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T1663 Impaired Gastric Motor Function Induced By Acute Restraint Stress Is No More Observed Following Repeated Chronic Stress in Mice
(74.1 +/- 2.3 %, n=6,). Conclusion: Gastric emptying and postprandial gastric motility were significantly impaired during acute restraint stress. In contrast, chronic restraint stress restored delayed gastric emptying. An adaptation mechanism is mediated, at least in part, by endogenous ghrelin following chronic stress in mice. T1664 Group-III Metabotropic Glutamate Receptors Are Expressed in Rodent Colon: A Role in Cholinergically Induced Electrolyte Secretion Marcela Julio-Pieper, Niall P. Hyland, John F. Cryan, Timothy G. Dinan Mood disorders are frequently associated with gastrointestinal (GI) dysfunction; therefore treatments addressing both central and GI components of chronic stress are highly desirable. Group-III metabotropic glutamate receptors (mGluRs 4, 6, 7 and 8) have been implicated in the pathophysiology of depression and anxiety, and growing evidence indicates them as possible target in the treatment of conditions involving chronic stress. However, there is little information about the role of group-III mGluRs outside the central nervous system. This study aimed to investigate the expression and possible role of group-III mGluRs in the rodent colon. mGluR 4 and 7 were analysed by western blot in colon samples from C57/ BL6 mice. mGluR 8 was detected by fluorescent immunohistochemistry in sections and submucosal wholemount preparations from Sprague Dawley colon. Immunohistochemistry analysis indicated that mGluR8 was present in the colon mucosa, submucosal and myenteric ganglia. Western blot studies showed the presence of mGluR4 in the mucosa and with less intensity in the layers containing the enteric nerves and muscles. Unexpectedly, mGluR7 was detected in colon mucosa, but not in the nerve and muscle layers. To further investigate this unique localization of mGluR7 and since colon mucosa regulates the passage of electrolytes between the intestinal lumen and the body, it was important to assess whether activation of mGluR7 modifies ion transport. Mucosal preparations of mouse colon were placed in Ussing chambers and voltage-clamped at 0mV. Tissues were pre-treated for 30 min with the selective agonist of mGluR7, AMN082 (0, 30nM, 100nM and 1uM), and then bethanecholinduced increase in short-circuit current (ISC) was studied, as a measure of the secretion of chloride and other electrolytes into the luminal space. AMN082 induced a dose-dependent increase in ISC, indicating that activation of mGluR7 could amplify cholinergically induced electrolyte secretion, and thus increase the secretion of water. Based on our functional data, mGluR7 could play a role in regulating the absorptive/secretory function in the colon, whereas according to their localization, mGluR4 and 8 might be implicated both in electrolyte transport and colon motility which needs to be further explored. These results suggest that targeting group-III mGluRs and particularly mGluR7 may not only be useful in the treatment of central components of chronic stress, but also of associated GI dysfunctions.
