The distribution of HCN2-positive cells in the gastrointestinal tract of mice

HCN2 channels are involved in the spontaneous rhythmic activities of some CNS neurons and act by generating I(f) current. The gastrointestinal (GI) tract is known to be capable of spontaneous rhythmic activity; however, the possible role of HCN2 channels in this organ has not yet been elucidated. This study investigated the distribution of HCN2-positive cells in the mouse GI tract using immunohistochemistry. To identify the nature of these HCN2 cells, anti-ChAT and anti-Kit antibodies were used to co-label neurons and the interstitial cells of Cajal (ICCs), respectively. Additionally, differences in the distribution of HCN2-positive cells within the GI tract were also analyzed. Our results showed that HCN2 channels were mainly located within the myenteric neurons of the enteric nervous system in the GI tract. Double-staining revealed that HCN2-positive neurons were labeled by ChAT, indicating that these HCN2-positive cells are also cholinergic neurons. Although the HCN2-positive cells were not stained by the anti-Kit antibody, their processes were in close proximity to ICCs around the myenteric plexus region. Moreover, several differences in the distribution of HCN2 in the stomach, small intestine and colon were partly consistent with the regional differences in the spontaneous rhythmic activities of these organs. Basing on the role HCN2, we suggested that HCN2 channels facilitate the release of Ach from cholinergic neurons to affect the GI peristalsis by acting on M receptors on the ICCs. However, the HCN2 channels are not directly involved in spontaneous slow-wave initiation by ICCs.     

An ultrastructural analysis of the developing enteric nervous system of the guinea-pig small intestine

Summary The developing enteric nervous system of the guinea-pig has been analysed ultrastructurally. In addition, electron microscope autoradiography, following incubation with tritiated 5-hydroxytryptamine ([³H]5-HT) or tritiated norepinephrine ([³H]NE) was used to locate the developing axons of enteric serotoninergic and adrenergic neurons respectively. Observations have been correlated with previous studies of the development of the various types of enteric neuron and the onset of intestinal neuromuscular function. Prior to 25 days of gestation no neurons can be recognized morphologically. Neurons first appear at 25 days' gestation, together with a primitive neuropil in neural islands within the outer gut mesenchyme. Ganglion cell precursors are primitive at first and resemble the cells in the surrounding mesenchyme. Growth cones are abundant but there are no terminal varicosities or synapses. The circular muscle also begins to form at this time. At 32 days' gestation the longitudinal layer of smooth muscle can be discerned and, within the myenteric plexus, terminal axonal varicosities appear containing small (about 50 nm in diameter) electron-lucent synaptic vesicles. The submucosal plexus appears to be derived from neurons and neurites that reach it from the earlier-developing myenteric plexus. The submucosal plexus can be recognized at 38 days of gestation but is not well developed until day 42. Synapses on ganglion cell somata first appear in the myenteric plexus on gestational day 38 and are numerous on day 42 when the first axo-dendritic synapses can be seen. Between days 42 and 48 the developing neural tissue and growing smooth muscle cells interdigitate but after day 48, the plexus becomes ensheathed by supporting cells and connective tissue and this interdigitation is lost. Prior to day 48 most varicosities contain small electron-lucent synaptic vesicles; however, after this time a variety of terminals appears. Between days 48 and 53 of gestation evidence of degenerating neuronal processes is common, indicating that cell death may occur. Electron microscopic autoradiography with [³H]5-HT reveals labelling of axons in the neuropil of the myenteric plexus at day 32 of gestation. Some primitive appearing cell bodies, however, are also labelled and these cells seem to be entering the myenteric plexus from the surrounding mesenchyme. After 42 days of gestation [³H]5-HT labels only axons of both nerve plexuses. Often, labelled terminals are apposed to ganglion cells or dendrites. In contrast, significant labelling by [³H]NE is not found until gestational day 48. Axons are labelled by [³H]NE and these tend to be located at the interface between the myenteric plexus and the surrounding connective tissue.     

