Journal of Histochemistry & Cytochemistry

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Microwave Staining of Enteric Neurons Using Cuprolinic Blue (Quinolinic Phthalocyanin) Combined with Enzyme Histochemistry and Peroxidase Immunohistochemistry Christa J. Van Ginneken, Marcel J. De Smet, Frans J. Van Meir and Andre A. Weyns J Histochem Cytochem 1999 47: 13 DOI: 10.1177/002215549904700103 The online version of this article can be found at: http://jhc.sagepub.com/content/47/1/13

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Volume 47(1): 13–21, 1999 The Journal of Histochemistry & Cytochemistry

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ARTICLE

Microwave Staining of Enteric Neurons Using Cuprolinic Blue (Quinolinic Phthalocyanin) Combined with Enzyme Histochemistry and Peroxidase Immunohistochemistry Christa J. Van Ginneken, Marcel J. De Smet, Frans J. Van Meir, and André A. Weyns Laboratory of Veterinary Anatomy and Embryology (CVG,MDS,AW) and Laboratory of Cell Biology and Histology (FVM), Faculty of Medicine, University of Antwerp, Antwerp, Belgium

Methods that visualize subsets as well as the entire enteric neuron population are not readily available or have proved to be unreliable. Therefore, we attempted to combine NADPH-d histochemistry, AChE histochemistry, and CGRP immunohistochemistry, techniques that mark subsets of enteric neurons, with a technique that appeared to visualize the entire enteric neuron population, the cuprolinic blue staining method. To guarantee representative staining results, the individual staining methods were modified by using microwaves. In addition, this preserved the characteristics of each of the individual techniques. The distribution of NADPH-d, AChE, and CGRP corresponded well with previous morphological and physiological reports. Consequently, the different combinations gave rise to rapid, useful, and ready-to-use double labeling techniques. Their main advantage is that they simultaneously visualize the total population as well as subsets of enteric neurons. (J Histochem Cytochem 47:13–21, 1999)

SUMMARY

The enteric nervous system (ENS) is very complex and its total number of neurons is believed to be of the same magnitude as the number of nerve cells in the spinal cord (Furness and Costa 1980). Many of the enteric neurons have been characterized in terms of neurotransmitter expression (Gershon et al. 1994). The neurotransmitters are co-stored in various combinations in these enteric neurons, which has given rise to the idea that particular combinations of peptides can be recognized as a “chemical code” and consequently characterize subsets of enteric neurons (Costa et al. 1986; Timmermans et al. 1990; Gershon et al. 1994). What makes the ENS even more complex is that there are indications that the expression of several neurotransmitters is adaptive and dynamic (Van Ginneken et al. in press). To quantify the subsets of enteric neurons revealed by their chemical codes and to evaluate neurotransmitter expression, it is necessary to relate the number of neurons expressing one or more Correspondence to: Christa J. Van Ginneken, Lab. of Veterinary Anatomy and Embryology, Slachthuislaan 68, 2060 Antwerp, Belgium. Received for publication March 26, 1998; accepted September 15, 1998 (8A4634). © The Histochemical Society, Inc.

KEY WORDS enteric nervous system cuprolinic blue NADPH-d acetylcholinesterase CGRP microwave double labeling

neurotransmitters to the total number of enteric neurons. To obtain a reliable estimate of the total number of neurons, a method is required that is neuron-specific and permits all enteric neurons to be visualized. Moreover, it would be very interesting if such a method could be combined with methods that visualize the expression of certain neurotransmitters. A variety of methods that stain the entire enteric neuron population have already been evaluated. However, they have proved to be nonselective, to have low reproducibility, to be not generally available, or to fall short of estimating the total number of enteric neurons (Björklund et al. 1984; Costa et al. 1987; Young et al. 1993; Parr and Sharkey 1994; Karaosmanoglu et al. 1996). Cuprolinic blue, an analogue of the well-known phthalocyanin dye Alcian Blue 8GX, has received renewed interest as a marker for the entire enteric neuron population (Scott 1972; Heinicke et al. 1987; Holst and Powley 1995; Karaosmanoglu et al. 1996). In combination with high salt concentrations, it has a remarkable specificity for single-stranded RNA. One of its advantages is that, under these specific conditions, cuprolinic blue does not bind to nuclear DNA

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Van Ginneken, De Smet, Van Meir, Weyns

or other compounds commonly found in tissues (Scott 1972; Mendelson et al. 1983; Tas et al. 1983). Consequently, some investigators have concluded that, given the high concentration of RNA in neurons, enteric neurons are the only structures that can be stained in gastrointestinalwhole-mountpreparations(Heinickeet al. 1987; Holst and Powley 1995; Karaosmanoglu et al. 1996). The aim of this study was to elaborate different combinations between cuprolinic blue staining and peroxidase immunohistochemistry and/or enzyme histochemistry to provide a readily usable procedure that simultaneously visualizes the total population as well as subsets of enteric neurons. Late fetal and neonatal jejunum was used to test whether this method is applicable to tissues of different developmental stages. Porcine jejunum was preferred to rodent tissue because the morphology and rate of development of the porcine intestine resembles that of humans.

