Supplementary results
Figure S1 During the elevated plus-maze, the benzodiazepine chlordiazepoxide (CDP) does
not modify the behavior of C57BL/6J mice tested in mildly anxiogenic conditions whilst it
increases the time spent in the open arms when the animals were tested under anxiogenic
conditions (F1,22=8.006, p<0.01; C57-Veh anxiogenic different from all other groups at °°
p<0.01). Non-anxiogenic conditions: Wt-veh=5, Wt-CDP=7; anxiogenic conditions, Wtveh=7, Wt-CDP=7.
-1-
Figure S2 Effects of Dox treatment on cell proliferation in the subventricular zone (SVZ) and
on long term survival of newly-born cells in the dentate gyrus (DG). (a) Density of HH3-IR
cells in the SVZ (t12=0.24, p=0.82). (b) Number of 4 months old IdU-IR cells in the DG of
bigenic-vehicle mice (BV, n=4) and bigenic-Dox mice (BD, n=5; t7=3.05, p=0.018). **:
p≤0.01 compared to control group. Bar scale= 10 µm. White bar =BV. Black bar=BD.
-2-
Figure S3 Confocal illustrations of Bax transgene expression after long-term Dox treatment
on. EYFPBax (green) cytoplasmic clusters were located in the Dentate Gyrus (DG) of
bigenic-Dox mice but not in the amygdala or the hypothalamus. Cells were counterstained
with DAPI (blue).
-3-
Figure S4 Two-factor structure of behavior during the elevated plus-maze for bigenic-vehicle
and bigenic-Dox mice. A factor analysis was performed to identify the relationship between
specific behavioral parameters quantified during the elevated plus-maze. For each
experimental group, factor loadings (left panels, 2d scatterplots; right panels, factor loadings
tables) show that independent behavioral measures could be accommodated by two factors. A
first factor interpreted as the locomotor /exploratory activity factor on which highly load: total
arm entries, total distance traveled, entries and distance traveled in closed arms. A second
factor interpreted as the anxiety-related factor on which highly load entries, distance and time
spent in open arms. Thus, we confirm the classic two-factor structure of the EPM that has
been described previously 1. Legends: Total entries (TotE, ), Entries in open arms (EOA, ),
-4-
Entries in closed arms (ECA, ), Total distance (TotD,
), Distance in open arms (DOA, ),
Distance in closed arms (DCA, ), Time spent in open arms (TOA, ). Factor loadings
highlighted in red are greater than 0.7 and thus indicate behavioral items that correlate highly
with a single factor.
-5-
a
b
c
Empty
d
Object
e
Figure S5 In vivo individual genomic integration of EYFPBax-TetO-ECFPBax or NestinrtTA constructs does not influence anxiety-related behaviors. Wild-type (Wt, n=18), Tet-Bax
(Bax, n=18) and nestin-rtTA (Nes, n=20) mice treated or not with Dox were compared to
Bigenic Nes/Bax mice treated with vehicle (Big-Veh n=10). (a) Time spent in the open arms
of the elevated plus-maze (F6,59=0.34, p>0.90). (b) Latency to emerge from the cylinder
during the emergence test (F6,59=0.60, p>0.72). (c) Time spent exploring the novel object
placed in the center zone of the open field (during the habituation phase (Empty): F6,59=0.15,
p>0.98; during the test phase (Object): F6,59=1.31, p>0.26). (d) Latency to retreat in the
cylinder during the predator exposure test (F6,59=0.48, p>0.81). (e) Time spent inside the
cylinder during the predator exposure test (F6,59=0.46, p>0.83).
-6-
Table S1 Morphology of the dendritic arbor of doublecortin neurons. The area of the soma,
the number of nodes and endings, and dendritic length of Big-Dox and Big-Veh Dcx-IR
neurons were similar.
-7-
Table S2 Effects of Dox treatment on Bax transgene expression. EYFPBax cell density and
caspase 3-IR cell density were estimated in the amygdala and hypothalamus after long-term
Dox treatment in bigenic-Dox mice and compared to bigenic-Vehicle mice.
-8-
Table S3 Parameters assessing locomotor activity during the elevated plus-maze test (EPM).
Inhibition of neurogenesis had no effect on any of the behavioral criteria that allow assessing
general activity in the EPM. Consequently, the avoidance of the open arm by bigenic-Dox
mice during the EPM cannot be explained by a reduced locomotor activity.
Reference List
1.
Rodgers, R. J. & Johnson, N. J. Factor analysis of spatiotemporal and ethological
measures in the murine elevated plus-maze test of anxiety. Pharmacol.Biochem.Behav
52, 297-303 (1995).
-9-