Supplementary Information

Antidepressants recruit new neurons to improve stress response regulation

Alexandre Surget, Arnaud Tanti, E. David Leonardo, Anthony Laugeray, Quentin Rainer, Chadi Touma, Rupert Palme, Guy Griebel, Yadira Ibarguen-Vargas, René Hen, Catherine Belzung.

 Supplementary Materials and Methods (p2)

 Supplementary Figure S1 (p12)

 Supplementary Figure S2 (p13)

 Supplementary Figure S3 (p14)

 Supplementary Figure S4 (p15)

 Supplementary Figure S5 (p16)

1 SUPPLEMENTARY MATERIALS AND METHODS

Animals

Male BALB/c mice were purchased from Taconic (Germantown, NY, USA) or from Centre d'Elevage Janvier (Le Genest Saint Isle, France), and were aged between 3 and 4 months at the time of the beginning of the UCMS exposure. Animals were group-housed (n = 4-5 per cage) until the onset of the UCMS regimen and maintained under standard laboratory conditions (12/12h light-dark cycle –on at 11:00/off at 23:00–, 22±2°C, food and water were provided ad libitum). All animal care and treatment were in accordance with the European Community Council directive 86/609/EEC and with the Guide and Use of Laboratory Animals established by the US National Institute of Health.

Drugs

The SSRI Fluoxetine-HCl (Eli Lilly, Indianapolis, IN) and the corticotropin-releasing factor receptor 1 (CRF1) receptor antagonist SSR125543 (Sanofi-Aventis, Bagneux, France) were dissolved in a saline solution (0.9% NaCl) or suspended in a saline solution (0.9% NaCl) containing 5% dimethyl sulphoxide (DMSO) and 5% Cremophor EL respectively. To assess the HPA axis negative feedback and the sensitivity of DG granule cells to glucocorticoid effects, the glucocorticoid receptor agonist dexamethasone-phosphate (DEX-P, Sigma- Aldrich, St Louis, MO, USA) was prepared for a single intraperitoneal (i.p.) administration (0.1 mg/kg) in a saline solution (0.9% NaCl). To assess the ability of the hippocampus to inhibit the HPA axis, dexamethasone (DEX, Sigma-Aldrich) was prepared for a single intrahippocampal infusion (50 ng in 0.6 µl) in a saline solution (0.9% NaCl) containing 0.2% ethanol. To assess cell proliferation and to label new adult-born cells in the DG, the thymidine analogous 5-bromo-2-deoxyuridine (BrdU, Sigma-Aldrich) was used as it can incorporate into DNA of proliferating cells. BrdU was dissolved in 0.9% NaCl. For the cell proliferation analysis, mice received four administrations of BrdU (4 x 75 mg/kg, i.p.), one injection every 2hr; mice were perfused 24hr after the last BrdU administration. For the neurogenesis analysis, mice were administered with BrdU during 3 days (twice daily, 150 mg/kg, i.p.), 8hr between the 2 injections each day; mice were perfused 4 weeks later. When

2 immunohistochemistry was performed, mice were anaesthetized with a mixture of ketamine/xylazine dissolved in 0.9% NaCl before perfusion.

Unpredictable Chronic Mild Stress (UCMS)

The stress regimen has been previously presented in detail1 and is a variant of the chronic mild stress procedures described by Willner in rats.2 Mice were repeatedly subjected to various socio-environmental stressors according to an unpredictable schedule for a total period of 6-8 weeks.1 UCMS-exposed mice were maintained under the same standard laboratory conditions but were single housed in small individual cages (24x11x12 cm), while non-stressed control mice were also single housed but in larger cages (42x28x18 cm) with plastic tunnel and shelter. From the third week of UCMS onward, mice were administered ip, once a day, with either vehicle, either fluoxetine (20 mg/kg/day) or SSR125543 (20 mg/kg/day). Doses have been based on our previous experiment showing activity at these concentrations.1

Body weight and coat state assessment

Body weight and coat state were assessed weekly, as markers of the progression of the UCMS-evoked syndrome.1, 3 The total score of the state of the coat resulted from the sum of scores obtained from seven different body parts: head, neck, dorsal coat, ventral coat, tail, forepaws and hindpaws. For each body area, a score of 0 was given for a well-groomed coat and 1 for an unkempt coat.

