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Localisation of intermediate conductance calcium-activated potassium channel (IK)-like immunoreactivity in rat and human neurones
which turns off 50% of the conductance at rest (-60 mV). Substituting CsF for CsCl in the imracellular solution shifted the voltage-dependant parameters of TTX-R INa leftward by -20 mV. Under these conditions, TTX-R INa had voltage-dependant properties similar to those reported previously for NaN/NaV1.9 in DRG neurons. Consistent with this, RT-PCR, single-cefl profiling and immunostaiinng experiments indicated that NaV1.9 transcripts/ subunits, but not NaV1.8, were expressed in the enteric nervous system and restricted to myemeric sensory neurons. A TTX-resistant current is co-expressed with the NaV1.9 subuint within myenteric sensory neurons. Due to its biophysical properties, TTX-R INa may play an important role in regulating subthreshold electrogenesis and favouring synaptic inputs to the soma, thereby confering to myenteric sensory neurons distinct integrative properties that could be involved in shaping enteric reflexes.
M1272
Enteric Dopaminergic Neurons: Definition, Developmental Lineage, and Effects of Extrinsic Denervation Zhishan Li, Tuan D. Pham, Jason J. Chen, Scott F. Hall, Michael D. Gershon Dopaminergic neurons have been suspected to be present in the enteric nervous system (ENS), but the innervation of the gut by sympathetic nerves, in which dopamine (DA) is a precursor to norepinephrine (NE), complicates analyses of enteric DA. We now report that mRNA transcripts (quantified by real-time RT-PCR) encoding tyrosine hydroxylase (TH) and the DA transporter (DAT) are present in the routine bowel (ileum > duodenum; proximal colon :> distal colon). These observations support the idea that the gut contains dopaminergic neurons because sympathetic neurons are extrinsic. Enteric DAT was identical in sequence to brain DAT and mediated the specific uptake of 3H-DAwhen the DAT cloned from the gut was expressed in exogenous (COS-7) cells. TH protein was demonstrated immunocytochemicaUy in enteric neuronal perikarya (submucosal >:> myenteric plexus; ileum >. duodenum > colon) that did not contain the immunoreactiv/ty of dopamine [3hydroxylase. Extrinsic denervation of the bowel led to increases m both TH-immunoreactive neurons and enteric expression of mRNA encoding TH. TH immunoreactivity was coincident with those of DA and DAT in subsets of neurons (submucosal :> myenteric) in guinea pig and mouse intestines in situ and in cultured ganglia from the guinea pig gut. The presence of TH, DA, and DAT in intrinsic neurons confirms the presence of dapoaninergic neurons in the ENS. A population of proliferating transiently catecholaminergic (TC) cells is present in the fetal gut, which dLsappears after E14 (mouse) or E15 (rat). TC ceils disappear because they are the precursors of non-catecholaminergic terminally differentiated neurons. TC ceils and their successors are lacking in mash-1 null mice. In contrast, the late-appealing intrinsic TH-expressmg neurons of the murine bowel develop normally in mash-1 null mice. We conclude that the ENS contains intrinsic dopaminergic neurons, which arise from a mash1-independent lineage of non-catecholaminergic precursors. The upregulation of TH in these cells following sympathetic denervation may help to compensate for the loss of noradrenertic inhibition.
