Intracisternal TRH analog induces Fos expression in gastric myenteric neurons and glia in conscious rats

Activation of gastric myenteric cells by intracisternal injection of the stable thyrotropin-releasing hormone (TRH) analog RX-77368, at a dose inducing near maximal vagal cholinergic stimulation of gastric functions, was investigated in conscious rats. Fos immunoreactivity was assessed in gastric longitudinal muscle-myenteric plexus whole mount preparations 90 min after intracisternal injection. Fos-immunoreactive cells were rare in controls (~1 cell/ganglion), whereas intracisternal RX-77368 (50 ng) increased the number to 24.8 +/- 1.8 and 26.8 +/- 2.2 cells/ganglion in the corpus and antrum, respectively. Hexamethonium (20 mg/kg sc) prevented Fos expression by 90%, whereas atropine (2 mg/kg sc) had no effect. The neuronal marker protein gene product 9.5 and the glial markers S-100 and glial fibrillary acidic proteins showed that RX-77368 induced Fos in both myenteric neurons and glia. Vesicular ACh transporter and calretinin were detected around the activated myenteric neurons. These results indicated that central vagal efferent stimulation by intracisternal RX-77368 activates gastric myenteric neurons as well as glial cells mainly through nicotinic ACh receptors in conscious rats.     

Central vagal stimulation activates enteric cholinergic neurons in the stomach and VIP neurons in the duodenum in conscious rats

The influence of central vagal stimulation induced by 2h cold exposure or intracisternal injection of thyrotropin-releasing hormone (TRH) analog, RX-77368, on gastro-duodenal enteric cholinergic neuronal activity was assessed in conscious rats with Fos and peripheral choline acetyltransferase (pChAT) immunoreactivity (IR). pChAT-IR was detected in 68%, 70% and 73% of corpus, antrum and duodenum submucosal neurons, respectively, and in 65% of gastric and 46% of duodenal myenteric neurons. Cold and RX-77368 induced Fos-IR in over 90% of gastric submucosal and myenteric neurons, while in duodenum only 25-27% of submucosal and 50-51% myenteric duodenal neurons were Fos positive. In the stomach, cold induced Fos-IR in 93% of submucosal and 97% of myenteric pChAT-IR neurons, while in the duodenum only 7% submucosal and 5% myenteric pChAT-IR neurons were Fos positive. In the duodenum, cold induced Fos in 91% of submucosal and 99% of myenteric VIP-IR neurons. RX-77368 induces similar percentages of Fos/pChAT-IR and Fos/VIP-IR neurons. These results indicate that increased central vagal outflow activates cholinergic neurons in the stomach while in the duodenum, VIP neurons are preferentially stimulated.     

Brain-gut interactions between central vagal activation and abdominal surgery to influence gastric myenteric ganglia Fos expression in rats

We previously showed that medullary thyrotropin-releasing hormone (TRH) or the stable TRH agonist, RX-77368 administered intracisternally induces vagal-dependent activation of gastric myenteric neurons and prevents post surgery-induced delayed gastric emptying in rats. We investigated whether abdominal surgery alters intracisternal (ic) RX-77368 (50 ng)-induced gastric myenteric neuron activation. Under 10 min enflurane anesthesia, rats underwent an ic injection of saline or RX-77368 followed by a laparotomy and a 1-min cecal palpation, or no surgery and were euthanized 90 min later. Longitudinal muscle/myenteric plexus whole-mount preparations of gastric corpus and antrum were processed for immunohistochemical detection of Fos alone or double labeled with protein gene-product 9.5 (PGP 9.5) and vesicular acetylcholine transporter (VAChT). In the non surgery groups, ic RX-77368 induced a 17 fold increase in Fos-expression in both gastric antrum and corpus myenteric neurons compared to saline injected rats. PGP 9.5 ascertained the neuronal identity of myenteric cells expressing Fos. In the abdominal surgery groups, ic RX-77368 induced a significant increase in Fos-expression in both the corpus and antrum myenteric ganglia compared with ic saline injected rats which has no Fos in the gastric myenteric ganglia. However, the response was reduced by 73-78% compared with that induced by ic RX 77368 without surgery. Abundant VAChT positive nerve fibers were present around Fos positive neurons. These results indicate a bidirectional interaction between central vagal stimulation of gastric myenteric neurons and abdominal surgery. The modulation of gastric vagus-myenteric neuron activity could play an important role in the recovery phase of postoperative gastric ileus.     

