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The pig is a good model for studying intestinal functions and disorders for its homologies with humans such as microbiome composition, size, nutrition being both omnivores and colon fermenters. The enteric nervous system (ENS) of pigs and humans has a multilayered submucosal plexus with an inner submucous plexus (ISP) near the mucosa and an outer plexus (OSP) near the circular muscle in addition to the myenteric plexus (MP) between the muscle layers. We have shown differences in the density and distribution of functionally distinct neurons in different regions and plexuses of the porcine colon. Aim: This study focused on Substance P, a peptide that modulates many functions in the gastrointestinal (GI) tract and plays an important role in neurogenic inflammation. We tested whether there were differences in the density and neurochemical profile of SP neurons in the ISP, OSP and MP of the ascending (AC) and descending (DC) colon of 15 Yucatan minipigs (12M, 3F, 7-months-old, body weight 25-30 kg). We processed colonic wholemounts for multiple labeling immunofluorescence using the HuCD, choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) as neuronal markers with high resolution confocal microscopy and Imaris software to quantify the number of neurons/mm2 and the % of total neurons identified by the pan-neuronal marker HuCD. Results: HuCD/SP-immunoreactive (IR) neurons were most abundant in the ISP vs. OSP and MP in both AC and DC (p Supported by NIH SPARC OT2OD24899 & NIH-P30DK41301. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The porcine gut is increasingly regarded as a useful translational model. The enteric nervous system in the colon coordinates diverse functions. However, knowledge of the molecular profiling of porcine enteric nerve system and its similarity to that of human is still lacking. We identified the distinct transcriptional programs associated with functional characteristics between inner submucosal and myenteric ganglia in porcine proximal and distal colon using bulk RNA and single-cell RNA sequencing. Comparative transcriptomics of myenteric ganglia in corresponding colonic regions of pig and human revealed highly conserved programs in porcine proximal and distal colon, which explained >96% of their transcriptomic responses to vagal nerve stimulation, suggesting that porcine proximal and distal colon could serve as predictors in translational studies. The conserved programs specific for inflammatory modulation were displayed in pigs with vagal nerve stimulation. This study provides a valuable transcriptomic resource for understanding of human colonic functions and neuromodulation using porcine model.

The enteric nervous system (ENS) controls gastrointestinal functions. In large mammals’ intestine, it comprises an inner (ISP) and outer (OSP) submucous plexus and a myenteric plexus (MP). This study quantifies enteric neurons in the ISP, OSP, and MP of the pig ascending (AC) and descending colon (DC) using the HuC/D, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) neuronal markers in whole mount preparations with multiple labeling immunofluorescence. We established that the ISP contains the highest number of HuC/D neurons/mm2, which were more abundant in AC vs. DC, followed by OSP and MP with similar density in AC and DC. In the ISP, the density of ChAT immunoreactive (IR) neurons was very similar in AC and DC (31% and 35%), nNOS-IR neurons were less abundant in AC than DC (15% vs. 42%, P < 0.001), and ChAT/nNOS-IR neurons were 5% and 10%, respectively. In the OSP, 39–44% of neurons were ChAT-IR in AC and DC, while 45% and 38% were nNOS-IR and 10–12% were ChAT/nNOS-IR (AC vs. DC P < 0.05). In the MP, ChAT-IR neurons were 44% in AC and 54% in DC (P < 0.05), nNOS-IR neurons were 50% in both, and ChAT/nNOS-IR neurons were 12 and 18%, respectively. The ENS architecture with multilayered submucosal plexuses and the distribution of functionally distinct groups of neurons in the pig colon are similar to humans, supporting the suitability of the pig as a model and providing the platform for investigating the mechanisms underlying human colonic diseases.

Made up of millions of enteric neurons and glial cells, the enteric nervous system (ENS) is in a key position to modulate the secretomotor function and visceral pain of the gastrointestinal tract. The early life developmental period, through which most of the ENS development occurs, is highly susceptible to microenvironmental perturbation. Over the past decade, accumulating evidence has shown the impact of stress and early life adversity (ELA) on host gastrointestinal pathophysiology. While most of the focus has been on alterations in brain structure and function, limited experimental work in rodents suggest that the enteric nervous system can also be directly affected, as shown by changes in the number, phenotype, and reactivity of enteric nerves. The work of Medland et al. in the current issue of this journal demonstrates that such alterations also occur in pigs, a larger mammalian species with high translational value to human. This work also highlights a sex-differential susceptibility of the ENS to the effect of ELA, which could contribute to the higher prevalence of GI disorders in women. In this mini-review, we will discuss the development and composition of the ENS and related gastrointestinal sensory motor and secretory functions. We will then focus on the influence of stress on the enteric nervous system, with a particular emphasis on neurodevelopmental changes. Finally, we will discuss the influence of sex on those parameters.

BACKGROUND: Corticotropin-releasing factor (CRF) pathways coordinate behavioral, endocrine, autonomic and visceral responses to stress. Convergent anatomical, molecular, pharmacological and functional experimental evidence supports a key role of brain CRF receptor (CRF-R) signaling in stress-related alterations of gastrointestinal functions. These include the inhibition of gastric acid secretion and gastric-small intestinal transit, stimulation of colonic enteric nervous system and secretory-motor function, increase intestinal permeability, and visceral hypersensitivity. Brain sites of CRF actions to alter gut motility encompass the paraventricular nucleus of the hypothalamus, locus coeruleus complex and the dorsal motor nucleus while those modulating visceral pain are localized in the hippocampus and central amygdala. Brain CRF actions are mediated through the autonomic nervous system (decreased gastric vagal and increased sacral parasympathetic and sympathetic activities). The activation of brain CRF-R2 subtype inhibits gastric motor function while CRF-R1 stimulates colonic secreto-motor function and induces visceral hypersensitivity. CRF signaling is also located within the gut where CRF-R1 activates colonic myenteric neurons, mucosal cells secreting serotonin, mucus, prostaglandin E2, induces mast cell degranulation, enhances mucosal permeability and propulsive motor functions and induces visceral hyperalgesia in animals and humans. CRF-R1 antagonists prevent CRF- and stress-related gut alterations in rodents while not influencing basal state. DISCUSSION: These preclinical studies contrast with the limited clinical positive outcome of CRF-R1 antagonists to alleviate stress-sensitive functional bowel diseases such as irritable bowel syndrome. CONCLUSION: The translational potential of CRF-R1 antagonists in gut diseases will require additional studies directed to novel anti-CRF therapies and the neurobiology of brain-gut interactions under chronic stress.

