Babu et al. Synaptic activity induces neurogenesis ELECTROPHYSIOLOGY Whole-cell recordings were performed on cultured neurons at similar time points to that of cell biological experiments (10-14 days in vitro). The cell cultures were transferred to the recording chamber and continuously superfused with extracellular saline at a ﬂow rate of ∼5 ml/min. The intracellular solution contained K-Gluconate, 110 mM; KCl, 25 mM; EGTA, 0.1 mM; Hepes, 10 mM; K2ATP, 4 mM and GTP, 0.3 mM; adjusted to pH 7.3 with KOH; osmolarity was 290–300 mOsm. Patch pipettes were pulled from borosilicate glass tubing (2.0 mm outer diameter, 0.5 mm wall thickness; Hilgenberg, Germany). An Axopatch 200A ampliﬁer (Axon Instruments) was used for voltage-clamp recordings. Recorded currents were ﬁltered at 5 kHz with a four-pole low pass Bessel ﬁlter and digitized at 10 kHz with a 1401plus interface (CED). Patch pipette resistance was 3–5 MΩ. The cells were voltage clamped at −80 mV to detect mEPSCs. Tetrodotoxin (TTX) was added at a concentration of 1 µM to select for mEPSCs. Data were analyzed using C-Stimﬁt (Christoph Schmidt-Hieber, University College London) based on template matching. Series resistance was not compensated and checked every 5 min. Experiments with more than 10% change in series resistance were discarded. Voltage-clamp recordings were made at 22–24°C. CALCIUM IMAGING Measurements of the intracellular Ca2+ signals were performed as described previously (Normann et al., 2000). Fura2-AM (Molecular Probes, Eugene, OR, USA) was dissolved in DMSO to generate a stock solution of 5 mM. The cells were incubated for ∼30 min in a solution with a ﬁnal concentration of 10 µM Fura2-AM. Afterwards the cells were washed and the dye was allowed to de-esterify for ∼10 min before ﬂuorescence was recorded in the presence of the extracellular solution as described before. The excitation light source (Polychrome II, TILL Photonics) was coupled to the epif- luorescent port of the microscope (Axioskop FS2, Zeiss; 60× water immersion objective, Olympus) via a light guide. Light intensity was reduced to 10%. The ﬁlter combination for excitation and emission comprised a beam splitter (BSP410) and emission ﬁlters (LWP420, KP600) from Delta Light & Optics (Lyngby). The ﬂuo- rescence was measured with a backilluminated frame-transfer CCD camera (EBFT 512, Princeton Instruments). Images with full spatial resolution were taken with exposure times of 5 s. For high-speed Ca2+ measurements (100 Hz repetition rate) we usually deﬁned two rectangular regions of interest (ROIs) of about 5 × 5 µm at the soma of cultured neurons. The pixels included in the ROIs were binned on-chip and digitized subsequently by the controller (Micromax, 1 MHz, Princeton Instruments). The ﬂuorescence signals were cor- rected for background, which was obtained simultaneously from ROIs shifted by 10–15 µm with respect to the original ROIs (Neher and Augustine, 1992). To convert the ﬂuorescence signals into Ca2+ concentrations, we used the isosbestic ratioing method (Grynkiewicz et al., 1985). The activity-induced ﬂuorescence change was recorded at an excitation wavelength of 380 nm. The Ca2+-insensitive isosbestic ﬂuorescence was measured immediately before and after this sweep, using an excitation wavelength of 356 nm. The ratio of the background cor- rected ﬂuorescence signals R = F356/F380 was calculated, and con- verted into the Ca2+ concentration using the equation (Schiller et al., 1995). [Ca2+] = Keff (R − Rmin)/(Rmax − R), where Rmin is the ratio in Ca2+-free solution and Rmax the ratio when Fura-2 is completely saturated with Ca2+. These values were determined by recording from hippocampal pyramidal cells with internal solutions containing either 30 mM EGTA (Rmin = 0.70 ± 0.01, n = 5) or 50 mM CaCl (Rmax = 6.97 ± 0.06, 2 n = 5). Keff was calculated according to Neher and Augustine (1992) as Keff = Kd (Rmax/Rmin), with the dissociation constant Kd = 250 nM (Balkowiec and Katz, 2002). All values are given as mean ± SEM, error bars in the ﬁgures also represent SEM. Derivatives of Ca2+ signals and cross-correlations were calculated using IGOR Pro (WaveMetrics, Lake Oswego, OR, USA). Statistical signiﬁcance was assessed by a two-tailed Wilcoxon test or Students t-test at the signiﬁcance level (P) indicated. WESTERN BLOTTING Cells were lysed in lysis buffer consisting of 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100 and protease and phosphatase inhibitors.The proteins were quantiﬁed using Bradford reagent (Bio- Rad). The proteins were separated with 10% SDS–polyacrylamide gel electrophoresis after boiling the samples in Laemmli’s buffer. After electrophoresis the proteins were blotted onto a nitrocellulose or PVDF membrane and then probed with primary and horseradish peroxidase (HRP) conjugated secondary antibodies. DETERMINATION OF BDNF RELEASE INTO THE MEDIUM BDNF protein was assessed with a direct ELISA method as described previously but with slight modiﬁcations (Balkowiec and Katz, 2002). Sterile 96-well plates were treated with poly-D-lysine (50 µg/ml) for 1 h at room temperature, rinsed with sterile double distilled water and air-dried. Equal amounts of the supernatant from the various time points after stimulations were adsorbed onto the plates by incubating the medium for 1 h at 37°C. The plates were then washed with tris buffer saline (TBS) with 0.05% Tween- 20 and blocked for 45 min with 5% bovine serum albumin (BSA). Subsequently, anti-BDNF antibody (Santa Cruz) was added for 1 h at room temperature. After washing, the HRP coupled secondary antibody was added for 1 h at room temperature in blocking solu- tion. After several washings the substrate orthophenylenediamine was added in Phosphate Citrate buffer. The plates were read at 490 nm with the reference wavelength of 405 nm in a multi-plate spectrophotometer (Wallac). RESULTS ADULT HIPPOCAMPAL PRECURSOR CELLS UNDERGO NEURONAL DIFFERENTIATION IN CO-CULTURES WITH PRIMARY NEURONS In the DG, neural precursor cells are located in close proximity to the granule cell neurons (Figures 1A,B). The precursor cells have complex morphologies and a subpopulation of them (type-1 cells) shows highly arborized processes that fan into the molecular layer (Filippov et al., 2003), where the primary input ﬁbers to the DG arrive from the entorhinal cortex (Figures 1C,D). In the ﬁgure, the inner molecular layer is visualized with the help of a calretinin staining. The cell bodies of type-1 cells and the highly proliferative type-2 cells are found in the subgranular zone, a ﬁber-rich border zone between the granule cell layer and the hilus. For more detailed www.frontiersin.org September 2009 | Volume 3 | Article 49 | 3