CCK-A receptor activation induces fos expression in myenteric neurons of rat small intestine

Cholecystokinin (CCK), a hormone secreted from endocrine cells of the small intestine, participates in the control of motility and secretion in the gastrointestinal tract, and in the control of food intake. At least some of the effects of CCK on intestinal function appear to be mediated via activation of intrinsic neurons in the myenteric plexus. However, the distribution of CCK-responsive enteric neurons within the rat small intestine is not known. Neither has the role of CCK-A receptors in the activation of rat myenteric neurons been investigated. Therefore, to determine the distribution of CCK-responsive neurons in the small intestinal myenteric plexus we utilized immunohistochemical detection of Fos, the protein product of the immediate early gene c-fos, to identify neurons that were activated by exogenous CCK. We also monitored Fos expression in the dorsal hindbrain, and examined CCK-induced Fos expression in the presence or absence of a receptor antagonist for the type-A CCK receptor. We found that CCK significantly increased Fos expression in the hindbrain and in myenteric neurons of the duodenum and jejunum, but not the ileum. Neuronal Fos responsiveness in both brain and myenteric neurons was mediated by CCK-A receptors, as CCK-induced Fos expression was eliminated in rats pretreated with a CCK-A receptor antagonist. We conclude that CCK activates small intestinal myenteric neurons, via CCK-A receptors. Activation of these intrinsic intestinal neurons may participate in reflexes and behaviors that are mediated by CCK.     

Cannabinoid CB2 receptors in the enteric nervous system modulate gastrointestinal contractility in lipopolysaccharide-treated rats

Enhanced intestinal transit due to lipopolysaccharide (LPS) is reversed by cannabinoid (CB)2 receptor agonists in vivo, but the site and mechanism of action are unknown. We have tested the hypothesis that CB2 receptors are expressed in the enteric nervous system and are activated in pathophysiological conditions. Tissues from either saline- or LPS-treated (2 h; 65 microg/kg ip) rats were processed for RT-PCR, Western blotting, and immunohistochemistry or were mounted in organ baths where electrical field stimulation was applied in the presence or absence of CB receptor agonists. Whereas the CB2 receptor agonist JWH133 did not affect the electrically evoked twitch response of the ileum under basal conditions, in the LPS-treated tissues JWH133 was able to reduce the enhanced contractile response in a concentration-dependent manner. Rat ileum expressed CB2 receptor mRNA and protein under physiological conditions, and this expression was not affected by LPS treatment. In the myenteric plexus, CB2 receptors were expressed on the majority of neurons, although not on those expressing nitric oxide synthase. LPS did not alter the distribution of CB2 receptor expression in the myenteric plexus. In vivo LPS treatment significantly increased Fos expression in both enteric glia and neurons. This enhanced expression was significantly attenuated by JWH133, whose action was reversed by the CB2 receptor antagonist AM630. Taking these facts together, we conclude that activation of CB2 receptors in the enteric nervous system of the gastrointestinal tract dampens endotoxin-induced enhanced intestinal contractility.     

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.     

Gonadotropin-releasing hormone analog buserelin causes neuronal loss in rat gastrointestinal tract

Gonadotropin-releasing hormone (GnRH) analogs are given to women undergoing in vitro fertilization. Case reports describing the development of chronic intestinal pseudo-obstruction and auto-antibodies against GnRH after such treatment suggest a strong association between intestinal dysfunction and GnRH analogs. No experimental model for studying such a relationship is currently at hand. Our main goal was to investigate possible enteric neurodegeneration and titers of GnRH antibodies in response to repeated administration of the GnRH analog buserelin in rat. Rats were treated for 1–4 sessions with daily subcutaneous injections of buserelin or saline for 5 days, followed by 3 weeks of recovery. Buserelin treatment caused significant loss of submucous and myenteric neurons in the fundus, ileum, and colon. The loss of enteric neurons can, at least partly, be explained by increased apoptosis. No GnRH- or GnRH-receptor-immunoreactive (IR) enteric neurons but numerous luteinizing hormone (LH)-receptor-IR neurons were detected. After buserelin treatment, the relative number of enteric LH-receptor-IR neurons decreased, whereas that of nitric-oxide-synthase-IR neurons increased. No intestinal inflammation or increased levels of circulating interleukins/cytokines were noted in response to buserelin treatment. Serum GnRH antibody titers were undetectable or extremely low in all rats. Thus, repeated administrations of buserelin induce neurodegeneration in rat gastrointestinal tract, possibly by way of LH-receptor hyperactivation. The present findings suggest that enteric neurodegenerative effects of GnRH analog treatment in man can be mimicked in rat. However, in contrast to man, no production of GnRH auto-antibodies has been noted in rat.     

