Ca²⁺ transients in myenteric glial cells during the colonic migrating motor complex in the isolated murine large intestine

Abstract

Non-technical summary A ganglionated neural plexus, the myenteric plexus, within the colon generates a propagating contraction called the colonic migrating motor complex (CMMC) that underlies faecal pellet propulsion. Neurons in the myenteric plexus are surrounded by a network of enteric glia cells (EGCs), which were traditionally thought to be the glue that held the neurons together. Using imaging techniques, we demonstrate that 36% of EGCs respond with prolonged Ca 2+ transients following their activation by excitatory nerve fibres that generate the CMMC, suggesting that EGCs are innervated and are activated during the CMMC.Abstract Enteric glia cells (EGCs) form a dense network around myenteric neurons in a ganglia and are likely to have not only a supportive role but may also regulate or be regulated by neural activity. Our aims were to determine if EGCs are activated during the colonic migrating motor complex (CMMC) in the isolated murine colon. Strips of longitudinal muscle were removed and Ca 2+ imaging (Fluo-4) used to study activity in EGCs within myenteric ganglia during CMMCs, followed by post hoc S100 staining to reveal EGCs. The cell bodies of EGCs and their processes formed caps and halos, respectively, around some neighbouring myenteric neurons. Some EGCs (36%), which were largely quiescent between CMMCs, exhibited prolonged tetrodotoxin (TTX; 1 μM)-sensitive Ca 2+ transients that peaked ∼39 s following a mucosal stimulus that generated the CMMC, and often outlasted the CMMC (duration ∼23 s). Ca 2+ transients in EGCs often varied in duration within a ganglion; however, the duration of these transients was closely matched by activity in closely apposed nerve varicosities, suggesting EGCs were not only innervated but the effective innervation was localized. Furthermore, all EGCs, even those that were quiescent, responded with robust Ca 2+ transients to KCl, caffeine, nicotine, substance P and GR 64349 (an NK2 agonist), suggesting they were adequately loaded with indicator and that some EGCs may be inhibited by substances released by neighbouring neurons. Intracellular Ca 2+ waves were visualised propagating between closely apposed glia and from glial cell processes to the soma (velocity 12 μm s −1 ) where they produced an accumulative rise in Ca 2+ , suggesting that the soma acts as an integrator of Ca 2+ activity. In conclusion, Ca 2+ transients in EGCs occur secondary to nerve activity; their activation is driven by intrinsic excitatory nerve pathways that generate the CMMC.

Clostridium difficile-related postinfectious IBS: a case of enteroglial microbiological stalking and/or the solution of a conundrum?

“…Enteric glial cells play an active role within the neural circuits that control gut motility [34]. To exert this role, EGC strongly interact with the enteric neurons and neurotransmitters [27,29].…”

Optical Tools to Investigate Cellular Activity in the Intestinal Wall

“…It has been shown that enteric glial cells tune in to neuronal activity 108,109 and take part in ENS signaling that underlies colonic motility. 110,111 Also, our understanding of myogenic and other mesenchymal control elements present in the gut wall, has improved based on imaging results. For example, recent reports show that interstitial cells of Cajal can operate independently from enteric neurons to control segmentation motor activity, 112 and need the Ca 2+ -activated Cl − channel Ano1 to coordinate slow waves in the smooth muscle.…”

<em>In Situ</em> Ca²⁺ Imaging of the Enteric Nervous System

“…In situ Ca 2+ imaging has yielded great insight into the signaling mechanisms of enteric neurons and glia and possesses several distinct advantages over cell culture models 6,24 . First, in situ preparations maintain the native matrix environment of neurons and glia and leave the bulk of their connections to target tissue intact.…”

“…Changes in fluorescence are recorded by a CCD camera and analyzed digitally 6 . The advent of Ca 2+ provided the opportunity to monitor neuron and glia cell interactions, responsiveness to various stimuli, and the involvement of these cell types in gastrointestinal processes in real time.…”

“…However, recent studies strongly suggest that neuron-glia interactions are essential for ENS functions 5,6 . For example, data show that enteric glia "listen" to neuronal activity 7 and modulate neuronal circuits 6,8 , protect enteric neurons from oxidative stress 9 and are capable of generating new enteric neurons in response to injury 10,11 . The protocol presented in this technical review provides a simple and robust method to examine the complex interplay between neurons and enteric glia using in situ intracellular Ca 2+ imaging.…”