGA-HRP tracing developed by tyramide–biotin amplification in red, anti-calretinin immunohistochemistry in green, and anti-CGRP immunohistochemistry in blue. Superposition of all three colors renders an axon (arrow) running at the boundary between the epithelium and the lamina propria almost totally bright white. Tiny white granules can also be observed in the net-like fiber penetrating deeply and reaching nearly the surface of the epithelium. This image demonstrates a population of vagal epithelial afferent fibers containing both calretinin and CGRP. The localization is in the dorsal wall of the esophagus opposite the cricoid cartilage. The thickness of the epithelium is marked by two arrowheads.

Figure 4

Single anterogradely traced nerve fiber in the duodenal wall running between the inner circular (cm) and outer longitudinal (lm) muscle layers

Figure 5

Nerve fibers running between the bundles of smooth muscles (sm) in the region of the lower esophageal sphincter at the gastroesophageal junction. Vagal afferent fibers (red) can be detected in the innermost layer directly opposite the mucosa (m). Calretininimmunoreactive nerve fibers are shown in green. Vagal afferents in the smooth muscle portion of the rat esophagus are not immunoreactive for calretinin.

Figure 6

Myenteric ganglion within the wall of the esophagus. Calretinin-immunoreactive large-caliber nerve fibers (green) can be observed entering the ganglion and arborizing in profuse terminal endings on the surface of the ganglion. Digital superposition of tyramide-biotin reaction product in red results in a yellow color in regions at which both labels are co-concentrated. Vagal afferent terminals marked by anterogradely transported WGA–HRP contain calretinin.

Figure 7

Anterograde tracing by WGA–biotin processed by tyramide–biotin amplification in combination with immunohistochemistry for calretinin. Superposition of HRP activity in red and calretinin immunoreactivity in green is shown. Tiny red and yellow granules are localized within a calretinin-immunoreactive nerve fiber and its terminal endings on an esophageal myenteric ganglion. The large-caliber calretinin-positive nerve fiber at the lower border of the picture can be observed entering the ganglion and arborizing in profuse intraganglionic laminar endings.

Figure 8

Detection of anterogradely transported WGA–HRP by tyramide–FITC amplification (red) in conjunction with immunohistochemistry for calbindin (green) and CGRP (blue). HRP reaction product can be detected in calbindin-positive fibers and countless endings on a myenteric ganglion of the esophagus. The CGRP-positive fiber (arrow) runs independently of the vagal afferents and comes into close contact with a calbindin-positive neuron in the center of the ganglion.

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 (Berthoud 1995,1996). Another alternative is the comparison between two consecutive sections, one stained for the tracer and the other by immunohistochemistry (Bentivoglio and Chen 1993). However, these methods are time-consuming and are more difficult in terms of data analysis. Anterogradely transported HRP or conjugates of HRP with WGA or CTB have therefore been visualized by indirect immunofluorescence using antisera raised against HRP or its conjugates. This procedure could be combined with immunohistochemistry, but the sensitivity appeared to be inferior to the original TMB method (Lindh et al. 1989; Tanaka et al. 1993).

Previous studies using the highly sensitive tracers DiI or WGA–HRP visualized by TMB histochemistry described two types of vagal afferent endings in the musculature of the gastrointestinal tract: intramuscular afferents and IGLEs (Berthoud and Powley 1992b; Berthoud and Neuhuber 1994; Kressel at al. 1994; Berthoud et al. 1997). Vagal afferents were also observed in the submucosa and mucosa of the esophagus in rat and cat, some penetrating deeply into the epithelium (Clerc and Condamin 1987; Neuhuber 1987; Neuhuber and Clerc 1990). These different types of vagal afferents were also detected by tyra-mide–biotin amplification and subsequent incubation with streptavidin–FITC. In contrast, tyramide amplification by the direct method was not sensitive enough compared to the results gained by DiI or TMB histochemistry. Furthermore, the use of WGA–biotin as a tracer and detection by tyramide–biotin amplification and streptavidin–FITC were inferior compared to WGA–HRP and the indirect method. However, it can be hypothesized that WGA–biotin is not as efficiently anterogradely transported in vagal afferents as WGA–HRP.

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 Kuramoto and Kuwano (1995) and their origins identified in the nodose and spinal ganglia by retrograde tracing experiments. Anterograde tracing now proved the vagal origin of the calbindin- and calretinin-positive laminar endings in the esophagus and made spinal afferents with this morphology less likely. CGRP immunohistochemistry, which was used as a marker for spinal afferents, never revealed large-caliber fibers with comparable profuse arborizations at the esophageal myenteric ganglia. In contrast, intramuscular vagal afferents at the gastroesophageal junction were not found positive for calretinin. Because a role of Ca binding proteins in neuronal signaling has been implied (Miller 1995), this might point to different electro-physiological and functional properties of these two morphologically distinct types of vagal afferents.

Footnotes

Acknowledgements

Supported by the Deutsche Forschungsgemeinschaft (KR 1665/2-1).

I thank Ms Anita Hecht for diligently preparing the cryocuts.

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