Dante Heredia - Academia.edu (original) (raw)

Papers by Dante Heredia

American Journal of Physiology-gastrointestinal and Liver Physiology, 2010

Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased state... more Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased states, including slow-transit constipation. However, the mechanisms underlying the CMMCs are controversial because it has been proposed that disinhibition (turning off of inhibitory neurotransmission) or excitatory nerve activity generate the CMMC. Therefore, our aims were to reexamine the mechanisms underlying the CMMC in the colon of wild-type and neuronal nitric oxide synthase (nNOS)(-/-) mice. CMMCs were recorded from the isolated murine large bowel using intracellular recordings of electrical activity from circular muscle (CM) combined with tension recording. Spontaneous CMMCs occurred in both wild-type (frequency: 0.3 cycles/min) and nNOS(-/-) mice (frequency: 0.4 cycles/min). CMMCs consisted of a hyperpolarization, followed by fast oscillations (slow waves) with action potentials superimposed on a slow depolarization (wild-type: 14.0 +/- 0.6 mV; nNOS(-/-): 11.2 +/- 1.5 mV). Both atropine (1 microM) and MEN 10,376 [neurokinin 2 (NK2) antagonist; 0.5 microM] added successively reduced the slow depolarization and the number of action potentials but did not abolish the fast oscillations. The further addition of RP 67580 (NK1 antagonist; 0.5 microM) blocked the fast oscillations and the CMMC. Importantly, none of the antagonists affected the resting membrane potential, suggesting that ongoing tonic inhibition of the CM was maintained. Fecal pellet propulsion, which was blocked by the NK2 or the NK1 antagonist, was slower down the longer, more constricted nNOS(-/-) mouse colon (wild-type: 47.9 +/- 2.4 mm; nNOS(-/-): 57.8 +/- 1.4 mm). These observations suggest that excitatory neurotransmission enhances pacemaker activity during the CMMC. Therefore, the CMMC is likely generated by a synergistic interaction between neural and interstitial cells of Cajal networks.

Gastroenterology, 2009

The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of feca... more The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of fecal matter through a rhythmic sequence of electrical activity and/or contractions along the large bowel. CMMCs have largely been studied in empty preparations; we investigated whether local reflexes generated by a fecal pellet modify the CMMC to initiate propulsive activity. Methods: Recordings of CMMCs were made from the isolated murine large bowel, with or without a fecal pellet. Transducers were placed along the colon to record muscle tension and propulsive force on the pellet and microelectrodes were used to record electrical activity from either side of a fecal pellet, circular muscle cells oral and anal of a pellet, and in colons without the mucosa. Results: Spontaneous CMMCs propagated in both an oral or anal direction. When a pellet was inserted, CMMCs increased in frequency and propagated anally, exerting propulsive force on the pellet. The amplitude of slow waves increased during the CMMC. Localized mucosal stimulation/circumferential stretch evoked a CMMC, regardless of stimulus strength. The serotonin (5-hydroxytryptamine-3) receptor antagonist ondansetron reduced the amplitude of the CMMC, the propulsive force on the pellet, and the response to mucosal stroking, but increased the apparent conduction velocity of the CMMC. Removing the mucosa abolished spontaneous CMMCs, which still could be evoked by electrical stimulation. Conclusions: The fecal pellet activates local mucosal reflexes, which release serotonin (5-hydroxytryptamine) from enterochromaffin cells, and stretch reflexes that determine the site of origin and propagation of the CMMC, facilitating propulsion.

American Journal of Physiology-gastrointestinal and Liver Physiology, 2010

The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine... more The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine colon. We have previously shown that 5-hydroxytryptamine (5-HT) released from enterochromaffin cells activates 5-HT(3) receptors on the mucosal processes of myenteric Dogiel type II neurons to initiate the events underlying the CMMC. Our aims were to further investigate the roles of 5-HT(1A), 5-HT(3), and 5-HT(7) receptor subtypes in generating and propagating the CMMC using intracellular microelectrodes or tension recordings from the circular muscle (CM) in preparations with and without the mucosa. Spontaneous CMMCs were recorded from the CM in isolated murine colons but not in preparations without the mucosa. In mucosaless preparations, ondansetron (3 microM; 5-HT(3) antagonist) plus hexamethonium (100 microM) completely blocked spontaneous inhibitory junction potentials, depolarized the CM. Ondansetron blocked the preceding hyperpolarization associated with a CMMC. Spontaneous CMMCs and CMMCs evoked by spritzing 5-HT (10 and 100 microM) or nerve stimulation in preparations without the mucosa were blocked by SB 258719 or SB 269970 (1-5 microM; 5-HT(7) antagonists). Both NAN-190 and (S)-WAY100135 (1-5 microM; 5-HT(1A) antagonists) blocked spontaneous CMMCs and neurally evoked CMMCs in preparations without the mucosa. Both NAN-190 and (S)-WAY100135 caused an atropine-sensitive depolarization of the CM. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP) (10 microM), and 5-carboxamidotryptamine (5-CT) (5 microM; 5-HT(1/5/7) agonist) increased the frequency of spontaneous CMMCs. 5-HTP and 5-CT also induced CMMCs in preparations with and without the mucosa, which were blocked by SB 258719. 5-HT(1A), 5-HT(3), and 5-HT(7) receptors, most likely on Dogiel Type II/AH neurons, are important in initiating, generating, and propagating the CMMC. Tonic inhibition of the CM appears to be driven by ongoing activity in descending serotonergic interneurons; by activating 5-HT(7) receptors on AH neurons these interneurons also contribute to the generation of the CMMC.

Gastroenterology, 2010

1. Heredia DJ, Dickson EJ, Bayguinov PO, et al. Localized release of serotonin (5-hydroxytryptami... more 1. Heredia DJ, Dickson EJ, Bayguinov PO, et al. Localized release of serotonin (5-hydroxytryptamine) by a fecal pellet regulates migrating motor complexes in murine colon. Gastroenterology 2009; 136:1328 -1338. 2. Keating DJ, Spencer NJ. Release of 5-hydroxytryptamine from the mucosa is not required for the generation or propagation of colonic migrating motor complexes. Gastroenterology 2010;138:659 -670. GASTROENTEROLOGY 2010;138:1213-1216 3. Dickson EJ, Heredia DJ, Hennig GW, et al. The importance of 5-HT 1A and 5-HT 7 receptors during murine colonic migrating motor complex. Neurogastroenterol Motil 2009;21(Suppl 1):48. 4. Meedeniya ACB, Brookes SJH, Costa M. Sources of 5-hydroxytryptamine immunoreactivity in the myenteric plexus of the guinea-pig small intestine. Proc Aust Neurosci Soc 1994;5:176.

Gastroenterology, 2007

Transit of fecal material through the human colon takes >30 hours, whereas transit through the sm... more Transit of fecal material through the human colon takes >30 hours, whereas transit through the small intestine takes 24 hours. The mechanisms underlying colonic storage and slow transit have yet to be elucidated. Our aim was to determine whether an intrinsic neural mechanism underlies these phenomena. Methods: Recordings were made from circular muscle (CM) cells and myenteric neurons in the isolated guinea pig distal colon using intracellular recordings and Ca 2؉ imaging techniques. Video imaging was used to determine the effects of colonic filling and pellet transit. Results: Circumferential stretch generated ongoing oral excitatory and anal inhibitory junction potentials in the CM. The application of longitudinal stretch inhibited all junction potentials. N-nitro-L-arginine (100 mol/L) completely reversed the inhibitory effects of longitudinal stretch suggesting that nitric oxide (NO) inhibited interneurons controlling peristaltic circuits. Ca 2؉ imaging in preparations that were stretched in both axes revealed ongoing firing in nNOS ؉ve descending neurons, even when synaptic transmission was blocked. Inhibitory postsynaptic potentials were evoked in mechanosensitive interneurons that were blocked by N--nitro-L-arginine (100 mol/L). Pellet transit was inhibited by longitudinal stretch. Filling the colon with fluid led to colonic elongation and an inhibition of motility. Conclusions: Our data support the novel hypothesis that slow transit and accommodation are generated by release of NO from descending (nNOS ؉ve) interneurons triggered by colonic elongation. We refer to this powerful inhibitory reflex as the intrinsic occult reflex (hidden from observation) because it withdraws motor activity from the muscle.

