the enteric nervous system ( ens ) is composed of the cell bodies and fibers that are localized between the muscle layers throughout the entire gastrointestinal tract .
the ens integrates the motility , secretion , blood flow and immune response of the digestive tract , independent of the extrinsic autonomic inputs .
the function of the ens is to coordinate the complex interactions of the enteric networks , which consist of sensory , inter , motor and secremotor neurons .
the myenteric plexus is positioned between the outer longitudinal and inner circular muscle layers throughout the digestive tract .
since the myenteric plexus modulates the contraction and relaxation of the smooth muscles , this plexus is an endogenous source for motor innervation to the muscular layers and secremotor innervation to the mucosa .
similar to other nervous systems , the functions of the myenteric plexus are mediated by various neurochemical substances .
indeed , substance p ( sp ) was isolated from the gut   and can be detected in a dense nerve fiber network within the myenteric plexus .
sp - containing nerve fibers are an intrinsic contractor of the longitudinal muscle layer . in addition , calcitonin gene - related peptide ( cgrp ) , which was originally identified as a splicing product of the alternative rna processing of the calcitonin gene in the rat brain , affects a variety of biological activities in the ens , such as release of gastrointestinal hormone , co - ordination of gastrointestinal motility , excitation of myenteric neurons and vasodilatation .
on the other hand , vitamin - d dependent calcium - binding proteins ( cabps ) were first isolated from the small intestine of the chicken , thus they were presumed to play a primary role in the absorption of dietary calcium .
thereafter , it was discovered that cabps transfer calcium across the membranes and regulate free intracellular calcium .
thus , cabps act as " buffer / transport " proteins in various cell types . among them , calbindin d-28k ( cb ) and calretinin ( cr ) are observed in ens neurons characterized by a distinct morphology and specific electrophysiological properties .
the ruminant stomach serves as a large fermentation chamber , and the motility patterns therein maintain vigorous mixing of the ingested food .
neuronal regulation is therefore crucial for the regulation and coordination of region - specific motility in the ruminant stomach .
as the exogenous autonomic nervous system is known to play a role in controlling stomach movement , it has been implied that the endogenous ens may also be involved in regulating the local forestomach functions . however , little data are available to elucidate the neurochemical properties of the ens in ruminants .
thus , we investigated the localization of cb , cr , cgrp and sp immunoreactivity in the myenteric plexus of the goat stomach to characterize the ens in ruminants .
twelve goats ( capra hircus , 10 - 16 months , 15 - 20 kg b.w . ) were used in this study .
all experiments were carried out in accordance with the nih guide for the care and use of laboratory animals .
the animals were anesthetized with ketamine - xylazine mixture and perfused via the common carotid artery with 3 l of 0.9% normal saline followed by 8 l of 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . the stomach was immediately removed and dissected .
the stomach contents were washed by rinsing in ice - cold 0.1 m phosphate buffer .
the tissues used for cryostat sectioning were stretched and pinned flat on pieces of balsa wood .
the tissues were then fixed in 0.1 m phosphate buffer containing 4% paraformaldehyde for 12 h at 4 with the mucosal surface facing up .
the fixed tissues were washed in 0.1 m phosphate buffer and cryoprotected in 0.1 m phosphate buffer containing 30% sucrose .
serial sections of 12 m were cut using cryostat ( reichert - jung , germany ) and mounted on gelatin - coated slides .
the sections were pre - incubated in phosphate - buffered saline ( pbs ) containing 10% normal goat serum ( santa cruz , usa ) for 30 min to reduce nonspecific background staining .
the primary antibodies were diluted in 0.1 m pbs containing 0.3% triton x-100 and 2% normal goat serum .
the tissues were incubated for 48 h at 4 in the solution containing primary antibodies .
the following antisera were used at the indicated concentrations : mouse anti - cb ( 1 : 1,000 ; swant , switzerland ) , rabbit anti - cr ( 1 : 1,000 ; chemicon , usa ) , rabbit anti - sp ( 1 : 2,000 ; peninsula , usa ) and rabbit anti - cgrp ( 1 : 2,000 ; peninsula , usa ) .
after the specimens were incubated with the primary antibodies , they were washed three times for 10 min with pbs and incubated for 2 h in buffer solution containing affinity - purified secondary anti - rabbit or -mouse antibodies conjugated to indocarbocyanine ( cy3 ) .
