Leptin and CCK modulate complementary background conductances to depolarize cultured nodose neurons

We have previously reported that intraceliac infusion of leptin induces a reduction of meal size that depends on intact vagal afferents. This effect of leptin is enhanced in the presence of cholecystokinin (CCK). The mechanisms by which leptin and CCK activate vagal afferent neurons are not known. In the present study, we have begun to address this question by using patch-clamp electrophysiological techniques to examine the mechanisms by which leptin and CCK activate cultured vagal afferents from adult rat nodose ganglia. We found that leptin depolarized 41 (60%) of 68 neurons. The magnitude of membrane depolarization was dependent on leptin concentration and occurred in both capsaicin-sensitive and capsaicin-insensitive neurons. We also found that a majority (16 of 22; 73%) of nodose neurons activated by leptin were also sensitive to CCK. CCK-induced depolarization was primarily associated with the increase of an inward current (11 of 12), whereas leptin induced multiple changes in background conductances through a decrease in an outward current (7 of 13), an increase in an inward current (3 of 13), or both (3 of 13). However, further isolation of background currents by recording in solutions that contained only sodium or only potassium revealed that both leptin and CCK were capable of increasing a sodium-dependent conductance or inhibiting a potassium-dependent conductance. Our results support the hypothesis that vagal afferents are a point of convergence and integration of leptin and CCK signaling for control of food intake and suggest multiple ionic mechanisms by which leptin and CCK activate vagal afferent neurons. cholecystokinin; vagal afferents; capsaicin; satiation     

Anti-inflammatory effects of leptin and cholecystokinin on acetic acid-induced colitis in rats: role of capsaicin-sensitive vagal afferent fibers

Leptin and cholecystokinin (CCK) have a synergistic interaction in the suppression of food intake, and afford similar gastroprotective activity. The present study was designed to investigate the putative protective effects of CCK and leptin on acute colonic inflammation. Leptin or CCK-8s was injected to rats intraperitoneally immediately before and 6 h after the induction of colitis with acetic acid. CCK-A receptor antagonist (L-364,718) or CCK-B receptor antagonist (L-365,260) was injected intraperitoneally 15 min before leptin or CCK treatments. In a group of rats, vagal afferent fibers were denervated by topical application of capsaicin on the cervical vagi. Rats were decapitated at 24 h, and the distal 8 cm of the colon were removed for macroscopic scoring, determination of tissue wet weight index (WWI), histologic assessment and tissue myeloperoxidase (MPO) activity. All inflammation parameters were increased by acetic acid-induced colitis compared to control group. Leptin or CCK-8s treatment reduced these parameters in a similar manner, while co-administration of leptin and CCK was found to be more effective in reducing the macroscopic score and WWI. CCK-8s-induced reduction in the score and WWI was prevented by CCK-A, but not by CCK-B receptor antagonist, whereas neither antagonist altered the inhibitory effect of leptin on colitis-induced injury. On the other hand, perivagal capsaicin prevented the protective effects of both CCK-8s and leptin on colitis. Our results indicate that leptin and CCK have anti-inflammatory effects on acetic acid-induced colitis in rats, which appear to be mediated by capsaicin-sensitive vagal afferent fibers involving the reduction in colonic neutrophil infiltration.     

Leptin-induced satiation mediated by abdominal vagal afferents

Leptin is a hormone secreted into the systemic blood primarily by white adipose tissue. However, leptin also is synthesized and stored by cells in the gastric mucosa. Because gastric mucosal leptin is secreted in response to ingestion of a meal, we hypothesized that it might contribute to satiation (meal termination) by acting on gastrointestinal vagal afferent neurons. To test whether leptin is capable of acutely reducing short-term food intake, we measured consumption of a liquid meal (15% sucrose) following low-dose leptin administration via the celiac artery, which perfuses the upper gastrointestinal tract. Leptin (1, 3, 10 mug) was infused via a chronically implanted, nonocclusive celiac arterial catheter or via a jugular vein catheter with its tip in the right cardiac atrium. Fifteen percent sucrose intake was then measured for 30 min. We found that leptin dose dependently inhibited sucrose intake when infused through the celiac catheter but not when infused into the general circulation via a jugular catheter. Plasma leptin concentrations in the general circulation following celiac arterial or jugular leptin infusions were not significantly different. Celiac arterial leptin infusion did not reduce meal size in vagotomized or capsaicin-treated rats. Finally, we also found that reduction of meal size by celiac leptin infusion was markedly enhanced when coinfused with cholecystokinin, a gastrointestinal satiety peptide whose action depends on vagal afferent neurons. Our results support the hypothesis that leptin contributes to satiation by a mechanism dependent on gastrointestinal vagal afferent innervation of the upper gastrointestinal tract.     

