bowel syndrome with a prevalence of around 10% [18]. Interestingly, this disease is known to be frequently complicated by psychiatric illness and mood disorders. Materials and Methods Animals Animals used in the present study were treated ethically. All procedures were approved by the Institutional Animal Care and Use Committee. BALB/c (wild-type) and W/Wv mice (3–6 weeks after birth) were killed by cervical dislocation, after being anaesthetized with diethyl ether. Unless otherwise stated, BALB/ c mice were used in all experiments. Ca2+ imaging Cell cluster preparations were used in Ca2+ imaging [16,19]. Although we detected 5-HT-augmentation of ICC Ca2+ activity in isolated ileal musculature segments containing the myenteric plexus (Figure S1; Video S1 and Video S2), we used cell cluster preparations in Ca2+ imaging to examine numerous drugs related to 5-HT, because it was difficult to stably load Ca2+ indicators in many intact muscle segments. The musculature along with the myenteric plexus were carefully dissected from the ileum, and incubated in Ca2+-free Hanks’ solution containing collagenase (1 mg/ml, Wako Chemical, Osaka, Japan), trypsin inhibitor (2 mg/ml, type I-S, Sigma, St Louis, MO, USA), ATP (0.3 mg/ml, Seikagakukogyo, Tokyo, Japan), and bovine serum albumin (2 mg/ml, Sigma) for 40 min at 37uC. The musculature preparation was then triturated with fire-blunted glass pipettes. The resultant cell clusters were plated onto a lab-made culture dish, and kept in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% foetal bovine serum (Sigma) and antibiotics (30 mg/ml streptomycin and 30 units/ml penicillin; Sigma) at 37uC for 2–3 days. The cultured cell cluster preparations were incubated in ‘normal’ solution containing approximately 8 mM Fluo3-AM (acetoxymethly ester of Fluo-3: Dojindo, Kumamoto, Japan) and detergents (0.02% Pluronic F-127: Dojindo; 0.02% cremophor EL, Sigma) for 3–4 h at room temperature. A CCD camera system (Argus HiSCA, Hamamatsu Photonics, Hamamatsu, Japan) was used to continu- ously monitor digital images of Fluo-3 emission light. The cell clusters were illuminated at 488 nm and emission light of 515– 565 nm was detected. The temperature of the recording chamber was kept at 35uC using a micro-warm plate system (MP10DM, Kitazato Supply, Fujinomiya, Japan). Digital images (0.963 mm/ pixel) were normally collected at 300 ms intervals. Changes in fluorescence emission intensity (F) were expressed as Ft/F0, where F0 is the basal fluorescence intensity. Ratio-images were constructed by dividing each Ca2+ image with a Ca2+ image acquired at a basal [Ca2+]i time after subtracting background fluorescence. The frequency of spontaneous [Ca2+]i oscillations in the presence of nifeipine (and TTX) did not differ from that of spontaneous oscillatory inward currents previously measured by the patch clamp technique [20]. We thus judged that the procedure for loading fluo- 3 was appropriate, and the spontaneous [Ca2+]i activity reflected pacemaker activity in ICC. This notion also agrees well with previous reports in which dihydropyridine (DHP) Ca2+ antagonists selectively suppress [Ca2+]i activity in smooth muscle by blocking L- type Ca2+ channels [13,19]. In preliminary experiments, we checked the effects of several concentrations of 5-HT receptor agonists and antagonists in order to assess which subtypes of 5-HT receptors are responsible for the augmentation of [Ca2+]i oscillations in ICC. First, 5-HT was examined at 1, 3, 10 and 50 mM (n = 3–4). The active area of Serotonin Augments Gut Pacemaker Activity [Ca2+]i oscillations was nearly the same (102% of the control) at 1– 3 mM, and increased to ,145% at 10 mM and to ,149% at 50 mM. We thus compared the effects of 5-HT and other 5-HT receptor agonists at 10 mM (Fig. 1 and 2). LY-278584, a 5-HT3 antagonist, did not significantly suppress [Ca2+]i oscillations below 10 mM in 10 min. In order to minimize the deterioration of fluo-3 fluorescence during illumination, 10 mM of LY-278584 was normally applied (Fig. 2A–B). Also, according to previous experiments in enteric neurones and ICC, 10 mM GR113808 [21] and 40 mM SK&F96365 [17] were used to inhibit 5-HT4 receptors and Ca2+-permeable transmembrane channels, respec- tively (Fig. 2C–D). Electrical recording An 868 planar microelectrode array (150 mm in polar distance) connected to a 64-channel amplifier (MED 64 System: Alpha Med Science, Osaka, Japan) was used to simultaneously record electrical field potentials of ,1 mm2 square [22,23]. Ileal musculature segments (,5 mm620 mm) containing the myenteric plexus were fixed using a brain slice anchor (SDH series, Harvard Apparatus Japan, Tokyo, Japan) in the recording chamber kept at 35uC on a heater, and were superfused with ‘normal’ extracellular solution at a constant rate of 2 ml/min. The extracellular solution contained nifedipine (Sigma) and TTX (LKT Laboratories: St Paul, MN, USA) in order to isolate ICC pacemaker activity by suppressing smooth muscle and neural activities. Slow electrical potentials were recorded by applying a high-pass filter of 0.1 Hz to stabilize the baseline drift [24]. A sampling rate of 20 kHz was applied. In field potential data processing, the digital resolution was reduced to 50 ms (20 Hz) by thinning out the recording points at 1:1000, and an FFT-based digital band-pass filter (0.04–0.5 Hz) was additionally applied. The effects of 5-HT and 2-Me-5-HT on ICC pacemaker electrical activity were evaluated using a power spectrum (9.4– 27.0 cpm). Two-dimensional field potential images were constructed by calculating the values at the desired location via spline interpolation (with 50 points between each electrode), using the MATLAB software package (Mathworks: Natick, MA, USA) [25]. RT-PCR A longer (50 min) enzymatic incubation and more complete trituration with glass pipettes were performed to obtain isolated cells. The digestive enzymes used were the same as described for cell cluster preparations. The resultant cell suspension was incubated in a ‘normal’ extracellular solution containing phyco- erythrin-conjugated anti-mouse CD117 (c-Kit) antibody (PE- ACK2, eBioscience, San Diego, CA, USA) in 1/100 v/v for 10 min, and then centrifuged and rinsed with ‘normal’ solution three times. About 5–10 isolated smooth muscle cells and c-Kit- immunopositive cells were separately collected into sterile tubes, using patch pipettes (GC150-15, Harvard Apparatus, Kent, U.K.) with 10–20 mm tip diameter under a fluorescent microscope. The samples were kept at 280uC until use. The procedures for subsequent RT-PCR were the essentially the same as previously described [26]. The following PCR primers were used: 5-HT2B (NM_008311, 1340-1443, amplicon = 104 bp): 59-GATCAACCCTGCCATG- TACCA-39 (+) and 59-CGCCATCGTTTTCAGTGAGA-39 (-); 5-HT3A (NM_013561, 363-463, amplicon = 101 bp): 59-GAC- TCCTGAGGACTTCGACAATG-39 (+) and 59-ACTTCCC- CACGTCCACAAACT-39 (-); 5-HT3B (NM_020274, 607-728, amplicon = 122 bp): 59-ACTCTTCTGGCACCATTAGAACC- 39 (+) and 59-GAGGCTGCAGTTCTGGATATCA-39 (-); 5-HT4 (NM_008313, 1100-1222, amplicon = 123 bp): 59-CTTTCC- TCTGGCTTGGCTATATCA-39 (+) and 59-GTCTTTTGT- PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24928