Generation of mouse lines

The generation and characterization of the Nes-CreERT2 transgenic mouse line is described in detail elsewhere (A. Dranovsky et al., manuscript in preparation). The colony of NCff mice (that is, Nes-CreERT2; Baxf/f mice) was maintained by interbreeding Nes-CreERT2; Baxf/f mice and Baxf/f mice. The Baxf/f mice generated from these crosses were used to assess the CreERT2-independent effects of TAM on mouse behaviour. An EYFP reporter line (ROSA26 f stopEYFP)31 was used in all experiments in which adult-born neurons were inducibly genetically labelled. Specifically, NCffY and NCY mice were generated as littermates by interbreeding Nes-CreERT2; Baxf/+; ROSA26 f stopEYFP/+ mice and Baxf/+; ROSA26 f stopEYFP/+ mice. NCff mice were maintained on a mixed (C57BL/6 and 129/SvEv) genetic background. The Pomc-τ-EGFP transgenic mouse line was obtained from GENSAT (http://www.gensat.org) and used to generate NCff; Pomc-τ-EGFP mice. To induce CreERT2-mediated recombination of Bax and/or EYFP in neural stem cells in the adult brain, mice (Nes-CreERT2; Baxf/f mice, Nes-CreERT2; Baxf/f; ROSA26 f stopEYFP/+ mice, Nes-CreERT2; ROSA26 f stopEYFP/+ mice or Nes-CreERT2; Baxf/f; Pomc-τ-EGFP mice) of at least 8 weeks of age were given 2 mg TAM intraperitoneally, once a day for 5 consecutive days. TAM (10 mg ml−1, Sigma, T-5648) solution was prepared in corn oil containing 10% ethanol. For vehicle, an identical volume of corn oil with 10% ethanol was injected intraperitoneally, once a day for 5 consecutive days. Mice were housed four to five per cage in a 12 h (6 a.m. to 6 p.m.) light–dark colony room at 22 °C and had free access to food and water. For the voluntary exercise regimen, four to five mice were housed per cage (29.2 cm × 19 cm × 12.7 cm), and each cage was equipped with two running wheels. Experimental protocols were approved by the Institutional Animal Care and Use Committee at Columbia University and the New York State Psychiatric Institute.

Electrophysiological recordings

Electrophysiological recordings in the dentate gyrus were performed as previously described23,24. Brains were collected from animals after inducing deep anaesthesia with halothane followed by decapitation, and transverse hippocampal slices (400 µm) were prepared using a vibratome. The slices were incubated in an interface chamber at 32 °C and perfused with oxygenated artificial cerebrospinal fluid (ACSF) (119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO 4 , 2.5 mM CaCl 2 , 26.2 mM NaHCO 3 , 1 mM NaH 2 PO 4 and 11 mM glucose). Slices were allowed to equilibrate for 2 h before positioning the electrodes and beginning stimulation. To record from the dentate gyrus, the medial perforant path (MPP) was stimulated using a stimulation isolation unit and a bipolar tungsten electrode (World Precision Instruments). Evoked potentials were recorded in the molecular layer above the upper blade of the dentate gyrus using a glass capillary microelectrode filled with ACSF (and with a tip resistance of 1–3 MΩ). Isolation of the MPP was confirmed by assessing paired-pulse depression of the MPP–dentate gyrus synaptic connection at 50 ms, which generated the highest level of depression. Input–output curves were obtained after recordings had been stable for 10 min. The stimulation intensity that produced one-third of the maximal response was used for the test pulses and tetanus. After a stable baseline response to test stimulation (once every 20 s) had been observed for 15 min, the ability to elicit LTP was assessed. LTP was induced with a weak stimulation protocol consisting of four trains of 1 s each, at 100 Hz within the train, repeated every 15 s23. Responses were recorded every 20 s for 60 min after LTP induction. A similar protocol was used to elicit and record LTP of mature dentate granule neurons except that 10 μM bicuculline (bicuculline methobromide, Sigma, B7561) was added to the ACSF to block GABA A -receptor-mediated inhibition.

