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Polarized neuroimmune signaling in the submucosal plexus of the guinea pig colon
A668 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
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3665
POLARIZED NEUROIMMUNE SIGNALING IN THE SUBMUCO· SAL PLEXUS OF THE GUINEA PIG COLON. Eckhard Weber, Michael Schemann, Thomas Frieling, Heinrich-HeineUniversity of Duesseldorf, Duesseldorf, Germany; Sch of Veterinary Medicine, Hannover, Germany.
MOTOR ACTIVITY OF THE GUINEA·PIG ILEUM INDUCED BY MAINTAINED DISTENSION AND CYCLOOXYGENASE INHIBI· TION. Diego Curro', Marcello Costa, Simon JH Brookes, The Flinders Univ of South Australia, Adelaide, SA, Australia; Flinders Univ, Adelaide, SA, Australia.
We have previously shown that the guinea pig colonic mucosa is innervated by descending VIPergic and ascending cholinergic neurons (J Physiol 1998). This study was aimed to investigate the functional correlate of these polarized projections. Method. Muscle-stripped segments of the guinea pig proximal colon were mounted in modified Ussing chambers that allowed measurement of short-circuit current (Isc) after electrical field stimulation (8V, 10Hz, O.5ms) in defined 2mm-wide windows up to 20mm orally and 20mm aborally from a separately perfused recording chamber. The recording chamber was perfused with Krebs solution and the stimulation chamber with low Ca z+ 1 high Mg z+ Krebs solution to block synaptic transmission. Results. (1) Amplitudes of electrically evoked Isc decreased with distance from the recording chamber. No responses to electrical stimulation were observed at stimulation distances greater than IOrnm orally and 6mm aborally. (2) Increase in Isc was significantly higher during oral compared to aboral stimulation. (3) With the VIP antagonist VIP 6-28 (lp,M) present in the recording chamber only the orally evoked Isc was significantly reduced whereas atropine (IJ.loM) significantly reduced aborally and orally evoked Isc. Atropine and VIP 6-28 together abolished the electrically evoked Isc. (4) Hexamethonium (100p,M) in the recording chamber significantly diminished orally evoked Isc, indicative of descending cholinergic interneurons projecting into the recording chamber where they synapse onto secretomotor neurons. The projection length of these interneurons was 4-6mm. (5) Prostaglandins (PGD z, PGE z; IOp,M) significantly increased aborally evoked Isc only when applied to the recording chamber. In addition, PG increased the sensitivity to distant aboral stimulations since increases in Isc could now be recorded from as far as 16mm. 5-HT,p or 5-HT 3 antagonists failed to block the PG effects. In contrast, application of capsaicin (10J.loM) or hexamethonium (100p,M) to the recording chamber blocked the increase of electrically evoked Isc by PG; application to the stimulation chamber was without effect. This indicated that PG activate or sensitize pathways consisting of long-projecting ascending capsaicin-sensitive primary afferents which transmit to enteric secretomotor pathways. Conclusion. The study provides functional evidence for polarized neuroimmune signaling to enteric mucosa and for interactions and plasticity between extrinsic sensory and intrinsic secretomotor pathways. (DFG Fr 733/13-1)
Fluid infusion into small intestine elicits peristalsis at a threshold volume and, if the distension is maintained, peristalsis becomes intermittent (1). Cyclooxygenase (COX) inhibitors increase the spontaneous activity of the intestinal circular smooth muscle (2). We investigated the effect of various degrees of infused volume, with or without piroxicam, a COX inhibitor, on intestinal mechanical activity. Segments of small intestine, taken from guinea-pigs killed by cervical dislocation, were recorded on video to perform spatio-temporal analysis of intestinal motor activity (3). The threshold volume of fluid to trigger peristalsis at 4-6 min intervals was established in controls by slow intraluminal infusion and thereafter segments emptied against gravity. When 30 % of the threshold volume was infused, there were no propulsive motor events. Piroxicam (50 p,M) induced episodes of irregular activity alternating with quiescent periods. Contractions propagated aborally (64.7±3.4 %) or orally (24.6±3.4 %) or did not propagate (lO.7±2.2%, n=4). Tetrodotoxin (I p,M) did not affect this pattern. Contractions propagating aborally went further (l9.0±3.5 mm) than non-propagating (10.5±3.1 mm) or orally-propagating contractions (8.5± 1.9 mm). Orally-propagating contractions were faster (l5.1±0.8 mrnls) than aboral ones (10.5±1.0 mrnls). TTX (1 p,M) abolished both of these differences, increasing both the length of the nonpropagating and orally-propagating contractions and the speed of aborallypropagating contractions. Segments infused with 90% (n=4) or 120 % (n=4) of threshold volume showed intermittent clusters of peristaltic contractions (2.7±0.6 and 5.3±O.7 peristaltic contractions per cluster, respectively, P<0.05) at intervals of 9.3±3.4 and 8.1 ±4.1 min. In segments infused with 120% of threshold volume, the threshold diameter and the speed of propagation of peristaltic contractions were both reduced, compared to controls. Thus, the threshold for the initiation of peristalsis undergoes cyclic variation and the mechanism underlying these cyclic changes may involve neuromodulatory processes. Inhibition of prostaglandin synthesis reveals myogenic activity that generates polarised, propagating contractions. Neural activity contributes to the length and speed of propagation of these contractions. 1) Henderson, V. E. (1923) Am. J. Physiol. 66: 380-390, 2) Bennett, A. et al. (1975) Br. J. Pharmacol. 54:197-204.3) Hennig, G.W. et al. (1999) 1. Physiol. 517: 575-590.
3664
3666
ELECTRICAL REFLEX RESPONSES OF MYENTERIC NEU· RONS, CIRCULAR AND LONGITUDINAL MUSCLE TO MUCO· SAL STIMULATION IN ISOLATED GUINEA·PIG DISTAL CO· LON. Nick J. Spencer, Terence K. Smith, Univ of Nevada Sch of Medicine, Reno, NY.
ORGANIZATION OF MYENTERIC NEURAL PATHWAYS IN· NERVATING SUBMUCOSAL VASODILATOR NEURONS IN GUINEA PIG ILEUM. Stephen 1. Vanner, Acad Univ, Kingston, ON, Canada.
Although the reflex responses of the guinea-pig small intestine have been thoroughly investigated, there have been relatively few studies of the intracellular electrical reflex responses of myenteric neurons, circular (CM) and longitudinal muscle (LM) of the colon. In this study, we used intracellular recording techniques to record synaptic responses of myenteric neurons and junction potentials in the LM and CM of the guinea-pig distal colon, following mucosal stimulation. To do this, we dissected off the CM layer to form a window, where by we could visualize the myenteric plexus, LM and CM, but preserve the mucosa over regions immediately oral, anal and circumferential to the dissected window. All electrical recordings were made in the presence of nicardipine (5p,M). Under depolarising current clamp (50-600pA), neurons fired bursts of action potentials (tonic firing) (14 out of 26 cells), or fired only a few action potentials (1-3 spikes; phasic cell) throughout prolonged current pulses (12 out of 26 cells). In phasic firing neurons, local brush stroking the mucosa with an fine artists brush, elicited fast excitatory post synaptic potentials (fEPSPs) and occassional action potentials from oral (6 out of 11 cells), anal (7 out of 11 cells) and circumferential pathways (2 out of 3 cells). In tonic firing neurons, fEPSPs and/or action potentials were elicited in 3 out of 11 cells from oral stimuli, lout of 9 cells from anal stimuli; and 0 out of 2 cells tested from circumferential stimuli. Essentially all phasic and tonic firing neurons received ongoing fEPSPs and in 3 out of 26 impalements, neurons discharged rhythmical bursts of action potentials. When intracellular recordings were made from LM cells, brush stroking the mucosa from oral extremities elicited compound inhibitory junction potentials (UPs) (22 cells; n=4 animals). Similarly, in the CM, oral stimuli elicited compound UPs consisting of nitrergic and aparnin-sensitive components. In the LM and CM, anal stimuli elicited excitatory junction potentials (EJPs). In conclusion, mucosal stimulation is a powerful stimulus to excite myenteric neurons in the colon, from both oral, anal and circumferential pathways. In addition, oral and anal mucosal stimuli evoke similar polarized reflex pathways in both the LM and CM, that occur with a similar latency. These pathways appear to be activated primarily by fEPSPs. (Support: NIH ROI DK457 13).