Leptin resistance in vagal afferent neurons inhibits cholecystokinin signaling and satiation in diet induced obese rats

Background and Aims

The gastrointestinal hormone cholecystokinin (CCK) plays an important role in regulating meal size and duration by activating CCK1 receptors on vagal afferent neurons (VAN). Leptin enhances CCK signaling in VAN via an early growth response 1 (EGR1) dependent pathway thereby increasing their sensitivity to CCK. In response to a chronic ingestion of a high fat diet, VAN develop leptin resistance and the satiating effects of CCK are reduced. We tested the hypothesis that leptin resistance in VAN is responsible for reducing CCK signaling and satiation.

Results

Lean Zucker rats sensitive to leptin signaling, significantly reduced their food intake following administration of CCK8S (0.22 nmol/kg, i.p.), while obese Zucker rats, insensitive to leptin, did not. CCK signaling in VAN of obese Zucker rats was reduced, preventing CCK-induced up-regulation of Y2 receptor and down-regulation of melanin concentrating hormone 1 receptor (MCH1R) and cannabinoid receptor (CB1). In VAN from diet-induced obese (DIO) Sprague Dawley rats, previously shown to become leptin resistant, we demonstrated that the reduction in EGR1 expression resulted in decreased sensitivity of VAN to CCK and reduced CCK-induced inhibition of food intake. The lowered sensitivity of VAN to CCK in DIO rats resulted in a decrease in Y2 expression and increased CB1 and MCH1R expression. These effects coincided with the onset of hyperphagia in DIO rats.

Conclusions

Leptin signaling in VAN is required for appropriate CCK signaling and satiation. In response to high fat feeding, the onset of leptin resistance reduces the sensitivity of VAN to CCK thus reducing the satiating effects of CCK.     

Capsaicin-sensitive vagal afferents and CCK in inhibition of gastric motor function induced by intestinal nutrients

The role of vagal afferent pathways and cholecystokinin (CCK) in mediating changes in gastric motor function after a meal was investigated in urethane-anesthetized rats. Proximal gastric motor function was measured manometrically, and nutrients were infused into an isolated segment of duodenum. Inhibition of gastric motility in response to duodenal infusion of protein (peptone or casein), but not carbohydrate (glucose), was significantly attenuated by administration of the CCK antagonist, L364,718. Selective ablation of vagal afferents by perineural treatment with the sensory neurotoxin, capsaicin, significantly reduced responses to both duodenal protein and glucose. These results suggest that protein in the duodenum decreases proximal gastric motor function via release of CCK and a vagal capsaicin-sensitive afferent pathway. In contrast, glucose acts via a capsaicin-sensitive vagal pathway not involving CCK. Thus separate neural and hormonal mechanisms mediate the effects of different nutrients in the duodenal feedback regulation of gastric motor function.     

Comparative pharmacology of cholecystokinin induced activation of cultured vagal afferent neurons from rats and mice

Cholecystokinin (CCK) facilitates the process of satiation via activation of vagal afferent neurons innervating the upper gastrointestinal tract. Recent findings indicate CCK acts on these neurons via a ruthenium red (RuR) sensitive pathway that involves members of the vanilloid (V) subfamily of transient receptor potential (TRP) channels. To further test this mechanism, the mouse provides an ideal model in which genetic tools could be applied. However, whether CCK acts by similar mechanism(s) in mice has not been determined. In the present study we explored the actions of CCK on nodose neurons isolated from Sprague Dawley (SD) rat and two strains of mice; C57BL/6 and BalbC using fluorescence-based calcium imaging. With minor exceptions nodose neurons isolated from all species/strains behaved similarly. They all respond to brief depolarization with a large calcium transient. A significant subset of neurons responded to capsaicin (CAP), a TRPV1 agonist, although neurons from C57BL/6 were 10-fold more sensitive to CAP than SD rats or BalbC mice, and a significantly smaller fraction of neurons from BalbC mice responded to CAP. CCK-8 dose-dependently activated a subpopulation of neurons with similar dose dependency, percent responders, and overlap between CCK and CAP responsiveness. In all species/strains CCK-8 induced activation was significantly attenuated (but not completely blocked) by pretreatment with the TRPV channel blocker RuR. Surprisingly, the CCK analogue JMV-180, which is reported to have pure antagonistic properties in rat but mixed agonist/antagonist properties in mice, behaved as a pure antagonist to CCK in both rat and mouse neurons. The pure antagonistic action of JMV-180 in this in vitro preparation suggests that prior reported differential effects of JMV-180 on satiation in rats versus mouse must be mediated by a site other than vagal afferent activation.     

