The aim of this study was to investigate the contribution of the TRPV1 receptor to jejunal afferent sensitivity in the murine
intestine. Multiunit activity was recorded in vitro from mesenteric ...afferents supplying segments of mouse jejunum taken from wild-type (WT) and TRPV1 knockout (TRPV1 â/â ) animals. In WT preparations, ramp distension of the gut (up to 60 mmHg) produced biphasic changes in afferent activity so
the pressureâresponse curve had an initial rapid increase in afferent discharge followed by a second phase of slower increase
in activity. Afferent response to distension was significantly lower in TRPV1 â/â than in WT mice. Single-unit analysis revealed three functional types of afferent fibres: (1) low-threshold fibres (2) wide
dynamic range fibres and (3) high-threshold fibres. There was a marked downward shift of the pressureâresponse curve for wide
dynamic range fibres in the TRPV1 â/â mice as compared to the WT controls. The afferent response to intraluminal hydrochloric acid (20 m m ) was also attenuated in the TRPV1 â/â mice. In contrast, the response to bath application of bradykinin (1 μ m , 3 ml) was not significantly different between the two groups. The TRPV1 antagonist capsazepine (10 μ m ) significantly attenuated the nerve responses to distension, intraluminal acid and bradykinin, as well as the spontaneous
discharge in WT mice. The WT jejunal afferents responded to capsaicin with rapid increases in afferent activity, whereas TRPV1 â/â afferents were not at all sensitive to capsaicin. Previous evidence indicates that TRPV1 is not mechanosensitive, so the
results of the present study suggest that activation of TRPV1 may sensitize small intestinal afferent neurones.
Background & Aims: Visceral hypersensitivity, a hallmark of irritable bowel syndrome, is generally considered to be mechanosensitive in nature and mediated via spinal afferents. Both stress and ...inflammation are implicated in visceral hypersensitivity, but the underlying molecular mechanisms of visceral hypersensitivity are unknown. Methods: Mice were infected with Nippostrongylus brasiliensis (Nb) larvae, exposed to environmental stress and the following separate studies performed 3–4 weeks later. Mesenteric afferent nerve activity was recorded in response to either ramp balloon distention (60 mm Hg), or to an intraluminal perfusion of hydrochloric acid (50 mmol/L), or to octreotide administration (2 μmol/L). Intraperitoneal injection of cholera toxin B-488 identified neurons projecting to the abdominal viscera. Fluorescent neurons in dorsal root and nodose ganglia were isolated using laser-capture microdissection. RNA was hybridized to Affymetrix Mouse whole genome arrays for analysis to evaluate the effects of stress and infection. Results: In mice previously infected with Nb, there was no change in intestinal afferent mechanosensitivity, but there was an increase in chemosensitive responses to intraluminal hydrochloric acid when compared with control animals. Gene expression profiles in vagal but not spinal visceral sensory neurons were significantly altered in stressed Nb-infected mice. Decreased afferent responses to somatostatin receptor 2 stimulation correlated with lower expression of vagal somatostatin receptor 2 in stressed Nb-infected mice, confirming a link between molecular data and functional sequelae. Conclusions: Alterations in the intestinal brain–gut axis, in chemosensitivity but not mechanosensitivity, and through vagal rather than spinal pathways, are implicated in stress-induced postinflammatory visceral hypersensitivity.
Tetrodotoxin-resistant (TTX-R) sodium currents have been proposed to underlie sensory neuronal hyperexcitability in acute
inflammatory models, but their role in chronic models is unknown. Since no ...pharmacological tools to separate TTX-R currents
are available, this study employs Na v 1.8 and Na v 1.9 null mice to evaluate these currents roles in a chronic hyperexcitability model after the resolution of an inflammatory
insult. Transient jejunitis was induced by infection with Nippostrongylus brasiliensis (Nb) in Na v 1.9 and Na v 1.8 null, wild-type and naïve mice. Retrogradely labelled dorsal root ganglia (DRG) neurons were harvested on day 20â24 post-infection
for patch clamp recording. Rheobase and action potential (AP) parameters were recorded as measures of excitability, and Na v 1.9 and Na v 1.8 currents were recorded. DRG neuronal excitability was significantly increased in post-infected mice compared to sham animals,
despite the absence of ongoing inflammation (sham = 1.9 ± 0.3, infected = 3.6 ± 0.7 APs at 2à rheobase, P
= 0.02). Hyperexcitability was associated with a significantly increased amplitude of TTX-R currents. Hyperexcitability
was maintained in Na v 1.9 â/â mice, but hyperexcitability was absent and APs were blunted in Na v 1.8 â/â mice. This study identifies a critical role for Na v 1.8 in chronic post-infectious visceral hyperexcitability, with no contribution from Na v 1.9. Nb infection-induced hyperexcitability is not observed in Na v 1.8 â/â mice, but is still present in Na v 1.9 â/â mice. It is not clear whether hyperexcitability is due to a change in the function of Na v 1.8 channels or a change in the number of Na v 1.8 channels.