Our previous study demonstrated that 5-HT activated myenteric neural pathways innervating submucosal vasodilator neurons (Gastro. A:3501,1998) but responses evoked by this stimulus were too inconsistent to establish the organization of these pathways. The aim of this study was to examine whether electrically stimulated myenteric neurons reproducibily activate submucosal cholinergic vasodilator neurons and if so, what the organization of these reflex pathways was. An in vitro preparation of combined submucosal-myenteric plexus from guinea pig ileum was employed. Exposed myenteric ganglia on one end of the preparation were electrically stimulated with a bipolar tungsten electrode and changes in the diameter of submucosal arterioles (n=67) were monitored in the opposite end of the preparation by videomicroscopy. Arterioles were preconstricted with PGF2a to 80-95 % maximum to enable dilator responses to be examined. Stimulation of myenteric ganglia evoked TTX-sensitive frequency (2-30 Hz) and train duration (1-9s) dependent dilations. Stimulation at 20Hz for 6 s produced large, reproducible dilations in a single preparation and these parameters were used in subsequent studies. Stimulation up to 18 mm from the recording site evoked large dilations in both the orad and abord directions. In double chamber baths, which chemically isolated the stimulating myenteric from the recording submucosal chamber, the nicotinic antagonist hexamethonium or the muscarinic antagonist, 4-DAMP almost completely blocked dilations when superfused in the submucosal chamber. (Submucosal vasodilator neurons have nicotinic receptors on their cell bodies and dilate arterioles by activating muscarinic receptors on the vessels {Neild et al. J. PhysioI.420:247,1990}). When hexamethonium was placed in the myenteric chamber ~50% of nerveevoked dilator responses were hexamethonium-sensitive, in both the oral (56%) and aboral (58%) orientations. The combination of 4-DAMP and hexamethonium or the substitution of 0 Ca2 +, 12 mM Mg2 +, which blocks synaptic transmission, did not result in greater inhibition than that seen with hexamethonium alone. These results show that nicotinic polysynaptic and long monosynaptic pathways in the myenteric plexus, projecting orad and aborad, can activate cholinergic submucosal vasodilator neurons. These pathways could co-ordinate intestinal blood flow with local motor responses in the intestine.
GASTROENTEROLOGY Vol. 118, No.4
3663
3665
POLARIZED NEUROIMMUNE SIGNALING IN THE SUBMUCO· SAL PLEXUS OF THE GUINEA PIG COLON. Eckhard Weber, Michael Schemann, Thomas Frieling, Heinrich-HeineUniversity of Duesseldorf, Duesseldorf, Germany; Sch of Veterinary Medicine, Hannover, Germany.
MOTOR ACTIVITY OF THE GUINEA·PIG ILEUM INDUCED BY MAINTAINED DISTENSION AND CYCLOOXYGENASE INHIBI· TION. Diego Curro', Marcello Costa, Simon JH Brookes, The Flinders Univ of South Australia, Adelaide, SA, Australia; Flinders Univ, Adelaide, SA, Australia.
We have previously shown that the guinea pig colonic mucosa is innervated by descending VIPergic and ascending cholinergic neurons (J Physiol 1998). This study was aimed to investigate the functional correlate of these polarized projections. Method. Muscle-stripped segments of the guinea pig proximal colon were mounted in modified Ussing chambers that allowed measurement of short-circuit current (Isc) after electrical field stimulation (8V, 10Hz, O.5ms) in defined 2mm-wide windows up to 20mm orally and 20mm aborally from a separately perfused recording chamber. The recording chamber was perfused with Krebs solution and the stimulation chamber with low Ca z+ 1 high Mg z+ Krebs solution to block synaptic transmission. Results. (1) Amplitudes of electrically evoked Isc decreased with distance from the recording chamber. No responses to electrical stimulation were observed at stimulation distances greater than IOrnm orally and 6mm aborally. (2) Increase in Isc was significantly higher during oral compared to aboral stimulation. (3) With the VIP antagonist VIP 6-28 (lp,M) present in the recording chamber only the orally evoked Isc was significantly reduced whereas atropine (IJ.loM) significantly reduced aborally and orally