Ca(2+) permeability of human heteromeric nAChRs expressed by transfection in human cells

The distribution of the calcium binding protein neurocalcin a has been examined in the enteric nervous system of young adult (3 months) and aged (24+ months) male rats by immunofluorescence. Neurocalcin-immunoreactive (NC-ir) neurons were observed in the submucous and myenteric plexuses throughout the gastrointestinal tract from the oesophagus to the distal large intestine. NC-ir nerve terminals were also seen on NC-ir and NC-negative neurons. Semiquantitative estimates revealed fewer NC-ir neurons in the submucous plexus than in the myenteric plexus. The greatest occurrence of NC-ir neurons was in the small and large intestine. NC-ir axons were seen in the mucosa and also in between the ganglia of the myenteric plexus. In the aged rats, there were no discernible changes in the numbers of NC-ir neurons in th e oesophagus and stomach, with an increase in the pylorus and slight decreases in the small and large intestines. No decrease in NC-ir was observed in the distal large intestine. NC-ir neurons never contained lipofuscin age pigment and many enteric neuro ns devoid of NC-ir contained age pigment. Like other previously investigated calcium-binding proteins in enteric neurons, the distribution of NC shows much variability from one part of the intestine to another. The observed slight decreases in the number of NC-ir enteric neurons in aged rats may compromise the regulation of calcium in these neurons.     

Neuronal and glial localization of GABA transporter immunoreactivity in the myenteric plexus

The neurotransmitter gamma-aminobutyric acid (GABA) is removed from the extracellular space by sodium and chloride dependent high affinity plasma membrane transporters. In the rat central nervous system, three GABA transporters, GAT1, GAT2 and GAT3, have been cloned and localized by immunohistochemistry. The purpose of this study was to examine the distribution of these transporters within the myenteric plexus of the rat gastrointestinal tract. We investigated their cellular locations using GAT1-3 specific antisera in lightly fixed segments of rat duodenum, ileum and colon. Immunohistochemistry revealed a large number of GAT2-immunoreactive structures that surrounded neurons within each ganglion of the myenteric plexus. GAT2 was colocalized in these structures with the glial cell marker p75(NTR), suggesting that the predominant high affinity GABA transporter within enteric glia is GAT2. GAT3 immunoreactivity was localized within many nerve cell bodies, and no labeling for GAT1 was detected, although it was present in retina, which was used as a control. Double labeling for calretinin and nitric oxide synthase (NOS) revealed colocalization of GAT3 with approximately 75% of calretinin-immunoreactive neurons and 15% of NOS-immunoreactive neurons. This suggests that a small proportion of inhibitory motor neurons and at least some putative intrinsic primary afferent neurons within the rat gastrointestinal tract express GAT3. Thus NOS neurons, which appear to utilize GABA as a transmitter, and calretinin-immunoreactive neurons, which do not appear to be GABAergic, both express immunoreactivity for GABA transporters.     

Immunohistochemical and in situ hybridization studies of gonadotropin releasing hormone (GnRH) and its receptor in rat digestive tract

GnRH(LH-RH) is first discovered in the hypothalamus and found to have a role in regulation of reproduction. With the study on it deepening, GnRH was demonnstrated that it also exists in a number of organs beyond the hypothalamus and acts on extrapituitary organs. To study whether digestive tract synthesizes GnRH and its receptor and, if it does, by what cells. In the experiment, the locallizations of GnRH and its receptors in rat digestive tract were studied using immunohistochemistry and in situ hybridization. The parietal cells of gastric gland, the villous and glandular epithelium in small and large intestine and parasympathetic ganglion cells of myenteric plexus showed GnRH immunoreactivity; GnRH mRNA hybridization signal was detected. The epithelium of gastric pit and the cells above in digestive tract showed GnRH receptor immunoreactivity; GnRH receptor mRNA hybridization signal was detected. The immunoreactive and signal materials distributed in cytoplasm of all positive cells, with nuclei being immunonegative and with no hybridization signal. These results suggested that the digestive tract can produce GnRH and express GnRH receptor; GnRH may also be a gastrointestinal hormone.     

Novel cyclic azo-bridged analogs of gonadotropin-releasing hormone.