Author(s): Moussaoui, Nabila; Jacobs, Jonathan P; Larauche, Muriel; Biraud, Mandy; Million, Mulugeta; Mayer, Emeran; Tache, Yvette | Abstract: Background/aimsWistar rat dams exposed to limited nesting stress (LNS) from post-natal days (PND) 2 to 10 display erratic maternal behavior, and their pups show delayed maturation of the hypothalamic-pituitary-adrenal axis and impaired epithelial barrier at PND10 and a visceral hypersensitivity at adulthood. Little is known about the impact of early life stress on the offspring before adulthood and the influence of sex. We investigated whether male and female rats previously exposed to LNS displays at weaning altered corticosterone, intestinal permeability, and microbiota.MethodsWistar rat dams and litters were maintained from PND2 to 10 with limited nesting/bedding materials and thereafter reverted to normal housing up to weaning (PND21). Control litters had normal housing. At weaning, we monitored body weight, corticosterone plasma levels (enzyme immunoassay), in vivo intestinal to colon permeability (fluorescein isothiocyanate-dextran 4 kDa) and fecal microbiota (DNA extraction and amplification of the V4 region of the 16S ribosomal RNA gene).ResultsAt weaning, LNS pups had hypercorticosteronemia and enhanced intestinal permeability with females g males while body weights were similar. LNS decreased fecal microbial diversity and induced a distinct composition characterized by increased abundance of Gram positive cocci and reduction of fiber-degrading, butyrate-producing, and mucus-resident microbes.ConclusionsThese data indicate that chronic exposure to LNS during the first week post-natally has sustained effects monitored at weaning including hypercorticosteronemia, a leaky gut, and dysbiosis. These alterations may impact on the susceptibility to develop visceral hypersensitivity in adult rats and have relevance to the development of irritable bowel syndrome in childhood.

Background Recent evidence from rat neuron-free mucosa study suggests that the membrane bile acid receptor TGR5 decreases colonic secretion under basal and stimulated conditions. As submucosal neurons are key players in secretory processes and highly express TGR5, we investigated their role in TGR5 agonist-induced inhibition of secretion and the pathways recruited. Methods TGR5 expression and localization were assessed in rat proximal (pC) and distal (dC) colon by qPCR and immunohistochemistry with double labeling for cholinergic neurons in whole-mount preparations. The influence of a selective (INT-777) or weak (ursodeoxycholic acid, UDCA) TGR5 agonist on colonic secretion was assessed in Ussing chambers, in dC preparation removing seromuscular ± submucosal tissues, in the presence of different inhibitors of secretion pathways. Key Results TGR5 mRNA is expressed in full thickness dC and pC and immunoreactivity is located in colonocytes and pChAT-positive neurons. Addition of INT-777, and less potently UDCA, decreased colonic secretion in seromuscular stripped dC by −58.17± 2.6%. INT-777 effect on basal secretion was reduced in neuron-free and TTX-treated mucosal-submucosal preparations. Atropine, hexamethonium, indomethacin, and L-NAME all reduced significantly INT-777's inhibitory effect while the 5-HT4 antagonist, RS-39604, and lidocaine abolished it. INT-777 inhibited stimulated colonic secretion induced by nicotine, but not cisapride, carbachol or PGE2. Conclusions & Inferences TGR5 activation inhibits basal and stimulated distal colonic secretion in rats by acting directly on epithelial cells and also inhibiting submucosal neurons. This could represent a counter-regulatory mechanism, at the submucosal level, of the known prosecretory effect of bile acids in the colon.

A few studies indicate that limited nesting stress (LNS) alters maternal behavior and the hypothalamic pituitary adrenal (HPA) axis of dams and offspring in male Sprague Dawley rats. In the present study, we evaluated the impact of LNS on maternal behavior in Wistar rats, and on the HPA axis, glycemia and in vivo intestinal permeability of male and female offspring. Intestinal permeability is known to be elevated during the first week postnatally and influenced by glucocorticoids. Dams and neonatal litters were subjected to LNS or normal nesting conditions (control) from days 2 to 10 postnatally. At day 10, blood was collected from pups for determination of glucose and plasma corticosterone by enzyme immunoassay and in vivo intestinal permeability by oral gavage of fluorescein isothiocyanate-dextran 4kDa. Dams exposed to LNS compared to control showed an increase in the percentage of time spent building a nest (118%), self-grooming (69%), and putting the pups back to the nest (167%). LNS male and female pups exhibited a reduction of body weight by 5% and 4%, adrenal weights/100g body weight by 17% and 18%, corticosterone plasma levels by 64% and 62% and blood glucose by 11% and 12% respectively compared to same sex control pups. In male LNS pups, intestinal permeability was increased by 2.7-fold while no change was observed in females compared to same sex control. There was no sex difference in any of the parameters in control pups except the body weight. These data indicate that Wistar dams subjected to LNS during the first postnatal week have an altered repertoire of maternal behaviors which affects the development of the HPA axis in both sexes and intestinal barrier function in male offspring.