evoked Isc. Atropine and VIP 6-28 together abolished the electrically evoked Isc. (4) Hexamethonium (100p,M) in the recording chamber significantly diminished orally evoked Isc, indicative of descending cholinergic interneurons projecting into the recording chamber where they synapse onto secretomotor neurons. The projection length of these interneurons was 4-6mm. (5) Prostaglandins (PGD z, PGE z; IOp,M) significantly increased aborally evoked Isc only when applied to the recording chamber. In addition, PG increased the sensitivity to distant aboral stimulations since increases in Isc could now be recorded from as far as 16mm. 5-HT,p or 5-HT 3 antagonists failed to block the PG effects. In contrast, application of capsaicin (10J.loM) or hexamethonium (100p,M) to the recording chamber blocked the increase of electrically evoked Isc by PG; application to the stimulation chamber was without effect. This indicated that PG activate or sensitize pathways consisting of long-projecting ascending capsaicin-sensitive primary afferents which transmit to enteric secretomotor pathways. Conclusion. The study provides functional evidence for polarized neuroimmune signaling to enteric mucosa and for interactions and plasticity between extrinsic sensory and intrinsic secretomotor pathways. (DFG Fr 733/13-1)
Fluid infusion into small intestine elicits peristalsis at a threshold volume and, if the distension is maintained, peristalsis becomes intermittent (1). Cyclooxygenase (COX) inhibitors increase the spontaneous activity of the intestinal circular smooth muscle (2). We investigated the effect of various degrees of infused volume, with or without piroxicam, a COX inhibitor, on intestinal mechanical activity. Segments of small intestine, taken from guinea-pigs killed by cervical dislocation, were recorded on video to perform spatio-temporal analysis of intestinal motor activity (3). The threshold volume of fluid to trigger peristalsis at 4-6 min intervals was established in controls by slow intraluminal infusion and thereafter segments emptied against gravity. When 30 % of the threshold volume was infused, there were no propulsive motor events. Piroxicam (50 p,M) induced episodes of irregular activity alternating with quiescent periods. Contractions propagated aborally (64.7±3.4 %) or orally (24.6±3.4 %) or did not propagate (lO.7±2.2%, n=4). Tetrodotoxin (I p,M) did not affect this pattern. Contractions propagating aborally went further (l9.0±3.5 mm) than non-propagating (10.5±3.1 mm) or orally-propagating contractions (8.5± 1.9 mm). Orally-propagating contractions were faster (l5.1±0.8 mrnls) than aboral ones (10.5±1.0 mrnls). TTX (1 p,M) abolished both of these differences, increasing both the length of the nonpropagating and orally-propagating contractions and the speed of aborallypropagating contractions. Segments infused with 90% (n=4) or 120 % (n=4) of threshold volume showed intermittent clusters of peristaltic contractions (2.7±0.6 and 5.3±O.7 peristaltic contractions per cluster, respectively, P<0.05) at intervals of 9.3±3.4 and 8.1 ±4.1 min. In segments infused with 120% of threshold volume, the threshold diameter and the speed of propagation of peristaltic contractions were both reduced, compared to controls. Thus, the threshold for the initiation of peristalsis undergoes cyclic variation and the mechanism underlying these cyclic changes may involve neuromodulatory processes. Inhibition of prostaglandin synthesis reveals myogenic activity that generates polarised, propagating contractions. Neural activity contributes to the length and speed of propagation of these contractions. 1) Henderson, V. E. (1923) Am. J. Physiol. 66: 380-390, 2) Bennett, A. et al. (1975) Br. J. Pharmacol. 54:197-204.3) Hennig, G.W. et al. (1999) 1. Physiol. 517: 575-590.
3664
3666
ELECTRICAL REFLEX RESPONSES OF MYENTERIC NEU· RONS, CIRCULAR AND LONGITUDINAL MUSCLE TO MUCO· SAL STIMULATION IN ISOLATED GUINEA·PIG DISTAL CO· LON. Nick J. Spencer, Terence K. Smith, Univ of Nevada Sch of Medicine, Reno, NY.
ORGANIZATION OF MYENTERIC NEURAL PATHWAYS IN· NERVATING SUBMUCOSAL VASODILATOR NEURONS IN GUINEA PIG ILEUM. Stephen 1. Vanner, Acad Univ, Kingston, ON, Canada.
