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IN SITU RECORDING OF GUT PACEMAKER CELLS
B Wang, Y Zhu, W Kunze, JD Huizinga
Intestinal Disease Research Program, Department of Medicine, McMaster University, Hamilton, Ontario
Interstitial cells of Cajal (ICC) are crucial players in gut physiology performing pacemaker functions and assisting in neurotransmission. ICC have been studied after chemical isolation or under culture conditions, but concerns that these methods affect the intrinsic properties have hindered substantial progress in our understanding of ICC. We have developed a method to obtain electrophysiological recordings from ICC in situ. We modified the longitudinal muscle myenteric plexus (LMMP) preparation by removing only small circular muscle bundles in various areas and cleaning the tissue with mild protease (0.01%) treatment. The subsequent electro-physiological recordings were performed on an up-right microscope with patch clamp techniques. ICC in the myenteric plexus area of mouse intestine (pacemaker ICC) were preserved after tissue dissection and the network was revealed by c-kit immunostaining or dye (Lucifer Yellow) injection. The characteristic pacemaker activity, i.e. rhythmic inward currents and slow waves were recorded from ICC in situ. At the single-channel level, we have recorded a dominant chloride channel in the cell attached configuration. We revealed cooperative gating behavior of multiple channels with a dominant conductance level of 126.3 ± 8.6 pS (n=10). In order to study mechanism of innervation of ICC we applied substance P (SP, 0.1 µM), while we were recording channel activity from ICC. SP caused activation or markedly increased open probability of a 116 pS intermediate conductance potassium channel. We report here that we have established an in situ model for studying the properties of ICC and its innervation in situ. This model is ideal for the study of cell-cell communication, such as enteric neuron-ICC- smooth muscle cell interaction.