Serotonin Augments Gut Pacemaker Activity Figure 4. Immunohistochemistry for c-KIT and 5-HT3. Smooth muscle layer of the ileum including the myenteric plexus was double-labelled with anti-c-KIT antibody (ACK4, red) and anti-5-HT3 antibody (sc-28958, green). Left panel (A: merged image) shows myenteric neurons (plexus) (green) surrounded by network-forming cells expressing both c-KIT and 5-HT3. Bar, 50 mm. An ICC-like cell is shown expanded in right panels (B: merged image; C: c-KIT; D: 5-HT3). Bar, 10 mm. The network-like structure and large nucleus are known histological features of ICCs. doi:10.1371/journal.pone.0024928.g004 application of 2-Me-5-HT caused no recovery (n = 5) (Fig. 2D). The responses of pacemaker [Ca2+]i activity to the drugs relating to 5-HT receptors and Ca2+ influx are summarized in Fig. 2E. 5-HT3 receptors in ICC To confirm expression of 5-HT receptors in ileal ICC, we performed RT-PCR. After enzymatic dispersion, c-Kit immuno- positive interstitial cells (equivalent to ICC) and smooth muscle cells were individually collected. A transcript for 5-HT3A was detected only in ICC, while 5-HT2B was detected in both ICC and smooth muscle cells (Fig. 3A). We also confirmed that neither CD68 nor Cma1 (mast cell markers) was detected in c-Kit- immunopositive interstitial cells (n = 4), unlike in spleen samples (n = 4) (Fig. 3B). In addition, transcripts for 5-HT3B and 5-HT4 were detected in ICC, but not in smooth muscle cells (not shown). Immunohistochemistry was also performed. Double-labelled immunostaining with anti-c-Kit and anti-5-HT3 antibodies (Fig. 4) revealed that network-forming interstitial cells expressing both 5- HT3 and c-Kit (orange cells in left panel) exist near the myenteric plexus, which contains neurons with only 5-HT3 immunoreactivity (green). In addition, these network-forming cells have large nuclei (a single cell is shown expanded in the right panels). The network- like structure and large nuclei are known features of ICC [2,6]. Augmentation of electrical activity In order to confirm the excitatory effect of 5-HT on ICC pacemaker activity, we next measured electrical activity. Isolated musclature of the mouse ileum was placed on an 868 microelectrode array (MEA) with a polar distance of 150 mm, and field potentials of a ,1 mm2 area were simultaneously monitored through a multi-channel amplifier and recording system (See Materials and Methods). To suppress smooth muscle and neural activities, extracellular solution contained nifedipine (1 mM)) and TTX (250 nM), respectively. A potential map was constructed by using the simultaneous recordings at 64 channels and spline interpolation (Fig. 5A–B). In this preparation, ICC electrical activity propagated from the left bottom to the right top in normal solution (A), and application of 5-HT (10 mM) potentiated ICC electrical activity (B). Represen- tative field potentials of three channels (Fig. 5C–D) show ICC electrical activities for a long duration. 5-HT significantly increased the amplitude of the field potentials, but did not alter the frequency (Video S3 and Video S4). Also, power spectra over the recording area were constructed from field potentials of all 64 channels for approximately 40 s. This analysis shows a marked increase in the spectral power without shifting the peak frequency. The effect of 2-Me-5-HT (10 mM), a 5-HT3 receptor agonist (Figure S3), on ICC electrical activity was also examined. Essentially similar enhancement was observed. Graphs in Fig. 6 summarise the enhancement of 5-HT and 2-Me-5-HT on ICC electrical activity. The spectral power between 9.4-27.0 cpm (Pw9.4-27.0cpm) was used to evaluate the ICC electrical activity, based on a comparison between wild-type and W/Wv mice [23]. In the control of normal ileum, Pw9.4–27.0cpm (from 64 channels) ranged from 1.0 to 19.061023 mV2 (n = 24). Application of 5-HT increased Pw9.4–27.0cpm to 168645% (P,0.01, n = 12), and 2-Me- 5-HT increased it to 153635% (P,0.01, n = 12). Discussion ICC act as gut pacemaker cells. Moreover, fairly recent studies suggest that these cells also coordinate peristaltic movements through their network-forming structure [30]. In the light of the important roles of ICC, any hormones and neurotransmitters that modulate ICC activity are considered to have a significant influence upon gut motility. The present finding that 5-HT augments ICC activity implies that this signalling molecule in particular plays a crucial role in regulating gut motility, because the gut contains a majority of 5-HT in the body [8,11]. The fact that 2-Me-5-HT causes similar effects to 5-HT implies that 5-HT3 receptors are responsible for the 5-HT-mediated enhancement of ICC Ca2+ activity. Lines of studies have suggested that the primary pacemaking activity is the spontaneous [Ca2+]i oscillations in ICC [13,15,31]. Namely, [Ca2+]i oscillations in ICC periodically activate plasmalemmal Ca2+-activated ion channels: Ca2+-activated Cl2 channels [32–35], and/or Ca2+-activated non- selective cation channels [36–38]. Ca2+ release from the intracellular Ca2+ stores, presumably endoplasmic reticulum (ER), appears to be the major Ca2+ source of [Ca2+]i oscillations PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24928