Five linear analogs of GnRH containing a p-aminophenylalanine (Pap) residue in their sequence and their six corresponding azo-bridged cyclic derivatives were synthesized. The precyclic peptides were prepared on solid-support, while azo-cyclization was performed in solution by diazotization of the p-aminophenylalanine residue followed by intramolecular coupling of the formed diazo salt with either tyrosine or histidine side chains present in the sequence. All peptides were examined for their binding ability to the GnRH receptor expressed on rat pituitary membranes and for their LH-release activity from dispersed rat pituitary cells. Linear analogs 1 i.e [Pap(5)] GnRH and 3, i.e. [Tyr(3), Pap(5)] GnRH, were found to bind to the GnRH receptors only slightly less avidly than native GnRH. Their cyclization, however, led to a marked reduction in the binding capacity, i.e. from IC(50) of 10(-9) M to the 10(-7) M range, and in biopotency, i.e. LH-release. All other linear and cyclic peptides were found to bind selectively to the GnRH receptor only in the low microM range. Only peptide 1 was found comparable to native GnRH in respect to LH-release activity and thus may potentially be a good agonist of the parent peptide. Peptides 1-4, the most potent GnRH receptor binders, were examined for their conformational properties using CD. Cyclic-azo peptides 2 and 4 were further evaluated by NMR spectroscopy in solution combined with molecular modeling. The structural information obtained explains in part the GnRH-like biological activity observed.     

Glutamate Receptor Subunit Expression in Primary Enteric Glia Cultures

Excitotoxicity, which is mediated via glutamate receptors, is also a phenomenon of the enteric nervous system. Whether enteric glial cells (EGCs), which resemble astrocytes of the central nervous system, express glutamate receptors and hence are involved in gut excitotoxicity is not yet known. To investigate glutamate receptor subunit expression in EGCs, primary EGC cultures of the myenteric plexus were analyzed by real-time PCR and Western blotting. These studies indeed showed that in EGC cultures, mRNA of the glutamate receptor subunits NR1, NR2A/B, GluR1, GluR3, and GluR5 and the protein bands of the glutamate receptor subunits NR2A/B, GluR1, GluR3, and GluR5 could be detected. Thus, in the enteric nervous system, glutamate receptor subunits are also expressed by EGCs, indicating that these cells might be involved in gut excitotoxicity.     

Glutamate receptor subunit expression in primary enteric glia cultures

Excitotoxicity, which is mediated via glutamate receptors, is also a phenomenon of the enteric nervous system. Whether enteric glial cells (EGCs), which resemble astrocytes of the central nervous system, express glutamate receptors and hence are involved in gut excitotoxicity is not yet known. To investigate glutamate receptor subunit expression in EGCs, primary EGC cultures of the myenteric plexus were analyzed by real-time PCR and Western blotting. These studies indeed showed that in EGC cultures, mRNA of the glutamate receptor subunits NR1, NR2A/B, GluR1, GluR3, and GluR5 and the protein bands of the glutamate receptor subunits NR2A/B, GluR1, GluR3, and GluR5 could be detected. Thus, in the enteric nervous system, glutamate receptor subunits are also expressed by EGCs, indicating that these cells might be involved in gut excitotoxicity.     

Cholecystokinin-58 and cholecystokinin-8 produce similar but not identical activations of myenteric plexus and dorsal vagal complex

The enteric nervous system (ENS: myenteric and submucosal plexuses) of the gastrointestinal tract may have a role in the reduction of food intake by cholecystokinin (CCK). Exogenous cholecystokinin-8 (CCK-8) activates the myenteric plexus and the feeding control areas of the dorsal vagal complex (DVC) of the brainstem. An increasing number of reports, however, have shown that CCK-58 is the sole or the major circulating form of CCK in rat, human and dog, and that it is qualitatively different from CCK-8 in evoking various gastrointestinal physiological responses (e.g., contraction of the gallbladder and exocrine pancreatic secretion). In the current report, we compared the abilities of exogenous CCK-58 to activate the myenteric plexus and the dorsal vagal complex with those of exogenous CCK-8 by quantifying Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation). We report that CCK-58 (1, 3, and 5 nmol/kg) increased Fos-LI in the myenteric plexus (p<0.001) and in the DVC (p<0.001) compared to the saline vehicle. The highest dose of CCK-58 increased Fos-LI more than an equimolar dose of CCK-8 in the myenteric plexus and the area postrema. Thus, CCK-8 and CCK-58 produce the same qualitative pattern of activation of central and peripheral neurons, but do not provoke identical quantitative patterns at higher doses. The different patterns produced by the two peptides at higher doses, in areas open to the circulation (myenteric plexus and area postrema) may reflect endocrine actions not observed at lower doses.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

Proteomics of the rat gut: analysis of the myenteric plexus-longitudinal muscle preparation