Cookie Test (CT)

This test required a device containing three aligned compartments with the same dimension (20x20x20 cm). Only the colors of the walls and the floor were different between the compartments: white for the first one, grey for the second one and black for the third one (Figure 2a). The three compartments communicated by two openings controlled by the experimenters. The apparatus was illuminated with 200-lux white light. Starting four weeks and a half before the first session, a small portion (2 g ± 1) of a chocolate cookie (Pepito, Lu, France) was placed in the home cage twice daily during 2.5 weeks in order to familiarize the mice with the palatable stimulus. The last two weeks before the first session were cookie-free. One hour before testing, food was removed in order to avoid inter-individual differences in the drive for feeding (hunger). At the time of testing, a small amount (2 g ± 1) of chocolate

3 cookie (or of regular food in a supplemental control experiment) was positioned at the center of the black compartment. The white compartment was the start compartment, where the mouse was placed with the head facing the opposite side to the opening. Each session of the test lasted five minutes; the door of the first opening was closed after the transition of the mouse within a maximum time of 2 minutes (the mouse was gently guided to the second room if required). The number of bites from the cookie was recorded within the 5min test period. Four sessions of testing were performed within 10 days, each session being separated from the previous one by 3 days. All the sessions were performed during the light phase (from 15:00).

X-ray irradiation procedure

For some experiments, mice were exposed to three targeted X-irradiations focusing on the hippocampus. All mice were anesthetized with ketamine/xylazine (100 mg/kg and 7 mg/kg respectively), placed in a stereotaxic frame and then, only for irradiated mice, exposed to cranial irradiation using a Siemens Stabilopan X-ray system (Hamburg, Germany) as previously described.4, 5 Animals were protected with a lead shield that covered the body but left unshielded for a 3.22 x 11 mm treatment field above the hippocampus (interaural 3.00 to . 00). The corrected dose rate was approximately 1.8 Gy/min at a source to skin distance of 30 cm. The procedure lasted 2 min 47 sec, delivering a total of 5 Gy. Three 5 Gy doses were given over the course of one week (day 0, 4, and 8). This procedure was performed at Columbia University in New York. Mice were then shipped for the UCMS procedure at the U930 of the Inserm and Université F. Rabelais in Tours. Because irradiation induces transient inflammation, mice were allowed to recover for 4 weeks before initiating the UCMS regimen and between 10 and 12 weeks before testing, a period after which the inflammatory effects of hippocampal irradiation are largely completed.6 Thanks to the lead shield, our procedure let the major part of the brain free of radiation. It has been shown that the number of proliferating cells in other neurogenic areas like the subventricular zone remains unaltered and that no consequence on the anatomy, physiology and behavioral functions of the other hippocampal subregions and underlying brain structures such as amygdala or hypothalamus was found from one month after irradiation.4

4 Immunohistochemistry

After anesthesia with ketamine/xylazine (100 mg/kg and 7 mg/kg respectively), mice were transcardially perfused: phosphate-buffered saline (PBS, pH=7.4) for 3min and 4% paraformaldehyde (PFA) for 5min. Brains were then collected, post-fixed overnight in 4% PFA and stored in 30% sucrose 0.1% sodium azide at least 24h before being processed.

Immunohistochemistry for subgranular zone (SGZ) proliferation was performed as previously described.5 Serial coronal sections through the rostro-caudal brain extent were cut (45µm), every third section from each brain was collected. Free-floating sections were treated with 3%

H202 / 50% ethanol for 20min, denatured in 2N HCl (30min). Sections were then incubated with rat anti-BrdU antibody (1:500, Oxford Biotechnology, Oxfordshire, UK) for 40h at 4°C followed by rabbit anti-rat biotinylated antibody (1:200, Vector Laboratories, Burlingame, CA, USA) for 2.5h at room temperature. The staining was amplified with an avidin-biotin complex (Elite ABC kit, Vector Laboratories) and visualized with DAB (Sigma-Aldrich).

To label the immediate early gene (IEG) Fos in several subcortical areas after intrahippocampal injection of dexamethasone (figure 3), immunohistochemistry was performed as below except that sections were not denatured with 2N HCl and were incubated with a rabbit anti-Fos antibody (1:5000, PC38, Calbiochem, San Diego, CA, USA) for 48h at 4°C followed by a donkey anti-rabbit biotinylated antibody (1:500, Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 2h at room temperature.