Materials and Methods Tissue Preparation Animals. Two fetal pigs (Piétrain 3 White Large) from the second half of the gestational period were obtained from a local slaughterhouse. The time interval between the death of the sow and the removal of the fetuses was approximately 15 min and, at that moment, the fetuses were already dead. The age of the fetal pigs was estimated by measuring the crown–rump length and by converting this into fetal age according to the method described by Evans and Sack (1973). For this study, two fetal pigs of respectively 84 (220 mm crown–rump length) and 109 (290 mm crown–rump length) days of age were used. Two neonatal pigs (Piétrain 3 White Large) of respectively 2 days and 4 days were used. These animals were sacrificed by severing the carotid arteries under deep barbiturate anesthesia (sodium pentobarbital).

Preparation. The intestinal tracts were removed through a midline ventral incision and were rinsed with 0.01 M PBS.

which contained the submucosal plexuses, was separated from the muscle layers and was transferred to PBS. From the muscle layers, the circular muscle layer’s muscle bundles were peeled off. The remaining slab contained the serosal layer and the longitudinal muscle layer bearing the myenteric plexus and was placed in PBS.

Microwave Applications Most of the subsequent incubations were performed in a 2,450-Hz 900-W Bosch microwave oven with multiple energy levels and a rotating plate (type HMT 862L/02; Bosch, Berlin, Germany), a microwave oven for domestic use. All the parameters, i.e., surrounding water volume (SWV), fixed position, energy level, and time of irradiation, were experimentally set in function of the critical temperature of 45C of an incubation medium with a volume (IM) of 1 or 5 ml (Van Ginneken et al. in press). In the present study, the critical final irradiation temperature was set at 45C because higher temperatures caused irreversible damage to the wholemount preparations, such as excessive shrinkage (Evers and Uylings 1994). The temperature of the incubation medium was measured during the different standardization trials with an electronic temperature meter and probe after the irradiation bursts and subsequent stirring of the incubation medium (Van Ginneken et al. in press). The standardization of the parameters resulted in the following set-up (Van Ginneken et al. in press) (Figure 1). The open vessels filled with 1 ml (Eppendorf tube) or 5 ml (25 ml glass vial) IM were placed at fixed positions in a glass Petri dish ([ in case of Eppendorf tubes 10 cm; [ in case of glass vials 15 cm), that was filled with 150 ml tapwater (SWV) on the rotating plate of the microwave. The Eppendorf tubes were situated 2.5 cm outside the center of the rotating plate and the glass vials 4 cm outside its center. This set-up is further referred to in the text as “standardized conditions.”

Nicotinamide Adenine DinucleotidephosphateDiaphorase (NADPH-d) Histochemistry The whole-mount preparations were placed in glass vials filled with aNADPH-d incubation medium containing 10 mg NADPH (Serva; Polylab, Antwerp, Belgium), 1.25 mg nitroblue tetrazolium (NBT) (Sigma Aldrich; Bornem, Belgium)

Fixation. The intestines were fixed by immersion using 4% freshly prepared phosphate-buffered (0.1 M) paraformaldehyde, pH 7.4 for 2 hr at 4C. The fixative was washed out with PBS over 24 hr. While awaiting further dissection, the intestines were stored at 4C in PBS enriched with 0.01% sodium azide.

Whole-Mount Preparation Whole-mount preparations were made according to Costa and Furness (1983). In short, segments approximately 2 cm in length were cut from the intestine. These segments were cut open along the line of mesenteric attachment. The mucosa was scraped off and the residues of mesentery were removed. Then the slabs were rinsed in PBS. At this stage of the preparation, the slabs could be stored in PBS enriched with 0.01% sodium azide at 4C. The subsequent dissection was performed with watchmaker’s forceps and very fine scissors. The submucosal layer,

Figure 1 Schematic of the experimental set-up during the different incubations. The open recipients, Eppendorf tube (ET) or glass vial (GV), filled with 1 ml or 5 ml incubation medium (IM), were placed at experimentally fixed positions in a glass Petri dish (PD) ([ in case of Eppendorf tubes 10 cm; [ in case of glass vials 15 cm) filled with 150 ml of tapwater (SWV) on the rotating plate (RP) of the microwave. The Eppendorf tubes were situated 25 mm outside the center of the rotating plate; the glass vials were placed 40 mm outside its center.