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.

Acute in vivo measurements are often the initial, most practicable approach used to investigate the effects of novel compounds or genetic manipulations on the regulation of gastric motility. Such acute methods typically involve either surgical implantation of devices or require intragastric perfusion of solutions, which can substantially alter gastric activity and may require extended periods of time to allow stabilization or recovery of the preparation. We validated a simple, non-invasive novel method to measure acutely gastric contractility, using a solid-state catheter pressure transducer inserted orally into the gastric corpus, in fasted, anesthetized rats or mice. The area under the curve of the phasic component (pAUC) of intragastric pressure (IGP) was obtained from continuous manometric recordings of basal activity and in responses to central or peripheral activation of cholinergic pathways, or to abdominal surgery. In rats, intravenous ghrelin or intracisternal injection of the thyrotropin-releasing hormone agonist, RX-77368, significantly increased pAUC while coeliotomy and caecal palpation induced a rapid onset inhibition of phasic activity lasting for the 1-h recording period. In mice, RX-77368 injected into the lateral brain ventricle induced high-amplitude contractions, and carbachol injected intraperitoneally increased pAUC significantly, while coeliotomy and caecal palpation inhibited baseline contractile activity. In wild-type mice, cold exposure (15-min) increased gastric phasic activity and tone, while there was no gastric response in corticotorpin releasing factor (CRF)-over-expressing mice, a model of chronic stress. Thus, the novel solid-state manometric approach provides a simple, reliable means for acute pharmacological studies of gastric motility effects in rodents. Using this method we established in mice that the gastric motility response to central vagal activation is impaired under chronic expression of CRF.

Visceral pain modulation by chronic stress in mice has been little studied. Electromyography (EMG) recording of abdominal muscle contractions, as a proxy to the visceromotor response (VMR), requires electrode implantation and post-surgical single housing (SH) which could affect the VMR to stress. To test this hypothesis, male mice had electrode implantation surgery (S) plus SH, or no surgery and were group housed (NS-GH) or single housed (NS-SH) and exposed to either water avoidance stress (WAS, 1 h/day) or left undisturbed in their home cages for 10 days. The VMR to phasic ascending colorectal distension (CRD) was assessed before (basal) and 24 h after 10 days of WAS or no stress using a surgery-free method of intraluminal colonic pressure (ICP) recording (solid-state manometry). WAS heightened significantly the VMR to CRD at 30, 45, and 60 mmHg in S-SH vs. NS-GH, but not compared to NS-SH conscious mice. Compared to basal CRD, WAS increased VMR at 60 mmHg in the S-SH group and decreased it at 30–60 mmHg in NS-GH mice, while having no effect in NS-SH mice. The average defecation during the hour of repeated WAS over 10 days was 1.9 and 2.4 fold greater in S-SH vs. NS-GH and NS-SH mice, respectively. These data indicate that the combination of S-SH required for VMR monitoring with EMG is an important component of repeated WAS-induced post-stress visceral hypersensitivity and defecation in mice.

Chronic stress and persistently high glucocorticoid levels can induce brain atrophy. Corticotropin-releasing factor (CRF)-overexpressing (OE) mice are a genetic model of chronic stress with elevated brain CRF and plasma corticosterone levels and Cushing's syndrome. The brain structural alterations in the CRF-OE mice, however, are not well known. We found that adult male and female CRF-OE mice had significantly lower whole brain and cerebellum weights than their wild type (WT) littermates (347.7+/-3.6mg vs. 460.1+/-4.3mg and 36.3+/-0.8mg vs. 50.0+/-1.3mg, respectively) without sex-related difference. The epididymal/parametrial fat mass was significantly higher in CRF-OE mice. The brain weight was inversely correlated to epididymal/parametrial fat weight, but not to body weight. Computerized image analysis system in Nissl-stained brain sections of female mice showed that the anterior cingulate and sensorimotor cortexes of CRF-OE mice were significantly thinner, and the volumes of the hippocampus, hypothalamic paraventricular nucleus and amygdala were significantly reduced compared to WT, while the locus coeruleus showed a non-significant increase. Motor functions determined by beam crossing and gait analysis showed that CRF-OE mice took longer time and more steps to traverse a beam with more errors, and displayed reduced stride length compared to their WT littermates. These data show that CRF-OE mice display brain size reduction associated with alterations of motor coordination and an increase in visceral fat mass providing a novel animal model to study mechanisms involved in brain atrophy under conditions of sustained elevation of brain CRF and circulating glucocorticoid levels.

Visceral hypersensitivity has been implicated as an important pathophysiological mechanism in functional gastrointestinal disorders. In this study, we investigated whether the sustained visceral hyperalgesia induced by repeated psychological stress in rats involves the activation of CRF1 signaling system using two different antagonists. Male Wistar rats were exposed to 10 consecutive days of water avoidance stress (WAS) or sham stress for 1 h/day, and the visceromotor response to phasic colorectal distension (CRD) was assessed before and after the stress period. Animals were injected subcutaneously with the brain penetrant CRF1 antagonist, CP-154,526, acutely (30 min before the final CRD) or chronically (via osmotic minipump implanted subcutaneously, during stress) or with the peripherally restricted, nonselective CRF1 and CRF2 antagonist, astressin, chronically (15 min before each stress session). Repeated WAS induced visceral hypersensitivity to CRD at 40 and 60 mmHg. CP-154,526 injected acutely significantly reduced stress-induced visceral hyperalgesia at 40 mmHg but not at 60 mmHg. Chronic subcutaneous delivery of astressin reduced the stress-induced visceral hyperalgesia to baseline at all distension pressures. Interestingly, chronically administered CP-154,526 eliminated hyperalgesia and produced responses below baseline at 40 mmHg and 60 mmHg, indicating a hypoalgesic effect of the compound. These data support a major role for CRF1 in both the development and maintenance of visceral hyperalgesia induced by repeated stress and indicate a possible role of peripheral CRF receptors in such mechanisms.

