Animals were group-housed with free access to water and food in the established animal houses, with a 12 hours light/dark cycle and a thermo-regulated environment. The use and care of animals complied with the guideline of the Biomedical Research Ethics Committee at the Shanghai Institutes for Biological Science, CAS. Adult (6–7 weeks old) male C57BL/6 mice (SLAC Laboratory Animal) were used in all experiments except electrophysiology trials which were performed on male Sprague Dawley rats (Animal House center, Kunming General Hospital, Kunming), weighing 220–250 g. mecp2
mice (005439) and nestin-cre mice (003771) were purchased from Jackson lab; floxed mecp2 (011918) were purchased from MMRRC at UC Davis.
For DMS treatment, mice with their cage were placed in DMS machine. Metal parts of cages were removed prior to DMS treatment. The applied program was described in Supplementary Figures. Briefly, twenty minutes successive trains of DMS were administered daily for different consecutive days depending on the purpose of experiments.14 days DMS were administered on retrovirus injected mice and 4 or 7 days DMS on Brdu labeled mice while 5 days DMS on acute induced depression mice. The control group conditions were identical to their DMS group but received sham stimulation. For electrophysiology experiments, control group received no treatment, Restraint stress (RS) group were restrained in the fixing cage 20 min each day for 7 days, RS + DMS group received 20 min DMS treatment in the fixing cage for 7 days. Electrophysiology studies were carried out 0.5 h later after the last restraint stress or DMS treatment.
In vivo electrophysiology
Experiments were carried out on rats anesthetized with pentobarbital (60 mg/kg, i.p.), and core temperature was maintained at 37 ± 0.5°C. Recordings of field excitatory postsynaptic potentials (EPSPs) were made from the CA1 stratum radiatum of the hippocampus in response to ipsilateral stimulation of the Schaffer collateral/commissural pathway using techniques described previously [28, 47]. Recording and stimulating electrodes were made by gluing together a pair of twisted Teflon-coated 90% platinum and 10% iridium wires (50 μm inner diameter, 75 μm outer diameter; World Precision Instruments, Sarasota, FL). The recording electrode was inserted 3.8 mm posterior to bregma and 2.8 mm right of the midline, and the stimulating electrode was inserted 4.8 mm posterior to bregma and 3.8 mm right of the midline. The optimal depth of the wire electrodes in the stratum radiatum of the CA1 area of the dorsal hippocampus was determined by electrophysiological criteria. Test EPSPs were evoked at a frequency of 0.033 Hz and at a stimulus intensity adjusted to give an EPSP amplitude 50% maximum response. The high frequency stimulation (HFS) protocol for inducing LTP consisted of 10 stimulus trains of 20 pulses at 200 Hz, with 2 s intertrain intervals. EPSP amplitude was expressed as mean ± S.E.M% of the baseline EPSP amplitude recorded over a 40-min baseline period, and amplitudes in the last 10-min of recording were averaged in one animal and then across animals to give a value for the group.
Preparation and stereotaxic injection of retrovirus
We thank Drs. Hongjun Song (Johns Hopkins University School of Medicine) and Zhengang Yang (Institutes of Brain Science, Fudan University) for providing retroviral constructs and supernatant containing retrovirus. Cell debris were removed from supernatant through 0.22 μm filter and the filtered supernatant was centrifuged at 65000 g at 4°Cfor 2 h. Then supernatant was removed and 50 μl PBS was used to re-suspend virus. We seed 293 T to determine the titer of virus. High titer virus of 108vg/ml was necessary to label new-born neurons in the adult DG.
For stereotaxic injection, it is critical to locate the exact hippocampus neurogenic area. The mouse was mounted onto the stereotaxic frame, orienting the head straight in terms of anterior-posterior axis and horizontally. Then we shaved a small area by a trimmer, cut the skin over the scull, cleaned up the blood in the wound.
The needle was firstly moved to the bregma and then moved posteriorly 2.0 mm and laterally 1.5 mm to the injection position. A small hole was made on the scull using an electric drill and the needle tip was moved down by 2.25 mm from the water level of the hole. 1 μl retrovirus solution was injected to one side.
For both Brdu and retrovirus stereotaxic injection statistics, each group (control, program1 and program5) has 4–5 mice. Each mouse was collected 9–12 slices from dorsal to ventral region of DG. And 1 or 2 intact neurons were chosen from each slice for analysis. ImageJ software (http://rsbweb.nih.gov/ij/) was used to trace the dendrites of new-born neuron. Then the dendritic length and tips number were auto-analyzed.