M1275 A Critical Effect of Temperature on Action Potential and Afterhyperpolarisation in AH/Sensory Myenteric Neurones Clare A. Jones, Francois Rugiero, Bruno Mazet, Maurice Gola, John B. Furness, Gareth A. Hicks, Nadine Clerc Emetic sensory (AH) neurones are characterized by action potentials (AP) with a Ca2+ componem, a long afterhyperpolarization (AHP) and a large range of excitability. The excitability of certain groups of central and peripheral neurones is known to be highly temperature-dependent in vitro, but little is known about the effect of temperature on AH neurones. Because there is a discontinuity in the way in which the AP is affected by temperature, we have investzgated, the effect of small changes (2 to 4~ in temperature in the range that is classically used for in vitro studies of non dissocmted enteric neurons. lntraceflular recordings were obtained from myenteric AH neurones of the guinea pig ileum, using the longitudinal mnscle/myenteric plexus preparation maintained in Krebs solution perfnsed at 3 ml/min. Temperature changes of 2 or 4~ were applied slowly (2-4 rain) and recordings were performed for 10 rain before and after the changes. Increasing temperature from 33~ to 35~ induced a decrease in AP half duration (9 + 0.7%, Ql0 = 1.6) and overshoot (29 +- 1.4%, Q~o = 4.5). In addition, the duration of the AHP which follows a directly evoked train of APs decreased dramatically (37 -+ 2.2%, Q~o= 20). These changes were reversible on return to 33~ Quantitatively similar changes were obtained when temperature was increased from 33~ to 37~ but they were not always reversible. The observed variations were not dependent on membrane potential which was found to be unaffected by temperature variation in the range 33-37~
M1273 Neurokinin 1 and 2 Receptors in Murine Enteric Primary Afferent Neurons Yn Kant Mao, Lu Wang, Yu Fang Wang, Nigel Bunnett, Gervais Tougas Background: Small intestinal inflammation induced by acute T.spiralis infection elicits a persistem hyperalgesic response to colorectal distention in the absence of colonic inflammation (Gastro 118:A701, 120:A1708). This hyperalgesic response involves NK-1 receptors (NK-1R) as the specific NK-1R antagonist SR140333 blocks it. NeuN antibody is a marker for rautine enteric sensory neurons (Gastro 2002, 122:A733). In this study we used double staining immunohistochemistry to colocalize NK-1R, NK-2R and NeuN within the gut sensory neurons of NIH Swiss mice (25-30g) to determine if their location is compatible with our electrophysiological observations. Methods: Adult NiH/Swiss mice were sacrificed, jejunum and colon tissues harvested and fixed for immunohistochemistry in 4% paraformaldehyde. Whole mount tissues (longitudinal muscle with myemeric plexuses) were processed for immunocytochemistry using anti-NK-1R (1:300) rabbit polyclonal antibody (Chemicon Inc.), anti-NK-2R (1:300) rabbit polyclonal amibody, NeuN (1:300) mouse antibody and Donkey-anti rabbit lgG-cy3 (1:100), anti rabbit IgG-FITC (1:50). The M.O.M kit was used for NeuN amibody. These tissues were then examined using by laser scanning confocal microscopy. Results: Abundant NK-1R and NK-2R were detected on myentetic neurons of the jejunum and colon. Almost all NK-1Rs were co-localized with NeuN immunostaiinng neuronal cell bodies, but only 30-50 % of NeuN immunostaining ceils also had NK-1R. Only very few ( < 1%) N K-1R staining neuronal cell bodies were without NeuN immunostainint. Most NeuN staining neuronal cell bodies had NK-2Rs immunostaining as well. However there were also frequent NK-2R positive neuronal cell bodies without NeuN immunostaining. These results mdicate that while almost all NK1R are located on enteric sensory neurons, NK-2R are not only found on enteric sensory neurons but also on NeuN negative neuronal cell bodies, probably enteric inter-neurons or motor neurons. Conclusions: In the murine enteric nervous system, NK-1 receptors are localized on enteric sensory neurons while NK2R are not only localized in mouse emeric sensory but also on inter-neurons and motor neurons. These observations have therapeutic implications for the developmem of new therapeutic approaches using NK receptor agonists and antagomsts.
We conclude that changes in temperature as little as 2-4~ can significantly affect the electrophysiological properties of AH/sensory neurons. These small temperature changes result in profound alterations to HVA Ca2+ and Ca2+ activated K+ (AHP) currents both of which are crucial in patterning the excitability of these neurons. Several mechanisms may contribute to such effects. In particular, changes in Na + and K+ conductances might condition the entry of Ca2§. As changes in Ca2+ entry are insufficient to explain the fact that the AHP decay is markedly affected, it is likely that the temperature dependence of the AHP results from some process subsequent to the entry of Ca2+. This work was supported by Glaxo SmithKline and CNRS-France.
