Purinergic Signalling (2010) 6:429–445 Bath solution was applied by gravity feed from a storage syringe through a funnel drug applicator to the recording chamber. In the experiments with slices of the anterior telencephalon, the purinergic agonists were pipetted directly into the funnel (placed above the anterior telencephalon) without stopping the flow. In all experiments with the nose– brain preparation, odorants were pipetted directly into the funnel drug applicator placed above the intact olfactory epithelium. An additional bath solution applicator for application of purinergic agonists and antagonists was positioned above the OB (see Fig. 8). Outflow was through a syringe needle placed at the height of the olfactory nerves in both preparations. Ca2+ imaging and data evaluation For calcium imaging, Fura-2-stained preparations were fixed with a grid in a recording chamber and placed on the stage of an upright microscope (Axioskop 2, Zeiss, Göttingen, Germany). Fluorescence images (excitation at 380 nm; emission >505 nm) were recorded using a frame- transfer, back-illuminated CCD camera (16 bits/pixel, Micromax; Visitron, München, Germany) and a custom- built monochromator. With this configuration intracellular calcium concentration [Ca2+]i increases are accompanied by a decrease of the Ca2+-dependent fluorescence. Images were acquired at 200 to 500 ms exposure time per image using the commercial image acquisition software Winview (Visitron). Before starting the imaging experiments, the slices were rinsed with bath solution for at least 5 min. Image analysis was performed using custom programs written in MATLAB (MathWorks, Natick, USA). To facilitate selection of regions of interest, a “pixel correlation map” was obtained by calculating the cross-correlation between the fluorescence signals of a pixel to that of its immediate neighbors and then the resulting values were displayed as a grayscale map. The fluorescence changes for individual regions of interest are given as ΔF/F values. For more detailed information see our previous work [22]. Because some slices showed a linear loss of fluorescence intensity over time (bleaching artifacts) the ΔF/F values used to present all averaged data, presented as mean ± SEM (standard error of the mean), were corrected to retain comparability. On the basis of the first ten and the last four data points of each recording a linear fitting was calculated using MATLAB functions and subtracted from the raw data. For the calculation of the proliferation rate, series of brain slices (70 μm thickness, two to four slices per animal) were prepared, and multiple fields of view were visualized to include the whole PVZ of both sides. Cell nuclei labeled with propidium iodide and BrdU were counted automati- cally using CellProfiler [23]. Counting results were verified by inspecting overlays of automatically identified objects on the original images. 431 BrdU injections and histology To stain cells in the S phase of their cell cycle, larval X. laevis (stages 51 to 54) were anesthetized, and 200 μM BrdU, a synthetic nucleoside that is an analogue of thymidine [24], was injected intraperitoneally. After 16– 18 h additional 200 μM BrdU were injected to sustain high BrdU levels. After 24 h the animals were sacrificed and a tissue block containing the anterior part of the brain was removed. For BrdU-immunohistochemistry, the removed tissue blocks were fixed in 4% formaldehyde, washed in phosphate buffer saline (PBS), embedded in 5% low melting point agarose (Sigma) and sectioned on the vibratome at 70 μm. Sections were washed in PBS containing 0.2% Triton X-100 (PBST), and non-specific binding was blocked with 2% normal goat serum (NGS; ICN, Aurora, Ohio, USA) in PBST for 1 h at room temperature. The slices were then incubated in 1 N HCl at 37°C for 45 min to denature DNA. Subsequently, the slices were incubated overnight at 4°C with primary antibodies [anti BrdU (B2531, monoclonal, derived from mouse, Sigma) diluted in 2% NGS/PBST (1:1,000)]. The primary antibodies were washed off with PBS and Alexa 488 conjugated goat anti mouse secondary antibodies (Molecular Probes) were applied at a dilution of 1:250 in 1% NGS/PBS for 2 h at room temperature. The secondary antibodies were washed off in several changes of PBS. Cell nuclei were stained with propidium iodide. For purinergic receptor immunohistochemistry, form- aldehyde fixed brains were washed in PBS, embedded in 5% low melting point agarose (Sigma), and subsequently sectioned on the vibratome at 70 μm. Sections were then washed in PBST, and non-specific binding was blocked with 2% NGS in PBST for 1 h at room temperature. Tissue was then incubated overnight at 4°C with the primary antibodies [anti rat P2X1, anti rat P2X2, anti rat P2X4, anti rat P2X7, anti rat P2Y2, and anti rat P2Y4 (1:200, raised in rabbit, Alomone Labs, Jerusalem, Israel] diluted in 2% NGS/PBST. Primary antibodies were washed off with PBS, and Alexa 488 conjugated goat anti rabbit secondary antibodies (Molecular Probes) were applied at a dilution of 1:250 in 1% NGS/PBS for 2 h at room temperature. The secondary antibody was washed off in several changes of PBS. The specificity of the anti P2 receptor antibodies used was previously assessed by the producer and evaluated in X. laevis by incubating slices with the anti P2 antibodies (1:200) preadsorbed, 1 h at room temperature, with the immunizing peptide provided by the producer. Slices were then transferred to slides and mounted in mounting medium (Daco, Hamburg, Germany). Images were acquired with a confocal laser-scanning microscope (Zeiss LSM 510/Axiovert 100 M, Jena, Germany).