has been controversy over use of selective serotonin reuptake inhibitors (SSRIs) to treat affective disorders in children and adolescents due to clinical reports of increased risk for suicidal ideation and behavior during treatment and animal studies showing changes in adult anxiety- and depressive-like behaviors after repeated treatment during adolescence. (10 mg/kg) stimulated greater increases in c-Fos expression across the extended amygdala in adults than in adolescents and 8-OH DPAT (0.5 mg/kg) produced greater increases in c-Fos in the lateral orbital cortex and central nucleus of the amygdala in adults. These data show that lower anxiogenic effects of acute SSRIs in adolescents are associated with lesser activation of cortical and amygdala brain regions. This immaturity could contribute to the different profile of behavioral effects observed in CHIR-98014 adolescents and adults treated with SSRIs. probe recovery and extracellular serotonin concentration (Justice 1993 The syringe contents were analyzed each day to obtain 5-HTin. Seven adult and nine adolescent rats were used for this experiment. 2.7 Fluoxetine dose response Animals were sequentially injected with 2.5 5 and 10 mg/kg fluoxetine doses previously shown to increase extracellular serotonin with two hours between each dose (Rutter and Auerbach 1993 Samples were collected at 20 minute intervals. Thirteen adult and thirteen adolescent rats were used for this experiment 2.8 Fluoxetine infusion Fluoxetine (30 μM) was infused through the microdialysis probe to investigate the effects of uptake inhibition without the influence of fluoxetine metabolism or somatodendritic 5-HT1A autoreceptors. The aCSF in the syringe during baseline collection was replaced with aCSF containing 30 ?蘉 fluoxetine a half-maximal dose for increasing extracellular serotonin in the prefrontal cortex (Hervas and Artigas 1998 Samples were collected at 20 minute intervals for four hours during fluoxetine infusion. Ten adult and seven adolescent rats were used for this experiment. 2.9 Verification of probe placement Brains were removed and postfixed in 10% formalin cut into 30 μm sections on a cryostat and stained with cresyl violet to CHIR-98014 confirm probe placement (Fig. S1). Animals with probes placed greater than ± 0.5 mm away from the target of +3.2 mm AP were excluded from further analysis (two adults and three adolescents). 2.1 HPLC detection for microdialysis Dialysates were injected onto a 2.1 × 100 mm reversed phase C18 column (Phenomenex Torrance CA). The mobile phase was run at 0.2 mL/min and consisted of 150 mM NaH2PO4 4.8 mM citric acid 3 mM SDS 50 μM EDTA CHIR-98014 (Sigma Aldrich) with 11% methanol and 17% acetonitrile (EMD Chemicals Philadelphia PA) pH=5.6. Serotonin was measured using an electrochemical detector set to 0.55V (BASi). The sensitivity was 1 fmol of serotonin in a 15 μL injection. Samples were quantitated with an external standard curve run each day. 2.11 3 DPAT Binding Samples of prefrontal cortex amygdala and hippocampus from adult and adolescent rats were dissected using a brain block frozen on dry ice and stored at ?80°C. A single point binding analysis was performed for each sample with 1 nM 3H-8-OH DPAT (Perkin Elmer Waltham MA) so VAV1 that age differences in either the affinity or CHIR-98014 total number of binding sites could be detected (Xu et al. 2002 Samples were thawed and homogenized with a dounce homogenizer in 20 volumes of Tris buffer (50 mM Tris 2 mM MgCl2 2 mM Sodium Ascorbate pH 8.0) prior to incubation (25 μg of protein per tube) with 3H-8-OH DPAT for 1 hour at room temperature. Serotonin (400 μM) was used for determination of nonspecific binding. The reactions were terminated by the addition of 3 mL of ice cold buffer and filtered onto glass fiber filters (Cambridge Technology Watertown MA) presoaked in 0.05% polyethylenimine. No age differences were detected in single point binding so saturation binding assays were not conducted. A total of 16 rats were used for this experiment (8 per age group). 2.12 c-Fos Immunostaining Adult and adolescent rats were treated with vehicle (saline or distilled water) 10 mg/kg fluoxetine or 0.5 mg/kg 8-OH DPAT and transcardially perfused two hours later with phosphate buffered saline followed by 10% formalin. Injections and perfusions were performed between 8am and 2pm. Animals treated with saline and distilled water..