M1276 Localisation of Intermediate Conductance Calcium-activated Potassium Channel (IK)-like Immunoreactivity in Rat and Human Neurones Inger-Sofie Selmer, David R. Robinson, Stephen J. Arnold, Mark X. Chen, Praveen Anand, Gareth A. Hicks Ca2+ activated K+ channels with imermediate conductance have been suggested to underlie the afterhyperpolarisation in guinea pig intrinsic primary afferent neurones (IPANs; Vogalis et al., 2002 J. Physiol. 538.2, 421-33). However, the precise molecular identity of the channels in these neurones is unknown (see Furness et al., this meeting). IK-like immunoreactivity (IR) has been detected in human dorsal root ganglia (DRG) suggesting the presence of such channels in neurones (Boettger eta[, 2002, Brain 125,252-63). Therefore, using immunohistochemieal techniques we have investigated IK-like IR in rat colonic sensory DRG, and rat and human enteric neurones. Methods: Injections of the retrograde tracer Fast Blue (FB, 25% in saline) were made into the wall of descending colon of two adult male Sprague Dawley rats (150-200g). Two weeks later, animals were sacrificed and DRG removed. Myenteric plexus wholemounts from rat small intestine, and full thickness sections of human colon, were prepared using standard techniques and used for immunohistochemistry with an antibody raised against a peptide from the N-terminus of human IK1, having 14 out of 15 amino acids conserved between rat and human. Results and Conclusions: IK-like 1R was detected in rat DRG neurones, and in 10 % of those retrogradely labelled from the colon. IK-like IR was found in neurones of the myenteric plexus in both rat and human tissue. Preliminary charactetisation of the IK IR neurones in the rat was carried out with NeuN, a putative marker of IPANs, however no co-localisation was observed. Further studies are needed to more clearly define the identity of enteric neurones containing IK channels in both rat and human. The results suggest that IK currents may be involved in the regulation of excitability of DRG neurons, including a sub population of those innervating the colon, and also provide further evidence for their role in the emetic nervous system.
M1274
Selective Expression of a Persistent Ttx-Resistant Na4. Current and Navl.9 Subunit in Myenteric Sensory Neurons Francois Rugiero, Nadine Clerc, Mohiin Mistry, Dominique Sage, Joel Black, Stephen Waxman, Marcel Crest, Patrick Delmas, Maurice Gola Enteric sensory neurons prime enteric reflexes controhng gut motility and secretions in the absence of a central command. The state of excitability of these neurons is likely to play a critical role in gut motility. Voltage-gated Na+ currents are crucial in setting neuronal excitability. Here, we have identified a TTX-resistam Na + current (TTX-R INa) in duodenum myemeric neurons of guinea pig and rat and have sought evidence regarding the molecular identity of the channel producing this current from the expression of Na + channel a subunits and the biophysical and pharmacological properties of TTX-R 1Na. Whole-cell patch clamp recording was performed in situ on the myemeric plexus of the guinea pig and the rat. RT-PCR using degenerate primers designed to amplify guinea pig TTX-R Na + channels (NaV1.8 and NaV1.9) and immunnlabeling for NaV1.9 and NaV1.8 subunits were performed on guinea pig and rat myemeric ganglia and DRGs. Wbole-cefl patch clamp recording revealed the presence of a voltage-gated Na + current that was highly resistant to TTX (IC50 - 200 raM) and selectively distributed in myemetic sensory neurons but not in interneurons and motor neurons. TTX-R lNa activated slowly in response to depolarization and exhibited a threshold for activation at -50 inV. V1/2 values of activation and steadystate inactivation were -32 and -31 mV respectively, which, as predicted from the window current, generated persistent currents. TTX-R lNa also had prominent ultraslow inactivation
A-343
AGA Abstracts
M1272
Enteric Dopaminergic Neurons: Definition, Developmental Lineage, and Effects of Extrinsic Denervation Zhishan Li, Tuan D. Pham, Jason J. Chen, Scott F. Hall, Michael D. Gershon Dopaminergic neurons have been suspected to be present in the enteric nervous system (ENS), but the innervation of the gut by sympathetic nerves, in which dopamine (DA) is a precursor to norepinephrine (NE), complicates analyses of enteric DA. We now report that mRNA transcripts (quantified by real-time RT-PCR) encoding tyrosine hydroxylase (TH) and the DA transporter (DAT) are present in the routine bowel (ileum > duodenum; proximal colon :> distal colon). These observations support the idea that the gut contains dopaminergic neurons because sympathetic neurons are extrinsic. Enteric DAT was identical in sequence to brain DAT and mediated the specific uptake of 3H-DAwhen the DAT cloned from the gut was expressed in exogenous (COS-7) cells. TH protein was demonstrated immunocytochemicaUy in enteric neuronal perikarya (submucosal >:> myenteric plexus; ileum >. duodenum > colon) that did not contain the immunoreactiv/ty of dopamine [3hydroxylase. Extrinsic denervation of the bowel led to increases m both TH-immunoreactive neurons and enteric expression of mRNA encoding TH. TH immunoreactivity was coincident with those of DA and DAT in subsets of neurons (submucosal :> myenteric) in guinea pig and mouse intestines in situ and in cultured ganglia from the guinea pig gut. The presence of TH, DA, and DAT in intrinsic neurons confirms the presence of dapoaninergic neurons in the ENS. A population of proliferating transiently catecholaminergic (TC) cells is present in the fetal gut, which dLsappears after E14 (mouse) or E15 (rat). TC ceils disappear because they are the precursors of non-catecholaminergic terminally differentiated neurons. TC ceils and their successors are lacking in mash-1 null mice. In contrast, the late-appealing intrinsic TH-expressmg neurons of the murine bowel develop normally in mash-1 null mice. We conclude that the ENS contains intrinsic dopaminergic neurons, which arise from a mash1-independent lineage of non-catecholaminergic precursors. The upregulation of TH in these cells following sympathetic denervation may help to compensate for the loss of noradrenertic inhibition.
