- Email: [email protected]
20 Pharmacological and Genetic Identification of Neurons of the Nucleus Tractus Solitarius Pars Centralis
19
made polyethylene bag (2 cm long,1cm diameter) was used for rectal visceral stimulation.Small electrodes were attached to the left forepaw for delivery of the somatic stimulus2 mA,3 ms,10 Hz electric current was applied in a block paradigm fashion before and after phasic barostat controlled distensions(40,60,80,100 mmHg).We used a 9.4 Tesla MRI scanner (Bruker Biospin) utilizing blood oxygen level dependant contrast (gradient echo EPI,slice thickness 1.5 mm) for functional brain imaging.All fMRI image reconstruction and statistical tests were performed in AFNI,employing deconvolution analyses (p=10-3). RESULTS:All tested animals exhibited cortical activity with visceral and somatic stimulation.The number of fMRI activated voxels in response to forepaw stimulation following visceral sensitization was significantly greater than baseline somatic stimulation (p< 0.05, paired T test with two tailed distribution).The cerebral representation of the somatic stimulation was localized in the rat's primary motor and primary somatosensory cortex,while visceral stimulation was noticed to have increased fMRI representation in the cingulate and secondary motor cortex. CONCLUSIONS: Repeated visceral stimulation in rat model increases registration of somatic sensory stimuli as evidenced by recruitment of more activated voxels in the same cerebral cortical regions as the initial somatic stimulus. Since forepaw innervation has no spinal convergence with the colon this finding suggests supraspinal sensitization.
AGA Abstracts
A Previous Cholecystectomy Increases the Risk of Developing Advanced Adenomas of the Colon Ramalinga Kedika, Amar Mahgoub, Mahir Patel, Vijay Bapat, Ali Siddiqui Introduction: Cholecystectomy has been implicated as a risk factor for colorectal cancer (CRC). However, there are limited data assessing the relationship between cholecystectomy and colorectal adenomatous polyps (AP). Aim: The aim of our study was to determine if cholecystectomy is associated with increased prevalence of AP, especially advanced AP in male veterans. Methods: The relationship of whether prior cholecystectomy modified the biological behavior of AP was investigated in a case-control study. Cases were patients with any number of AP and a prior cholecystectomy. Controls were patients with any number of AP without prior cholecystectomy. The cases and the controls selected were matched for age. Factors in each group associated with advanced AP (diameter ≥1 cm and/or a villous component and/or high grade dysplasia) were examined by univariate analysis (UA) (Chisquare analysis and Mann-Whitney U Test). Next, significant variables by UA were included in a stepwise logistic regression analysis to determine independent predictors of aggressive clinical characteristics of polyps. All values are presented as means ± SE, statistic significance was determined at a p ≤ 0.05. Results: We identified a total of 1234 with AP (cases=127, controls=1107). The mean age of patients was 64.1±1.9 years. By univariate analysis, those with a prior cholecystectomy had a greater mean number of AP (4.2 vs. 3.5; p = 0.04) and larger mean AP size (7.9mm vs. 7.1mm; p = 0.04) than those without a cholecystectomy. Right sided lesions occurred in 34.9% of cases with prior cholecystectomy compared to 41.2% of those without a prior cholecystectomy (p=0.21). By logistic regression, prior cholecystectomy was associated with more advanced AP (OR=1.5 [1.0-2.2]; p=0.04). Patients who had a cholecystectomy were 51% more likely to have advanced AP. This relationship was significant even when adjusted for NSAID use, age, and BMI. There appeared to be a trend towards increased time since cholecystectomy being associated with advanced polyps (9.69 yrs vs. 8.99 years, p=0.056). Conclusions: A prior cholecystectomy is independently associated with an increased risk of developing advanced AP. Also, there appears to be a trend toward a greater prevalence of advanced lesions as time since cholecystectomy increases. We speculate that cholecystectomy may be a risk factor for CRC because it increases the development of advanced APs.