To assess the effects of chronic stress and fluoxetine on neurogenesis, we labeled 4-week old new neurons by means of immunodetection of the neuronal-specific nuclear protein (NeuN) and BrdU. Serial coronal sections through the rostro-caudal brain extent were cut (30µm). Free-floating sections were denatured with 2N HCl (30min), incubated with primary antibodies for 20h at 4°C followed by fluorochrome-labelled secondary antibodies for 2.5h at room temperature and then mounted on slides with the mounting medium Hard-Set Vectashield (Vector Laboratories). The primary antibodies used were: a mouse anti-NeuN monoclonal antibody (1:1000, Chemicon International, Billerica, MA, USA) and a rat anti- BrdU monoclonal antibody (1:500 Oxford Biotechnology). The secondary antibodies used were: Alexa-488nm anti-mouse and Alexa-633nm anti-rat antibodies (1:200, Molecular Probe, Eugene, OR, USA).

5 In order to assess the sensitivity of 4-week-old new neurons and of the whole granule cell population to novelty and glucocorticoid effect, we labeled cells simultaneously for the neuronal-specific nuclear protein (NeuN), Fos and BrdU. Immunohistochemistry was similar to the one described above, with the addition of a rabbit anti-Fos monoclonal antibody (1:1000 Oxford Biotechnology) as primary antibody and an Alexa-546nm anti-rabbit (1:200, Molecular Probe) as secondary antibody.

Image analysis and cell quantification

When staining was visualized with DAB, the number of positive cells was counted using x20 and x40 objective lens with a conventional light microscope. For SGZ cell proliferation, we counted all BrdU-labeled cells in the granule cell layer (GCL) including the SGZ, where the neural progenitors are located. The SGZ was defined as the deepest row of cells in the GCL bordering the hilus, although it does not strictly represent a well-differentiated cell layer. A cell was counted as being in the SGZ if it was touching or in the deepest row of the GCL. The cells that were located two cells or more away from the SGZ were considered as hilar cells and were not counted. This counting was performed on every third 45µm-section throughout the entire hippocampus. This spacing ensured that the same neurons were not counted in two sections.

For Fos-labeled cell counting of regions of interest and data analysis, the nomenclature and nuclei boundaries used were those defined by Paxinos and Franklin’s mouse brain atlas.7 Fos+ cells within each region were counted bilaterally in consecutive sections starting from bregma 0.62 to bregma -1.58. For every animals and regions, the same number of sections was used and the volume of each area controlled. Regions included and corresponding coordinates were the lateral septum (LS - bregma [0.62 to 0.14]), anteromedial bed nucleus of the stria terminalis (amBST - bregma [0.62 to 0.02]), posteromedial bed nucleus of the stria terminalis (pmBST - bregma [-0.10 to -0.34]), anteroventral bed nucleus of the stria terminalis (avBST - bregma [0.62 to 0.02]), ventrolateral preoptic area (vlPOA -bregma [0.26 to -0.10]), medial preoptic area (mPOA - bregma [0.14 to -0.46]), dorsomedial hypothalamic nucleus (DMH - bregma [-1.34 to -1.58]), lateral hypothalamic nucleus (LH - bregma [-0.34 to -0.70]), and the paraventricular nucleus (PVN) of the hypothalamus (bregma [-0.58 to -1.22]).

When staining was visualized with fluorochrome-labeled secondary antibodies, the number of positive cells was counted using a x40 objective lens with a confocal microscope and Fluoview Software, v5.0 (Olympus) for NeuN/BrdU/Fos labeling and with an epifluorescence

6 microscope for DCX/Fos labeling. Eight sections (16 hippocampus) along the rostro-caudal extent of the hippocampus were examined, approximately bregma: -1.46, -1.82, -2.18, -2.54, -2.80, -3.08, -3.28, -3.52 according to Paxinos and Franklin (2001).7 We counted all the cells double- or triple-labeled for NeuN/BrdU, NeuN/Fos, NeuN/BrdU/Fos within the entire GCL of the eight sections for each mouse.

However, these BrdU+ cells represent only a small part of the neurons born during the last weeks. We therefore aimed to determine activity in a broader population of young adult-born neurons. For this purpose, we counted the number of NeuN+/Fos+ cells in the inner part of the GCL containing the SGZ, a reliable method previously used.8, 9 All the NeuN+/Fos+ cells touching or in the SGZ (i.e. the two deepest rows of the GCL plus cells touching the last row at the interface with the hilus) was considered to belong to the inner part of the GCL.

Cells were counted as positive when the signal stood out clearly when compared with the surrounding tissue – at least 20% higher than the background intensity, confirmed with ImageJ software (NIH) when required. Colocalization was examined carefully by checking the shape of the signal (particularly when several antibodies stained the nuclei such as NeuN, BrdU and Fos) and by taking z-stacks with 10 focal plans.