T1662 Importance of Corticosteroid Receptors Within the Amygdala On PostInflammatory Colonic Hyperalgesia Anthony C. Johnson, Kristen Campbell-Dittmeyer, Beverley Greenwood-Van Meerveld Background: A subgroup of patients develop irritable bowel syndrome following an acute colitis combined with a significant life stress. In preclinical models we found an up-regulation of stress hormone expression in the rat brain following an acute colitis. Moreover, in rodents modulation of the amygdala with corticosterone (CORT) induced anxiety-like behavior coupled with a hypersensitive colon which involves both mineralocorticoid (MR) and glucocorticoid (GR)-mediated mechanisms. We hypothesized that MR and/or GR receptors in the amygdala modulate post-inflammatory colonic hypersensitivity. Methods: Distal colitis was induced in male Fischer 344 rats via a 0.5 ml intra-rectal enema of 2,4,6-trinitrobenzenesulphonic acid (TNBS, 50 mg) in 25% ethanol. At 23 days post-TNBS enema, rats were randomly assigned to groups in which a micropellet containing a selective GR antagonist (mifepristone, 15 μg) or a MR antagonist (spironolactone, 15 μg) was implanted stereotaxically bilaterally on the dorsal margin of the amygdala. After recovery from the colitis (day 30 post-TNBS), colonic sensitivity to graded levels of isobaric colorectal distension (CRD) at 0-60 mmHg was assessed by measuring a visceromotor response (VMR) to CRD, quantified as the number of abdominal contractions during a 10 min recording period. Control groups included rats that underwent sham micropellet surgery and rats that received a saline enema. The location of the micropellet was confirmed at the end of the experiment. Results: In rats with or with-out sham surgery, post-inflammatory colonic hyperalgesia was confirmed at 30-days post-TNBS enema by an elevation of the VMR to CRD at 40 and 60 mmHg compared to the saline enema-control group (19.9±1.7 vs. 12.9±1.5, p<0.01 at 40 mmHg; 27.2±2.5 vs. 18.9±1.7, p<0.001 at 60 mmHg, n=15/group). Neither the GR nor MR antagonist altered the exaggerated VMR to CRD induced by 40 mmHg CRD. At 60 mmHg, while rats that received micropellets of mifepristone remained hypersensitive to CRD (27.3±1.9, p<0.01 vs. saline, n=7), rats with spironolactone were not significantly different from the saline controls (23.9±2.0, p>0.05 vs. saline, n=7). Summary and Conclusions: Stereotaxic delivery of a selective MR but not a GR antagonist, at doses shown previously to inhibit the VMR to CRD in rats implanted with amygdaloid CORT, induced a small though significant inhibition of post-inflammatory colonic hyperalgesia. These findings suggest that postinflammatory colonic hypersensitivity involves at least in part the interaction of endogenous corticosteroids with MR at the level of the amygdala but appears independent of the interaction with GR.
T1665 5-HT4-Stimulated Neuroprotection and Neurogenesis in the Enteric Nervous System (ENS): Significance in Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice Mintsai Liu, Jingwen Wang, Rene Hen, Michael D. Gershon We have previously shown that 5-HT4 stimulation promotes survival and neurite extension in cultured enteric neurons. In 5-HT4 knockout mice (KO), gastric emptying is delayed, intestinal motility is slowed, enteric neurons are smaller and their age-related loss accelerates. We now test the hypotheses that Akt mediates 5-HT4-promoted enteric neuronal survival and that 5-HT4-mediated neuroprotection and neurogenesis is significant In Vivo. Neurons were isolated from adult gut, cultured, and exposed to a 5-HT4 agonist, RS67506 or tegaserod (10 nM). In-cell ELISA was used to quantify pAkt. RS67506 and tegaserod each concentrationdependently increased the pAkt/Akt ratio (p < 0.05). This increase was reduced (p < 0.05) by the 5-HT4 antagonist, SB204070 (10 nM), the adenyl cyclase inhibitor, SQ22536 (10 μM), and the PI3-K inhibitor, LY294002 (10 μM). RS67506 and tegaserod also increased pCREB nuclear translocation. 5-HT4-induced neuroprotection may thus be mediated by pAkt (Epac 1/2, which couples 5-HT4 stimulation to pAkt was detected) and/or by pCREB. To test the In Vivo significance of 5-HT4-mediated neuroprotection, DSS (5%) was included in drinking water for 7 days. The severity of the resulting DSS-induced colitis was confirmed by measuring neutrophil (myeloperoxidase) and macrophage (F4/80) infiltration. Nerve terminal degeneration, manifested by the presence of retraction bulbs, visualized by electron micrograph (EM) and peripherin immunoreactivity, which was observed in the inflamed colon of wild-type (WT) and KO mice; however, nerve terminal degeneration also occurred spontaneously in KO animals. These changes were accompanied by enhanced autophagy in neurites and perikarya, which was confirmed by quantifying LC3B-immunoreactivity and by EM. Colitis also promoted cell proliferation in the myenteric plexus (evaluated with Ki67 immunoreactivity) and increased the numbers of small sized neurons in myenteric ganglia. Proliferation was decreased in KO mice (p < 0.01). Administration of BrdU (7 day infusion) revealed that inflammation, tegaserod, and RS67506 each stimulated the generation of new HuC/D-immunoreactive neurons in germinal niches adjacent to myenteric ganglia in WT mice, but not in KO mice. Inflammation and deletion of 5-HT4 receptors thus are accompanied in the ENS by autophagy and neurodegeneration. These adverse changes are linked to the promotion of neurogenesis by stem cells that are retained in specialized regions (germinal niches) in adult animals. Neurodegeneration is facilitated while neuronal replacement is diminished by 5-HT4 receptors deletion.