Gastroenterology, 2008

Background: Habitual, non-retentive constipation in children is one of the most common diagnoses ... more Background: Habitual, non-retentive constipation in children is one of the most common diagnoses in the general pediatric clinic and the pediatric gastroenterology clinic. In recent years, an increased incidence of this disease has been reported from both specialty clinics. Although the genetic nature of this disease is not known, recent data from the adult medical literature showed a tendency towards familial clustering. There are no data reported from the pediatric population. Aim: To investigate familial clustering of habitual constipation in the pediatric patients. Methods: Pediatric patients, who attended the pediatric gastroenterology and general pediatric clinics at Marshall University School of Medicine were prospectively recruited to the study. Children with the diagnosis of functional, non-retentive constipation constituted our study population (test group), and children with no constipation were the control group. Constipation symptoms were analyzed in the family members of each participant, utilizing a standard questionnaire (Rome criteria). Exclusion criteria included children younger than 6 months, children from broken families, i.e.: single parent, multiple parents, or divorced families, and/or children with various neuromuscular diseases. Results: A total of 78 families were recruited, of whom 27 had constipated probands (test group) and 51 families had no constipation (control group). A total of 216 family members completed the questionnaires. Significantly more family members from the test group had constipation compared to the control group (p<0.0001) ( ) Conclusion: Habitual constipation in children seemed to cluster in families. The patho-physiology behind this phenomenon is yet to be discovered. Constipation and familial clustering * Student t-test or Chi-Square analyses W1341 Background: Chronic diabetes causes complications such as altered colonic motility which leads to significant morbidity. We have previously demonstrated that hyperglycemia-induced apoptosis is associated with increased oxidative stress in enteric neurons. Aim: We examined the role of oxidative stress and TLR4 signaling in hyperglycemia-induced apoptosis of enteric neurons. Methods:In Vitro experiments were performed in the recently characterized enteric neuronal cell line IM-PEN. Glucose-induced (5-40mM, 24h) TLR4 expression was assessed using immunocytochemistry. Enteric neurons were cultured in the presence or absence of LPS (0.1-10µg/ml) and assessed for apoptosis using the TUNEL method. LPS-induced chemokine (KC) release was measured using ELISA. Cells were treated with vehicle or H 2 O 2 (10µM) in the presence of TLR4siRNA or control siRNA and assessed for apoptosis by cleaved caspase-3 expression. The effect of LPS on colonic motility In Vivo was assessed in the WT and TLR4 mutant mice using the bead expulsion test. Proximal colon relaxation was assessed by isometric muscle recording in conjunction with electrical field stimulation. Results: IM-PEN cells exposed to 20-40mM glucose for 24 h have a significantly higher level of apoptosis (cleaved caspase3 expression) compared to 5mM glucose (p <0.05). Exposure of enteric neurons to 20 mM glucose resulted in increased TLR4 expression. Activation of TLR4 with LPS, induced apoptosis in enteric neurons in a dose depended fashion (% TUNEL+/PGP9.5+: Vehicle:2.2+0.4; LPS(0.1):3.4+0.6; LPS(1):7.01+0.7; LPS(10):8.38+0.5). 24 h exposure to LPS increased the release of KC from enteric neurons showing the presence of a functional TLR4 receptor in these neurons (KC(pg/ml): Vehicle: 639+12; LPS (0.1):1862+62; LPS(1) 2825+339,p<0.0001). H 2 O 2 (10µM) resulted in a 3 fold increase in cleaved caspase 3 expression compared to vehicle and this was reduced to 0.65 fold in the presence of TLR4 siRNA but not control siRNA. In Vivo analysis of motility in WT mice treated with LPS showed a delay in bead expulsion after injection of LPS (p<0.05).This effect was not seen in LPS treated TLR4 KO mice.Isometric muscle recording showed a reduction in relaxation in LPS treated WT mice (p<0.05). Conclusions:We have demonstrated a role for TLR4 signaling in hyperglycemia and oxidative stress induced apoptosis in enteric neurons. Knock down of TLR4 ameliorates oxidative stress induced apoptosis in enteric neurons. Activation of TLR4 with LPS induces changes in colonic motility In Vivo. Alteration in TLR4 signaling in enteric neurons can contribute to the motility changes seen in diabetes and is a potential therapeutic target.

Journal of Physiology-london, 2008

Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despi... more Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despite this, transit through the shorter colon is at least 10 times slower. Recently we demonstrated that elongating a segment of colon releases nitric oxide (NO) to inhibit peristalsis. The aims of this study were to determine if colonic elongation was physiologically significant, and whether elongation activated polarized intrinsic neural reflexes. Video imaging monitored fecal pellet evacuation from isolated guinea-pig colons full of pellets. Recordings were made from the circular muscle (CM) and longitudinal muscle (LM) in flat sheet preparations using either intracellular microelectrode or Ca 2+ imaging techniques. Full colons were 158.1 ± 6.1% longer than empty colons. As each pellet was expelled, the colon shortened and pellet velocity increased exponentially (full 0.34, empty 1.01 mm s −1 ). In flat sheet preparations, maintained circumferential stretch generated ongoing peristaltic activity (oral excitatory and anal inhibitory junction potentials) and Ca 2+ waves in LM and CM. Colonic elongation (140% of its empty slack length) applied oral to the recording site abolished these activities, whereas anal elongation significantly increased the frequency and amplitude of ongoing peristaltic activity. Oral elongation inhibited the excitation produced by anal elongation; this inhibitory effect was reversed by blocking NO synthesis. Pelvic nerve stimulation elicited polarized responses that were also suppressed by NO released during colonic elongation. In conclusion, longitudinal stretch excites specific mechosensitive ascending and descending interneurons, leading to activation of polarized reflexes. The dominance of the descending inhibitory reflex leads to slowed emptying of pellets in a naturally elongated colon.

Journal of Physiology-london, 2010

The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pa... more The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pattern. Although the CMMC spontaneously propagates along an empty colon it is responsible for faecal pellet propulsion in the murine large bowel. Unlike the peristaltic reflex, the CMMC is an &amp;amp;amp;amp;amp;amp;#39;all or none&amp;amp;amp;amp;amp;amp;#39; event that appears to be dependent upon Dogiel Type II/AH neurons for its regenerative slow propagation down the colon. A reduction in the amplitude of CMMCs or an elongated colon have both been thought to underlie slow transit constipation, although whether these phenomena are related has not been considered. In this study we examined the mechanisms by which colonic elongation might affect the CMMC using video imaging of the colon, tension and electrophysiological recordings from the muscle and Ca(2+) imaging of myenteric neurons. As faecal pellets were expelled from the murine colon, it shortened by up to 29%. Elongation of the colon resulted in a linear reduction in the velocity of a faecal pellet and the amplitude of spontaneous CMMCs. Elongation of the oral end of a colonic segment reduced the amplitude of CMMCs, whereas elongation of the anal end of the colon evoked a premature CMMC, and caused the majority of CMMCs to propagate in an anal to oral direction. Dogiel Type II/AH sensory neurons and most other myenteric neurons responded to oral elongation with reduced amplitude and frequency of spontaneous Ca(2+) transients, whereas anal elongation increased their amplitude and frequency in most neurons. The inhibitory effects of colonic elongation were reduced by blocking nitric oxide (NO) production with l-NA (100 mum) and soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 mum); whereas, l-arginine (1-2 mm) enhanced the inhibitory effects of colonic elongation. In conclusion, polarized neural reflexes can be triggered by longitudinal stretch. The dominant effect of elongation is to reduce CMMCs primarily by inhibiting Dogiel Type II/AH neurons, thus facilitating colonic accommodation and slow transit.

American Journal of Physiology-gastrointestinal and Liver Physiology, 2010

Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased state... more Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased states, including slow-transit constipation. However, the mechanisms underlying the CMMCs are controversial because it has been proposed that disinhibition (turning off of inhibitory neurotransmission) or excitatory nerve activity generate the CMMC. Therefore, our aims were to reexamine the mechanisms underlying the CMMC in the colon of wild-type and neuronal nitric oxide synthase (nNOS)(-/-) mice. CMMCs were recorded from the isolated murine large bowel using intracellular recordings of electrical activity from circular muscle (CM) combined with tension recording. Spontaneous CMMCs occurred in both wild-type (frequency: 0.3 cycles/min) and nNOS(-/-) mice (frequency: 0.4 cycles/min). CMMCs consisted of a hyperpolarization, followed by fast oscillations (slow waves) with action potentials superimposed on a slow depolarization (wild-type: 14.0 +/- 0.6 mV; nNOS(-/-): 11.2 +/- 1.5 mV). Both atropine (1 microM) and MEN 10,376 [neurokinin 2 (NK2) antagonist; 0.5 microM] added successively reduced the slow depolarization and the number of action potentials but did not abolish the fast oscillations. The further addition of RP 67580 (NK1 antagonist; 0.5 microM) blocked the fast oscillations and the CMMC. Importantly, none of the antagonists affected the resting membrane potential, suggesting that ongoing tonic inhibition of the CM was maintained. Fecal pellet propulsion, which was blocked by the NK2 or the NK1 antagonist, was slower down the longer, more constricted nNOS(-/-) mouse colon (wild-type: 47.9 +/- 2.4 mm; nNOS(-/-): 57.8 +/- 1.4 mm). These observations suggest that excitatory neurotransmission enhances pacemaker activity during the CMMC. Therefore, the CMMC is likely generated by a synergistic interaction between neural and interstitial cells of Cajal networks.

