RESUMEN
Osteoporosis and bone fractures occur at higher frequency in patients with inflammatory bowel disease (IBD), and decreased bone mass is observed in animal models of colitis. Another consistent feature of colitis is increased serotonin (5-HT) availability in the intestinal mucosa. Since gut-derived 5-HT can decrease bone mass, via activation of 5-HT1B receptors on pre-osteoblasts, we tested the hypothesis that 5-HT contributes to bone loss in colitis. Colitis was chronically induced in mice by adding dextran sodium sulfate (DSS) to their drinking water for 21 days. At day 21, circulating 5-HT levels were elevated in DSS-inflamed mice. Micro-computed tomography of femurs showed a decrease in trabecular bone volume fraction, formation, and surface area, due largely to decreased trabecular numbers in DSS-treated mice. The colitis-induced loss of trabecular bone was significantly suppressed in mice treated with the 5-HT synthesis inhibitor, p-chloro-DL-phenylalanine (PCPA; 300 mg/kg/day IP daily), and in mice treated with the 5-HT1B receptor antagonist GR55562 (1 mg/Kg/day SC daily). The 5-HT reuptake transporter (SERT) is critical for moving 5-HT from the interstitial space into enterocytes and from serum into platelets. Mice lacking SERT exhibited significant deficits in trabecular bone mass that are similar to those observed in DSS-inflamed mice, and these deficits were not extensively worsened by DSS-induced colitis in the SERT-/- mice. Taken together, findings from both the DSS and SERT-/- mouse models support a contributing role for 5-HT as a significant factor in bone loss induced by colitis.
Asunto(s)
Resorción Ósea/metabolismo , Colitis/metabolismo , Serotonina/metabolismo , Animales , Resorción Ósea/diagnóstico por imagen , Colitis/inducido químicamente , Colitis/patología , Sulfato de Dextran , Fémur/diagnóstico por imagen , Fémur/patología , Mucosa Intestinal/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Microtomografía por Rayos XRESUMEN
BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that, in addition to motor, sensory, and cognitive symptoms, also causes constipation, which is poorly understood. Here, we characterize gastrointestinal (GI) dysmotility in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and evaluate whether autoantibodies target the enteric nervous system (ENS) and cause dysmotility. METHODS: EAE was induced in male SJL and B6 mice. GI motility was assessed in vivo and ex vivo in wild type (WT) and B cell-deficient mice. MS and EAE serum was used to survey potential targets in the ENS and changes in the ENS structure were characterized using immunohistochemistry. KEY RESULTS: EAE mice developed accelerated gastric emptying and delayed whole GI transit with reduced colonic motility. Fecal water content was reduced, and colonic migrating myoelectrical complexes (CMMC) and slow waves were less frequent. Colons from EAE mice exhibited decreased GFAP levels in glia. Sera from MS patients and from EAE mice targeted ENS neurons and glia. B-cell deficiency in EAE protected against colonic dysmotility. CONCLUSIONS & INFERENCES: Consistent with symptoms experienced in MS, we demonstrate that EAE mice widely exhibit features of GI dysmotility that persisted in the absence of extrinsic innervation, suggesting direct involvement of ENS neurocircuitry. The absence of GI dysmotility in B cell-deficient mice with EAE together with EAE and MS serum immunoreactivity against ENS targets suggests that MS could be classified among other diseases known to induce autoimmune GI dysmotility.
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Autoanticuerpos/inmunología , Estreñimiento/inmunología , Encefalomielitis Autoinmune Experimental/complicaciones , Encefalomielitis Autoinmune Experimental/inmunología , Motilidad Gastrointestinal/inmunología , Animales , Sistema Nervioso Entérico/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/inmunología , Neuroglía/inmunología , Neuronas/inmunologíaRESUMEN
Persistent changes in gastrointestinal motility frequently accompany the resolution of colitis, through mechanisms that remain to be determined. Trinitrobenzene sulfonic acid (TNBS) colitis in the guinea pig decreases the rate of propulsive motility, causes hyperexcitability of AH neurons, and induces synaptic facilitation. The changes in motility and AH neurons are sensitive to cyclooxygenase-2 (COX-2) inhibition. The aim of this investigation was to determine if the motility and neurophysiological changes persist following recovery from colitis. Evaluations of inflammation, colonic motility and intracellular electrophysiology of myenteric neurons 8 weeks after TNBS administration were performed and compared to matched control conditions. Myeloperoxidase levels in the colons were comparable to control levels 56 days after TNBS treatment. At this time point, the rate of colonic motility was decreased relative to controls following treatment with TNBS alone or TNBS plus a COX-2 inhibitor. Furthermore, the electrical properties of AH neurons and fast synaptic potentials in S neurons were significantly different from controls and comparable to those detected during active inflammation. Collectively, these data suggest that altered myenteric neurophysiology initiated during active colitis persists long term, and provide a potential mechanism underlying altered gut function in individuals during remission from inflammatory bowel disease.