Interactions between gastrointestinal signals are a part of integrated systems regulating food intake (FI). We investigated whether cholecystokinin (CCK)-8 and urocortin systems potentiate each other to inhibit FI and gastric emptying (GE) in fasted mice. Urocortin 1 and urocortin 2 (1 μg/kg) were injected ip alone or with CCK (3 μg/kg) in lean, diet-induced obese (DIO) or corticotropin-releasing factor receptor-2 (CRF2)-deficient mice. Gastric vagal afferent activity was recorded from a rat stomach-vagus in vitro preparation. When injected separately, urocortin 1, urocortin 2, or CCK did not modify the 4-h cumulative FI in lean mice. However, CCK plus urocortin 1 or CCK plus urocortin 2 decreased significantly the 4-h FI by 39 and 27%, respectively, compared with the vehicle + vehicle group in lean mice but not in DIO mice. Likewise, CCK-urocortin-1 delayed GE in lean but not DIO mice, whereas either peptide injected alone at the same dose had no effect. CCK-urocortin 2 suppression of FI was observed in wild-type but not CRF2-deficient mice. Gastric vagal afferent activity was increased by intragastric artery injection of urocortin 2 after CCK at a subthreshold dose, and the response was reversed by devazepide. These data establish a peripheral synergistic interaction between CCK and urocortin 1 or urocortin 2 to suppress FI and GE through CRF2 receptor in lean mice that may involve CCK modulation of gastric vagal afferent responsiveness to urocortin 2. Such synergy is lost in DIO mice, suggesting a resistance to the satiety signaling that may contribute to maintain obesity.

Intraperitoneal (i.p.) corticotropin releasing factor (CRF) induced a CRF(1) receptor-dependent stimulation of myenteric neurons and motility in the rat proximal colon. We characterize the colonic enteric nervous system response to CRF in conscious rats. Laser capture microdissection combined with reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry in longitudinal muscle myenteric plexus whole-mount colonic preparations revealed CRF(1) receptor expression in myenteric neurons. CRF (i.p., 10 microg kg(-1)) induced Fos immunoreactivity (IR) (cells per ganglion) selectively in myenteric plexus of proximal (18.3 +/- 2.4 vs vehicle: 0.0 +/- 0.0) and distal colon (16.8 +/- 1.2 vs vehicle: 0.0 +/- 0.0), but not in that of gastric corpus, antrum, duodenum, jejunum and ileum. The selective CRF(1) agonist, stressin(1)-A (i.p., 10 microg kg(-1)) also induced Fos IR in myenteric but not in submucosal plexus of the proximal and distal colon. Fos IR induced by CRF was located in 55 +/- 1.9% and 53 +/- 5.1% of CRF(1) receptor-IR myenteric neurons and in 44 +/- 2.8% and 40 +/- 3.9% of cholinergic neurons with Dogiel type I morphology, and in 20 +/- 1.6% and 80 +/- 3.3% of nitrergic neurons in proximal and distal colon respectively. CRF and stressin(1)-A elicit defecation and diarrhoea. These data support that one mechanism through which peripherally injected CRF ligands stimulate colonic function involves a direct action on colonic cholinergic and nitrergic myenteric neurons expressing CRF(1) receptor.

Stress stimulates colonic motor function and plays a role in functional bowel disorders, prevalently in women. We examined, in conscious female rats, the influence of water avoidance stress for 60 min on colonic myenteric neuron activity using immunohistochemical detection of Fos as a marker of neuronal activity. In control rats, Fos immunoreactive nuclei were rare in proximal and distal colon and no defecation was observed. Water avoidance stimulated fecal pellet output, which was associated with Fos expression in myenteric ganglia of proximal and distal colon including in a population of peripheral choline acetyltransferase-immunoreactive neurons. Atropine blocked fecal pellet output but not Fos expression in myenteric ganglia. These results indicate that psychological stress stimulates the activity of colonic cholinergic myenteric neurons.

BACKGROUND:Corticotropin-releasing factor (CRF) peptides exert profound effects on the secretomotor function of the gastrointestinal tract. Nevertheless, despite the presence of CRF peptides and receptors in colonic tissue, their influence on colonic blood flow (CBF) is unknown. AIM:To determine the effect and mechanism of members of the CRF peptide family on CBF in isoflurane-anesthetized rats. METHODS:Proximal CBF was measured with laser-Doppler flowmetry simultaneously with mean arterial blood pressure (MABP) measurement. Rats were injected with intravenous human/rat CRF (CRF1 > CRF2 affinity), mouse urocortin 2 (mUcn2, selective CRF2 agonist), or sauvagine (SVG, CRF2 > CRF1 affinity) at 1-30 µg/kg. The nitric oxide (NO) synthase inhibitor, L-NAME (3 mg/kg, iv), the cyclooxygenase inhibitor, indomethacin (Indo, 5 mg/kg, ip), or selective CRF2 antagonist, astressin2-B (Ast2B, 50 µg/kg, iv) was given before SVG injection (10 µg/kg, iv). RESULTS:SVG and mUcn2 dose-dependently increased CBF while decreasing MABP and colonic vascular resistance (CVR). CRF had no effect on CBF, but increased CVR. The hyperemic effect of SVG was inhibited by L-NAME but not by Indo, whereas hypotension was partially reduced by L-NAME. Sensory denervation had no effect on SVG-induced changes. Ast2B inhibited SVG-induced hyperemia and decreased CVR, and partially reduced the hypotension. CONCLUSIONS:Peripheral CRF2 activation induces colonic hyperemia through NO synthesis, without involving prostaglandin synthesis or sensory nerve activation, suggesting a direct action on the endothelium and myenteric neurons. Members of the CRF peptide family may protect the colonic mucosa via the activation of the CRF2 receptor.