Gastroenterology, 2009

The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of feca... more The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of fecal matter through a rhythmic sequence of electrical activity and/or contractions along the large bowel. CMMCs have largely been studied in empty preparations; we investigated whether local reflexes generated by a fecal pellet modify the CMMC to initiate propulsive activity. Methods: Recordings of CMMCs were made from the isolated murine large bowel, with or without a fecal pellet. Transducers were placed along the colon to record muscle tension and propulsive force on the pellet and microelectrodes were used to record electrical activity from either side of a fecal pellet, circular muscle cells oral and anal of a pellet, and in colons without the mucosa. Results: Spontaneous CMMCs propagated in both an oral or anal direction. When a pellet was inserted, CMMCs increased in frequency and propagated anally, exerting propulsive force on the pellet. The amplitude of slow waves increased during the CMMC. Localized mucosal stimulation/circumferential stretch evoked a CMMC, regardless of stimulus strength. The serotonin (5-hydroxytryptamine-3) receptor antagonist ondansetron reduced the amplitude of the CMMC, the propulsive force on the pellet, and the response to mucosal stroking, but increased the apparent conduction velocity of the CMMC. Removing the mucosa abolished spontaneous CMMCs, which still could be evoked by electrical stimulation. Conclusions: The fecal pellet activates local mucosal reflexes, which release serotonin (5-hydroxytryptamine) from enterochromaffin cells, and stretch reflexes that determine the site of origin and propagation of the CMMC, facilitating propulsion.

American Journal of Physiology-gastrointestinal and Liver Physiology, 2010

The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine... more The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine colon. We have previously shown that 5-hydroxytryptamine (5-HT) released from enterochromaffin cells activates 5-HT(3) receptors on the mucosal processes of myenteric Dogiel type II neurons to initiate the events underlying the CMMC. Our aims were to further investigate the roles of 5-HT(1A), 5-HT(3), and 5-HT(7) receptor subtypes in generating and propagating the CMMC using intracellular microelectrodes or tension recordings from the circular muscle (CM) in preparations with and without the mucosa. Spontaneous CMMCs were recorded from the CM in isolated murine colons but not in preparations without the mucosa. In mucosaless preparations, ondansetron (3 microM; 5-HT(3) antagonist) plus hexamethonium (100 microM) completely blocked spontaneous inhibitory junction potentials, depolarized the CM. Ondansetron blocked the preceding hyperpolarization associated with a CMMC. Spontaneous CMMCs and CMMCs evoked by spritzing 5-HT (10 and 100 microM) or nerve stimulation in preparations without the mucosa were blocked by SB 258719 or SB 269970 (1-5 microM; 5-HT(7) antagonists). Both NAN-190 and (S)-WAY100135 (1-5 microM; 5-HT(1A) antagonists) blocked spontaneous CMMCs and neurally evoked CMMCs in preparations without the mucosa. Both NAN-190 and (S)-WAY100135 caused an atropine-sensitive depolarization of the CM. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP) (10 microM), and 5-carboxamidotryptamine (5-CT) (5 microM; 5-HT(1/5/7) agonist) increased the frequency of spontaneous CMMCs. 5-HTP and 5-CT also induced CMMCs in preparations with and without the mucosa, which were blocked by SB 258719. 5-HT(1A), 5-HT(3), and 5-HT(7) receptors, most likely on Dogiel Type II/AH neurons, are important in initiating, generating, and propagating the CMMC. Tonic inhibition of the CM appears to be driven by ongoing activity in descending serotonergic interneurons; by activating 5-HT(7) receptors on AH neurons these interneurons also contribute to the generation of the CMMC.

Gastroenterology, 2010

1. Heredia DJ, Dickson EJ, Bayguinov PO, et al. Localized release of serotonin (5-hydroxytryptami... more 1. Heredia DJ, Dickson EJ, Bayguinov PO, et al. Localized release of serotonin (5-hydroxytryptamine) by a fecal pellet regulates migrating motor complexes in murine colon. Gastroenterology 2009; 136:1328 -1338. 2. Keating DJ, Spencer NJ. Release of 5-hydroxytryptamine from the mucosa is not required for the generation or propagation of colonic migrating motor complexes. Gastroenterology 2010;138:659 -670. GASTROENTEROLOGY 2010;138:1213-1216 3. Dickson EJ, Heredia DJ, Hennig GW, et al. The importance of 5-HT 1A and 5-HT 7 receptors during murine colonic migrating motor complex. Neurogastroenterol Motil 2009;21(Suppl 1):48. 4. Meedeniya ACB, Brookes SJH, Costa M. Sources of 5-hydroxytryptamine immunoreactivity in the myenteric plexus of the guinea-pig small intestine. Proc Aust Neurosci Soc 1994;5:176.

Gastroenterology, 2007

Transit of fecal material through the human colon takes >30 hours, whereas transit through the sm... more Transit of fecal material through the human colon takes >30 hours, whereas transit through the small intestine takes 24 hours. The mechanisms underlying colonic storage and slow transit have yet to be elucidated. Our aim was to determine whether an intrinsic neural mechanism underlies these phenomena. Methods: Recordings were made from circular muscle (CM) cells and myenteric neurons in the isolated guinea pig distal colon using intracellular recordings and Ca 2؉ imaging techniques. Video imaging was used to determine the effects of colonic filling and pellet transit. Results: Circumferential stretch generated ongoing oral excitatory and anal inhibitory junction potentials in the CM. The application of longitudinal stretch inhibited all junction potentials. N-nitro-L-arginine (100 mol/L) completely reversed the inhibitory effects of longitudinal stretch suggesting that nitric oxide (NO) inhibited interneurons controlling peristaltic circuits. Ca 2؉ imaging in preparations that were stretched in both axes revealed ongoing firing in nNOS ؉ve descending neurons, even when synaptic transmission was blocked. Inhibitory postsynaptic potentials were evoked in mechanosensitive interneurons that were blocked by N--nitro-L-arginine (100 mol/L). Pellet transit was inhibited by longitudinal stretch. Filling the colon with fluid led to colonic elongation and an inhibition of motility. Conclusions: Our data support the novel hypothesis that slow transit and accommodation are generated by release of NO from descending (nNOS ؉ve) interneurons triggered by colonic elongation. We refer to this powerful inhibitory reflex as the intrinsic occult reflex (hidden from observation) because it withdraws motor activity from the muscle.

Gastroenterology, 2008

Background: Habitual, non-retentive constipation in children is one of the most common diagnoses ... more Background: Habitual, non-retentive constipation in children is one of the most common diagnoses in the general pediatric clinic and the pediatric gastroenterology clinic. In recent years, an increased incidence of this disease has been reported from both specialty clinics. Although the genetic nature of this disease is not known, recent data from the adult medical literature showed a tendency towards familial clustering. There are no data reported from the pediatric population. Aim: To investigate familial clustering of habitual constipation in the pediatric patients. Methods: Pediatric patients, who attended the pediatric gastroenterology and general pediatric clinics at Marshall University School of Medicine were prospectively recruited to the study. Children with the diagnosis of functional, non-retentive constipation constituted our study population (test group), and children with no constipation were the control group. Constipation symptoms were analyzed in the family members of each participant, utilizing a standard questionnaire (Rome criteria). Exclusion criteria included children younger than 6 months, children from broken families, i.e.: single parent, multiple parents, or divorced families, and/or children with various neuromuscular diseases. Results: A total of 78 families were recruited, of whom 27 had constipated probands (test group) and 51 families had no constipation (control group). A total of 216 family members completed the questionnaires. Significantly more family members from the test group had constipation compared to the control group (p<0.0001) ( ) Conclusion: Habitual constipation in children seemed to cluster in families. The patho-physiology behind this phenomenon is yet to be discovered. Constipation and familial clustering * Student t-test or Chi-Square analyses W1341 Background: Chronic diabetes causes complications such as altered colonic motility which leads to significant morbidity. We have previously demonstrated that hyperglycemia-induced apoptosis is associated with increased oxidative stress in enteric neurons. Aim: We examined the role of oxidative stress and TLR4 signaling in hyperglycemia-induced apoptosis of enteric neurons. Methods:In Vitro experiments were performed in the recently characterized enteric neuronal cell line IM-PEN. Glucose-induced (5-40mM, 24h) TLR4 expression was assessed using immunocytochemistry. Enteric neurons were cultured in the presence or absence of LPS (0.1-10µg/ml) and assessed for apoptosis using the TUNEL method. LPS-induced chemokine (KC) release was measured using ELISA. Cells were treated with vehicle or H 2 O 2 (10µM) in the presence of TLR4siRNA or control siRNA and assessed for apoptosis by cleaved caspase-3 expression. The effect of LPS on colonic motility In Vivo was assessed in the WT and TLR4 mutant mice using the bead expulsion test. Proximal colon relaxation was assessed by isometric muscle recording in conjunction with electrical field stimulation. Results: IM-PEN cells exposed to 20-40mM glucose for 24 h have a significantly higher level of apoptosis (cleaved caspase3 expression) compared to 5mM glucose (p <0.05). Exposure of enteric neurons to 20 mM glucose resulted in increased TLR4 expression. Activation of TLR4 with LPS, induced apoptosis in enteric neurons in a dose depended fashion (% TUNEL+/PGP9.5+: Vehicle:2.2+0.4; LPS(0.1):3.4+0.6; LPS(1):7.01+0.7; LPS(10):8.38+0.5). 24 h exposure to LPS increased the release of KC from enteric neurons showing the presence of a functional TLR4 receptor in these neurons (KC(pg/ml): Vehicle: 639+12; LPS (0.1):1862+62; LPS(1) 2825+339,p<0.0001). H 2 O 2 (10µM) resulted in a 3 fold increase in cleaved caspase 3 expression compared to vehicle and this was reduced to 0.65 fold in the presence of TLR4 siRNA but not control siRNA. In Vivo analysis of motility in WT mice treated with LPS showed a delay in bead expulsion after injection of LPS (p<0.05).This effect was not seen in LPS treated TLR4 KO mice.Isometric muscle recording showed a reduction in relaxation in LPS treated WT mice (p<0.05). Conclusions:We have demonstrated a role for TLR4 signaling in hyperglycemia and oxidative stress induced apoptosis in enteric neurons. Knock down of TLR4 ameliorates oxidative stress induced apoptosis in enteric neurons. Activation of TLR4 with LPS induces changes in colonic motility In Vivo. Alteration in TLR4 signaling in enteric neurons can contribute to the motility changes seen in diabetes and is a potential therapeutic target.

