Data analysis of proliferation, migration and early differentiation assays.

<p>(A–B) Cells in S-phase 1 hour after BrdU injection. Bar graph depicting the BrdU+ cells/mm shows a significant decrease in the proliferative rate of the SVZ (A) and DG (B) in ENU animals. (C–D) SVZ or SGZ-derived cells 30 days after BrdU injection protocol. Bar graph depicting significant decrease in the numbers of BrdU+ cells in the OB (C) and GCL of the DG (D) in ENU-exposed animals. (E–F) Early differentiation of neurons in OB. Bar graph depicting the area fraction (E) and integrated density (F) of doublecortin (Dcx) positive cells in 4 different regions of OB, showing a reduction in all of them after ENU-treatment. *<i>p</i><0.05, **<i>p</i><0.01.</p

Ultrastructural characterization of the RMS in ENU animals.

<p>Detail of a typical neuroblasts chain in the RMS of ENU treated animal, surrounded by astrocytic cells. Scale bar: 5 µm.</p

Ultrastructural characterization of the DG.

<p>(A–B) Ultrastructural images of the SGZ from DG. (A) The control SGZ showed niches formed by precursor cells (arrow heads). (B) The ENU SGZ did not present the typical niches. (C) Cell quantification of the cell population in SGZ under electron microscopy, measured as cells/mm, resulted in a significantly decrease of total cells, due to a reduction in the number of type D cells. Scale bar: 10 µm *<i>p</i><0.01.</p

Evaluation of GAPS to Dcx expression in the DG of control and ENU animals.

<p>*<i>p</i><0.01.</p

ENU treatment impairs olfactory discrimination.

<p>During the habituation-dishabituation test a cotton swab was repeatedly presented to the mice above a target area and changed every minute. Exploration of the target area was examined. After 5 presentations without odorant, the swab was impregnated with an odorant (Odor A), and presented 6 times. Then, another swab was impregnated with a different odorant, (Odor B), which was also presented 6 times. The dotted lines represent the 1 min bins of exploration time of the target area. Notice that both groups similarly detected Odor A (last No Odor presentation Vs first Odor A presentation; * <i>p</i><0.01 for ENU-treated group; # <i>p</i><0.01 for control group), but when Odor B was presented only control animals discriminated the odor difference, responding to the new stimulus (last Odor A presentation Vs first Odor B presentation; # <i>p</i><0.01).</p

Decrease in proliferation after ENU-exposure as decrease in BrdU immunostaining.

<p>Micrograph of the BrdU+ cells immunolabeled with DAB showed a decrease in the proliferation in the SVZ (B) and DG (D) of treated animals, compared with control group (A and C, respectively). Delimited areas in C and D images are enlarged as C′ and D′, respectively, showing a detail of BrdU+ cells in DG. Scale bar: A 50 µm, C 100 µm.</p

Cell quantification in the SVZ after ENU-exposure.

<p>*<i>p</i><0.05,</p><p>**<i>p</i><0.01.</p

Dcx-expression in OB and DG of ENU-animals.

<p>Micrographs of Dcx+ cells immunolabeled with DAB. (A) High magnification of an OB sections from control mouse, where the 4 different regions under study are detailed. Zones 1 and 4 correspond to part of the OB central region, and of the granular layer. Zones 2 and 3 correspond to part of granular and glomerular layers. (B) In control animals Dcx-expression was relatively constant across the SGZ, showing scarce and short gaps (arrowheads). (C) The SGZ in treated animals presented wide and frequent gaps (arrows), compared with the control group. GCL: granular cell layer, GL: glomerular layer, RMS: rostral migratory stream. Scale bar: A 200 µm, B 100 µm.</p

Ultrastructural characterization of the SVZ from ENU-treated animals.

<p>The SVZ of treated animals was altered after ENU-exposure. (A) The type A cells located in the dorsal horn of the SVZ in ENU animals were drastically reduced, and substituted by astrocytic expansions. (B) High magnification of the SVZ dorsal horn with expansions rich in intermediate filaments (asterisks) in ENU animals. (C) Ependymal cell and neuroblasts frequently presented direct contact (arrow heads) in SVZ of treated animals. (D) Synaptic contacts located next to ependymal cell in animals exposed to ENU. (E) Large portions of basal membranes were observed between ependymal cells (arrows). (F) Myelinated and unmyelynated axons (arrows) were located between type A cells that composed chains, in ENU animals. Lv: lateral ventricle. Scale bar: A,F 10 µm, B 500 nm, C–E 2 µm.</p

Ultrastructural quantification of cell population in SVZ after ENU-exposure.

<p>The bar graph represents the number of cells/mm for the different cell types in the SVZ. The total cell number was reduced significantly by ENU treatment. A reduction was also detected in the number of type C and A cells. However, type B and E cell compartments remained constant. There were not changes in the number of astrocytes contacting with ventricle lumen (Bv) and neurons (N). *<i>p</i><0.05, **<i>p</i><0.01.</p