Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.     

CaMKII Is Essential for the Function of the Enteric Nervous System

Background

Ca²⁺/calmodulin-dependent protein kinases (CaMKs) are major downstream mediators of neuronal calcium signaling that regulate multiple neuronal functions. CaMKII, one of the key CaMKs, plays a significant role in mediating cellular responses to external signaling molecules. Although calcium signaling plays an essential role in the enteric nervous system (ENS), the role of CaMKII in neurogenic intestinal function has not been determined. In this study, we investigated the function and expression pattern of CaMKII in the ENS across several mammalian species.

Methodology/Principal Findings

CaMKII expression was characterized by immunofluorescence analyses and Western Blot. CaMKII function was examined by intracellular recordings and by assays of colonic contractile activity. Immunoreactivity for CaMKII was detected in the ENS of guinea pig, mouse, rat and human preparations. In guinea pig ENS, CaMKII immunoreactivity was enriched in both nitric oxide synthase (NOS)- and calretinin-containing myenteric plexus neurons and non-cholinergic secretomotor/vasodilator neurons in the submucosal plexus. CaMKII immunoreactivity was also expressed in both cholinergic and non-cholinergic neurons in the ENS of mouse, rat and human. The selective CaMKII inhibitor, KN-62, suppressed stimulus-evoked purinergic slow EPSPs and ATP-induced slow EPSP-like response in guinea pig submucosal plexus, suggesting that CaMKII activity is required for some metabotropic synaptic transmissions in the ENS. More importantly, KN-62 significantly suppressed tetrodotoxin-induced contractile response in mouse colon, which suggests that CaMKII activity is a major determinant of the tonic neurogenic inhibition of this tissue.

Conclusion

ENS neurons across multiple mammalian species express CaMKII. CaMKII signaling constitutes an important molecular mechanism for controlling intestinal motility and secretion by regulating the excitability of musculomotor and secretomotor neurons. These findings revealed a fundamental role of CaMKII in the ENS and provide clues for the treatment of intestinal dysfunctions.     

Limited Impact of 6-Mercaptopurine on Inflammation-Induced Chemokines Expression Profile in Primary Cultures of Enteric Nervous System

Increasing evidences indicate that the enteric nervous system (ENS) and enteric glial cells (EGC) play important regulatory roles in intestinal inflammation. Mercaptopurine (6-MP) is a cytostatic compound clinically used for the treatment of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn’s disease. However, potential impacts of 6-MP on ENS response to inflammation have not been evaluated yet. In this study, we aimed to gain deeper insights into the profile of inflammatory mediators expressed by the ENS and on the potential anti-inflammatory impact of 6-MP in this context. Genome-wide expression analyses were performed on ENS primary cultures exposed to lipopolysaccharide (LPS) and 6-MP alone or in combination. Differential expression of main hits was validated by quantitative real-time PCR (qPCR) using a cell line for EGC. ENS cells expressed a broad spectrum of cytokines and chemokines of the C-X-C motif ligand (CXCL) family under inflammatory stress. Induction of Cxcl5 and Cxcl10 by inflammatory stimuli was confirmed in EGC. Inflammation-induced protein secretion of TNF-α and Cxcl5 was partly inhibited by 6-MP in ENS primary cultures but not in EGC. Further work is required to identify the cellular mechanisms involved in this regulation. These findings extend our knowledge of the anti-inflammatory properties of 6-MP related to the ENS and in particular of the EGC-response to inflammatory stimuli.

     

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca²⁺) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca²⁺ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca²⁺ indicator such as Fluo-4 permits measurements of Ca²⁺ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca²⁺ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca²⁺ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca²⁺ responses in enteric neurons and glial cells.     

The Orexin System in the Enteric Nervous System of the Bottlenose Dolphin (Tursiops truncatus)

This study provides a general approach to the presence and possible role of orexins and their receptors in the gut (three gastric chambers and intestine) of confined environment bottlenose dolphin. The expression of prepro-orexin, orexin A and B and orexin 1 and 2 receptors were investigated by single immunostaining and western blot analysis. The co-localization of vasoactive intestinal peptide and orexin 1 receptor in the enteric nervous system was examined by double immunostaining. Also, orexin A concentration were measured in plasma samples to assess the possible diurnal variation of the plasma level of peptide in this species. Our results showed that the orexin system is widely distributed in bottlenose dolphin enteric nervous system of the all gastrointestinal tract examined. They are very peculiar and partially differs from that of terrestrial mammals. Orexin peptides and prepro-orexin were expressed in the main stomach, pyloric stomach and proximal intestine; while orexin receptors were expressed in the all examined tracts, with the exception of main stomach where found no evidence of orexin 2 receptor. Co-localization of vasoactive intestinal peptide and orexin 1 receptor were more evident in the pyloric stomach and proximal intestine. These data could suggest a possible role of orexin system on the contractility of bottlenose dolphin gastrointestinal districts. Finally, in agreement with several reports, bottlenose dolphin orexin A plasma level was higher in the morning during fasting. Our results emphasize some common features between bottlenose dolphin and terrestrial mammals. Certainly, further functional investigations may help to better explain the role of the orexin system in the energy balance of bottlenose dolphin and the complex interaction between feeding and digestive physiology.     