肠易激综合征早期生活事件模型下的大鼠结肠肠神经可塑性研究

目的从神经可塑性角度探讨肠易激综合征的可能发病机制、临床特征及治疗方法。方法雄性SD大鼠出生后连续13 d每天分离3 h。腹壁撤退反射实验用来检测内脏痛觉过敏。肠神经系统结构可塑性可以通过铺片技术和免疫荧光技术,比较近端结肠神经节(HuD阳性细胞)的大小和数目以及胶质细胞(GFAP)的变化来检测。检测近端结肠多组肌间神经丛和黏膜下神经丛肠神经递质类型(ChAT-、VIP-、nNOS-、calbindin-TrKA-、P75-阳性细胞),分析神经递质的可塑性变化。结果新生期应激可致成年鼠内脏敏感性增高,新生期母婴分离可以诱导肠神经结构改变,导致神经元肥大和增生、胶质细胞与神经元的比例增高。神经递质方面,新生期母婴分离组P75和TrkA表达(黏膜下神经丛、肌间神经丛)较正常组均明显增加。ChAT在肌间神经丛表达明显增加,VIP在黏膜下神经丛表达降低,nNOS在肌间神经丛表达增高,Cabindin表达未见明显异常。结论新生期母婴分离可以引起大鼠结肠肠神经可塑性的长期改变。新生期母婴分离诱导的内脏高敏感性中存在肠神经系统可塑性。早期生活事件是引起成年后肠神经系统可塑性改变的重要原因。     

Immunohistochemical identification of cholinergic neurons in the myenteric plexus of guinea-pig small intestine.

It is well established that acetylcholine is a neurotransmitter at several distinct sites in the mammalian enteric nervous system. However, identification of the cholinergic neurons has not been possible due to an inability to selectively label enteric cholinergic neurons. In the present study an immunohistochemical method has been developed to localize choline acetyltransferase, the synthetic enzyme for acetylcholine, in order that cholinergic neurons can be visualized. The morphology, neurochemical coding and projections of cholinergic neurons in the guinea-pig small intestine were determined using double-labelling immunohistochemistry. These experiments have revealed that many myenteric neurons are cholinergic and that they can be distinguished by their specific combinations of immunoreactivity for neurochemicals such as calretinin, neurofilament protein triplet, substance P, enkephalin, somatostatin, 5-hydroxytryptamine, vasoactive intestinal peptide and calbindin. On the basis of their previously described projections, functional roles could be attributed to each of these populations. The identified cholinergic neurons are: motorneurons to the longitudinal muscle (choline acetyltransferase/calretinin); motorneurons to the circular muscle (choline acetyltransferase/neurofilament triplet protein/substance P, choline acetyltransferase/substance P and choline acetyltransferase alone); orally directed interneurons in the myenteric plexus (choline acetyltransferase/calretinin/enkephalin); anally directed interneurons in the myenteric plexus (choline acetyltransferase/somatostatin, choline acetyltransferase/5-hydroxytryptamine, choline acetyltransferase/vasoactive intestinal peptide); secretomotor neurons to the mucosa (choline acetyltransferase/somatostatin); and sensory neurons mediating myenteric reflexes (choline acetyltransferase/calbindin). This information provides a unique opportunity to identify functionally distinct populations of cholinergic neurons and will be of value in the interpretation of physiological and pharmacological studies of enteric neuronal circuitry.     

豚鼠胃肠壁内神经丛的磷酸醋、醋类和神经递质代谢有关的酶组织化学观察

本文报道用光镜半定量和显微光度计定量分析研究了豚鼠胃肠壁内神经丛神经元的几种酶的组织化学反应。结果表明,神经元的碱性磷酸酶(AlP)、酸性磷酸酶(AcP)、5′-核苷酸酶(5′-Nase)、硫胺素焦磷酸酶(TPPase)、非特异性酯酶(NsE)和胆碱乙酰转移酶(ChAT)反应强弱明显不等。消化道不同节段或不同部位神经元的单胺氧化酶(MAO)、氨基肽酸(AP)和乙酰胆碱酯酶(AChE)反应虽有差别,但却显阳性反应,同一神经节内各神经元的反应比较近似。胃肠各段壁内神经丛中50～66%神经元呈ChAT强阳性反应,这些细胞可能为胆碱能神经元。整个消化道粘膜下丛与肠肌丛神经元相比,除NsE外,另几种酶均有高度显著差异。粘膜下丛神经元AcP和AP反应较强,肠肌丛神经元AlP、5′-Nase、TPPase、MAO、ChAT和AChE反应较强,胃壁内神经丛不如肠道的发达。尤其是胃粘膜下丛只见少数单个散在的神经元,它们的各种酶组织化学反应均较弱。各段肠中,以十二指肠和近端结肠壁内神经丛神经元的各种酶组织化学反应较强。上述结果表明,消化道不同部位以及同一部位不同类型的神经元在代谢和功能上有明显的差别。     