The corticotropin-releasing factor (CRF) signaling systems encompass CRF and the structurally related peptide urocortin (Ucn) 1, 2, and 3 along with 2 G-protein coupled receptors, CRF₁ and CRF₂. CRF binds with high and moderate affinity to CRF₁ and CRF₂ receptors, respectively while Ucn1 is a high-affinity agonist at both receptors, and Ucn2 and Ucn3 are selective CRF₂ agonists. The CRF systems are expressed in both the brain and the colon at the gene and protein levels. Experimental studies established that the activation of CRF₁ pathway in the brain or the colon recaptures cardinal features of diarrhea predominant irritable bowel syndrome (IBS) (stimulation of colonic motility, activation of mast cells and serotonin, defecation/watery diarrhea, and visceral hyperalgesia). Conversely, selective CRF1 antagonists or CRF₁/CRF₂ antagonists, abolished or reduced exogenous CRF and stress-induced stimulation of colonic motility, defecation, diarrhea and colonic mast cell activation and visceral hyperalgesia to colorectal distention. By contrast, the CRF₂ signaling in the colon dampened the CRF₁ mediated stimulation of colonic motor function and visceral hyperalgesia. These data provide a conceptual framework that sustained activation of the CRF₁ system at central and/or peripheral sites may be one of the underlying basis of IBS-diarrhea symptoms. While targeting these mechanisms by CRF₁ antagonists provided a relevant novel therapeutic venue, so far these promising preclinical data have not translated into therapeutic use of CRF₁ antagonists. Whether the existing or newly developed CRF₁ antagonists will progress to therapeutic benefits for stress-sensitive diseases including IBS for a subset of patients is still a work in progress.

Obestatin is a new peptide for which anorexigenic effects were recently reported in mice. We investigate whether peripheral injection of obestatin or co-injection with cholecystokinin (CCK) can modulate food intake, gastric motor function (intragastric pressure and emptying) and gastric vagal afferent activity in rodents. Obestatin (30, 100 and 300 microg/kg, i.p.) did not influence cumulative food intake for the 2h post-injection in rats or mice nor gastric emptying in rats. In rats, obestatin (300 microg/kg) did not modify CCK (1 microg/kg, i.p.)-induced significant decrease in food intake (36.6%) and gastric emptying (31.0%). Furthermore, while rats injected with CCK (0.3 microg/kg, i.v.) displayed gastric relaxation, no change in gastric intraluminal pressure was elicited by obestatin (300 microg/kg, i.v.) pre- or post-CCK administration. In in vitro rat gastric vagal afferent preparations, 20 units that had non-significant changes in basal activity after obestatin at 30 microg responded to CCK at 10 ng by a 182% increase. These data show that obestatin neither influences cumulative food intake, gastric motility or vagal afferent activity nor CCK-induced satiety signaling.

In rodents, maternal pup interactions play an important role in programming the stress responsiveness of the adult organism. The aims of this study were 1) to determine the effect of different neonatal rearing conditions on acute and delayed stress-induced visceral sensitivity as well as on other measures of stress sensitivity of the adult animal; and 2) to determine the role of corticotropin-releasing factor receptor (CRF-R) subtype 1 (CRF1R) in mediating visceral hypersensitivity. Three groups of male Long-Evans rat pups were used: separation from their dam for 180 min daily from postnatal days 2–14 (MS180), daily separation (handling) for 15 min (H), or no handling. The visceromotor responses (VMR) to colorectal distension, stress-induced colonic motility, and anxiety-like behavior were assessed in the adult rats. The VMR was assessed at baseline, immediately after a 1-h water avoidance (WA) stress, and 24 h poststress. Astressin B, a nonselective CRF-R antagonist, or CP-154,526, a selective CRF1R antagonist, was administered before the stressor and/or before the 24-h measurement. MS rats developed acute and delayed stress-induced visceral hyperalgesia. In contrast, H rats showed hypoalgesia immediately after WA and no change in VMR on day 2. MS rats with visceral hyperalgesia also exhibited enhanced stress-induced colonic motility and increased anxiety-like behavior. In MS rats, both CRF-R antagonists abolished acute and delayed increases in VMR. Rearing conditions have a significant effect on adult stress responsiveness including immediate and delayed visceral pain responses to an acute stressor. Both acute and delayed stress-induced visceral hypersensitivity in MS rats are mediated by the CRF/CRF1R system.

1. The characterization of corticotropin releasing factor (CRF) and, more recently, the discovery of additional CRF-related ligands, urocortin 1, urocortin 2 and urocortin 3, the cloning of two distinct CRF receptor subtypes, 1 (CRF(1)) and 2 (CRF(2)), and the development of selective CRF receptor antagonists provided new insight to unravel the mechanisms of stress. Activation of brain CRF(1) receptor signaling pathways is implicated in stress-related endocrine response and the development of anxiety-like behaviors. 2. Compelling evidence in rodents showed also that both central and peripheral injection of CRF and urocortin 1 mimic acute stress-induced colonic response (stimulation of motility, transit, defecation, mucus and watery secretion, increased ionic permeability and occurrence of diarrhea) in rodents. Central CRF enhances colorectal distention-induced visceral pain in rats. Peripheral CRF reduced pain threshold to colonic distention and increased colonic motility in humans. 3. Nonselective CRF(1)/CRF(2) antagonists and selective CRF(1) antagonists inhibit exogenous (central or peripheral) CRF- and acute stress-induced activation of colonic myenteric neurons, stimulation of colonic motor function and visceral hyperalgesia while selective CRF(2) antagonists have no effect. None of the CRF antagonists influence basal or postprandial colonic function in nonstressed animals. 4. These findings implicate CRF(1) receptors in stress-related stimulation of colonic function and hypersensitivity to colorectal distention. Targeting CRF(1)-dependent pathways may have potential benefit against stress or anxiety-/depression-related functional bowel disorders.