Distribution of NADPH Diaphorase-Positive Neurons in the Enteric Nervous System of the Rabbit Intestine

Nitric oxide (NO) has been proposed as an inhibitory transmitter in gastrointestinal muscle relaxation. We analyzed the distribution of nitric-oxide producing neurons in the rabbit intestine through nicotinamide-adenine-dinucleotide-phosphate-diaphorase histochemistry. By this reliable and convenient method, we visualized neuronal nitric-oxide-synthase, the enzyme responsible for nitric oxide generation, in the rabbit intestine. In the ileum and rectum, nitric-oxide-synthase-related diaphorase activity was present in the myenteric plexus ganglion cells, and in the nerve fibers in the internodal strand, secondary, and tertiary plexuses. These fibers were particularly abundant in the deep circular rather than in the outer longitudinal muscle layer. In the inner submucosal plexus, we found scarce labeled neurons. Labeled neural somata showed a range of sizes and shapes suggesting different functional roles. The present basic information is required to use the rabbit as an experimental animal in neurochemical NO enteric research.     

Binding of Isolectin IB4 to Neurons of the Mouse Enteric Nervous System

The plant lectin, IB4, binds to primary afferent neurons of dorsal root and trigeminal ganglia, where it is selective for nociceptive neurons. In the enteric nervous system of the guinea-pig IB4 labels intrinsic primary afferent neurons, which are believed to have roles as nociceptors. Here we investigate whether IB4 binding is also a marker of intrinsic primary afferent neurons in the mouse. Neurons that bound IB4 were common in the enteric plexuses of the small intestine and colon. Labeled neurons were rare in the stomach, and absent from the esophagus and gallbladder. Binding was to the cell surface, initial parts of axons and to clumps in the cytoplasm. Similar binding occurred on small and medium sized neurons of dorsal root, nodose and trigeminal ganglia. In the enteric nervous system, IB4 revealed large round or oval (type II) neurons, type I neurons with prominent laminar dendrites and small neurons of myenteric ganglia. The type II neurons were immunoreactive for calretinin, and some type I neurons were immunoreactive for nitric oxide synthase. Most neurons in the submucosal ganglia bound IB4, and some of these were vasoactive intestinal peptide immunoreactive. Thus IB4 binds to specific subgroups of enteric neurons in the mouse. These include intrinsic primary afferent neurons, but other neurons, including secretomotor neurons, are labeled. The results suggest that IB4 is not a specific label for enteric nociceptive neurons.     

A Propionate-Inducible Expression System for Enteric Bacteria

A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P_prpB) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P_prpB system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P_prpB of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P_prpB system exhibited negligible basal expression by addition of glucose to the medium.     

Expression of Sulfated Gangliosides in the Central Nervous System

Abstract: Several sulfated lipids were detected in the ganglioside fraction isolated from a cell line of oligodendrocyte progenitors that had been metabolically labeled with [³⁵S]sulfate. Separation of the ganglioside fraction by two-dimensional TLC showed that, except for galactosylceramide-sulfate, none of the sulfate-labeled lipids comigrated with those glycosphingolipids visualized by orcinol staining, indicating that these sulfolipids were quantitatively minor components. At least eight sulfate-labeled lipid bands were susceptible to desialylation by Arthrobacter ureafaciens neuraminidase, which resulted in the formation of three new bands that retained the labeled sulfate. Six of the sulfate-labeled lipid bands containing sialic acid were also susceptible to Vibrio cholerae neuraminidase, which generated two labeled bands that appeared identical to the two major products formed after treatment with A. ureafaciens neuraminidase. In vivo labeling of lipids from 14-day-old rat brain with [³⁵S]sulfate demonstrated that the synthesis of sulfated lipids containing sialic acid also occurred in intact brain tissue. These results show that sulfated gangliosides are synthesized in the CNS and that oligodendrocytes are one cell type that contributes to this synthesis.     

Adenosine-Mediated Enteric Neuromuscular Function Is Affected during Herpes Simplex Virus Type 1 Infection of Rat Enteric Nervous System