Asunto(s)
Colitis/fisiopatología , Motilidad Gastrointestinal/fisiología , Plexo Mientérico/fisiopatología , Neuronas/metabolismo , Ácido Trinitrobencenosulfónico/toxicidad , Animales , Colitis/inducido químicamente , Electrofisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Cobayas , Masculino , Plexo Mientérico/efectos de los fármacos , Neuronas/efectos de los fármacosRESUMEN
The aim of this investigation was to characterize the electrical properties of neurons in the rat major pelvic ganglia (MPG) using intracellular recording techniques. MPG were dissected from male rats euthanized by isoflurane and thoracotomy. Neurons were classified as "phasic" or "tonic" according to their rate of accommodation during a 500-ms depolarizing current pulse. Phasic cells were further subdivided into rapidly or slowly adapting. The firing pattern of tonic cells was divided into regular high frequency, low frequency or irregular firing. In tonic cells, onset spikes showed TTX-resistant discharges; whereas sustained spikes were TTX sensitive. Changing the current pulse amplitude or the stimulation interval could alter the firing pattern in both types of neurons. Subthreshold membrane potential oscillations (SMPOs) were primarily observed when neurons were depolarized. SMPOs were Na(+) dependent and TTX sensitive. The majority of tonic and phasic neurons generated rebound spikes, most of which were partially Na(+) dependent. A small percentage (<6%) of neurons exhibited spontaneous activity. Taken together these findings are consistent with the concept that neurons in the MPG exhibit heterogeneous electrical properties.
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Potenciales de Acción/fisiología , Ganglios Autónomos/citología , Ganglios Autónomos/fisiología , Neuronas/fisiología , Diafragma Pélvico/inervación , Animales , Estimulación Eléctrica , Genitales Masculinos/inervación , Masculino , Ratas , Ratas Wistar , Vejiga Urinaria/inervaciónRESUMEN
BACKGROUND: Serotonin (5-hydroxytryptamine, 5-HT) is an important mediator of every major gut-related function. Recent investigations also suggest that 5-HT can influence the development and severity of inflammation within the gut, particularly in the setting of inflammatory bowel disease (IBD). AIM: To review the roles that the intestinal serotonin signalling system plays in gut function, with a specific focus on IBD. METHODS: We reviewed manuscripts from 1952 to 2017 that investigated and discussed roles for 5-HT signalling in gastrointestinal function and IBD, as well as the influence of inflammation on 5-HT signalling elements within the gut. RESULTS: Inflammation appears to affect every major element of intestinal 5-HT signalling, including 5-HT synthesis, release, receptor expression and reuptake capacity. Importantly, many studies (most utilising animal models) also demonstrate that modulation of selective serotonergic receptors (via agonism of 5-HT4 R and antagonism of 5-HT3 R) or 5-HT signal termination (via serotonin reuptake inhibitors) can alter the likelihood and severity of intestinal inflammation and/or its complicating symptoms. However, there are few human studies that have studied these relationships in a targeted manner. CONCLUSIONS: Insights discussed in this review have strong potential to lead to new diagnostic and therapeutic tools to improve the management of IBD and other related disorders. Specifically, strategies that focus on modifying the activity of selective serotonin receptors and reuptake transporters in the gut could be effective for controlling disease activity and/or its associated symptoms. Further studies in humans are required, however, to more completely understand the pathophysiological mechanisms underlying the roles of 5-HT in this setting.
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Enfermedades Inflamatorias del Intestino/fisiopatología , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Animales , Humanos , Mucosa Intestinal/metabolismo , Antagonistas de la Serotonina/farmacologíaRESUMEN
Alterations in motility, secretion and visceral sensation are hallmarks of irritable bowel syndrome. As all of these aspects of gastrointestinal function involve serotonin signalling between enterochromaffin cells and sensory nerve fibres in the mucosal layer of the gut, potential alterations in mucosal serotonin signalling have been explored as a possible mechanism of altered function and sensation in irritable bowel syndrome. Literature related to intestinal serotonin signalling in normal and pathophysiological conditions has been searched and summarized. Elements of serotonin signalling that are altered in irritable bowel syndrome include: enterochromaffin cell numbers, serotonin content, tryptophan hydroxylase message levels, 5-hydroxyindoleacedic acid levels, serum serotonin levels and expression of the serotonin-selective reuptake transporter. Both genetic and epigenetic factors could contribute to decreased serotonin-selective reuptake transporter in irritable bowel syndrome. A serotonin-selective reuptake transporter gene promoter polymorphism may cause a genetic predisposition, and inflammatory mediators can induce serotonin-selective reuptake transporter downregulation. While a psychiatric co-morbidity exists with IBS, changes in mucosal serotonin handling support the concept that there is a gastrointestinal component to the aetiology of irritable bowel syndrome. Additional studies will be required to gain a more complete understanding of changes in serotonin signalling that are occurring, their cause and effect relationship, and which of these changes have pathophysiological consequences.