22 Differential Expression of CRFR1 and CRFR2 mRNA in the Amygdala of Two Animal Models of Irritable Bowel Syndrome (IBS): Relevance to Visceral Pain Processing Javier A. Bravo, Timothy G. Dinan, John F. Cryan There is a high comorbidity between functional bowel disorders such as IBS and stress related psychiatric conditions. Thus alterations in the bidirectional communication between the brain and the gut are thought to underlie some of the symptoms of IBS. This brain gut axis can be affected by several stressors, as it has been shown in two animal models: early life stress (Maternal Separation [MS]) and the Wistar-Kyoto strain of rats. Both models show high levels of anxiety, altered hypothalamus pituitary adrenal (HPA) axis function and increased visceral hypersensitivity. It is noteworthy that pain, including visceral pain, carries a negative affective load closely related to anxiety and depression. Accumulating evidence suggests that the amygdala is an important player in the emotional-affective dimension of visceral pain. Corticotrophin releasing factor (CRF) is the primary hypothalamic peptide in the regulation of the HPA axis and thus the stress response. Moreover, the amygdala is a major extrahypothalamic site for CRF expression and function, contributing to anxiety and depression. Additionally the amygdala has been described as a site of CRF pain modulation. CRF binds to two receptors CRFR1 and CRFR2, both of which mediate different and often opposing functional effects. We hypothesise that the level of expression of CRF, CRFR1 and CRFR2 within the amygdala would be altered in stress-related animal models of IBS. To test this, Sprague Dawley rats were subjected to MS for 3h daily during post natal days 212. As control, age matched rats were not separated (NS) from their dams. These animals were analyzed when they reached adulthood. Additionally adult male Wistar-Kyoto rats were compared to age matched Sprague Dawley control rats. To analyze the amygdala expression of CRF, CRFR1 and CRFR2 mRNAs in situ hybridisation was used. Densitometric analysis showed that MS rats had an increased level of CRFR1 mRNA in basolateral and central areas of the amygdala whilst reduced CRFR2 mRNA was observed only in the basolateral amygdala in comparison to NS. Wistar-Kyoto rats also showed higher levels of CRFR1 in the basolateral amygdala and lower levels of CRFR2 in central amygdala when compared to Sprague Dawley rats. The results suggest that in the amygdala of these two animal models of IBS the difference in receptor expression promotes a preferential signalling through CRFR1, thus potentially contributing not only to the behavioural changes associated to stress related disorders, but also to the affective component of visceral pain modulation, which is enhanced in IBS patients.
20 Pharmacological and Genetic Identification of Neurons of the Nucleus Tractus Solitarius Pars Centralis ShuXia Wan, R. Alberto Travagli Neurons of the nucleus tractus solitarius (NTS) receive sensory inputs from cardiovascular, gastrointestinal and respiratory viscera. Our recent studies showed a differential distribution of metabotropic glutamate receptors (mGluR) in terminals of NTS neurons impinging on gastric-projecting DMV neurons , namely group II mGluR are localized on GABAergic and glutamatergic NTS terminals, while group III mGluR are present only on glutamatergic synapses. Further, recent studies have shown that capsaicin and purinergic agonists can distinguish sensory inputs carried by C- and A-delta fibers, respectively, impinging upon identified baroreceptive medialis NTS neurons. Since the requirements of the cardiovascular and gastrointestinal systems vary gre atly in the type,timing and duration of responses, we tested whether neurons in the centralis NTS (cNTS), which receive subdiaphragmatic visceral inputs, are similarly organized. Whole cell recordings were conducted in cNTS neurons in thin slices of brainstem from juvenile rats. Single cell RT-PCR was conducted on pharmacologically identified cNTS neurons. Perfusion with ATP or capsaicin increased the frequency of mEPSCs (P<0.05). The excitatory effects of ATP were mimicked by 10μM α-β-met-ATP (N=6) and blocked by 10μM PPADS (N=5) indicating actions at P2X receptors. The effects of capsaicin were antagonized by 1μM capsazepine (N=5; P<0.05) indicating actions via VR-1 receptors. All cNTS neurons responded to capsaicin. Fifteen neurons also responded to ATP while 30 neurons responded to capsaicin alone. EPSCs were evoked in cNTS neurons via stimulation of the tractus solitarius. The release characteristics of impinging synaptic inputs were analyzed by varying the extracellular calcium concentratiojn (V-M analysis). The response to α-βMeATP was then tested. Two groups of cNTS neurons could be distinguished according to whether they responded to α-β-MeATP or not, and whether their data points were tightly distributed(α-β-MeATP responsive) or scattered (α-β-MeATP unresponsive). scRT-PCR indicated that α-β-MeATP-responsive neurons expressed group III mGluR but not GAD-67 (an indicator of GABAergic phenotype), while α-β-MeATP unresponsive neurons expressed GAD-67 but not group III mGluRs. Our data indicate that 1) NTS neurons controlling subdiaphragmatic viscera (cNTS) have a different synaptic organization than medialis NTS neurons controlling baroreceptor reflexes; 2) the mGluR phenotype of cNTS neurons responsive to α-β-MeATP is different from that of unresponsive neurons. Taken together these data suggest that cNTS fibers responsive to α-β-MeATP may be involved in non-essential gastrointestinal motility functions.
