MATERIALS AND METHODS

Animals
Experiments were carried out on male (12–15 weeks old) HAB and normal depression/anxiety animals (NAB) mice bred for their innate level of anxiety-related behavior at the Department of Pharmacology and Toxicology (University of Innsbruck, Austria) (for details see Sartori et al, 2011a). Animals were group-housed under standard laboratory conditions (12 : 12 light/dark cycle with lights on at 07:00 hours, 22±2 °C, 50–60% humidity) with pelleted food and water available ad libitum. All experiments were designed to minimize animal suffering as well as the number of animals used. Experiments comprising this study were approved by the national ethical committee on animal care and use (Bundesministerium für Wissenschaft und Forschung) in compliance with international laws and policies.

Drug Treatments
Reboxetine (40 mg/kg per day; Ochem, Chicago, IL), desipramine (30 mg/kg per day; Sigma-Aldrich, Vienna, Austria), fluoxetine (18 mg/kg per day; Eubio, Vienna, Austria), paroxetine (10 mg/kg per day; Ochem), and citalopram (10 mg/kg per day, kindly provided by Lundbeck, Denmark) were administered via the drinking water for 3 weeks. A drug intake of the dose indicated above was achieved by adapting the concentrations of the drug in the drinking solutions according to mean drinking volume and body weight per cage (Sartori et al, 2012). Mice were kept on drug treatment until completion of all experiments. Control mice received tap water. 5-Bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) was injected at a dose of 100 mg/kg (intraperitoneally) once per day for 4 days for the labeling of newly born cells.

Deep Brain Stimulation
Before surgery, animals were handled for 3 days to habituate them to the experimenter and the stimulation procedure. Using a stereotaxic frame, anesthetized animals (ketamine/xylazine 80/5 mg/kg, intraperitoneally) were implanted with electrodes (MS303-3-B-SPC, bipolar, twisted, 8 mm; Plastics One, Roanoke, VA) reaching the NAcb core (coordinates: 1.10 mm anterior, 1.45 mm lateral, and 4.65 mm ventral to bregma). After surgery, animals were individually caged, and received buprenorphine (0.1 mg/kg intraperitoneally) every 8 h over 3 days to minimize pain. Following 4–5 days of recovery, animals received high-frequency stimulations daily (130 Hz, 100 μA, 60 μs pulse width; A310 Accupulser and A365 Stimulus Isolator; World Precision Instruments, Berlin, Germany) for 1 h per day (NAcb-DBS). Control animals (NAcb-sham) were connected to the stimulator without current being applied. At the end of the experiments, localization of electrodes in the NAcb was confirmed on cresyl violet-stained coronal sections (see Figure 2b). Animals with misplaced electrodes were excluded from all data sets.

Experiments
In Experiment 1, the sensitivity of HAB mice to the antidepressant efficacy of different selective serotonin reuptake inhibitors (SSRIs) was explored in the forced swim test (FST) (Figure 1). Experiments 2–4 (Figure 2a) were designed to investigate the effect of single and repeated NAcb-DBS (i) on the anxiety- and depression-related behavior displayed by HAB mice, using a battery of established tests, (ii) on adult hippocampal neurogenesis, and (iii) on challenge-induced neuronal activation patterns in different brain regions, using immunohistochemistry.

Behavior
All behavioral tests were performed on HAB and NAB mice 1 h after completion of NAcb-DBS or NAcb-sham. The FST and tail suspension test (TST) were used to assess depression-like behavior (for a review see Cryan and Mombereau, 2004; Cryan et al, 2005). Anxiety-related behavior was investigated using the novelty-suppressed feeding paradigm, while general locomotor activity was measured in the open field. All behavioral tests were performed according to previous protocols established in our laboratory (Sartori et al, 2012; Whittle et al, 2011), details of which are provided in the Supplementary Information.

Immunohistochemistry
At 2 h after the last challenge, when expression levels of c-Fos protein are at their peak (Zangenehpour and Chaudhuri, 2002), mice were deeply anesthetized with an overdose of sodium pentobarbital (200 mg/kg) and transcardially perfused with saline followed by 4% paraformaldehyde (in 0.1 mol/l phosphate buffer, pH 7.4), as described previously (Muigg et al, 2009). One-in-eight of the free-floating coronal sections (50 μm) throughout the entire murine HPC were processed for BrdU, doublecortin (DCX), and c-Fos immunohistochemistry, respectively, following established protocols (Couillard-Despres et al, 2009; Muigg et al, 2009). Sections were incubated with one of the following primary antibodies: rat anti-BrdU (1 : 350; Serotec, Dusseldorf, Germany), goat anti-DCX C18 (1 : 250; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti c-Fos (1 : 10 000; Santa Cruz Biotechnology). Subsequently, they were incubated with the corresponding biotinylated goat anti-rat, rabbit anti-goat, or goat anti-rabbit secondary antibody (all 1 : 200; Vector laboratories, Burlingame, CA). The formed antigen-antibody-complexes were visualized by the avidin-biotin-horseradish peroxidase procedure (Vectastain Elite ABC kit; Vector Laboratories) using 3,3′-diaminobenzidine as the chromogen.
Immunoreactive cells were quantified in the regions of interest using a light-optical microscope (Olympus, Vienna, Austria) and a computer-assisted image analysis system (cellSens Dimension; Olympus). A cell was considered as labeled (positive) for c-Fos, DCX, or BrdU when the brown-black DAB staining was unambiguously darker than the background, and this included all cells from low to high intensities of staining.

Data Analysis
Data are presented as mean±standard error of the mean (SEM). Exact n numbers are given in the table and figure legends. Statistical analysis was performed using STATISTICA 8.0 (StatSoft, Tulsa, OK) after data had been screened for outliers using the Grubb's test. All data were further tested for homoscedasticity using Levene's test. Data were statistically analyzed using one-way ANOVA (post hoc Bonferroni) or unpaired Student's t-test. Statistical significance was set at P<0.05.