RESUMO
BACKGROUND & AIMS: Patients with cholestatic disease have increased systemic concentrations of bile acids (BAs) and profound pruritus. The G-protein-coupled BA receptor 1 TGR5 (encoded by GPBAR1) is expressed by primary sensory neurons; its activation induces neuronal hyperexcitability and scratching by unknown mechanisms. We investigated whether the transient receptor potential ankyrin 1 (TRPA1) is involved in BA-evoked, TGR5-dependent pruritus in mice. METHODS: Co-expression of TGR5 and TRPA1 in cutaneous afferent neurons isolated from mice was analyzed by immunofluorescence, in situ hybridization, and single-cell polymerase chain reaction. TGR5-induced activation of TRPA1 was studied in in HEK293 cells, Xenopus laevis oocytes, and primary sensory neurons by measuring Ca(2+) signals. The contribution of TRPA1 to TGR5-induced release of pruritogenic neuropeptides, activation of spinal neurons, and scratching behavior were studied using TRPA1 antagonists or Trpa1(-/-) mice. RESULTS: TGR5 and TRPA1 protein and messenger RNA were expressed by cutaneous afferent neurons. In HEK cells, oocytes, and neurons co-expressing TGR5 and TRPA1, BAs caused TGR5-dependent activation and sensitization of TRPA1 by mechanisms that required Gßγ, protein kinase C, and Ca(2+). Antagonists or deletion of TRPA1 prevented BA-stimulated release of the pruritogenic neuropeptides gastrin-releasing peptide and atrial natriuretic peptide B in the spinal cord. Disruption of Trpa1 in mice blocked BA-induced expression of Fos in spinal neurons and prevented BA-stimulated scratching. Spontaneous scratching was exacerbated in transgenic mice that overexpressed TRG5. Administration of a TRPA1 antagonist or the BA sequestrant colestipol, which lowered circulating levels of BAs, prevented exacerbated spontaneous scratching in TGR5 overexpressing mice. CONCLUSIONS: BAs induce pruritus in mice by co-activation of TGR5 and TRPA1. Antagonists of TGR5 and TRPA1, or inhibitors of the signaling mechanism by which TGR5 activates TRPA1, might be developed for treatment of cholestatic pruritus.
Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase/metabolismo , Prurido/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Colestase/complicações , Modelos Animais de Doenças , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Peptídeo Liberador de Gastrina/metabolismo , Células HEK293 , Humanos , Camundongos Knockout , Peptídeos Natriuréticos/metabolismo , Neurônios Aferentes/citologia , Neurônios Aferentes/metabolismo , Nociceptores/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Cultura Primária de Células , Prurido/etiologia , Receptores Acoplados a Proteínas G/genética , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/genética , Xenopus laevisRESUMO
TRPA1 is an ion channel that detects specific chemicals in food and also transduces mechanical, cold and chemical stimulation. Its presence in sensory nerve endings is well known and recent evidence indicates that it is expressed by some gastrointestinal enteroendocrine cells (EEC). The purpose of the present work is to identify and quantify EEC that express TRPA1 in the mouse gastrointestinal tract. Combined in situ hybridisation histochemistry for TRPA1 and immunofluorescence for EEC hormones was used. TRPA1 expressing EEC were common in the duodenum and jejunum, were rare in the distal small intestine and were absent from the stomach and large intestine. In the duodenum and jejunum, TRPA1 occurred in EEC that contained both cholecystokinin (CCK) and 5-hydroxytryptamine (5HT) and in a small number of cells expressing 5HT but not CCK. TRPA1 was absent from CCK cells that did not express 5HT and from EEC containing glucagon-like insulinotropic peptide. Thus TRPA1 is contained in very specific EEC populations. It is suggested that foods such as garlic and cinnamon that contain TRPA1 stimulants may aid digestion by facilitating the release of CCK.