Neuronal activation of brain vagal-regulatory pathways and upper gut enteric plexuses by insulin hypoglycemia

Neuronal activation of brain vagal-regulatory nuclei and gastric/duodenal enteric plexuses in response to insulin (2 U/kg, 2 h) hypoglycemia was studied in rats. Insulin hypoglycemia significantly induced Fos expression in the paraventricular nucleus of the hypothalamus, locus coeruleus, dorsal motor nucleus of the vagus (DMN), and nucleus tractus solitarii (NTS), as well as in the gastric/duodenal myenteric/submucosal plexuses. A substantial number of insulin hypoglycemia-activated DMN and NTS neurons were choline acetyltransferase and tyrosine hydroxylase positive, respectively, whereas the activated enteric neurons included NADPH- and vasoactive intestinal peptide neurons. The numbers of Fos-positive cells in each above-named brain nucleus or in the gastric/duodenal myenteric plexus of insulin-treated rats were negatively correlated with serum glucose levels and significantly increased when glucose levels were lower than 80 mg/dl. Acute bilateral cervical vagotomy did not influence insulin hypoglycemia-induced Fos induction in the brain vagal-regulatory nuclei but completely and partially prevented this response in the gastric and duodenal enteric plexuses, respectively. These results revealed that brain-gut neurons regulating vagal outflow to the stomach/duodenum are sensitively responsive to insulin hypoglycemia.     

CCK enhances response to gastric distension by acting on capsaicin-insensitive vagal afferents

Capsaicin treatment destroys vagal afferent C fibers and markedly attenuates reduction of food intake and induction of hindbrain Fos expression by CCK. However, both anatomical and electrophysiological data indicate that some gastric vagal afferents are not destroyed by capsaicin. Because CCK enhances behavioral and electrophysiological responses to gastric distension in rats and people, we hypothesized that CCK might enhance the vagal afferent response to gastric distension via an action on capsaicin-insensitive vagal afferents. To test this hypothesis, we quantified expression of Fos-like immunoreactivity (Fos) in the dorsal vagal complex (DVC) of capsaicin-treated (Cap) and control rats (Veh), following gastric balloon distension alone and in combination with CCK injection. In Veh rats, intraperitoneal CCK significantly increased DVC Fos, especially in nucleus of the solitary tract (NTS), whereas in Cap rats, CCK did not significantly increase DVC Fos. In contrast to CCK, gastric distension did significantly increase Fos expression in the NTS of both Veh and Cap rats, although distension-induced Fos was attenuated in Cap rats. When CCK was administered during gastric distension, it significantly enhanced NTS Fos expression in response to distension in Cap rats. Furthermore, CCK's enhancement of distension-induced Fos in Cap rats was reversed by the selective CCK-A receptor antagonist lorglumide. We conclude that CCK directly activates capsaicin-sensitive C-type vagal afferents. However, in capsaicin-resistant A-type afferents, CCK's principal action may be facilitation of responses to gastric distension.     

Inhibitory effects of beta-alanine on the vagal efferent and afferent excitatory responses of the stomach to stimulation of vagal trunk in cats.