Morphology and distribution of nitric oxide synthase-, neurokinin-1 receptor-, calretinin-, calbindin-, and neurofilament-M-immunoreactive neurons in the myenteric and submucosal plexuses of the rat small intestine

Characterization of the enteric neurons is vital for understanding their physiological role. We have used single and dual label fluorescence and peroxidase-based immunohistochemistry in myenteric and submucosal whole mounts from the rat small intestine to evaluate the morphology and distribution of enteric neurons immunoreactive for the following phenotypic antigens: neuronal nitric oxide synthase (NOS), neurokinin-1 receptor (NK-1R), calretinin (Calr), calbindin (Cal), and neurofilament-M (NF-M). NOS-immunoreactive neurons had Dogiel type I morphology, were abundant in the myenteric plexus compared to the submucosal plexus, and never coexpressed NK-1R immunoreactivity. NK-1R- and Calr-immunoreactive neurons had Dogiel type II morphology and were distributed comparably in both plexuses. NK-1R and Calr-immunoreactivity were coexpressed in many of the same neurons. Calbindin-immunoreactive neurons exhibited four distinct morphologies: small and large Dogiel type II neurons, Dogiel type I neurons, and small elongated neurons. These neurons were significantly fewer in number in the myenteric plexus compared to the submucosal plexus. Neurofilament-M-immunoreactive neurons had three morphologies, Dogiel type II neurons, small Dogiel type II neurons, and a less common subpopulation of small, elongated, multipolar neurons. These neurons were also fewer in number in the myenteric plexus compared to the submucosal plexus. The distribution of these phenotypic markers may assist future work that elucidates the functional activities of these enteric neurons such as control of intestinal motility and adaptation to the entry of gastric contents.     

Transplantation of postnatal rat enteric ganglia into denervated adult rat hippocampus

These experiments explore the possible value of the myenteric plexus as a source of donor cells for autografting into the central nervous system. Neurons and glia from 10-12-day postnatal rat myenteric plexus survive for at least one month after transplantation into cholinergically denervated syngeneic adult rat hippocampus. A population of donor cholinergic neurons has acetylcholinesterase-positive processes, but these appear not to innervate host tissue. Host gliosis in response to these implants seems to be less than that seen with other peripheral ganglia, and unlike Schwann cells, the enteric glia form end-feet on brain capillaries.     

Involvement of prostaglandin E(2) derived from enteric glial cells in the action of bradykinin in cultured rat myenteric neurons

We characterized bradykinin (BK)-induced changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) and membrane potential in cultured rat myenteric neurons using ratiometric Ca(2+) imaging with fura-2 and the whole-cell patch-clamp technique, respectively. BK evoked a dose-dependent increase of [Ca(2+)]i that was abolished by HOE 140, a B2 receptor antagonist but not by [Lys-des-Arg(9)]-BK, a B1 receptor antagonist. [Lys-des-Arg(9)]-HOE140, a B1 receptor agonist, failed to cause a [Ca(2+)]i response. Double staining with antibodies against the B2 receptor together with PGP9.5 or S100 indicated that B2 receptors were expressed in neurons and glial cells. The BK-evoked [Ca(2+)]i increase was suppressed by indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and potentiated by prostaglandin E(2) (PGE(2)). The release of PGE(2) from cultured myenteric plexus cells was increased by BK. BK induced a large increase in [Ca(2+)]i in neurons when myenteric plexus cells were cultured at the high density but not at the low density, and caused a small increase in [Ca(2+)]i in neurons when proliferation of enteric glial cells was suppressed. BK evoked a slow and sustained depolarization in myenteric neurons, which was sensitive to indomethacin. These results indicated that BK caused a [Ca(2+)]i increase and depolarization in rat myenteric neurons through the activation of B2 receptors, which was partly associated with PGE(2) released from glial cells in response to BK. It is suggested that a neuron-glial interaction plays an important role in the effect of BK in the rat myenteric plexus.     