Assuntos
Células Enteroendócrinas/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Extratos Celulares , Células Enteroendócrinas/citologia , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/genéticaRESUMO
BACKGROUND & AIMS: Transient receptor potential ankyrin (TRPA) 1, an excitatory ion channel expressed by sensory neurons, mediates somatic and visceral pain in response to direct activation or noxious mechanical stimulation. Although the intestine is routinely exposed to irritant alimentary compounds and inflammatory mediators that activate TRPA1, there is no direct evidence for functional TRPA1 receptors on enteric neurons, and the effects of TRPA1 activation on intestinal function have not been determined. We characterized expression of TRPA1 by enteric neurons and determined its involvement in the control of intestinal contractility and transit. METHODS: TRPA1 expression was characterized by reverse-transcription polymerase chain reaction and immunofluorescence analyses. TRPA1 function was examined by Ca(2+) imaging and by assays of contractile activity and transit. RESULTS: We detected TRPA1 messenger RNA in the mouse intestine and TRPA1 immunoreactivity in enteric neurons. The cecum and colon had immunoreactivity for neuronal TRPA1, but the duodenum did not. TRPA1 immunoreactivity was also detected in inhibitory motoneurons and descending interneurons, cholinergic neurons, and intrinsic primary afferent neurons. TRPA1 activators, including cinnamaldehyde, allyl isothiocyanate (AITC), and 4-hydroxynonenal, increased [Ca(2+)](i) in myenteric neurons. These were reduced by a TRPA1 antagonist (HC-030031) or deletion of Trpa1. TRPA1 activation inhibited contractility of the segments of colon but not stomach or small intestine of Trpa1(+/+) but not Trpa1(-/-) mice; this effect was reduced by tetrodotoxin or N(G)-nitro-l-arginine methyl ester. Administration of AITC by gavage did not alter gastric emptying or small intestinal transit, but luminal AITC inhibited colonic transit via TRPA1. CONCLUSIONS: Functional TRPA1 is expressed by enteric neurons, and activation of neuronal TRPA1 inhibits spontaneous neurogenic contractions and transit of the colon.
Assuntos
Esvaziamento Gástrico/fisiologia , Motilidade Gastrointestinal/fisiologia , Interneurônios/metabolismo , Neurônios Motores/metabolismo , Neurônios Aferentes/metabolismo , RNA Mensageiro/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Potencial de Receptor Transitório/fisiologia , Acroleína/análogos & derivados , Acroleína/farmacologia , Aldeídos/farmacologia , Animais , Carbacol/farmacologia , Ceco/efeitos dos fármacos , Ceco/inervação , Ceco/metabolismo , Ceco/fisiologia , Colo/efeitos dos fármacos , Colo/inervação , Colo/metabolismo , Colo/fisiologia , Duodeno/efeitos dos fármacos , Duodeno/inervação , Duodeno/metabolismo , Duodeno/fisiologia , Células Epiteliais/metabolismo , Feminino , Imunofluorescência , Gânglios/metabolismo , Mucosa Gástrica/metabolismo , Motilidade Gastrointestinal/efeitos dos fármacos , Íleo/efeitos dos fármacos , Íleo/inervação , Íleo/metabolismo , Íleo/fisiologia , Interneurônios/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Isotiocianatos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estômago/efeitos dos fármacos , Estômago/inervação , Estômago/fisiologia , Substância P/farmacologia , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/agonistasRESUMO
Ghrelin, a peptide hormone from the stomach, has been recently discovered to reduce sodium excretion from the kidney. Although the effects on the kidney suggest actions in the distal nephron, the sites of expression of ghrelin receptors have not been localised. In the present work we have used a mouse that expresses green fluorescent protein under the control of the ghrelin receptor promoter to locate sites of receptor expression in the kidney. Receptor expression was confined to the straight parts of the distal tubules and the thin limbs of the loops of Henle. No expression was detected in other structures, including the glomeruli, proximal tubules and collecting ducts. Ghrelin receptors were not found in extra-renal or intra-renal arteries, despite observations that ghrelin is a vasodilator. The distribution revealed by in situ hybridisation histochemistry was the same as that revealed by the reporter. In conclusion, ghrelin receptors have a restricted distribution in the kidney. The location in the straight parts of the distal tubules accords with observations that ghrelin promotes sodium retention.