Asunto(s)
Mucosa Intestinal/metabolismo , Síndrome del Colon Irritable/metabolismo , Serotonina/metabolismo , Transducción de Señal/fisiología , Motilidad Gastrointestinal , Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Inflamación , Mucosa Intestinal/inmunología , Síndrome del Colon Irritable/genética , Síndrome del Colon Irritable/inmunología , Polimorfismo Genético , Serotonina/genéticaRESUMEN
Serotonin-selective reuptake transporter (SERT) expression is decreased in animal models of intestinal inflammation and in individuals with inflammatory bowel disease (IBD) or irritable bowel syndrome (IBS), and it is possible that resultant changes in intestinal serotonin signalling contribute to the manifestation of clinical features associated with these disorders. The objective of this investigation was to determine whether inhibition of SERT function leads to changes in gut motility and sensitivity. Mice underwent a 14-day treatment with the SERT inhibitor, paroxetine (20 mg kg(-1)), or vehicle (saline/propylene glycol). Gastrointestinal (GI) transit following charcoal gavage, colonic motility, stool frequency and visceromotor responses to colorectal distension were evaluated. In mice treated with paroxetine, stool output was decreased, upper GI transit was delayed, and colonic sensitivity to a nociceptive stimulus was attenuated. These results demonstrate that reduced SERT function (via pharmacological blockade) significantly alters GI motility and sensitivity in mice, and support the concept that altered SERT expression and function could contribute to symptoms associated with IBS and IBD.
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Colon/metabolismo , Motilidad Gastrointestinal/fisiología , Umbral del Dolor/fisiología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Animales , Colon/efectos de los fármacos , Electromiografía , Motilidad Gastrointestinal/efectos de los fármacos , Enfermedades Inflamatorias del Intestino/fisiopatología , Síndrome del Colon Irritable/fisiopatología , Masculino , Ratones , Umbral del Dolor/efectos de los fármacos , Paroxetina/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacologíaRESUMEN
UNLABELLED: mu-Opiate receptor agonists, such as loperamide, influence biliary excretion and suppress cholecystokinin (CCK)-induced gallbladder contraction. Loperamide decreases cholinergic mechanisms, like pancreatic polypeptide (PP) release, while muscarinic agonist (bethanechol)-induced PP release remains unaffected. The effects of loperamide on gallbladder contraction and peptide release were performed to resolve this discrepancy. METHODS: Six subjects (27.6 +/- 2.0 years) received bethanechol (12.5, 25 and 50 microg kg(-1) h(-1) continuously over 40 min) after oral 16 mg loperamide (vs placebo) in a crossover design. Gallbladder volume and plasma levels of CCK, PP, motilin, gastrin, neurotensin, cholylglycine were measured regularly. RESULTS: Bethanechol significantly reduced gallbladder volume (26.7 +/- 1.9 to a nadir of 15.3 +/- 2.2 mL, P < or = 0.05), and this action was inhibited by loperamide. Basal CCK levels increased significantly after loperamide. Incremental integrated CCK release after bethanechol was higher under loperamide (P < or = 0.05), as placebo CCK release was significantly decreased under bethanechol (2.0 +/- 0.4-0.8 +/- 0.3 pmol L(-1)). In both settings, PP levels were significantly increased after bethanechol, while release of neurotensin, motilin, gastrin and cholylglycine was unaffected. CONCLUSION: The mu-opiate receptor agonist loperamide inhibits bethanechol-induced gallbladder contraction. This effect is not mediated by inhibition of CCK release, as loperamide even enhances basal CCK plasma levels. As cholinergic mechanisms, like bethanechol-induced incremental PP release, were unaffected, mu-opiate agonists might influence gallbladder contraction via vagal-cholinergic pathways.