Release of endogenous 5-hydroxytryptamine from resting and stimulated enteric neurons

Physiological and biochemical evidence has indicated that there may be serotoninergic neurons in the enteric nervous system. A critical step in the identification of a neurotransmitter is the demonstration of the release of the substance upon nerve stimulation. We now report the release of endogenous 5-hydroxytryptamine from enteric neurons. Segments of guinea-pig small intestine were everted and perfused in vitro through the newly created serosal lumen. Tests with [³H]5-hydroxytryptamine revealed the existence of a tissue barrier preventing diffusion of mucosal (enteroendocrine cell) 5-hydroxytryptamine into the perfusate; thus, all 5-hydroxytryptamine in the perfusate was of neural origin. The gut was stimulated electrically. 5-Hydroxytryptamine in the perfusate and in the myenteric plexus was assayed by a specific radioenzymatic method. 5-Hydroxytryptamine was present in the myenteric plexus; it was released into the perfusate spontaneously and the release was enhanced by electrical stimulation. The stimulated, but not the spontaneous, release of the amine was Ca²⁺-dependent. Comparison with the release of newly taken up [³H]5-hydroxytryptamine showed that the specific radioactivity of electrically released 5-hydroxytryptamine was higher than that of either the spontaneously released or tissue amine. Stimulation also increased the release of 5-hydroxytryptamine more than that of its metabolites in the perfusate.These results indicate that 5-hydroxytryptamine is an endogeneous constituent of the enteric nervous system, that it is released by electrical field stimulation of enteric nerves, and that newly taken up 5-hydroxytryptamine is released preferentially by these neurons.     

The neurochemistry and innervation patterns of extrinsic sensory and sympathetic nerves in the myenteric plexus of the C57Bl6 mouse jejunum

In vitro anterograde tracing of axons in mesenteric nerve trunks using biotinamide in combination with immunohistochemical labelling was used to characterize the extrinsic nerve projections in the myenteric plexus of the mouse jejunum. Anterogradely-labelled spinal sensory fibres innervating the enteric nervous system were identified by their immunoreactivity for calcitonin gene-related peptide (CGRP), while sympathetic noradrenergic fibres were detected with tyrosine hydroxylase (TH), using confocal microscopy. The presence of these markers has been previously described in the spinal sensory and sympathetic fibres. Labelled extrinsic nerve fibres in the myenteric plexus were identified apposing enteric neurons that were immunoreactive for either calretinin (CalR), calbindin (CalB) or nitric oxide synthase (NOS). Of the total anterogradely labelled axons in the myenteric plexus, 20% were CGRP-immunoreactive. Labelled CGRP-immunoreactive varicosities were closely apposed to CalR-immunoreactive myenteric cells, many of which were Dogiel type I (40%; interneurons) or type II (20%; intrinsic sensory) neurons. Labelled CGRP-immunoreactive varicosities were also observed in close appositions to CalB-immunoreactive myenteric cell bodies, of which a small subset had type II morphology (18%; intrinsic sensory neurons). A further 43% of all biotinamide-filled fibres were immunoreactive for TH and these fibres were apposed to CalR-immunoreactive cell bodies (small-sized; excitatory motor neurons) and NOS-immunoreactive cell bodies (either type I or small neurons; inhibitory motor neurons and interneurons) in the myenteric plexus. The results provide a neurochemical and neuroanatomical basis for connections between dorsal root afferent neurons and myenteric neurons and suggest an anatomical substrate for the well-known modulation of enteric circuits from sympathetic nerves. No anterogradely-labelled fibres were stained for NOS-immunoreactivity, despite more than 60% of dorsal root ganglion (DRG) neurons retrogradely labelled from the jejunum showing NOS-immunoreactivity. This was due to a substantial, time-dependent, and apparently selective, loss of NOS from extrinsic axons under in vitro conditions. Lastly, a small population of non-immunoreactive biotinamide-filled fibres (<1%) gave rise to dense terminal structures around individual myenteric cell bodies lacking CalR, CalB or NOS. These specialized endings may represent vagal fibres or a subset of spinal sensory neurons that do not contain CGRP.     