T1663 Impaired Gastric Motor Function Induced By Acute Restraint Stress Is No More Observed Following Repeated Chronic Stress in Mice Reji Babygirija, Zheng Jun, Kirk A. Ludwig, Toku Takahashi Background: Physical and psychological stress contributes to the pathogenesis of functional dyspepsia. We have previously demonstrated that acute stress delays gastric emptying and impairs gastric motility via central CRF and peripheral sympathetic pathways in rats (Am J Physiol 288, R427-432, 2005). In contrast, a recent study suggests that gastric emptying is restored following chronic stress loading (Life Sci 82,862-868, 2008). Ghrelin accelerates gastric emptying and stimulates postprandial gastric contractions in rats. A recent study suggests that plasma ghrelin levels and gastric ghrelin mRNA expression are significantly increased following chronic stress in mice (Nature Neurosci 11,752-3, 2008). We compared the effects of acute and chronic restraint stress on solid gastric emptying and postprandial gastric motility in mice. We also studied whether endogenous ghrelin is involved in mediating gastric emptying and motility following chronic stress in mice. Methods: Adult male Swiss Webster mice were used for the study. After 16 hr fasting, mice were given 0.2 g of preweighed pellet to measure solid gastric emptying. A strain gauge transducer was implanted on the serosal surface of antrum to record the circular muscle contractions. Postprandial gastric motility was recorded following acute (90 minute of single stress) or chronically restraint stress (repeated stress for 5 consecutive days). Motility index (area under the curve) was calculated using a computer-assisted system. Ghrelin antagonist (D-lys 3 GHRP-6, 0.28 mg/kg) was administered intraperitoneally 30 minutes before feeding. Results: In acute stress loading, gastric emptying was significantly delayed (43.1 +/- 7.9 %), compared to that of control mice (76.7 +/- 4.7 % n=6, P<0.05). However, following chronic stress, gastric emptying returned to control levels (75.8 +/- 3.0 %). Acute stress also significantly impaired postprandial gastric motility to 67. 3 +/- 3.5% (n=4, P<0.001) of controls, while chronic stress had no more inhibitory effects on postprandial gastric motility. Ghrelin receptor antagonist has no significant effects on gastric emptying in control mice (71.2 +/- 2.2 %). Restored gastric emptying following chronic stress was significantly antagonized by ghrelin receptor antagonist (46.7 +/- 2.8 %, n=6, P<0.01), compared to that of saline-injected mice
T1666 Immunohistochemical Characterization of Transient Receptor Potential Vanilloid (TRPV) 1 and Sensory Neuropeptides in Mouse Rectum: Clear Differences of the Subpopulations in Mucosal, Submucosal and Muscle Layer Kenjiro Matsumoto, Takuji Hosoya, Kimihito Tashima, Syunji Horie BACKGROUND & AIMS: In the gut, TRPV1 modulates physiological functions such as motility, secretion and circulation. TRPV1 activation leads to release neuropeptides including tachykinins and CGRP. These neuropeptides are distributed in the enteric nervous system
A-553
AGA Abstracts
AGA Abstracts
reported that intracerebroventricular injection of CART peptide inhibits food intake and decreases gastrointestinal motility in rodents. Aim: To determine whether peripheral CCK affects neuronal activity in CART positive neurons of the PVN. Methods: Non-fasted male Sprague-Dawley rats (n=4/group) were injected intraperitoneally (IP) with CCK-8S or vehicle solution (0.15 M NaCl) during the light phase and brains were removed 90 min after injection. Fos immunohistochemistry and double staining with anti-CART were performed using the free-floating technique. The number of Fos positive neurons was determined in the PVN