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 ne urons. In DiI-labelled colonic myenteric neurons, the feasibility of record ing ionic events was determined by applying DiI either to the mucosa or the circular muscle, dispersing neurons after 48 h organotypic culture, and pa tch-clamping/calcium imaging labeled neurons after 3-7 days in culture. Mye nteric neurons with diffuse DiI fluorescence were typically smooth and agra nular. Neurons labeled after DiI was applied to circular muscle, fired in e ither a phasic or a tonic manner, and exhibited fast afterhyperpolarization s (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 prol onged afterhyperpolarization (AH, 4-6 s). The offset of a suprathreshold de polarizing step elicited a prolonged outward tail current that approximated the timecourse of the prolonged AH. In addition, in response to membrane d epolarization in DiI-labeled neurons loaded with fura-2, robust Ca2+ transi ents were recorded using the perforated patch technique. These results demo nstrate that DiI labeling of cultured myenteric neurons is feasible, and pa tch clamp/Ca2+ fluorescence recordings can be made from specific population s of cultured DiI-labeled colonic myenteric neurons. (C) 2000 Elsevier Scie nce B.V. All rights reserved.