Immunohistochemistry and confocal microscopy

To assess the survival of adult-born neurons in the dentate gyrus, BrdU was administered intraperitoneally, once a day in 0.9% NaCl for 2 or 10 days at 150 mg kg−1 body weight. Mice were anaesthetized with ketamine or xylazine (100 and 7 mg kg−1 body weight, respectively) and transcardially perfused (with cold saline, followed by 4% cold paraformaldehyde in PBS). Brains were postfixed overnight in 4% paraformaldehyde at 4 °C, then cryoprotected in 30% sucrose and stored at 4 °C. Coronal serial sections (40 μm) of the entire hippocampus and sagittal sections (40 μm) of the olfactory bulb were obtained using a cryostat and stored in PBS. For BrdU or NeuN immunohistochemistry, sections were mounted onto SuperFrost Plus charged glass slides. Following pretreatment with 10 mM citrate buffer, sections were subjected to antigen retrieval in 10 mM citrate buffer, using a boiling protocol. After cooling to room temperature, sections were rinsed three times in PBS and blocked in PBS with 0.3% Triton X-100 and 10% normal donkey serum (NDS) for 2 h at room temperature. Incubation with primary antibodies was carried out at 4 °C overnight (for BrdU, rat anti-BrdU antibody at 1/100 dilution, Serotec; for NeuN, mouse, 1/500, Chemicon). Fluorescent-label-coupled secondary antibodies (Jackson ImmunoResearch) were used at a final concentration of 1/400 in PBS. For GFAP, NeuN and EYFP triple immunohistochemistry, floating sections were used. Briefly, sections were washed three times in PBS, blocked in PBS buffer containing 0.3% Triton X-100 and 10% NDS, and incubated in primary antibodies overnight, with shaking at 4 °C (GFAP, rabbit, 1/2000, DAKO; NeuN, mouse, 1/500, Chemicon; GFP, chicken, 1/500, Abcam). The next day, sections were washed three times in PBS and incubated with fluorescent-label-coupled secondary antibodies (Jackson ImmunoResearch) for 2 h at room temperature. For GFP immunohistochemistry alone (rabbit, 1/500, Invitrogen) was used. For calbindin immunohistochemistry, a similar protocol was used (mouse, 1/5,000, Swant). For DCX and Ki67 immunohistochemistry, floating sections were first quenched to remove endogenous peroxidase activity (with 1% H 2 O 2 in 1:1 PBS:methanol). Sections were then washed in PBS, blocked (in PBS containing 0.3% Triton X-100 and 10% NDS) and incubated with primary antibody overnight at 4 °C (DCX, goat, 1/500, SantaCruz Biotechnology; Ki67, rabbit, 1/100, Vector Labs). Following washes in PBS, sections were incubated with horse-radish-peroxidase-coupled, biotinylated secondary antibodies. Following incubation with ABC solution (Vector Labs), the colour reaction was carried out using a DAB kit (Vector Labs). An unbiased and blinded quantification protocol was used to quantify DCX+ and BrdU+ cells in the granule cell layer of the dentate gyrus along the septotemporal axis24. For quantification of survival of adult-born cells in the main olfactory bulb, two high magnification (×20) images of randomly selected regions in the granule cell layer were obtained from six matched sagittal sections for each mouse. BrdU+ cells were quantified using a cell counter plug-in for the software ImageJ (NIH), and surface density was computed. Bright-field images were obtained using an Axioplan-2 upright microscope (Zeiss). For quantification of EYFP+ neurons in NCY and NCffY mice, five to six (dorsal) and three (ventral) matched sections were selected, and the mean number of EYFP+ neurons per section was computed. Type I neural stem cells expressing EYFP were not included in the analysis. All analyses of mice with the inducible genetic reporter ROSA26 f stopEYFP were performed at 6 weeks post injection of vehicle or TAM. Phenotyping of BrdU-expressing cells in the granule cell layer of the dentate gyrus entailed the scanning of at least 80 cells from the dorsal and ventral hippocampus of each mouse using an LSM 510 META scanning confocal microscope (Zeiss). To determine the number of BrdU+ cells expressing GFAP or NeuN, z-stack analysis was performed using the LSM 510 image browser. To compute the percentage of DCX-expressing cells that also expressed EYFP in NCffY mice, approximately 120 DCX+ neurons per mouse dentate gyrus were scanned using a FluoView 1000 confocal microscope (Olympus) (×40 magnification and numerical aperture (NA) of 1.3). To determine the number of DCX+ neurons expressing EYFP, z-stack analysis was performed using FluoView 1000 v1.5 software. Three mice were used for the analysis. A one-in-six series of adjacent sections stained with nuclear fast red (Vector Labs) was used to measure the volume of the granule cell layer in the dentate gyrus.