Adenosine plays an important role in regulating intestinal motility and inflammatory processes. Previous studies in rodent models have demonstrated that adenosine metabolism and signalling are altered during chronic intestinal inflammatory diseases. However, the involvement of the adenosinergic system in the pathophysiology of gut dysmotility associated to a primary neurodysfunction is still unclear. Recently, we showed that the neurotropic Herpes simplex virus type-1 (HSV-1), orally inoculated to rodents, infects the rat enteric nervous system (ENS) and affects gut motor function without signs of systemic infection. In this study we examined whether changes in purinergic metabolism and signaling occur during permanent HSV-1 infection of rat ENS. Using isolated organ bath assays, we found that contraction mediated by adenosine engagement of A₁ or A_2A receptors was impaired at 1 and 6 weeks post-viral administration. Immunofluorescence studies revealed that viral infection of ENS led to a marked redistribution of adenosine receptors: A₁ and A_2B receptors were confined to the muscle layers whereas A_2A and A₃ receptors were expressed mainly in the myenteric plexus. Viral-induced ENS neurodysfunction influenced adenosine metabolism by increasing adenosine deaminase and CD73 levels in longitudinal muscle-myenteric plexus with no sign of frank inflammation. This study provides the first evidence for involvement of the adenosinergic system during HSV-1 infection of the ENS. As such, this may represent a valid therapeutic target for modulating gut contractility associated to a primary neurodysfunction.     

Cytokine Gene Expression Within the Central Nervous System

1. The identification of cytokine genes expressed in the central nervous system is critical to understanding the immune network in various diseases of brain, such as infection, degeneration, and malignancy.2. Expression of cytokine genes in human astrocytoma cell lines and in fresh brain specimens was studied by the reverse-transcribed/polymerase chain reaction method.3. The correlation between clinical malignancy and cytokine gene expression within malignant glioma was examined, especially regarding the relevancy of inhibitory cytokines, such as transforming growth factor- and interleukin-10.     

Immunohistochemical Localization of Glycogen Phosphorylase Isozymes in the Rat Gastrointestinal Muscle Layers and Enteric Nervous System

Glycogen represents the major brain energy reserve though its precise functions are still under debate. Glycogen has also been found in different cell types of the enteric nervous system (ENS), the largest and most complex component of the peripheral nervous system. In the present work we have demonstrated, by application of isozyme-specific antibodies, the presence of isozymes of glycogen phosphorylase (GP), one of the major control sites in glycogen metabolism, in the rat ENS. Immunohistochemistry revealed that isoform BB (brain) is the predominant isozyme expressed in enteric glial cells (EGC) and rare neurons of the myenteric and submucosal plexuses. Isoform MM (muscle) appears in cells which are, according to their location and morphology, probably interstitial cells of Cajal (ICC). In addition, both GP isoforms are expressed in longitudinal and circular intestinal smooth muscle layers. As GP BB is mainly regulated by the cellular AMP level, a special function of glycogen in the energy supply of neural gut functions is suggested.     

Developmental Expression of the Myelin-Associated Glycoprotein in the Peripheral Nervous System Is Different from That in the Central Nervous System

We recently characterized two developmentally regulated myelin-associated glycoprotein (MAG) polypeptides synthesized by mouse brain mRNA in vitro. We now extended these studies to include the peripheral nervous system (PNS). Total cytoplasmic RNA was isolated from the sciatic nerves of 7-, 12-, and 17-day-old and adult rats and translated in vitro in a rabbit reticulocyte lysate system. In contrast to results in the CNS, it appears that only one MAG polypeptide, p67MAG, is synthesized by PNS mRNA at all ages. The implications of these findings are discussed with respect to recent observations concerning both the localization of MAG and the synthesis of MAG in the PNS of dysmyelinating mutant mice.     

Divergent Dimethylarginine Dimethylaminohydrolase Isoenzyme Expression in the Central Nervous System

Cellular and Molecular Neurobiology - The endogenous methylated derivative of ʟ-arginine, Nω,Nω′-dimethyl-ʟ-arginine (asymmetric dimethylarginine, ADMA), an independent...     

研究报告：缓激肽上调豚鼠肠道黏膜下神经丛环氧合酶2的表达

目的缓激肽和缓激肽B2受体在肠神经系统中起重要作用。缓激肽通常参与肠道的炎症反应和神经保护,这种作用取决于缓激肽诱导前列腺素的形成。环氧合酶1 (COX1)和环氧合酶2 (COX2)催化花生四烯酸转化为前列腺素。本研究旨在探讨缓激肽刺激对豚鼠肠神经前列腺素E2 (p GE2)释放和COX2表达的影响及信号机制。方法本文通过免疫荧光检测肠神经细胞中COX2与神经细胞标志物Anti-Hu和ch AT的表达;采用PCR及蛋白质印迹(Western blot)检测不同条件下缓激肽刺激对COX2表达的影响;使用缓激肽B1受体的选择性拮抗剂Leu-8和B2受体的选择性拮抗剂HOE-140,研究缓激肽影响COX2表达的信号机制;利用COX2选择性拮抗剂NS398和COX1拮抗剂FR12207,观察COX2在缓激肽诱导p EG2释放的作用。结果 COX2与神经细胞标志物Anti-Hu和ch AT在肠神经细胞上共同表达,缓激肽可通过B2受体诱导肠神经细胞COX2的表达。缓激肽刺激引起的肠神经细胞p GE2的释放与COX2表达升高密切相关。结论缓激肽通过B2R影响肠道黏膜下神经丛COX2的表达,肠道缓激肽...