23 A Central Serotonin 2C Receptor Pathway Regulates Gastrointestinal Motor Activity via Ghrelin-Neuropeptide Y Signaling, But Does Not Require Downstream Activation of Melanocortin 3/4 Receptors in Rats Akihiro Asakawa, Naoki Fujitsuka, Mizuki Hayashi, Marie Sameshima, Haruka Amitani, Shinya Kojima, Mineko Fujimiya, Akio Inui
21
BACKGROUND: Serotonin (5-HT) is involved in the modulation of most of the neuronal circuits in the body and plays a key role in the regulation of appetite and emotion in the brain. 5-HT2c receptor agonists exert most of their anorectic effects through downstream melanocortin 4 receptors (MC4R) in the hypothalamus. We hypothesized that a central 5HT2cR pathway regulates physiological gastrointestinal (GI) motor activity, as well as feeding. METHODS: The 5-HT2cR agonist, m-chlorophenylpiperazine hydrochloride (m-CPP; 50 μg) or fenfluramine (50 μg) were administered intracerebroventricularly (i.c.v.) to rats that had been fasted for 24h. GI motility was measured in conscious, freely moving rats by a strain gauge force transducer method. The effects of SB242084 (5-HT2cR antagonist; 20 μg, i.c.v. or 0.2-10 mg/kg, p.o.), HS014 (melanocortin 4 receptor (MC4R) antagonist; 2 μg, i.c.v.), SHU9119 (MC3/4R antagonist; 1 nmol, i.c.v.), neuropeptide Y (NPY: 1 nmol, i.c.v.), ghrelin (3nmol, i.v.), phentolamine hydrochloride (alpha-adrenergic receptor blocker; 40 μg/kg/min, i.v.) and hesperidine (the active flavonoid ingredient in Rikkunshito; 100 mg/ kg, p.o.), on the GI motility induced by fenfluramine were examined. Plasma acyl ghrelin
Visceral Stimulation Enhances Cortical Registration of Somatic Afferent Signals in Rats Venelin Kounev, Ranjit S. Makar, Rupeng Li, James S. Hyde, Reza Shaker INTRODUCTION:In recent studies it has been demonstrated that visceral and somatic stimulation can be quantified by measuring the BOLD fMRI signal of cerebral cortex.It is well known that perception of noxious somatic and visceral stimuli differs considerably. However,it is not well established whether repeated non-noxious visceral stimuli can influence somatic sensory function.It has been proposed that altered cortical modulation may underlie many of the chronic somatic pain syndromes.Our aim was to assess whether visceral stimuli can alter somatic sensory cerebral cortical response using fMRI technique in a rat model. METHODS:Adult Sprague-Dawley rats (n=5,120-420g) anesthetized by a continuous infusion of medetomidine (100mcg/kg/hr) and pancoronium(2mg/Kg/Hr) were studied. A custom
AGA Abstracts
A-2
made polyethylene bag (2 cm long,1cm diameter) was used for rectal visceral stimulation.Small electrodes were attached to the left forepaw for delivery of the somatic stimulus2 mA,3 ms,10 Hz electric current was applied in a block paradigm fashion before and after phasic barostat controlled distensions(40,60,80,100 mmHg).We used a 9.4 Tesla MRI scanner (Bruker Biospin) utilizing blood oxygen level dependant contrast (gradient echo EPI,slice thickness 1.5 mm) for functional brain imaging.All fMRI image reconstruction and statistical tests were performed in AFNI,employing deconvolution analyses (p=10-3). RESULTS:All tested animals exhibited cortical activity with visceral and somatic stimulation.The number of fMRI activated voxels in response to forepaw stimulation following visceral