Extrinsic Fibers Research Articles

Both afferent and efferent nerves are implicated in cholecytokinin motor actions in the small intestine of the rat

Cholecystokinin (CCK) regulates intestinal motility after being released by several luminal nutrients. However the mechanism of action of CCK is still not well known. The aim of our study was to establish the mechanism of action of CCK in the rat intestine using an in vivo model and focusing on the nervous pathways involved in the response as well as type of receptors. Anesthetized rats were prepared with two strain-gauges, in duodenum and jejunum, to record circular muscle motor activity. A group of animals was also prepared with a catheter to infuse capsaicin inside the duodenum. Responses to CCK-octapeptide (CCK-8) as well as to CCK agonists were studied. CCK-8 was also infused after CCK antagonists, atropine, hexamethonium or L-nitroarginine. Results show that duodenal response to CCK-8 is excitatory although inhibitory responses can be induced by gastrin. In the jejunum, CCK-8 induces an inhibitory response that is mediated by both CCK-A and -B receptors. Excitatory responses to CCK-8 are due to stimulation of preganglionic receptors while inhibitory responses are NO mediated through stimulation of postganglionic CCK-B receptors. Capsaicin locally applied in duodenal mucosa significantly decreased CCK-8 response, whereas mucosal exposure to lidocaine completely blocked CCK-8 response. In conclusion our results show that CCK response varies along the intestine according to the predominance of excitatory or inhibitory efferent innervation. Moreover, CCK-8 actions are mediated through both extrinsic and intrinsic afferent fibres.

Root resorption and signs of repair in Papillon-Lefèvre syndrome. A case study

The aim of this investigation was to describe some tooth-related histological features of prepubertal periodontitis. Teeth extracted during treatment of two Papillon-Lefèvre syndrome patients were processed by means of the sawing and grinding technique. Light microscopy examination revealed little or no cementum in the coronal parts of the roots. Resorptions of various depths (0.02 to 1.5 mm) and to various extents (affecting up to 1/3 of the root surface) were observed in the 5 investigated teeth. Some resorptive defects on 1 of the examined incisors showed signs of spontaneous repair. Extrinsic fibers were inserted into the new cellular intrinsic fiber cementum which had formed directly on the bottom of the defect. Intact acellular extrinsic fiber cementum was found where fibers were still attached. Here, the characteristic of pristine cementum, a hyaline layer of peripheral dentin, could be identified. If resorption was not present, the cementum did not show any signs of hypoplasia. Thus, histological features of prepubertal periodontitis in the current material were (i) areas of extensive resorption, (ii) signs of spontaneous repair, and (iii) healthy cementum.

Excitatory and inhibitory modulation of taste responses in the hamster brainstem.

The rostral portion of the nucleus of the solitary tract (NST) contains second-order gustatory neurons, sends projections to the parabrachial complex and brainstem reticular formation, and receives descending projections from several nuclei of the ascending gustatory pathway. Electrophysiological responses of NST neurons can be modulated by several factors, including blood glucose and insulin levels and taste aversion conditioning. We are using extracellular electrophysiological recording in vivo, combined with local microinjection of neurotransmitter agonists and antagonists, to study the mechanisms by which taste responses of cells in the hamster NST can be modulated. Afferent fibers of the chorda tympani (CT) nerve make excitatory synaptic contact with NST neurons; this excitation is probably mediated by the excitatory amino acid glutamate. Microinjection of kynurenic acid, a nonspecific glutamate receptor antagonist, into the NST completely and reversibly blocks afferent input from the CT nerve, produced by either anodal electrical or chemical stimulation of the anterior tongue. The non-NMDA ((RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate) receptor antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) also completely blocks gustatory input to these cells, whereas the N-methyl-D-aspartate (NMDA) antagonist DL-2-amino-5-phosphonovalerate (APV) produces only a small effect. There are many gamma-aminobutyric acid (GABA)-containing neurons within the NST and taste-responsive NST cells are maintained under a tonic GABAergic inhibition. Microinjection of the GABAA receptor antagonist bicuculline methiodide increases the taste responsiveness of NST neurons, whereas application of GABA inhibits taste responses in these cells. Preliminary data show that GABAergic inhibition can be produced by stimulation of the gustatory cortex. There are both intrinsic substance P (SP)-containing neurons and extrinsic SP-immunoreactive fibers in the rostral NST. Microinjection of SP into the NST enhances the responses of many NST cells to gustatory stimulation; NaCl-best neurons are preferentially excited by SP.