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Betanecol/farmacología , Colecistoquinina/sangre , Vesícula Biliar/fisiología , Loperamida/farmacología , Contracción Muscular/efectos de los fármacos , Receptores Opioides mu/agonistas , Adulto , Estudios Cruzados , Vesícula Biliar/efectos de los fármacos , Humanos , Masculino , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Polipéptido Pancreático/sangre , Polipéptido Pancreático/metabolismo , PlacebosRESUMEN
Regulated release of serotonin (5-HT) from enterochromaffin (EC) cells activates neural reflexes that are involved in gut motility, secretion, vascular perfusion and sensation. The 5-HT-selective reuptake transporter (SERT) terminates serotonergic signalling in the intestinal mucosa. The aim of this investigation was to determine whether mucosal 5-HT content, release, and/or reuptake are altered in a murine model of immune cell-mediated colitis. Experiments were conducted 6 days after colitis was induced by 2,4,6-trinitrobenzene sulfonic acid, a time point when macroscopic and histological damage scores indicated significant inflammation. During inflammation, SERT transcript levels and immunoreactivity were reduced, and the uptake of [3H] 5-HT was impaired. Increases in mucosal 5-HT content and the number of 5-HT-immunoreactive mast cells in the lamina propria were also detected in the inflamed region, whereas EC cell numbers did not change. Mucosal 5-HT released under basal and stimulated conditions was unchanged in animals with colitis. These data suggest that murine colitis alters 5-HT signalling by increasing 5-HT availability through decreased 5-HT uptake by mucosal epithelial cells. These findings support the concept that altered 5-HT signalling could be a contributing factor in altered gut function and sensitivity in inflammatory bowel disease.
Asunto(s)
Colitis/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Serotonina/metabolismo , Animales , Colitis/inducido químicamente , Colitis/patología , Modelos Animales de Enfermedad , Células Enterocromafines/metabolismo , Inmunohistoquímica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Mastocitos/metabolismo , Ratones , ARN Mensajero/análisis , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Transcripción Genética , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
The most common functional disorders of the biliary tract and pancreas are associated with disordered motility of the sphincter of Oddi (SO). The SO is a neuromuscular structure located at the junction of the bile and pancreatic ducts with the duodenum. The primary functions of the SO are to regulate the delivery of bile and pancreatic juice into the duodenum, and to prevent the reflux of duodenal contents into the biliary and pancreatic systems. Disordered motility of the SO leads to the common and painful clinical conditions of SO dysfunction and acute pancreatitis. In order to understand normal SO motility, studies have been performed addressing SO function, control of spontaneous SO activity, responses to bioactive agents, SO innervation, and reflexes with other gastrointestinal organs. These studies have led to the current understanding of how the SO functions and may permit the development of targeted therapy for SO dysfunction and acute pancreatitis. This review summarizes the current knowledge regarding the control and regulation of SO motility, highlighting laboratory based and clinical research performed over the last 5 years.
Asunto(s)
Esfínter de la Ampolla Hepatopancreática/anatomía & histología , Esfínter de la Ampolla Hepatopancreática/fisiología , Animales , Motilidad Gastrointestinal/fisiología , Humanos , Esfínter de la Ampolla Hepatopancreática/fisiopatologíaRESUMEN
This investigation was conducted to establish whether guinea-pig trinitrobenzene sulfonic acid (TNBS)-colitis was associated with a change in the number of neurones of the myenteric plexus, and, if so, whether select subpopulations of neurones were affected. Total neurones were quantified with human (Hu) antiserum, and subpopulations were evaluated with antisera directed against choline acetyltransferase, nitric oxide synthase, calretinin, neuronal nuclear protein or vasoactive intestinal peptide (VIP). Colitis was associated with a loss of 20% of the myenteric neurones, most of which occurred during the first 12 h past-TNBS administration. During this period, myenteric ganglia were infiltrated with neutrophils while lymphocytes appeared at a later time-point. The neuronal loss persisted at a 56-day time-point, when inflammation had resolved. The decrease in myenteric neurones was not associated with a decrease in any given subpopulation of neurones, but the proportion of VIP-immunoreactive neurones increased 6 days following TNBS administration and returned to the control range at the 56 days. These findings indicate that there is an indiscriminant loss of myenteric neurones that occurs during the onset of TNBS-colitis, and the loss of neurones may be associated with the appearance of neutrophils in the region.
