Volume 152, Issue 8, Pages 1831-1833.e4 (June 2017) Micro-Coordination of Pacemaker Potentials in the Intestine of the Mouse  Hirotaka Morishita, Naoko Iwata, Chiho Takai, Naoto Mochizuki, Noriyuki Kaji, Masatoshi Hori, Shunichi Kajioka, Shinsuke Nakayama  Gastroenterology  Volume 152, Issue 8, Pages 1831-1833.e4 (June 2017) DOI: 10.1053/j.gastro.2017.04.016 Copyright © 2017 AGA Institute Terms and Conditions

Figure 1 Photograph showing a dialysis membrane utilized for MEA recordings. A muscle sheet was mounted on an 8 × 8 sensing electrode array. Gastroenterology 2017 152, 1831-1833.e4DOI: (10.1053/j.gastro.2017.04.016) Copyright © 2017 AGA Institute Terms and Conditions

Figure 2 Coordination patterns of basal spontaneous electric activity in the ileum: ‘bumpy/localized’, ‘expanding’ and ‘migrating’. Representative field potential images (left) and illustration summarizing the pattern characteristics (right) are shown for each type. Gastroenterology 2017 152, 1831-1833.e4DOI: (10.1053/j.gastro.2017.04.016) Copyright © 2017 AGA Institute Terms and Conditions

Supplemental Figure 1 Illustration showing the electric current components of the field potentials propagating over the membrane electrode assembly (MEA) sensing regions. In this model, each cell represents an electric syncytium of pacemaker interstitial cells and smooth muscle cells located at a different microelectrode region, i.e. ME(A), ME(B), ME(C) and ME(D). The waveform of the spontaneous electric activity recorded by the MEA in the presence of nifedipine is mostly defined by a transient negative potential (period A: fast down-stroke) followed by a slowly oscillating potential (period S), which are considered to reflect the depolarization and repolarization phases of the pacemaker potential, respectively. Some MEA recordings, also show a transient positive potential of sizeable amplitude preceding the transient negative potential (period C). Careful comparison between the field potential traces and images indicates that this preceding positive potential may reflect the intercellular propagation of a pacemaker current that charges the capacitances of the membranes of interstitial cells and smooth muscle cells (electrically connected via gap junction channels) in the adjacent microelectrode region to subsequently activate an inward (pacemaker) current. The propagation of a light-colored area of activity behind a dark-colored area implies that these areas represent the source and sink regions, respectively, of a local circuit current (a volume conductor) that propagates pacemaker activity. It is noted that both the transient positive and negative potentials contribute to the depolarization phase, the former passively and the latter actively. This propagation feature agrees well with an important role for an atypical voltage-gated current in pacemaker interstitial cells.1 Note that each capacitor in the figure represents the capacitance of all the electrically connected cellular components in that ME region (i.e. interstitial cells and smooth muscle cells). Gastroenterology 2017 152, 1831-1833.e4DOI: (10.1053/j.gastro.2017.04.016) Copyright © 2017 AGA Institute Terms and Conditions

Supplemental Figure 2 An example of the shift of micro-coordination pattern from ‘expanding’ to ‘migrating’. A-C: control; D-F: after application of 5-hydroxytryptamine (5-HT, 100 μM). A and D: Continuous recordings of field potential made by three different microelectrodes (MEs). B and E: Field potential images acquired during the periods shown by the lines in A and C, respectively. The active area expanded from the lower left region in B, but progressively migrated from the middle-left region to the lower-right region in E. C and F: The traces represent changes in field potential recorded at four different MEs, shown on an expanded scale. The colors of the circles below the potential images are matched to those of the traces. For the potential images (B, E) and superimposed field potentials (C, F), the amplitude of the field potential recorded by each ME was corrected by a normalizing factor (FN). The MEA measurement under control conditions showed that the basal electric activity exhibited the ‘expanding’ pattern as illustrated in Figure 2 (the middle row), although there was variation in the initializing region between cycles (See, Video 2). The shift of the pattern to ‘migrating’ in the presence of 5-HT implies a contribution of pacemaker cells to propulsion-like motions of the small intestine.1,2 Since the oral and anal ends of the muscle sheet were placed carefully toward the upper and lower ends of the MEA, respectively, the diagonal orientation of the migrating waves in E indicates only minor contributions of the circular (horizontal) and longitudinal (vertical) smooth muscle layers. Gastroenterology 2017 152, 1831-1833.e4DOI: (10.1053/j.gastro.2017.04.016) Copyright © 2017 AGA Institute Terms and Conditions