sensitization was significantly greater than baseline somatic stimulation (p< 0.05, paired T test with two tailed distribution).The cerebral representation of the somatic stimulation was localized in the rat's primary motor and primary somatosensory cortex,while visceral stimulation was noticed to have increased fMRI representation in the cingulate and secondary motor cortex. CONCLUSIONS: Repeated visceral stimulation in rat model increases registration of somatic sensory stimuli as evidenced by recruitment of more activated voxels in the same cerebral cortical regions as the initial somatic stimulus. Since forepaw innervation has no spinal convergence with the colon this finding suggests supraspinal sensitization.
AGA Abstracts
A Previous Cholecystectomy Increases the Risk of Developing Advanced Adenomas of the Colon Ramalinga Kedika, Amar Mahgoub, Mahir Patel, Vijay Bapat, Ali Siddiqui Introduction: Cholecystectomy has been implicated as a risk factor for colorectal cancer (CRC). However, there are limited data assessing the relationship between cholecystectomy and colorectal adenomatous polyps (AP). Aim: The aim of our study was to determine if cholecystectomy is associated with increased prevalence of AP, especially advanced AP in male veterans. Methods: The relationship of whether prior cholecystectomy modified the biological behavior of AP was investigated in a case-control study. Cases were patients with any number of AP and a prior cholecystectomy. Controls were patients with any number of AP without prior cholecystectomy. The cases and the controls selected were matched for age. Factors in each group associated with advanced AP (diameter ≥1 cm and/or a villous component and/or high grade dysplasia) were examined by univariate analysis (UA) (Chisquare analysis and Mann-Whitney U Test). Next, significant variables by UA were included in a stepwise logistic regression analysis to determine independent predictors of aggressive clinical characteristics of polyps. All values are presented as means ± SE, statistic significance was determined at a p ≤ 0.05. Results: We identified a total of 1234 with AP (cases=127, controls=1107). The mean age of patients was 64.1±1.9 years. By univariate analysis, those with a prior cholecystectomy had a greater mean number of AP (4.2 vs. 3.5; p = 0.04) and larger mean AP size (7.9mm vs. 7.1mm; p = 0.04) than those without a cholecystectomy. Right sided lesions occurred in 34.9% of cases with prior cholecystectomy compared to 41.2% of those without a prior cholecystectomy (p=0.21). By logistic regression, prior cholecystectomy was associated with more advanced AP (OR=1.5 [1.0-2.2]; p=0.04). Patients who had a cholecystectomy were 51% more likely to have advanced AP. This relationship was significant even when adjusted for NSAID use, age, and BMI. There appeared to be a trend towards increased time since cholecystectomy being associated with advanced polyps (9.69 yrs vs. 8.99 years, p=0.056). Conclusions: A prior cholecystectomy is independently associated with an increased risk of developing advanced AP. Also, there appears to be a trend toward a greater prevalence of advanced lesions as time since cholecystectomy increases. We speculate that cholecystectomy may be a risk factor for CRC because it increases the development of advanced APs.