Dual (excitatory and inhibitory) calretinin innervation of AMPA receptor-containing neurons in the rat lateral septum

A recent study demonstrated both an extrinsic and an intrinsic calretinin (CR) innervation of the rat septal complex and that a population of the extrinsic calretinin fibers is aspartate/glutamate-containing. The aim of this study was to determine which types (GluR1, GluR2/3, or both) of AMPA receptor-containing lateral septal area neurons are innervated by extrinsic and intrinsic CR neurons and whether the intrinsic CR cells are GABAergic. Light- and electron-microscopic single immunostaining for CR, GluR1, and GluR2/3, as well as light- and electron-microscopic-double immunostaining experiments for CR plus GluR1 and CR plus GluR2/3 were performed in the lateral septal area. Furthermore, the "mirror" colocalization technique was employed on consecutive vibratome sections of the septal complex to investigate whether the intrinsic septal CR neurons are GABAergic. The results are summarized as follows: (1) both GluR1- and GluR2/3-immunoreactive neurons are innervated by CR-containing fibers; (2) the majority of these synapses, observed mainly on the soma and, to a lesser extent, on proximal dendrites of AMPA receptor-containing neurons, represent asymmetric synaptic membrane specializations; (3) a minority of CR-containing axon terminals associated with both GluR1- and GluR2/3-immunoreactive neurons form symmetric contacts, predominantly on their soma; and (4) 93% of the lateral septal area CR cells are GABAergic. These observations indicate that both GluR1- and GluR2/3-containing lateral septal area neurons receive a dual intrinsic and extrinsic CR innervation. The former (intrinsic) CR boutons are GABAergic, while the latter form asymmetric synaptic contacts, are excitatory, and probably originate in the supramammillary area, since previous work has demonstrated that a population of supramammillo-septal fibers contain aspartate and/or glutamate.

Extrinsic intestinal reinnervation after canine small bowel autotransplantation

Background: The process of extrinsic reinnervation after small bowel transplantation is poorly understood. Methods: Jejunal and ileal specimens, obtained from the dogs that underwent intestinal autotransplantation by an end-to-end (E-E) or end-to-side arterial reconstruction, were analyzed at 1 (n = 7), 3 (n = 6), 6 (n = 6), 12 (n = 6), or 24 (n = 2) months and compared with control specimens (n = 7). Tissue catecholamine levels and indirect immunohistochemistry results for extrinsic neuropeptides, calcitonin gene–related peptide, neuropeptide Y, substance P, and tyrosine hydroxylase (TH) were examined. Results: Catecholamine levels in the grafts were undetectable until 6 months but increased significantly after 12 months, particularly in the E-E group. Immunohistochemistry results showed no significant indication of extrinsic reinnervation until 12 months, when TH fibers were observed in five of six dogs. The E-E group revealed some TH fibers extending across the arterial anastomosis toward the graft mesentery. Examination of the intestinal anastomosis at 12 months showed abundant peptidergic and TH extrinsic fibers in the host side, whereas there were few or none on the graft side. Conclusions: These results suggest that extrinsic reinnervation of the graft intestinal wall does occur but requires a prolonged period, and the major route of extrinsic reinnervation is along the arterial axis of the intestinal graft, not beyond the enteric anastomosis. (Surgery 1998;123:25-35.)

