Cultured neurons from embryonic (E15.5) C57Bl6 mouse cortex were maintained in 24-well plates coated with polyornithine and laminin at a density of 3 × 105
cells/well. Culture medium was B27-supplemented Neurobasal (Invitrogen, Carlsbad, CA) containing 1 mM glutamine, penicillin and streptomycin. Cells were grown in 0.5 ml of medium per well and the medium was not changed. All experiments were conducted at 4 –5 div. Although the cultured cells at this age are postmitotic neurons, which have elaborated extensive processes and express neuron-specific immunohistochemical markers, synaptogenesis has not yet begun (c.f., Hardingham and Bading, 2002
; Han and Stevens, 2009
). Cortical astrocyte cultures from E15.5 C57Bl6 mouse embryos were maintained in poly-L-lysine-coated T75 flasks in DMEM/F12 medium (1/1) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and penicillin and streptomycin. Prior to transfection, astrocytes were replated into 24-well plates (5 × 105
cells/well; 0.5 ml medium/well).
Transfection, plasmids and luciferase reporter assays
Cortical neurons were transfected on 3 div by the Ca2+
-phosphate method (Xia et al., 1996
). Astrocytes were transfected 1– 2 days after replating using Lipofectamine 2000 (Invitrogen) diluted 1:33 in Opti-MEM without penicillin/streptomycin. The 4xCRE
reporter consisted of four CRE
s inserted in pCIC-CK vector (Stratagene, La Jolla, CA) upstream of the luciferase reporter gene. In control experiments, empty pCIC-CK plasmid was not stimulated by any of the agonists used in this study (not shown; and see Kingsbury et al., 2003
). Co-transfection with constitutively active TK Renilla
plasmid (Dual-Luciferase Reporter Assay System, Promega, Madison, WI) served as a control for variability in transfection efficiency and cell density. Experiments were conducted 24 hr after transfection. Luciferase activity was measured with a Centro LB 960 microplate luminometer (Berthold Technologies, Oak Ridge, TN). The ratio of 4xCRE
luciferase and Renilla
activities was computed for each sample and the mean of 3 determinations was expressed as a fraction of unstimulated activity. The result for each experimental condition is reported as mean ± sem (n = 3 –5), where n is the number of independent experiments. Statistical analyses were conducted with ANOVA followed by Tukey test using InStat (GraphPad, San Diego, CA).
Intracellular cAMP levels were measured by ELISA (Assay Designs, Ann Arbor, MI); cAMP levels are reported as pmoles/106 cells.
Quantitative real-time PCR (Q-PCR)
Neurons were cultured for 3 –4 days in 35mm culture dishes at a density of 1.2 × 106 cells/dish and then stimulated for 1, 3 or 24 hours. Total RNA was purified with Rneasy Mini Kit (Qiagen) including the on-column DNA digestion step. This procedure regularly gave high purity RNA (260/280 nm absorbance ratio ~ 2.0). cDNA was reverse-transcribed (Transcriptor Kit, Roche) using random primers. Reactions were run in a LightCycler 480 (Roche) using SYBR Green I Master Mix (Roche) with the following settings: pre-incubation (5 min @ 95°C); amplification, 45 cycles (10 sec @ 95°C; 15 sec @ 58°C; 1 min @ 72°C); and a melting curve (5 sec @ 95°C followed by 1 min ramp from 65°C to 97°C). All samples generated single melting curve peaks. Primers were used at 0.5 μM concentration and had the following sequences. GAPDH: 5-TGATGACATCAAGAAGGTGGTGAAG-3 (forward) and 5-TCCTTGGAGGCCATGTAGGCCAT-3 (reverse), c-fos: 5-AAGTTGGCACTAGAGACGGACAGA-3 (forward) and 5-ACTTCGACCATGATGTTCTCGGGT-3 (reverse), BDNF-exon I: 5-GGACAGCAAAGCCACAATGTTCCA-3 (forward) and 5-TTGCCTTGTCCGTGGACGTTTACT-3 (reverse) and BDNF-exon IV: 5-ACCAGGTGAGAAGAGTGATGACCA-3 (forward) and 5-GTTGCCTTGTCCGTGGACGTTTA-3 (reverse).