finally , the specimens were washed in pbs and coverslipped with a solution of pbs containing 80% glycerol .
the slide preparations were observed under an olympus bx51 microscope ( olympus , japan ) attached to an imt2000 digital camera ( imtechnology , korea ) with the appropriate filters ( green filter ; exciter filter 513~565 nm ; beam filter 580 nm ; barrier filter 590 nm ) .
the images of immunoreactivity in the myenteric plexus were captured using adobe photoshop version 6.0 software via imt2000 .
twelve goats ( capra hircus , 10 - 16 months , 15 - 20 kg b.w . ) were used in this study .
all experiments were carried out in accordance with the nih guide for the care and use of laboratory animals .
the animals were anesthetized with ketamine - xylazine mixture and perfused via the common carotid artery with 3 l of 0.9% normal saline followed by 8 l of 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . the stomach was immediately removed and dissected .
the stomach contents were washed by rinsing in ice - cold 0.1 m phosphate buffer .
the tissues used for cryostat sectioning were stretched and pinned flat on pieces of balsa wood .
the tissues were then fixed in 0.1 m phosphate buffer containing 4% paraformaldehyde for 12 h at 4 with the mucosal surface facing up .
the fixed tissues were washed in 0.1 m phosphate buffer and cryoprotected in 0.1 m phosphate buffer containing 30% sucrose .
serial sections of 12 m were cut using cryostat ( reichert - jung , germany ) and mounted on gelatin - coated slides .
the sections were pre - incubated in phosphate - buffered saline ( pbs ) containing 10% normal goat serum ( santa cruz , usa ) for 30 min to reduce nonspecific background staining .
the primary antibodies were diluted in 0.1 m pbs containing 0.3% triton x-100 and 2% normal goat serum .
the tissues were incubated for 48 h at 4 in the solution containing primary antibodies .
the following antisera were used at the indicated concentrations : mouse anti - cb ( 1 : 1,000 ; swant , switzerland ) , rabbit anti - cr ( 1 : 1,000 ; chemicon , usa ) , rabbit anti - sp ( 1 : 2,000 ; peninsula , usa ) and rabbit anti - cgrp ( 1 : 2,000 ; peninsula , usa ) .
after the specimens were incubated with the primary antibodies , they were washed three times for 10 min with pbs and incubated for 2 h in buffer solution containing affinity - purified secondary anti - rabbit or -mouse antibodies conjugated to indocarbocyanine ( cy3 ) .
finally , the specimens were washed in pbs and coverslipped with a solution of pbs containing 80% glycerol .
the slide preparations were observed under an olympus bx51 microscope ( olympus , japan ) attached to an imt2000 digital camera ( imtechnology , korea ) with the appropriate filters ( green filter ; exciter filter 513~565 nm ; beam filter 580 nm ; barrier filter 590 nm ) .
the images of immunoreactivity in the myenteric plexus were captured using adobe photoshop version 6.0 software via imt2000 .
in the present study , cb , cr , sp , and cgrp immunoreactivities were observed in the nerve cell bodies or fibers of the stomach myenteric plexuses of the korean native goat .
cb immunoreactivity was found in the nerve cell bodies and fibers of the rumen , reticulum and abomasum ; however , only cb - immunoreactive ( cb - ir ) nerve fibers were observed in the omasum ( fig .
cb - ir neurons were oval - shaped with generally smooth margins and were classified as dogiel type ii cells based on dogiel 's classification for enteric neurons .
the average number of cb - ir cell bodies in the myenteric plexus was 7.8 cells / ganglion .
cr - immunoreactive ( cr - ir ) fibers were found in all subregions of the stomach ; in particular , varicosities of cr - ir fibers were dominantly observed in the reticulum ( fig .
sp - immunoreactive ( sp - ir ) neurons and fibers were observed in all stomachs , except in the omasum .
cgrp - immunoreactive ( cgrp - ir ) fibers were found in all subregions of the stomach , and varicosities of cgrp - ir fibers were prominently observed in the myenteric plexus of goat stomach , compared to cb- , cr- , and sp - ir fibers ( fig .
in the present study , we observed cb- , cr- , cgrp- and sp - ir myenteric neurons within the goat stomach .
it is known that there are several histologically distinct types of intrinsic neurons in the ens .
