Babu et al. Synaptic activity induces neurogenesis between these two has hardly been addressed. It has been reported, though, that systems level LTP might increase adult hippocampal neurogenesis (Bruel-Jungerman et al., 2006). However, it remained unknown whether synaptic plasticity in neurons could induce adult neurogenesis on the level of the precursor cells. To determine the relationship between synaptic network plastic- ity and precursor cell behavior we developed a co-culture model consisting of hippocampal neurons and adult hippocampal pre- cursor cells. Hippocampal neurons are an excellent model to study network activity and its synaptic relevance. In culture, they show relatively low synaptic release probability, which increases after brief calcium (Ca2+) rises mediated by NMDA receptor activation (Malenka, 2003; Malenka and Bear, 2004). Although the short-term cellular effects of such stimuli on mature functional neurons are well documented, the effects of such changes on neural precursor cells are not known. Using this model we addressed the following questions: (1) Can neural precursor cells detect neuronal network activity? (2) How do precursor cells respond to changes in synaptic activity levels? (3) What are the signaling mechanisms by which synaptically active neurons inﬂuence precursor cells? MATERIALS AND METHODS ADULT HIPPOCAMPAL PRECURSOR CELLS CULTURE All institutional guidelines regarding animal handling and killing were followed. Precursor cells were cultured as described previously with slight modiﬁcations (Babu et al., 2007). C57Bl6 mice were killed by an overdose of ketamine. The brains were removed and placed in Hepes buffer Hanks balanced salt saline solution (pH 7.3). The DG was isolated and digested in an enzyme cocktail contain- ing 10 U/ml Papain (Worthington), 2 mg/ml dispase (Roche), and 1 U/ml DNase (Worthington) at 37°C for 40 min. Cell suspen- sions were washed with DPBS and enriched for precursor cells on a 22% percoll gradient (Amersham). The cells were plated on polystyrene surfaces coated with Poly-lysine (Sigma; 10 µg/ml) and Laminin (Roche; 5 µg/ml). The medium was serum free with added Neurobasal (Invitrogen), 2% B27 (Invitrogen) and 2 mM Glutamax (Invitrogen) and 20 ng/ml EGF (Peprotech) and 20 ng/ml bFGF (Peprotech). For differentiation, growth factors were withdrawn and the cells were allowed to differentiate for 4–6 days. In some of the differentiating cultures we also added 5 ng/ml of FGF-2. For green ﬂuorescent protein (EGFP) viral labeling, precursor cells were incubated with viral supernatant containing retrovirus with EGFP vector overnight for transducing and expressing the virus. The cells were washed subsequently and puriﬁed by ﬂuo- rescence activated cell sorting (FACS) for EGFP expression and subsequent propagation. Puriﬁcation was tested by immunostain- ing for EGFP expression. PRIMARY POSTNATAL HIPPOCAMPAL NEURONAL CULTURE For cultures of primary hippocampal neurons, postnatal mice (P 0–1) were killed by decapitation and the hippocampus was dissected and subjected to digestion by Papain (20 U/ml) for 30–40 min at 37°C followed by Trypsin inhibitor (1 mg/ml). Cells were plated on to poly-lysine/laminin-coated coverslips at 5 × 104 cells/cm2 with medium containing Neurobasal, B27 (2%) and Glutamax (2 mM). Under these conditions about 80% of the cells were MAP2ab positive neurons. The co-culture was established by trypsinizing the EGFP-labeled precursor cells and overlaying the primary hip- pocampal neurons with the precursor cell suspension at a density of 5 × 105 cells/cm2, replacing 75% of the medium. To sequester endogenously secreted BDNF recombinant Fc-trkB (R&D) and bioneutralizing antibody against BDNF (Santa-Cruz) were added at a concentration of 5–10 µg/ml. Rabbit anti-NT3 (Santa-Cruz) was used at a concentration of 5–10 µg/ml for neutralizing endog- enously secreted neurotrophin-3 (NT3). IMMUNOCYTOCHEMISTY AND IMMUNOHISTOCHEMISTRY Cells were ﬁxed in 4% paraformaldehyde for 20 min, washed with PBS, and blocked and permeabilized by incubating in PBS con- taining 0.25% Triton X-100 and 10% donkey serum. The primary antibodies were applied in 3% donkey serum in PBS overnight at 4°C. As primary antibodies we used mouse anti-βIII-tubulin (1:1000; Promega), rabbit anti-βIII-tubulin (1:2000; Covance), mouse anti-Synaptophysin (1:50; Abcam) mouse anti-MAP2ab (1:500; Sigma) and goat anti-GFP (1:1000; Axis). Primary antibodies were detected by ﬂuorophore-conjugated secondary antibodies (Dianova) incubated for 2 h at room tem- perature. The nuclei were stained with Hoechst 33258 (Sigma). For imaging we used a confocal microscope (TCS-SP2 Leica, Bensheim) in sequential scanning mode or an epiﬂuorescence microscope (Zeiss Zeiss Axioplan2 epiﬂuorescence microscope) with appropri- ate ﬁlters to prevent cross talk. Identiﬁcation of precursor cells in tissue sections derived from Nestin-GFP mice were performed as previously was reported (Kempermann et al., 2003). Expression of neuronal markers was revealed with speciﬁc antibodies that recognized Calretinin and NeuN cells. Antibodies used were: rabbit anti-Calretinin antibody 1:250 (Swant), monoclonal mouse anti-NeuN antibody 1:100 (Chemicon) monoclonal mouse anti-MAP2ab (1:500; Sigma). All secondary antibodies were raised in donkey and diluted 1:250 (Jackson Immunoresearch, Dianova). Sections were mounted in polyvinyl alcohol with diazabicyclo-octane as anti-fading agent (DABCO). LTP IN CULTURED HIPPOCAMPAL CELLS On the day following the initiation of the co-culture the medium was replaced by Hepes-buffered extracellular saline (ECS) solu- tions consisting of: NaCl, 140 mM; KCl, 5 mM; CaCl2, 1 mM; Hepes, 25 mM; Glucose, 33 mM; adjusted to pH 7.4 with NaOH. To block GABAergic receptors Bicuculline (20 µM; Sigma) or SR95531 (10 µM; Gabazine; Tocris) was added to the extracellu- lar solution. To stimulate opening of synaptic glutamate (NMDA) receptors and induce synaptic plasticity in the neuronal network glycine was added at 100 µM. Strychnine (Sigma) was added at 1 µM to prevent the stimulation of glycinergic receptors. In the control solution, MgCl was added instead of glycine at 1 mM to 2 maintain the voltage-dependent block of NMDA receptors. The stimulations were done at room temperature. The NMDA receptor inhibitor APV (50 µM) and the CaMK inhibitor KN-93 (1 µM) were added directly to the glycine containing solution. After stimu- lation the cultures were replaced with fresh medium and returned to the CO2 incubator. Frontiers in Neuroscience | Neurogenesis September 2009 | Volume 3 | Article 49 | 2