5 Days Treatment
B
160
Immobility (sec)
1400
1200
1000
800
600
400
140
120
100
*
80
60
40
20
0
VEH CIT10 CIT20
C
200
0
VEH RS1 RS5 SB1 SB5 CIT10 CIT20
160
Swimming (sec)
Total Distance Traveled (cm)
A
12 Days Treatment
140
120
100
60
40
20
0
Climbing (sec)
D
*
80
VEH CIT10 CIT20
160
140
120
100
80
60
40
20
0
VEH CIT10 CIT20
Total Distance Traveled (cm)
E
1400
1200
1000
800
600
400
200
0
VEH CIT10 CIT20
Supplemental Figure 1. Effect of 5-HT2C antagonists and citalopram on locomotion. (a)
Subchronic treatment with RS 102221, SB 242084, or citalopram did not affect locomotor
activity. (b) Chronic treatment (12 days) with 10 mg/kg/day citalopram reduced immobility.
ANOVA F(2,40) = 3.83, *P < .05. (c) 10 mg/kg/day citalopram increased swimming behavior.
ANOVA F(2,40) = 6.52, *P < .05. (d) 10 mg/kg/day or 20 mg/kg/day citalopram did not affect
time spent climbing. (e) 10 mg/kg/dayor 20 mg/kg/day did not affect locomotor activity. n = 1215 mice per group for all experiments here; error bars represent s.e.m.
Supplemental Figure 2. Effect of 5-HT2C antagonists and citalopram in the olfactory
bulbectomy model. (a) Subchronic treatment with 5 mg/kg/day SB242084 did not affect
locomtor activity; ANOVA (b) Subchronic treatment with 1 mg/kg/day RS 102221 did not affect
locomtor activity; ANOVA. (c) Subchronic treatment with 10 mg/kg/day citalopram did not
affect locomtor activity. n = 12-15 mice per group for all experiments here; error bars represent s.e.m.
Total Consumption (mL)
A
Control
Stress
2
*
1.75
1.5
*
1.25
1
.75
.5
.25
0
BL
week 1 week 2 week 3 week 4
Total Consumption (mL)
B
Control
2
Stress
1.75
1.5
1.25
1
.75
.5
.25
0
VEH
SB1
RS3
CIT10
5 Days Treatment
Supplemental Figure 3. Subchronic 5-HT2C antagonist treatment does not affect total
consumption in the sucrose preference test. (a) Chronic mild stress only affected total
consumption of water and 2% sucrose solution on weeks 2 and 3; AOVA F(4,276) = 9.473, *P <
.0001 Control vs. Stress; Student-Newman-Keuls. (b) Subchronic treatment with 1 mg/kg/day
SB 242084, 3 mg/kg/day RS 102221, or 10 mg/kg/day citalopram did not affect total
consumption. n = 12-15 mice per group for all experiments here; error bars represent s.e.m.
Supplemental Figure 4. Subchronic treatment with 5-HT2C antagonists produces differential
effects on neurotrophic protein expression in medial prefrontal cortex (mPFC). (a) Subchronic
treatment with 5-HT2C antagonists and chronic treatment with citalopram had no effects on
pERK expression; ANOVA F(4,33) = .707, P = .59. (b) Subchronic treatment with 1 mg/kg/day
RS102221 significantly increased pAKT expression; ANOVA F(4,33) = 5.51, *P < .01, StudentNewman-Keuls post-hoc analysis. (c) Subchronic treatment with 10 mg/kg/day citalopram
significantly increased pGSK3 expression; ANOVA F(4,33) = 13.06, *P < .0001, StudentNewman-Keuls post-hoc analysis. (d) Subchronic treatment with 5-HT2C antagonists and
chronic treatment with citalopram had no effects on pP70S6K expression; ANOVA F(4,33) = .708,
P = .59.