Depletion of endogenous dopamine stores and shift in beta-adrenoceptor subtypes in cardiac tissue following five weeks of chronic denervation.

Surgical ablation of extrinsic cardiac nerve fibers results in a chronically denervated state of the left ventricle of the heart. The present study was performed to elucidate the effect of a period of 5 weeks of chronic denervation on cardiac catecholamine levels in general and dopamine in particular. Moreover, the possible effect on cardiac beta-adrenoceptor subtypes was investigated. Experiments were performed on adult dogs. In addition to adrenaline and noradrenaline the tissue levels of dopamine were found to be severely depressed. A significant shift from beta1- to beta2-adrenoceptor subtype was observed, while the total beta-adrenoceptor density remained unaffected. The present findings indicate that catecholamine synthesis in chronically denervated hearts is impaired upstream of dopamine and that a shift in beta-adrenoceptor subtype occurs already within a relatively short period of five weeks of denervation, and suggest that the lack of endogenous catecholamines influence the relative expression levels of the two subtypes of beta-adrenoceptors present in cardiac tissue.

Metabolic alterations in the chronically denervated dog heart.

Previous studies have shown that chronic cardiac denervation impairs myocardial glucose oxidation. To investigate this further we tested whether the tissue content of glucose transporters, activity of glycolytic enzymes or metabolic capacity of pyruvate dehydrogenase were altered. Moreover, we investigated whether the decline in glucose utilization was associated with an upregulation of proteins and enzymes involved in fatty acid handling. Chronic cardiac denervation results also in decreased left ventricular efficiency. We explored whether alterations in mitochondrial properties could be held responsible for this phenomenon. Twelve adult dogs were included in the study. In 6 of them chronic cardiac denervation was accomplished by surgical ablation of the extrinsic nerve fibers. The other 6 dogs were sham-operated. Biopsies were obtained from the left ventricle after 4-5 weeks of denervation. The content or enzymatic activity of proteins involved in fatty acid and glucose handling was assessed. Features of glutamate oxidation were measured in freshly isolated mitochondria. The content or activity of a set of fatty acid handling proteins did not change during chronic cardiac denervation. In contrast GLUT1 content significantly increased in the chronically denervated left ventricle, while the active form of pyruvate dehydrogenase declined (p < 0.05). Glutamate oxidation characteristics in freshly isolated mitochondria were not affected by chronic denervation. The impairment of glucose oxidation in the chronically denervated myocardium is most likely caused by a decline of pyruvate dehydrogenase in its active form. It is unlikely that the decrease in work efficiency is caused by alterations in mitochondrial properties.

Impaired expression of neural cell adhesion molecule L1 in the extrinsic nerve fibers in Hirschsprung's disease

Immunohistochemical studies on the ganglionic and aganglionic segment in Hirschsprung's disease (HD) were carried out using antibodies against three neural membrane proteins, Thy-1, Integrin α5, and L1. Enteric neural elements were immunostained with antibodies against neurofilament, which is the neuronal cytoskeletal protein. In ganglionic segments, neurofilament-immunoreactivity was detected in neuronal cell bodies and fine nerve fibers of the myenteric and submucosal plexuses. All of these neural elements were immunopositive for Thy-1, integrin α5, and L1. In aganglionic segments, no intrinsic neurons were detected, and instead, hypertrophied nerve bundles were observed in intermuscular space, in submucosa, and in circular muscle layer by immunochemistry for neurofilament. These hypertrophied nerve bundles were immunopositive with anti-Thy-1 and anti-integrin α5 antibodies. However, they were not immunostained with anti-L1 in all five cases. These findings indicate that the expression of L1 molecule, which plays an important role in cell adhesion, neural cell migration, and neurite outgrowth, is impaired in the extrinsic nerve fibers in aganglionic colon. And this may perturb neural creast migration and adequate neurite outgrowth, with resulting aganglionic segment and abnormal nerve bundles of extrinsic fibers in HD.