Journal of Physiology-london, 2008

Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despi... more Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despite this, transit through the shorter colon is at least 10 times slower. Recently we demonstrated that elongating a segment of colon releases nitric oxide (NO) to inhibit peristalsis. The aims of this study were to determine if colonic elongation was physiologically significant, and whether elongation activated polarized intrinsic neural reflexes. Video imaging monitored fecal pellet evacuation from isolated guinea-pig colons full of pellets. Recordings were made from the circular muscle (CM) and longitudinal muscle (LM) in flat sheet preparations using either intracellular microelectrode or Ca 2+ imaging techniques. Full colons were 158.1 ± 6.1% longer than empty colons. As each pellet was expelled, the colon shortened and pellet velocity increased exponentially (full 0.34, empty 1.01 mm s −1 ). In flat sheet preparations, maintained circumferential stretch generated ongoing peristaltic activity (oral excitatory and anal inhibitory junction potentials) and Ca 2+ waves in LM and CM. Colonic elongation (140% of its empty slack length) applied oral to the recording site abolished these activities, whereas anal elongation significantly increased the frequency and amplitude of ongoing peristaltic activity. Oral elongation inhibited the excitation produced by anal elongation; this inhibitory effect was reversed by blocking NO synthesis. Pelvic nerve stimulation elicited polarized responses that were also suppressed by NO released during colonic elongation. In conclusion, longitudinal stretch excites specific mechosensitive ascending and descending interneurons, leading to activation of polarized reflexes. The dominance of the descending inhibitory reflex leads to slowed emptying of pellets in a naturally elongated colon.

Journal of Physiology-london, 2010

The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pa... more The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pattern. Although the CMMC spontaneously propagates along an empty colon it is responsible for faecal pellet propulsion in the murine large bowel. Unlike the peristaltic reflex, the CMMC is an &amp;amp;amp;amp;amp;amp;#39;all or none&amp;amp;amp;amp;amp;amp;#39; event that appears to be dependent upon Dogiel Type II/AH neurons for its regenerative slow propagation down the colon. A reduction in the amplitude of CMMCs or an elongated colon have both been thought to underlie slow transit constipation, although whether these phenomena are related has not been considered. In this study we examined the mechanisms by which colonic elongation might affect the CMMC using video imaging of the colon, tension and electrophysiological recordings from the muscle and Ca(2+) imaging of myenteric neurons. As faecal pellets were expelled from the murine colon, it shortened by up to 29%. Elongation of the colon resulted in a linear reduction in the velocity of a faecal pellet and the amplitude of spontaneous CMMCs. Elongation of the oral end of a colonic segment reduced the amplitude of CMMCs, whereas elongation of the anal end of the colon evoked a premature CMMC, and caused the majority of CMMCs to propagate in an anal to oral direction. Dogiel Type II/AH sensory neurons and most other myenteric neurons responded to oral elongation with reduced amplitude and frequency of spontaneous Ca(2+) transients, whereas anal elongation increased their amplitude and frequency in most neurons. The inhibitory effects of colonic elongation were reduced by blocking nitric oxide (NO) production with l-NA (100 mum) and soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 mum); whereas, l-arginine (1-2 mm) enhanced the inhibitory effects of colonic elongation. In conclusion, polarized neural reflexes can be triggered by longitudinal stretch. The dominant effect of elongation is to reduce CMMCs primarily by inhibiting Dogiel Type II/AH neurons, thus facilitating colonic accommodation and slow transit.

dialécticamente pasa por distintas etapas, al inicio, al recibir la noticia criminal se encuentra... more dialécticamente pasa por distintas etapas, al inicio, al recibir la noticia criminal se encuentra en un estado de IMPROBABILIDAD, para salir de ésta incertidumbre ordena una INVESTIGACIÓN PRELIMINAR disponiendo diligencias urgentes o diligencias preliminares y luego de un plazo correspondiente (por ejemplo hay un plazo legal para investigar de 20 días, salvo que se produzca la detención en cuyo caso solo tendrá 24 horas y si se trata de un caso de drogas, espionaje, o terrorismo su investigación no puede pasar de los 15 días; pero podría fijar un plazo distinto atendiendo a la complejidad del caso y las circunstancias del hecho, pudiendo fijar un plazo fiscal no mayor a los 120 días por contraste con la norma contenida en el artículo 342 del Código Procesal Penal), cumpliendo actos de investigación, puede llegar a una imputación penal, esto es, subsume los hechos con una norma determinada del ordenamiento penal sustantivo y por tanto ya se ubica en lo que es PROBABLE, y que da origen a una "causa probable" que se expresa en su Disposición de Formalización de la Investigación Preparatoria, acá el Fiscal ya tiene a un presunto autor identificado, un delito y un bien jurídico lesionado, una víctima afectada, tiene conciencia que el hecho imputado no ha prescrito, que no hay requisitos de procedibilidad o procesabilidad y por tanto ese mismo Fiscal está convencido que el inculpado ha cometido un delito determinado y merece una condena, su razonamiento llegó a una CERTEZA POSITIVA que se expresa en la formulación de un requerimiento de acusación ( y cuando su razonamiento llega a una CERTEZA NEGATIVA se expresa en la formulación de un requerimiento de Sobreseimiento; finalmente también puede ser MIXTA es decir existir Certeza Negativa y Certeza Positiva a la vez, eso se da cuando emite un requerimiento que contiene una Acusación MIXTA ). De modo tal que el la ETAPA INTERMEDIA, no solo está destinada al control de la ACUSACION, sino también eventualmente al control del SOBRESEIMIENTO que pueda pedir el mismo Fiscal.

dialécticamente pasa por distintas etapas, al inicio, al recibir la noticia criminal se encuentra... more dialécticamente pasa por distintas etapas, al inicio, al recibir la noticia criminal se encuentra en un estado de IMPROBABILIDAD, para salir de ésta incertidumbre ordena una INVESTIGACIÓN PRELIMINAR disponiendo diligencias urgentes o diligencias preliminares y luego de un plazo correspondiente (por ejemplo hay un plazo legal para investigar de 20 días, salvo que se produzca la detención en cuyo caso solo tendrá 24 horas y si se trata de un caso de drogas, espionaje, o terrorismo su investigación no puede pasar de los 15 días; pero podría fijar un plazo distinto atendiendo a la complejidad del caso y las circunstancias del hecho, pudiendo fijar un plazo fiscal no mayor a los 120 días por contraste con la norma contenida en el artículo 342 del Código Procesal Penal), cumpliendo actos de investigación, puede llegar a una imputación penal, esto es, subsume los hechos con una norma determinada del ordenamiento penal sustantivo y por tanto ya se ubica en lo que es PROBABLE, y que da origen a una "causa probable" que se expresa en su Disposición de Formalización de la Investigación Preparatoria, acá el Fiscal ya tiene a un presunto autor identificado, un delito y un bien jurídico lesionado, una víctima afectada, tiene conciencia que el hecho imputado no ha prescrito, que no hay requisitos de procedibilidad o procesabilidad y por tanto ese mismo Fiscal está convencido que el inculpado ha cometido un delito determinado y merece una condena, su razonamiento llegó a una CERTEZA POSITIVA que se expresa en la formulación de un requerimiento de acusación ( y cuando su razonamiento llega a una CERTEZA NEGATIVA se expresa en la formulación de un requerimiento de Sobreseimiento; finalmente también puede ser MIXTA es decir existir Certeza Negativa y Certeza Positiva a la vez, eso se da cuando emite un requerimiento que contiene una Acusación MIXTA ). De modo tal que el la ETAPA INTERMEDIA, no solo está destinada al control de la ACUSACION, sino también eventualmente al control del SOBRESEIMIENTO que pueda pedir el mismo Fiscal.

