PKA-CREB-BDNF signaling regulated long lasting antidepressant activities of Yueju but not ketamine

Yueju confers antidepressant effects in a rapid and long-lasting manner, similar to ketamine. CREB (cAMP-response element binding protein) signaling is implicated in depression pathology and antidepressant responses. However, the role of CREB and associated brain derived neurotrophic factor (BDNF) signaling in rapid and long-lasting antidepressant effects remains unclear. Here, we demonstrated that ICR and Kunming strain mice conferred antidepressant responses lasting for 1 and 5 days, respectively, following a single dose of Yueju. One day post Yueju in Kunming but not ICR strain mice, expression of total and phosphorylated CREB, as well as the CREB signaling activator, PKA (protein kinase A) was up-regulated in the hippocampus. Although BDNF gene expression increased at 3 hours in both strains, it remained up-regulated at 1 day only in Kunming mice. Ketamine showed similar strain-dependent behavioral effects. However, blockade of PKA/CREB signaling blunted the antidepressant effects and reversed the up-regulation of BDNF gene expression by Yueju, but not ketamine. Conversely, blockade of mammalian target of rapamycin signaling led to opposite effects. Taken altogether, prolonged transcriptional up-regulation of hippocampal BDNF may account for the stain-dependent enduring antidepressant responses to Yueju and ketamine, but it was mediated via PKA/CREB pathway only for Yueju.


Discussion
The present study aimed to dissect molecular and neurobiological mechanisms responsible for long-lasting antidepressant responses after Yueju. Antidepressant effects of Yueju or ketamine last for 1 or 5 days in ICR and KM mice, respectively. One day after Yueju and ketamine treatment, there was increased expression of total and phosphorylated CREB protein in KM but not ICR mice. Importantly, the CREB upstream regulator, PKA, and the downstream effector, BDNF, also showed a similar strain-dependent expression pattern. Although PKA/CREB/BDNF expression were up-regulated 1 day post administration of either ketamine or Yueju, inhibition of PKA-CREB signaling only reversed BDNF gene expression and the antidepressant effect of Yueju, but not ketamine, indicating that the PKA/CREB/BDNF pathway was required for the lasting antidepressant effects of Yueju but not ketamine.
CREB signaling in the hippocampus has been implicated in affective and cognitive behaviors, and previous studies have shown strain dependent differences in CREB signaling that may contribute to differential learning and memory related behaviors [25][26][27][28][29] . CREB activation is a hallmark of the neural plasticity responsible for antidepressant effects after chronic SSRI administration 30 . Here we demonstrated the strain-dependent differences in CREB signaling linked to a persistent antidepressant response after a single dose of Yueju. pCREB signaling was up-regulated 1 day post-Yueju and sustained at least for one more day in KM mice, contrasting to no change in CREB signaling in ICR mice, which paralleled the longer duration of antidepressant response in KM mice. The increased pCREB expression is partly attributable to increased gene and protein expression of total CREB as well as increased activation of PKA. It is notable that CREB signaling in the hippocampus was not up-regulated by 30 minutes post Yueju, whereas both strains showed antidepressant effects, indicating that CREB signaling was not responsible for their immediate antidepressant responses. Importantly, up-regulated expression of CREB was detected 3 hours post Yueju, with increased CREB and BDNF mRNA expression in KM mice. Inhibition of CREB signaling abolished up-regulation of BDNF gene expression and antidepressant response at 1 day post Yueju, supporting the hypothesis that CREB-BDNF signaling is required for the maintenance of antidepressant response to Yueju.
Although Yueju and ketamine both activated the PKA/CREB pathway, our further studies showed the role of the pathway was different. In Yueju-treated mice, up-regulation of BDNF mRNA expression was temporally aligned with increased total and phosphorylated CREB expression, and blockade of PKA/CREB signaling reversed the up-regulation of BDNF expression as well as the antidepressant effect of Yueju. In contrast, after treatment with ketamine, the increase in CREB expression was only transitory whereas the BDNF gene expression continued to be up-regulated, suggesting the two events may be independent. Furthermore, blockade of PKA/CREB signaling failed to change BDNF expression or antidepressant effect of ketamine. These findings suggest that CREB signaling does not play a primary role in antidepressant actions of ketamine, unlike Yueju. This finding is agreement with reports that hippocampal CREB and PKA protein expression is not associated with antidepressant response of ketamine 31 . Conversely, we found that the mTOR inhibitor rapamycin could reverse the antidepressant effect of ketamine, but not Yueju, supporting the hypothesis that long-lasting antidepressant action of ketamine may rely mainly on mTOR-associated signaling 4 . As Yueju is able to reverse the deficient mTOR-related signaling in chronically stressed animals 24 , there may exist a cross-talk between PKA/CREB and mTOR signaling, which warrants further investigation.
The present study demonstrates for the first time that up-regulation of BDNF expression may be universally involved in the persistent antidepressant response of Yueju and ketamine in both KM and ICR mouse strains. This is in contrast to the specific role of CREB in the persistent antidepressant response to Yueju. It has been well documented that chronic SSRI treatment activates transcription factors, including CREB, leading to an increase in the expression of neurotrophic factors, including BDNF, and their receptors 30,32 . For ketamine, the requirement of BDNF for initiation of the antidepressant response has been demonstrated previously 5 . Our studies showed that, without changes in CREB activation or BDNF mRNA expression, BDNF protein in the hippocampus is up-regulated quickly after ketamine or Yueju treatment in both KM and ICR mice. This non-transcriptional BDNF up-regulation was thus responsible for the antidepressant response immediately post Yueju or ketamine administration. This is followed by up-regulation of BDNF mRNA expression, likely via CREB signaling for Yueju and mTOR signaling for ketamine. ICR mice with a shorter time course of BDNF mRNA expression displayed a shorter antidepressant time course than KM mice. The transcriptional up-regulation of BDNF may regulate the cellular signaling governing the persistent antidepressant responses, as reversal of BDNF gene expression by blocking either CREB or mTOR signaling was associated with a blunted antidepressant response 1 day post Yueju or ketamine administration, respectively. Although the molecular link from the initial non-transcriptional BDNF protein upregulation to the following transcriptional BDNF up-regulation remains to be determined, it has been shown that BDNF, via activating its receptor TrkB, can induce the activation of mTOR signaling and CREB signaling 4,33 . Furthermore, a recent study demonstrates that in the cultured neurons, BDNF can initiate self-amplification of BDNF mRNA expression via multiple signaling pathways including PKA-CREB pathway 34 . Therefore, the initial non-transcriptional up-regulation of BDNF may still be of importance in inducing the cellular signaling that promotes BDNF gene expression for long-lasting antidepressant responses. This can be elucidated by manipulating BDNF gene expression in a time-dependent manner.
The present study identified for the first time strain-dependent PKA/CREB/BDNF signaling that regulates long-lasting antidepressant responses to Yueju. Our results also indicate that continuous up-regulation of hippocampal BDNF mRNA is essential for the persistent antidepressant response, either for Yueju or ketamine. Further studies should address the genetic variants and gene networks that perturb BDNF and other signaling commonly responsible for individual differences in antidepressant responses after ketamine or Yueju treatment. As long-lasting antidepressant efficacy is implicated in sustained therapeutic outcomes, elucidation of the underlying mechanisms and genetic variants influencing the responses may shed new light on effective personalized medicines for depression. . The herbal mixture was powdered, immersed in 95% of ethanol with constant shaking and filtered. This procedure was repeated three times, and the collected solvent was evaporated at low pressure and medium temperature (< 55 °C) until ethanol was completely eliminated. The extract of Yueju was dispersed in Tween 80 solution (0.5%, w/v in saline) and administrated intragastrically (270 mg/ml, i.g.). Quality control of the preparation was performed as described previously using HPLC fingerprint analysis. Different samples of Yueju preparation were revealed very similar and suitable 8 . The doses of ketamine (50 mg/kg in ICR mice 8 and 30 mg/kg in KM mice 24 ) and Yueju 8,24 were optimized based on a pilot assessment of a dose-response relationship in the strain of mice as we described in previous studies. 0.5%, w/v Tween 80 in saline solution via i.g. and saline via i.p. served as the vehicle controls, and their behavioral data or western blot were collapsed as there were no statistical differences between them. H-89 at 10 mg/kg (Sigma, St. Louis, MO, USA) and rapamycin at 5 mg/kg (Sigma, St. Louis, MO, USA), were dissolved in 0.5% DMSO (dimethyl sulfoxide) and distilled water, respectively, and were injected i.p. 30 minutes before ketamine, Yueju, or vehicle administration.