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Histochemical Combinations in the Microwave (50 ml stock solution 2.5% NBT, dissolved in a solution of methanol:dimethylformamide 1:1) and 50 ml stock solution 10% DMSO (Across Chimica; Beerse, Belgium) in 5 ml PBS containing 0.3% Triton X-100 (Merck Belgalabo; Overijse, Belgium). Once the open glass vials, filled with the NADPH-d incubation medium, were correctly placed under the standardized conditions, an irradiation burst of 90 W was given for 5 min. This burst was followed by a prolonged incubation for 10 min in the warm SWV. Afterwards, the whole-mount preparations were rinsed four times for 5 min each in PBS.

Acetylcholinesterase (AChE) Histochemistry The protocol used, was based on the protocols of Baljet and Drukker (1975) and Hanker and colleagues (1973) but modified in our laboratory. Three stock solutions were made. Stock Solution A was made under continuous stirring and controlled pH. A starting volume of 800 ml distilled water was used. To this starting volume other reagents are added. First, 10 ml Triton X-100 (Merck Belgalabo) was added. Subsequently, 3.28 g sodium acetate anhydride was dissolved in the starting volume. Afterwards, 100% acetic acid was added until the pH reached 5.3. To increase the pH to 5.6, 1.468 g sodium citrate.2 H2O was dissolved in the starting volume. By adding dropwise 0.506 g CuSO4 anhydride, previously dissolved in 30 ml distilled water, the pH decreased to 5.31. Afterwards, 0.171 g Iso-OMPA (tetra-iso-propyl-pyro-phosphor-amide) (KochLight Lab. Interlaboratoire; Brussels, Belgium) was dissolved. Finally, the starting volume was adjusted to 1.000 liter with distilled water (pH 5.4). Stock Solution A could be stored for 1 year at 4C. Stock-Solution B consisted of a 5% S-acetylthiocholine iodide (Sigma Aldrich) solution in distilled water and remained stable for 1 month when stored at 4C. Stock Solution C consisted of a saturated (33%) K 3Fe(CN)6 solution in distilled water and remained stable for 1 week when stored at 4C. After a rinse for 5 min in distilled water, the tissues were rinsed twice for 5 min each in 0.05 M sodium acetate buffer (pH 5.2). Then the whole-mount preparations were placed for 5 min in stock Solution A. Meanwhile, the incubation medium was prepared as follows: 5 ml stock Solution A, 50 ml stock Solution B, and 50 ml stock Solution C. The whole-mount preparations were brought into the glass vials filled with 5 ml of incubation medium. Once the open vessels were correctly placed under the standardized conditions, an irradiation burst of 90 W was given for 5 min. This burst was followed by standing for 15 min in the warm SWV, during which the IM temperature stabilized at room temperature (RT). Afterwards a second burst of 90 W for 5 min was given. Finally, the reaction was stopped by transferring the tissues for 5 min to 0.05 M sodium acetate buffer (pH 5.2), followed by a rinse in distilled water for 5 min.

Cuprolinic Blue Staining The protocol used was based on the protocol described by Holst and Powley (1995), but modified in our laboratory (Van Ginneken et al. in press). The whole-mount preparations were rinsed three times for 5 min each in distilled water, followed by rinsing twice for 5 min each in 0.05 M sodium acetate buffer (pH 5.6). Then, the whole-mount preparations were incubated in 5 ml of

0.05 M sodium acetate buffer (pH 5.6) enriched with 100 ml of a saturated (33% in distilled water) K3Fe(CN)6 solution for 30 min at RT. After triple rinses in distilled water for 1 min each, the whole-mount preparations were placed in 5 ml of 0.05 M sodium acetate buffer (pH 5.6) enriched with 1 M MgCl2, Triton X-100 (final concentration 0.3%) (Merck Belgalabo), and DMSO (final concentration 0.1%) (Across Chimica) for 10 min at RT. Subsequently, the whole-mount preparations were blotted onto filter paper. From this moment on, direct contact between metal tools and the 0.5% cuprolinic blue incubation medium was avoided. This 0.5% incubation medium consisted of 20 ml 0.05 M sodium acetate buffer (pH 5.6), 0.1 g quinolinic phthalocyanin (cat. no. 17052; Polysciences, Warrington, PA), and 4.07 g MgCl2 (final pH becomes 4.6). It could be stored for several months at 4C as 1-ml portions in Eppendorf tubes or as 5-ml portions in glass vials and could be reused several times. The whole-mount preparations were placed in these vessels (Eppendorf tubes or glass vials) for the incubation. The open vessels were correctly placed under the standardized conditions and an irradiation burst of 90 W was given for 5 min. This burst was followed by standing for 15 min in the warm SWV. Differentiation was carried out by transferring the tissues to 0.05 M sodium acetate buffer (pH 5.6), enriched with 1 M MgCl2 for 2 min. Finally, the whole-mount preparations were rinsed three times for 5 min each in distilled water.

