©1996-2002 All Rights Reserved. Online Journal of Veterinary Research. You may not store these pages in any form except for your own personal use. All other usage or distribution is illegal under international copyright treaties. Permission to use any of these pages in any other way besides the before mentioned must be gained in writing from the publisher. This article is exclusively copyrighted in its entirety to OJVR publications. This article may be copied once but may not be, reproduced or re-transmitted without the express permission of the editors.

Department of Anatomy, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon, Kyunggi-Do, 441-744, South Korea.*Department of Anatomy, College of Medicine, Hallym University. Chunchon, Kangwon-Do, 200-702, South Korea.+Corresponding Author, Dr. In Se Lee, Department of Anatomy, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University, Suwon, Kyunggi-Do, 441-744, South Korea, TEL: +82-31-290-2743, FAX: +82-31-290-2717, E-mail: inselee@snu.ac.kr

Immunohistochemical and double immunofluorescent was used to identify the localization of calbindin D-28k (CB), calretinin (CR), parvalbumin (PA), substance P (SP), calcitonin gene-related peptide (CGRP) and galanin (GAL) in the goat small intestine. Localization of CB with SP, CGRP or GAL in the myenteric and submucosal plexuses suggests that CB may serve a neuromodulatory role for SP-, CGRP-, and GAL-immunoreactive neurons in intestinal wall motility. Distributions of CB, CR and SP of the goat small intestine differ from guinea-pig and the pig. This difference may be relevant to morphological characteristics.

The enteric nervous system (ENS) is one of the three portions of the autonomic nervous system and can perform its function independently of the central nervous system. It consists of local nerve networks embedded in the gut wall and is subdivided into two ganglionated plexuses, i.e., the submucosal and myenteric plexuses. In large animals, however, a further division of the submucous plexus may be present, i.e., an inner submucosal nerve network, located close to the abluminal side of the laminal muscularis mucosae, and an outer one, lying adjacent to the luminal side of the circular smooth muscle layer. In the human small intestine, even a third ganglionated plexus, the so-called intermediate ganglionic plexus, can be distinguished (Timmermans et al, 1997).

The ENS mediates complex reflex activities involving intestinal motility, mucosal transportation and blood flow. These functions are achieved by complex interactions within the enteric networks, which consist of sensory neurons, interneurons, and motor neurons, each of which is likely to synthesize a diverse set of neurotransmitters (Hens et al., 2000).

Recent immunohistochemical investigations have revealed that ranges of neurotransmitters or neuromodulators are contained within the enteric neurons. Thus, the concept of neurochemical coding is considered an important aspect of the understanding of enteric microcircuits and their functions. Moreover, the colocalization of these chemical substances has contributed to identifying the roles of the enteric neurons. In addition, a variety of biologically active peptides have been recognized in nerves of the gut wall of the intestine, and it has also been reported that substance P (SP), calcitonin gene-related peptide (CGRP) and galanin (GAL) mainly modulate the motility and/or the sensation of the gut in mammalians.

Calbindin D-28k (CB), calretinin (CR) and parvalbumin (PA) are members of the EF-hand calcium binding protein family, and specifically bind intracellular free calcium in the micromolar range. CB may act either as a calcium transporter or as an intracellular calcium buffer, promoting or restricting calcium-dependent events in the cellular metabolism (Persechini, 1989). Moreover, these calcium-binding proteins may modulate the release or action of SP, CGRP and GAL (Resibois et al., 1988).

Significant species differences may exist in terms of the anatomical structure and the range of neurotransmitters or neuromodulators, even between closely related species. A number of studies have been performed upon the peptide-containing neurons of a wide variety of species, including man, the guinea-pig, the pig and the horse (Hens et al., 2000; Li and Furness, 1998; Bagnol et al., 1997; Costa et al., 1996; Dhatt et al., 1994; Song et al., 1994; Kirchgessner and Gershon, 1991; Song et al., 1991; Dahlstrand et al., 1988; Resibois et al., 1988; Furness and Costa, 1980). However, few studies have been undertaken on the neurons of the goat enteric nervous system. The aim of this study was to identify the localization of CB, CR, PA, SP, CGRP and GAL in the goat small intestine. The present study also attempts to elucidate the anatomical differences of ENS between the ruminant and the guinea-pig, dog, horse and pig.

