Differential Ca(2+) signaling characteristics of inhibitory and excitatory myenteric motor neurons in culture.

Physiological studies on functionally identified myenteric neurons are scarce because of technical limitations. We combined retrograde labeling, cell culturing, and fluorescent intracellular Ca(2+) concentration ([Ca(2+)](i)) signaling to study excitatory neurotransmitter responsiveness of myenteric motor neurons. 1, 1-Didodecyl-3,3,3',3'-tetramethyl indocarbocyanine (DiI) was used to label circular muscle motor neurons of the guinea pig ileum. DiI-labeled neurons were easily detectable in cultures prepared from these segments. The excitatory neurotransmitters (10(-5) M) acetylcholine, substance P, and serotonin induced a transient rise in [Ca(2+)](i) in subsets of DiI-labeled neurons (66.7, 56.5, and 84. 3%, respectively). DiI-labeled motor neurons were either inhibitory (23.8%) or excitatory (76.2%) as assessed by staining for nitric oxide synthase or choline acetyltransferase. Compared with excitatory motor neurons, significantly fewer inhibitory neurons in culture responded to acetylcholine (0 vs. 69%) and substance P (12.5 vs. 69.2%). We conclude that combining retrograde labeling and Ca(2+) imaging allows identification of differential receptor expression in functionally identified neurons in culture.