American Journal of Physiology-gastrointestinal and Liver Physiology, 2010

Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased state... more Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased states, including slow-transit constipation. However, the mechanisms underlying the CMMCs are controversial because it has been proposed that disinhibition (turning off of inhibitory neurotransmission) or excitatory nerve activity generate the CMMC. Therefore, our aims were to reexamine the mechanisms underlying the CMMC in the colon of wild-type and neuronal nitric oxide synthase (nNOS)(-/-) mice. CMMCs were recorded from the isolated murine large bowel using intracellular recordings of electrical activity from circular muscle (CM) combined with tension recording. Spontaneous CMMCs occurred in both wild-type (frequency: 0.3 cycles/min) and nNOS(-/-) mice (frequency: 0.4 cycles/min). CMMCs consisted of a hyperpolarization, followed by fast oscillations (slow waves) with action potentials superimposed on a slow depolarization (wild-type: 14.0 +/- 0.6 mV; nNOS(-/-): 11.2 +/- 1.5 mV). Both atropine (1 microM) and MEN 10,376 [neurokinin 2 (NK2) antagonist; 0.5 microM] added successively reduced the slow depolarization and the number of action potentials but did not abolish the fast oscillations. The further addition of RP 67580 (NK1 antagonist; 0.5 microM) blocked the fast oscillations and the CMMC. Importantly, none of the antagonists affected the resting membrane potential, suggesting that ongoing tonic inhibition of the CM was maintained. Fecal pellet propulsion, which was blocked by the NK2 or the NK1 antagonist, was slower down the longer, more constricted nNOS(-/-) mouse colon (wild-type: 47.9 +/- 2.4 mm; nNOS(-/-): 57.8 +/- 1.4 mm). These observations suggest that excitatory neurotransmission enhances pacemaker activity during the CMMC. Therefore, the CMMC is likely generated by a synergistic interaction between neural and interstitial cells of Cajal networks.

Gastroenterology, 2009

The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of feca... more The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of fecal matter through a rhythmic sequence of electrical activity and/or contractions along the large bowel. CMMCs have largely been studied in empty preparations; we investigated whether local reflexes generated by a fecal pellet modify the CMMC to initiate propulsive activity. Methods: Recordings of CMMCs were made from the isolated murine large bowel, with or without a fecal pellet. Transducers were placed along the colon to record muscle tension and propulsive force on the pellet and microelectrodes were used to record electrical activity from either side of a fecal pellet, circular muscle cells oral and anal of a pellet, and in colons without the mucosa. Results: Spontaneous CMMCs propagated in both an oral or anal direction. When a pellet was inserted, CMMCs increased in frequency and propagated anally, exerting propulsive force on the pellet. The amplitude of slow waves increased during the CMMC. Localized mucosal stimulation/circumferential stretch evoked a CMMC, regardless of stimulus strength. The serotonin (5-hydroxytryptamine-3) receptor antagonist ondansetron reduced the amplitude of the CMMC, the propulsive force on the pellet, and the response to mucosal stroking, but increased the apparent conduction velocity of the CMMC. Removing the mucosa abolished spontaneous CMMCs, which still could be evoked by electrical stimulation. Conclusions: The fecal pellet activates local mucosal reflexes, which release serotonin (5-hydroxytryptamine) from enterochromaffin cells, and stretch reflexes that determine the site of origin and propagation of the CMMC, facilitating propulsion.

American Journal of Physiology-gastrointestinal and Liver Physiology, 2010

The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine... more The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine colon. We have previously shown that 5-hydroxytryptamine (5-HT) released from enterochromaffin cells activates 5-HT(3) receptors on the mucosal processes of myenteric Dogiel type II neurons to initiate the events underlying the CMMC. Our aims were to further investigate the roles of 5-HT(1A), 5-HT(3), and 5-HT(7) receptor subtypes in generating and propagating the CMMC using intracellular microelectrodes or tension recordings from the circular muscle (CM) in preparations with and without the mucosa. Spontaneous CMMCs were recorded from the CM in isolated murine colons but not in preparations without the mucosa. In mucosaless preparations, ondansetron (3 microM; 5-HT(3) antagonist) plus hexamethonium (100 microM) completely blocked spontaneous inhibitory junction potentials, depolarized the CM. Ondansetron blocked the preceding hyperpolarization associated with a CMMC. Spontaneous CMMCs and CMMCs evoked by spritzing 5-HT (10 and 100 microM) or nerve stimulation in preparations without the mucosa were blocked by SB 258719 or SB 269970 (1-5 microM; 5-HT(7) antagonists). Both NAN-190 and (S)-WAY100135 (1-5 microM; 5-HT(1A) antagonists) blocked spontaneous CMMCs and neurally evoked CMMCs in preparations without the mucosa. Both NAN-190 and (S)-WAY100135 caused an atropine-sensitive depolarization of the CM. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP) (10 microM), and 5-carboxamidotryptamine (5-CT) (5 microM; 5-HT(1/5/7) agonist) increased the frequency of spontaneous CMMCs. 5-HTP and 5-CT also induced CMMCs in preparations with and without the mucosa, which were blocked by SB 258719. 5-HT(1A), 5-HT(3), and 5-HT(7) receptors, most likely on Dogiel Type II/AH neurons, are important in initiating, generating, and propagating the CMMC. Tonic inhibition of the CM appears to be driven by ongoing activity in descending serotonergic interneurons; by activating 5-HT(7) receptors on AH neurons these interneurons also contribute to the generation of the CMMC.

Gastroenterology, 2010

1. Heredia DJ, Dickson EJ, Bayguinov PO, et al. Localized release of serotonin (5-hydroxytryptami... more 1. Heredia DJ, Dickson EJ, Bayguinov PO, et al. Localized release of serotonin (5-hydroxytryptamine) by a fecal pellet regulates migrating motor complexes in murine colon. Gastroenterology 2009; 136:1328 -1338. 2. Keating DJ, Spencer NJ. Release of 5-hydroxytryptamine from the mucosa is not required for the generation or propagation of colonic migrating motor complexes. Gastroenterology 2010;138:659 -670. GASTROENTEROLOGY 2010;138:1213-1216 3. Dickson EJ, Heredia DJ, Hennig GW, et al. The importance of 5-HT 1A and 5-HT 7 receptors during murine colonic migrating motor complex. Neurogastroenterol Motil 2009;21(Suppl 1):48. 4. Meedeniya ACB, Brookes SJH, Costa M. Sources of 5-hydroxytryptamine immunoreactivity in the myenteric plexus of the guinea-pig small intestine. Proc Aust Neurosci Soc 1994;5:176.

Gastroenterology, 2007

Transit of fecal material through the human colon takes >30 hours, whereas transit through the sm... more Transit of fecal material through the human colon takes >30 hours, whereas transit through the small intestine takes 24 hours. The mechanisms underlying colonic storage and slow transit have yet to be elucidated. Our aim was to determine whether an intrinsic neural mechanism underlies these phenomena. Methods: Recordings were made from circular muscle (CM) cells and myenteric neurons in the isolated guinea pig distal colon using intracellular recordings and Ca 2؉ imaging techniques. Video imaging was used to determine the effects of colonic filling and pellet transit. Results: Circumferential stretch generated ongoing oral excitatory and anal inhibitory junction potentials in the CM. The application of longitudinal stretch inhibited all junction potentials. N-nitro-L-arginine (100 mol/L) completely reversed the inhibitory effects of longitudinal stretch suggesting that nitric oxide (NO) inhibited interneurons controlling peristaltic circuits. Ca 2؉ imaging in preparations that were stretched in both axes revealed ongoing firing in nNOS ؉ve descending neurons, even when synaptic transmission was blocked. Inhibitory postsynaptic potentials were evoked in mechanosensitive interneurons that were blocked by N--nitro-L-arginine (100 mol/L). Pellet transit was inhibited by longitudinal stretch. Filling the colon with fluid led to colonic elongation and an inhibition of motility. Conclusions: Our data support the novel hypothesis that slow transit and accommodation are generated by release of NO from descending (nNOS ؉ve) interneurons triggered by colonic elongation. We refer to this powerful inhibitory reflex as the intrinsic occult reflex (hidden from observation) because it withdraws motor activity from the muscle.