The GCL area was measured with ImageJ software from pictures at x20 objective lens. To obtain the GCL volume, the area was multiplied by the section thickness for images or by the total z-series thickness for images from the confocal microscope. It is noteworthy that cell quantification was performed by experimenters unaware of the slide codes.

Fecal sample collection

Feces were collected according to the schedule represented in Figure 5b. For each fecal collection, the litter of the cage was collected and substituted by new sawdust. The feces were then extracted from the litter in another room, this procedure allowed a minimal disturbance of the animals during collection. Each sample contained therefore the feces emitted between two collection time points. It is noteworthy that the animals were handled several times each day in order to familiarize them to be manipulated. Moreover, two weeks before the feces collection, the animals from this experiment were subjected to a similar schedule of sawdust changes in order to familiarize them to this procedure.

Extraction and analysis of fecal corticosterone metabolites

7 Fecal samples were analyzed for immunoreactive corticosterone metabolites using a 5α- pregnane-3β,11β,21-triol-20-one enzyme-immunoassay (EIA) as previously described.10 Details regarding development, biochemical characteristics and physiological validation of this assay have previously been described.11, 12 Before EIA analysis, the fecal samples were dried and homogenized. Aliquots of 0.05 g were extracted with 1 ml of 80% methanol. A detailed description of the extraction procedure and the assay performance has been published elsewhere.11 The intra- and inter-assay coefficients of variation were 9.1% and 14.0%, respectively. As a caveat, fecal corticosterone metabolite levels cannot provide a temporal resolution as precise as plasma corticosterone levels. However, this method is largely noninvasive compared with multiple blood collections or indwelling catheters and avoids examining multiple groups of animals at different time points. Accordingly, although fecal corticosterone metabolite measures may slightly lessen temporal resolution, this method has the major advantage to circumvent important methodological bias induced by invasive procedures, and to comply with higher ethical standards.

DEX administration procedures

To test the effects of irradiation, UCMS and fluoxetine on the negative feedback of the HPA axis, we carried out a DEX suppression test. For this purpose, mice were i.p. injected with either DEX-P (0.1mg/kg, 5 mice per group) or saline (0.9% NaCl, 5 mice per group). All fecal boli that were voided between 8h and 10h after injection were collected in order to measure the level of fecal corticosterone metabolites.

To assess the sensitivity of granule cells to novelty and glucocorticoid effects, mice were i.p. injected with either DEX-P (0.1mg/kg, 5 mice per group) or saline (0.9% NaCl, 5 mice per group). Thirty minutes later, mice were placed in a circular open-field (diameter 35 cm) for 5min. Then, 90 min later (120 min after injection), mice were anesthetized with ketamine/xylazine (120 mg/kg and 10 mg/kg respectively), transcardially perfused and brains collected for immunohistochemistry.

To determine the ability of the hippocampus to regulate subcortical relay sites toward the PVN and to inhibit directly HPA axis activity, we administered the glucocorticoid receptor agonist DEX directly into the hippocampus. The infusion procedure was derived from the method previously described.13 Mice were stereotaxically and bilaterally implanted with guide cannulas (6mm long, 0.6mm outer diameter, 0.36mm inner diameter). The following coordinates were used for the guide cannula implantation according to Paxinos and Franklin’s

8 atlas: bregma = -3.08, lateral = ±2.3, vertical = -1.4.7 The next days, mice were familiarized to a mock infusion protocol to minimize any stress associated with the procedure before the start of the infusion experiments. After a 1-week recovery period from the surgery, infusion cannulas (7 mm long, 0.3 mm outer diameter, 0.17 mm inner diameter) were inserted and extended 1 mm below the guide cannula. Infusion was carried out directly in the home cage where a small portion of a chocolate cookie was placed in the cage in order to decrease locomotor activity during infusion. The animals received bilateral infusions during 1.5min of either vehicle (0.9% NaCl / 0.2% ethanol) or DEX (50 ng in 0.6 µl). The dose of DEX was determined earlier by preliminary experiments using doses of DEX ranging from 1 to 500 ng. We also attested earlier by methylene blue infusions that such volume of solution is confined close to the infusion site and does not diffuse toward other brain structures. The cannulas remained in place for 2.25 min after injection. In one experiment, brains were collected 2h following the infusion to assess DEX-induced c-fos changes in downstream brain structures and PVN (figure 3). In another experiment, we aimed to assess intrahippocampal DEX- induced changes in plasma corticosterone levels. For this purpose, the animals were killed by