Ultrasonomicrometry measures distance between piezoelectric crystals based on transmission time of ultrasound bursts. It allows monitoring of coordinated motion of small and delicate tissues, including gastrointestinal sphincters. Its suitability for motility studies in small animals such as mice suggests that its use in gastrointestinal studies will increase in coming years.

Peripheral corticotropin-releasing factor (CRF) receptor ligands inhibit gastric acid secretion and emptying while stimulating gastric mucosal blood flow in rats. Endogenous CRF ligands are expressed in the upper gastrointestinal (GI) tissues pointing to local expression of CRF receptors. We mapped the distribution of CRF receptor type 1 (CRF1) and 2 (CRF2) in the rat upper GI. Polyclonal antisera directed against the C-terminus of the CRF receptor protein were generated in rabbits and characterized by western blotting and immunofluorescence using CRF1- and CRF2-transfected cell lines and in primary cultured neurons from rat brain cortex. A selective anti-CRF1 antiserum (4467a-CRF1) was identified and used in parallel with another antiserum recognizing both CRF1 and CRF2 (4392a-CRF1&2) to immunostain gastric tissue sections. Antiserum 4467a-CRF1 demonstrated specific immunostaining in a narrow zone in the upper oxyntic gland within the stomach corpus. Conversely, 4392a-CRF1&2 labeled cells throughout the oxyntic gland and submucosal blood vessels. Pre-absorption with the specific antigen peptide blocked immunostaining in all experiments. Doublestaining showed co-localization of 4392a-CRF1&2 but not 4467a-CRF1 immunoreactivity with H/K-ATPase and somatostatin immunostaining in parietal and endocrine cells of the oxyntic gland. No specific staining was observed in the antrum with either antisera, whereas only antiserum 4392a-CRF1&2 showed modest immunoreactivity in the duodenal mucosa. Finally, co-localization of CRF2 and urocortin immunoreactivity was found in the gastric glands. These results indicate that both CRF receptor subtypes are expressed in the rat upper GI tissues with a distinct pattern and regional differences suggesting differential function.

CRF injected intraperitoneally (i.p.) stimulates colonic motor function and induces Fos expression in colonic myenteric neurons. We investigated central and spinal Fos expression and changes in colonic motility in response to i.p. injection of CRF and urocortin. Ovine CRF(9-33) that is devoid of intrinsic activity at the CRF receptors, was used as control peptide. Myoelectrical activity was monitored for 1 h before and after peptide injection (10 microg/kg, i.p.) in conscious non fasted rats with chronically implanted intraparietal electrodes in the cecum and proximal colon. Brain and lumbosacral spinal cord were processed for Fos immunohistochemistry at 1 h postinjection. CRF and urocortin elicited defecation and a new pattern of ceco-colonic clustered spike bursts that peaked within 15 min and lasted for the 1 h experimental period while CRF(9-33) did not modify baseline myoelectrical activity and defecation. CRF increased significantly Fos expression in the central nucleus of the amygdala (lateral part), parabrachial nucleus (external lateral subnucleus), area postrema, nucleus tractus solitarius, locus coeruleus, paraventricular nucleus of the hypothalamus, the intermediolateral column and area I-VII, X at the L6-S1 level of the spinal cord by 11-, 6.5-, 5.3-, 5.0-, 4.7-, 2.7- and 1.4-fold, respectively compared with i.p. CRF(9-33) injected rats that had little Fos expression. Urocortin induced a similar pattern of Fos response in the brain and the spinal cord. These results indicate that i.p. CRF and urocortin induce a peptide specific activation of brain nuclei receiving viscerosensory inputs and involved in autonomic circuitries whose effector limbs may impact on visceral function.

1. Corticotropin-releasing factor (CRF) acts in the brain to inhibit thyrotropin-releasing hormone (TRH) analogue, RX-77368-induced vagal stimulation of gastric motility. We investigated CRF receptor-mediated actions of rat urocortin (rUcn) injected intracisternally (ic) on gastric motor function. 2. Urethane-anaesthetized rats with strain gauges on the gastric corpus were injected i.c. with rUcn and 20 min later, with i.c. RX-77368. CRF antagonists were injected i.c. 10 min before rUcn. 3. RX-77368 (1.5, 3, 10, 30 and 100 ng, i.c.) dose-dependently increased corpus contractions, expressed as total area under the curve (AUC, mV min(-1)) to 2.6+/-2.5, 6.1+/-5.9, 9.8+/-2.6, 69.7+/-21.7 and 74.9+/-28.7 respectively vs 0.2+/-0.1 after i.c. saline. Ucn (1, 3 or 10 microg) inhibited RX-77368 (30 ng)-induced increase in total AUC by 28, 62 and 93% respectively vs i.c. saline+RX-77368. 4. The CRF(1)/CRF(2) antagonist, astressin-B (60 microg, i.c.) completely blocked i.c. rUcn (3 microg, i.c.)-induced inhibition of gastric motility stimulated by RX-77368 (30 ng). 5. The selective CRF(2) antagonist, astressin(2)-B (30, 60 or 100 microg, i.c. ) dose-dependently prevented i.c. rUCn action while the CRF(1) antagonist, NBI-27914 did not. 6. In conscious rats, rUcn (0.6 or 1 microg, i.c.) inhibited gastric emptying of an ingested chow meal by 61 and 92% respectively. rUcn action was antagonized by astressin(2)-B. 7. These data show that i.c. rUcn acts through CRF(2) receptors to inhibit central vagal gastric contractile response and postoprandial emptying.