Gastroenterology, 2008

Background: Habitual, non-retentive constipation in children is one of the most common diagnoses ... more Background: Habitual, non-retentive constipation in children is one of the most common diagnoses in the general pediatric clinic and the pediatric gastroenterology clinic. In recent years, an increased incidence of this disease has been reported from both specialty clinics. Although the genetic nature of this disease is not known, recent data from the adult medical literature showed a tendency towards familial clustering. There are no data reported from the pediatric population. Aim: To investigate familial clustering of habitual constipation in the pediatric patients. Methods: Pediatric patients, who attended the pediatric gastroenterology and general pediatric clinics at Marshall University School of Medicine were prospectively recruited to the study. Children with the diagnosis of functional, non-retentive constipation constituted our study population (test group), and children with no constipation were the control group. Constipation symptoms were analyzed in the family members of each participant, utilizing a standard questionnaire (Rome criteria). Exclusion criteria included children younger than 6 months, children from broken families, i.e.: single parent, multiple parents, or divorced families, and/or children with various neuromuscular diseases. Results: A total of 78 families were recruited, of whom 27 had constipated probands (test group) and 51 families had no constipation (control group). A total of 216 family members completed the questionnaires. Significantly more family members from the test group had constipation compared to the control group (p<0.0001) ( ) Conclusion: Habitual constipation in children seemed to cluster in families. The patho-physiology behind this phenomenon is yet to be discovered. Constipation and familial clustering * Student t-test or Chi-Square analyses W1341 Background: Chronic diabetes causes complications such as altered colonic motility which leads to significant morbidity. We have previously demonstrated that hyperglycemia-induced apoptosis is associated with increased oxidative stress in enteric neurons. Aim: We examined the role of oxidative stress and TLR4 signaling in hyperglycemia-induced apoptosis of enteric neurons. Methods:In Vitro experiments were performed in the recently characterized enteric neuronal cell line IM-PEN. Glucose-induced (5-40mM, 24h) TLR4 expression was assessed using immunocytochemistry. Enteric neurons were cultured in the presence or absence of LPS (0.1-10µg/ml) and assessed for apoptosis using the TUNEL method. LPS-induced chemokine (KC) release was measured using ELISA. Cells were treated with vehicle or H 2 O 2 (10µM) in the presence of TLR4siRNA or control siRNA and assessed for apoptosis by cleaved caspase-3 expression. The effect of LPS on colonic motility In Vivo was assessed in the WT and TLR4 mutant mice using the bead expulsion test. Proximal colon relaxation was assessed by isometric muscle recording in conjunction with electrical field stimulation. Results: IM-PEN cells exposed to 20-40mM glucose for 24 h have a significantly higher level of apoptosis (cleaved caspase3 expression) compared to 5mM glucose (p <0.05). Exposure of enteric neurons to 20 mM glucose resulted in increased TLR4 expression. Activation of TLR4 with LPS, induced apoptosis in enteric neurons in a dose depended fashion (% TUNEL+/PGP9.5+: Vehicle:2.2+0.4; LPS(0.1):3.4+0.6; LPS(1):7.01+0.7; LPS(10):8.38+0.5). 24 h exposure to LPS increased the release of KC from enteric neurons showing the presence of a functional TLR4 receptor in these neurons (KC(pg/ml): Vehicle: 639+12; LPS (0.1):1862+62; LPS(1) 2825+339,p<0.0001). H 2 O 2 (10µM) resulted in a 3 fold increase in cleaved caspase 3 expression compared to vehicle and this was reduced to 0.65 fold in the presence of TLR4 siRNA but not control siRNA. In Vivo analysis of motility in WT mice treated with LPS showed a delay in bead expulsion after injection of LPS (p<0.05).This effect was not seen in LPS treated TLR4 KO mice.Isometric muscle recording showed a reduction in relaxation in LPS treated WT mice (p<0.05). Conclusions:We have demonstrated a role for TLR4 signaling in hyperglycemia and oxidative stress induced apoptosis in enteric neurons. Knock down of TLR4 ameliorates oxidative stress induced apoptosis in enteric neurons. Activation of TLR4 with LPS induces changes in colonic motility In Vivo. Alteration in TLR4 signaling in enteric neurons can contribute to the motility changes seen in diabetes and is a potential therapeutic target.

Journal of Physiology-london, 2008

Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despi... more Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despite this, transit through the shorter colon is at least 10 times slower. Recently we demonstrated that elongating a segment of colon releases nitric oxide (NO) to inhibit peristalsis. The aims of this study were to determine if colonic elongation was physiologically significant, and whether elongation activated polarized intrinsic neural reflexes. Video imaging monitored fecal pellet evacuation from isolated guinea-pig colons full of pellets. Recordings were made from the circular muscle (CM) and longitudinal muscle (LM) in flat sheet preparations using either intracellular microelectrode or Ca 2+ imaging techniques. Full colons were 158.1 ± 6.1% longer than empty colons. As each pellet was expelled, the colon shortened and pellet velocity increased exponentially (full 0.34, empty 1.01 mm s −1 ). In flat sheet preparations, maintained circumferential stretch generated ongoing peristaltic activity (oral excitatory and anal inhibitory junction potentials) and Ca 2+ waves in LM and CM. Colonic elongation (140% of its empty slack length) applied oral to the recording site abolished these activities, whereas anal elongation significantly increased the frequency and amplitude of ongoing peristaltic activity. Oral elongation inhibited the excitation produced by anal elongation; this inhibitory effect was reversed by blocking NO synthesis. Pelvic nerve stimulation elicited polarized responses that were also suppressed by NO released during colonic elongation. In conclusion, longitudinal stretch excites specific mechosensitive ascending and descending interneurons, leading to activation of polarized reflexes. The dominance of the descending inhibitory reflex leads to slowed emptying of pellets in a naturally elongated colon.

Journal of Physiology-london, 2010

The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pa... more The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pattern. Although the CMMC spontaneously propagates along an empty colon it is responsible for faecal pellet propulsion in the murine large bowel. Unlike the peristaltic reflex, the CMMC is an &amp;amp;amp;amp;amp;amp;#39;all or none&amp;amp;amp;amp;amp;amp;#39; event that appears to be dependent upon Dogiel Type II/AH neurons for its regenerative slow propagation down the colon. A reduction in the amplitude of CMMCs or an elongated colon have both been thought to underlie slow transit constipation, although whether these phenomena are related has not been considered. In this study we examined the mechanisms by which colonic elongation might affect the CMMC using video imaging of the colon, tension and electrophysiological recordings from the muscle and Ca(2+) imaging of myenteric neurons. As faecal pellets were expelled from the murine colon, it shortened by up to 29%. Elongation of the colon resulted in a linear reduction in the velocity of a faecal pellet and the amplitude of spontaneous CMMCs. Elongation of the oral end of a colonic segment reduced the amplitude of CMMCs, whereas elongation of the anal end of the colon evoked a premature CMMC, and caused the majority of CMMCs to propagate in an anal to oral direction. Dogiel Type II/AH sensory neurons and most other myenteric neurons responded to oral elongation with reduced amplitude and frequency of spontaneous Ca(2+) transients, whereas anal elongation increased their amplitude and frequency in most neurons. The inhibitory effects of colonic elongation were reduced by blocking nitric oxide (NO) production with l-NA (100 mum) and soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 mum); whereas, l-arginine (1-2 mm) enhanced the inhibitory effects of colonic elongation. In conclusion, polarized neural reflexes can be triggered by longitudinal stretch. The dominant effect of elongation is to reduce CMMCs primarily by inhibiting Dogiel Type II/AH neurons, thus facilitating colonic accommodation and slow transit.

American Journal of Physiology-gastrointestinal and Liver Physiology, 2010

Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased state... more Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased states, including slow-transit constipation. However, the mechanisms underlying the CMMCs are controversial because it has been proposed that disinhibition (turning off of inhibitory neurotransmission) or excitatory nerve activity generate the CMMC. Therefore, our aims were to reexamine the mechanisms underlying the CMMC in the colon of wild-type and neuronal nitric oxide synthase (nNOS)(-/-) mice. CMMCs were recorded from the isolated murine large bowel using intracellular recordings of electrical activity from circular muscle (CM) combined with tension recording. Spontaneous CMMCs occurred in both wild-type (frequency: 0.3 cycles/min) and nNOS(-/-) mice (frequency: 0.4 cycles/min). CMMCs consisted of a hyperpolarization, followed by fast oscillations (slow waves) with action potentials superimposed on a slow depolarization (wild-type: 14.0 +/- 0.6 mV; nNOS(-/-): 11.2 +/- 1.5 mV). Both atropine (1 microM) and MEN 10,376 [neurokinin 2 (NK2) antagonist; 0.5 microM] added successively reduced the slow depolarization and the number of action potentials but did not abolish the fast oscillations. The further addition of RP 67580 (NK1 antagonist; 0.5 microM) blocked the fast oscillations and the CMMC. Importantly, none of the antagonists affected the resting membrane potential, suggesting that ongoing tonic inhibition of the CM was maintained. Fecal pellet propulsion, which was blocked by the NK2 or the NK1 antagonist, was slower down the longer, more constricted nNOS(-/-) mouse colon (wild-type: 47.9 +/- 2.4 mm; nNOS(-/-): 57.8 +/- 1.4 mm). These observations suggest that excitatory neurotransmission enhances pacemaker activity during the CMMC. Therefore, the CMMC is likely generated by a synergistic interaction between neural and interstitial cells of Cajal networks.