Quantification of the granule cell layer volume and mossy fibre length in the dentate gyrus

The surface area of the granule cell layer was traced in ImageJ from ×10 images of hippocampal sections spanning the septotemporal axis, and the volume was determined by multiplying the surface area of the granule cell layer by the distance between sections sampled (240 μm). Four mice per group were used for this analysis. To measure the length of axons of young adult-born neurons, we used NCff; Pomc-τ-EGFP mice, in which axons are genetically labelled with EGFP. Mossy fibre length was determined by tracing the stratum lucidum along the inner edge of the stratum pyramidale. For measurements, the starting point was the intersection of the trace and a line between the tip of the inner and outer blades of the dentate gyrus32. Four dorsal sections from each mouse were used for these measurements.

Sholl analysis

Five to six EYFP+ neurons with complex dendritic trees were chosen from each mouse (from both the dorsal and ventral dentate gyrus) and scanned using a FluoView 1000 (Olympus) (×40, 1.3 NA or ×60, 1.42 NA). Images of collapsed z-stacks were imported into Adobe Illustrator CS3, and dendritic trees were reconstructed using the tracing tool. Dendritic complexity was analysed from 8-bit images by using the ImageJ Sholl Analysis plug-in (http://www-biology.ucsd.edu/labs/ghosh/software/). The centre of all concentric circles was defined as the centre of the cell’s soma. The parameters used were starting radius (10 μm), ending radius (300 μm from the centre) and interval between consecutive radii (10 μm). Three to four mice per group were used.

Focal X-ray irradiation of the hippocampus

Ten-week-old Baxf/f mice were anaesthetized with sodium pentobarbital (administered intraperitoneally at 42 mg kg−1 body weight, once per day on each of three days that were spaced apart by 3–4 days), placed in a stereotaxic frame and exposed to cranial irradiation using a Stabilopan X-ray system (Siemens) operated at 300 kVp and 20 mA. Animals were protected with a lead shield that covered the entire body, but a 3.22 × 11 mm2 treatment field above the hippocampus (interaural 3.00 to 0.00) was left unshielded and exposed to X-rays. Dosimetry was done using an electrometer ionization chamber (model PR-06G, Capintec) and Ready Pack Radiographic XV films (Kodak). The corrected dose rate was approximately 1.8 Gy min−1 at a source-to-skin distance of 30 cm. The procedure lasted 2 min 47 s, delivering a total of 5 Gy. Three 5-Gy doses were delivered, on days 1, 4 and 8. Behavioural testing was carried out 4 months after hippocampal X-ray irradiation.

Behavioural testing

Behavioural testing was performed using male and female mice that were 14–18 weeks of age at the time of testing, unless otherwise specified. All experiments and analyses were performed blind to genotype or treatment.

Tests for anxiety-like and depression-like behaviours

Testing in the open field test, light–dark test, elevated plus maze, novelty-suppressed feeding and forced swim tests was carried out at 8 or 10 weeks after vehicle or TAM treatment.

The open field test is a standard test of both anxiety and locomotor behaviour. It consists of a simple square enclosure that is equipped with infrared detectors to track animal movement in the horizontal and vertical planes. Measures of total distance travelled and rearing events are used as an index of exploratory activity33, whereas the proportion of time or distance spent in the centre is construed as a measure of anxiety-like behaviour. Mice were placed in the corner of the open field, and activity was recorded for 30 or 60 min. Testing took place either under low light (200 lx) or bright light (1,000–1,200 lx) conditions.