Asunto(s)
Colon/efectos de los fármacos , Enfermedades del Colon/inducido químicamente , Plexo Mientérico/efectos de los fármacos , Neuronas/patología , Ácido Trinitrobencenosulfónico/toxicidad , Animales , Colon/fisiopatología , Enfermedades del Colon/patología , Cobayas , Inflamación , Plexo Mientérico/patología , Neuronas/efectos de los fármacosRESUMEN
Although a well-developed plexus of nerves and ganglia is known to be present in the wall of the gallbladder, little has previously been learned about the function or organization of this innervation. The current study was undertaken in order to evaluate the hypothesis that the ganglionated plexus of the gallbladder is analogous to elements of the enteric nervous system (ENS). The ganglionated plexus of the gallbladder was found to resemble closely the submucosal plexus of the small intestine in its organization into two irregular anastomosing and interwoven networks of ganglia, in the numbers of neurons per ganglion, and in the manifestation of histochemically demonstrable acetylcholinesterase activity in virtually all ganglion cells. In common with enteric ganglia, laminin immunoreactivity was observed to be excluded from the interiors of gallbladder ganglia, which were surrounded by a periganglionic laminin-immunoreactive sheath. As in the submucosal plexus, intrinsic substance P-, vasoactive intestinal polypeptide (VIP)-, and neuropeptide Y (NPY)-immunoreactive neurons were seen in the ganglionated plexus of the gallbladder. Extrinsic nerves in the gallbladder that degenerated following chemical sympathectomy with 6-hydroxydopamine (6-OHDA), and which contained NPY, tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH) immunoreactivities, formed a perivascular plexus closely associated with blood vessels. Endogenous catecholamines could also be demonstrated in these perivascular nerves by aldehyde-induced histofluorescence. In addition to perivascular nerves, paravascular nerve bundles were observed that were loosely associated with vessels, did not degenerate following administration of 6-OHDA, and contained NPY immunoreactivity. Other paravascular nerves, probably visceral sensory axons, coexpressed substance P and calcitonin-gene-related peptide (CGRP) immunoreactivities. The ganglionated plexus of the gallbladder resembled enteric ganglia in having intrinsic 5-hydroxytryptamine (5-HT)-immunoreactive cells and highly varicose nerve fibers. The 5-HT-immunoreactive gallbladder axons were, like those of the gut, resistant to 6-OHDA, and separate from fibers that expressed TH immunoreactivity. Differences between the ganglionated plexus of the gallbladder and enteric ganglia of the small intestine included in the gallbladder are 1) the presence of TH-immunoreactive cells that contain an endogenous catecholamine, but not DBH; 2) DBH-immunoreactive neurons, some of which coexpress substance P immunoreactivity, but which contain neither a catecholamine nor TH immunoreactivity; 3) an apparent absence of CGRP-immunoreactive cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Vesícula Biliar/anatomía & histología , Cobayas/anatomía & histología , Intestinos/inervación , Plexo Mientérico/anatomía & histología , Neuropéptidos/fisiología , Plexo Submucoso/anatomía & histología , Nervio Vago/anatomía & histología , Acetilcolinesterasa/fisiología , Vías Aferentes/anatomía & histología , Animales , Dopamina beta-Hidroxilasa/fisiología , Técnica del Anticuerpo Fluorescente , Ganglios Parasimpáticos/anatomía & histología , Ganglios Espinales/anatomía & histología , Laminina/fisiología , Masculino , Fibras Nerviosas/anatomía & histología , Neuronas/ultraestructura , Serotonina/fisiología , Tirosina 3-Monooxigenasa/fisiologíaRESUMEN
The cytochemical technique for the demonstration of cytochrome oxidase has been used to locate the sites of chronically active neurons in the CNS. The current experiments were undertaken to validate the use of this technique for the identification of active neurons and ganglia in the myenteric plexus of the gut. The excitotoxin veratridine was used to cause repetitive firing of action potentials and to depolarize myenteric neurons over an 8-hour test period in vitro. The effects of veratridine were monitored electrophysiologically and correlated with the ability of the drug to affect the density of the cytochrome oxidase reaction product, as well as the proportion of reactive myenteric neurons. The action of veratridine on cytochrome oxidase activity was evaluated by means of video microdensitometry and computer-assisted morphometry. Effects of veratridine were considered to be specific if they were blocked by the inclusion of tetrodotoxin (TTX), which antagonizes the action of veratridine on voltage-dependent Na+ channels. Veratridine (1.0 microM) depolarized the membranes of myenteric type II neurons and greatly increased both the average density of the cytochrome oxidase reaction product in individual neuronal cell bodies and the proportion that were reactive. The concentration-effect relationship for veratridine was biphasic. As the concentration of veratridine was raised, neurons became sufficiently depolarized such that they ceased to fire action potentials. This absence of spiking was associated with a decline in the stimulation of cytochrome oxidase activity by veratridine. At still higher concentrations of veratridine the degree of neuronal depolarization continued to increase. This further depolarization was associated with a secondary increase in cytochrome oxidase activity. All effects of veratridine were blocked by TTX. It is concluded that the cytochemical demonstration of cytochrome oxidase reflects the recent metabolic history and thus the physiological activity of myenteric neurons. In control preparations, fixed without exposure to veratridine, considerable variation in cytochrome oxidase was observed between ganglia and between individual neurons within ganglia. This suggests that there are significant differences in the activity of individual ganglia and neurons of the myenteric plexus. These observations support the hypothesis that there is a functional heterogeneity that corresponds to an anatomical heterogeneity (previously demonstrated) of the myenteric plexus.