Assuntos
Regulação da Expressão Gênica/fisiologia , Túbulos Renais Distais/metabolismo , Alça do Néfron/metabolismo , Receptores de Grelina/biossíntese , Animais , Transporte de Íons/fisiologia , Túbulos Renais Distais/citologia , Alça do Néfron/citologia , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/fisiologia , Receptores de Grelina/genética , Sódio/metabolismoRESUMO
BACKGROUND: Sleep/wake disturbances (e.g., insomnia and sleep fragmentation) are common in neurodegenerative disorders, especially Alzheimer's disease (AD) and frontotemporal dementia (FTD). These symptoms are somewhat reminiscent of narcolepsy with cataplexy, caused by the loss of orexin-producing neurons. A bidirectional relationship between sleep disturbance and disease pathology suggests a detrimental cycle that accelerates disease progression and cognitive decline. The accumulation of brain tau fibrils is a core pathology of AD and FTD-tau and clinical evidence supports that tau may impair the orexin system in AD/FTD. This hypothesis was investigated using tau mutant mice. OBJECTIVE: To characterize orexin receptor mRNA expression in sleep/wake regulatory brain centers and quantify noradrenergic locus coeruleus (LC) and orexinergic lateral hypothalamus (LH) neurons, in tau transgenic rTg4510 and tau-/- mice. METHODS: We used i n situ hybridization and immunohistochemistry (IHC) in rTg4510 and tau-/- mice. RESULTS: rTg4510 and tau-/- mice exhibited a similar decrease in orexin receptor 1 (OX1R) mRNA expression in the LC compared with wildtype controls. IHC data indicated this was not due to decreased numbers of LC tyrosine hydroxylase-positive (TH) or orexin neurons and demonstrated that tau invades TH LC and orexinergic LH neurons in rTg4510 mice. In contrast, orexin receptor 2 (OX2R) mRNA levels were unaffected in either model. CONCLUSION: The LC is strongly implicated in the regulation of sleep/wakefulness and expresses high levels of OX1R. These findings raise interesting questions regarding the effects of altered tau on the orexin system, specifically LC OX1Rs, and emphasize a potential mechanism which may help explain sleep/wake disturbances in AD and FTD.
Assuntos
Nível de Alerta , Locus Cerúleo/metabolismo , Receptores de Orexina/metabolismo , Proteínas tau/metabolismo , Animais , Feminino , Região Hipotalâmica Lateral/metabolismo , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/metabolismoRESUMO
1. Previous work indicates that agonists of ghrelin receptors can act within the spinal cord to stimulate autonomic outputs to the colorectum and to blood vessels. Because of the close relationship between colorectal and urinary bladder control, we have investigated whether ghrelin receptor agonists also stimulate spinal centres that influence the bladder. 2. The ghrelin receptor agonist capromorelin (10 mg/kg), injected intravenously in anaesthetized male rats, disrupted the ongoing cycle of micturition reflexes and caused phasic oscillations in pressure that averaged approximately 20 mmHg. Fluid output from the bladder was diminished. The effects of capromorelin were inhibited by hexamethonium (10 mg/kg bolus followed by 4 mg/kg per h infusion, i.v.) and were further reduced by atropine (5 mg/kg bolus followed by 2.5 mg/kg per h infusion, i.v.). Capromorelin (250 microg) injected directly into the spinal cord at the lumbosacral level also increased contractile activity of the bladder. However, capromorelin, up to 0.1 mmol/L, had no effect on the tension of isolated muscle strips from the bladder. Effects of intravenous capromorelin (10 mg/kg) on bladder pressure were still observed after the descending pathways in the spinal cord were disrupted at the thoracic level. 3. In situ hybridization studies revealed ghrelin receptor gene expression in neurons of the autonomic intermediolateral (IML) cell columns. Following a series of micturition reflexes elicited by infusion of saline into the bladder, the immediate early gene product c-Fos was observed in neurons of the lumbosacral IML and approximately 20% of these also expressed ghrelin receptor gene transcripts. 4. It is concluded that ghrelin receptors are expressed by lumbosacral autonomic preganglionic neurons of the micturition reflex pathways and that ghrelin receptor agonists stimulate these neurons.