M1275 A Critical Effect of Temperature on Action Potential and Afterhyperpolarisation in AH/Sensory Myenteric Neurones Clare A. Jones, Francois Rugiero, Bruno Mazet, Maurice Gola, John B. Furness, Gareth A. Hicks, Nadine Clerc Emetic sensory (AH) neurones are characterized by action potentials (AP) with a Ca2+ componem, a long afterhyperpolarization (AHP) and a large range of excitability. The excitability of certain groups of central and peripheral neurones is known to be highly temperature-dependent in vitro, but little is known about the effect of temperature on AH neurones. Because there is a discontinuity in the way in which the AP is affected by temperature, we have investzgated, the effect of small changes (2 to 4~ in temperature in the range that is classically used for in vitro studies of non dissocmted enteric neurons. lntraceflular recordings were obtained from myenteric AH neurones of the guinea pig ileum, using the longitudinal mnscle/myenteric plexus preparation maintained in Krebs solution perfnsed at 3 ml/min. Temperature changes of 2 or 4~ were applied slowly (2-4 rain) and recordings were performed for 10 rain before and after the changes. Increasing temperature from 33~ to 35~ induced a decrease in AP half duration (9 + 0.7%, Ql0 = 1.6) and overshoot (29 +- 1.4%, Q~o = 4.5). In addition, the duration of the AHP which follows a directly evoked train of APs decreased dramatically (37 -+ 2.2%, Q~o= 20). These changes were reversible on return to 33~ Quantitatively similar changes were obtained when temperature was increased from 33~ to 37~ but they were not always reversible. The observed variations were not dependent on membrane potential which was found to be unaffected by temperature variation in the range 33-37~
M1273 Neurokinin 1 and 2 Receptors in Murine Enteric Primary Afferent Neurons Yn Kant Mao, Lu Wang, Yu Fang Wang, Nigel Bunnett, Gervais Tougas Background: Small intestinal inflammation induced by acute T.spiralis infection elicits a persistem hyperalgesic response to colorectal distention in the absence of colonic inflammation (Gastro 118:A701, 120:A1708). This hyperalgesic response involves NK-1 receptors (NK-1R) as the specific NK-1R antagonist SR140333 blocks it. NeuN antibody is a marker for rautine enteric sensory neurons (Gastro 2002, 122:A733). In this study we used double staining immunohistochemistry to colocalize NK-1R, NK-2R and NeuN within the gut sensory neurons of NIH Swiss mice (25-30g) to determine if their location is compatible with our electrophysiological observations. Methods: Adult NiH/Swiss mice were sacrificed, jejunum and colon tissues harvested and fixed for immunohistochemistry in 4% paraformaldehyde. Whole mount tissues (longitudinal muscle with myemeric plexuses) were processed for immunocytochemistry using anti-NK-1R (1:300) rabbit polyclonal antibody (Chemicon Inc.), anti-NK-2R (1:300) rabbit polyclonal amibody, NeuN (1:300) mouse antibody and Donkey-anti rabbit lgG-cy3 (1:100), anti rabbit IgG-FITC (1:50). The M.O.M kit was used for NeuN amibody. These tissues were then examined using by laser scanning confocal microscopy. Results: Abundant NK-1R and NK-2R were detected on myentetic neurons of the jejunum and colon. Almost all NK-1Rs were co-localized with NeuN immunostaiinng neuronal cell bodies, but only 30-50 % of NeuN immunostaining ceils also had NK-1R. Only very few ( < 1%) N K-1R staining neuronal cell bodies were without NeuN immunostainint. Most NeuN staining neuronal cell bodies had NK-2Rs immunostaining as well. However there were also frequent NK-2R positive neuronal cell bodies without NeuN immunostaining. These results mdicate that while almost all NK1R are located on enteric sensory neurons, NK-2R are not only found on enteric sensory neurons but also on NeuN negative neuronal cell bodies, probably enteric inter-neurons or motor neurons. Conclusions: In the murine enteric nervous system, NK-1 receptors are localized on enteric sensory neurons while NK2R are not only localized in mouse emeric sensory but also on inter-neurons and motor neurons. These observations have therapeutic implications for the developmem of new therapeutic approaches using NK receptor agonists and antagomsts.