these include excitatory and inhibitory motor neurons to the muscle , vasomotor neurons , secremotor neurons , interneurons and sensory neurons .
many investigators have tried to relate these physiological functions to individual cytoarchitecturally defined enteric neurons .
although the ens can function independently of the cns , the latter has an important role in the coordination of the diverse functions of the ens .
the ens is well connected to the central autonomic neural network in the cns through both motor and sensory pathways of the sympathetic and the parasympathetic nervous system .
the parasympathetic motor pathways consist of the vagus and sacral nerves that control motor and secremotor functions .
these neurons are located in the smooth muscle layer and are sensitive to mechanical distention of the gut . in the goat stomach , the cb - ir neurons observed in the myenteric plexus were identified as morphological dogiel type ii cells featuring smooth perikarya with long multiple processes .
cb - ir cells are known to have the electrophysiological characteristics of after - hyperpolarization ( ah ) , and may be considered to be intrinsic sensory neurons .
moreover , the cb - ir dogiel type ii neurons located in the myenteric plexus of the guinea - pig ileum are known to be cholinergic and to activate the intrinsic primary afferent neurons . in the guinea - pig gi tract ,
the somata of cb - containing primary afferent neurons in the intestine of several species are located within the myenteric plexus and project to the epithelium .
based on these previous studies , cb - ir neurons in the myenteric neurons of goat stomach may also be sensory neurons that project to the epithelium .
these findings are similar to another study detailing the profiles of in the equine stomach .
therefore , our findings indicate that the localization / functionality of cb in myenteric plexus of the goat may be similar to that of other animals . in the present study , cr - immunoreactivity
, cr is known to be present in the myenteric neurons , and related with sensory function of circular muscle layer and villi .
moreover , cr - ir neurons are identified as small dogiel type i neurons and sensory intrinsic neurons .
however , cr - ir was only found in the myenteric nerve fibers of goat stomach in this study .
these data show that there are differences in the distributions and functions of cr in goat stomach compared to that of other animals . in a variety of species
, sp has been shown to be the major excitatory neurotransmitter of motor neurons in the gastrointestinal tract . however , a significant difference in the density of sp has been reported in the various regions of the digestive tract ; sp - ir neurons were found in the submucosal and myenteric plexuses of small animals , while sp - ir neurons were rarely observed in large animals .
indeed , in the horse , sp - immunoreactivity was not observed in the submucosal plexus , and was only weakly detected in the myenteric plexus . in the pig
this discrepancy might be related to the differential ganglionic organization between small and large animals . in the present study ,
since sp affects the contraction of the smooth muscle of the stomach , this peptide stimulates gastric motility and increases the intragastric pressure .
cgrp is a ' marker peptide ' of the dogiel type ii cell population in the ganglionated plexuses of the porcine small intestine and has a sensory function .
however , we did not observe cgrp - ir myenteric neurons in the goat stomach . although the function of cgrp - ir neurons in the goat stomach is unclear ,
our findings indicate that there is species - specific localization of cgrp - ir cells in the myenteric plexus .
similar to cb , the distribution of cgrp - ir fibers indicate that cgrp may be involved in the sensory functions of the stomach , which initiate mixing of the ingesta , regurgitation of the forestomach ingesta , and signal satiety . in conclusion ,
the immunohistochemical localization of cb , cr , cgrp and sp in the myenteric plexus of the goat stomach displayed species - specific patterns .
these findings suggest that these substances may be directly and/or indirectly related to the gastric functions of the goat stomach .
 to understand the neurochemical properties of the gastric myenteric plexus of ruminants , the expression patterns of calbindin d-28k ( cb ) , calretinin ( cr ) , substance p ( sp ) and calcitonin gene - related peptide ( cgrp ) were explored in the korean native goat . in gastric myenteric plexus , cb and sp immunoreactivity were observed in round- or oval - shaped neurons . 
 cr and cgrp immunoreactivity were detected only in the nerve fibers . 
 this immunohistochemical localization of cb , cr , cgrp and sp in the myenteric plexus of the goat stomach exhibited species - specific patterns . 
 these findings suggest that these substances may be directly or indirectly related to the gastric functions of the goat stomach . 
Introduction
Materials and Methods
Animal and Tissue Preparation
Immunohistochemistry
Results
Discussion