Assuntos
Fibras Autônomas Pré-Ganglionares/fisiologia , Receptores de Grelina/metabolismo , Micção/fisiologia , Animais , Expressão Gênica , Masculino , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-fos/isolamento & purificação , Proteínas Proto-Oncogênicas c-fos/metabolismo , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Grelina/agonistas , Receptores de Grelina/genética , Reflexo , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiologia , Traumatismos da Medula Espinal , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/imunologia , Bexiga Urinária/fisiologiaRESUMO
Damage following ischemia and reperfusion (I/R) is common in the intestine and can be caused during abdominal surgery, in several disease states and following intestinal transplantation. Most studies have concentrated on damage to the mucosa, although published evidence also points to effects on neurons. Moreover, alterations of neuronally controlled functions of the intestine persist after I/R. The present study was designed to investigate the time course of damage to neurons and the selectivity of the effect of I/R damage for specific types of enteric neurons. A branch of the superior mesenteric artery supplying the distal ileum of anesthetised guinea pigs was occluded for 1 h and the animals were allowed to recover for 2 h to 4 weeks before tissue was taken for the immunohistochemical localization of markers of specific neuron types in tissues from sham and I/R animals. The dendrites of neurons with nitric oxide synthase (NOS) immunoreactivity, which are inhibitory motor neurons and interneurons, were distorted and swollen by 24 h after I/R and remained enlarged up to 28 days. The total neuron profile areas (cell body plus dendrites) increased by 25%, but the sizes of cell bodies did not change significantly. Neurons of type II morphology (intrinsic primary afferent neurons), revealed by NeuN immunoreactivity, were transiently reduced in cell size, at 24 h and 7 days. These neurons also showed signs of minor cell surface blebbing. Calretinin neurons, many of which are excitatory motor neurons, were unaffected. Thus, this study revealed a selective damage to NOS neurons that was observed at 24 h and persisted up to 4 weeks, without a significant change in the relative numbers of NOS neurons.
Assuntos
Plexo Mientérico/patologia , Neurônios/patologia , Traumatismo por Reperfusão/patologia , Animais , Contagem de Células , Feminino , Cobaias , Íleo/irrigação sanguínea , Íleo/inervação , Imuno-Histoquímica , Masculino , Microscopia Confocal , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase/metabolismo , Traumatismo por Reperfusão/metabolismoRESUMO
1. The best characterized mammalian circadian rhythms follow a light-entrained central master pacemaker in the suprachiasmatic nucleus and are associated with fluctuations in the activities of clock genes, including Clock, Bmal1, Per and Cry, the products of which bind to sequences in the promoters of effector genes. This is the central clock. 2. In the present review, we discuss evidence for an independent, but interacting, gut-associated circadian clock, the peripheral clock, which is entrained by food. 3. Disruption of circadian rhythms is associated with a wide range of pathologies, most prominently metabolism linked, but the effects of disruption of circadian rhythms on the digestive system are less well studied, although also likely to lead to functional consequences. There are clues suggestive of links between gastrointestinal disorders related to inflammation, cancer and motility and disruption of peripheral rhythms. Research aimed at understanding these links is still in its infancy. 4. We also discuss practical aspects of the presence of circadian rhythms in gastrointestinal tissues for researchers related to experimental design, data interpretation and the choice of animal models. 5. There is currently sufficient evidence to suggest that circadian rhythms are important to gut function, metabolism and mucosal defence and that further investigation will uncover connections between disordered rhythms and gastrointestinal malfunction.
Assuntos
Ritmo Circadiano/fisiologia , Digestão/fisiologia , Motilidade Gastrointestinal/fisiologia , Animais , Fenômenos Fisiológicos do Sistema Digestório , Gastroenterite/imunologia , Gastroenterite/fisiopatologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/inervação , Trato Gastrointestinal/patologia , Trato Gastrointestinal/fisiopatologia , Humanos , Sistema Imunitário/fisiopatologia , Modelos Biológicos , Fatores de TempoRESUMO
Spinal motor neurons must extend their axons into the periphery through motor exit points (MEPs), but their cell bodies remain within spinal motor columns. It is not known how this partitioning is established in development. We show here that motor neuron somata are confined to the CNS by interactions with a neural crest subpopulation, boundary cap (BC) cells that prefigure the sites of spinal MEPs. Elimination of BC cells by surgical or targeted genetic ablation does not perturb motor axon outgrowth but results in motor neuron somata migrating out of the spinal cord by translocating along their axons. Heterologous neural crest grafts in crest-ablated embryos stop motor neuron emigration. Thus, before the formation of a mature transitional zone at the MEP, BC cells maintain a cell-tight boundary that allows motor axons to cross but blocks neuron migration.