The effects of beta-alanine on the electrically evoked vagal efferent (hexamethonium-sensitive initial excitatory response) and afferent (hexamethonium-resistant delayed excitatory response) responses of the cat stomach were studied. beta-alanine (30 to 300 micrograms/kg, i.v.) dose-dependently inhibited both the efferent and afferent response. The IC50 values of beta-alanine on the efferent and afferent response were 296 +/- 65 micrograms/kg and 128 +/- 35 microgram/kg, respectively. Maximal inhibitory effects of beta-alanine (300 micrograms/kg, i.v.) appeared about 1 hr after the injection. Glycine and taurine (100 to 10,000 micrograms/kg) did not affect these responses. Treatment with hexamethonium (10 mg/kg, i.v.) prevented the efferent response, but augmented the afferent response. The treatment with hexamethonium abolished the inhibitory effect of beta-alanine on the afferent response. Both picrotoxin (100 and 500 micrograms/kg, i.v.) and bicuculline (2000 micrograms/kg, i.v.) antagonized the inhibitory effects of beta-alanine on the vagal efferent and afferent responses of the stomach. The present experiments clearly demonstrated that beta-alanine inhibited both the vagal efferent and afferent excitatory responses of stomach to electrical stimulation of vagal trunk in cats.     

Vagus nerve stimulation preferentially induces Fos expression in nitrergic neurons of rat esophagus

To identify neurochemical phenotypes of esophageal myenteric neurons synaptically activated by vagal preganglionic efferents, we immunohistochemically detected the expression of Fos, an immediate early gene product, in whole-mount preparations of the entire esophagus of rats following electrical stimulation of the vagus nerves. When electrical stimulation was applied to either the cervical left (LVN) or right vagus nerve (RVN), neurons with nuclei showing Fos immunoreactivity (IR) were found to comprise approximately 10% of the total myenteric neurons in the entire esophagus. These neurons increased from the oral toward the gastric end of the esophagus, with the highest frequency in the abdominal portion of the esophagus. A significant difference was not found in the number of Fos neurons between the LVN-stimulated and RVN-stimulated esophagus. Double-immunolabeling showed that nitric oxide synthase (NOS)-IR occurred in most (86% and 84% in the LVN-stimulated and RVN-stimulated esophagus, respectively) of the Fos neurons in the entire esophagus. Furthermore, the stimulation of either of the vagus nerves resulted in high proportions (71%-90%) of Fos neurons with NOS-IR, with respect to the total Fos neurons in each segment, in the entire esophagus. However, a small proportion (8% and 7% in the LVN-stimulated and RVN-stimulated esophagus, respectively) of the Fos neurons in the esophagus exhibited choline acetyltransferase (ChAT)-IR. The occurrence-frequency of Fos neurons with ChAT-IR was less than 4% of the total Fos neurons in any segment of the LVN-stimulated and RVN-stimulated esophagus. Some of the Fos neurons with ChAT-IR appeared to be innervated by numerous varicose ChAT-positive nerve terminals. The present results showing that electrical stimulation of the vagus nerves induces a high proportion of Fos neurons with NOS-IR suggests the preferential activation of NOS neurons in the esophagus by vagal preganglionic efferents. This connectivity between the vagal efferents and intrinsic nitrergic neurons might be involved in inhibitory actions on esophageal motility.This study was supported by Grant-in Aids for Scientific Research from Ministry of Education, Sports, and Culture of Japan to H.K. (no. 15500236) and to M.K. (no. 14570065).     

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.     

Vanilloid receptor (VR1) expression in vagal afferent neurons innervating the gastrointestinal tract

