Recording ionic events from cultured, DiI-labelled myenteric neurons in the guinea-pig proximal colon.
To date investigations of enteric neurons by patch clamping/calcium imaging have been limited by studying unidentified heterogeneous populations of neurons. In DiI-labelled colonic myenteric neurons, the feasibility of recording ionic events was determined by applying DiI either to the mucosa or the circular muscle, dispersing neurons after 48 h organotypic culture, and patch-clamping/calcium imaging labeled neurons after 3-7 days in culture. Myenteric neurons with diffuse DiI fluorescence were typically smooth and agranular. Neurons labeled after DiI was applied to circular muscle, fired in either a phasic or a tonic manner, and exhibited fast afterhyperpolarizations (100-300 ms duration) at the end of a depolarizing pulse. They expressed a fast inward current and at least three different outward currents. Action potentials elicited in DiI-labeled sensory neurons were followed by a prolonged afterhyperpolarization (AH, 4-6 s). The offset of a suprathreshold depolarizing step elicited a prolonged outward tail current that approximated the timecourse of the prolonged AH. In addition, in response to membrane depolarization in DiI-labeled neurons loaded with fura-2, robust Ca(2+) transients were recorded using the perforated patch technique. These results demonstrate that DiI labeling of cultured myenteric neurons is feasible, and patch clamp/Ca(2+) fluorescence recordings can be made from specific populations of cultured DiI-labeled colonic myenteric neurons.
Vogalis
F
F
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
Hillsley
K
K
Smith
T
T
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D002232:Carbocyanines; D002470:Cell Survival; D002478:Cells, Cultured; D003106:Colon / Q000294:innervation*; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods; D016257:Fura-2 / Q000031:analogs & derivatives; D006168:Guinea Pigs; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods*; D009197:Myenteric Plexus / Q000166:cytology* / Q000502:physiology; D009475:Neurons, Afferent / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D018408:Patch-Clamp Techniques
D002232:Carbocyanines; D005456:Fluorescent Dyes; C049925:fura-2-am; C072024:carbocyanine dye DiIC12(3); D002118:Calcium; D016257:Fura-2