SUPPLEMENTARY FIGURES
Supplementary figure S1: Additional behavioral parameters assessed at the end of the uCMS
protocol. (a) Weight gain variation along the 6 weeks of uCMS exposure. uCMS-exposed
animals gained significantly less weight than control animals (F(1,16)=13.566, p=0.002,
η2=0.459). Not surprisingly, uCMS_FLX animals gained significantly less weight than uCMS
(F(4,40)=3.961, p=0.008, η2=0.284) . (b) The SDT apparatus consisted in a black arena divided
by transparent perforated walls that define 4 separated chambers: a pre-chamber, which is
connected by a trap-door to a middle chamber that leads to a right- (RC) and a left-chamber
(LC). Sweet pellets (Cheerios®, Nestlé) were placed in the LC, and regular food pellets
(Mucedola 4RF21-GLP) were placed in the RC. For the test, each animal was allowed to move
freely during 10 min to reach and eat the pellets. Average number of incursions in the left and
right chambers of the Sweet Drive Test (SDT) box was assessed during the test and no
differences were observed among experimental groups (Left chamber: F(5,42)=0.8258, p=0.5385;
Right chamber: F(5,42)=2.179, p=0.0746). (c) Food consumption in a 10 min period assessed
immediately after Novelty Suppressed Feeding (NSF) test; average food intake did not differ
among experimental groups (F(5,54)=1.557, p=0.1880). Abbreviations: Control = CT;
unpredictable chronic mild stress = uCMS; uCMS rats treated with fluoxetine = uCMS_FLX;
uCMS rats treated with imipramine = uCMS_IMIP; uCMS rats treated with tianeptine =
1
uCMS_TIAN;
uCMS
rats
treated
with
agomelatine
=
uCMS_AGOM.
2
Supplementary Figure S2: Corticosterone levels measured in the serum of rats between 8:00
and 9:00 (basal levels; nadir, N) and between 20:00 and 21:00 (peak levels; zenith, Z). (a) At
the 4th week of the uCMS protocol, control (CT) animals presented a functional circadian
regulation of the corticosterone production (nadir vs zenith: t22=5.628, p<0.0001, η2=0.59019)
whereas uCMS-exposed animals presented significantly higher basal (nadir, N) levels of
corticosterone than CTs (t68=1.766, p=0.0409, d=0.04387), and no circadian regulation (nadir vs
zenith: t114=1.847, p=0.0673). (b) At the 6th week, the CT (nadir vs zenith: t22=4.724, p<0.0001,
d=0.5035) and uCMS-exposed (nadir vs zenith: t22=0.08231, p=0.9351) animals presented the
same profile as at the 4th week (CTnadir vs uCMSnadir: t22=3.132, p=0.0048, d=0.3084). All ADs
except tianeptine, could revert the uCMS effects, presenting a circadian regulation of the
corticosterone secretion (nadir vs zenith: FLX: t16=3.980, p=0.0011, d=0.4975; IMIP: t22=2.148,
p=0.0430, d=0.1734; AGOM: t22=2.853, p=0.0093, d=0.2701). Although not statistically
significant, tianeptine showed a strong trend to induce a reestablishment of the circadian
corticosterone secretion pattern (t18=1.821, p=0.0853). Error bars denote SEM. * Denotes
differences between nadir and zenith measurements. # Denotes differences between basal levels
(Nadir, N) of corticosterone, after ADs treatment comparing to uCMS untreated animals. n.s.not statistically significant. */# p<0.05; **/## p<0.01; ***/### p<0.001. n=8-12 animals per
group. Abbreviations: N= Nadir; Z= Zenith; Control = CT; unpredictable chronic mild stress =
uCMS; uCMS rats treated with fluoxetine = uCMS_FLX; uCMS rats treated with imipramine =
uCMS_IMIP; uCMS rats treated with tianeptine = uCMS_TIAN; uCMS rats treated with
agomelatine = uCMS_AGOM.
3
Supplementary Figure S3: Additional morphological data on the hippocampal DG granule
cells. (a) Sholl analysis of reconstructed granule neurons of the dorsal DG shows higher
complexity of neurons from control (uCMS vs CT: F(1,7)= 16.264, p=0.005, η2=0.699),
imipramine-treated and tianeptine-treated animals compared to uCMS-exposed (F(4,18)=15.03,
p<0.0001, η2=0.4550, post-hoc uCMS_IMIP/TIAN vs uCMS: p<0.0001). (b) Sholl analysis of
reconstructed granule neurons of the ventral DG. Control animals, and animals treated with
fluoxetine or agomelatine present significantly less complex granule neurons than uCMSexposed animals (F(4,21)=18.63, p<0.0001, η2=0.4701; post-hoc FLX: p<0.05; post-hoc AGOM:
p<0.0001). Error bars denote SEM. * Denotes the effect of uCMS-exposure; # Denotes the
effect of antidepressants comparing to untreated uCMS-exposed animals. */# p<0.05; **/##
p<0.01; ***/### p<0.001. n=5/6 animals per group. Abbreviations: Control = CT; unpredictable
chronic mild stress = uCMS; uCMS rats treated with fluoxetine = uCMS_FLX; uCMS rats
treated with imipramine = uCMS_IMIP; uCMS rats treated with tianeptine = uCMS_TIAN;
uCMS rats treated with agomelatine = uCMS_AGOM.
4
Supplementary Figure S4: Confirmation of the macrodissections’ accuracy. Gene expression
analysis of three genes that are predominantly expressed in the DG of CT animals compared to
the remainder tissue of the hippocampus: NeuroD1, DSP and Prox1. This specificity was
confirmed by an enrichment in the expression of these three genes in the DG comparing to the
remaining hippocampus (NeuroD1: t4=5.436, p=0.0056, d=0.8808; DSP: t4=11.12, p=0.0004,
d=0.9687; Prox1: t4=8.583, p=0.001, d=0.9485). Bars represent the relative expression of each
target gene after normalization to B2M mRNA levels; error bars denote SEM. *Denotes
statistical differences between groups after normalization to B2M mRNA levels; **p<0.01;
***p<0.001; n=3 per group.
5
Supplementary Figure S5: Common transcriptional regulation of fluoxetine, imipramine and
tianeptine. Predicted gene network (IPA software) including some of the co-regulated genes
between fluoxetine, imipramine and tianeptine. The functions associated to this network are
tissue morphology and connective tissue development and function. Green symbols:
significantly down-regulated genes; red symbols: significantly up-regulated genes. Genes in
white color were not significantly regulated in the analysis. Significance was set at p<0.01.
6
Supplementary Figure S6: Neural cell type enrichment heatmaps obtained from Genes2mind
online resource (genes2mind.org), using as input the significantly regulated (p<0.01) transcripts
in each comparison. Grey color indicates genes for which there is no available data.
7