Calcitonin Gene-related peptide (CGRP) Immunohistochemistry Polyclonal CGRP antiserum raised in rabbits was purchased from Amersham (Gent, Belgium). All other immunoreagents were obtained from Dako (Glostrup, Denmark): normal swine serum (X0901 065), biotinylated F(ab9)2 fragment of swine anti-rabbit serum (E0431), and peroxidase-conjugated streptavidin (P0397). After three rinses in PBS for 5 min each, the whole-mount preparations were incubated in 3% H2O2 in PBS for 30 min to block endogenous peroxidase. After four rinses in PBS for 5 min each, nonspecific tissue reactivity was blocked with 5% normal swine serum (Dako) in PBS containing 1% Triton X-100 and 5% nonfat dry milk powder (Nestle). Once the open vessels that contained the whole-mount preparations and the incubation medium were correctly placed under the standardized conditions, an irradiation burst of 90 W was given for 5 min. After standing for 15 min in the warm SWV, the whole-mount preparations were blotted on filter paper. Next, the tissues were incubated in the different antisera. The polyclonal primary antiserum against CGRP (dilution 1:400), the secondary swine anti-rabbit biotinylated antiserum (dilution 1:200), and the tertiary peroxidase-conjugated streptavidin antiserum (dilution 1:200) were diluted in PBS containing 1% Triton X-100, 5% normal swine serum, and 5% nonfat dry milk powder. The incubations in the different antisera consisted of three irradiation bursts of 90 W for 5 min each under the standardized conditions. After each burst, the whole-mount preparations were left to incubate for 15 min in the warm SWV. Before another irradiation burst was given, the whole-mount preparations were slightly stirred in the tubes to improve homogeneous penetration of the medium and the SWV was replaced with fresh SWV RT. After the


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third incubation in the warm SWV, antisera were removed by rinsing six times for 5 min each in PBS, followed by blotting of the whole-mount preparations on filter paper. After removal of the tertiary antiserum with PBS, the whole-mount preparations were rinsed twice for 5 min each in 0.05 M sodium acetate buffer, pH 5.2. Subsequently, the sites of antibody binding were visualized with a 0.05% AEC (aminoethylcarbozol; Sigma Chemie, Deisenhofen, Germany) solution made with 5 ml 0.05 M sodium acetate buffer (pH 5.2), 50 ml 5% AEC stock solution in dimethylformamide, and 50 ml 3% H2O2. Before the incubation, the AEC solution was filtered through Millipore 0.22 mm. The incubation in the AEC solution consisted of an irradiation burst for 5 min at 90 W under the standardized conditions, followed by standing in the warm SWV for 2 to a maximum of 5 minutes (determined empirically by microscopic examination). The reaction was stopped by rinsing the whole-mount preparations three times for 5 min each in 0.01 M PBS.

Combinations NADPH-d Histochemistry/Cuprolinic Blue Staining. Immediately after performing NADPH-d enzyme-histochemistry, overnight fixation in 4% freshly prepared paraformaldehyde solution in 0.1 M PBS, pH 7.4, was carried out at 4C. Then, the whole-mount preparations were rinsed three times for 5 min each in distilled water. Subsequently, cuprolinic blue staining was performed according to the above-mentioned protocol. Finally, the whole-mount preparations were mounted on slides in a 3:1 glycerin:PBS mixture.

AChE Histochemistry/Cuprolinic Blue Staining. Cuprolinic blue staining could be performed immediately after AChE enzyme histochemistry without any modifications in either of the two protocols described above. Finally, the wholemount preparations were mounted on slides in a 3:1 glycerin:PBS mixture. Cuprolinic Blue Staining/CGRP Immunohistochemistry. After the cuprolinic blue staining according to the standardized protocol, the whole-mount preparations were rinsed three times for 5 min each in PBS. Subsequently, CGRP immunohistochemistry could be performed according to the above-mentioned protocol. Finally, the whole-mount preparations were mounted on slides in a 3:1 glycerin:PBS mixture.

