Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC176
Activation of enteric glia during the colonic migrating motor complex (CMMC) in mice
M. J. Broadhead1, P. O. Bayguinov1, K. M. Paul1, G. W. Hennig1, T. K. Smith1
1. Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada, United States.
Introduction: Enteric glial cells (EGCs) are astrocyte-like cells found in the enteric nerve plexuses. EGCs may modulate neural activity (Bush, 2002). The CMMC is a neurogenic series of contractile and electrical events that have been shown to underlie propulsion of fecal pellets in the mouse (Heredia et al., 2010). The aim of this study was to determine EGCs are activated during the CMMC. Methods: Adult C57/BL6 mice were killed by inhalation of 5% isoflurane in 97% O2 + 3% CO2 followed by cervical dislocation. The entire colon was removed and pinned serosal side up and perfused with oxygenated Krebs’ solution at 37°C. Strips of longitudinal muscle was removed to expose myenteric ganglia and the tissue loaded with Fluo-4. Ca2+ imaging was used to examine the activity of myenteric neurons and EGC during the CMMC (Bayguinov et al., 2010). CMMCs were evoked either by stimulating the mucosa under the recording site with puffs of nitrogen or by brushing the mucosa at the anal end of the colon (Bayguinov et al., 2010). Following Ca2+ imaging experiments, tissues were dual-stained for neuronal Nitric Oxide Synthase (nNOS) and S-100. Results: S100 staining revealed a dense glial cell network around myenteric neurons; whereas, NOS immunoreactivity stained ~40% of all neurons in the myenteric ganglia. Glia loaded with Fluo-4 looked like tear dropped or triangular shaped cells that formed caps on the apical parts of neurons; whereas, their processes formed distinctive halos around myenteric neurons. Between CMMCs, Ca2+ activity in EGCs was variable, with most (83%, n=4) cells remaining quiescent, although slow fluctuations in Ca2+ were observed in a small number of cells. In contrast, many varicose nerve fibers and myenteric neurons, including NOS-positive inhibitory motor neurons, displayed uncoordinated fast Ca2+ transients (frequency 1.3 ± 0.2Hz). During CMMCs, which had duration of 27.9 ± 3.0 s, many varicosities and neurons displayed coordinated, prolonged phasic bursts of activity, while NOS-positive inhibitory motor neurons decreased their activity (Bayguinov et al., 2010). However, during a CMMC, EGCs exhibited a coordinated sustained, slow rise in calcium (duration: 24.2 ± 1.5 s; n=4) that slowly declined following the CMMC. Calcium transients in EGCs, which were observed to propagate along their processes, correlated with net increases in neuronal activity, as well as varicosities that were closely apposed to or upon the glial cells. In the presence of TTX (1µM), high KCl (50mM), spritzing substance P (1µM) or sodium nitroprusside (10µm; nitric oxide donor) activated the glial cell network. Conclusion: EGCs are activated presumably by excitatory neurotransmitters during the CMMC, and possibly by nitric oxide between complexes. However, their role in modulating neuronal activity needs to be further elucidated.
Where applicable, experiments conform with Society ethical requirements