Assuntos
Neurônios Motores/fisiologia , Crista Neural/citologia , Crista Neural/embriologia , Medula Espinal/citologia , Medula Espinal/embriologia , Fatores de Transcrição , Animais , Axônios/fisiologia , Movimento Celular/fisiologia , Embrião de Galinha , Proteínas de Ligação a DNA/genética , Denervação , Camundongos , Camundongos Mutantes , Microcirurgia , Neurônios Motores/ultraestrutura , Crista Neural/transplante , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Codorniz , Raízes Nervosas Espinhais/citologia , Raízes Nervosas Espinhais/embriologiaRESUMO
Insulin-like peptide 5 (INSL5) is a newly discovered gut hormone expressed in colonic enteroendocrine L-cells but little is known about its biological function. Here, we show using RT-qPCR and in situ hybridisation that Insl5 mRNA is highly expressed in the mouse colonic mucosa, colocalised with proglucagon immunoreactivity. In comparison, mRNA for RXFP4 (the cognate receptor for INSL5) is expressed in various mouse tissues, including the intestinal tract. We show that the human enteroendocrine L-cell model NCI-H716 cell line, and goblet-like colorectal cell lines SW1463 and LS513 endogenously express RXFP4. Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr202/Tyr204), AKT (Thr308 and Ser473) and S6RP (Ser235/236) and inhibited cAMP production but did not stimulate Ca2+ release. Acute INSL5 treatment had no effect on GLP-1 secretion mediated by carbachol or insulin, but modestly inhibited forskolin-stimulated GLP-1 secretion in NCI-H716 cells. However, chronic INSL5 pre-treatment (18 h) increased basal GLP-1 secretion and prevented the inhibitory effect of acute INSL5 administration. LS513 cells were found to be unresponsive to INSL5 despite expressing RXFP4 Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of Rxfp4 and were unresponsive to INSL5. This study provides novel insights into possible autocrine/paracrine roles of INSL5 in the intestinal tract.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Insulina/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Colo/metabolismo , AMP Cíclico/biossíntese , Perfilação da Expressão Gênica , Células Caliciformes/metabolismo , Humanos , Insulina/genética , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismoRESUMO
BACKGROUND AND PURPOSE: To better understand opioid signalling in visceral nociceptors, we examined the expression and selective activation of µ and δ opioid receptors by dorsal root ganglia (DRG) neurons innervating the mouse colon. EXPERIMENTAL APPROACH: DRG neurons projecting to the colon were identified by retrograde tracing. δ receptor-GFP reporter mice, in situ hybridization, single-cell RT-PCR and µ receptor-specific antibodies were used to characterize expression of µ and δ receptors. Voltage-gated Ca2+ currents and neuronal excitability were recorded in small diameter nociceptive neurons (capacitance <30 pF) by patch clamp and ex vivo single-unit afferent recordings were obtained from the colon. KEY RESULTS: In situ hybridization of oprm1 expression in Fast Blue-labelled DRG neurons was observed in 61% of neurons. µ and δ receptors were expressed by 36-46% of colon DRG neurons, and co-expressed by ~25% of neurons. µ and δ receptor agonists inhibited Ca2+ currents in DRG, effects blocked by opioid antagonists. One or both agonists inhibited action potential firing by colonic afferent endings. Incubation of neurons with supernatants from inflamed colon segments inhibited Ca2+ currents and neuronal excitability. Antagonists of µ, but not δ receptors, inhibited the effects of these supernatant on Ca2+ currents, whereas both antagonists inhibited their actions on neuronal excitability. CONCLUSIONS AND IMPLICATIONS: A significant number of small diameter colonic nociceptors co-express µ and δ receptors and are inhibited by agonists and endogenous opioids in inflamed tissues. Thus, opioids that act at µ or δ receptors, or their heterodimers may be effective in treating visceral pain.