22 Differential Expression of CRFR1 and CRFR2 mRNA in the Amygdala of Two Animal Models of Irritable Bowel Syndrome (IBS): Relevance to Visceral Pain Processing Javier A. Bravo, Timothy G. Dinan, John F. Cryan There is a high comorbidity between functional bowel disorders such as IBS and stress related psychiatric conditions. Thus alterations in the bidirectional communication between the brain and the gut are thought to underlie some of the symptoms of IBS. This brain gut axis can be affected by several stressors, as it has been shown in two animal models: early life stress (Maternal Separation [MS]) and the Wistar-Kyoto strain of rats. Both models show high levels of anxiety, altered hypothalamus pituitary adrenal (HPA) axis function and increased visceral hypersensitivity. It is noteworthy that pain, including visceral pain, carries a negative affective load closely related to anxiety and depression. Accumulating evidence suggests that the amygdala is an important player in the emotional-affective dimension of visceral pain. Corticotrophin releasing factor (CRF) is the primary hypothalamic peptide in the regulation of the HPA axis and thus the stress response. Moreover, the amygdala is a major extrahypothalamic site for CRF expression and function, contributing to anxiety and depression. Additionally the amygdala has been described as a site of CRF pain modulation. CRF binds to two receptors CRFR1 and CRFR2, both of which mediate different and often opposing functional effects. We hypothesise that the level of expression of CRF, CRFR1 and CRFR2 within the amygdala would be altered in stress-related animal models of IBS. To test this, Sprague Dawley rats were subjected to MS for 3h daily during post natal days 212. As control, age matched rats were not separated (NS) from their dams. These animals were analyzed when they reached adulthood. Additionally adult male Wistar-Kyoto rats were compared to age matched Sprague Dawley control rats. To analyze the amygdala expression of CRF, CRFR1 and CRFR2 mRNAs in situ hybridisation was used. Densitometric analysis showed that MS rats had an increased level of CRFR1 mRNA in basolateral and central areas of the amygdala whilst reduced CRFR2 mRNA was observed only in the basolateral amygdala in comparison to NS. Wistar-Kyoto rats also showed higher levels of CRFR1 in the basolateral amygdala and lower levels of CRFR2 in central amygdala when compared to Sprague Dawley rats. The results suggest that in the amygdala of these two animal models of IBS the difference in receptor expression promotes a preferential signalling through CRFR1, thus potentially contributing not only to the behavioural changes associated to stress related disorders, but also to the affective component of visceral pain modulation, which is enhanced in IBS patients.
20 Pharmacological and Genetic Identification of Neurons of the Nucleus Tractus Solitarius Pars Centralis ShuXia Wan, R. Alberto Travagli Neurons of the nucleus tractus solitarius (NTS) receive sensory inputs from cardiovascular, gastrointestinal and respiratory viscera. Our recent studies showed a differential distribution of metabotropic glutamate receptors (mGluR) in terminals of NTS neurons impinging on gastric-projecting DMV neurons , namely group II mGluR are localized on GABAergic and glutamatergic NTS terminals, while group III mGluR are present only on glutamatergic synapses. Further, recent studies have shown that capsaicin and purinergic agonists can distinguish sensory inputs carried by C- and A-delta fibers, respectively, impinging upon identified baroreceptive medialis NTS neurons. Since the requirements of the cardiovascular and gastrointestinal systems vary gre atly in the type,timing and duration of responses, we tested whether neurons in the centralis NTS (cNTS), which receive subdiaphragmatic visceral inputs, are similarly organized. Whole cell recordings were conducted in cNTS neurons in thin slices of brainstem from juvenile rats. Single cell RT-PCR was conducted on pharmacologically identified cNTS neurons. Perfusion with ATP or capsaicin increased the frequency of mEPSCs (P<0.05). The excitatory effects of ATP were mimicked by 10μM α-β-met-ATP (N=6) and blocked by 10μM PPADS (N=5) indicating actions at P2X receptors. The effects of capsaicin were antagonized by 1μM capsazepine (N=5; P<0.05) indicating actions via VR-1 receptors. All cNTS neurons responded to capsaicin. Fifteen neurons also responded to ATP while 30 neurons responded to capsaicin alone. EPSCs were evoked in cNTS neurons via stimulation of the tractus solitarius. The release characteristics of impinging synaptic inputs were analyzed by varying the extracellular calcium concentratiojn (V-M analysis). The response to α-βMeATP was then tested. Two groups of cNTS neurons could be distinguished according to whether they responded to α-β-MeATP or not, and whether their data points were tightly distributed(α-β-MeATP responsive) or scattered (α-β-MeATP unresponsive). scRT-PCR indicated that α-β-MeATP-responsive neurons expressed group III mGluR but not GAD-67 (an indicator of GABAergic phenotype), while α-β-MeATP unresponsive neurons expressed GAD-67 but not group III mGluRs. Our data indicate that 1) NTS neurons controlling subdiaphragmatic viscera (cNTS) have a different synaptic organization than medialis NTS neurons controlling baroreceptor reflexes; 2) the mGluR phenotype of cNTS neurons responsive to α-β-MeATP is different from that of unresponsive neurons. Taken together these data suggest that cNTS fibers responsive to α-β-MeATP may be involved in non-essential gastrointestinal motility functions.