Low-affinity CCK-A receptors are coexpressed with leptin receptors in rat nodose ganglia: implications for leptin as a regulator of short-term satiety

The paradigm for the control of feeding behavior has changed significantly. Research has shown that leptin, in the presence of CCK, may mediate the control of short-term food intake. This interaction between CCK and leptin occurs at the vagus nerve. In the present study, we aimed to characterize the interaction between CCK and leptin in the vagal primary afferent neurons. Single neuronal discharges of vagal primary afferent neurons innervating the gastrointestinal tract were recorded from rat nodose ganglia. Three groups of nodose ganglia neurons were identified: group 1 responded to CCK-8 but not leptin; group 2 responded to leptin but not CCK-8; group 3 responded to high-dose CCK-8 and leptin. In fact, the neurons in group 3 showed CCK-8 and leptin potentiation, and they responded to gastric distention. To identify the CCK-A receptor (CCKAR) affinity states that colocalize with the leptin receptor OB-Rb, we used CCK-JMV-180, a high-affinity CCKAR agonist and low-affinity CCKAR antagonist. As expected, immunohistochemical studies showed that CCK-8 administration significantly potentiated the increase in the number of c-Fos-positive neurons stimulated by leptin in vagal nodose ganglia. Administration of CCK-JMV-180 eliminated the synergistic interaction between CCK-8 and leptin. We conclude that both low- and high-affinity CCKAR are expressed in nodose ganglia. Many nodose neurons bearing low-affinity CCKAR express OB-Rb. These neurons also respond to mechanical distention. An interaction between CCKAR and OB-Rb in these neurons likely facilitates leptin mediation of short-term satiety.     

Leptin inhibits gastric emptying in rats: role of CCK receptors and vagal afferent fibers

Leptin regulates energy homeostasis and body weight by balancing energy intake and expenditure. It was recently reported that leptin, released into the gut lumen during the cephalic phase of gastric secretion, is capable of initiating intestinal nutrient absorption. Vagal afferent neurons also express receptors for both CCK and leptin, which are believed to interact in controlling food intake. The present study was undertaken to investigate the central and peripheral effects of leptin on gastric emptying rate. Under anesthesia, male Sprague-Dawley rats (250-300 g) were fitted with gastric Gregory cannulas (n=12) and some had additional cerebroventricular cannulas inserted into their right lateral ventricles. Following recovery, the rate of gastric emptying of saline (300 mOsm/kg H(2)O) was determined after instillation into the gastric fistula (3 ml, 37 degrees C, containing phenol red, 60 mg/l as a non-absorbable dilution marker). Gastric emptying rate was determined from the volume and phenol red concentrations recovered after 5 min. Leptin, injected intraperitoneally (i.p.; 10, 30, 60, 100 microg/kg) or intracerebroventricularly (i.c.v.; 5, 15 microg/rat) 15 min before the emptying, delayed gastric emptying rate of saline at the dose of 30 microg/kg or 15 microg/rat (p<0.001). When CCK(1) receptor blocker L-364,718 (1 mg/kg, i.p.), CCK(2) receptor blocker L-365,260 (1 mg/kg, ip) or adrenergic ganglion blocker bretylium tosylate (15 mg/kg, i.p.) was administered 15 min before ip leptin (30 microg/kg) injections, leptin-induced delay in gastric emptying was abolished only by the CCK(1) receptor blocker (p<0.001). However, the inhibitory effect of central leptin on gastric emptying was reversed by adrenergic blockade, but not by either CCK antagonists. Our results demonstrated that leptin delays gastric emptying. The peripheral effect of leptin on gastric motility appears to be mediated by CCK(1) receptors, suggesting the release of CCK and the involvement of vagal afferent fibers. On the other hand, the central effect of leptin on gastric emptying is likely to be mediated by adrenergic neurons. These results indicate the existence of a functional interaction between leptin and CCK receptors leading to inhibition of gastric emptying and short-term suppression of food intake, providing an additional feedback control in producing satiety.     