CO2 asphyxiation 4h following infusion and then decapitated (figure 6). The trunk blood was collected, plasma separated and stored at -20°C until corticosterone radioimmunoassay. Plasma was analyzed for total corticosterone levels using a 125I-labeled corticosterone double- antibody radioimmunoassay kit (MP Biomedicals, NY) according to the manufacturer's protocol. To avoid inter-assay variability, all the samples were run in a single assay (the intra- assay variability was 4.5%). According to the manufacturer, the assay sensitivity is 7.7 ng/ml and the percentage of cross-reactivity with steroids was corticosterone 100%, desoxycorticosterone 0.34%, testosterone 0.1%, cortisol 0.05%, aldosterone 0.03%, progesterone 0.02%, androstenedione 0.01%, 5α-dihydrotestosterone 0.01%, others <0.01%. For histological verification of the cannula position, 30-µm brain sections were cut in a cryostat. We included only those animals in which the correct position of the cannula was demonstrated.

Statistics

Considering that relatively small sample sizes (n ≤ 14/group) were used and that assumptions for parametric statistics could not be ensured (normality and homoscedasticity), data were analyzed using the non-parametric Kruskal–Wallis “ANOVA by ranks” H-test. Significant effects (i.e., P< 0.05) were followed up with post-hoc tests (Holm-Bonferroni method) when appropriate. P-values that are indicated in the Results section always derived from the

9 between-groups comparisons using the Kruskal-Wallis H-test, while P-values resulting from post-hoc comparisons are indicated in the figures. The sample sizes are provided in the figure legends.

REFERENCES

1. Surget A, Wang Y, Leman S, Ibarguen-Vargas Y, Edgar N, Griebel G et al. Corticolimbic transcriptome changes are state-dependent and region-specific in a rodent model of depression and of antidepressant reversal. Neuropsychopharmacology 2009; 34(6): 1363-1380.

2. Willner P. Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology (Berl) 1997; 134(4): 319-329.

3. Alonso R, Griebel G, Pavone G, Stemmelin J, Le Fur G, Soubrie P. Blockade of CRF(1) or V(1b) receptors reverses stress-induced suppression of neurogenesis in a mouse model of depression. Mol Psychiatry 2004; 9(3): 278-286, 224.

4. Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S et al.Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 2003; 301(5634): 805-809.

5. Surget A, Saxe M, Leman S, Ibarguen-Vargas Y, Chalon S, Griebel G et al. Drug- dependent requirement of hippocampal neurogenesis in a model of depression and of antidepressant reversal. Biol Psychiatry 2008; 64(4): 293-301.

6. Meshi D, Drew MR, Saxe M, Ansorge MS, David D, Santarelli L et al. Hippocampal neurogenesis is not required for behavioral effects of environmental enrichment. Nat Neurosci 2006; 9(6): 729-731.

7. Paxinos G, Franklin KBJ. The mouse brain in stereotaxic coordinates. Academic Press: San Diego, CA, 2001, 124pp.

8. Snyder JS, Ramchand P, Rabbett S, Radik R, Wojtowicz JM, Cameron HA. Septo- temporal gradients of neurogenesis and activity in 13-month-old rats. Neurobiol Aging 2009.

9. Snyder JS, Radik R, Wojtowicz JM, Cameron HA. Anatomical gradients of adult neurogenesis and activity: young neurons in the ventral dentate gyrus are activated by water maze training. Hippocampus 2009; 19(4): 360-370.

10 10. Ibarguen-Vargas Y, Surget A, Touma C, Palme R, Belzung C. Multifaceted strain- specific effects in a mouse model of depression and of antidepressant reversal. Psychoneuroendocrinology 2008; 33(10): 1357-1368.

11. Touma C, Palme R, Sachser N. Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones. Horm Behav 2004; 45(1): 10-22.

12. Touma C, Sachser N, Mostl E, Palme R. Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice. Gen Comp Endocrinol 2003; 130(3): 267-278.

13. Mizoguchi K, Ishige A, Aburada M, Tabira T. Chronic stress attenuates glucocorticoid negative feedback: involvement of the prefrontal cortex and hippocampus. Neuroscience 2003; 119(3): 887-897.