The enteric nervous system (ENS)--present all along the gastrointestinal tract - is the largest and most complicated division of the peripheral nervous system that can function independently of the brain. The peripheral nerve cells are organized in two separate but interconnected meshworks, called the myenteric and submucous plexus. The nervous control of intestinal motility is primarily governed by the myenteric plexus (MP), which lies in-between the longitudinal- (LM) and circular-muscle layers and regulates their functions. To determine whether the proteomic technology is adapted to the analysis of specific gut tissues, we dissected the MP-LM layers from the jejunum, ileum, and colon of Long Evans rats, homogenized them, and separated the proteins using two-dimensional gel electrophoresis. A subset of all the visualized protein spots, covering the entire range of molecular weights and isoelectric points, was then selected and further analyzed by matrix-assisted laser desorption/ionization-time of flight and liquid chromatography mass spectrometry. We identified around 80 proteins in each gut segment, and among those, five were segment-specific. Most of the proteins identified were derived from muscle cells, but we also detected some neuron-specific proteins. This study represents, to our knowledge, the first extensive protein catalog of a neuromuscular layer of the rat intestine and it may constitute the basis to understand pathophysiological mechanisms related to the ENS.     

Transient expression of the calcitonin receptor by enteric neurons of the embryonic and early post-natal mouse

Calcitonin receptor-immunoreactivity (CTR-ir) was found in enteric neurons of the mouse gastrointestinal tract from embryonic day 13.5 (E13.5) to post-natal day 28 (P28). CTR-ir occurred in cell bodies in ganglia of the myenteric plexus extending from the esophagus to the colon and in nerve cells of the submucosal ganglia of the small and large intestines. CTR-ir was also found in vagal nerve trunks and mesenteric nerves. Counts in the ileal myenteric plexus revealed CTR-ir in 80% of neurons. CTR-ir was clearly evident in the cell bodies of enteric neurons by E15.5. The immunoreactivity reached maximum intensity between P1.5 and P12 but was weaker at P18 and barely detectable at P28. The receptor was detected in nerve processes in the intestine for only a brief period around E17.5, when it was present in one to two axonal processes per villus in the small intestine. In late gestation and soon after birth, CTR-ir was also evident in the mucosal epithelium. The perinatal expression of CTR within the ENS suggests that the calcitonin/CTR system may have a role in the maturation of enteric neurons. Signals may reach enteric neurons in milk, which contains high levels of calcitonin.     

Distribution and co-localization of calbindin D28k with VIP and neuropeptide Y but not somatostatin, galanin and substance P in the enteric nervous system of the rat

Calbindin D28k, previously demonstrated in the mammalian central nervous system, has been localized to discrete neurons in the enteric nervous system of the rat. Calbindin D28k is present in cell bodies in both the myenteric and submucous plexi and in interganglionic nerve fibers in all regions of the gastrointestinal tract. Immunoreactive nerve fibers were also detected in the mucosal region, although none were observed in the pyloric sphincter, circular or longitudinal muscle layers. The highest concentration of immunoreactivity was present in the submucosal plexus and mucosa of the colon. Western blot analysis of the protein detected by the antiserum confirmed that it comigrated with purified calbindin D28k and the single immunoreactive band seen in extracts from rat brain. The colocalization of calbindin D28k with components of the peptidergic innervation was also investigated. Of the peptides studied the neurons containing both vasoactive intestinal polypeptide and neuropeptide Y in the submucous plexus were seen to exhibit calbindin D28k immunoreactivity. The neurons containing somatostatin, galanin and substance P did not demonstrate co-localization. In the stomach, calbindin D28k was detected within a small number of epithelial cells which were found to correspond to a sub-population of the somatostatin-immunoreactive endocrine cells.     

Basic fibroblast growth factor, neurofilament, and glial fibrillary acidic protein immunoreactivities in the myenteric plexus of the rat esophagus and colon

