Figure legends
Figure 1 . Experimental design (A) and the location of
intracerebroventricular injection sites (B).
Figure 2 . ICV-STZ induced depressive- and anxiety-like
behaviors without cognitive impairment, with no significant difference
in Aβ, Tau, pTauSer199 protein levels and
pTauSer199/Tau ratio in the mPFC. Immobility time in
the FST (A), sucrose preference in the SPT (B) and latency time in the
NSFT (C, D) were used to evaluate depressive-like behaviors. Time in
center in the OFT (E) was used to evaluate anxiety-like behavior. Total
distance in the OFT (F) was used to detect locomotor activity.
Discrimination ratio in the NORT (G) was used to detect recognition
memory and discrimination ability (n1, n2 = 6). Western blots (H)
and quantification of Aβ (I), Tau (J), pTauSer199 (K)
and pTauSer199/Tau ratio in the mPFC. β-actin is shown
as a quantitative loading control (n1, n2 = 6). The data shown
are individual values with means ± SEM. *P < 0.05,
significantly different from vehicle group; A-G, I-L, unpaired Student’s
t-test.
Figure 3 . ICV-STZ induced IDO activation in the PrL and IL,
increased the level of pro-inflammatory cytokines in the IL. Location of
the PrL and IL injection sites (A). Intra-PrL or IL administration of
1-MT had no effect on body weight (B) (n=6 per group). Levels of Trp (C)
and Kyn (D) examined by HPLC–MS/MS and the Kyn/Trp ratio (E) were used
as a measure of IDO activity in PrL (n1, n2, n3, n4 = 6). Levels of Trp
(F) and Kyn (G) and the Kyn/Trp ratio (H) in the IL (n1, n2, n3,
n4 = 6). Levels of IL-1β (I) and IL-6 (J) examined by ELISA were used to
evaluate the neuroinflammatory response in the PrL (n1, n2, n3, n4 = 6).
Levels of IL-1β (K) and IL-6 (L) in the IL (n1, n2, n3, n4 = 6). The
data shown are individual values with means ± SEM. *P < 0.05,
significantly different from vehicle group; #P < 0.05,
significantly different from ICV-STZ group; C-L, two-way ANOVA followed
by Tukey’s multiple comparison test.
Figure 4 . Intra-PrL or IL administration of 1-MT prevented
depressive-like behaviors induced by ICV-STZ. Immobility time in the FST
(A), sucrose preference in the SPT (B) and latency time in the NSFT (C,
D) were used to evaluate depressive-like behaviors. Time in center in
the OFT (E) was used to evaluate anxiety-like behavior. Total distance
in the OFT (F) was used to detect locomotor activity (G) (n1, n2,
n3, n4 = 6). The data shown are individual values with means ± SEM.
*P < 0.05, significantly different from vehicle group;
#P < 0.05, significantly different from ICV-STZ group; A-F,
one-way ANOVA followed by Tukey’s multiple comparison test.
Figure 5 . ICV-STZ and intra-PrL administration of 1-MT had no
effects on microglia-related indicators in the PrL. Fluorescent images
of immunostaining for Iba1 (A) and the count of microglia (B) were used
to evaluate the expression of microglia in the PrL. Iba1 was labelled
with Alexa Fluor 647 (red) and the cell nucleus was counterstained with
DAPI (blue), scale bars=100 μm, n1, n2, n3, n4 = 6. Western blots
and quantification of Iba1 (C) in the PrL, β-actin is shown as a
quantitative loading control (n1, n2, n3, n4 = 6). Morphological
images (D), the count of branches (E) and the number of end-points (F)
were used to evaluate the state of microglia in the PrL (n1, n2,
n3, n4 = 6). Levels of 3-HK (G) examined by HPLC–MS/MS in PrL
(n1, n2, n3, n4 = 6). The data shown are individual values with
means ± SEM. *P < 0.05, significantly different from vehicle
group; #P < 0.05, significantly different from ICV-STZ group;
B, C, E-G, two-way ANOVA followed by Tukey’s multiple comparison test.