Although the reflex responses of the guinea-pig small intestine have been thoroughly investigated, there have been relatively few studies of the intracellular electrical reflex responses of myenteric neurons, circular (CM) and longitudinal muscle (LM) of the colon. In this study, we used intracellular recording techniques to record synaptic responses of myenteric neurons and junction potentials in the LM and CM of the guinea-pig distal colon, following mucosal stimulation. To do this, we dissected off the CM layer to form a window, where by we could visualize the myenteric plexus, LM and CM, but preserve the mucosa over regions immediately oral, anal and circumferential to the dissected window. All electrical recordings were made in the presence of nicardipine (5p,M). Under depolarising current clamp (50-600pA), neurons fired bursts of action potentials (tonic firing) (14 out of 26 cells), or fired only a few action potentials (1-3 spikes; phasic cell) throughout prolonged current pulses (12 out of 26 cells). In phasic firing neurons, local brush stroking the mucosa with an fine artists brush, elicited fast excitatory post synaptic potentials (fEPSPs) and occassional action potentials from oral (6 out of 11 cells), anal (7 out of 11 cells) and circumferential pathways (2 out of 3 cells). In tonic firing neurons, fEPSPs and/or action potentials were elicited in 3 out of 11 cells from oral stimuli, lout of 9 cells from anal stimuli; and 0 out of 2 cells tested from circumferential stimuli. Essentially all phasic and tonic firing neurons received ongoing fEPSPs and in 3 out of 26 impalements, neurons discharged rhythmical bursts of action potentials. When intracellular recordings were made from LM cells, brush stroking the mucosa from oral extremities elicited compound inhibitory junction potentials (UPs) (22 cells; n=4 animals). Similarly, in the CM, oral stimuli elicited compound UPs consisting of nitrergic and aparnin-sensitive components. In the LM and CM, anal stimuli elicited excitatory junction potentials (EJPs). In conclusion, mucosal stimulation is a powerful stimulus to excite myenteric neurons in the colon, from both oral, anal and circumferential pathways. In addition, oral and anal mucosal stimuli evoke similar polarized reflex pathways in both the LM and CM, that occur with a similar latency. These pathways appear to be activated primarily by fEPSPs. (Support: NIH ROI DK457 13).
Our previous study demonstrated that 5-HT activated myenteric neural pathways innervating submucosal vasodilator neurons (Gastro. A:3501,1998) but responses evoked by this stimulus were too inconsistent to establish the organization of these pathways. The aim of this study was to examine whether electrically stimulated myenteric neurons reproducibily activate submucosal cholinergic vasodilator neurons and if so, what the organization of these reflex pathways was. An in vitro preparation of combined submucosal-myenteric plexus from guinea pig ileum was employed. Exposed myenteric ganglia on one end of the preparation were electrically stimulated with a bipolar tungsten electrode and changes in the diameter of submucosal arterioles (n=67) were monitored in the opposite end of the preparation by videomicroscopy. Arterioles were preconstricted with PGF2a to 80-95 % maximum to enable dilator responses to be examined. Stimulation of myenteric ganglia evoked TTX-sensitive frequency (2-30 Hz) and train duration (1-9s) dependent dilations. Stimulation at 20Hz for 6 s produced large, reproducible dilations in a single preparation and these parameters were used in subsequent studies. Stimulation up to 18 mm from the recording site evoked large dilations in both the orad and abord directions. In double chamber baths, which chemically isolated the stimulating myenteric from the recording submucosal chamber, the nicotinic antagonist hexamethonium or the muscarinic antagonist, 4-DAMP almost completely blocked dilations when superfused in the submucosal chamber. (Submucosal vasodilator neurons have nicotinic receptors on their cell bodies and dilate arterioles by activating muscarinic receptors on the vessels {Neild et al. J. PhysioI.420:247,1990}). When hexamethonium was placed in the myenteric chamber ~50% of nerveevoked dilator responses were hexamethonium-sensitive, in both the oral (56%) and aboral (58%) orientations. The combination of 4-DAMP and hexamethonium or the substitution of 0 Ca2 +, 12 mM Mg2 +, which blocks synaptic transmission, did not result in greater inhibition than that seen with hexamethonium alone. These results show that nicotinic polysynaptic and long monosynaptic pathways in the myenteric plexus, projecting orad and aborad, can activate cholinergic submucosal vasodilator neurons. These pathways could co-ordinate intestinal blood flow with local motor responses in the intestine.