Twelve goats (Capra hircus, 10-16months, 15-20 kg B.W.) were used in this study. Goats were obtained from Samtaco Animal Center (Korea). The animals were anesthetized with ketamine-xylazine mixture and perfused via the common carotid artery with 3 L of 0.85 % normal saline. Small intestines were opened along the mesenteric attachment, and the tissues were cut out from the intestinal wall at three portions – the sigmoid flexure of the duodenum, and middle parts of the jejunum and ileum. The tissues were stretched and pinned flat on pieces of balsa wood. The tissues were then fixed in 4% paraformaldehyde in phosphate buffered saline (PBS, pH 7.4) with mucosal surface facing up, and cryoprotected in 30% sucrose in PBS. Serial sections of 14m were cut using Cryostat (Reichert-Jung, Germany) and mounted on gelatin-coated slides. The sections were stored at –70 ? for 1-3 weeks before processing for immunohistochemistry and double immunofluorescence.

The sections were treated with 3% H2O2 in methanol for 15 minutes followed by 5% normal goat serum (Dako, USA) for 30 minutes to reduce nonspecific background staining. Some sections were then incubated in primary antibodies (Table 1) containing 0.3% triton X-100 and 2% normal goat serum overnight at room temperature. After washing three times for 10 minutes with PBS, sections were sequentially incubated, in goat anti-rabbit IgG (Vector, USA) and streptavidin (Vector, USA), diluted 1:200 in the same solution as primary antisera. Between the sequential incubations, the tissues were washed with PBS three times for 10 minutes each. The sections were visualized with DAB (3,3’-diaminobenzidine) in 0.1M Tris buffer, and mounted on gelatin-coated slides. Immunoreactions were observed under an Axioplan microscope (Carl Zeiss, Germany).

For co-localization studies, the tissues were incubated with mixtures of mouse monoclonal CB and rabbit polyclonal CR, SP, CGRP, GAL antibodies. The antibody-antigen complexes were visualized using secondary antibodies conjugated with differenent fluorochromes (Table 1), i.e., indocarbocyanine (Cy3)-conjugated anti-mouse IgG for CB, and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit Igg for CR, SP, CGRP and GAL. Flourescence labeling was examined under an Axioplan microscope (Carl Zeiss, Germany), using appropriate filters.
 
 

CB-, CR-, SP-, CGRP-, and GAL-like immunoreactivities (IR) were demonstrated in both nerve cell bodies and fibers of the neurons of the myenteric and submucosal plexuses of the goat small intestine. The complete histological images are very large (6mb each) and sections are shown herein in Figures 3-6. If you wish to view the complete image click on complte image at the bottom of each figure, respectively.

SP-IR was observed in the myenteric plexus as well as in the submucosal plexus, but the number of SP-immunoreactive neurons was more numerous in the submucosal plexus than in the myenteric plexus (Figure 1). CB-IR was prominent in both plexuses, but CB-immunoreactive neurons were more abundant in the submucosal plexus than in the myenteric plexus. CR-IR cells and their immunoreactivities were more abundant in the myenteric plexus than in the submucosal plexus and PA-IR was observed only in the epithelia of villi and the intestinal crypt but not in the plexuses. The CB-IR of the submucosal plexus gradually decreased on progressing from the duodenum to the ileum in both plexuses and the number of CR positive neurons was similar in the three portions of the small intestine.
 
 

Figure 1. Ratio of immunoreactive cells for calcium binding proteins in neurons of the myenteric and submucosal plexuses in the Korean native goat small intestine.

