Yueju conferred a longer duration of antidepressant response in KM compared to ICR mice

The time course of depression-like behavior following a single dose of Yueju or ketamine in KM (Fig. 1A) and ICR (Fig. 1B) mice is illustrated. To avoid potential confounding effects of repeated testing, individual animals were tested once. In both strains, there were significant decreases in the time spent immobile at 30 minutes (F (2,32) = 17.061, p < 0.05), 3 hours (F (2,32) = 9.065, p < 0.05), 1 day (F (2,32) = 11.551, p < 0.05), 2 days (F (2,32) = 10.713, p < 0.05), 3 days (F (2,32) = 10.152, p < 0.05), and 5 days (F (2,32) = 5.89, p < 0.05) post Yueju or ketamine administration in KM mice (Fig. 1A). There were also significant decreases in the time spent immobile at 30 min (F (2,29) = 9.67, p < 0.05), 3 hours (F (2,29) = 18.652, p < 0.05), and 1 day (F (2,29) = 20.607, p < 0.05) post Yueju or ketamine administration in ICR mice (Fig. 1B). The antidepressant-like effect was no longer apparent in KM mice at 7 days (F (2,32) = 1.353, p = 0.274), or in ICR mice at 2 days (F (2,29) = 0.054, p = 0.948) post Yueju administration. This pattern was also evident in ketamine-treated animals. In the open field test, neither Yueju nor ketamine (30 min post-drug) altered the total distance traveled (F (2, 32) = 0.352, p = 0.706) or the total time spent in center (F (2, 32) = 0.036, p = 0.965) in KM mice, similar to ICR mice8.

Figure 1: Time course of Yueju (YJ) and ketamine (KET) mediated antidepressant-like behavioral effects. Independent groups of mice were used at each time point and for each drug treatment, to avoid behavioral habituation. (A) Antidepressant activities in KM mice. Analysis of variance (ANOVA) F (2,20) = 60.161, p < 0.0001 for treatment; F (6,210) = 3.374, p < 0.01 for duration of response; F (12,231) = 1.188, p = 0.293 for treatment-duration interaction. Therefore, we examined treatment effects by time point. n = 11/group. (B) Antidepressant activities in ICR mice. Analysis of variance (ANOVA) F (2,11) = 33.893, p < 0.0001 for treatment; F (3,108) = 14.033, p < 0.0001 for duration of response; F (6,120) = 4.471, p < 0.001 for treatment-duration interaction. n = 10/group. *p < 0.05, **p < 0.01, ***p < 0.001, YJ compared to control; #p < 0.05, ##p < 0.01, ###p < 0.001, KET compared to control. Data represent means ± SEM. Full size image

Yueju treatment induced a long-lasting up-regulation of BDNF expression in KM mice

BDNF mRNA expression did not alter 30 minutes post-Yueju in KM strain mice (F (2,17) = 1.493, p = 0.256). However, there was a significant increase in BDNF mRNA levels at 3 hours (F (2,17) = 108.764, p < 0.05), 1 day (F (2,17) = 17.708, p < 0.05), and 2 days (F (2,17) = 9.499, p < 0.05) after Yueju and ketamine treatment in KM mice (Fig. 2A). In contrast, BDNF mRNA increased only at 3 hours (F (2,17) = 43.605, p < 0.05), but not 1 day (F (2,17) = 1.779, p = 0.203) post Yueju or ketamine in ICR mice (Fig. 2B). There was a significant increase in BDNF protein expression in KM mice 1 day (F (2,17) = 6.568, p < 0.05) after Yueju (Fig. 2C). Despite the lack of change at a transcriptional level, BDNF protein expression significantly increased 30 min after Yueju treatment in KM mice (F (2,17) = 41.119, p < 0.05, Fig. 2C), similar to ICR strain mice8. Acute ketamine showed a similar pattern of BDNF mRNA and protein expression in both strains (Fig. 2).

Figure 2: BDNF mRNA in KM or ICR mice as well as protein expression in KM mice after an acute dose of Yueju (YJ) and Ketamine (KET). (A) BDNF mRNA expression significantly increased in KM mice at 3 hours (F (2,17) = 108.764, p < 0.05), 1 day (F (2,17) = 17.708, p < 0.05) and 2 days (F (2,17) = 9.499, p < 0.05) after Yueju and ketamine administration. n = 6/group. (B) BDNF mRNA expression increased in ICR mice at 3 hours (F (2,17) = 43.605, p < 0.05) but not at 1 day (F (2,17) = 1.779, p = 0.203) after Yueju or ketamine administration. n = 6/group. (C) BDNF protein expression in KM mice 30 minutes (p < 0.01) or 1 day (p < 0.05) post Yueju and ketamine. n = 5/group. *p < 0.05, **p < 0.01, ***p < 0.001, compared to control. Data represent means ± SEM. Full size image

Yueju induced long-term up-regulation of CREB mRNA and protein expression in KM mice