Gastroenterology, 2009

The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of feca... more The colonic migrating motor complex (CMMC) is a motor pattern that regulates the movement of fecal matter through a rhythmic sequence of electrical activity and/or contractions along the large bowel. CMMCs have largely been studied in empty preparations; we investigated whether local reflexes generated by a fecal pellet modify the CMMC to initiate propulsive activity. Methods: Recordings of CMMCs were made from the isolated murine large bowel, with or without a fecal pellet. Transducers were placed along the colon to record muscle tension and propulsive force on the pellet and microelectrodes were used to record electrical activity from either side of a fecal pellet, circular muscle cells oral and anal of a pellet, and in colons without the mucosa. Results: Spontaneous CMMCs propagated in both an oral or anal direction. When a pellet was inserted, CMMCs increased in frequency and propagated anally, exerting propulsive force on the pellet. The amplitude of slow waves increased during the CMMC. Localized mucosal stimulation/circumferential stretch evoked a CMMC, regardless of stimulus strength. The serotonin (5-hydroxytryptamine-3) receptor antagonist ondansetron reduced the amplitude of the CMMC, the propulsive force on the pellet, and the response to mucosal stroking, but increased the apparent conduction velocity of the CMMC. Removing the mucosa abolished spontaneous CMMCs, which still could be evoked by electrical stimulation. Conclusions: The fecal pellet activates local mucosal reflexes, which release serotonin (5-hydroxytryptamine) from enterochromaffin cells, and stretch reflexes that determine the site of origin and propagation of the CMMC, facilitating propulsion.

American Journal of Physiology-gastrointestinal and Liver Physiology, 2010

The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine... more The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine colon. We have previously shown that 5-hydroxytryptamine (5-HT) released from enterochromaffin cells activates 5-HT(3) receptors on the mucosal processes of myenteric Dogiel type II neurons to initiate the events underlying the CMMC. Our aims were to further investigate the roles of 5-HT(1A), 5-HT(3), and 5-HT(7) receptor subtypes in generating and propagating the CMMC using intracellular microelectrodes or tension recordings from the circular muscle (CM) in preparations with and without the mucosa. Spontaneous CMMCs were recorded from the CM in isolated murine colons but not in preparations without the mucosa. In mucosaless preparations, ondansetron (3 microM; 5-HT(3) antagonist) plus hexamethonium (100 microM) completely blocked spontaneous inhibitory junction potentials, depolarized the CM. Ondansetron blocked the preceding hyperpolarization associated with a CMMC. Spontaneous CMMCs and CMMCs evoked by spritzing 5-HT (10 and 100 microM) or nerve stimulation in preparations without the mucosa were blocked by SB 258719 or SB 269970 (1-5 microM; 5-HT(7) antagonists). Both NAN-190 and (S)-WAY100135 (1-5 microM; 5-HT(1A) antagonists) blocked spontaneous CMMCs and neurally evoked CMMCs in preparations without the mucosa. Both NAN-190 and (S)-WAY100135 caused an atropine-sensitive depolarization of the CM. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP) (10 microM), and 5-carboxamidotryptamine (5-CT) (5 microM; 5-HT(1/5/7) agonist) increased the frequency of spontaneous CMMCs. 5-HTP and 5-CT also induced CMMCs in preparations with and without the mucosa, which were blocked by SB 258719. 5-HT(1A), 5-HT(3), and 5-HT(7) receptors, most likely on Dogiel Type II/AH neurons, are important in initiating, generating, and propagating the CMMC. Tonic inhibition of the CM appears to be driven by ongoing activity in descending serotonergic interneurons; by activating 5-HT(7) receptors on AH neurons these interneurons also contribute to the generation of the CMMC.

Gastroenterology, 2010

1. Heredia DJ, Dickson EJ, Bayguinov PO, et al. Localized release of serotonin (5-hydroxytryptami... more 1. Heredia DJ, Dickson EJ, Bayguinov PO, et al. Localized release of serotonin (5-hydroxytryptamine) by a fecal pellet regulates migrating motor complexes in murine colon. Gastroenterology 2009; 136:1328 -1338. 2. Keating DJ, Spencer NJ. Release of 5-hydroxytryptamine from the mucosa is not required for the generation or propagation of colonic migrating motor complexes. Gastroenterology 2010;138:659 -670. GASTROENTEROLOGY 2010;138:1213-1216 3. Dickson EJ, Heredia DJ, Hennig GW, et al. The importance of 5-HT 1A and 5-HT 7 receptors during murine colonic migrating motor complex. Neurogastroenterol Motil 2009;21(Suppl 1):48. 4. Meedeniya ACB, Brookes SJH, Costa M. Sources of 5-hydroxytryptamine immunoreactivity in the myenteric plexus of the guinea-pig small intestine. Proc Aust Neurosci Soc 1994;5:176.

Gastroenterology, 2007

Transit of fecal material through the human colon takes >30 hours, whereas transit through the sm... more Transit of fecal material through the human colon takes >30 hours, whereas transit through the small intestine takes 24 hours. The mechanisms underlying colonic storage and slow transit have yet to be elucidated. Our aim was to determine whether an intrinsic neural mechanism underlies these phenomena. Methods: Recordings were made from circular muscle (CM) cells and myenteric neurons in the isolated guinea pig distal colon using intracellular recordings and Ca 2؉ imaging techniques. Video imaging was used to determine the effects of colonic filling and pellet transit. Results: Circumferential stretch generated ongoing oral excitatory and anal inhibitory junction potentials in the CM. The application of longitudinal stretch inhibited all junction potentials. N-nitro-L-arginine (100 mol/L) completely reversed the inhibitory effects of longitudinal stretch suggesting that nitric oxide (NO) inhibited interneurons controlling peristaltic circuits. Ca 2؉ imaging in preparations that were stretched in both axes revealed ongoing firing in nNOS ؉ve descending neurons, even when synaptic transmission was blocked. Inhibitory postsynaptic potentials were evoked in mechanosensitive interneurons that were blocked by N--nitro-L-arginine (100 mol/L). Pellet transit was inhibited by longitudinal stretch. Filling the colon with fluid led to colonic elongation and an inhibition of motility. Conclusions: Our data support the novel hypothesis that slow transit and accommodation are generated by release of NO from descending (nNOS ؉ve) interneurons triggered by colonic elongation. We refer to this powerful inhibitory reflex as the intrinsic occult reflex (hidden from observation) because it withdraws motor activity from the muscle.

Gastroenterology, 2008

Background: Habitual, non-retentive constipation in children is one of the most common diagnoses ... more Background: Habitual, non-retentive constipation in children is one of the most common diagnoses in the general pediatric clinic and the pediatric gastroenterology clinic. In recent years, an increased incidence of this disease has been reported from both specialty clinics. Although the genetic nature of this disease is not known, recent data from the adult medical literature showed a tendency towards familial clustering. There are no data reported from the pediatric population. Aim: To investigate familial clustering of habitual constipation in the pediatric patients. Methods: Pediatric patients, who attended the pediatric gastroenterology and general pediatric clinics at Marshall University School of Medicine were prospectively recruited to the study. Children with the diagnosis of functional, non-retentive constipation constituted our study population (test group), and children with no constipation were the control group. Constipation symptoms were analyzed in the family members of each participant, utilizing a standard questionnaire (Rome criteria). Exclusion criteria included children younger than 6 months, children from broken families, i.e.: single parent, multiple parents, or divorced families, and/or children with various neuromuscular diseases. Results: A total of 78 families were recruited, of whom 27 had constipated probands (test group) and 51 families had no constipation (control group). A total of 216 family members completed the questionnaires. Significantly more family members from the test group had constipation compared to the control group (p<0.0001) ( ) Conclusion: Habitual constipation in children seemed to cluster in families. The patho-physiology behind this phenomenon is yet to be discovered. Constipation and familial clustering * Student t-test or Chi-Square analyses W1341 Background: Chronic diabetes causes complications such as altered colonic motility which leads to significant morbidity. We have previously demonstrated that hyperglycemia-induced apoptosis is associated with increased oxidative stress in enteric neurons. Aim: We examined the role of oxidative stress and TLR4 signaling in hyperglycemia-induced apoptosis of enteric neurons. Methods:In Vitro experiments were performed in the recently characterized enteric neuronal cell line IM-PEN. Glucose-induced (5-40mM, 24h) TLR4 expression was assessed using immunocytochemistry. Enteric neurons were cultured in the presence or absence of LPS (0.1-10µg/ml) and assessed for apoptosis using the TUNEL method. LPS-induced chemokine (KC) release was measured using ELISA. Cells were treated with vehicle or H 2 O 2 (10µM) in the presence of TLR4siRNA or control siRNA and assessed for apoptosis by cleaved caspase-3 expression. The effect of LPS on colonic motility In Vivo was assessed in the WT and TLR4 mutant mice using the bead expulsion test. Proximal colon relaxation was assessed by isometric muscle recording in conjunction with electrical field stimulation. Results: IM-PEN cells exposed to 20-40mM glucose for 24 h have a significantly higher level of apoptosis (cleaved caspase3 expression) compared to 5mM glucose (p <0.05). Exposure of enteric neurons to 20 mM glucose resulted in increased TLR4 expression. Activation of TLR4 with LPS, induced apoptosis in enteric neurons in a dose depended fashion (% TUNEL+/PGP9.5+: Vehicle:2.2+0.4; LPS(0.1):3.4+0.6; LPS(1):7.01+0.7; LPS(10):8.38+0.5). 24 h exposure to LPS increased the release of KC from enteric neurons showing the presence of a functional TLR4 receptor in these neurons (KC(pg/ml): Vehicle: 639+12; LPS (0.1):1862+62; LPS(1) 2825+339,p<0.0001). H 2 O 2 (10µM) resulted in a 3 fold increase in cleaved caspase 3 expression compared to vehicle and this was reduced to 0.65 fold in the presence of TLR4 siRNA but not control siRNA. In Vivo analysis of motility in WT mice treated with LPS showed a delay in bead expulsion after injection of LPS (p<0.05).This effect was not seen in LPS treated TLR4 KO mice.Isometric muscle recording showed a reduction in relaxation in LPS treated WT mice (p<0.05). Conclusions:We have demonstrated a role for TLR4 signaling in hyperglycemia and oxidative stress induced apoptosis in enteric neurons. Knock down of TLR4 ameliorates oxidative stress induced apoptosis in enteric neurons. Activation of TLR4 with LPS induces changes in colonic motility In Vivo. Alteration in TLR4 signaling in enteric neurons can contribute to the motility changes seen in diabetes and is a potential therapeutic target.