In the myenteric and submucosal plexuses, most of the CB immunoreactive neurons were immunoreactive for SP. The colocalization of CB with CR, CGRP and GAL was also confirmed in both plexuses, but the number of these double-immunoreactive cells was much fewer than that of cells doubly immunoreactive for CB and SP. SP containing neurons gradually decreased in number on progressing from duodenum to the ileum in the submucosal plexus, but increased in the myenteric plexus as shown in Figure 2.

Figure 2. Ratio of immunoreactive cells for substance P, CGRP and galanin in neurons of the myenteric and submucosal plexuses in the Korean native goat small intestine.

Figure 3. One section of micrographs of longitudinal sections of the submucosal (upper plates) and myenteric (lower plates) plexuses in Korean native goat small intestine. The sections were immunostained with one of anti-calbindin D-28k (CB), calretinin (CR), parvalbumin. a, b : duodenum  c, d : jejunum   e, f : ileum (PA a, b only a in Fig. 3). To view the complete image (6 megabytes) click here. This may take several minutes
 

Fig. 4. Micrographs of longitudinal sections of the submucosal (upper plates) and myenteric (lower plates) plexuses in Korean native goat small intestine. ), substance P (SP), calcitonin gene-related peptide (CGRP) and galanin (GAL), and visualized by DAB (×200). a, b : duodenum  c, d : jejunum   e, f : ileum. To view the complete image (6 megabytes) click here. This may take several minutes.
 

Fig. 5. Micrographs of double-immunostained sections of the submucosal (upper plates) and myenteric (lower plates) plexuses of the duodenum Korean native goat small intestine (duodenum). The sections were double-immunoreacted with the mixture of anti-mouse CB and one of anti-rabbit CR, SP and then fluoresced by Cy3-conjugated IgG (CB; left plates) and FITC-conjugated IgG (CR and SP; right plates) (×200).  a, b : submucosal plexus  c, d : myenteric plexus. To view the complete image (6 megabytes) click here. This may take several minutes.

Fig. 6. Micrographs of double-immunostained sections of the submucosal (upper plates) and myenteric (lower plates) plexuses of the duodenum Korean native goat small intestine (duodenum). The sections were double-immunoreacted with the mixture of anti-mouse CB and one of anti-rabbit CGRP and GAL, and then fluoresced by Cy3-conjugated IgG (CB; left plates) and FITC-conjugated IgG (CGRP and GAL,; right plates) (×200).  a, b : submucosal plexus  c, d : myenteric plexus. To view the complete image (6 megabytes) click here. This may take several minutes.
 

Recently, many studies have provided data on the chemical coding within the myenteric and submucosal plexuses in various animals (Rasmussen et al., 2001;Hens et al., 2000). We report for the first time on the results of a similar approach on the myenteric and submucosal neurons in the goat small intestine. Based on their neurochemical coding, we identified the distributions of some neuropeptides and calcium binding proteins. We found four subpopulations of co-localization, i.e., CB/CR, CB/SP, CB/CGRP and CB/GAL, which is a further indication of the multiple transmitter mechanisms in enteric nerves in general.

Our chemical coding evaluation is based on the relative counts of antigen-immunoreactive cell bodies. Neuron specific enolase (NSE)-IR was used as a nonspecific marker for cell bodies, because NSE antibodies have been previously used as a tool for locating enteric ganglia and nerve fibers in the intestine (Vento et al., 2001).

The distribution of CB, CR, PA, SP, CGRP and GAL immunoreactivity has been demonstrated immunohistochemically in neurons and their processes in the goat small intestine. It is known that there are several histologically distinct intrinsic neuronal types in the ENS. These include excitatory and inhibitory motor neurons to the muscle, vasomotor neurons, secretomotor neurons, interneurons and sensory neurons. Many investigators have tried to relate these physiological functions to individual cytoarchitecturally defined enteric neurons (Furness et al., 1987; Furness et al., 1988; Gabella, 1971). Classically, Dogiel classified enteric neurons into three types. He suggested that type-I cells were motor neurons as their axons extended towards muscle. Moreover, because many of the CR, SP and GAL-positive neurons were Dogiel type-I neurons and their fibers were dense in both the muscular layer and the myenteric plexuses, it was suggested that CR, SP and GAL-immunoreactive neurons in the myenteric plexus may be associated with the control of the gut motility.
Although the patterns of staining in the submucosal and myenteric plexuses were quite different in some neuronal subpopulations compared with other animals, there are few striking similarities.