Results The elaboration of combinations among cuprolinic blue, NADPH-d enzyme histochemistry, AChE enzyme histochemistry, and CGRP immunohistochemistry resulted in useful double labeling techniques. Furthermore, the staining results did not differ between the late fetal and neonatal jejunal whole-mount preparations. In each of these combinations, the enteric neurons that stained for cuprolinic blue alone appeared as blue-green cells with nonreacting nuclei (Figures 2– 13). At high magnification, the cytoplasm contained a fine blue-green granulation and in the nucleus, the nucleolus or nucleoli appeared as blue-iridescent round

structures (Figures 2–9 and 13). The neuronal processes lacked staining because of lower RNA concentrations. In each of the combinations, a light blue background staining occurred. However, this did not prevent the recognition of the distinct meshworks of the different enteric plexuses. In the whole-mount preparation of the myenteric plexus, more or less rectangular ganglia that ran parallel to the circular muscle bundles were clearly visible (Figures 2, 6, and 10). The background staining was mainly due to the cuprolinic blue-stained fibroblasts, which appeared as spindleshaped cells along the muscle cells of the longitudinal muscle layer. However, the staining result in the fibroblasts was much weaker. On the basis of their location and appearance, fibroblasts were easily distinguished from perikarya. In the whole-mount preparation of the submucosal plexuses, background staining was generally more pronounced owing to the higher fibroblast content of this tissue layer (Figures 7, 9, and 11– 13). Nevertheless, a distinction between the inner and outer submucous plexus could be made. The outer submucous plexus consisted of large ganglia that were situated far apart and contained rather large cuprolinic blue-stained perikarya (Figures 3, 5, 7, 9, 11, and 13). The inner submucous plexus consisted of small polygonal ganglia that were situated close to each other and contained rather small cuprolinic bluestained perikarya (Figures 4, 8, and 12). NADPH-d Histochemistry/Cuprolinic Blue Staining

Only a small number of the enteric neurons population were stained with NADPH-d histochemistry, appearing as dark blue-stained perikarya that gave rise to dark blue neuronal processes. In the nonreacting nucleus, the nucleolus or nucleoli were stained bluegreen by the cuprolinic blue staining technique. In each of the enteric plexuses NADPH-d-expressing neurons were easily distinguishable from the cuprolinic blue-stained perikarya by the difference in color and staining-intensity (Figures 2, 3, and 5). In ganglia of the myenteric plexus, NADPH-d-expressing neurons of variable size and shape were scattered (Figure 2), whereas they appeared to be clustered in those of the outer submucous plexus (Figures 3 and 5). In the inner submucous plexus, rather small NADPH-d-expressing neurons were very rarely seen (Figure 4). The nerve fibers revealed by NADPH-d enzyme histochemistry facilitated the recognition of the cuprolinic blue-stained perikarya. These NADPH-d-expressing varicose nerve fibers were observed in the paravascular plexus, in the interconnecting nerve strands, in the ganglia, and parallel to smooth muscle bundles. In the ganglia, NADPH-d-expressing nerve fibers encircled both NADPH-d-expressing neurons and cuprolinic blue, non-NADPH-d-expressing perikarya (Figures 2 and 3).


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Histochemical Combinations in the Microwave AChE Histochemistry/Cuprolinic Blue Staining

In this sequential staining technique, the cuprolinic blue-stained neurons appeared to be more pronounced, compared to the combination with NADPH-d enzyme histochemistry. Furthermore, there was a remarkable color shift from blue to green in the cells that were stained with cuprolinic blue alone. AChE histochemistry revealed a subset of the enteric neuron population larger than that revealed by NADPH-d histochemistry. AChE-expressing neurons were stained with different intensities. In the perikaryon and in the nerve fibers of these enteric neurons a brown granulation filled the cytoplasm (Figures 7–9). However, the AChE histochemical reaction did not reveal the fine structure of the processes. The nucleus remained unstained, except for the nucleolus or nucleoli, which were stained by the cuprolinic blue staining technique (Figures 6, 7, and 9). The slightly colored background did not affect the evaluation of the plexuses because the brownish color of the AChE-expressing perikarya and nerve fibers contrasted well with the background and with the bluegreencuprolinicblue-stainedperikarya.AChE-expressing neurons of variable size and shape were scattered throughout the ganglia of the different enteric plexuses. In these ganglia, AChE-expressing nerve fibers encircled both AChE-expressing neurons and cuprolinic blue, non-AChE-expressing perikarya. Furthermore, AChEexpressing varicose nerve fibers were observed in the paravascular plexus, in the interconnecting nerve strands, and parallel to smooth muscle bundles. Cuprolinic Blue Staining/CGRP Immunohistochemistry