The novelty-suppressed feeding test has been validated as a model that is sensitive to chronic, but not acute, antidepressant treatment8. Mice were food deprived in their home cages for 24–26 h before testing. The testing apparatus consisted of a plastic arena (45 cm long, 15 cm high and 30 cm wide) whose floor was covered with an approximately 2-cm depth of wood-chip bedding. A single food pellet (familiar laboratory mouse chow) was placed on a circular piece of white filter paper (12 cm in diameter) positioned in the centre of the arena. The test began with a mouse being placed in a corner of the arena, and the latency to approach the pellet and begin feeding was recorded (for a maximum time of 10 min). Testing was carried out under bright light conditions. Each mouse was weighed before food deprivation and just before testing to assess changes in body weight. Immediately after the test, each mouse was transferred to its home cage, and the amount of food consumed within 5 min was measured. When appropriate, survival analysis was performed, and statistical differences between the latencies were determined using the Kaplan–Meier product-limit method.

The elevated plus maze23 and the light–dark test34 were done as described previously.

For the forced swim test, mice were placed for 6 min in transparent plastic buckets (19 cm in diameter and 23 cm deep) that had been filled with water at 23–25 °C, and their behaviour was recorded using an automated video-tracking system. Testing was carried out over two consecutive days, with the first day serving the purpose of pre-exposure. Mobility (swimming and climbing behaviour) on the second day was analysed using ViewPoint Life Sciences Software.

Object recognition test

At 8 weeks after Bax ablation in neural stem cells, separate cohorts of mice were tested for similar and novel object recognition behaviour. NCff mice were tested 8 weeks after TAM or vehicle treatment. Testing entailed placing mice in an arena (45 cm long, 15 cm high and 30 cm wide) with two distinct objects, for seven sessions (each of 7 min) spaced apart by a 3-min intertrial interval. Mice became habituated to the objects during sessions one to six, and one of the objects was then replaced with a novel or a similar object in session seven. Objects and object positions were counterbalanced during testing. The objects that were selected for testing elicited comparable levels of exploration and were categorized as novel or similar based on the exploration levels evoked in NCff mice in pilot experiments. Sessions were videorecorded, and videos were manually scored for locomotion (grid crossings) and object exploration (when an animal’s snout was 2 cm or less from the object).

Spatial and reversal learning

A cohort of mice was tested at 8 weeks after Bax ablation in neural stem cells. Testing using the reference version of the Morris water maze was performed as described elsewhere35. The task was performed with three training phases executed in succession: visible platform (2 days); acquisition phase (4 days), with a hidden platform in the training quadrant (Q3); and transfer/reversal phase (reversal learning, 4 days), with a hidden platform in the opposite quadrant (Q1). Each phase comprised four trials (120 s maximum and 15-min intertrial interval) per day. The start location was in a different quadrant in each trial so that no single start location was used in consecutive trials. Shaping was carried out before the first trial of the visible platform and the acquisition phases. A probe trial (60 s and no platform) was performed 24 h after the last trial of the acquisition and transfer phase. The animals’ trajectories were recorded with a videotracking system (HVS Image Analysing VP-118).

Active place avoidance

Spatial learning was also tested using an active place avoidance task, which is sensitive to hippocampal dysfunction36. The place avoidance training apparatus consists of a slowly rotating (clockwise at 1 r.p.m.) circular platform (40 cm in diameter) within which a non-rotating 60° region of the room is a shock zone (Supplementary Fig. 12, delineated in red). Visual cues are located on the walls of the room. Mice walk freely on the rotating platform and learn to avoid the shock zone based on the visual cues. When the mouse enters the shock zone, it receives a brief foot shock at a constant current (500 ms, 60 Hz, 0.2 mA) that is scrambled across pairs of parallel rods located on the platform floor. Additional shocks of the same intensity and duration are administered every 1.5 s until the mouse leaves the shock zone. The position of the mouse is tracked by PC-based software that analyses images from an overhead camera and delivers shocks appropriately (Tracker, Bio-Signal Group). Track analysis software is used to compute the number of times each animal enters the shock zone and the number of shocks administered. On the first day of the experiment, mice walked freely on the rotating platform for 10 min while the shock device was turned off (pretraining). Then the shock device was turned on, and mice were given three 10-min training trials with an intertrial interval of 50 min, for two days (trials one to six).

