Hippocampal cell culture
Hippocampal neurons from newborn C57Black6 mice were prepared as described previously [9, 22, 23]. Neurons were plated onto either 12 mm glass coverslips or 35 mm dishes at a density between 400 and 600 cells per mm2 and cultured in Neurobasal media (Invitrogen, Gaithersburg, MD, USA) containing 1% rat serum and B27 (Invitrogen), and penicillin and streptomycin (Sigma). On days in vitro (DIV) 3, 2.4 μM cytosine D-arabinofuranoside (Sigma) was added to each dish to prevent proliferation of non-neuronal cells. At DIV 8, medium was replaced with transfection medium (TM)  which consists of a salt-glucose-glycine (SGG) solution and minimum Eagle’s medium (9:1; v/v) with sodium selenite 10 μg/ml, insulin 15 μg/ml, transferring 8.25 μg/ml, and penicillin-streptomycin 0.5%. SGG includes (in mM): NaCl 114, NaHCO3 26, KCl 6.3, MgCl2 1, CaCl2 2, Hepes 10, glycine 1, glucose 30, sodium pyruvate 0.5, Phenol Red 0.2%. All the experiments were done after a culturing period of 10 to 13 days during which hippocampal neurons develop a rich network of processes, express function NMDA-type and AMPA/kainite-type glutamate receptors, and form synaptic contacts [7, 24].
Recombinant adeno-associated viruses
The vectors used to construct and package rAAVs have been described previously [9, 10, 25]. The recombinant virus for the expression of the humanized Renilla reniformis green fluorescent (hrGFP) was generated as described in previous work . A recombinant adeno-associated virus vector containing 1 kbp cytomegalovirus enhancer (CMV)/chicken β actin hybrid promoter (CBA) was used to express CREB and mCREB. Both rAAV-CREB and rAAV-mCREB contain triple Flag epitope tag. All the vectors were generated by standard molecular biology techniques and verified by sequencing. Viral particles were produced and purified as described previously [9, 10, 26, 27]. For viral infection, neurons were infected with 1011 rAAV particles/μL at DIV 4. Infection efficiencies were determined immunocytochemically at DIV 9 or 10 by using antibodies to the Flag tag, or by analyzing the fluorescence of hrGFP; they ranged from 80%-95% of the viable neurons.
Mouse hippocampal neurons were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X 100 in PBS for 10 min. PFA was blocked by 10 min incubation in 1.25 M Glycine. After washing in PBS, the cells were blocked with blocking buffer (2% BSA, 10% normal goat serum in PBS) at room temperature for 1 h. The cells were exposed to primary antibody (anti-Flag M2 Flag antibody, Sigma, MO, US) in dilution buffer (2% BSA, 0.1% Triton X 100 in PBS) at 4°C overnight, washed in PBST (0.1% Tween 20 in PBS) and exposed to secondary antibodies in dilution buffer. After washing with PBST, the cells were incubated with Hoechst 33258 for 5 min, washed in PBS and mounted with mowiol and glycerol mounting medium, and dried at room temperature overnight. The cells were examined by fluorescent microscopy using a Leica SP2 confocal microscope (Leica, Wetzlar, Germany).
Hippocampal neurons incubated under appropriate conditions were washes with ice-cold PBS and cell lysates were prepared. The lysates were mixed with 5 × Laemmli sample buffer and boiled for 5 min. The proteins were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose (NC) membranes. The blots were blocked with Tris-buffered saline with Tween-20 (TBST; 20 mM Tris–HCl [PH 7.6], 0.136 M NaCl and 0.5% Tween-20 [vol./vol.]) containing 5% skim milk at room temperature for 1 h followed by incubation of primary antibody in TBST containing 5.0% BSA at 4°C overnight. Expression of CREB and phospho-CREB (pCREB) were measured by immunoblotting using the antibodies to the CREB (rabbit monoclonal antibody; Cell Signaling Technology, MA, USA) and pCREB (rabbit polyclonal antibody; Upstate, MA, USA); hrGFP expression levels were detected using antibody to the hrGFP (rabbit polyclonal antibody; Stratagene); tubulin (mouse monoclonal antibody; Sigma, MO, US) was chosen as the loading control. After washing with TBST, goat-anti-rabbit or mouse HRP conjugated IgG (Promega, WI, US) were added for 1 h at room temperature. Blots were then washes with TBST and exposed to X-ray film. The blots were quantitatively analyzed using ImageJ (http://rsb.info.nih.gov/ij/). All results are given as means ± SEM; statistical significance was determined by One-way ANOVA, Bonferroni post hoc test.
Quantitative reverse transcriptase PCR
To determine the mRNA levels of the potential pro-survival genes regulated by CREB, QRT-PCR was performed using real-time TaqMan technology with a sequence detection system model 7300 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). Total RNA was extracted using RNeasy kit (Qiagen GmbH, Germany) with additional on-column DNase I digestion during RNA purification. cDNA was generated from 1.5 μg of total RNA using High Capacity cDNA Reverse Transcription kit (Applied Biosystems). QRT-PCR was carried out using TaqMan Universal PCR Master Mix (Applied Biosystems). The following TaqMan gene expression assays were used in this study: atf3 (Mm00476032), bdnf (Mm00432069), btg2 (Mm00476162), gadd45β (Mm00435123), gadd45γ (Mm00442225), gusb (Mm00446953_m1). The thermal cycling conditions comprised 10 min at 95°C, and 45 cycles of 15 s for denaturation at 95°C and 60 s for annealing and extension at 60°C. The expression levels of the target mRNA were normalized to the relative ratio of the expression of gusb mRNA. Each QRT-PCR assay was performed three times. All results are given as means ± SEM; statistical significance was determined by One-way ANOVA, Bonferroni post hoc test.
Assessment of cell death
The induction and analysis of NMDA induced neuronal cell death assay was done as described with slight changes [11, 27, 28]. Briefly, the cells were treated with 20 μM NMDA for 10 min at 37°C, washed three times with TM and incubated at 37°C for 20 h. The percentage of dead cells was determined by analyzing Hoechst 33258 stained nuclei, and the percentage of dead cells was determined. The Staurosporine induced and growth factor withdrawal induced apoptosis assay have been described previously [9–11]. Briefly, 36 h after staurosporine (10 nM) exposure, or 72 h after keeping hippocampal neurons in TM medium minus the growth and trophic factors, all in the presence of 1 μM tetrodotoxin (TTX, Tocris Bioscience), cell death was assessed by determining the percentage of hippocampal neurons with shrunken cell body and large round chromatin clumps. All the cell death assays were done at DIV 10-13, at least 20 visual fields from each coverslip (corresponding to 1500–2000 cells per coverslip) were counted. All results are given as means ± SEM; statistical significance was determined by Two-way ANOVA, Bonferroni post hoc test.