Figure 6 . Intra-IL administration of 1-MT blocked the
activation of microglia in the IL induced by ICV-STZ. Fluorescent images
of immunostaining for Iba1 (A) and the count of microglia (B) were used
to evaluate the expression of microglia in the IL. Iba1 was labelled
with Alexa Fluor 647 (red) and the cell nucleus was counterstained with
DAPI (blue), scale bars=100 μm, n1, n2, n3, n4 = 6. Western blots
and quantification of Iba1 (C) in the IL, β-actin is shown as a
quantitative loading control (n1, n2, n3, n4 = 6). Morphological
images (D), the count of branches (E) and the number of end-points (F)
were used to evaluate the state of microglia in the IL (n1, n2,
n3, n4 = 6). Levels of 3-HK (G) examined by HPLC–MS/MS in IL
(n1, n2, n3, n4 = 6). The data shown are individual values with
means ± SEM. *P < 0.05, significantly different from vehicle
group; #P < 0.05, significantly different from ICV-STZ group;
B, C, E-G, two-way ANOVA followed by Tukey’s multiple comparison test.
Figure 7 . Intra-PrL administration of 1-MT prevented astrocyte
defects in the PrL induced by ICV-STZ. Fluorescent images of
immunostaining for GFAP (A) and the count of astrocytes (B) were used to
evaluate the expression of astrocytes in the PrL. GFAP was labelled with
Alexa Fluor 488 (green) and the cell nucleus was counterstained with
DAPI (blue), scale bars=100 μm, n1, n2, n3, n4 = 6. Sholl
analysis (C) and the line plot (D) revealed the number of intersections
per 3 μm of astrocytes in the PrL, n1, n2, n3, n4 = 6. Western
blots (E) and quantification of GLT-1 (F), GLAST (G) and GFAP (H) in
PrL, β-actin is shown as a quantitative loading control (n1, n2,
n3, n4 = 6). Levels of KA (I) examined by HPLC–MS/MS in PrL (n1,
n2, n3, n4 = 6). The data shown are individual values with means ± SEM.
*P < 0.05, significantly different from vehicle group;
#P < 0.05, significantly different from ICV-STZ group; B,
F-I, two-way ANOVA followed by Tukey’s multiple comparison test.
Figure 8 . ICV-STZ and intra-IL administration of 1-MT had no
effects on astrocyte-related indicators in the IL. Fluorescent images of
immunostaining for GFAP (A) and the count of astrocytes (B) were used to
evaluate the expression of astrocytes in the IL. GFAP was labelled with
Alexa Fluor 488 (green) and the cell nucleus was counterstained with
DAPI (blue), scale bars=100 μm, n1, n2, n3, n4 = 6. Sholl
analysis (C) and the line plot (D) revealed the number of intersections
per 3 μm of astrocytes in the IL, n1, n2, n3, n4 = 6. Western
blots (E) and quantification of GLT-1 (F), GLAST (G) and GFAP (H) in the
IL, β-actin is shown as a quantitative loading control (n1, n2,
n3, n4 = 6). Levels of KA (I) examined by HPLC–MS/MS in IL (n1,
n2, n3, n4 = 6). The data shown are individual values with means ± SEM.
*P < 0.05, significantly different from vehicle group;
#P < 0.05, significantly different from ICV-STZ group; B,
F-I, two-way ANOVA followed by Tukey’s multiple comparison test.
Figure 9 . Intra-PrL or IL administration of 1-MT improved
synaptic deficits in the PrL and IL induced by ICV-STZ. Golgi staining
of PrL and IL pyramidal neurons (A). Representative images of dendritic
spines in the PrL (B), scale bars=2 μm. Total dendritic spine number (C)
and the proportion of each type (D) were used to evaluate the spine
morphology alterations in the PrL, n1, n2, n3, n4 =18 dendrites
from 6 brains in each group. Representative images of dendritic spines
in the IL (E), scale bars=2 μm. Total dendritic spine number (F) and the
proportion of each type (G) were used to evaluate the spine morphology
alterations in the IL, n1, n2, n3, n4 =18 dendrites from 6 brains
in each group. Western blots (H) and quantification of BDNF (I) in the
PrL (n1, n2, n3, n4 = 6). Western blots (J) and quantification of
BDNF (K) in the IL (n1, n2, n3, n4 = 6). The data shown are
individual values with means ± SEM. *P < 0.05, significantly
different from vehicle group; #P < 0.05, significantly
different from ICV-STZ group; C, D, F, G, I, K, two-way ANOVA followed
by Tukey’s multiple comparison test.
Figure S1. The location of intra-PrL (A) and IL (B) injection
sites. Food consumption (C) and water intake across the groups (D) (n=6
per group).