The vanilloid receptor VR1 is a nonselective cation channel activated by capsaicin as well as increases in temperature and acidity, and can be viewed as molecular integrator of chemical and physical stimuli that elicit pain. The distribution of VR1 receptors in peripheral and central processes of rat primary vagal afferent neurons innervating the gastrointestinal tract was investigated by immunohistochemistry. Forty-two percent of neurons in the nodose ganglia retrogradely labeled from the stomach wall expressed low to moderate VR1 immunoreactivity (VR1-IR). VR1-IR was considerably lower in the nodose ganglia as compared to the jugular and dorsal root ganglia. In the vagus nerve, strongly VR1-IR fibers ran in separate fascicles that supplied mainly cervical and thoracic targets, leaving only weakly VR1-IR fibers in the subdiaphragmatic portion. Vagal afferent intraganglionic laminar endings (IGLEs) in the gastric and duodenal myenteric plexus did not express VR1-IR. Similarly, VR1-IR was contained in fibers running in perfect register with vagal afferents, but was not colocalized with horseradish peroxidase in the same varicosities of intramuscular arrays (IMAs) and vagal afferent fibers in the duodenal submucosa anterogradely labeled from the nodose ganglia. Only in the gastric mucosa did we find evidence for colocalization of VR1-IR in vagal afferent terminals. In contrast, many nerve fibers coursing through the myenteric and submucosal plexuses contained detectable VR1-IR, the majority of which colocalized calcitonin gene-related peptide immunoreactivity. In the dorsal medulla there was a dense plexus of VR1-IR varicose fibers in the commissural, dorsomedial and gelatinosus subnuclei of the medial NTS and the lateral aspects of the area postrema, which was substantially reduced, but not eliminated on the ipsilateral side after supranodose vagotomy. It is concluded that about half of the vagal afferents innervating the gastrointestinal tract express low levels of VR1-IR, but that presence in most of the peripheral terminal structures is below the immunohistochemical detection threshold.     

Mucosal acid challenge activates nitrergic neurons in myenteric plexus of rat stomach

We tested the hypothesis that intrinsic neurons of the rat gastric myenteric plexus can be activated by an acid (HCl) challenge of the mucosa. Activated neurons were visualized by immunohistochemical detection of c-Fos, a marker for neuronal excitation. The neurochemical identity of the neurons activated by the HCl challenge was determined by colocalizing c-Fos with a marker for excitatory pathways, choline acetyltransferase (ChAT), and a marker for inhibitory pathways, nitric oxide synthase (NOS). Two hours after intragastric administration of HCl or saline, stomachs were removed and immunofluorescence triple labeling of myenteric neurons was carried out on whole mount preparations. Treatment with 0.35, 0.5, and 0.7 M HCl induced c-Fos in 8%, 56%, and 64%, respectively, of NOS-positive but not ChAT-positive neurons. c-Fos was also seen in glial cells of HCl-treated rats, whereas in saline-treated animals c-Fos was absent from the myenteric plexus. HCl treatment did not change the proportion of ChAT- and NOS-immunoreactive neurons in the myenteric ganglia. It is concluded that gastric acid challenge concentration-dependently stimulates a subpopulation of nitrergic, but not cholinergic, myenteric plexus neurons, which may play a role in muscle relaxation, vasodilatation, and/or secretion.     

Effect of sympathectomy and demedullation on increased myenteric and dorsal vagal complex Fos-like immunoreactivity by cholecystokinin-8

Chemical sympathectomy with daily, intraperitoneal (IP) injections of guanethidine sulfate to adult rats, attenuated myenteric, but not dorsal vagal complex (DVC) Fos-like immunoreactivity (Fos-LI) by cholecystokinin-8 (CCK). This technique destroys only 60-70% of the sympathetic neurons, and spares the hormonal source of catecholamines, the adrenal medulla. The goal of the current study is to evaluate the effect of complete sympathectomy or destroying 100% of the sympathetic neurons by injecting guanethidine to 1-day-old pups (40 mg/kg daily for 5 weeks), and surgically removing the adrenal medulla. In the DVC, demedullation and sympathectomy-demedullation increased Fos-LI by CCK in the area postrema and nucleus of the solitary tract, but sympathectomy-demedullation increased it only in the area postrema. In the myenteric plexus, sympathectomy increased this response in the duodenum, and demedullation increased it in the duodenum and jejunum. On the other hand, sympathectomy-demedullation attenuated myenteric Fos-LI in the jejunum. These results indicate that catecholamines may play an inhibitory role on the activation of the DVC neurons by CCK. In the myenteric neurons, however, catecholamines may have both inhibitory and excitatory roles depending on the level of the intestine e.g., duodenum vs. jejunum. This may also indicate that CCK activates the enteric neurons by different mechanisms or through different pathways.     