and arcuate nucleus (ARC) of the hypothalamus that served as control nucleus. Results: CCK-8S (6 and 10 μg/kg body wt, IP) dose-dependently increased the number of Fos positive neurons in the PVN (mean ± SEM: 219 ± 17 and 311 ± 24 neurons/section respectively) compared to the vehicle treated animals (27 ± 1, p<0.05). CCK-8S had no effect on Fos expression in the ARC. Double staining for CART and Fos revealed that CCK8S (6 and 10 μg /kg body wt, IP) results in dose-dependent increase in double labeling (11.2 ± 0.8% and 21.9 ± 2.3% respectively) while only few activated CART neurons in the PVN were observed in vehicle treated rats (1.6 ± 0.5%). Conclusion: These data show for the first time that peripherally injected CCK induces an increase of neuronal activity in CART neurons of the PVN suggesting the involvement of CART in the central mediation of CCK-induced reduction of food intake.
T1662 Importance of Corticosteroid Receptors Within the Amygdala On PostInflammatory Colonic Hyperalgesia Anthony C. Johnson, Kristen Campbell-Dittmeyer, Beverley Greenwood-Van Meerveld Background: A subgroup of patients develop irritable bowel syndrome following an acute colitis combined with a significant life stress. In preclinical models we found an up-regulation of stress hormone expression in the rat brain following an acute colitis. Moreover, in rodents modulation of the amygdala with corticosterone (CORT) induced anxiety-like behavior coupled with a hypersensitive colon which involves both mineralocorticoid (MR) and glucocorticoid (GR)-mediated mechanisms. We hypothesized that MR and/or GR receptors in the amygdala modulate post-inflammatory colonic hypersensitivity. Methods: Distal colitis was induced in male Fischer 344 rats via a 0.5 ml intra-rectal enema of 2,4,6-trinitrobenzenesulphonic acid (TNBS, 50 mg) in 25% ethanol. At 23 days post-TNBS enema, rats were randomly assigned to groups in which a micropellet containing a selective GR antagonist (mifepristone, 15 μg) or a MR antagonist (spironolactone, 15 μg) was implanted stereotaxically bilaterally on the dorsal margin of the amygdala. After recovery from the colitis (day 30 post-TNBS), colonic sensitivity to graded levels of isobaric colorectal distension (CRD) at 0-60 mmHg was assessed by measuring a visceromotor response (VMR) to CRD, quantified as the number of abdominal contractions during a 10 min recording period. Control groups included rats that underwent sham micropellet surgery and rats that received a saline enema. The location of the micropellet was confirmed at the end of the experiment. Results: In rats with or with-out sham surgery, post-inflammatory colonic hyperalgesia was confirmed at 30-days post-TNBS enema by an elevation of the VMR to CRD at 40 and 60 mmHg compared to the saline enema-control group (19.9±1.7 vs. 12.9±1.5, p<0.01 at 40 mmHg; 27.2±2.5 vs. 18.9±1.7, p<0.001 at 60 mmHg, n=15/group). Neither the GR nor MR antagonist altered the exaggerated VMR to CRD induced by 40 mmHg CRD. At 60 mmHg, while rats that received micropellets of mifepristone remained hypersensitive to CRD (27.3±1.9, p<0.01 vs. saline, n=7), rats with spironolactone were not significantly different from the saline controls (23.9±2.0, p>0.05 vs. saline, n=7). Summary and Conclusions: Stereotaxic delivery of a selective MR but not a GR antagonist, at doses shown previously to inhibit the VMR to CRD in rats implanted with amygdaloid CORT, induced a small though significant inhibition of post-inflammatory colonic hyperalgesia. These findings suggest that postinflammatory colonic hypersensitivity involves at least in part the interaction of endogenous corticosteroids with MR at the level of the amygdala but appears independent of the interaction with GR.