An ultrastructural study of neurons and non-neuronal cells in the myenteric plexus of the rabbit colon

The myenteric plexus of the rabbit colon showed a degree of structural organization that was unusually high for the peripheral nervous system, providing a basis for the complex integrative activity which is known to occur. It resembled central nervous tissue in several respects: a wide range of neuron types was present; the proportion of glial cells to neurons was about 2:1; and there was a densely packed, avascular neuropil, not penetrated by connective tissue. Most neurons had at least one surface exposed to the extraganglionic space. Clear evidence was obtained for spontaneous neuronal degeneration. Three types of non-neuronal (glial) cells were observed: Type 1, which was most common, contained many 10 nm ‘gliofilaments’ and resembled enteric glial cells or astrocytes in the central nervous system; Type 2, composing about 5% of the glial cells, had few filaments; Type 3 was seen only rarely, had a small dark nucleus, little cytoplasm, may have been of extraganglionic origin and resembled microglia of the central nervous system. Fibroblast-like cells were also present in extraganglionic sites. Schwann cells could not be identified within the myenteric ganglia.     

Enteric Neurogenesis During Life Span Under Physiological and Pathophysiological Conditions

The enteric nervous system (ENS) controls gastrointestinal key functions and is mainly characterized by two ganglionated plexus located in the gut wall: the myenteric plexus and the submucous plexus. The ENS harbors a high number and diversity of enteric neurons and glial cells, which generate neuronal circuitry to regulate intestinal physiology. In the past few years, the pivotal role of enteric neurons in the underlying mechanism of several intestinal diseases was revealed. Intestinal diseases are associated with neuronal death that could in turn compromise intestinal functionality. Enteric neurogenesis and regeneration is therefore a crucial aspect within the ENS and could be revealed not only during embryogenesis and early postnatal periods, but also in the adulthood. Enteric glia and/or enteric neural precursor/progenitor cells differentiate into enteric neurons, both under homeostatic and pathologic conditions beyond the perinatal period. The unique role of the intestinal microbiota and serotonin signaling in postnatal and adult neurogenesis has been shown by several studies in health and disease. In this review article, we will mainly focus on different recent studies, which advanced the concept of postnatal and adult ENS neurogenesis. Moreover, we will discuss the key factors and underlying mechanisms, which promote enteric neurogenesis. Finally, we will shortly describe neurogenesis of transplanted enteric neural progenitor cells. Anat Rec, 302:1345–1353, 2019. © 2019 Wiley Periodicals, Inc.     

Ca2+-activated K+ Current in Freshly Isolated c-Kit Positive Cells in Guinea-pig Stomach

This study was designed to isolate Ca²⁺-activated K⁺ current (I_KCa) and elucidate its physiological significance in freshly isolated interstitial cells of Cajal (ICCs) of guinea-pig stomach. Single ICC was freshly isolated by enzymatically dissociating from myenteric border of gastric antrum free of circular muscles, and conventional whole-cell voltage clamp technique including immunohistochemical techniques were employed to characterize the cells: In myenteric border of gastric antrum, ICC-MY (ICCs from myenteric border) were detected by immunohistochemical reactivity, and single ICC-MY which has many branches was immunohistochemically c-Kit positive. Under K⁺-rich and 0.1 mM ethylene glycol-bis (2-aminoethyl ether)-N,N,N'',N''-tetraacetic acid pipette solution, ICC produced spontaneous inward current (-256±92.2 pA). When step-depolarizing pulse from -80 to +80 mV was applied at holding potential (V_h) of -80 mV, voltage-dependent outward currents were recorded with superimposed spontaneous transient outward currents (STOCs). Both STOCs and outward currents were reversibly affected by tetraethylammonium chloride (TEA) and iberiotoxin (IbTX); 2 mM TEA and 200 nM IbTX completely abolished STOCs and significantly inhibited outward K⁺ current over the whole potential range tested for current/voltage (I/V) relationship. In addition, TEA delayed repolarization phase of spontaneous inward current. The present results indicate the presence of I_KCa in a single ICC, and it might be involved in regulation of repolarizing phase of spontaneous inward current in guinea-pig stomach.     

