SUPPLEMENTARY INFORMATION. All experiments were conducted according to policies on the care and use of laboratory

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1 SUPPLEMENTARY INFORMATION Supplementary Methods: All experiments were conducted according to policies on the care and use of laboratory animals of the Society of Neurosciences, with respect of the national laws on animal use. Generation of TREK-1 deficient mice. TREK-1 genomic clones were isolated from a 129 mouse genomic library by using a TREK-1 cdna probe and subcloned into pbluescript SK (Stratagene). The Floxed targeting vector was generated from a 7.5 kb Bgl2/EcoR1 restriction fragment containing exon 1-3 of the KCNK2 gene. The vector was designed to allow CRE-mediated deletion of exon 3 which encodes the TM1 domain of the channel. A first loxp sequence was inserted in the 5 flanking intron of exon 3. Similarly the PGKneomycin resistance cassette (neo) was inserted together with a second loxp sequence in the 3 flanking intron of exon 3. Both loxp sequences were in the same orientation to allow CRE-mediated simultaneous excision of Exon 3 and neo cassette. A copy of the diphteric toxin gene was subcloned adjacent to the homologous region for negative selection of the ES clone. The targeting vector (50 g) was linearized prior to electroporation into 129-derived embryonic stem cells. After drug selection (G-418, 350 g/ml), one positive clone (1/288) was identified by Southern blot and PCR analysis. Five highly chimeric males were generated by injection of the targeted ES cells into C57Bl/6J blastocysts weeks old male TREK-1 +/+ and TREK-1 -/- used for behavioral experiments were from N6 and N11 C57Bl/6J derived +/- F2 intercross littermates. TREK-1 +/- (N6 and N11 backcross to C57Bl/6J) were crossed to generate +/+ and -/- male and female littermates (N6F1). Male and female of the same genotype (either +/+ or -/- ) were then crossed to each other to generate large number of animals used for behavioral experiment. The results arising from experiments performed on N6F2 and N11F2 mice were comparable. A/ /07/06

2 Corticosterone Assay. Mice were individually housed for at least 12 hours before blood sample collection. Serum samples, collected in the morning were obtained by retroorbital puncture before and 30 min after the end of a 10-min tube restraint. Levels of corticosterone were measured by radioimmunoassay using a commercially available kit (MP Biomedicals). TREK-1, Tryptophane hydroxylase, BrdU and Doublecortin (DCX) immunostaining. For counting of BrdU or DCX-positive cells, mice were injected with BrdU (4 x 75 mg per kg every 2 hours) on the final day of the 21-day vehicle or fluoxetine treatment. Twenty-four hours after the last injection of BrdU, mice were euthanized and transcardially perfused with 4% cold paraformaldehyde. To ascertain that BrdU positive cells differenciate and mature into neurons, another group of mice were euthanized 3 weeks after BrdU (a time at which the cells have matured). Serial sections of the brains were cut (40 m) throughout the entire hippocampus on a vibratome (Leica). Every sixth section throughout the hippocampus was processed for BrdU or doublecortin (DCX) immunohistochemistry as previously described 35,37. For each immunodetection, slides were first incubated overnight at 4 C with monoclonal mouse anti-brdu (1:200; Becton-Dickinson), goat α-dcx (1:400, Santa Cruz Laboratories), polyclonal rabbit anti-trek-1 17 (diluted 1:5000) or monoclonal mouse antitryptophan hydroxylase (diluted 1:1000; Sigma). For chromogenic immunodetection, sections were then incubated for 1 hour in biotin-conjugated species-specific secondary antibodies (diluted 1:100; Vector Laboratories) followed by a peroxidase-avidin complex solution according to the manufacturer s protocol. The peroxidase activity of immune complexes was visualized with DAB staining using the VectaStain ABC kit (Vector Laboratories). For fluorescent double-labeling, performed to determine the cell phenotype, sections were incubated overnight at 4 C with anti-sheep BrdU (1:200, Interchim), anti-goat DCX (1:200, Santa Cruz Laboratories), anti-mouse NeuN (neuron specific nuclear protein, marker for mature neurons, 1:250, Chemicon), GFAP (Glial Fibrillary acidic protein, marker for astrocytes, 1:250, Dako), anti-trek 1:3000 or anti-thp (1:1000). They were revealed with A/ /07/06