CGRP immunohistochemistry revealed a small portion of the enteric neuron population. In the perikaryon and in the nerve fibers, the immunohistochemical reaction resulted in a red granulation of the cytoplasm. However, the reaction was unable to reveal the fine structure of the neuronal processes. The nucleus remained unstained, except for the cuprolinic blue-stained nucleolus or nucleoli. Evaluation of the enteric plexuses was facilitated by the contrast between the red immunohistochemical reaction product and the cuprolinic blue staining. In the ganglia of the myenteric and outer submucous plexus, CGRP-expressing neurons were located mainly in clusters on the outside of the ganglia (Figures 10 and 11). Some of them were observed in the interconnecting nerve strands (Figure 10). In the inner submucous plexus, scattered CGRP-expressing neurons were rarely found (Figure 12). These sparse CGRP-expressing neurons were rather large compared with the cuprolinic blue-stained perikarya that were seen in the outer submucous plexus (Figure 13).

In the ganglia, CGRP-expressing nerve fibers encircled both CGRP-expressing neurons and cuprolinic blue, non-CGRP-expressing perikarya. In addition to ganglia, CGRP-expressing varicose nerve fibers were observed in the paravascular plexus, in the interconnecting nerve strands, and parallel to smooth muscle bundles.

Discussion The individual staining techniques, i.e., NADPH-d histochemistry, AChE histochemistry, and CGRP immunohistochemistry, proved to be highly compatible with the cuprolinic blue staining. Nevertheless, some modifications were carried out to optimize the staining results: the incorporation of microwave applications into the different staining methods and an additional fixation in combining NADPH-d histochemistry. Encouraged by the knowledge that microwaves have a beneficial effect on chemical reactions, on the retrieval of antigens, and on the penetration of substances, we incorporated microwave applications into the different staining methods (Boon and Kok 1989a,b; Shi et al. 1995). However, before the different incubations were begun, special attention had to be paid to the fixation times because the requirements and limitations vary among the methods that were used. Fixation times were restricted to 2 hr when NADPH-d histochemistry was performed. However, when combined with long preservation times, these short fixation times markedly reduced the quality of the cuprolinic blue staining. Therefore, after NADPH-d histochemistry and before the cuprolinic blue staining technique, tissues were fixed overnight. When cuprolinic blue is combined with methods other than NADPH-d histochemistry, the initial fixation times can be longer and no additional fixation is needed. By incorporating the above-mentioned modifications, the properties of the individual techniques were preserved. Therefore, it can be safely assumed that the different techniques do not influence their respective extent of staining. A one-to-one co-localization of NADPH-d and bNOS in enteric plexuses has been demonstrated, implying that both enzyme histochemistry and immunohistochemistry can be used to visualize nitrergic neurons (Dawson et al. 1991, Timmermans et al. 1994). In the pig jejunum, NADPH-d histochemistry revealed a rather small enteric neuron subpopulation. The nitrergic neurons were confined mainly to the outer layers of the intestinal wall: outer submucous plexus, myenteric plexus, and muscle layers. This observation corresponds well with previous descriptions of the distribution and projections of nitrergic neurons in the porcine small intestine (Ekblad et al. 1994; Timmermans et al. 1994; Brehmer et al. 1998). Lesion studies have revealed that NADPH-d-expressing nerve fibers


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Histochemical Combinations in the Microwave

that encircle nerve cell bodies in the myenteric plexus project from myenteric perikarya, whereas in the submucous plexus they arise from both submucosal and myenteric nitrergic perikarya (Costa et al 1992; Ekblad et al. 1994; Timmermans et al 1994). On the ba-

sis of these morphological findings, these submucosal and myenteric nitrergic neurons most probably fulfill the role of interneurons. This is substantiated by Yuan and colleagues (1995), who have clearly demonstrated that some interneurons are capable of producing NO.

