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).