The distribution and co-localization of nitric oxide synthase and vasoactive intestinal polypeptide in nerves of the colons with Hirschsprung's disease.

The distribution and co-localization of nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) were examined by means of immunohistochemistry and NADPH diaphorase (NADPH-d) histochemistry in the gut of patients with Hirschsprung's disease. In the normoganglionic segment, many nitrergic nerve cells were localized in Auerbach's plexus and nerve fibres were observed preferentially in the circular muscle. The submucosal nitrergic nerve cells were mainly situated in Schabadasch's plexus with occasional cells demonstrable in Meissner's plexus. NOS and VIP were co-localized in most ganglion cells of Auerbach's plexus. In the oligoganglionic segment, a marked reduction of NOS- and VIP- positive nerve cells and fibres was noticed in both the myenteric and submucosal plexuses, and nitrergic fibres had disappeared in the inner layer of the circular muscle. In the aganglionic segment, NOS and VIP were revealed only in extrinsic nerve fasciculi and rami and co-localized in a few fibres. From these observations, the inner layer of the circular muscle of the oligoganglionic segment and the whole of the muscularis propria of the aganglionic segment were considered to be totally lacking in nitrergic innervation. Nitrergic nerves of the human colon comprise both intrinsic and extrinsic elements and the majority of intrinsic nitrergic nerve cells contain VIP. Very low numbers of extrinsic nitrergic fibres contain VIP.

Distribution of peptide-containing neurons in the developing rat right atrium, studied using immunofluorescence and confocal laser scanning.

The developmental pattern and distribution of peptide-containing neurons in the rat heart right atrium has been studied by indirect immunofluorescence. Antibodies against neuropeptide Y (NPY), substance P (SP), and vasoactive intestinal polypeptide (VIP) were applied to whole-mount stretch preparations of the right atria from hearts of newborn to 40 day-old animals. NPY-like immunoreactivity (L1) was compared with the synaptic vesicle marker SV2 in double immunoincubation studies. The distribution of immunofluorescence was studied by confocal laser scanning microscopy. NPY-L1 and SP-L1 were present throughout the atria already at birth, in contrast to VIP-L1 that was observed at day 10. The postnatal changes of innervation were basically quantitative, with an increase in density of nerve fibres and number of varicosities, while the basic pattern of innervation was essentially established during the first 1-10 days. NPY- and SP-positive bundles of fibres appeared to enter the right atrium along the superior caval vein, having extrinsic origins. Nerve fibres with NPY-L1 colocalized in most nerve terminals with SV2-L1, and showed a developmental pattern similar to that observed for adrenergic neurons earlier. These NPY/SV2 positive fibres probably represent the extrinsic NPY innervation. In addition, NPY-L1 was identified in large intrinsic nerve cells bodies located near the atrioventricular (AV) region. Most of the VIP-L1 was observed in short nerve fibres originating in intrinsic VIP-positive cell bodies, but a few apparently extrinsic VIP-positive fibres were found, probably representing preganglionic parasympathetic neurons. SP in the atria was probably of extrinsic (sensory) origin and no nerve cell bodies with SP-L1 were detected. The results show that the peptidergic innervation in the developing rat right atrium involves both extrinsic and intrinsic peptidergic neurons which may participate in the regulation of neurotransmission in local neuronal circuits.

Vesicular monoamine transporter 2 expression in enteric neurons and enterochromaffin-like cells of the rat

The cellular localization of the vesicular monoamine transporter 2 (VMAT2) in the rat digestive tract was investigated with immunohistochemistry. VMAT2-immunoreactivity (IR) was localized to neurons and fibers of enteric and pancreatic ganglia, to processes supplying the gut wall, the pancreas and blood vessels, and to enterochromaffin-like (ECL) cells in the gastric corpus, which contained calbindin-IR. Few VMAT2-IR cells were also found in the gastric antrum, but they did not contain gastrin-IR. VMAT2-IR was expressed in extrinsic sympathetic fibers as demonstrated by the elimination of a portion of VMAT2-IR processes by sympathectomy. The VMAT2-IR pattern is consistent with the overall distribution of biogenic amine cell groups in the digestive tract. Our results provide further evidence that VMAT2 is the vesicular amine transporter responsible for accumulation of monoamines into secretory vesicles of monoaminergic neurons and ECL cells.

