Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells.

BACKGROUND: The intimate association between glial cells and neurons within the enteric nervous system has confounded careful examination of the direct responsiveness of enteric glia to different neuroligands. Therefore, we aimed to investigate whether neurotransmitters known to elicit fast excitatory potentials in enteric nerves also activate enteric glia directly. METHODS: We studied the effect of acetylcholine (ACh), serotonin (5-HT), and adenosine triphosphate (ATP) on intracellular Ca(2+) signaling using aequorin-expressing and Fluo-4 AM-loaded CRL-2690 rat and human enteric glial cell cultures devoid of neurons. The influence of these neurotransmitters on the proliferation of glia was measured and their effect on the expression of c-Fos as well as glial fibrillary acidic protein (GFAP), Sox10, and S100 was examined by immunohistochemistry and quantitative RT-PCR. KEY RESULTS: Apart from ATP, also ACh and 5-HT induced a dose-dependent increase in intracellular Ca(2+) concentration in CRL-2690 cells. Similarly, these neurotransmitters also evoked Ca(2+) transients in human primary enteric glial cells obtained from mucosal biopsies. In contrast with ATP, stimulation with ACh and 5-HT induced early gene expression in CRL-2690 cells. The proliferation of enteric glia and their expression of GFAP, Sox10, and S100 were not affected following stimulation with these neurotransmitters. CONCLUSIONS & INFERENCES: We provide evidence that enteric glial cells respond to fast excitatory neurotransmitters by changes in intracellular Ca(2+). On the basis of our experimental in vitro setting, we show that enteric glia are not only directly responsive to purinergic but also to serotonergic and cholinergic signaling mechanisms.

ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.

The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

Cannabinoid receptor 1 signalling dampens activity and mitochondrial transport in networks of enteric neurones.

Cannabinoid (CB) receptors are expressed in the enteric nervous system (ENS) and CB(1) receptor activity slows down motility and delays gastric emptying. This receptor system has become an important target for GI-related drug development such as in obesity treatment. The aim of the study was to investigate how CB(1) ligands and antagonists affect ongoing activity in enteric neurone networks, modulate synaptic vesicle cycling and influence mitochondrial transport in nerve processes. Primary cultures of guinea-pig myenteric neurones were loaded with different fluorescent markers: Fluo-4 to measure network activity, FM1-43 to image synaptic vesicles and Mitotracker green to label mitochondria. Synaptic vesicle cluster density was assessed by immunohistochemistry and expression of CB(1) receptors was confirmed by RT-PCR. Spontaneous network activity, displayed by both excitatory and inhibitory neurones, was significantly increased by CB(1) receptor antagonists (AM-251 and SR141716), abolished by CB(1) activation (methanandamide, mAEA) and reduced by two different inhibitors (arachidonylamide serotonin, AA-5HT and URB597) of fatty acid amide hydrolase. Antagonists reduced the number of synaptic vesicles that were recycled during an electrical stimulus. CB(1) agonists (mAEA and WIN55,212) reduced and antagonists enhanced the fraction of transported mitochondria in enteric nerve fibres. We found immunohistochemical evidence for an enhancement of synaptophysin-positive release sites with SR141716, while WIN55,212 caused a reduction. The opposite effects of agonists and antagonists suggest that enteric nerve signalling is under the permanent control of CB(1) receptor activity. Using inhibitors of the endocannabinoid degrading enzyme, we were able to show there is endogenous production of a CB ligand in the ENS.