Supplemental Figure 5. Subchronic treatment with selective serotonin reuptake inhibitors
increases mTOR expression in medial prefrontal cortex (mPFC). Systemic treatment with 10
mg/kg/day citalopram and 10 mg/kg/day fluoxetine significantly increased pmTOR in mPFC;
ANOVA F(2,17) = 5.652; *P < .05, Student-Newman-Keuls post-hoc analysis. Relative optical
densities were calculated for each band of interest and normalized to optical density of GAPDH
to account of differences in amount of protein loaded. n = 6-8 mice per group; error bars
represent s.e.m.
A
B
Supplemental Figure 6. VTA cannulae placement. (a) Schematic representations of successive
coronal sections (rostral to caudal) of the mouse brain, showing histological verification of
cannulae placements in the ventral tegmental area. Dots indicate cannulae placement for animals
infused with vehicle, 0.3 ug/day SB 242084, and 1.0 ug/day SB 242084. (b) Bilateral cannulae
placement into VTA coronal tissue section.
Supplemental Figure 7. Bilateral infusion of 1 ug/mg/day SB 242084 into the substantia nigra
does not significantly affect BDNF expression in the medial prefrontal cortex; ANOVA.
A
B
Supplemental Figure 8. SN cannulae placement. (a) Bilateral cannulae placement into SN
coronal tissue section. (b) Schematic representations of successive coronal sections (rostral to
caudal) of the mouse brain, showing histological verification of cannulae placements in the
substantia nigra. Dots indicate cannulae placement for animals infused with vehicle or 1.0 ug/day
SB 242084.
Supplemental Figure 9. Subchronic treatment with 5-HT2C antagonist increases apical spine
density in the mPFC. (a) Subchronic treatment with 1 mg/kg/day SB 242084 increased distal
apical spine density on mPFC pyramidal neurons across stressed and non-stressed mice;
ANOVA F(3,40) = 2.95; *P < .05, Student-Newman-Keuls post-hoc analsyis. (b) Representative
neuronal reconstructions showing effects of stress and 1 mg/kg/day SB 242084 treatment on
dendritic spine density. n = 6 mice per group for all experiments here; error bars represent s.e.m.
Supplemental Figure 10. Subchronic treatment with 5-HT2C antagonists and citalopram alters
apical dendritic spine type in layer II/III pyramidal neurons in the mPFC. (a) 1 mg/kg/day SB
242084 reduced the number of thin proximal apical spines across stressed and non-stressed mice;
ANOVA *P < .05 stress vs non-stress, Student-Newman Keuls post-hoc analysis. (b) In nonstressed mice, 1 mg/kg/day SB 242084 and 10 mg/kg/day citalopram increased the number of
mushroom proximal apical spines; ANOVA #P < .05 VEH vs drug, Student-Newman Keuls
post-hoc analysis. (c) 1 mg/kg SB 242084 reduced the ratio of thin/mushroom proximal apical
spines across stress and no stress conditions; ANOVA *P < .05 stress vs non-stress, StudentNewman Keuls post-hoc analysis. (d) No main effects or interactions were observed for number
of thin distal apical spines. (e) In non-stressed mice, 10 mg/kg/day citalopram increased the
number of mushroom distal spines; ANOVA #P < .05 VEH vs drug, Student-Newman Keuls
post-hoc analysis. (f) A main effect of condition [F(1,40) = 5.23; p < .05] revealed that stress
increased the ratio of thin/mushroom distal apical spines. n = 6 mice per group for all
experiments here; error bars represent s.e.m.
Supplemental Figure 11. 5-HT2C Antagonists and citalopram reverse stress-induced changes in
dendritic arborization. (a) Chronic mild stress reduced the number of intersections of dendritic
processes 20-500μm from the cell body. In stressed mice, 3 mg/kg/day RS 102221 and 1
mg/kg/day SB 242084 reversed the reduction of dendritic intersections. (b) In stressed mice, 10
mg/kg/day citalopram reversed the reduction of dendritic intersections. Statistics listed in figure
caption in main manuscript.