3 anti-igg Alexa 488 or 594-coupled antibodies (1:400; Molecular Probes). For quantification of BrdU or DCX labeling, a modified stereology protocol was used 35. All BrdU or DCXlabeled cells in the granular cell layer and subgranular zone were counted in each section (n = 10 and 5 mice per group) at 400x and 1000x under a light microscope (Olympus) by an experimenter blinded to the study code. The total number of BrdU or DCX-positive cells per section was multiplied by 6 to obtain the total number of cells per dentate gyrus. For double BrdU/NeuN or BrdU/GFAP labeling, around 50 BrdU-positive cells per mouse were analyzed using a Laser Scanning Confocal Microscope (TCS SP, Leica) equipped with a DMIRBE inverted microscope. Patch-clamp recordings in transfected cells. COS cells were seeded at a density of cells/35-mm dish 24 h before transfection. Cells were transiently transfected by the classical DEAE-dextran method with human TREK1 or TRAAK plasmids and co-transfected with EYFP, a fluorescent marker. Transfected cells were visualized 48 to 72 h after transfection using fluorescence. For whole-cell experiments the bath solution contained (in mm): 150 NaCl, 5 KCl, 1 CaCl 2, 3 MgCl 2, 10 HEPES, adjusted to ph 7.4 with NaOH; the patch pipette solution contained (in mm): 155 KCl, 3 MgCl 2, 5 EGTA, 10 HEPES, adjusted to ph 7.2 with KOH. Cells were clamped at 80 mv and voltage changes were either applied by ramp (-120 to +50 mv) or by step (-100 to +40 mv). Cells were continuously superfused with a microperfusion system. Recordings were done at room temperature and whole cell currents measured at 0 mv. To obtain IC 50 values for concentration-dependent inhibition experimental data were averaged and than fitted with a standard sigmoidal function. Fluoxetine concentrations in mouse whole-brain. Mice received a single dose of fluoxetine (i.p., 3 mg per kg) for acute treatment or 1 mg per kg daily for 21 days (chronic treatment), whereas the control group of animals received saline injections. Mice were then euthanized at different times following the last injection (0, 0.5, 3, 6 and 24 hours). Brains were rapidly A/ /07/06

4 removed, weighted and homogeneized (10 ml/g) in cold acetone-1n formic acid (85:15 v/v) and centrifuged (11000 rpm, 30 min). The supernatant was shaken twice with n-heptanechloroform (90:10 v/v), the organic phase was discarded. The samples of aqueous phase were evaporated and diluted in 100 l methanol. Determination of whole-brain fluoxetine concentrations were performed using a gas chromatographic/mass spectrometric analysis with a capillary column (Varian) according to standard methods. The chromatographic system was connected to a mass spectrometer (Agilent HP5973). Quantification of fluoxetine brain concentrations were obtained from measuring of peak areas of 3 ions (104, 148 and 309) compared to calibration peaks prepared from a solution of fluoxetine (10 mg/l) diluted in methanol. Statistical Analyses. The behavioral studies were undertaken by investigators blind to the genotype of mice. Data were expressed as mean ± s.e.m. Statistical analysis of differences between groups was performed by using unpaired t test or ANOVA (with one or two factors and with or without repeated measured where appropriate). Where F ratios were significant, statistical analyses were extended and post-hoc comparisons made by using LSD, Newman- Keuls or Tukey s test multiple comparison tests. The level of significance was set at P < Supplementary Data: Suppl Figure 1. TREK-1 immunolocalization throughout the network of limbic, striatal and prefrontal cortical neuronal circuits. Sections were stained with the polyclonal antibody anti-trek TREK-1 was highly expressed in the accumbens nucleus (Nac), the amygdalo-hippocampal area (AHi), the hippocampal CA3 field (CA3), the medial prefrontal cortex (mpfc), the dorsal raphe nucleus (DRN) and the paraventricular nucleus (PVN) but not or poorly in the pontine nucleus (PN) and the ventral tegmental area (VTA). A/ /07/06

5 Suppl Figure 2. TRAAK expression in 5-HT neurons of the dorsal raphe nucleus Sections were stained with the polyclonal antibody anti TRAAK 30. TRAAK is highly expressed in the DRN and present in 5-HT neurons identified by the tryptophane hydroxylase antibody as for TREK-1 (see legend Fig. 5b). Suppl Figure 3. 5-HT selective reuptake inhibitors (SSRIs) inhibit human TREK-1 but not TRAAK currents. a) Whole cell currents were evoked by voltage ramp and steps in absence (Control) and in presence of 10 µm paroxetine. b) The inhibition of TREK-1 currents by SSRIs is concentration-dependent. Dose-response curves are represented for fluoxetine (IC 50 = 6.52 µm, n = 15), paroxetine (IC 50 = 5.47 M, n = 18), sertraline (IC 50 = 3.19 M, n = 7) and fluvoxamine (IC 50 = 8.54 M, n = 8). Suppl Figure 4. Mean whole-brain fluoxetine concentrations after acute and chronic fluoxetine treatment. Data are expressed as mean ± s.e.m. (n = 3 per time point) in nmol per g brain. A/ /07/06