Distribution of peptide-containing nerve fibres in achalasia of the oesophagus.

In this study the innervation of the normal human oesophagus was compared with samples taken from 12 patients undergoing Heller's cardiomyotomy for achalasia. The distribution of all nerve fibres in the oesophageal wall was revealed by immunoreactivity to neuron specific enolase and subpopulations of nerve fibres were revealed by immunoreactivity to vasoactive intestinal peptide, neuropeptide Y, enkephalin and substance P. In healthy oesophagus, many nerve fibres immunoreactive for vasoactive intestinal peptide and neuropeptide Y were present in the circular and longitudinal muscle layers of the oesophageal wall and in the cardia of the stomach, whereas fibres immunoreactive for enkephalin and substance P were uncommon. Neuropeptide Y-reactive fibres were commonly seen around blood vessels. In the myenteric plexus cell bodies reactive for vasoactive intestinal peptide and neuropeptide Y were prevalent, as were varicose and non-varicose fibres. In contrast, samples from patients with achalasia revealed few nerve fibres immunoreactive for vasoactive intestinal peptide or neuropeptide Y in either circular or longitudinal muscle, suggesting damage to the inhibitory motor neurons to the muscle layers. Very few fibres were found that were reactive for neuron-specific enolase, indicating that other fibre population (e.g. excitatory cholinergic motor neurons) are also damaged in achalasia. These abnormalities were observed in biopsies from both the constricted and dilated portions of the oesophagus, but the pattern of innervation in the gastric cardia was normal. Myenteric ganglion cells were seen in the oesophagus in only two patients and varicose nerve fibres in the myenteric plexus were uncommon. Neuropeptide Y-reactive perivascular nerve fibres were still found in achalasia as well as non-varicose nerve fibres in the myenteric plexus. These findings indicate damage to all intrinsic neurons in the oesophageal wall in achalasia; however, extrinsic nerve fibres appear to be intact.

HISTOPATHOLOGY OF THE ENTERIC NEUROPATHIES: From Silver Staining to Immunohistochemistry

The gut is abundantly supplied with neurons, extrinsic and intrinsic nerve fibers. Knowledge regarding the structure of the enteric nervous system derives principally from the classic silver-staining methods. Because silver stains do not provide information on the molecular constituents of neurons, these data only facilitate classification and may have diagnostic significance. Studies using histochemistry and immunohistochemistry are now completing the morphologic picture and laying the groundwork for the formulation of therapeutic strategies based upon demonstrable chemical defects in enteric disease.

Enhanced extrinsic innervation of nasal and oral chemosensory mucosae in keratin 14-NGF transgenic mice

The role of nerve growth factor (NGF) in neurotrophic support for the extrinsic innervation of the nasal and oral mucosae was investigated in keratin 14 (K14) - NGF transgenic mice in which NGF was overexpressed in K14-synthesizing cells. K14 immunoreactivity was localized in the epithelial basal cells of the whisker pad skin, the hard palate, the floor of the ventral meatus, and the anterior tongue that are stratified squamous epithelia, and also in basal cells of the vomeronasal, olfactory, and respiratory epithelia that are non-stratified epithelia. In transgenic mice, NGF expression was identified and confined primarily to the basal cells of stratified epithelia. The nasal mucosae including the vomeronasal, olfactory, and respiratory mucosae, and the glands associated with the vomeronasal organ received a greater innervation of protein gene product 9.5-immunoreactive extrinsic fibers in transgenic animals than nontransgenic controls. An increased density of calcitonin gene-related peptide-immunoreactive extrinsic fibers was observed in the nonsensory epithelia of the vomeronasal organ, the olfactory sensory and respiratory epithelia in transgenic animals. Our results indicated that the hyperinnervation of the nasal and oral mucosae by extrinsic neurons is due at least partially to target-derived NGF synthesis and release by K14-expressing basal cells.