Tail suspension test (TST).
Mice were assessed in the TST, which was performed with a computerized device that allowed four animals to be tested at one time. In a chamber that was acoustically and visually isolated, an individual mouse was suspended 50 cm above the floor by adhesive tape placed approximately 1 cm from the tip of the tail. The activities of the animal were videotaped. ANY-maze software (Stoeling Co.Ltd., USA) was used to calculate the total time spent immobile during the last 4 min in a 6-min testing period 39 . Open field Test (OFT). The OFT assesses locomotor activity and anxiety-like behavior in a bright-lit open area. Testing was performed for 5 min in a well-illuminated (∼ 300 lux) transparent acrylic cage (40 × 40 × 15 cm). The mice were gently placed on the center and left to explore the area for 5 min. The digitized image of the path taken by each mouse was tracked by camera, and the total running distances (locomotor activity) and spending times in center were analyzed using ANY-maze software. The testing apparatus was thoroughly cleaned with 70% ethanol and then dried between each animal. Western blots. Mice were sacrificed by decapitation at the designated time points. The hippocampus was dissected out and put into ice cold tubes containing an enzyme inhibitor. Brain tissue was homogenized and western blot analysis was carried out, using primary antibodies for rabbit pCREB (1:500), rabbit CREB (1:1000), rabbit PKAC-α (1:1000) and rabbit β -tubulin (1: 5000) (all from Cell Signal Inc., CA, USA), BDNF (1:200, Santa Cruz). A secondary antibody (1:2000) conjugated with horseradish peroxidase was used. Immunoreactivity was visualized by ECL reagent. Blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.) and were shown as density relative to β -tubulin. All experiments were performed 3 times.
Statistical analyses. All data were presented as means ± S.E.M. Differences among groups were determined using one-way ANOVA or two-way ANOVA, followed by a Bonferroni post hoc analysis if appropriate. p < 0.05 was the accepted level of significance.