VIP is highly expressed in the colon and regulates motility, vasodilatation, and sphincter relaxation. However, its role in the development and progress of colitis is still controversial. Our aim was to determine the participation of VIP on dextran sodium sulfate (DSS)-induced colonic mucosal inflammation using VIP-/-and WT mice treated with VIP antagonists. Colitis was induced in 32 adult VIP-/-and 14 age-matched WT litter-mates by giving 2.5 % DSS in the drinking water. DSS-treated WT mice were injected daily with VIP antagonists, VIPHyb (n = 22), PG 97-269 (n = 9), or vehicle (n = 31). After euthanasia, colons were examined; colonic cytokines mRNA were quantified. VIP-/-mice were remarkably resistant to DSS-induced colitis compared to WT. Similarly, DSS-treated WT mice injected with VIPHyb (1 μM) or PG 97-269 (1 nM) had significantly reduced clinical signs of colitis. Furthermore, colonic expression of IL-1Ï, TNF-α, and IL-6 was significantly lower in VIP-/-and VIPHyb or PG 97-269 compared to vehicle-treated WT. Genetic deletion of VIP or pharmacological inhibition of VIP receptors resulted in resistance to colitis. These data demonstrate a pro-inflammatory role for VIP in murine colitis and suggest that VIP antagonists may be an effective clinical treatment for human inflammatory bowel diseases. © 2014 Springer Science+Business Media New York (outside the USA).

Alterations of gastrointestinal (GI) motor function are part of the visceral responses to stress. Inhibition of gastric emptying and stimulation of colonic motor function are the commonly encountered patterns induced by various stressors. Activation of brain corticotropin-releasing factor (CRF) receptors mediates stress-related inhibition of upper GI and stimulation of lower GI motor function through interaction with different CRF receptor subtypes. CRF subtype 1 receptors are involved in the colonic and anxiogenic responses to stress and may have clinical relevance in the comorbidity of anxiety/depression and irritable bowel syndrome.

We assessed the role of central corticotropin-releasing factor (CRF) in stress-induced worsening of colitis in inbred rat strains with hypo (Lewis/N) and hyper (Fischer344/N) CRF responses to stress. Intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNB) induced colitis of similar severity in both strains as assessed on day 7 by macroscopic scoring, histological evaluation, tissue myeloperoxidase (MPO) activity, and decrease in food intake and body weight. Colitis was inhibited by daily intracerebroventricular injections of CRF in both strains. Chronic stress (3 h/day, water avoidance or wrap restraint on alternate days for 6 days) aggravated colitis more in Lewis than Fischer rats (71 and 22% further increase in MPO activity, respectively). The CRF antagonist astressin injected intracerebroventricularly enhanced the colitis response to stress and caused mortality in both strains. Fischer rats had higher plasma corticosterone levels 20 min after stress alone on day 1 and after TNB plus stress on days 1 and 3 compared with Lewis. These data show that central CRF restrains the proinflammatory action of stress in experimental colitis.

Visceral hypersensitivity is an important pathophysiological factor in irritable bowel syndrome (IBS). Pre-clinical studies suggest that the alpha(2)delta ligand pregabalin reduces both visceral allodynia and hyperalgesia, but is inactive on basal sensitivity.To assess the effect of pregabalin on the perception of rectal distension in hypersensitive IBS patients.Twenty-six patients with Rome-II-defined IBS (aged 18-46 years, 7 male) were included in a randomized, double-blind, placebo-controlled, parallel-group study in which they received either 3 weeks oral pregabalin (titrated: 50 mg tid days 1-3, 100 mg tid days 4-7, 150 mg tid days 8-11; fixed 200 mg tid days 12-21 +/-4) or placebo control. Rectal sensitivity was assessed using a barostat technique, in which sensory thresholds were determined using the ascending method of limits, followed by tracking both before and after treatment. Only patients with a pain threshold ofor=28 mmHg were included in the study.Pregabalin significantly increased the sensory thresholds from baseline for first sensation (p = 0.045), desire to defecate (p = 0.008) and pain (p = 0.048) compared with placebo control. In addition, pregabalin significantly increased rectal compliance (p0.0001), although this appeared to be unrelated to the changes in sensitivity. Despite the occurrence of mild dizziness and somnolence, pregabalin was generally well tolerated.Pregabalin increased distension sensory thresholds to normal levels in IBS patients with rectal hypersensitivity. A concomitant increase in rectal compliance appeared to be unrelated to the reduction in sensitivity. These data suggest that alpha(2)delta ligands are worthy of further investigation in the treatment of visceral pain disorders, including IBS.