Peripheral versus central modulation of gastric vagal pathways by metabotropic glutamate receptor 5

Metabotropic glutamate receptors (mGluR) are classified into group I, II, and III mGluR. Group I (mGluR1, mGluR5) are excitatory, whereas group II and III are inhibitory. mGluR5 antagonism potently reduces triggering of transient lower esophageal sphincter relaxations and gastroesophageal reflux. Transient lower esophageal sphincter relaxations are mediated via a vagal pathway and initiated by distension of the proximal stomach. Here, we determined the site of action of mGluR5 in gastric vagal pathways by investigating peripheral responses of ferret gastroesophageal vagal afferents to graded mechanical stimuli in vitro and central responses of nucleus tractus solitarius (NTS) neurons with gastric input in vivo in the presence or absence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). mGluR5 were also identified immunohistochemically in the nodose ganglia and NTS after extrinsic vagal inputs had been traced from the proximal stomach. Gastroesophageal vagal afferents were classified as mucosal, tension, or tension-mucosal (TM) receptors. MPEP (1-10 microM) inhibited responses to circumferential tension of tension and TM receptors. Responses to mucosal stroking of mucosal and TM receptors were unaffected. MPEP (0.001-10 nmol icv) had no major effect on the majority of NTS neurons excited by gastric distension or on NTS neurons inhibited by distension. mGluR5 labeling was abundant in gastric vagal afferent neurons and sparse in fibers within NTS vagal subnuclei. We conclude that mGluR5 play a prominent role at gastroesophageal vagal afferent endings but a minor role in central gastric vagal pathways. Peripheral mGluR5 may prove a suitable target for reducing mechanosensory input from the periphery, for therapeutic benefit.     

Nesfatin-1 inhibits gastric acid secretion via a central vagal mechanism in rats

Nesfatin-1, a novel hypothalamic peptide, inhibits nocturnal feeding behavior and gastrointestinal motility in rodents. The effects of nesfatin-1 on gastrointestinal secretory function, including gastric acid production, have not been evaluated. Nesfatin-1 was injected into the fourth intracerebral ventricle (4V) of chronically cannulated rats to identify a nesfatin dose sufficient to inhibit food intake. Nesfatin-1 (2 μg) inhibited dark-phase food intake, in a dose-dependent fashion, for >3 h. Gastric acid production was evaluated in urethane-anesthetized rats. Nesfatin-1 (2 μg) was introduced via the 4V following endocrine stimulation of gastric acid secretion by pentagastrin (2 μg·kg(-1)·h(-1) iv), vagal stimulation with 2-deoxy-d-glucose (200 mg/kg sc), or no stimulus. Gastric secretions were collected via gastric cannula and neutralized by titration to determine acid content. Nesfatin-1 did not affect basal and pentagastrin-stimulated gastric acid secretion, whereas 2-deoxy-d-glucose-stimulated gastric acid production was inhibited by nesfatin-1 in a dose-dependent manner. c-Fos immunofluorescence in brain sections was used to evaluate in vivo neuronal activation by nesfatin-1 administered via the 4V. Nesfatin-1 caused activation of efferent vagal neurons, as evidenced by a 16-fold increase in the mean number of c-Fos-positive neurons in the dorsal motor nucleus of the vagus (DMNV) in nesfatin-1-treated animals vs. controls (P < 0.01). Finally, nesfatin-induced Ca(2+) signaling was evaluated in primary cultured DMNV neurons from neonatal rats. Nesfatin-1 caused dose-dependent Ca(2+) increments in 95% of cultured DMNV neurons. These studies demonstrate that central administration of nesfatin-1, at doses sufficient to inhibit food intake, results in inhibition of vagally stimulated secretion of gastric acid. Nesfatin-1 activates DMNV efferent vagal neurons in vivo and triggers Ca(2+) signaling in cultured DMNV neurons.     