Assuntos
Colo/metabolismo , Nociceptores/metabolismo , Receptores Opioides delta/biossíntese , Receptores Opioides mu/biossíntese , Animais , Perfilação da Expressão Gênica , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Opioides delta/genética , Receptores Opioides mu/genéticaRESUMO
The cornea is innervated by three main functional classes of sensory neurons: polymodal nociceptors, pure mechano-nociceptors and cold-sensing neurons. Here we explored transient receptor potential cation channel subfamily V member 1 (TRPV1) expression in guinea pig corneal sensory neurons, a widely used molecular marker of polymodal nociceptors. We used retrograde tracing to identify corneal afferent neurons in the trigeminal ganglion (TG) and double label in situ hybridization and/or immunohistochemistry to determine their molecular profile. In addition, we used immunohistochemistry to reveal the neurochemistry and structure of TRPV1 expressing nerve endings in the corneal epithelium. Approximately 45% of corneal afferent neurons expressed TRPV1, 28% expressed Piezo2 (a marker of putative pure mechano-nociceptors) and 8% expressed the transient receptor potential cation channel subfamily M member 8 (TRPM8; a marker of cold-sensing neurons). There was no co-expression of TRPV1 and Piezo2 in corneal afferent neurons, but 6% of TRPV1 neurons co-expressed TRPM8. The TRPV1 expressing corneal afferent neurons could be divided into three subpopulations on the basis of calcitonin gene-related peptide (CGRP) and/or or glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3) co-expression. In the corneal epithelium, the TRPV1 axons that co-expressed CGRP and GFRα3 ended as simple unbranched endings in the wing cell layer. In contrast, those that only co-expressed GFRα3 had ramifying endings that branched and terminated in the squamous cell layer, whereas those that only co-expressed CGRP had simple endings in the basal epithelium. This study shows that the majority of TRPV1 expressing corneal afferent neurons (>90%) are likely to be polymodal nociceptors. Furthermore, TRPV1 expressing corneal afferent neurons can be subdivided into specific subpopulations based on their molecular phenotype, nerve terminal morphology and distribution in the corneal epithelium.
RESUMO
Recently, a novel class of mechanically sensitive channels has been identified and have been called Piezo channels. In this study, we explored Piezo channel expression in sensory neurons supplying the guinea pig corneal epithelium, which have well-defined modalities in this species. We hypothesized that a proportion of corneal afferent neurons express Piezo2, and that these neurons are neurochemically distinct from corneal polymodal nociceptors or cold-sensing neurons. We used a combination of retrograde tracing to identify corneal afferent neurons and double label in situ hybridization and/or immunohistochemistry to determine their molecular and/or neurochemical profile. We found that Piezo2 expression occurs in â¼26% of trigeminal ganglion neurons and 30% of corneal afferent neurons. Piezo2 corneal afferent neurons are almost exclusively non-calcitonin gene-related peptide (CGRP)-immunoreactive (-IR), medium- to large-sized neurons that are NF200-IR, suggesting they are not corneal polymodal nociceptors. There was no coexpression of Piezo2 and transient receptor potential cation channel subfamily M member 8 (TRPM8) transcripts in any corneal afferent neurons, further suggesting that Piezo2 is not expressed in corneal cold-sensing neurons. We also noted that TRPM8-IR or CGRP-IR corneal afferent neurons are almost entirely small and lack NF200-IR. Piezo2 expression occurs in a neurochemically distinct subpopulation of corneal afferent neurons that are not polymodal nociceptors or cold-sensing neurons, and is likely confined to a subpopulation of pure mechano-nociceptors in the cornea. This provides the first evidence in an in vivo system that Piezo2 is a strong candidate for a channel that transduces noxious mechanical stimuli.
Assuntos
Córnea/anatomia & histologia , Canais Iônicos/genética , Canais Iônicos/metabolismo , Neurônios Aferentes/metabolismo , Amidinas , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Córnea/inervação , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Feminino , Cobaias , Masculino , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Gânglio Trigeminal/metabolismoRESUMO
There is ambiguity concerning the distribution of neurons that express the ghrelin receptor (GHSR) in the medulla oblongata. In the current study we used a sensitive nonradioactive method to investigate GHSR mRNA distribution by in situ hybridization. Strong expression of the GHSR gene was confirmed in neurons of the facial nucleus (FacN, 7), the dorsal vagal complex (DVC), and the semicompact (but not compact) nucleus ambiguus (AmbSC and AmbC). In addition, expression of GHSR was found in other regions, where it had not been described before. GHSR-positive neurons were observed in the gustatory rostral nucleus tractus solitarius and in areas involved in vestibulo-ocular processing (such as the medial vestibular nucleus and the nucleus abducens). GHSR expression was also noted in ventral areas associated with cardiorespiratory control, including the gigantocellular reticular nucleus, the lateral paragigantocellular nucleus, the rostral and caudal ventrolateral medulla, the (pre)-Bötzinger complex, and the rostral and caudal ventrolateral respiratory group. However, GHSR-positive neurons in ventrolateral areas did not express markers for cardiovascular presympathetic vasomotor neurons, respiratory propriobulbar rhythmogenic neurons, or sensory interneurons. GHSR-positive cells were intermingled with catecholamine neurons in the dorsal vagal complex but these populations did not overlap. Thus, the ghrelin receptor occurs in the medulla oblongata in 1) second-order sensory neurons processing gustatory, vestibulo-ocular, and visceral sensation; 2) cholinergic somatomotor neurons of the FacN and autonomic preganglionic neurons of the DMNX and AmbSC; 3) cardiovascular neurons in the DVC, Gi, and LPGi; 4) neurons of as yet unknown function in the ventrolateral medulla.