Morphology and Neurochemistry of Descending and Ascending Myenteric Plexus Neurons of Sheep Ileum

The specific patterns of gastrointestinal motility in large herbivores may relate to differences in the organization of enteric nerve circuits, compared with other mammals. To investigate this possibility, we characterized the morphologies, chemical phenotypes, and projections of myenteric plexus (MP) neurons of the sheep ileum. Morphologies and projections were investigated after application of the carbocyanine dye (1,1′, di‐octadecyl‐3,3,3′,3′,‐tetramethylindo‐carbocyanine perchlorate, DiI) to fixed tissues. To study chemical phenotypes, the fluorescent tracer Fast Blue (FB) was injected into the wall of the ileum, in vivo, 12–14 cm oral to the ileo‐caecal junction. Over 80% of the descending and ascending DiI‐labeled neurons had typical Dogiel type I morphology, whereas only a few Dogiel type II neurons were observed. Nevertheless, there were long projections (up to 10 cm) of Dogiel type II neurons in both directions. Both type II and type I neurons were neurofilament immunoreactive (IR). We observed long projections of descending (up to 18 cm) and ascending (up to 12–14 cm) FB‐labeled MP neurons. Nitric oxide synthase (NOS)‐IR, peripheral choline acetyltransferase (pChAT)‐IR, and substance P (SP)‐IR occurred in both descending and ascending myenteric neurons. NOS‐IR was in approximately 60% of FB‐labeled descending and ascending neurons, whereas those expressing pChAT‐IR were 67 ± 15% and 60 ± 14%, respectively. Descending neurons expressing SP‐IR were 48 ± 15% and ascending were 56 ± 12%. NOS‐IR and pChAT‐IR, and SP‐IR and pChAT‐IR were commonly colocalized in both ascending and descending pathways. In descending pathways, almost all SP‐IR neurons were also pChAT‐IR (98 ± 3%) and NOS‐IR (99 ± 2 NOS⁺/SP⁺/pChAT^?). Many FB‐labeled descending neurons showed both NOS‐ and pChAT‐IR. Descending neurons may represent inhibitory motor neurons (NOS⁺/SP⁺/pChAT^?) and two classes of interneurons (pChAT⁺/NOS^?, and pChAT⁺/NOS⁺/SP⁺). In ascending pathways, most neurons are pChAT⁺/NOS⁺/SP⁺. Thus, in sheep, ascending interneurons and ascending excitatory motor neurons both have the same phenotype, and other markers are needed to distinguish them. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

人直肠肌间神经丛透射电镜的观察

目的研究人直肠肌间神经丛形态学的微观结构。方法采用国人成年直肠标本,经LKB-V型超薄切片机制成半薄切片,在直肠半薄切片上定位肌间神经丛后,进行超薄切片,厚度为50～70nm。醋酸铀、柠檬酸铅染色,JEOL-100CX透射电镜观察。结果在典型的肌间丛神经节可观察到3种细胞成分,即被囊细胞、胶质细胞和神经节细胞。根据神经节细胞间电子密度不同,可将神经节细胞分为明细胞和暗细胞两类。结论人直肠肌间神经丛含有与中枢神经系统相似的细胞成分,神经节内含有丰富神经胶质细胞,其与神经元的比例为2∶1,神经元发出“翼翅”状突起。     

Projections of intestinal neurons showing immunoreactivity for vasoactive intestinal polypeptide are consistent with these neurons being the enteric inhibitory neurons.

Experiments were performed to determine if the distribution of vasoactive intestinal peptide(VIP)-like immunoreactivity in nerve cell bodies and axons of the myenteric plexus and circular muscle of the small intestine is consistent with VIP being the transmitter of enteric inhibitory neurons. Immunoreactivity for VIP was found in nerve cell bodies of the myenteric plexus and in axons within the myenteric plexus and circular muscle. When the axons in the myenteric plexus were interrupted, there was accumulation of material showing reactivity for VIP on the oral side, indicating that the neurons project in an anal direction. The VIP-like immunoreactivity in axons which supply the circular muscle disappeared after a myectomy in which the overlying myenteric plexus was removed, but remained intact when extrinsic nerves were served. The projections of VIP neurons from the myenteric plexus to the circular muscle correspond to the expected projections of enteric inhibitory neurons determined by functional studies.     