1 Johnson and Johnson Pharmaceutical Research and Development, Department of Internal Medicine, Beerse, Belgium
2 Holburn Group of Companies, Bowmanville, Ontario, Canada
3 Johnson and Johnson ...Pharmaceutical Research and Development, Department of Functional Genomics, Beerse, Belgium
Vagal afferent neurons are thought to convey primarily physiological information, whereas spinal afferents transmit noxious signals from the viscera to the central nervous system. To elucidate molecular identities for these different properties, we compared gene expression profiles of neurons located in nodose ganglia (NG) and dorsal root ganglia (DRG) in mice. Intraperitoneal administration of Alexa Fluor-488-conjugated cholera toxin B allowed enrichment for neurons projecting to the viscera. Fluorescent neurons in DRG (from T10 to T13) and NG were isolated using laser-capture microdissection. Gene expression profiles of these afferent neurons, obtained by microarray hybridization, were analyzed using multivariate spectral map analysis, significance analysis of microarrays (SAM) algorithm, and fold-difference filtering. A total of 1,996 genes were differentially expressed in DRG vs. NG, including 41 G protein-coupled receptors and 60 ion channels. Expression profiles obtained on laser-captured neurons were contrasted to those obtained on whole ganglia, demonstrating striking differences and the need for microdissection when studying visceral sensory neurons because of dilution of the signal by somatic sensory neurons. Furthermore, we provide a detailed catalog of all adrenergic and cholinergic, GABA, glutamate, serotonin, and dopamine receptors; voltage-gated potassium, sodium, and calcium channels; and transient receptor potential cation channels present in afferents projecting to the peritoneal cavity. Our genome-wide expression profiling data provide novel insight into molecular signatures that underlie both functional differences and similarities between NG and DRG sensory neurons. Moreover, these findings will offer novel insight into mode of action of pharmacological agents modulating visceral sensation.
microarray; vagal and spinal afferents; nociception; spectral map analysis
Vagal influences over mast cells Stead, Ronald H.; Colley, Elizabeth C.; Wang, Bingxian ...
Autonomic neuroscience,
04/2006, Letnik:
125, Številka:
1
Journal Article
Recenzirano
The established microanatomical association of rat intestinal mucosal mast cells (IMMC) and mucosal nerves raises the possibility that there is crosstalk between mast cells and extrinsic nerves that ...connect to the CNS. The idea of mast cell–CNS interactions is supported by the demonstration that rat mast cell protease II (RMCPII), found predominantly in IMMC, can be conditionally released by pairing an audio–visual cue with antigen challenge. That the vagus nerve is involved in the IMMC–nerve axis was further demonstrated in a series of our studies showing that: (a) vagal afferents penetrate the small intestinal mucosa and contact IMMC; (b) vagotomy causes a reduction in IMMC density, suggesting a trophic relationship (typical of nerve–target interactions); and (c) stimulation of the cervical vagus causes an increase in histamine and serotonin in IMMC. To further investigate the IMMC-nerve axis in a model of post-inflammatory bowel disorders, infection with
Nippostrongylus brasiliensis (Nb) was used to demonstrate an increase in mast cell numbers in the intestinal mucosa and mucosal nerve remodelling with hyperinnervation. Administration of Nb antigen resulted in dramatic increases in mesenteric afferent nerve firing in Nb infected rats, that was absent in sham animals. Moreover, challenge of post-Nb rats with 2-methyl-5HT caused increased mesenteric afferent firing, indicating that vagal afferent innervation remains intact in the post-infection state. These data suggest a functional connection between mast cells and extrinsic afferent nerves. Nb infection provides a useful model of altered communication between IMMCs, peripheral nerves and the CNS, as may occur in post-inflammatory disease states. Since a close anatomical relationship has also previously been demonstrated between nerves and IMMC in humans, further understanding the mast cell–nerve axis may be of critical importance in the development of treatments for various human disease states, including functional bowel disorders.