Assuntos
Expressão Gênica/genética , Bulbo/citologia , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Receptores de Grelina/genética , Animais , Masculino , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/fisiologia , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Receptores de Grelina/metabolismoRESUMO
An acute enteritis is commonly followed by intestinal neuromuscular dysfunction, including prolonged hyperexcitability of enteric neurons. Such motility disorders are associated with maintained increases in immune cells adjacent to enteric ganglia and in the mucosa. However, whether the commonly used animal model, trinitrobenzene sulphonate (TNBS)-induced enteritis, causes histological and immune cell changes similar to human enteric neuropathies is not clear. We have made a detailed study of the mucosal damage and repair and immune cell invasion following intralumenal administration of TNBS. Intestines from untreated, sham-operated and TNBS-treated animals were examined at 3 h to 56 days. At 3 h, the mucosal surface was completely ablated, by 6 h an epithelial covering was substantially restored and by 1 day there was full re-epithelialisation. The lumenal epithelium developed from a squamous cell covering to a fully differentiated columnar epithelium with mature villi at about 7 days. Prominent phagocytic activity of enterocytes occurred at 1-7 days. A surge of eosinophils and T lymphocytes associated with the enteric nerve ganglia occurred at 3 h to 3 days. However, elevated immune cell numbers occurred in the lamina propria of the mucosa until 56 days, when eosinophils were still three times normal. We conclude that the disruption of the mucosal surface that causes TNBS-induced ileitis is brief, a little more than 6 h, and causes a transient immune cell surge adjacent to enteric ganglia. This is much briefer than the enteric neuropathy that ensues. Ongoing mucosal inflammatory reaction may contribute to the persistence of enteric neuropathy.
Assuntos
Enterite/patologia , Eosinófilos/fisiologia , Íleo/patologia , Mucosa Intestinal/patologia , Linfócitos T/fisiologia , Doença Aguda , Animais , Enterite/imunologia , Feminino , Cobaias , Íleo/imunologia , Mucosa Intestinal/imunologia , Masculino , Fagócitos/fisiologia , Regeneração , Ácido TrinitrobenzenossulfônicoRESUMO
BACKGROUND: In developing neurons, somal migration and initiation of axon outgrowth often occur simultaneously and are regulated in part by similar classes of molecules. When neurons reach their final destinations, however, somal translocation and axon extension are uncoupled. Insights into the mechanisms underlying this process of disengagement came from our study of the behaviour of embryonic spinal motor neurons following ablation of boundary cap cells. These are neural crest derivatives that transiently reside at motor exit points, central nervous system (CNS):peripheral nervous system (PNS) interfaces where motor axons leave the CNS. In the absence of boundary cap cells, motor neuron cell bodies migrate along their axons into the periphery, suggesting that repellent signals from boundary cap cells regulate the selective gating of somal migration and axon outgrowth at the motor exit point. Here we used RNA interference in the chick embryo together with analysis of null mutant mice to identify possible boundary cap cell ligands, their receptors on motor neurons and cytoplasmic signalling molecules that control this process. RESULTS: We demonstrate that targeted knock down in motor neurons of Neuropilin-2 (Npn-2), a high affinity receptor for class 3 semaphorins, causes their somata to migrate to ectopic positions in ventral nerve roots. This finding was corroborated in Npn-2 null mice, in which we identified motor neuron cell bodies in ectopic positions in the PNS. Our RNA interference studies further revealed a role for Plexin-A2, but not Plexin-A1 or Plexin-A4. We show that chick and mouse boundary cap cells express Sema3B and 3G, secreted semaphorins, and Sema6A, a transmembrane semaphorin. However, no increased numbers of ectopic motor neurons are found in Sema3B null mouse embryos. In contrast, Sema6A null mice display an ectopic motor neuron phenotype. Finally, knockdown of MICAL3, a downstream semaphorin/Plexin-A signalling molecule, in chick motor neurons led to their ectopic positioning in the PNS. CONCLUSION: We conclude that semaphorin-mediated repellent interactions between boundary cap cells and immature spinal motor neurons regulates somal positioning by countering the drag exerted on motor neuron cell bodies by their axons as they emerge from the CNS at motor exit points. Our data support a model in which BC cell semaphorins signal through Npn-2 and/or Plexin-A2 receptors on motor neurons via a cytoplasmic effector, MICAL3, to trigger cytoskeletal reorganisation. This leads to the disengagement of somal migration from axon extension and the confinement of motor neuron cell bodies to the spinal cord.