There were strain dependent differences in total CREB and pCREB protein expression in the hippocampus 1 day and 2 days after Yueju and ketamine administration. In ICR mice, pCREB, total CREB protein expression and the ratio of pCREB/CREB was unchanged 1 day (Fig. 3A,B) or 2 days (Fig. 3C,D) after Yueju or ketamine administration. In KM mice, pCREB significantly increased 1 day after Yueju and ketamine (F (2,11) = 14.050, p < 0.05, Fig. 3A). Additionally, total CREB expression significantly increased 1 day after Yueju and ketamine (F (2,11) = 39.725, p < 0.05, Fig. 3B), whereas the ratio of phosphorylated CREB to total CREB (pCREB/CREB) was unchanged by Yueju or ketamine (F (2,11) = 0.943, p = 0.425). In the following day, only Yueju, but not ketamine, continued to significantly increase the expression of pCREB (F (2,11) = 6.560, p < 0.05, Fig. 3C) and total CREB (F (2,11) = 22.774, p < 0.05, Fig. 3D) in KM mice. These findings suggest that the increased pCREB expression is contributed by increased expression of total CREB.

Figure 3: CREB mRNA and protein expression after an acute dose of Yueju (YJ) and Ketamine (KET) in the hippocampus of KM or ICR mice. pCREB (A) and total CREB (B) protein expression in KM and ICR mice 1 day after YJ and KET treatment. pCREB (C) and total CREB (D) protein expression in KM mice and ICR mice 2 days after YJ and KET treatment. CREB mRNA expression in KM and ICR mice 3 hours (E) and 1 day (F) after Yueju treatment. *p < 0.05, **p < 0.01, ***p < 0.001, compared to control, n = 4~6. Data represent means ± SEM. Full size image

The increase in CREB protein expression may be due to transcriptional up-regulation, and thus we also assessed CREB gene expression in the hippocampus at 30 minutes (F (2,17) = 0.103, p = 0.903), 3 hours (F (2,17) = 0.652, p = 0.535) and 1 day (F (2,17) = 0.920, p = 0.420) post-drug in ICR mice. There was no change in CREB mRNA expression at any time point measured post-Yueju or -ketamine in ICR mice. Although neither Yueju nor ketamine altered CREB gene expression in KM mice at 30 min (F (2,17) = 0.303, p = 0.743), there was significant increase 3 hours (F (2,17) = 39.936, p < 0.05, Fig. 3E) after Yueju or ketamine in KM mice. Additionally, only Yueju (p < 0.01), but not ketamine, continued to increase CREB mRNA expression 1 day after administration (F (2,17) = 9.862, p < 0.05, Fig. 3F).

Blockade of PKA-CREB signaling blunted antidepressant effects and up-regulation of BDNF gene expression by Yueju, but not ketamine

Consistent with CREB expression, PKA protein expression was up-regulated 1 day post Yueju and ketamine (F (2,17) = 13.228, p < 0.001) treatment in KM mice, but not in ICR mice (F (2,17) = 0.541, p = 0.593, Fig. 4A).

Figure 4: PKA protein expression and the effect a PKA antagonist on antidepressant response and BDNF/CREB gene expression in the hippocampus after a single Yueju/ketamine treatment. (A) PKA protein expression 1 day after Yueju (YJ) and Ketamine (KET) treatment in KM and ICR mice. PKA protein significantly increased in KM mice (F (2,17) = 13.228, p < 0.001) but not in ICR mice (F (2,17) = 0.541, p = 0.593). n = 6/group. *p < 0.05, ***p < 0.001, compared to control. (B) The effect of saline (F (2,23) = 13.413, p < 0.001) or H-89 (F (2,23) = 24.307, p < 0.001) pretreatment on immobility time in the tail suspension test 1 day after Yueju and ketamine treatment in KM mice. n = 8/group. **p < 0.01, ***p < 0.001, compared to vehicle (VEH, saline); ###p < 0.001, compared to vehicle (H-89); &&&p < 0.001, compared to YJ. (C) The effect of saline (F (2,11) = 15.173, p < 0.01) or H-89 (F (2,11) = 9.746, p < 0.01) pretreatment on BDNF mRNA expression 1 day after Yueju and ketamine treatment in KM mice. n = 4/group. **p < 0.01, compared to vehicle (saline); ##p < 0.01, compared to vehicle (H-89); &&p < 0.01, compared to YJ. (D) The effect of saline (F (2,11) = 11.688, p < 0.01) or H-89 (F (2,11) = 0.033, p = 0.968) pretreatment on CREB mRNA expression 1 day after Yueju and ketamine treatment in KM mice. n = 4/group. **p < 0.01, compared to vehicle (saline); &&p < 0.01, compared to YJ. Data represent means ± SEM. Full size image