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Complejo IV de Transporte de Electrones/metabolismo , Plexo Mientérico/enzimología , Animales , Cobayas , Histocitoquímica , Masculino , Plexo Mientérico/citología , Plexo Mientérico/fisiología , Tetrodotoxina/farmacología , Veratridina/farmacologíaRESUMEN
This study was undertaken to examine the morphological features of cells within ganglia of the guinea pig gallbladder, and to examine the ultrastructure of the ganglionated plexus. Gallbladder neurons are large, with a relatively simple form, having only one or two major processes. Neurobiotin often filled axons to their varicose arbors on smooth muscle in close proximity to the interganglionic connectives. With the exception of connective tissue clefts that sometimes penetrated into them, ganglia were devoid of intercellular spaces, capillaries, or connective tissue elements such as collagen and basal laminae. However, ganglia were surrounded by a single, continuous basal lamina that was enclosed within a fibroblast and collagen capsule. Within ganglia, neurons were insulated by the processes of cells that resembled the astrocyte-like glia of enteric ganglia. Although few classical synapses were observed, numerous sites of direct apposition were identified between vesicle-rich profiles and processes of gallbladder neurons. Direct appositions between vesicle-rich profiles and the ganglion-limiting basal laminae were also observed. Vesiculated profiles contained small clear vesicles and large dense-core vesicles. Within interganglionic connectives, axons were unmyelinated and were isolated from one another by processes of glia that resembled Schwann cells. As was seen in the ganglia, direct appositions between vesicle-rich profiles and the connective-limiting basal laminae were observed. The results of this study demonstrate that gallbladder ganglia are similar, ultrastructurally, to enteric ganglia in the CNS-like composition of the neuropil. However, the greater degree of glial investment, lesser degree of innervation, and simpler neurons indicated differences from the enteric nervous system that may be functionally significant.
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Vesícula Biliar/inervación , Ganglios Parasimpáticos/ultraestructura , Neuronas/ultraestructura , Animales , Axones/ultraestructura , Biomarcadores , Biotina/análogos & derivados , Biotina/fisiología , Ganglios Parasimpáticos/citología , Cobayas , Peroxidasa de Rábano Silvestre , Iontoforesis , Microscopía Electrónica , Neuroglía/ultraestructuraRESUMEN
The sphincter of Oddi is a smooth muscle sphincter that regulates the flow of bile into the duodenum. To identify potential chemical coding in sphincter of Oddi neurons, immunohistochemistry and histochemistry were employed to assay for putative neurotransmitters and related synthetic enzymes in wholemount preparations, with and without colchicine treatment. Immunoreactivities for enkephalin-endorphin (ENK-END), substance P (SP), nitric oxide synthase, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) were demonstrated within the ganglionated plexus. Roughly half of the neurons in the sphincter of Oddi expressed immunoreactivity for both SP and ENK-END, but not for nitric oxide synthase. About 25% of the neurons expressed nitric oxide synthase immunoreactivity as well as NADPH-diaphorase activity. This contingent of neurons was made up of two subgroups: one that expressed immunoreactivity for VIP, the other for NPY. Neurons that expressed CGRP immunoreactivity were sparse in sphincter of Oddi ganglia; however, many axons immunoreactive for both CGRP and SP were present in the ganglionated plexus. The CGRP/SP fibers are probably visceral afferents that may influence ganglionic output through axon reflex circuits. These results, along with studies of the actions of these neuroactive compounds on sphincter tone, support the view that ganglia of the sphincter of Oddi are largely comprised of excitatory (SP/ENK-END-immunoreactive) and inhibitory (nitric oxide synthase/VIP- or NPY-immunoreactive) neurons, and that sphincter of Oddi tone is controlled by the regulation of the outputs of these two groups of cells.