T1665 5-HT4-Stimulated Neuroprotection and Neurogenesis in the Enteric Nervous System (ENS): Significance in Dextran Sulfate Sodium (DSS)-Induced Colitis in Mice Mintsai Liu, Jingwen Wang, Rene Hen, Michael D. Gershon We have previously shown that 5-HT4 stimulation promotes survival and neurite extension in cultured enteric neurons. In 5-HT4 knockout mice (KO), gastric emptying is delayed, intestinal motility is slowed, enteric neurons are smaller and their age-related loss accelerates. We now test the hypotheses that Akt mediates 5-HT4-promoted enteric neuronal survival and that 5-HT4-mediated neuroprotection and neurogenesis is significant In Vivo. Neurons were isolated from adult gut, cultured, and exposed to a 5-HT4 agonist, RS67506 or tegaserod (10 nM). In-cell ELISA was used to quantify pAkt. RS67506 and tegaserod each concentrationdependently increased the pAkt/Akt ratio (p < 0.05). This increase was reduced (p < 0.05) by the 5-HT4 antagonist, SB204070 (10 nM), the adenyl cyclase inhibitor, SQ22536 (10 μM), and the PI3-K inhibitor, LY294002 (10 μM). RS67506 and tegaserod also increased pCREB nuclear translocation. 5-HT4-induced neuroprotection may thus be mediated by pAkt (Epac 1/2, which couples 5-HT4 stimulation to pAkt was detected) and/or by pCREB. To test the In Vivo significance of 5-HT4-mediated neuroprotection, DSS (5%) was included in drinking water for 7 days. The severity of the resulting DSS-induced colitis was confirmed by measuring neutrophil (myeloperoxidase) and macrophage (F4/80) infiltration. Nerve terminal degeneration, manifested by the presence of retraction bulbs, visualized by electron micrograph (EM) and peripherin immunoreactivity, which was observed in the inflamed colon of wild-type (WT) and KO mice; however, nerve terminal degeneration also occurred spontaneously in KO animals. These changes were accompanied by enhanced autophagy in neurites and perikarya, which was confirmed by quantifying LC3B-immunoreactivity and by EM. Colitis also promoted cell proliferation in the myenteric plexus (evaluated with Ki67 immunoreactivity) and increased the numbers of small sized neurons in myenteric ganglia. Proliferation was decreased in KO mice (p < 0.01). Administration of BrdU (7 day infusion) revealed that inflammation, tegaserod, and RS67506 each stimulated the generation of new HuC/D-immunoreactive neurons in germinal niches adjacent to myenteric ganglia in WT mice, but not in KO mice. Inflammation and deletion of 5-HT4 receptors thus are accompanied in the ENS by autophagy and neurodegeneration. These adverse changes are linked to the promotion of neurogenesis by stem cells that are retained in specialized regions (germinal niches) in adult animals. Neurodegeneration is facilitated while neuronal replacement is diminished by 5-HT4 receptors deletion.