Journal of Physiology-london, 2008

Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despi... more Propulsion in both small and large intestine is largely mediated by the peristaltic reflex; despite this, transit through the shorter colon is at least 10 times slower. Recently we demonstrated that elongating a segment of colon releases nitric oxide (NO) to inhibit peristalsis. The aims of this study were to determine if colonic elongation was physiologically significant, and whether elongation activated polarized intrinsic neural reflexes. Video imaging monitored fecal pellet evacuation from isolated guinea-pig colons full of pellets. Recordings were made from the circular muscle (CM) and longitudinal muscle (LM) in flat sheet preparations using either intracellular microelectrode or Ca 2+ imaging techniques. Full colons were 158.1 ± 6.1% longer than empty colons. As each pellet was expelled, the colon shortened and pellet velocity increased exponentially (full 0.34, empty 1.01 mm s −1 ). In flat sheet preparations, maintained circumferential stretch generated ongoing peristaltic activity (oral excitatory and anal inhibitory junction potentials) and Ca 2+ waves in LM and CM. Colonic elongation (140% of its empty slack length) applied oral to the recording site abolished these activities, whereas anal elongation significantly increased the frequency and amplitude of ongoing peristaltic activity. Oral elongation inhibited the excitation produced by anal elongation; this inhibitory effect was reversed by blocking NO synthesis. Pelvic nerve stimulation elicited polarized responses that were also suppressed by NO released during colonic elongation. In conclusion, longitudinal stretch excites specific mechosensitive ascending and descending interneurons, leading to activation of polarized reflexes. The dominance of the descending inhibitory reflex leads to slowed emptying of pellets in a naturally elongated colon.

Journal of Physiology-london, 2010

The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pa... more The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pattern. Although the CMMC spontaneously propagates along an empty colon it is responsible for faecal pellet propulsion in the murine large bowel. Unlike the peristaltic reflex, the CMMC is an &amp;amp;amp;amp;amp;amp;#39;all or none&amp;amp;amp;amp;amp;amp;#39; event that appears to be dependent upon Dogiel Type II/AH neurons for its regenerative slow propagation down the colon. A reduction in the amplitude of CMMCs or an elongated colon have both been thought to underlie slow transit constipation, although whether these phenomena are related has not been considered. In this study we examined the mechanisms by which colonic elongation might affect the CMMC using video imaging of the colon, tension and electrophysiological recordings from the muscle and Ca(2+) imaging of myenteric neurons. As faecal pellets were expelled from the murine colon, it shortened by up to 29%. Elongation of the colon resulted in a linear reduction in the velocity of a faecal pellet and the amplitude of spontaneous CMMCs. Elongation of the oral end of a colonic segment reduced the amplitude of CMMCs, whereas elongation of the anal end of the colon evoked a premature CMMC, and caused the majority of CMMCs to propagate in an anal to oral direction. Dogiel Type II/AH sensory neurons and most other myenteric neurons responded to oral elongation with reduced amplitude and frequency of spontaneous Ca(2+) transients, whereas anal elongation increased their amplitude and frequency in most neurons. The inhibitory effects of colonic elongation were reduced by blocking nitric oxide (NO) production with l-NA (100 mum) and soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 mum); whereas, l-arginine (1-2 mm) enhanced the inhibitory effects of colonic elongation. In conclusion, polarized neural reflexes can be triggered by longitudinal stretch. The dominant effect of elongation is to reduce CMMCs primarily by inhibiting Dogiel Type II/AH neurons, thus facilitating colonic accommodation and slow transit.

dialécticamente pasa por distintas etapas, al inicio, al recibir la noticia criminal se encuentra... more dialécticamente pasa por distintas etapas, al inicio, al recibir la noticia criminal se encuentra en un estado de IMPROBABILIDAD, para salir de ésta incertidumbre ordena una INVESTIGACIÓN PRELIMINAR disponiendo diligencias urgentes o diligencias preliminares y luego de un plazo correspondiente (por ejemplo hay un plazo legal para investigar de 20 días, salvo que se produzca la detención en cuyo caso solo tendrá 24 horas y si se trata de un caso de drogas, espionaje, o terrorismo su investigación no puede pasar de los 15 días; pero podría fijar un plazo distinto atendiendo a la complejidad del caso y las circunstancias del hecho, pudiendo fijar un plazo fiscal no mayor a los 120 días por contraste con la norma contenida en el artículo 342 del Código Procesal Penal), cumpliendo actos de investigación, puede llegar a una imputación penal, esto es, subsume los hechos con una norma determinada del ordenamiento penal sustantivo y por tanto ya se ubica en lo que es PROBABLE, y que da origen a una "causa probable" que se expresa en su Disposición de Formalización de la Investigación Preparatoria, acá el Fiscal ya tiene a un presunto autor identificado, un delito y un bien jurídico lesionado, una víctima afectada, tiene conciencia que el hecho imputado no ha prescrito, que no hay requisitos de procedibilidad o procesabilidad y por tanto ese mismo Fiscal está convencido que el inculpado ha cometido un delito determinado y merece una condena, su razonamiento llegó a una CERTEZA POSITIVA que se expresa en la formulación de un requerimiento de acusación ( y cuando su razonamiento llega a una CERTEZA NEGATIVA se expresa en la formulación de un requerimiento de Sobreseimiento; finalmente también puede ser MIXTA es decir existir Certeza Negativa y Certeza Positiva a la vez, eso se da cuando emite un requerimiento que contiene una Acusación MIXTA ). De modo tal que el la ETAPA INTERMEDIA, no solo está destinada al control de la ACUSACION, sino también eventualmente al control del SOBRESEIMIENTO que pueda pedir el mismo Fiscal.

dialécticamente pasa por distintas etapas, al inicio, al recibir la noticia criminal se encuentra... more dialécticamente pasa por distintas etapas, al inicio, al recibir la noticia criminal se encuentra en un estado de IMPROBABILIDAD, para salir de ésta incertidumbre ordena una INVESTIGACIÓN PRELIMINAR disponiendo diligencias urgentes o diligencias preliminares y luego de un plazo correspondiente (por ejemplo hay un plazo legal para investigar de 20 días, salvo que se produzca la detención en cuyo caso solo tendrá 24 horas y si se trata de un caso de drogas, espionaje, o terrorismo su investigación no puede pasar de los 15 días; pero podría fijar un plazo distinto atendiendo a la complejidad del caso y las circunstancias del hecho, pudiendo fijar un plazo fiscal no mayor a los 120 días por contraste con la norma contenida en el artículo 342 del Código Procesal Penal), cumpliendo actos de investigación, puede llegar a una imputación penal, esto es, subsume los hechos con una norma determinada del ordenamiento penal sustantivo y por tanto ya se ubica en lo que es PROBABLE, y que da origen a una "causa probable" que se expresa en su Disposición de Formalización de la Investigación Preparatoria, acá el Fiscal ya tiene a un presunto autor identificado, un delito y un bien jurídico lesionado, una víctima afectada, tiene conciencia que el hecho imputado no ha prescrito, que no hay requisitos de procedibilidad o procesabilidad y por tanto ese mismo Fiscal está convencido que el inculpado ha cometido un delito determinado y merece una condena, su razonamiento llegó a una CERTEZA POSITIVA que se expresa en la formulación de un requerimiento de acusación ( y cuando su razonamiento llega a una CERTEZA NEGATIVA se expresa en la formulación de un requerimiento de Sobreseimiento; finalmente también puede ser MIXTA es decir existir Certeza Negativa y Certeza Positiva a la vez, eso se da cuando emite un requerimiento que contiene una Acusación MIXTA ). De modo tal que el la ETAPA INTERMEDIA, no solo está destinada al control de la ACUSACION, sino también eventualmente al control del SOBRESEIMIENTO que pueda pedir el mismo Fiscal.