To further evaluate the role of PKA/CREB/BDNF signaling in antidepressant actions of Yueju and ketamine, animals were pretreated with a PKA inhibitor H-89. The pre-treatment of KM mice with H-89 was able to reverse the antidepressant-like effect of Yueju. A two-way ANOVA revealed significant differences for the H-89 pre-treatment (F (1,28) = 28.439, p < 0.001), Yueju treatment (F (1,28) = 4.636, p < 0.05) and H-89 × Yueju interaction (F (1,28) = 15.186, p < 0.001, Fig. 4B). The administration of H-89 alone did not affect the immobility in the tail suspension test in non-Yueju mice (p = 0.369), but significantly increased it in the Yueju-treated mice (p < 0.01). In contrast, H-89 couldn’t block the antidepressant-like effect of ketamine: a two-way ANOVA revealed the significant main effect of ketamine treatment (F (1,28) = 45.366, p < 0.001), but not H-89 pre-treatment (F (1,28) = 2.064, p = 0.162) or H-89 × ketamine interaction (F (1,28) = 0.06, p = 0.808).

Consistent with blocking the behavioral effects of Yueju, H-89 pretreatment also reversed up-regulation of BDNF gene expression induced by Yueju. A two-way ANOVA revealed significant differences for the H-89 pre-treatment (F (1,12) = 24.961, p < 0.001), Yueju treatment (F (1,12) = 32.005, p < 0.001), and H-89 × Yueju interaction (F (1,12) = 15.892, p < 0.01, Fig. 4C). The administration of H-89 alone did not affect the BDNF mRNA level in non-Yueju mice (p = 0.558), but significantly decreased it in the Yueju-treated mice (p < 0.01). In contrast, H-89 couldn’t block the increase of BDNF mRNA induced by ketamine: a two-way ANOVA revealed the significant main effect of ketamine treatment (F (1,12) = 48.365, p < 0.001), but not H-89 pre-treatment (F (1,12) = 0.38, p = 0.549) or H-89 × ketamine interaction (F (1,12) = 0.01, p = 0.921).

H-89 pretreatment also reversed up-regulation of CREB gene expression induced by Yueju. A two-way ANOVA revealed significant differences for the H-89 pre-treatment (F (1,12) = 13.041, p < 0.01), Yueju treatment (F (1,12) = 8.871, p < 0.05) and H-89 × Yueju interaction (F (1,12) = 9.936, p < 0.01, Fig. 4D). The administration of H-89 alone did not affect the CREB mRNA level in non-Yueju mice (p = 0.752), but significantly decreased it in the Yueju-treated mice (p < 0.01). Meanwhile, neither ketamine nor H-89 affected the CREB mRNA level (two way ANOVA, F (3,15) = 0.053, p = 0.983). These results suggest that the antidepressant effects of Yueju, but not ketamine, are dependent on the PKA/CREB/BDNF pathway.

Blockade of mTOR signaling blunted antidepressant effects and up-regulation of BDNF gene expression by ketamine, but not Yueju

Additionally, we assessed the role of mTOR signaling in the antidepressant effects of Yueju or ketamine. Animals were pretreated with an mTOR inhibitor rapamycin before administration of Yueju and ketamine. The pre-treatment of KM mice with rapamycin was able to reverse the antidepressant-like effect of ketamine: a two-way ANOVA revealed significant main effect of rapamycin pre-treatment (F (1,28) = 7.243, p < 0.05), ketamine treatment (F (1,28) = 14.328, p < 0.001) and rapamycin × ketamine interaction (F (1,28) = 6.003, p < 0.05, Fig. 5A). The administration of rapamycin alone did not affect the immobility in the tail suspension test in non-ketamine mice (p = 0.874), but significantly increased it in the ketamine-treated mice (p < 0.01). In contrast, rapamycin couldn’t block the antidepressant-like effect of Yueju: a two-way ANOVA revealed the significant main effect of Yueju treatment (F (1,28) = 38.210, p < 0.05), but not rapamycin pre-treatment (F (1,28) = 0.349, p = 0.56) or rapamycin × Yueju interaction (F (1,28) = 0.67, p = 0.42).

Figure 5: Effects of mTOR blockade with rapamycin pretreatment on immobility times in the tail suspension test in KM mice and BDNF gene expression 1 day post-Yueju and ketamine. (A) The effect of saline (F (2,23) = 18.818, p < 0.001) or rapamycin (F (2,23) = 10.734, p < 0.001) pretreatment on immobility time in the tail suspension test 1 day after Yueju and ketamine treatment in KM mice. n = 8/group. ***p < 0.001, compared to vehicle (VEH, saline); ###p < 0.001, compared to vehicle (rapamycin); &&p < 0.01, compared to KET. (B) The effect of saline (F (2,11) = 34.595, p < 0.001) or rapamycin (F (2,11) = 23.702, p < 0.001) pretreatment on BDNF mRNA expression 1 day after Yueju and ketamine treatment in KM mice. n = 4/group. ***p < 0.001, compared to vehicle (saline); ###p < 0.001, compared to vehicle (rapamycin); &&p < 0.01, compared to KET. Data represent means ± SEM. Full size image