We conclude that changes in temperature as little as 2-4~ can significantly affect the electrophysiological properties of AH/sensory neurons. These small temperature changes result in profound alterations to HVA Ca2+ and Ca2+ activated K+ (AHP) currents both of which are crucial in patterning the excitability of these neurons. Several mechanisms may contribute to such effects. In particular, changes in Na + and K+ conductances might condition the entry of Ca2§. As changes in Ca2+ entry are insufficient to explain the fact that the AHP decay is markedly affected, it is likely that the temperature dependence of the AHP results from some process subsequent to the entry of Ca2+. This work was supported by Glaxo SmithKline and CNRS-France.
M1276 Localisation of Intermediate Conductance Calcium-activated Potassium Channel (IK)-like Immunoreactivity in Rat and Human Neurones Inger-Sofie Selmer, David R. Robinson, Stephen J. Arnold, Mark X. Chen, Praveen Anand, Gareth A. Hicks Ca2+ activated K+ channels with imermediate conductance have been suggested to underlie the afterhyperpolarisation in guinea pig intrinsic primary afferent neurones (IPANs; Vogalis et al., 2002 J. Physiol. 538.2, 421-33). However, the precise molecular identity of the channels in these neurones is unknown (see Furness et al., this meeting). IK-like immunoreactivity (IR) has been detected in human dorsal root ganglia (DRG) suggesting the presence of such channels in neurones (Boettger eta[, 2002, Brain 125,252-63). Therefore, using immunohistochemieal techniques we have investigated IK-like IR in rat colonic sensory DRG, and rat and human enteric neurones. Methods: Injections of the retrograde tracer Fast Blue (FB, 25% in saline) were made into the wall of descending colon of two adult male Sprague Dawley rats (150-200g). Two weeks later, animals were sacrificed and DRG removed. Myenteric plexus wholemounts from rat small intestine, and full thickness sections of human colon, were prepared using standard techniques and used for immunohistochemistry with an antibody raised against a peptide from the N-terminus of human IK1, having 14 out of 15 amino acids conserved between rat and human. Results and Conclusions: IK-like 1R was detected in rat DRG neurones, and in 10 % of those retrogradely labelled from the colon. IK-like IR was found in neurones of the myenteric plexus in both rat and human tissue. Preliminary charactetisation of the IK IR neurones in the rat was carried out with NeuN, a putative marker of IPANs, however no co-localisation was observed. Further studies are needed to more clearly define the identity of enteric neurones containing IK channels in both rat and human. The results suggest that IK currents may be involved in the regulation of excitability of DRG neurons, including a sub population of those innervating the colon, and also provide further evidence for their role in the emetic nervous system.
M1274
Selective Expression of a Persistent Ttx-Resistant Na4. Current and Navl.9 Subunit in Myenteric Sensory Neurons Francois Rugiero, Nadine Clerc, Mohiin Mistry, Dominique Sage, Joel Black, Stephen Waxman, Marcel Crest, Patrick Delmas, Maurice Gola Enteric sensory neurons prime enteric reflexes controhng gut motility and secretions in the absence of a central command. The state of excitability of these neurons is likely to play a critical role in gut motility. Voltage-gated Na+ currents are crucial in setting neuronal excitability. Here, we have identified a TTX-resistam Na + current (TTX-R INa) in duodenum myemeric neurons of guinea pig and rat and have sought evidence regarding the molecular identity of the channel producing this current from the expression of Na + channel a subunits and the biophysical and pharmacological properties of TTX-R 1Na. Whole-cell patch clamp recording was performed in situ on the myemeric plexus of the guinea pig and the rat. RT-PCR using degenerate primers designed to amplify guinea pig TTX-R Na + channels (NaV1.8 and NaV1.9) and immunnlabeling for NaV1.9 and NaV1.8 subunits were performed on guinea pig and rat myemeric ganglia and DRGs. Wbole-cefl patch clamp recording revealed the presence of a voltage-gated Na + current that was highly resistant to TTX (IC50 - 200 raM) and selectively distributed in myemetic sensory neurons but not in interneurons and motor neurons. TTX-R lNa activated slowly in response to depolarization and exhibited a threshold for activation at -50 inV. V1/2 values of activation and steadystate inactivation were -32 and -31 mV respectively, which, as predicted from the window current, generated persistent currents. TTX-R lNa also had prominent ultraslow inactivation
A-343
AGA Abstracts