Supplementary Methods.
Dentate gyrus cell counts. Fluorescence of Hoechst staining of the fixed brain slices
(excitation 405 nm, emission 415 - 485 nm) was detected using a Zeiss LSM 710
Olympus FV1000 (Zeiss, United Kingdom) confocal system equipped with a Zeiss
Axio Observer A1 inverted microscope, 40x PlanFluor oil immersion objective lens
(NA 1.3), and a 25 mW 405 nm diode laser (Coherent BV, The Netherlands). The
system was equipped with a 405 nm primary dichroic mirror to separate excitation
and emission light. Excitation intensity was adjusted using AOTF. Axial series of 2D
fluorescence images (1024 x 1024 pixels) of optical sections through nuclei (z-stacks)
were collected using photomultiplier working in integration mode at 1.27 μs pixel
dwell time and 12-bit precision of signal digitization (4096 intensity levels). The zstacks were collected with confocal pinhole set to 1.0 airy units at the maximum of
DAPI emission, which corresponded to 700 nm of optical section thickness. The
voxel dimensions in the object space were 280 nm, 280 nm and 480 nm in the x, y
and z directions, respectively. Each section corresponded to 3 by 5 tiled images (850
x 1488 μm).
In order to estimate the number of nuclei in dentate gyrus maximum intensity
projections along z-axis were generated from the z-stacks and used in further analysis
steps. Background images were calculated using grayscale opening (kernel size 150
pixels) followed by uniform filtering (kernel size 250 pixels). The background images
were subtracted from their raw counterparts and the binary masks (corresponding to
nuclei) were generated from the corrected images using windowed Otsu algorithm
(window size 300 pixels). The mask were subjected to median filtering (kernel 7) to
remove noise. The areas corresponding to single nuclei were isolated from the mask
and the areas of the nuclei were measured. The total binary mask was subjected to
binary closing followed by dilation (kernel size 5) and filling of holes. These
operations were repeated with increasing number of closing rounds till the masks
corresponding to nuclei merged into a single structure (dentate gyrus). The total area
of the structure was measured and the number of nuclei which corresponded to it was
calculated using the formula:
Nnucl=log(Andg/ Atdg)/log(1-Anucl/ Atdg), where Andg was the area occupied by
nuclei within the structure of dente gyrus, Atdg was the total area of dente gyrus and
Anucl was the are of a single nucleus. The algorithm was implemented under Matlab
2007 (MathWorks) equipped with Delft Image Processing Toolbox.