Dopamine Transporter Genetic Reduction Induces Morpho-Functional Changes in the Enteric Nervous System

Antidopaminergic gastrointestinal prokinetics are indeed commonly used to treat gastrointestinal motility disorders, although the precise role of dopaminergic transmission in the gut is still unclear. Since dopamine transporter (DAT) is involved in several brain disorders by modulating extracellular dopamine in the central nervous system, this study evaluated the impact of DAT genetic reduction on the morpho-functional integrity of mouse small intestine enteric nervous system (ENS). In DAT heterozygous (DAT+/−) and wild-type (DAT+/+) mice (14 ± 2 weeks) alterations in small intestinal contractility were evaluated by isometrical assessment of neuromuscular responses to receptor and non-receptor-mediated stimuli. Changes in ENS integrity were studied by real-time PCR and confocal immunofluorescence microscopy in longitudinal muscle-myenteric plexus whole-mount preparations (). DAT genetic reduction resulted in a significant increase in dopamine-mediated effects, primarily via D1 receptor activation, as well as in reduced cholinergic response, sustained by tachykininergic and glutamatergic neurotransmission via NMDA receptors. These functional anomalies were associated to architectural changes in the neurochemical coding and S100β immunoreactivity in small intestine myenteric plexus. Our study provides evidence that genetic-driven DAT defective activity determines anomalies in ENS architecture and neurochemical coding together with ileal dysmotility, highlighting the involvement of dopaminergic system in gut disorders, often associated to neurological conditions.

Changes in the Neurochemical Coding of the Anterior Pelvic Ganglion Neurons Supplying the Male Pig Urinary Bladder Trigone after One-Sided Axotomy of Their Nerve Fibers

The present study investigated the effect of unilateral axotomy of urinary bladder trigone (UBT)-projecting nerve fibers from the right anterior pelvic ganglion (APG) on changes in the chemical coding of their neuronal bodies. The study was performed using male pigs with immunohistochemistry and quantitative real-time PCR (qPCR). The animals were divided into a control (C), a morphological (MG) or a molecular biology group (MBG). APG neurons supplying UBT were revealed using the retrograde tracing technique with Fast Blue (FB). Unilateral axotomy resulted in an over 50% decrease in the number of FB+ neurons in both APG ganglia. Immunohistochemistry revealed significant changes in the chemical coding of FB+ cells only in the right ganglion: decreased expression of dopamine-B-hydroxylase (DBH)/tyrosine hydroxylase (TH) and up-regulation of the vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT), galanin (GAL), vasoactive intestinal polypeptide (VIP) and brain nitric oxide synthase (bNOS). The qPCR results partly corresponded with immunofluorescence findings. In the APGs, genes for VAChT and ChAT, TH and DBH, VIP, and NOS were distinctly down-regulated, while the expression of GAL was up-regulated. Such data may be the basis for further studies concerning the plasticity of these ganglia under experimental or pathological conditions.

Involvement of Enteric Glia in Small Intestine Neuromuscular Dysfunction of Toll-Like Receptor 4-Deficient Mice

Enteric glial cells (EGCs) influence nitric oxide (NO)− and adenosine diphosphate (ADP)− mediated signaling in the enteric nervous system (ENS). Since Toll-like receptor 4 (TLR4) participates to EGC homoeostasis, this study aimed to evaluate the possible involvement of EGCs in the alterations of the inhibitory neurotransmission in TLR4−/− mice. Ileal segments from male TLR4−/− and wild-type (WT) C57BL/6J mice were incubated with the gliotoxin fluoroacetate (FA). Alterations in ENS morphology and neurochemical coding were investigated by immunohistochemistry whereas neuromuscular responses were determined by recording non-adrenergic non-cholinergic (NANC) relaxations in isometrically suspended isolated ileal preparations. TLR4−/− ileal segments showed increased iNOS immunoreactivity associated with enhanced NANC relaxation, mediated by iNOS-derived NO and sensitive to P2Y1 inhibition. Treatment with FA diminished iNOS immunoreactivity and partially abolished NO− and ADP− mediated relaxation in the TLR4−/− mouse ileum, with no changes of P2Y1 and connexin-43 immunofluorescence distribution in the ENS. After FA treatment, S100β and GFAP immunoreactivity in TLR4−/− myenteric plexus was reduced to levels comparable to those observed in WT. Our findings show the involvement of EGCs in the alterations of ENS architecture and in the increased purinergic and nitrergic-mediated relaxation, determining gut dysmotility in TLR4−/− mice.

Distribution and trafficking of the μ-opioid receptor in enteric neurons of the guinea pig

The μ-opioid receptor (MOR) is a major regulator of gastrointestinal motility and secretion and mediates opiate-induced bowel dysfunction. Although MOR is of physiological and therapeutic importance to gut function, the cellular and subcellular distribution and regulation of MOR within the enteric nervous system are largely undefined. Herein, we defined the neurochemical coding of MOR-expressing neurons in the guinea pig gut and examined the effects of opioids on MOR trafficking and regulation. MOR expression was restricted to subsets of enteric neurons. In the stomach MOR was mainly localized to nitrergic neurons (∼88%), with some overlap with neuropeptide Y (NPY) and no expression by cholinergic neurons. These neurons are likely to have inhibitory motor and secretomotor functions. MOR was restricted to noncholinergic secretomotor neurons (VIP-positive) of the ileum and distal colon submucosal plexus. MOR was mainly detected in nitrergic neurons of the colon (nitric oxide synthase positive, 87%), with some overlap with choline acetyltransferase (ChAT). No expression of MOR by intrinsic sensory neurons was detected. [d-Ala2, MePhe4, Gly(ol)5]enkephalin (DAMGO), morphiceptin, and loperamide induced MOR endocytosis in myenteric neurons. After stimulation with DAMGO and morphiceptin, MOR recycled, whereas MOR was retained within endosomes following loperamide treatment. Herkinorin or the δ-opioid receptor agonist [d-Ala2, d-Leu5]enkephalin (DADLE) did not evoke MOR endocytosis. In summary, we have identified the neurochemical coding of MOR-positive enteric neurons and have demonstrated differential trafficking of MOR in these neurons in response to established and putative MOR agonists.

Biomarkers in canine inflammatory bowel disease diagnostics

Canine inflammatory bowel disease (IBD) is a heterogeneous group of chronic gastrointestinal disorders. The etiology, similar to human IBD, remains unknown. Canine IBD is diagnosed by exclusion, which is a long, time and money-consuming process due to the need of elimination of other diseases presenting with similar symptoms. Therefore, a search for a specific and sensitive marker is needed to overcome these difficulties. The article is divided into 3 sections presenting up-to-date information about laboratory markers, immunohistochemical markers and changes in the neurochemical coding of the enteric nervous system, concentrating on their usefulness and future applications. Data concerning laboratory and immunohistochemical markers is based mainly on canine IBD, while the neuroimmunohistochemistry section presents knowledge from human IBD due to the lack of such studies in veterinary medicine.