Capsaicin-sensitive afferent nerves activate submucosal secretomotor neurons in guinea pig ileum

This study examined whether capsaicin-sensitive sensory nerves regulate intestinal ion transport using both Ussing chamber and intracellular recording techniques in in vitro submucosal preparations from the guinea pig ileum. In Ussing chamber studies, serosal application of capsaicin (20 nM-20 microM) evoked a biphasic dose-dependent increase in short-circuit current (Isc) (maximal effective concentration 200 nM and 2 microM, respectively). In chloride-free buffer, capsaicin responses were significantly reduced. Capsaicin evoked little or no response when extrinsic sensory nerve fibers had been surgically removed and tetrodotoxin and low-calcium and high-magnesium solutions blocked responses to capsaicin. In epithelial preparations devoid of submucosal neurons, capsaicin had virtually no effect, suggesting that responses evoked by capsaicin-sensitive nerves result from activation of submucosal secretomotor neurons. Intracellular recordings from single submucosal neurons demonstrated that superfusion with capsaicin (2 microM) depolarized neurons with an associated decreased conductance. Depolarizations were completely desensitized when capsaicin was reapplied, but synaptic inputs were unaffected. This study suggests that capsaicin-sensitive nerves can regulate ion transport in the gastrointestinal tract by release of neurotransmitter(s) that activate submucosal secretomotor neurons.

Immunohistochemical demonstration of enteric nervous distribution after syngeneic small bowel transplantation in rats

Small bowel transplantation causes a disturbance of the enteric neural networks after complete extrinsic denervation. The morphologic changes in the enteric nervous system after transplantation were immunohistochemically investigated in jejunal isografts at 10 days, 100 days, and 400 days after transplantation. No remarkable differences were revealed concerning the antibodies for general neural markers, vasoactive intestinal polypeptide, substance P, somatostatin, or galanin between controls and isografts. Identical differences were detected in the distribution of nerve fibers containing calcitonin gene-related peptide and catecholamines. In the isografts a partial reduction of calcitonin gene-related peptide-immunopositive fibers was shown. A complete elimination of catecholaminergic nerves was seen in the isografts at 10 and 100 days; however, a sparse distribution of catecholaminergic nerves was observed in the 400-day isograft. Most intrinsic neural elements are preserved; however, the extrinsic, sympathetic, and sensory nerves are completely disrupted as a consequence of transplantation. Reinnervation of extrinsic nerve fibers could occur in the transplanted small intestine.

The intra-adrenal distribution of intrinsic and extrinsic nitrergic nerve fibres in the rat

The intra-adrenal distribution of nitric oxide synthase (NOS)-immunoreactive nerve fibres was studied in rats subjected to various denervations. Splanchnic nerve section eliminated the NOS-immunoreactive nerve fibres which innervate adrenal chromaffin and neuronal cells. It did not affect those innervating blood vessels and zona glomerulosa, which instead were affected by adrenal demedullation. Guanethidine, 6-hydroxydopamine (6-OHDA) and capsaicin treatments, however, did not produce any change. These results suggest that nitrergic nerves which innervate adrenal medullary cells are extrinsic (largely preganglionic sympathetic), whilst those innervating the zona glomerulosa and the majority of adrenal vessels are intrinsic, and that they do not belong to nerves sensitive to the sympathetic nerve neurotoxins, guanethidine and 6-OHDA, or the sensory neurotoxin, capsaicin.

NADPH-diaphorase expression in neurones and glial cells of the locust brain.