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Ganglios/ultraestructura , Neuronas/ultraestructura , Esfínter de la Ampolla Hepatopancreática/ultraestructura , Animales , Antígenos , Endorfinas/metabolismo , Femenino , Cobayas , Inmunohistoquímica , Masculino , NADP/metabolismo , Péptidos , Sustancia P/metabolismoRESUMEN
The measurement of the density of the reaction product produced by the histochemical demonstration of cytochrome oxidase activity provides a method for the visual identification of physiologically active enteric neurons. The current study utilized the cytochrome oxidase technique in order to evaluate the metabolic history of neurons in different regions of the bowel and in chemically identified types of neuron. In addition, the effect of drugs or neurotoxins commonly used in the immunocytochemical identification of enteric neuronal phenotypes was also analyzed. Cytochrome oxidase activity was visualized with a blue-black reaction product resulting from the cobalt-intensified oxidation of 3,3'-diaminobenzidine. Peptides or 5-hydroxytryptamine (5-HT) were localized with biotinylated secondary antibodies and alkaline phosphatase-labeled avidin. Bound avidin or endogenous alkaline phosphatase was visualized with a red reaction product in the presence or absence, respectively, of levamisole. Use of measured without interference from a simultaneously demonstrated histo- or immunochemical marker. A multi-peptidergic class of cholinergic submucosal secretomotor neuron containing neuropeptide Y (NPY) and calcitonin gene related peptide (CGRP) immunoreactivities was found to be less metabolically active than the average of all submucosal neurons. In contrast, a non-cholinergic submucosal secretomotor neuron containing dynorphin (which is also known to contain vasoactive intestinal peptide) immunoreactivity was more metabolically active than submucosal neurons that do not contain this peptide. On average, submucosal neurons were more metabolically active than those of the myenteric plexus, and levels of metabolic activity in the myenteric plexus were found to be higher in the duodenum and the cecum than in the jejunum-ileum or colon. Myenteric neurons characterized by CGRP or NPY immunoreactivities or by endogenous alkaline phosphatase activity, were all less metabolically active than the average of all neurons in myenteric ganglia. Colchicine, which stimulates intestinal motility, was observed to increase cytochrome oxidase activity in enteric neurons, suggesting that an effect on the enteric nervous system contributes to its action on the bowel. The neurotoxins, 6-hydroxydopamine and 5,7-dihydroxytryptamine (5,7-DHT) were each found to stimulate neuronal metabolic activity. 5,7-DHT appeared to activate excitatory subtypes of 5-HT receptor since its effects were blocked or mimicked by compounds that act as antagonists or agonists, respectively, at these receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Complejo IV de Transporte de Electrones/análisis , Intestinos/inervación , Neuronas/enzimología , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/química , Animales , Calcitonina/análisis , Péptido Relacionado con Gen de Calcitonina/análisis , Colchicina/farmacología , Densitometría , Dinorfinas/análisis , Complejo IV de Transporte de Electrones/química , Cobayas , Inmunohistoquímica , Intestinos/enzimología , Masculino , Plexo Mientérico/enzimología , Neuronas/ultraestructura , Neuropéptido Y/análisis , Fragmentos de Péptidos/análisis , Serotonina/análisis , Coloración y Etiquetado , Transmisión Sináptica/efectos de los fármacosRESUMEN
Primary afferent input to the cat sacral parasympathetic nucleus (SPN) has been examined by injury filling sacral dorsal roots, ventral roots, or both with horseradish peroxidase (HRP). Appropriate spinal segments were processed for the demonstration of HRP with diaminobenzidine and prepared for sequential light (LM) and electron (EM) microscopy. At the LM level, a large fascicle of primary afferent fibers was observed passing ventrally along the lateral edge of the dorsal horn into the region of the SPN. Varicosities were seen throughout the course of the axons but were particularly abundant within the SPN. Injury filling of the ventral roots with HRP resulted in a Golgi-like labelling of preganglionic neurons and their dendritic arbors, as well as ventral root afferent fibers. Swellings on both dorsal and ventral root afferent axons were observed in close apposition to labelled preganglionic neurons and their dendrites. At the ultrastructural level, afferent terminals were found to contain clear spherical vesicles; 66% of these terminals also contained at least one dense-cored vesicle. Of particular interest was the presence of labelled dorsal and ventral root afferent terminals synapsing on labelled preganglionic neurons. Preganglionic neurons were also postsynaptic to unlabelled terminals containing clear spherical (79.7%) or pleomorphic vesicles (20.3%). These data indicate that preganglionic neurons receive direct input from several sources, and provide the first demonstration of direct input to these cells from sensory fibers in the dorsal and ventral roots. The connections described in the present study provide interesting and, as yet, unexplored possibilities for sensory and autonomic reflex integration.
Asunto(s)
Neuronas Aferentes/fisiología , Neuronas Eferentes/fisiología , Sistema Nervioso Parasimpático/fisiología , Animales , Gatos , Comunicación Celular , Dendritas/fisiología , Dendritas/ultraestructura , Ganglios Parasimpáticos/citología , Ganglios Parasimpáticos/fisiología , Ganglios Parasimpáticos/ultraestructura , Peroxidasa de Rábano Silvestre , Humanos , Lactante , Masculino , Microscopía Electrónica , Neuronas Motoras/fisiología , Neuronas Motoras/ultraestructura , Sistema Nervioso Parasimpático/citología , Región SacrococcígeaRESUMEN
In the cat, preganglionic neurons (PGNs) found in the dorsal portion of the sacral parasympathetic nucleus (dorsal band or DB cells) participate in bowel control, while those found along the lateral edge (lateral band or LB cells) influence bladder function. In order to determine whether differences in the synaptic inputs exist between these two populations, HRP was applied to the sacral ventral rootlets of cats, and the S2 cord segment was prepared for sequential light and electron microscopy. When measured with light microscope, the LB somata had greater cross-sectional areas than did the DB cells. Ultrastructurally, the LB cells had a significantly greater percentage of their membrane apposed by synaptic active sites than did the DB cells. Also, the proximal dendrities of the labelled neurons received greater synaptic input than did the somata. No difference was found in the proportion of terminals containing dense cored vesicles (DCVs) when comparing LB and DB somata; however, the LB proximal dendrites had a higher proportion of their surface contacted by DCV-containing terminals than did the DB dendrites. These ultrastructural results offer evidence that these two populations of preganglionic neurons differ with respect to their synaptic input as well as their peripheral targets.