23 A Central Serotonin 2C Receptor Pathway Regulates Gastrointestinal Motor Activity via Ghrelin-Neuropeptide Y Signaling, But Does Not Require Downstream Activation of Melanocortin 3/4 Receptors in Rats Akihiro Asakawa, Naoki Fujitsuka, Mizuki Hayashi, Marie Sameshima, Haruka Amitani, Shinya Kojima, Mineko Fujimiya, Akio Inui
21
BACKGROUND: Serotonin (5-HT) is involved in the modulation of most of the neuronal circuits in the body and plays a key role in the regulation of appetite and emotion in the brain. 5-HT2c receptor agonists exert most of their anorectic effects through downstream melanocortin 4 receptors (MC4R) in the hypothalamus. We hypothesized that a central 5HT2cR pathway regulates physiological gastrointestinal (GI) motor activity, as well as feeding. METHODS: The 5-HT2cR agonist, m-chlorophenylpiperazine hydrochloride (m-CPP; 50 μg) or fenfluramine (50 μg) were administered intracerebroventricularly (i.c.v.) to rats that had been fasted for 24h. GI motility was measured in conscious, freely moving rats by a strain gauge force transducer method. The effects of SB242084 (5-HT2cR antagonist; 20 μg, i.c.v. or 0.2-10 mg/kg, p.o.), HS014 (melanocortin 4 receptor (MC4R) antagonist; 2 μg, i.c.v.), SHU9119 (MC3/4R antagonist; 1 nmol, i.c.v.), neuropeptide Y (NPY: 1 nmol, i.c.v.), ghrelin (3nmol, i.v.), phentolamine hydrochloride (alpha-adrenergic receptor blocker; 40 μg/kg/min, i.v.) and hesperidine (the active flavonoid ingredient in Rikkunshito; 100 mg/ kg, p.o.), on the GI motility induced by fenfluramine were examined. Plasma acyl ghrelin
Visceral Stimulation Enhances Cortical Registration of Somatic Afferent Signals in Rats Venelin Kounev, Ranjit S. Makar, Rupeng Li, James S. Hyde, Reza Shaker INTRODUCTION:In recent studies it has been demonstrated that visceral and somatic stimulation can be quantified by measuring the BOLD fMRI signal of cerebral cortex.It is well known that perception of noxious somatic and visceral stimuli differs considerably. However,it is not well established whether repeated non-noxious visceral stimuli can influence somatic sensory function.It has been proposed that altered cortical modulation may underlie many of the chronic somatic pain syndromes.Our aim was to assess whether visceral stimuli can alter somatic sensory cerebral cortical response using fMRI technique in a rat model. METHODS:Adult Sprague-Dawley rats (n=5,120-420g) anesthetized by a continuous infusion of medetomidine (100mcg/kg/hr) and pancoronium(2mg/Kg/Hr) were studied. A custom
AGA Abstracts
A-2