Each sample was run in duplicate and a 3-point (400, 200 and 100 ng) cDNA dilution curve was constructed. The reaction efficiency was calculated for each treatment condition. RNA for each target gene was normalized to GAPDH
according to the following formula (Pfaffl, 2001
are the reaction efficiencies for the target and GAPDH PCR reaction respectively; CP is the crossing threshold for each reaction; and control and sample refers to untreated or treated samples, respectively. This equation takes into account the actual measured reaction efficiency for each treatment condition, rather than assuming a theoretical efficiency of 2.0.
Neurons grown in polyornithine/laminin-coated 35 mm plastic dishes were stimulated for 3 hours, washed in ice-cold PBS and lysed with Ripa Buffer (Sigma-Aldrich) supplemented with protease (Roche Applied Science, Indianapolis, IN) and phosphatase (Pierce, Rockford, IL) inhibitors. Samples were run in duplicate on pre-cast 4–12% Bis-Tris gels (Invitrogen) and transferred to PVDF membranes. Blots were blocked for 1 hour with tris-buffered saline containing 0.01% Tween-20, 5% powered milk and 5% bovine albumin (Sigma-Aldrich). Membranes were incubated overnight at 4°C with antibody to phospho-S133-CREB (Cell Signaling, Danvers, MA) diluted 1:1000 and incubated with secondary horse radish peroxidase-linked antibodies (GE Healthcare, Piscataway, NJ) at room temperature for 1 hr. Bands were visualized by enhanced chemiluminescence (Millipore, Billerica, MA) and images were acquired and analyzed on an Image Station 2000R (Kodak-Carestream Health, Rochester, NY). Uniform loading was verified by stripping and reprobing with antibodies to total CREB (1:1000; Cell Signaling).
Glutamate and aspartate scavenging systems
To degrade glutamate in the extracellular space, cultures were incubated with 5 units/ml glutamate-pyruvate transaminase (GPT) and 5 mM pyruvate for 20 min prior to stimulation with forskolin. To degrade both L-aspartate and glutamate, cultures were incubated with 5 units/ml glutamate-pyruvate transaminase (GPT), 10 mM pyruvate, 5 units/ml glutamate-oxaloacetate transaminase (GOT) and 3 mM α-ketoglutarate for 20 min prior to stimulation with forskolin. Omission of GPT and GOT served as controls.
Excitatory amino acid analysis
At 4 div, medium (500 μl) from neurons in 24 well plates was removed and replaced by 200 μl of fresh medium; cells were stimulated by addition of forskolin 4 hrs later. HPLC was used to quantify aspartate and glutamate in 250 μl of the combined culture medium from two duplicate wells. Samples were acidified with 0.14 M perchloric acid and neutralized with 0.25 M potassium bicarbonate. Samples were reacted with 2 volumes of o-phthaldehyde (OPA) for 2 minutes before injection on to a C18 reverse phase column (Beckman-Coulter, Fullerton, CA). Derivatized amino acids were detected by fluorescence. The elution was carried out at 0.7 ml/min with a linear gradient mixing 7.5% methanol prepared in 20 mM sodium acetate, pH 7 (solution A) with 30% methanol and 30% acetonitrile prepared in water (solution B). Over the range encompassing aspartate, the gradient ran for 15 min from 97% A and 3% B to 85% A and 15%B. The column was stripped with 100% B following the elution of aspartate and glutamate. The positions of aspartate and glutamate on the chromatogram were identified by running standards and confirmed by spiking experimental samples with these amino acids.
Drugs and reagents
Forskolin, 8-Br-cAMP, NMDA, DL-AP5, ifenprodil, SKF38393, SCH23390, pyruvate, glutamate-pyruvate transaminase (from porcine heart), glutamate-oxaloacetate transaminase type I (from porcine heart), L-glutamate, L-aspartate, OPA and α-ketoglutarate were obtained from Sigma-Aldrich (St. Louis, MO); H89, dideoxy-forskolin, and Bapta-AM from EMD-Calbiochem (San Diego, CA); Bapta-free acid from Invitrogen (Carlsbad, CA); TBOA and trans-PDC from Tocris-Cookson (Ellisville, MO) and EGTA-AM from AnaSpec (San Jose, CA). Antagonists were added 30 minutes before stimulation except for Bapta-AM and EGTA-AM which were added 60 before stimulation.