Synergistic interaction between CCK and leptin to regulate food intake

Leptin administered (either intracerebroventricularly, icv, or intraperitoneally, ip) acts in synergy with CCK to suppress food intake and body weight in lean mice or rats. The potentiating effect induced by the co-injection of ip CCK and leptin to inhibit food consumption in mice is mediated by the CCK-A receptor and capsaicin sensitive afferents. In vitro, studies in rats showed that a subset of gastric vagal afferent fibers responded to leptin injected directly into the gastric artery only after a prior intra-arterial CCK injection. Moreover, the tonic activity of gastric-related neurons in the nucleus tractus solitarius (NTS) increased when leptin was delivered into the gastric chamber of an in vitro stomach-brainstem preparation. CCK co-injected with leptin potentiated Fos expression selectively in the area postrema, NTS and paraventricular nucleus of the hypothalamus (PVN), which points to the PVN as part of the afferent and efferent limbs of the circuitry involved in the synergistic interaction between leptin and CCK. The dampening of CCK or leptin inhibitory action on ingestive behavior when either factor is not present or their receptors are non functional supports the notion that such leptin-CCK interaction may have a physiological relevance. These observations provide a mean through which leptin and CCK integrate short- and mid-term meal-related input signals into long-term control of energy balance.     

A chronic high fat diet alters the homologous and heterologous control of appetite regulating peptide receptor expression

Leptin, ghrelin and neuropeptide W (NPW) modulate vagal afferent activity, which may underlie their appetite regulatory actions. High fat diet (HFD)-induced obesity induces changes in the plasma levels of these peptides and alters the expression of receptors on vagal afferents. We investigated homologous and heterologous receptor regulation by leptin, ghrelin and NPW. Mice were fed (12 weeks) a standard laboratory diet (SLD) or HFD. Nodose ganglia were cultured overnight in the presence or absence of each peptide. Leptin (LepR), ghrelin (GHS-R), NPW (GPR7) and cholecystokinin type-1 (CCK1R) receptor mRNA, and the plasma leptin, ghrelin and NPW levels were measured. SLD: leptin reduced LepR, GPR7, increased GHS-R and CCK1R mRNA; ghrelin increased LepR, GPR7, CCK1R, and decreased GHS-R. HFD: leptin decreased GHS-R and GPR7, ghrelin increased GHS-R and GPR7. NPW decreased all receptors except GPR7 which increased with HFD. Plasma leptin was higher and NPW lower in HFD. Thus, HFD-induced obesity disrupts inter-regulation of appetite regulatory receptors in vagal afferents.     

Adenosine-induced activation of esophageal nociceptors

Clinical studies implicate adenosine acting on esophageal nociceptive pathways in the pathogenesis of noncardiac chest pain originating from the esophagus. However, the effect of adenosine on esophageal afferent nerve subtypes is incompletely understood. We addressed the hypothesis that adenosine selectively activates esophageal nociceptors. Whole cell perforated patch-clamp recordings and single-cell RT-PCR analysis were performed on the primary afferent neurons retrogradely labeled from the esophagus in the guinea pig. Extracellular recordings were made from the isolated innervated esophagus. In patch-clamp studies, adenosine evoked activation (inward current) in a majority of putative nociceptive (capsaicin-sensitive) vagal nodose, vagal jugular, and spinal dorsal root ganglia (DRG) neurons innervating the esophagus. Single-cell RT-PCR analysis indicated that the majority of the putative nociceptive (transient receptor potential V1-positive) neurons innervating the esophagus express the adenosine receptors. The neural crest-derived (spinal DRG and vagal jugular) esophageal nociceptors expressed predominantly the adenosine A(1) receptor while the placodes-derived vagal nodose nociceptors expressed the adenosine A(1) and/or A(2A) receptors. Consistent with the studies in the cell bodies, adenosine evoked activation (overt action potential discharge) in esophageal nociceptive nerve terminals. Furthermore, the neural crest-derived jugular nociceptors were activated by the selective A(1) receptor agonist CCPA, and the placodes-derived nodose nociceptors were activated by CCPA and/or the selective adenosine A(2A) receptor CGS-21680. In contrast to esophageal nociceptors, adenosine failed to stimulate the vagal esophageal low-threshold (tension) mechanosensors. We conclude that adenosine selectively activates esophageal nociceptors. Our data indicate that the esophageal neural crest-derived nociceptors can be activated via the adenosine A(1) receptor while the placodes-derived esophageal nociceptors can be activated via A(1) and/or A(2A) receptors. Direct activation of esophageal nociceptors via adenosine receptors may contribute to the symptoms in esophageal diseases.     