Figure 2 Neurons in a ganglion of the myenteric plexus of a neonatal pig display the typical cuprolinic blue staining pattern (arrow). NADPH-d-expressing neurons are scattered in the myenteric ganglia (arrowhead). NADPH-d-expressing varicose nerve fibers (small arrowhead) run parallel to smooth muscle bundles and are clearly visible in the interconnecting nerve strands and in the ganglia, where some are found to surround myenteric neurons. Bar 5 50 mm. Figure 3 Neurons in a ganglion of the outer submucous plexus of a neonatal pig display the typical cuprolinic blue staining pattern (arrow). NADPH-d-expressing neurons are clustered at a pole of the ganglion (arrowhead). NADPH-d-expressing varicose nerve fibers are clearly visible in the interconnecting nerve strands and in the ganglia, where some are found to surround enteric neurons (small arrowhead). In the background a blood vessel is visible that shows a weak reaction with NADPH-d enzyme histochemistry (small arrow). Bar 5 50 mm. Figure 4 A ganglion of the inner submucous plexus is clearly visible in the jejunum of a neonatal pig. This ganglion is filled with rather small cuprolinic blue-stained neurons (arrow). Some NADPH-d-expressing nerve fibers (small arrowhead) are seen in the ganglion and in the interconnecting nerve strands (small arrow). Bar 5 50 mm. Figure 5 Neurons that display the typical cuprolinic blue staining (arrow) fill a ganglion of the outer submucosal plexus. The nucleolus is clearly visible after staining with cuprolinic blue and appears as a round blue structure (small arrow). NADPH-d-expressing perikarya are concentrated on one side of the ganglion (arrowhead). In the ganglion, the NADPH-d-expressing nerve fibers contrast well with the cuprolinic blue-stained perikarya (small arrowhead). Bar 5 25 mm. Figure 6 Neurons in a ganglion of the myenteric plexus of a neonatal pig display the typical cuprolinic blue staining pattern (arrow). AChEexpressing neurons are scattered in the myenteric ganglia (arrowhead). Well-contrasting, brown AChE-expressing thin nerve fibers are abundant in the ganglia and interconnecting nerve strands (small arrowhead). A remnant of the circular muscle layer lies on top of the myenteric plexus (small arrow). Bar 5 50 mm. Figure 7 A ganglion of the outer submucous plexus is clearly visible in the jejunum of a neonatal pig. In this ganglion, dark brown AChEexpressing neurons (arrowhead) are scattered among cuprolinic blue-stained perikarya (arrow). Thin AChE-expressing nerve fibres run in the ganglia and in the interconnecting nerve strands (small arrowhead). Weak cuprolinic blue staining occurs in fibroblasts (small arrow). Bar 5 50 mm. Figure 8 In the inner submucous plexus of the jejunum of a neonatal pig, ganglia (asterisks) are clearly visible. These ganglia, which lie close to each other, are filled with rather small cuprolinic blue-stained neurons (arrow) and rather large but few AChE-expressing neurons (arrowhead). Some brown AChE-expressing nerve fibers (small arrowhead) can be seen in the ganglia and interconnecting nerve strands (small arrow). Bar 5 50 mm. Figure 9 Neurons that display the typical cuprolinic blue staining (arrow) and that express AChE (arrowhead) are scattered in a ganglion of the outer submucosal plexus of a piglet. The nucleolus is clearly visible by staining with cuprolinic blue and appears as a round structure in all perikarya (small arrows). In the ganglion, brown AchE-expressing nerve fibers (small arrowheads) contrast well with the cuprolinic bluestained perikarya. Bar 5 25 mm. Figure 10 Some cuprolinic blue-stained perikarya are found outside a myenteric ganglion of a neonatal pig in the plane of the longitudinal muscle layer (arrow). CGRP-expressing neurons are found mainly on the outside of the ganglion (arrowheads). Occasionally, a CGRPexpressing neuron is found in the plane of the longitudinal muscle layer (arrowheads). Well-contrasting red varicose nerve fibers are abundant in the ganglia and interconnecting nerve strands (small arrowheads). Many encircle cuprolinic blue stained perikarya (asterisk). Bar 5 50 mm. Figure 11 A ganglion of the outer submucous plexus is clearly visible in the jejunum of a neonatal pig. It is filled with perikarya that display the typical cuprolinic blue staining pattern (arrows). Red CGRP-expressing neurons are found mainly on the outside of the ganglion (arrowheads). CGRP-expressing nerve fibers (small arrowheads) run in the interconnecting nerve strands and in the ganglia, where some of them encircle cuprolinic blue stained perikarya (asterisk). In addition, in neurons a weak reaction with cuprolinic blue is also observed in fibroblasts (small arrows). Bar 5 50 mm. Figure 12 In the inner submucous plexus of the jejunum of a neonatal pig, ganglia are clearly visible. These ganglia, which lie close to each other, are filled with rather small cuprolinic blue-stained neurons (arrows) and relatively large CGRP-expressing neurons (arrowheads). Red CGRP-expressing varicosities (small arrowhead) can be seen in the interconnecting nerve strands and in the ganglia, where some of them encircle cuprolinic blue-stained perikarya (asterisk). Bar 5 50 mm. Figure 13 Neurons that display the typical cuprolinic blue staining (arrow) and that express CGRP (arrowhead) are scattered in a ganglion of the outer submucosal plexus of a neonatal pig. Weak staining with cuprolinic blue can also be observed in fibroblasts (small arrows). In the ganglion, red CGRP-expressing varicosities (small arrowheads) contrast well with the cuprolinic blue-stained perikarya. Some of these CGRP-expressing nerve fibers encircle nerve cell bodies (asterisk). Bar 5 25 mm.


