Fig. 5.

Laser confocal imaging of touch-induced purinergic Ca²⁺ signals in neurons of the intact microdissected submucous plexus of the rat distal colon. a Perfusion chamber used to record touch-induced Ca²⁺ responses in the neurons. It includes a recording chamber made of a 35-mm microwell dish with a 1-cm, number 0.0 cover glass in the center of the bottom of the dish for visualizing the fluorescent neurons. A portion of the dish is embedded with a 2-mm thick layer of Sylgard shaped around a half moon at one end of the dish. Perfusion with oxygenated buffer or drugs is exactly opposite to the suction. The microdissected M-SMP preparation is stretched and fixed with many (15-20) micropins along the Sylgard layer. The remaining SMP without mucosa is stretched over the glass cover slip of the dish and secured with magnetic metal feet (not shown). A large ganglion of the intermediate layer of the submucous plexus is viewed with a 40× oil immersion objective (N.A. 1.3). b Inset shows Fluo-4/AM loaded neurons representing second order neurons that are visualized. c Side view of the tissue: The submucous plexus layer is located flat on the bottom of the dish and the mucosa/submucosa lifts over the Sylgard ends at each side of the half moon. The tissue is touched for 20 s on the mucosal surface of M-SMP portion that is stretched across the Sylgard with a fire-polished glass pipette to evoke a Ca²⁺ response in the neurons—the tissue is free to move/stretch downward during the touch, since it is located a distance of 2 mm above the glass bottom. d–g The P2Y₁ antagonist MRS2179 or TTX abolish the touch-induced Ca²⁺ fluorescence response. A representative example of the effects of MRS2179 and TTX on touch-induced Ca²⁺ transients in a single ganglion (seven neurons in one ganglion). A 20-min exposure to the selective P2Y₁ antagonist MRS2179 (10 μM) abolished the touch response in the neurons. In the presence of 0.2 μM TTX, the touch response decreased to below baseline levels. Arrows indicate touch-responsive cells. h Pooled data for the peak effects of MRS2179 and TTX; averaged transients from 30 neurons from 3 to 4 separate ganglia. i Breaking down endogenous nucleotides reduces the touch-induced Ca²⁺ response in the neurons of the intact M-SMP. The touch response was determined in the presence of 5 units/ml apyrase exposed for 10 min. Apyrase reduced by 50% the control touch response (n = 4 neurons, p < 0.01). Values are means ± SEM. (Modified from [18] by permission)