Calcitonin gene-related peptide-containing neurons supplying the rat digestive system: differential distribution and expression pattern.

In the enteric nervous system, calcitonin gene-related peptide (CGRP) immunoreactivity is localized to a substantial number of capsaicin-sensitive afferent fibers and to intrinsic neurons and processes. CGRP immunoreactivity detected by immunohistochemistry represents the expression of two distinct genes, the calcitonin/alpha-CGRP and the beta-CGRP genes, which have different tissue distributions. In the present study, we used (1) in situ hybridization histochemistry and ribonucleic acid (RNA) blot hybridization with RNA probes complementary to the divergent sequences of alpha- and beta-CGRP messenger RNAs (mRNAs) to differentiate which CGRP gene was expressed in enteric and afferent neurons; and (2) axonal transport approaches in combination with CGRP immunohistochemistry to define the location of CGRP-containing afferent neurons supplying the digestive system. In situ hybridization histochemistry with [35S]-labeled RNA probes indicated that in the gastrointestinal tract beta-CGRP mRNA, but not alpha-CGRP mRNA, was expressed in enteric neurons confined to the myenteric and submucous plexuses of the small and large intestine. In dorsal root and vagal sensory ganglia, mRNAs for alpha-CGRP and beta-CGRP were both present in a vast population of neurons, with an overlapping pattern, even though the alpha-CGRP signal appeared more intense. RNA blot hybridization analysis showed a single band of hybridization at 1.2 Kb with the beta-CGRP RNA probe in RNA extracts from muscle layer-myenteric plexus and submucosal layer preparations of the ileum, and from dorsal root ganglia; it also showed a single band at 1.3 Kb with the alpha-CGRP RNA probe in extracts from dorsal root ganglia, but not from the intestine. These findings further support the differential expression of alpha- and beta-CGRP mRNAs. Retrograde transport of fast blue or fluorogold coupled with CGRP immunohistochemistry demonstrated that the vast majority of CGRP-containing afferent neurons supplying the stomach, proximal duodenum, and pancreas were located in dorsal root ganglia at the middle and lower thoracic and at the upper lumbar levels, and represented a major component of the afferent innervation of these viscera (up to 89%). Approximately 50% of CGRP-immunoreactive afferent neurons also expressed tachykinin (TK) immunoreactivity, as shown by triple labeling. Only a minor component of the afferent innervation of the stomach, duodenum, and pancreas derived from vagal CGRP-containing neurons (less than 8%). A large portion of these neurons (an average of 62%) also contained TK immunoreactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Apolipoprotein A-IV stimulates duodenal vagal afferent activity to inhibit gastric motility via a CCK1 pathway

Apolipoprotein A-IV (apo A-IV), a peptide expressed by enterocytes in the mammalian small intestine and released in response to long-chain triglyceride absorption, may be involved in the regulation of gastric acid secretion and gastric motility. The specific aim of the present study was to determine the pathway involved in mediating inhibition of gastric motility produced by apo A-IV. Gastric motility was measured manometrically in response to injections of either recombinant purified apo A-IV (200 microg) or apo A-I, the structurally similar intestinal apolipoprotein not regulated by triglyceride absorption, close to the upper gastrointestinal tract in urethane-anesthetized rats. Injection of apo A-IV significantly inhibited gastric motility compared with apo A-I or vehicle injections. The response to exogenous apo A-IV injections was significantly reduced by 77 and 55%, respectively, in rats treated with the CCK(1) receptor blocker devazepide or after functional vagal deafferentation by perineural capsaicin treatment. In electrophysiological experiments, isolated proximal duodenal vagal afferent fibers were recorded in vitro in response to close-arterial injection of vehicle, apo A-IV (200 microg), or CCK (10 pmol). Apo A-IV stimulated the discharge of duodenal vagal afferent fibers, significantly increasing the discharge in 4/7 CCK-responsive units, and the response was abolished by CCK(1) receptor blockade with devazepide. These data suggest that apo A-IV released from the intestinal mucosa during lipid absorption stimulates the release of endogenous CCK that activates CCK(1) receptors on vagal afferent nerve terminals initiating feedback inhibition of gastric motility.     

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.     

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.     