Cardiac and pulmonary vagal neurons receive excitatory chemoreceptor input

The effects of hypercapnia and hypocapnia on the activities of the cardiac and pulmonary vagal single fibers were examined in the decerebrated, unanesthetized, paralyzed, and vagotomized cats. The animals breathed 100% O2. Fractional end tidal CO2 concentration was raised to 9% by adding CO2 into the O2 inlet. Average discharge rate of efferent cardiac vagal units (n=10) increased from 1.0+/-0.3 to 2.2+/-0.3 Hz. Hypocapnia apnea was produced by hyperventilation. Activities of cardiac vagal units tested (n = 4) showed dramatic decrease (0.1+/-0.0 Hz). Mean arterial blood pressure did not change significantly under these conditions. In contrast, only instantaneous firing rate during inspiration was significantly increased for efferent pulmonary vagal units (n = 11) during hypercapnia. The activities of the 3 pulmonary vagal units tested with hypocapnia decreased significantly. We concluded that cardiac and pulmonary vagal neurons were excited by chemoreceptor input.     

Gastrin releasing peptides increase Fos-like immunoreactivity in the enteric nervous system and the dorsal vagal complex

We and others have shown that gastrin-releasing peptide (GRP) reduces food intake. In this study, we determined the activation of the gastrointestinal and dorsal vagal complex (DVC) neurons by various forms of GRP to determine the pathway involved in this reduction. We found the following: (1) GRP-10, -27 and -29 (2.1 nmol/kg, i.p.) increased the Fos-like immunoreactivity (Fos-LI, a marker for neuronal activation) in the myenteric neurons of the stomach and the area postrema (AP) of the DVC; (2) GRP-27 and GRP-29 increased the Fos-LI in the myenteric plexus of the duodenum; and (3) only GRP-29 increased the Fos-LI in the submucosal plexus of the duodenum. In conclusion, GRP may reduce food intake by activating the area postrema. The enteric neurons may have a potential role in this reduction through the direct activation of the AP or exerting local gut actions, such as the stimulation of gut motility or secretions.     

P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> purinoceptors in the rat enteric nervous system

Adenosine 5-triphosphate receptors are known to be involved in fast excitatory postsynaptic currents in myenteric neurons of the digestive tract. In the present study, the distribution of P2X₂ and P2X₃ receptor mRNA was examined by in situ hybridisation while P2X₂ and P2X₃ receptor protein was localised by immunohistochemical methods. In addition, P2X₂ and P2X₃ receptors were colocalised with calbindin and calretinin in the myenteric and submucosal plexus. P2X₂- and P2X₃-immunoreactive neurons were found in the myenteric and submucosal plexuses throughout the entire length of the rat digestive tract from the stomach to the colon. Approximately 60%, 70% and 50% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 56% and 45% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X₂ receptor. Approximately 10%, 2% and 15% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 62% and 40% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X₃ receptor. Double-labelling studies showed that about 10–25% of the neurons with P2X₂ immunoreactivity in myenteric plexus and 30–50% in the submucosal plexus were found to express calbindin or calretinin. About 80% of the neurons with P2X₃ receptor immunoreactivity in the myenteric plexus and about 40% in the submucosal plexus expressed calretinin. Approximately 30–75% of the neurons with P2X₃ receptor immunoreactivity in the submucosal plexus expressed calbindin, while none of them were found to express calbindin in the myenteric plexus.     

Spatial organization of neurons in the dorsal motor nucleus of the vagus synapsing with intragastric cholinergic and nitric oxide/VIP neurons in the rat