Using NADPH-diaphorase (NADPH-d) staining as a marker for the enzyme nitric oxide synthase (NOS) we investigated the possible sites of nitric oxide (NO) synthesis in the olfactory pathways of an insect brain. Staining of frozen sections revealed NADPH-d activity in neurones and in glial cells. A cluster of intensely stained interneurones innervates the neuropile of the antennal lobe. NADPH-d expression in the mushroom bodies showed a compartmentalized pattern. The mushroom body intrinsic Kenyon cells did not express NADPH-d. The pedunculus and lobes showed fine granular staining and were invaded by NADPH-d-positive mushroom body extrinsic fibres. The expression of NADPH-d in glial cells enclosing the mushroom bodies suggest that insect glial cells may release NO as a messenger molecule.

Immunohistochemical study of enteric nervous system after small bowel transplantation in humans

The neurohormonal structures of two human intestines removed due to rejection 22 months and eight months after intestinal transplantation were studied by an indirect immunohistochemical method and compared with normal ileum. The distribution and density of neurons immunoreactive for tyrosine hydroxylase, substance P, calcitonin gene-related peptide, neuropeptide Y, vasoactive intestinal peptide, galanin, gastrin-releasing peptide, L-enkephalin, and somatostatin were examined. Mucosal endocrine cells immunoreactive for somatostatin, peptide YY, and glucagon were also examined. Extrinsic adrenergic fibers and perivascular fibers were absent in all intestinal layers of the failed grafts. The distribution of intrinsic neurons was unchanged; however, the density was decreased by one rank. Distribution of endocrine cells of the first graft was similar to the normal. Extrinsic fibers were not detected by immunohistochemistry in human small intestinal grafts following long-term survival and eventual rejection, while the immunohistochemical expression of intrinsic neural and endocrine transmitters were well preserved.

Distribution of extrinsic enkephalin-containing nerve fibers in the rat rectum and their origin in the major pelvic ganglion

The distribution of nerve fibers containing enkephalin (ENK)-like immunoreactivity was examined in the rectum of aganglionosis rats (AGRs) which completely lack the intramural ganglion cells in the large intestine, and was compared with that of their normal littermates. Furthermore, Met 5-enkephalin-Arg 6-Gly 7-Leu 8 (MEAGL)-like immunoreactive neurons projecting to the rectum were examined using retrograde tracing combined with immunohistochemistry in the major pelvic ganglion of normal male rats. In the intermuscular space of the aganglionic rectum of AGRs, unlike the pattern of the normal intermuscular plexus, moderate numbers of ENK-like-immunoreactive fibers were arranged in an irregular, coarse network; greatly diminished numbers of immunoreactive fibers were found in the submucosa. No ENK-like-immunoreactive fibers were seen in the circular muscle layer and mucosa. In the normal rat rectum, ENK-like-immunoreactive fibers were seen throughout all layers, and immunoreactive nerve cells were found predominantly in the myenteric plexus of colchicine-treated animals. Fluoro-Gold injected into the upper rectum labelled numerous principal ganglion neurons in the major pelvic and inferior mesenteric ganglia. Less than 10% of tracer-labelled neurons were positive for fluorescein immunolabelling of MEAGL in the major pelvic ganglion; no immunoreactive neurons were found in the inferior mesenteric ganglion. In the major pelvic ganglion of the colchicine-treated normal rats, about 5% of principal ganglion neurons were immunoreactive for MEAGL. Comparison of serial paraffin sections of the major pelvic ganglion stained for tyrosine hydroxylase (TH), MEAGL and vasoactive intestinal polypeptide (VIP), respectively, revealed that more than half of MEAGL-like immunoreactive neurons were also positive for TH; there was no case showing co-existence of MEAGL with VIP in the principal neurons. These results indicate that a small number of enkephalin-containing neurons in the major pelvic ganglion project to the rectum, and that more than half of these neurons are postganglionic sympathetic. They may terminate mainly in the myenteric ganglia in the rectum.