Mice received four or seven days DMS treatment and Bronodeoxyuridine (Brdu) (50 mg/kg in saline, every two hours for four times, Sigma, flu/Ald, B5002) was intraperitoneally (i.p.) injected into the control or DMS group mice 12 hours after the last DMS treatment. Mice were killed two hours later after the last i.p. injection.
Tissue preparation, immunostaining and imaging
Mice were transcardially perfused with 0.1 M cold PBS followed by 4% paraformaldehyde (PFA) fixation. The brains were post-fixed for 12 hours in 4% PFA and dehydrated in 30% sugar solution for another 12 hours. Coronal sections of 40-μm thickness were cut on the freezing microtome (LEICA CM1950) and stored in PBS.
For Brdu immunostaining, free-floating sections were incubated in 2 N HCl for 15 min at 37 degree then neutralized in 0.1 M boric acid solution (pH 8.5) for 10 min and washed by PBS for 3 times (each time for 5 minutes). Sections were incubated in mouse anti-Brdu antibody (1:1500; MAB3510) at 4 degree overnight and washed again then incubated in CF555 conjugated donkey anti-mouse secondary antibody (1:500; Biotum,20037) for 2.5 hours at room temperature.
For GFP immunostaining, free-floating sections were incubated in mouse anti-GFP antibody (1:1000;Santa Cruz, 9996) at 4 degree overnight and washed again then incubated in CF488 conjugated goat anti-mouse secondary antibody (1:500;Biotum,20010) for 2.5 hours (dark) at room temperature.
Immunostaining sections were photographed in Z-series stacks using a Nikon A1 confocal microscope. NIH Image J with NeuronJ plugin was used to count the Brdu-immunoreactive cell number in the dentate gyrus and to quantify the area of the dentate gyrus, also to analyze total dendritic length of GFP+ new-born neurons.
The results were expressed as Mean ± S.E.M. Statistical significance (P < 0.05) was assessed using the two-tailed Student’s t-test.
Learned helplessness paradigm
Mice were placed into the inescapable shock chamber. 360 scrambled foot shocks (0.6 mA) with varying duration (1-3s) and interval-episodes (1-15s) were delivered over two consecutive days. Control group did not receive foot shocks but were placed to the shock chamber with equal time. On the first and second days, 8 hours after foot shocks, mice were administered DMS. Then mice were administered DMS for another 3 days.
Forced swim test
Mice were placed individually in the testing cylinder (33 cm high × 10 cm in diameter) containing water with 20 cm depth. The procedures were conducted as two minutes pretest followed by four minutes test and both sessions were videotaped. Observers scored the immobility (floating passively with slight movements) time in the test four minutes.
Mice were anesthetized with 0.7% Pentobarbital sodium at 10ul/g, and exposed to cranial irradiation at a field of 3.5×11mm above the hippocampus but other body parts were protected with a 75 mm lead shield. Caesium-137 was used as radioactive source operated by MDS Nordion GC-3000Elan. The dose rate was approximately 4 Gy per min. The procedure lasted 5 min and 30 sec, delivering a total of 20 Gy in the center.
mutant mice were carried out light–dark transition test after daily DMS stimulation for 5 month at the age of 5–6 month. The chamber is divided into two unequal compartments by an opaque shelter with a hole (of height 4 cm) at the floor level. The smaller compartment (18cm×27cm×30cm) is painted black and covered by an opaque lid while the larger compartment (27cm×27cm×30cm) is uncovered and is illuminated by ceiling room lights. Firstly, mice were gently placed into the dark compartment. Transition between the two compartments and time spent in the light compartment were automatically recorded by the Ethovision videotracking system in a 10 min session.
Quantitative real time PCR
Total RNA was collected from fresh hippocampus tissue for reverse transcription (Bio-Rad, 1708891). 2 × SYBR Green Master Mix (TOYOBO, QPK201) and QIAGEN Rotor-Gene Q machine were used in real time PCR experiments. Mouse MKP-1 real time primer: forward: CGCTTCTCGGAAGGATATGCT; reverse: GTCAATAGCCTCGTTGAACCAG. Fgf1b primers were used as reported .