Assuntos
Moléculas de Adesão Celular/metabolismo , Movimento Celular/genética , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Semaforinas/metabolismo , Medula Espinal/embriologia , Medula Espinal/metabolismo , Animais , Moléculas de Adesão Celular/genética , Diferenciação Celular/genética , Embrião de Galinha , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Inibidores do Crescimento/genética , Inibidores do Crescimento/metabolismo , Ligantes , Camundongos , Proteínas dos Microfilamentos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neuropilina-2/genética , Neuropilina-2/metabolismo , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/embriologia , Sistema Nervoso Periférico/metabolismo , Interferência de RNA/fisiologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Semaforinas/genética , Transdução de Sinais/genética , Medula Espinal/citologiaRESUMO
The chick embryo is widely used for the study of vertebrate development, but a general, reliable loss-of-function strategy for the analysis of gene function is currently not available. By using small inhibitory hairpin RNA (siRNA) molecules generated by the mouse U6 promoter, we have applied an RNA interference approach to achieve quantitative knockdown of the neuropilin-1 (Nrp-1) receptor in chick embryos. Functional knockdown was evident in the abolition of Sema3A-induced growth cone collapse in Nrp-1-siRNA but not Nrp-2-siRNA-expressing dorsal root ganglion (DRG) neurons. Two nervous system defects in Nrp-1 mutant mice were phenocopied in embryos treated with Nrp-1 siRNA. First, DRG axons prematurely entered the dorsal horn and projected inappropriately. Second, targeted early migrating neural crest cells destined for the sympathetic chain arrested ectopically within ventral spinal nerve roots. Localized knockdown induced by specific siRNA constructs will allow rapid functional analysis of genes regulating chick neural development whilst circumventing embryonic lethal effects often associated with global gene knockout in the mouse.
Assuntos
Galinhas/genética , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Neuropilina-1/deficiência , Neuropilina-1/genética , RNA Interferente Pequeno/metabolismo , Animais , Aves , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Gânglios Espinais/citologia , Gânglios Espinais/embriologia , Gânglios Espinais/metabolismo , Deleção de Genes , Vetores Genéticos/genética , Técnicas In Vitro , Camundongos , Camundongos Knockout , Sistema Nervoso/citologia , Crista Neural/citologia , Crista Neural/metabolismo , Neuropilina-1/metabolismo , Conformação de Ácido Nucleico , Fenótipo , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Nuclear Pequeno/genéticaRESUMO
We have analyzed signaling pathways involved in neurotrophic factor (NTF)-induced upregulation of nociceptive properties, specifically vanilloid receptor type 1 (VR1), by adult rat dorsal root ganglion neurons. Upregulation of VR1 by nerve growth factor and glial cell line-derived neurotrophic factor is partially blocked by a MEK inhibitor. Dominant negative Ras, but not Rap, blocks NTF-induced ERK activation and VR1 upregulation. Activated Ras mimics NTF-mediated induction of VR1 in dorsal root ganglion neurons. An inhibitor of phosphatidylinositol 3-kinase, LY294002, also inhibited NTF-induced VR1 upregulation. However, this may at least in part be due to a block of NTF-induced ERK activation. Constitutive simultaneous stimulation of both ERK and phosphatidylinositol 3-kinase is not sufficient for VR1 upregulation. Together, the data suggest that VR1 expression by dorsal root ganglion neurons is regulated by common Ras-dependent pathways.