In the goat, CB-immunoreactive neurons were more abundant in the submucosal plexus than in the myenteric plexus. This result is contradictory to that found in the rat, guinea pig, human and monkey (Clerc et al., 1998; Goodman and Iversen, 1986; Walters et al., 1993; Resibois et al., 1988). In contrast to CB, CR-immunoreactive neurons were more abundant in the myenteric plexus, as found in the guinea pig. However, though this result is similar to that of the guinea-pig, it does not agree with those of the human (Clerc et al., 1998; Goodman and Iversen, 1986).

CB-containing neurons have been reported to be present in the intestinal myenteric plexus of various species, including, the guinea pig, rat, pig, domestic fowl and the human (Furness et al., 1988; Scheuermann et al., 1991; Walters et al., 1993; Lunam, 1993). In the guinea pig small intestine, about one-third of all myenteric nerve cells have been reported to be immunoreactive for CB and believed to be Dogiel type II neurons (Furness et al., 1988). These cells have the electrophysiological characteristics of AH/type 2 neurons (Iyer et al., 1988), and may be considered to be intrinsic sensory neurons (Furness et al., 1988). In the goat, CB-IR cells also have the Dogiel type II morphology. Because CB-positive Dogiel type II neurons actually perform a sensory function in the small intestine, neurons doubly immunoreactive for CB and SP or other peptides are thought to have a sensory function that may modulate the sensation of the gut lumen. The results that CB and SP-IR neurons are more abundant in the duodenum than in the jejunum and ileum indicates that they have more important roles in the duodenum during absorption or digestion. On the other hand, CR-immunoreactive neurons with the morphological characteristics of Dogiel type I cells may modulate the motility of muscles in both the inner and outer layers.

Although the functions of SP-IR neurons have not been elucidated, the abundance of SP-IR varicose fibers within both muscle layers suggests that most of the SP cells might function as motor neurons. SP-immunoreacted neurons colabeled with CR may be motor neurons to the longitudinal muscle, as has been suggested for the small intestine (Brooks et al., 1992).

In a variety of species, SP has been shown to be the major excitatory neurotransmitters of motor neurons in the gastrointestinal tract (Holzer and Lippe, 1984; Maggi et al., 1994). However, a significant difference in the density of SP was noticed among the different regions of the digestive tract. In contrast to small animals in which SP-IR neurons were found in the submucosal and myenteric plexuses, SP-IR is absent or very weak in the plexuses of large animals. Pearson (1994) reported that in the horse, SP immunoreactivity was not observed in any of the neurons of the submucosal plexus and that only weak SP-immunoreactivity was seen in the myenteric plexus. In the pig, Hens et al (2000) reported SP-immunoreactive neurons and fibers in both plexuses, while Schmidt et al (1991) reported that only SP-IR fibers were evident in the myenteric plexus.