T1663 Impaired Gastric Motor Function Induced By Acute Restraint Stress Is No More Observed Following Repeated Chronic Stress in Mice Reji Babygirija, Zheng Jun, Kirk A. Ludwig, Toku Takahashi Background: Physical and psychological stress contributes to the pathogenesis of functional dyspepsia. We have previously demonstrated that acute stress delays gastric emptying and impairs gastric motility via central CRF and peripheral sympathetic pathways in rats (Am J Physiol 288, R427-432, 2005). In contrast, a recent study suggests that gastric emptying is restored following chronic stress loading (Life Sci 82,862-868, 2008). Ghrelin accelerates gastric emptying and stimulates postprandial gastric contractions in rats. A recent study suggests that plasma ghrelin levels and gastric ghrelin mRNA expression are significantly increased following chronic stress in mice (Nature Neurosci 11,752-3, 2008). We compared the effects of acute and chronic restraint stress on solid gastric emptying and postprandial gastric motility in mice. We also studied whether endogenous ghrelin is involved in mediating gastric emptying and motility following chronic stress in mice. Methods: Adult male Swiss Webster mice were used for the study. After 16 hr fasting, mice were given 0.2 g of preweighed pellet to measure solid gastric emptying. A strain gauge transducer was implanted on the serosal surface of antrum to record the circular muscle contractions. Postprandial gastric motility was recorded following acute (90 minute of single stress) or chronically restraint stress (repeated stress for 5 consecutive days). Motility index (area under the curve) was calculated using a computer-assisted system. Ghrelin antagonist (D-lys 3 GHRP-6, 0.28 mg/kg) was administered intraperitoneally 30 minutes before feeding. Results: In acute stress loading, gastric emptying was significantly delayed (43.1 +/- 7.9 %), compared to that of control mice (76.7 +/- 4.7 % n=6, P<0.05). However, following chronic stress, gastric emptying returned to control levels (75.8 +/- 3.0 %). Acute stress also significantly impaired postprandial gastric motility to 67. 3 +/- 3.5% (n=4, P<0.001) of controls, while chronic stress had no more inhibitory effects on postprandial gastric motility. Ghrelin receptor antagonist has no significant effects on gastric emptying in control mice (71.2 +/- 2.2 %). Restored gastric emptying following chronic stress was significantly antagonized by ghrelin receptor antagonist (46.7 +/- 2.8 %, n=6, P<0.01), compared to that of saline-injected mice
T1666 Immunohistochemical Characterization of Transient Receptor Potential Vanilloid (TRPV) 1 and Sensory Neuropeptides in Mouse Rectum: Clear Differences of the Subpopulations in Mucosal, Submucosal and Muscle Layer Kenjiro Matsumoto, Takuji Hosoya, Kimihito Tashima, Syunji Horie BACKGROUND & AIMS: In the gut, TRPV1 modulates physiological functions such as motility, secretion and circulation. TRPV1 activation leads to release neuropeptides including tachykinins and CGRP. These neuropeptides are distributed in the enteric nervous system
A-553
AGA Abstracts
AGA Abstracts
reported that intracerebroventricular injection of CART peptide inhibits food intake and decreases gastrointestinal motility in rodents. Aim: To determine whether peripheral CCK affects neuronal activity in CART positive neurons of the PVN. Methods: Non-fasted male Sprague-Dawley rats (n=4/group) were injected intraperitoneally (IP) with CCK-8S or vehicle solution (0.15 M NaCl) during the light phase and brains were removed 90 min after injection. Fos immunohistochemistry and double staining with anti-CART were performed using the free-floating technique. The number of Fos positive neurons was determined in the PVN and arcuate nucleus (ARC) of the hypothalamus that served as control nucleus. Results: CCK-8S (6 and 10 μg/kg body wt, IP) dose-dependently increased the number of Fos positive neurons in the PVN (mean ± SEM: 219 ± 17 and 311 ± 24 neurons/section respectively) compared to the vehicle treated animals (27 ± 1, p<0.05). CCK-8S had no effect on Fos expression in the ARC. Double staining for CART and Fos revealed that CCK8S (6 and 10 μg /kg body wt, IP) results in dose-dependent increase in double labeling (11.2 ± 0.8% and 21.9 ± 2.3% respectively) while only few activated CART neurons in the PVN were observed in vehicle treated rats (1.6 ± 0.5%). Conclusion: These data show for the first time that peripherally injected CCK induces an increase of neuronal activity in CART neurons of the PVN suggesting the involvement of CART in the central mediation of CCK-induced reduction of food intake.