Tetrodotoxin‐resistant (TTX‐R) sodium currents have been proposed to underlie sensory neuronal hyperexcitability in acute inflammatory models, but their role in chronic models is unknown. Since no ...pharmacological tools to separate TTX‐R currents are available, this study employs Na
v
1.8 and Na
v
1.9 null mice to evaluate these currents roles in a chronic hyperexcitability model after the resolution of an inflammatory insult. Transient jejunitis was induced by infection with
Nippostrongylus brasiliensis
(Nb) in Na
v
1.9 and Na
v
1.8 null, wild‐type and naïve mice. Retrogradely labelled dorsal root ganglia (DRG) neurons were harvested on day 20–24 post‐infection for patch clamp recording. Rheobase and action potential (AP) parameters were recorded as measures of excitability, and Na
v
1.9 and Na
v
1.8 currents were recorded. DRG neuronal excitability was significantly increased in post‐infected mice compared to sham animals, despite the absence of ongoing inflammation (sham = 1.9 ± 0.3, infected = 3.6 ± 0.7 APs at 2× rheobase,
P
= 0.02). Hyperexcitability was associated with a significantly increased amplitude of TTX‐R currents. Hyperexcitability was maintained in Na
v
1.9
−/−
mice, but hyperexcitability was absent and APs were blunted in Na
v
1.8
−/−
mice. This study identifies a critical role for Na
v
1.8 in chronic post‐infectious visceral hyperexcitability, with no contribution from Na
v
1.9. Nb infection‐induced hyperexcitability is not observed in Na
v
1.8
−/−
mice, but is still present in Na
v
1.9
−/−
mice. It is not clear whether hyperexcitability is due to a change in the function of Na
v
1.8 channels or a change in the number of Na
v
1.8 channels.
Tetrodotoxin-resistant (TTX-R) sodium currents have been proposed to underlie sensory neuronal hyperexcitability in acute inflammatory models, but their role in chronic models is unknown. Since no ...pharmacological tools to separate TTX-R currents are available, this study employs Na sub(v)1.8 and Na sub(v)1.9 null mice to evaluate these currents roles in a chronic hyperexcitability model after the resolution of an inflammatory insult. Transient jejunitis was induced by infection with Nippostrongylus brasiliensis (Nb) in Na sub(v)1.9 and Na sub(v)1.8 null, wild-type and naive mice. Retrogradely labelled dorsal root ganglia (DRG) neurons were harvested on day 20-24 post-infection for patch clamp recording. Rheobase and action potential (AP) parameters were recorded as measures of excitability, and Na sub(v)1.9 and Na sub(v)1.8 currents were recorded. DRG neuronal excitability was significantly increased in post-infected mice compared to sham animals, despite the absence of ongoing inflammation (sham = 1.9 plus or minus 0.3, infected = 3.6 plus or minus 0.7 APs at 2x rheobase, P = 0.02). Hyperexcitability was associated with a significantly increased amplitude of TTX-R currents. Hyperexcitability was maintained in Na sub(v)1.9 super(-/-) mice, but hyperexcitability was absent and APs were blunted in Na sub(v)1.8 super(-/-) mice. This study identifies a critical role for Na sub(v)1.8 in chronic post-infectious visceral hyperexcitability, with no contribution from Na sub(v)1.9. Nb infection-induced hyperexcitability is not observed in Na sub(v)1.8 super(-/-) mice, but is still present in Na sub(v)1.9 super(-/-) mice. It is not clear whether hyperexcitability is due to a change in the function of Na sub(v)1.8 channels or a change in the number of Na sub(v)1.8 channels.
To date investigations of enteric neurons by patch clamping/calcium imaging have been limited by studying unidentified heterogeneous populations of neurons. In DiI-labelled colonic myenteric neurons, ...the feasibility of recording ionic events was determined by applying DiI either to the mucosa or the circular muscle, dispersing neurons after 48 h organotypic culture, and patch-clamping/calcium imaging labeled neurons after 3–7 days in culture. Myenteric neurons with diffuse DiI fluorescence were typically smooth and agranular. Neurons labeled after DiI was applied to circular muscle, fired in either a phasic or a tonic manner, and exhibited fast afterhyperpolarizations (100–300 ms duration) at the end of a depolarizing pulse. They expressed a fast inward current and at least three different outward currents. Action potentials elicited in DiI-labeled sensory neurons were followed by a prolonged afterhyperpolarization (AH, 4–6 s). The offset of a suprathreshold depolarizing step elicited a prolonged outward tail current that approximated the timecourse of the prolonged AH. In addition, in response to membrane depolarization in DiI-labeled neurons loaded with fura-2, robust Ca
2+ transients were recorded using the perforated patch technique. These results demonstrate that DiI labeling of cultured myenteric neurons is feasible, and patch clamp/Ca
2+ fluorescence recordings can be made from specific populations of cultured DiI-labeled colonic myenteric neurons.
PDF