A possible explanation for this discrepancy might be that ganglionic organization is different between small and large animals. The small intestines of large animals such as the horse, pig and dog are relatively thick. In these animals, ENS is composed of three ganglia, whereas that of small animals such as the rat, mouse and guinea-pig is composed of two. The submucosal plexuses of large animals are subdivided into an inner submucosal plexus, which is located close to the abluminal side of the lamina musclularis mucosa, and an outer submucosal plexus lying adjacent to the luminal side of the circular muscle layer (Timmermann et al., 1997). In these animals, therefore, the SP-IR fibers of the inner circular muscle layer originated from the outer submucosal plexus rather than the myenteric plexus. Although the small intestine of the goat is composed of three ganglia (data not shown), the distribution parttern of SP-IR neurons is quite unlike that of the horse and the pig. This result indicates that the origins of the fibers located in the inner circular muscle may be the inner submucosal and myenteric plexuses. But the retrograde tracing studies remain to be elucidated.
The occurrence, function and frequency of CGRP fibers and neurons in the myenteric and submucosal ganglia have been reported to differ markedly from one species to another. Whereas the guinea-pig was more or less devoid of myenteric CGRP fibers in the intestine (Goodman and Iversen, 1986), such fibers and neurons were numerous in the human (Timmermann et al, 1992), pig (Hens et al., 2000) and dog (Sternini et al., 1992). This difference may be the result of the morphological characteristics of the small intestine.

In this study, the immunoreactivities of GAL were denser in the jejunum and ileum than in the duodenum. These results may reflect the fact that the major roles of the jejunum and ileum are peristaltic movement and rhythmic segmentation, and that these are more potent in the goat than in the other animals, because the role of GAL in the ENS is known to be excitatory (Korolkiewicz et al., 2000). The localization of CB, CR, SP, CGRP and GAL in the enteric neurons of the intestinal wall showed that these substances may have an important role in intestinal movement. The colocalization of CB with SP, CGRP or GAL in the myenteric and submucosal plexuses provides anatomical support that CB may serve a neuromodulatory role for SP-, CGRP-, and GAL-immunoreactive neurons in terms of intestinal wall motility. Distributions of CB, CR and SP in the goat small intestine differed from those of the guinea pig and pig. This suggests that the mechanisms and neurotransmitters involving enteric circuits in the goat differ from those in other animals. Moreover, this difference may be relevant to the morphological characteristics of digestive tracts.

In conclusion, this study demonstrates that some striking species differences may occur in the peptide and calcium binding protein distributions of the goat and the more extensively studied animals, like guinea-pig.

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Fig. 3. Micrographs of longitudinal sections of the submucosal (upper plates) and myenteric (lower plates) plexuses in Korean native goat small intestine. The sections were immunostained with one of anti-calbindin D-28k (CB), calretinin (CR), parvalbumin. a, b : duodenum  c, d : jejunum   e, f : ileum (PA a, b only a in Fig. 3).

Fig. 4. Micrographs of longitudinal sections of the submucosal (upper plates) and myenteric (lower plates) plexuses in Korean native goat small intestine. ), substance P (SP), calcitonin gene-related peptide (CGRP) and galanin (GAL), and visualized by DAB (×200). a, b : duodenum  c, d : jejunum   e, f : ileum.

Fig. 5. Micrographs of double-immunostained sections of the submucosal (upper plates) and myenteric (lower plates) plexuses of the duodenum Korean native goat small intestine (duodenum). The sections were double-immunoreacted with the mixture of anti-mouse CB and one of anti-rabbit CR, SP and then fluoresced by Cy3-conjugated IgG (CB; left plates) and FITC-conjugated IgG (CR and SP; right plates) (×200).  a, b : submucosal plexus  c, d : myenteric plexus.

Fig. 6. Micrographs of double-immunostained sections of the submucosal (upper plates) and myenteric (lower plates) plexuses of the duodenum Korean native goat small intestine (duodenum). The sections were double-immunoreacted with the mixture of anti-mouse CB and one of anti-rabbit CGRP and GAL, and then fluoresced by Cy3-conjugated IgG (CB; left plates) and FITC-conjugated IgG (CGRP and GAL,; right plates) (×200).  a, b : submucosal plexus  c, d : myenteric plexus.

©1996-2002 All Rights Reserved. Online Journal of Veterinary Research. You may not store these pages in any form except for your own personal use. All other usage or distribution is illegal under international copyright treaties. Permission to use any of these pages in any other way besides the before mentioned must be gained in writing from the publisher. This article is exclusively copyrighted in its entirety to OJVR publications. This article may be copied once but may not be, reproduced or re-transmitted without the express permission of the editors.