Asunto(s)
Ganglios Parasimpáticos/fisiología , Neuronas/fisiología , Sistema Nervioso Parasimpático/fisiología , Médula Espinal/fisiología , Sinapsis/fisiología , Animales , Gatos , Femenino , Ganglios Parasimpáticos/citología , Peroxidasa de Rábano Silvestre , Región Lumbosacra , Microscopía Electrónica , Neuronas/ultraestructura , Sistema Nervioso Parasimpático/citología , Médula Espinal/citologíaRESUMEN
Longitudinal muscle-myenteric plexus preparations of guinea pig intestines and sphincter of Oddi (SO) were immunostained for orphanin FQ/nociceptin. Orphanin FQ-immunoreactive (OFQ-IR) neurons and nerve fibers were relatively abundant in the SO, duodenum, ileum, cecum, and distal colon, with fewer neurons and nerve fibers observed in the proximal colon. Double staining with antibodies directed against the neuron-specific RNA binding protein Hu revealed that while the numbers of OFQ-IR neurons per ganglion decreased along the gut tube, similar proportions (7-9%) of neurons in these regions were OFQ-IR, whereas <1% of the neurons in the proximal colon were OFQ positive. In the ileum, where 8% of the myenteric neurons were OFQ-IR, all OFQ-IR neurons expressed choline acetyltransferase. In addition, multiple-label immunohistochemistry demonstrated that 58% of the OFQ-IR neurons were calretinin-IR, 52% were substance P-IR, and 28% were enkephalin-IR. Nitric oxide synthase immunoreactivity was observed in about 5% of OFQ-IR neurons, or 0.4% of the total population, and a similar proportion of the OFQ-IR neurons was positive for vasoactive intestinal peptide. No OFQ-IR neurons were immunoreactive for calbindin, somatostatin, or serotonin. These results, combined with previous studies of chemical coding and projection patterns in the guinea pig myenteric plexus, indicate that OFQ-IR is expressed preferentially in excitatory motor neurons projecting to the longitudinal and circular muscle layers, as well as a small subgroup of descending interneurons. Because OFQ is expressed by excitatory motor neurons, and because this peptide inhibits excitatory neurotransmission in the guinea pig ileum, it is likely that OFQ acts through a feedback autoinhibitory mechanism.
Asunto(s)
Cobayas/metabolismo , Intestinos/inervación , Plexo Mientérico/metabolismo , Neuronas/metabolismo , Péptidos Opioides/metabolismo , Esfínter de la Ampolla Hepatopancreática/inervación , Animales , Íleon/inervación , Inmunohistoquímica , Plexo Mientérico/citología , Distribución Tisular , NociceptinaRESUMEN
Several neurotransmitters have been reported to exist in the ganglionated plexus of the guinea pig gallbladder. These include substance P, neuropeptide Y (NPY), calcitonin gene-related peptide, vasoactive intestinal peptide (VIP), acetylcholine, norepinephrine, serotonin, and dopamine. To determine which neuropeptides are intrinsic to gallbladder ganglia, we performed immunohistochemistry on colchicine-treated preparations. In separate, single-labeled preparations, a majority of neurons contained substance P-, NPY-, or somatostatin-like immunoreactivity. In double-labeled preparations, a large majority of the neurons that contained substance P-like immunoreactivity also contained NPY-like immunoreactivity and somatostatin-like immunoreactivity. Immunoreactivity for VIP was present in a small percentage of the gallbladder neurons which did not contain substance P-like immunoreactivity. Additional experiments were done to test for the presence of other compounds, known to exist in the neurons of the gut. Although immunoreactivity was found in control preparations of small intestine, the ganglionated plexus of the gallbladder lacked immunoreactivity for galanin, dynorphin, enkephalin, gastrin-releasing peptide, or gamma-aminobutyric acid. We conclude that ganglia of the guinea pig gallbladder contain at least two populations of neurons, based on transmitter phenotype. One of these populations appears to contain substance P, NPY, and somatostatin. Another population, which represents a small contingent of the total population of neurons, contains VIP.