11 SUPPLEMENTARY FIGURE S1

Supplementary figure S1. Fluoxetine and the CRF1 antagonist SSR125543 reverse the UCMS-induced decrease of neurogenesis in the dentate gyrus. (a) Schematic representation of the experimental design. (b) Neurogenesis in the dentate gyrus assessed by the number of NeuN+/BrdU+ cells per mm3 of GCL, n = 8-10 per group.(c) Neurogenesis in the dentate gyrus (BrdU+/NeuN+ cells) is reduced by the UCMS exposure. This effect is reversed by SSR125543 (20 mg/kg/day, ip; n = 10 per group). #,p<0.05 UCMS-vehicle mice vs. control-vehicle mice; *, p<0.05 and **, p<0.01 UCMS-fluoxetine/SSR125543 mice vs. UCMS-vehicle mice. Data represent mean±sem.

12 SUPPLEMENTARY FIGURE S2

Supplementary figure S2. Fluoxetine, but not SSR125543, requires hippocampal neurogenesis to reverse the detrimental effects of UCMS on body weight gain and coat state. (a) A significant reduction of the body weight gain was observed in UCMS-exposed mice. Fluoxetine treatment (20 mg/kg/day, ip) was able to reverse this UCMS effect only in non-irradiated mice, n = 13-14 per group. (b) The weekly coat state evaluation revealed a UCMS-induced degradation of the coat which was reversed by fluoxetine treatment only in non-irradiated mice, n = 13-14 per group. (c) A significant reduction of the body weight gain was observed in UCMS-exposed mice. SSR125543 treatment (20 mg/kg/day, ip) was able to reverse this effect in both non-irradiated and irradiated mice, n = 10 per group. (d) UCMS provoked a deterioration of the coat which was improved by SSR125543 in both non- irradiated and irradiated mice, n = 10 per group. #, p<0.05 and ##, p<0.01 UCMS-vehicle mice vs. control-vehicle mice; *, p<0.05 and **, p<0.01 UCMS-fluoxetine/SSR125543 mice vs. UCMS-vehicle mice. Data represent mean±sem.

13 SUPPLEMENTARY FIGURE S3

Supplementary figure S3. UCMS and fluoxetine affect the number of Fos+ cells in different brain areas regulating the HPA axis after intrahippocampal dexamethasone (DEX) infusion. For the experimental design see figure 3b. Detailed results of the changes induced by intrahippocampal DEX infusion on the number of Fos+ cells in medial preoptic area (mPOA), ventrolateralpreoptic areas (vlPOA), anteromedial bed nucleus of striaterminalis (amBST), posteromedial bed nucleus of striaterminalis (pmBST), lateral septum, dorsomedial hypothalamic nuclei (DMH), lateral hypothalamic nuclei (LH), anteroventral BST (avBST), arcuate nucleus and paraventricular nucleus (PVN) in control, UCMS-vehicle and UCMS-fluoxetine (20 mg/kg/day, ip) mice, n = 4 per group.*, p<0.05 sham (veh) mice vs. DEX mice in each treatment group, or between line-connected groups. Data represent mean±sem.

14 SUPPLEMENTARY FIGURE S4

Supplementary figure S4. Chronic stress induces some changes in the circadian activity of the HPA axis. The 24h baseline circadian HPA activity was determined using the level of fecal corticosterone metabolites (CORT). Fluoxetine reversed two of these changes in non- irradiated mice. (a): non-irradiated mice,n = 9-11 per group. (b): irradiated mice, n = 8-10 per group. (c) in control mice, n = 10-11 per group.#,p<0.05 and ##, p<0.01 UCMS-vehicle mice vs. control-vehicle mice; *, p<0.05 UCMS-fluoxetine mice vs. UCMS-vehicle mice. Data represent mean±sem.

15 SUPPLEMENTARY FIGURE S5

Supplementary figure S5. Plasma corticosterone (CORT) concentration following intrahippocampal dexamethasone (DEX) infusion is affected by chronic stress, fluoxetine and neurogenesis. For the experimental design see figure 5a. Detailed results of the plasma CORT concentration following intrahippocampal (DEX) infusion 4h before. The intrahippocampal DEX infusion caused a significant decrease of the plasmatic corticosterone (CORT) concentrations in control mice. The ability of the hippocampus to reduce CORT levels following DEX was not altered by irradiation. In contrast, the UCMS exposure abolished the hippocampus-induced CORT suppression. Fluoxetine treatment (20 mg/kg/day, ip) was able to restore the CORT suppression to a level similar to the control mice in non- irradiated mice but not in irradiated mice (n = 7-8 per group). *, p<0.05 and **, p<0.01 sham mice vs. DEX mice, or between line-connected groups. Data represent mean±sem.

16