Activity in varicosities within the myenteric plexus between and during the colonic migrating motor complex in the isolated murine large intestine

Abstract

Background Neuronal communication within the myenteric plexus occurs when action potentials along nerve fibers produce Ca2+ transients in varicosities leading to exocytosis of vesicles and neurotransmitters release. We used Ca2+ transients in varicosities to monitor action potential activity in myenteric nerve pathways both between and during the colonic migrating motor complex (CMMC) in the isolated murine colon. Methods Strips of longitudinal muscle were removed to reveal the myenteric ganglia, which were then loaded with Fluo-4. Key Results Many varicosities, including synaptotagmin 1 labeled varicosities, exhibited ongoing Ca2+ transients (duration of unitary Ca2+ transient 3.9s). Between CMMCs, varicosities fired at a frequency of 0.6 Hz, which correlated with spontaneous inhibitory junction potentials in the circular muscle, suggesting they were mainly in inhibitory nerve pathways. During a CMMC other previously quiescent varicosities fired at 1.3 Hz (max 2.0 Hz) for the duration (24 s) of the CMMC, suggesting they were on excitatory nerve pathways. Activity in varicosities was correlated with Ca2+ transient responses in a number of neurons. Some varicosities appeared to release an inhibitory neurotransmitter that reduced activity in nNOS-positive neurons. Varicosities along the same nerve fiber exhibited identical patterns of activity that allowed nerve fibers to be traced throughout the myenteric plexus and intermodal strands. Activity in varicosities was reduced by hexamethonium (100μM), and blocked by ω-conotoxin GVIA (200 nM) and tetrodotoxin (1μM; TTX). Conclusions & Inferences Ca2+ imaging of varicosities allows for a determination of activity in neural pathways within the enteric nervous system.

Colonic Migrating Motor Complexes, High Amplitude Propagating Contractions, Neural Reflexes and the Importance of Neuronal and Mucosal Serotonin

“…27 Between CMMCs, varicosities fired at a frequency of about 0.6 Hz, which correlates with the frequency of spontaneous IJPs in the CM, suggesting they were mainly in inhibitory nerve pathways. 27 During a CMMC other previously quiescent varicosities fired up to 2 Hz for the duration of the CMMC and may have been part of excitatory nerve pathways. 27 Other varicosities decreased their activity during the CMMC suggesting may have been part of inhibitory nerve pathways.…”

“…Ca 2＋ imaging of activity of myenteric neurons and pacemaker ICC-MY in whole isolated colon demonstrated that IMNs were active but ICC-MY were inhibited between CMMCs. [26][27][28] Tetrodotoxin blocked the firing in myenteric neurons and increased the activity in ICC-MY. Similarly, L-NA also increased the activity in ICC-MY, suggesting they were inhibited by NO released from IMNs.…”

“…27 This is possible because as an action potential propagates down a nerve fiber it depolarizes all varicosities along a particular nerve fiber at the same time causing Ca 2＋ influx and potentially neurotransmitter release (Fig. 4).…”

“…27 During a CMMC other previously quiescent varicosities fired up to 2 Hz for the duration of the CMMC and may have been part of excitatory nerve pathways. 27 Other varicosities decreased their activity during the CMMC suggesting may have been part of inhibitory nerve pathways. Due to the large number of 5-HT varicosities in myenteric ganglia visualized with immunohistochemistry, many of these active varicosities may have been on 5-HT nerve fibers.…”

“…21 During the CMMC, there is a defined pattern switch that turns off IMNs to allow full excitation of the muscle and pacemaker ICC by EMNs. [26][27][28] Without appreciating the integrated nature of the HAPC/ CMMC and the unified nature of the ENS there has been a tendency to focus on one or other of the individual components to explain changes in motility and secretion. 29,30 An understanding of how motor and secretomotor activities are integrated and probably regulated by mucosal and neuronal 5-HT will give insights into basic physiology of these important behaviors and also allow better targeting of different 5-HT receptors in GI disorders to normalize these interactions.…”