Serotonin Augments Gut Pacemaker Activity Figure 1. 5-HT potentiates ICC pacemaker [Ca2+]i activity. A) Ca2+ images acquired from a cell cluster preparation in control (a-b) and 5 min after 5-HT (10 mM) application (c-d). B) Time course of pacemaker [Ca2+]i activity recorded in the three squares (x, y and z) indicated in Ba. Dotted lines correspond to the times when images (Ba-d) were acquired. Changes in fluorescence emission intensity (F) are expressed as Ft/F0, where F0 is the basal fluorescence intensity. C-E) Bars show changes in the peak amplitude (C), frequency (D), and active area of spontaneous pacemaker [Ca2+]i activity (E) (Mean 6 s.d., n = 22). Asterisks, P,0.05 compared to control. F-G) Immunohistochemistry of the ileal myentric plexus (MyP) layer in wild- type (F) and W/Wv mice (G) lacking ICC in this region of the small intestine. Red and green represent neurones (PGP9.5) and ICC (c-Kit), respectively. H) The lack of effect of 5-HT (10 mM) in a cell cluster preparation from W/Wv mice. doi:10.1371/journal.pone.0024928.g001 AGCGCTCATCATCAC-39 (-); c-Kit (Y00864, 2156-2256, amplicon = 101 bp): 59-GAGCCTTCCTGTGACAGTTCAAA- T-39 (+) and 59-TCTATTCTTGCGGATCTCCTCTTG-39 (-); glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (M32599, 730–833, amplicon = 104 bp): 59-CATGGCCTTCCGTGT- TCCT-39 (+) and 59-CCTGCTTCACCACCTTCTTGA-39 (-); CD68 (a marker for mast cells) (NM_009853, 574-830, amplicon = 257 bp): 59-CACCTGTCTCTCTCATTTCC-39(+) and 59- CTTAGAGAGAGCAGGTGAAG-39 (-); Cma1 (a marker for macrophages) (NM_010780, 332–668, amplicon = 337 bp): 59- CGGGAAGGTCTATAACAGTCCTCC-39 (+) and 59-CTGG- TGAAGTGTTTGCAGGCT-39(-). The pair of primers for 5- HT4 was also designed to cover 5-HT4A, 5-HT4B, 5-HT4E, and 5- HT4F. Immunohistochemistry Small segments (10 mm620 mm) of smooth muscle layers (including the myenteric plexus) isolated from the mouse ileum, were fixed with 4% paraformaldehyde (4uC) for 30 min, and permeabilized with 0.1% triton X-100 and 5% BSA (bovine serum albumin, fraction V: Sigma) for 1 h. The tissue was double stained sequentially with anti-5-HT3 antibody [SR-3 (H-138) sc-28958: Santa Cruz Biotechnology, Santa Cruz, CA, USA] and anti- mouse CD117 (c-Kit) antibody (ACK4: Acris antibodies, Ger- many) in 100 mM PBS (phosphate buffered-saline solution) overnight. The PBS contained 1% BSA in order to block non- specific reactions. This was followed by incubation with secondary antibodies, Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 555-conjugated anti-rat IgG (Molecular Probes, Eugene, OR, USA) at a concentration of 15 mg/ml for 1 h. Double-stained small segments were mounted on a slide glass with an anti-fading agent (ProLong: Molecular Probes) and scanned using a confocal microscope (TCS-SP2: Leica Microsystems, Tokyo, Japan). Controls were prepared by omitting the primary antibodies. The reactivity was negligible in network-forming cells in the myenteric plexus (i.e. ICC). The antibody used for staining the 5-HT3 receptor (sc-28958: Santa Cruz Biotechnology) is a rabbit polyclonal antibody raised against amino acids 341–478 mapping at the C-terminus of 5-HT3A of human origin, and is used in human, mouse and rat specimens. Solutions and drugs The composition of the ‘normal’ extracellular solution used in [Ca2+]i imaging and electrical recording was (in mM): NaCl, 125; KCl, 5.9; MgCl2 1.2; CaCl2 2.4; glucose 11; Tris-HEPES 11.8 (pH 7.4). Nifedipine, LY-278584, 2-methyl-5-HT (maleate salt), GR113808 and O-methyl-5-HT (hydrochloride) were purchased from Sigma. SK&F96365 was from Calbiochem (San Diego, CA, U.S.A.). Stock solutions of nifedipine were prepared by dissolving the drug in ethanol, while other drugs were dissolved in dimethyl sulfoxide (DMSO). The working concentrations of the solvents were less than 1%. In preliminary experiments, we observed that applications of this concentration of either solvent alone had little effect on [Ca2+]i oscillation in cell cluster preparations. DMEM and other reagents for cell culture were purchased from Sigma. Statistics Numerical data are expressed as mean6SD. Differences between means were evaluated by paired t-tests. The probability (P),0.05 was taken as a statistically significant difference. PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24928