Neurons in the rat lateral hypothalamic area integrate information from the gastric vagal nerves and the cerebellar interpositus nucleus

Previous investigations have demonstrated that the neuronal activity in the lateral hypothalamic area (LHA) is respectively modulated by afferent inputs from the gastric vagal nerves innervating the upper gastrointestinal tract, as well as the cerebellar interpositus nucleus (IN). The aim of this study was to examine whether the gastric vagal and cerebellar IN inputs converge onto single LHA neurons in rats, especially those sensitive to glycemia. Of the 114 LHA neurons recorded, 60 (52.6%) and 51 (44.7%) responded to gastric vagal and cerebellar IN stimulation, respectively. Of the 60 LHA neurons responsive to gastric vagal stimulation, 30 also responded to the cerebellar IN stimulus, indicating a convergence of gastric vagal and cerebellar inputs onto single hypothalamic cells. When the gastric vagal nerves and cerebellar IN were stimulated simultaneously, a summation of the responses was observed in all 6 neurons tested. Moreover, of 24 neurons that responded to both the gastric vagal and cerebellar IN stimuli, 15 (62.5%) were identified as glycemia-sensitive. These results demonstrate that the visceral information transmitted by the gastric vagal nerves and the somatic information forwarded by the cerebellar IN converge onto single LHA neurons, especially those sensitive to glycemia. The findings also suggest that integration of somatic-visceral responses related to short-term feeding regulation may take place in the LHA.     

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.     

Differential mechanism and site of action of CCK on the pancreatic secretion and growth in rats

Recent studies demonstrated that cholecystokinin (CCK) at physiological levels stimulates pancreatic enzyme secretion via a capsaicin-sensitive afferent vagal pathway. This study examined whether chemical ablation of afferent vagal fibers influences pancreatic growth and secretion in rats. Bilateral subdiaphragmatic vagal trunks were exposed, and capsaicin solution was applied. Pancreatic wet weight and pancreatic secretion and growth in response to endogenous and exogenous CCK were examined 7 days after capsaicin treatment. Perivagal application of capsaicin increased plasma CCK levels and significantly increased pancreatic wet weight compared with those in the control rats. Oral administration of CCK-1 receptor antagonist loxiglumide prevented the increase in pancreatic wet weight after capsaicin treatment. In addition, continuous intraduodenal infusion of trypsin prevented the increase in plasma CCK levels and pancreatic wet weight after capsaicin treatment. There were no significant differences in the expression levels of CCK-1 receptor mRNA and protein in the pancreas in capsaicin-treated and control rats. Intraduodenal administration of camostat or intravenous infusion of CCK-8 stimulated pancreatic secretion in control rats but not in capsaicin-treated rats. In contrast, repeated oral administrations of camostat or intraperitoneal injections of CCK-8 significantly increased pancreatic wet weight in both capsaicin-treated and control rats. Present results suggest that perivagal application of capsaicin stimulates pancreatic growth via an increase in endogenous CCK and that exogenous and endogenous CCK stimulate pancreatic growth not via vagal afferent fibers but directly in rats.     

Expression of 5-HT3 receptors by extrinsic duodenal afferents contribute to intestinal inhibition of gastric emptying

Intestinal perfusion with carbohydrates inhibits gastric emptying via vagal and spinal capsaicin-sensitive afferent pathways. The aim of the present study was to determine the role of 1) 5-hydroxytryptamine (5-HT)(3) receptors (5-HT(3)R) in mediating glucose-induced inhibition of gastric emptying and 2) 5-HT(3)R expression in vagal and spinal afferents in innervating the duodenum. In awake rats fitted with gastric and duodenal cannulas, perfusion of the duodenum with glucose (50 and 100 mg) inhibited gastric emptying. Intestinal perfusion of mannitol inhibited gastric emptying only at the highest concentration (990 mosm/kgH(2)O). Pretreatment with the 5-HT(3)R antagonist tropisetron abolished both glucose- and mannitol-induced inhibition of gastric emptying. Retrograde labeling of visceral afferents by injection of dextran-conjugated Texas Red into the duodenal wall was used to identify extrinsic primary afferents. Immunoreactivity for 5-HT(3)R, visualized with an antibody directed to the COOH terminus of the rat 5-HT(3)R, was found in >80% of duodenal vagal and spinal afferents. These results show that duodenal extrinsic afferents express 5-HT(3)R and that the receptor mediates specific glucose-induced inhibition of gastric emptying. These findings support the hypothesis that enterochromaffin cells in the intestinal mucosa release 5-HT in response to glucose, which activates 5-HT(3)R on afferent nerve terminals to evoke reflex changes in gastric motility. The primary glucose sensors of the intestine may be mucosal enterochromaffin cells.