The dorsal motor nucleus of the vagus (DMV) contains preganglionic neurons that control gastric motility and secretion. Stimulation of different parts of the DMV results in a decrease or an increase in gastric motor activities, suggesting a spatial organization of vagal preganglionic neurons in the DMV. Little is known about how these preganglionic neurons in the DMV synapse with different groups of intragastric motor neurons to mediate contraction or relaxation of the stomach. We used pharmacological and immunohistochemical methods to characterize intragastric neural pathways involved in mediating gastric contraction and relaxation in rats. Microinjections of L-glutamate (L-Glu) into the rostral or caudal DMV produced gastric contraction and relaxation, respectively, in a dose-related manner. Intravenous infusion of hexamethonium blocked these actions, suggesting mediation via preganglionic cholinergic pathways. Atropine inhibited gastric contraction by 85.5 +/- 4.5%. Gastric relaxation was reduced by intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 52.5 +/- 11.9%) or VIP antagonist (56.3 +/- 14.9%). Combined administration of L-NAME and VIP antagonist inhibited gastric relaxation evoked by L-Glu (87.8 +/- 4.3%). Immunohistochemical studies demonstrated choline acetyltransferase immunoreactivity in response to L-Glu microinjection into the rostral DMV in 88% of c-Fos-positive intragastric myenteric neurons. Microinjection of L-Glu into the caudal DMV evoked expression of nitric oxide (NO) synthase and VIP immunoreactivity in 81 and 39%, respectively, of all c-Fos-positive intragastric myenteric neurons. These data indicate spatial organization of the DMV. Depending on the location, microinjection of L-Glu into the DMV may stimulate intragastric myenteric cholinergic neurons or NO/VIP neurons to mediate gastric contraction and relaxation.     

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats

The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons; in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.     

大鼠下丘脑外侧区内微量注射胃动素对胃窦运动和迷走背核复合体神经元电活动的影响

采用清醒大鼠胃运动记录和玻璃微电极记录神经元活动的实验方法 ,研究下丘脑外侧区 (lateralhy pothalamicarea,LHA)微量注射胃动素 (motilin) ,对清醒大鼠胃窦运动和对麻醉大鼠迷走背核复合体 (dorsalvagalcomplex ,DVC)中胃扩张敏感神经元电活动的调节作用。LHA内微量注射胃动素 (0 37nmol/ 0 5 μl)可使胃窦运动增强 76 2 9± 4 0 9% (P <0 0 1)。DVC中 6 0个胃扩张 (gastricdistention ,GD)敏感神经元中 ,39(6 5 % )个GD刺激引起电活动增强 ,2 1(35 % )个电活动减弱 ,分别称之为GD兴奋型神经元和GD抑制型神经元。双侧LHA微量注射胃动素 0 37nmol/ 0 5 μl,14个GD抑制型神经元中有 12个单位放电频率增加 4 4 35± 7 89% (P <0 0 1) ;2 4个GD兴奋型神经元中有 15个单位放电频率减少 7 17± 7 89% (P <0 0 5 )。结果提示 ,中枢胃动素可能通过LHA-DVC-迷走神经实现对胃窦运动的调控     

Endogenous cholecystokinin reduces food intake and increases Fos-like immunoreactivity in the dorsal vagal complex but not in the myenteric plexus by CCK1 receptor in the adult rat

We hypothesized that endogenous CCK reduces food intake by activating the dorsal vagal complex (DVC) and the myenteric neurons of the gut. To test this hypothesis, adult rats were given camostat mesilate; a nonnutrient releaser of endogenous CCK, by orogastric gavage, and Fos-like immunoreactivity (Fos-LI) was quantified in the DVC and the myenteric plexus. The results for endogenous CCK were compared with those for exogenous CCK-8. Exogenous CCK-8 reduced food intake and stimulated Fos-LI in the DVC and in myenteric neurons of the duodenum and jejunum. In comparison, endogenous CCK reduced food intake and increased DVC Fos-LI but did not increase Fos-LI in the myenteric plexus. Similar to CCK-8, devazepide, a specific CCK(1) receptor antagonist, and not L365,260, a specific CCK(2) receptor antagonist, attenuated the reduction of food intake by camostat. In addition, Fos-LI in the DVC in response to both exogenous CCK-8 and camostat administration was significantly attenuated by vagotomy, as well as by blocking CCK(1) receptors. These results demonstrate for the first time that reduction of food intake in adult rats by endogenous CCK released by a nonnutrient mechanism requires CCK(1) receptors, the vagus nerve, and activation of the DVC, but not the myenteric plexus.