The enteric nervous system consists of a number of interconnected networks of neuronal cell bodies and fibers as well as satellite cells, the enteric glia. Basic fibroblast growth factor (bFGF) is a mitogen for a variety of mesodermal and neuroectodermal-derived cells and its presence has been described in many tissues. The present work employs immunohistochemistry to analyze neurons and glial cells in the esophageal and colic enteric plexus of the Wistar rat for neurofilament (NF) and glial fibrillary acidic proteins (GFAP) immunoreactivity as well as bFGF immunoreactivity in these cells. Rats were processed for immunohistochemistry; the distal esophagus and colon were opened and their myenteric plexuses were processed as whole-mount preparations. The membranes were immunostained for visualization of NF, GFAP, and bFGF. NF immunoreactivity was seen in neuronal cell bodies of esophageal and colic enteric ganglia. GFAP-immunoreactive enteric glial cells and processes were present in the esophageal and colic enteric plexuses surrounding neuronal cell bodies and axons. A dense net of GFAP-immunoreactive processes was seen in the ganglia and connecting strands of the myenteric plexus. bFGF immunoreactivity was observed in the cytoplasm of the majority of the neurons in the enteric ganglia of esophagus and colon. The two-color immunoperoxidase and immunofluorescence methods revealed bFGF immunoreactivity also in the nucleus of GFAP-positive enteric glial cells. The results suggest that immunohistochemical localization of NF and GFAP may be an important tool in the study of the plasticity in the enteric nervous system. The presence of bFGF in neurons and glia of the myenteric plexus of the esophagus and the colon indicates that this neurotrophic factor may exert autocrine and paracrine actions in the enteric nervous system.     

急性力竭运动对大鼠胃肠传输速率及回肠肌间神经丛氮能神经的影响

目的：研究力竭运动对大鼠胃肠动力的影响及其肠神经机制。方法：24只大鼠随机分成对照组和急性力竭运动组,建立力竭运动大鼠模型,测定胃肠传输速率,用酶组织化学方法和计算机图像分析技术对两组大鼠回肠肌间神经丛内氮能神经元的数目和一氧化氮合酶（NOS）的表达进行测定。结果：急性力竭运动组大鼠胃肠传输速率明显延迟,回肠肌间神经丛内氮能神经元的数目明显增多和NOS的表达显著增强（P〈0．05和P〈0．01）。结论：大鼠力竭运动后小肠肌问神经丛内氮能神经元的数目增多和NOS的表达增强可能是导致胃肠传输速率延迟的重要原因之一。     

Oral inoculation with herpes simplex virus type 1 infects enteric neuron and mucosal nerve fibers within the gastrointestinal tract in mice.

Herpes simplex virus type 1 (HSV-1) is commonly encountered first during childhood as an oral infection. After this initial infection resolves, the virus remains in a latent form within innervating sensory ganglia for the life of the host. We have previously shown, using a murine model, that HSV-1 placed within the lumen of the esophagus gains access to nerves within the gut wall and establishes a latent infection in sensory ganglia (nodose ganglia) of the tenth cranial nerve (R. M. Gesser, T. Valyi-Nagy, S. M. Altschuler, and N. W. Fraser, J. Gen. Virol. 75:2379-2386, 1994). Peripheral processes of neurons in these ganglia travel through the vagus nerve and function as primary sensory receptors in most of the gastrointestinal tract, relaying information from the gut wall and mucosal surface to secondary neurons within the brain stem. In the work described here, we further examined the spread of HSV-1 through the enteric nervous system after oral inoculation. By immunohistochemistry, HSV-1 was found to infect myenteric ganglia in Auerbach's plexus between the inner and outer muscle layers of the gut wall, submucosal ganglia (Meisner's plexus), and periglandular ganglion plexuses surrounding submucosal glands. Virus-infected nerve fibers were also seen projecting through the mucosal layer to interact directly with surface epithelial cells. These intramucosal nerve fibers may be a conduit by which intraluminal virus is able to gain access to the enteric nervous system from the gastrointestinal lumen.     

Comparison of opioid receptor distributions in the rat ileum

The cellular expression patterns of mu-, delta- and kappa-opioid receptors in the rat ileum were examined using fluorescence immunohistochemistry. Double-labelling was used to examine cellular receptor co-localisation as a pre-requisite for intracellular molecular interactions, such as heterodimerisation. Tissues were stained as whole-mount preparations. Strong, broadly distributed immunoreactivity (ir) was observed for each receptor in the myenteric and submucous plexuses. Although intracellular mu- and delta-ir patterns differed in ganglion neurons, mu/delta co-expression was extensive in these cells. mu/delta co-expression was also observed in interstitial cells, which were diffusely distributed in submucous plexus preparations but generally located adjacent to myenteric plexus structures. Punctate kappa-ir was seen broadly in nerve fibres in both plexuses, suggesting localisation in varicosities. Neuronal mu/kappa co-localisation was not apparent, although kappa-ir fibres were often apposed against mu-ir cells. mu/kappa co-localisation was detected in interstitial cells in submucous plexus preparations. Similarities in mu and delta expression patterns might reflect similar functional properties previously detected for these receptors. This study indicates that the rat gastrointestinal tract might provide a useful tool for the future study of molecular interactions between opioid receptor types.