Autoradiographic localization of [3H]gamma-aminobutyric acid in the myenteric plexus of the guinea-pig small intestine

Localization of [³H]GABA in the guinea-pig myenteric plexus has been studied using light microscopic autoradiography. In the presence of β-alanine, 10^?3 M, an inhibitor of glial cell high affinity GABA transport, [³H]GABA was transported by a high affinity uptake system into neuronal elements of the plexus. Scattered neurones accumulating [³H]GABA showed localization of silver grains over the soma and axonal processes. In addition a large population of uptake sites for [³H]GABA was found within the secondary and tertiary meshworks of the plexus so that dense accumulations of silver grains were observed localized over distinct ‘tracts’ within all three meshworks of the plexus. These results are considered to provide strong evidence for GABAergic neurones in the enteric nervous system.     

Enteric glial cells immunoreactive for P2X7 receptor are affected in the ileum following ischemia and reperfusion

The aim of this study was to analyze the effect of ischemia and reperfusion injury (IS) on enteric glial cells (EGCs) and neurons immunoreactive for the P2X7 receptor. Intestinal ischemia was induced by obstructing blood flow in the ileal vessels for 35 min. Afterwards, the vessels were reperfused for 14 days. Tissues were prepared for immunohistochemical labeling of P2X7 receptor, HuC/D (Hu) (pan-neuronal marker) and S100β (glial marker); HuC/D (Hu) and glial fibrillary acidic protein (GFAP, glial marker)/DAPI (nuclear marker); or S100β and GFAP/DAPI. Qualitative and quantitative analyses of colocalization, density, profile area and cell proliferation were performed via fluorescence and confocal laser scanning microscopy. The quantitative analyses revealed that a) neurons and EGCs were immunoreactive for P2X7 receptor; b) the P2X7 receptor immunoreactive cells and Hu immunoreactive neurons were reduced after 0 h and 14 days of reperfusion; c) the S100β and GFAP immunoreactive EGCs were increased; d) the profile area of S100β immunoreactive EGCs was increased by IS; e) few GFAP immunoreactive proliferated at 14 days of reperfusion; f) distinct populations of glial cells can be discerned: S100β⁺/GFAP⁺ cells, S100β⁺/GFAP^– cells and S100β^–/GFAP ⁺ cells; g) histological analysis revealed less alterations in the epithelium cells in the IS groups and h) myeloperoxidase reaction revealed increased of the neutrophils in the lamina propria in the IS groups. This study showed that IS is associated with significant neuronal loss, increase of glial cells and altered purinergic receptor expression and that these changes may contribute to intestinal disorders.     

Embryonic development of the ganglion plexuses and the concentric layer structure of human gut: a topographical study

In this study, we performed a detailed topographical study on the development of ganglion plexuses and the smooth muscle layers of human embryonic and fetal gut. Neuron and glia differentiation was investigated with anti-PGP9.5 and anti-S100 antibodies respectively. The differentiation of smooth muscle and interstitial cells of Cajal (ICC) was studied with anti-smooth muscle -actin and anti-C-Kit antibodies respectively. By week 7, rostro-caudal neural crest cell (NCC) colonization of the gut was complete, and NCCs have differentiated into neurons and glia. At the foregut, neurons and glia were aggregated into ganglion plexus in the myenteric region, and the longitudinal and circular muscle layers have started to differentiate; however, neurons and glia were not found in the submucosa. At the hindgut, neurons and glia were dispersed within the mesenchyme. Myenteric plexus, longitudinal and circular muscle layers formed along the entire gut by week 9. Scattered and individual neurons and glia, and small ganglion plexuses were detected in the foregut and midgut submucosa by week 12. Ganglion plexus was not seen in the hindgut submucosa until week 14. Muscularis mucosae was formed at the foregut and midgut by week 12 but was only discernible at the hindgut 2 weeks later. As the gut wall developed, ganglion plexus increased in size with more neurons and glia, and the formation of intra-plexus nerve fascicle. ICCs were localized in the ganglion plexus as early as week 7. ICCs were initially dispersed in the plexus and were preferentially localized at the periphery of the plexus by week 20. The specification of the annular layers of human embryonic and fetal gut follows a strict spatio-temporal pattern in a rostro-caudal and centripetal manner suggesting that interaction between (1) homotypic and/or heterotypic cells; and (2) cells and the extracellular matrix is critical for the embryonic development of the gut mesenchyme and the enteric nervous system.