One-trial contextual fear conditioning

Mice were tested at 8 weeks following Bax ablation in neural stem cells. Conditioning was conducted on one side of a shuttle box (Med-Associates, ENV-010MC; 20.3 cm × 15.9 cm × 21.3 cm) with a clear plexiglass wall, three aluminium walls and a stainless steel grid as a floor. The chamber was lit from above with a house light (CM1820 bulb), ventilated with a house fan and encased by a sound-dampening cubicle. On the days of testing, mice were brought out of the vivarium and allowed to habituate for 1 h outside the testing room before starting the experiment. Mouse behaviour was recorded by digital video cameras mounted above the conditioning chamber. FreezeFrame and FreezeView software (Actimetrics) were used for recording and analysing freezing behaviour, respectively. The one-trial contextual fear conditioning protocol entailed delivery of a single 2-s foot shock of 0.75 mA at 185 s after placement of the mouse in the training context. The mouse was taken out 15 s after termination of the foot shock and returned to its home cage. For the training context, A, the house fan and lights were switched on; stainless steel grids were exposed; and a mild lemon scent was used as an olfactory cue. Ethanol (70%) was used to clean grids between runs. For the distinct context, C, the stainless steel grid floor was covered with a plastic panel and cage bedding. The chamber walls were covered using plastic inserts, and the house fan and lights were turned off. The chamber door was left ajar during testing. A mild anise scent was used as an olfactory cue, and a non-alcoholic antiseptic was used to clean the chamber between runs. Mice were brought into the testing room in cardboard buckets by a different handler, and the testing room was dimly lit before placement of the mice in the testing chambers. The one-trial contextual fear conditioning protocol was used for extinction learning and memory-clearance experiments. Only males were used for these studies.

Contextual fear-discrimination learning

Mice were tested at 8 weeks after Bax ablation in neural stem cells. This test captures an animal’s ability to distinguish between two similar contexts, conditions that are most likely to recruit the dentate gyrus25. The shock-associated training context, A, and the similar (no-shock) context, B, shared many features, including an exposed stainless steel grid floor (a salient feature of the context) and roof. The similar context differed from the training context in that two plastic inserts were used to cover the walls; the house fan and lights were turned off; and the chamber door was left ajar during testing. A mild mint scent was used as an olfactory cue, and a non-alcoholic antiseptic was used to clean the grids between runs. Mice were brought into the testing room in buckets by the same experimenter who had handled the mice for the training context. In pilot experiments, the similar context was found to evoke comparable levels of freezing behaviour as that observed in the training context, indicative of extensive generalization (pattern completion) between the two contexts. For discrimination learning, mice were exposed to the training context in which they received a single 2-s foot shock of 0.75 mA at 185 s after placement in the chamber. Mice were taken out of the chamber 15 s after termination of the foot shock and returned to their home cage. After 1 h, mice were placed in the similar context, in which they were left for 180 s and were never shocked. Measurement of the freezing levels in both the training context (3-min pre-shock) and the similar context (3 min) each day allowed the assessment of discrimination between the two contexts and was computed as a discrimination ratio: (Freezing Training context − Freezing Similar context )/(Freezing Training context + Freezing Similar context ). A score of 0 indicates complete lack of discrimination: that is, freezing levels are the same in the similar and training contexts (Freezing Similar context = Freezing Training context ). A score of 1 indicates perfect discrimination: that is, freezing level in the similar context is zero (Freezing Similar context = 0). Only males were used for these experiments.

Home cage activity

Animal behaviour was recorded for 15 min in the home cage, and videos were manually scored for locomotion (grid crossings).

Statistical analysis

Statistical analysis was carried out using StatView software or Microsoft Excel. Statistical significance was assessed by unpaired two-tailed Student’s t-tests or ANOVA. Significant main effects or interactions were followed up with Fisher’s predicted least-square difference post hoc tests where appropriate: *, P < 0.05; **, P < 0.01.