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In addition to nitrergic interneurons, the myenteric plexus harbors nitrergic motor neurons which give rise to anally directed processes that terminate in the neighborhood of smooth muscle cells (Costa et al. 1992; Ekblad et al. 1994; Timmermans et al. 1994). NO, which is released by these neurons, is believed to mediate part of the nonadrenergic, noncholinergic relaxation of the gut (Kanada et al. 1992; Sanders and Ward, 1992). Whether or not the outer submucosal plexus harbors nitrergic motor neurons is still a matter of controversy (Timmermans et al. 1994; Brehmer et al. 1998). AChE histochemistry revealed the largest subpopulation of enteric neurons compared with NADPH-d histochemistry and CGRP immunohistochemistry. AChE-expressing neurons are abundant in the myenteric and outer submucous plexus. This is in agreement with previous reports on AChE expression in the porcine small intestine (De Ridder and Weyns 1994). Caution should be exercised in ascribing a function to AChE-expressing neurons because it is not unequivocally proven that they are cholinergic in nature (Lehmann and Fibiger 1979). Nevertheless, AChE histochemistry can be useful to classify enteric neurons morphologically and according to their chemical content (Lehmann and Fibiger 1979; De Ridder and Weyns 1994). Furthermore, there is growing evidence that acetylcholinesterase may function as a neuropeptide-processing enzyme (Small 1989). This may be all the more reason to use AChE histochemistry in combination with immunohistochemistry. The last protocol was developed to simultaneously visualize the enteric neurons with cuprolinic blue and CGRP immunoreactivity. Nevertheless, the protocol should be appropriate to use with the immunoperoxidase visualization of various neurotransmitters other than CGRP. Holst and Powley (1995) have already described the use of cuprolinic blue as a counterstain for Phaseolus vulgaris immunohistochemistry in gastrointestinal wholemount preparations. Our demonstration of CGRP expression corresponds well with previous reports that describe the distribution of CGRP-expressing nerve cell bodies and fibers in the porcine small intestine (Timmermans et al. 1990). The presence of CGRP-expressing perikarya in the enteric plexuses suggests that at least part of the observed CGRP-expressing nerve fibers are intrinsic in origin. However, several studies have reported that an important part of the CGRP-expressing nerve fibers originates from extrinsic sources (Lee et al. 1987; Sternini et al. 1987). The extrinsic CGRP-expressing nerve fibres principally contact submucosal blood vessels (Sternini et al. 1987). Most of these extrinsic CGRPexpressing nerve fibers have their cell bodies in dorsal root ganglia, proivding further evidence for their presumed sensory nature (Gibbins et al. 1987). These af-

ferent CGRP-expressing nerve fibers control intestinal circulation, probably via an axon reflex (Vanner 1993,1994). Regarding the intrinsic CGRP-expressing nerve supply, Sternini and colleagues (1987) have shown that these CGRP-expressing nerve fibers are mainly conveyed to the muscle layers and the enteric plexuses. This distribution suggests that CGRP is involved in the regulation of intestinal movement and that CGRP plays a role at the neuro–neural junction. In this context, CGRP is reported to have a dual effect on intestinal smooth muscle cells (Bartho et al. 1987; Holzer et al. 1989). The contractile effect of CGRP is most probably brought about by activation of cholinergic motor and/or interneurons, whereas the relaxation, which appears to be the predominant action, seems to be evoked directly by the intestinal smooth muscle cells (Bartho et al. 1987; Holzer et al. 1989; Bartho 1991). By incorporating the above-mentioned modifications, the elaboration of combinations among cuprolinicblue,NADPH-dhistochemistry,AChEhistochemistry and CGRP immunohistochemistry resulted in rapid, useful, and ready-to-use double labeling techniques. Their main advantage is that they simultaneously visualize the total population as well as subsets of enteric neurons.

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