Corticotropin releasing factor receptor 1 (CRF1) is the key receptor that mediates stress-related body responses. However to date there are no CRF1 antagonists that have shown clinical efficacy in stress-related diseases. We investigated the inhibitory effects of a new generation, topology 2 selective CRF1 antagonists, NGD 98-2 and NGD 9002 on exogenous and endogenous CRF-induced stimulation of colonic function and visceral hypersensitivity to colorectal distension (CRD) in conscious rats. CRF1 antagonists or vehicle were administered orogastrically (og) or subcutaneously (sc) before either intracerebroventricular (icv) or intraperitoneal (ip) injection of CRF (10 µg/kg), exposure to water avoidance stress (WAS, 60 min) or repeated CRD (60 mmHg twice, 10 min on/off at a 30 min interval). Fecal pellet output (FPO), diarrhea and visceromotor responses were monitored. In vehicle (og)-pretreated rats, icv CRF stimulated FPO and induced diarrhea in >50% of rats. NGD 98-2 or NGD 9002 (3, 10 and 30 mg/kg, og) reduced the CRF-induced FPO response with an inhibitory IC50 of 15.7 and 4.3 mg/kg respectively. At the highest dose, og NGD 98-2 or NGD 9002 blocked icv CRF-induced FPO by 67-87% and decreased WAS-induced-FPO by 23-53%. When administered sc, NGD 98-2 or NGD 9002 (30 mg/kg) inhibited icv and ip CRF-induced-FPO. The antagonists also prevented the development of nociceptive hyper-responsivity to repeated CRD. These data demonstrate that topology 2 CRF1 antagonists, NGD 98-2 and NGD 9002, administered orally, prevented icv CRF-induced colonic secretomotor stimulation, reduced acute WAS-induced defecation and blocked the induction of visceral sensitization to repeated CRD.

Brain corticotropin-releasing factor (CRF) acting on CRF receptor type 1 (CRF(1)) is a main signaling pathway in the stress response. CRF is also produced in a variety of peripheral sites and acts locally as a proinflammatory mediator. We investigated CRF(1) mRNA expression in the human gastrointestinal tract, and localized CRF(1) immunoreactive cells in the colonic mucosa of healthy subjects and patients with ulcerative colitis (UC). In 4 male healthy subjects (24-29 years), CRF(1) transcript was detected by RT-PCR throughout the gastrointestinal tract with the highest levels in the ileum and rectum and the lowest level in the colon. Immunohistochemistry on whole thickness sigmoid colon sections showed that CRF(1) was localized in the lamina propria and epithelial cells and enteric neurons. In sigmoid colonic biopsies, immunohistochemically double-labeled cells with CRF(1) and CD163, a marker for macrophages, represent 79% of total CRF(1) immunoreactive (IR) cells in healthy subjects. In 10 UC patients, the total number of CRF(1) IR cells and CRF(1)/CD163 double-labeled macrophages was increased by 4.2 and 4.0 folds respectively compared to healthy subjects. These findings indicate that CRF(1) is distributed throughout the GI tract of healthy human subjects. The increase of CRF(1) IR cells prominently in macrophages of the sigmoid colonic mucosa of UC patients provides anatomical support for a role of CRF(1) signaling in modulating the immune-inflammatory process of UC.

Corticotropin-releasing factor (CRF) signaling pathways are involved in the stress response, and there is growing evidence supporting hair growth inhibition of murine hair follicle in vivo upon stress exposure. We investigated whether the blockade of CRF receptors influences the development of hair loss in CRF over-expressing (OE)-mice that display phenotypes of Cushing's syndrome and chronic stress, including alopecia. The non-selective CRF receptors antagonist, astressin-B (5 µg/mouse) injected peripherally once a day for 5 days in 4-9 months old CRF-OE alopecic mice induced pigmentation and hair re-growth that was largely retained for over 4 months. In young CRF-OE mice, astressin-B prevented the development of alopecia that occurred in saline-treated mice. Histological examination indicated that alopecic CRF-OE mice had hair follicle atrophy and that astressin-B revived the hair follicle from the telogen to anagen phase. However, astressin-B did not show any effect on the elevated plasma corticosterone levels and the increased weights of adrenal glands and visceral fat in CRF-OE mice. The selective CRF₂ receptor antagonist, astressin₂-B had moderate effect on pigmentation, but not on hair re-growth. The commercial drug for alopecia, minoxidil only showed partial effect on hair re-growth. These data support the existence of a key molecular switching mechanism triggered by blocking peripheral CRF receptors with an antagonist to reset hair growth in a mouse model of alopecia associated with chronic stress.

Corticotropin-releasing factor (CRF) overexpressing (OE) mice are a genetic model that exhibits features of chronic stress. We investigated whether the adaptive feeding response to a hypocaloric challenge induced by food deprivation is impaired under conditions of chronic CRF overproduction. Food intake response to a 16-h overnight fast and ip injection of gut hormones regulating food intake were compared in CRF-OE and wild type (WT) littermate mice along with brain Fos expression, circulating ghrelin levels, and gastric emptying of a nonnutrient meal. CRF-OE mice injected ip with saline showed a 47 and 44% reduction of 30-min and 4-h cumulative food intake response to an overnight fast, respectively, compared with WT. However, the 30-min food intake decrease induced by ip cholecystokinin (3 μg/kg) and increase by ghrelin (300 μg/kg) were similar in CRF-OE and WT mice. Overnight fasting increased the plasma total ghrelin to similar levels in CRF-OE and WT mice, although CRF-OE mice had a 2-fold reduction of nonfasting ghrelin levels. The number of Fos-immunoreactive cells induced by fasting in the arcuate nucleus was reduced by 5.9-fold in CRF-OE compared with WT mice whereas no significant changes were observed in other hypothalamic nuclei. In contrast, fasted CRF-OE mice displayed a 5.6-fold increase in Fos-immunoreactive cell number in the dorsal motor nucleus of the vagus nerve and a 34% increase in 20-min gastric emptying. These findings indicate that sustained overproduction of hypothalamic CRF in mice interferes with fasting-induced activation of arcuate nucleus neurons and the related hyperphagic response. Sustained over-production of brain corticotrophin-releasing factor interferes with fasting-activated neuronal activation in the arcuate nucleus and results in reduction of food intake dissociated from gastric emptying.