Fast acting antidepressants that target glutamatergic receptors represent an important breakthrough in the therapy for depression31. Previous studies have shown the important role of mTOR and related signaling in rapid antidepressant effects in non-stressed animals6. The present study focused on examining changes in glutamatergic receptors and mTOR signaling affected by chronic stress and therapeutic mechanisms for two distinct rapid-acting antidepressants, ketamine and Yueju. Both ketamine and Yueju rapidly reversed the depression-like responses in the CMS exposed mice and remained effective at 5 and 6 days post-drug administration, respectively. CMS compromised ERK/Akt signaling and downstream mTOR effectors in the PFC of CMS mice, which were resumed by Yueju or ketamine treatment. The normalization of Akt signaling was particularly associated with the Yueju’s lasting antidepressant effect. Both ketamine and Yueju quickly reversed the down-regulated expressions of the GluR1 subunit and selectively up-regulated expression of NR1 subunit in the CMS mice, whereas Yueju showed a longer duration in reversing NR1 level. These effects may increase the AMPAR/NMDAR ratio for antidepressant responses of Yueju and ketamine. Taken together, a rapid and lasting remedy of mTOR-related signaling and glutamatergic function in the PFC may, at least in part, be mechanistically linked with rapid and lasting antidepressant actions in depression-like subjects.

CMS is a well-developed and widely-used rodent model of depression32. A variety of procedures have been employed for CMS in rats and mice. Here, we used a CMS procedure leading to a full spectrum of depression-like and/or anxiety-like symptoms in mice, including decreased sucrose preference, increased immobility time in both TST and FST and increased latency for eating and decreased food consumption in the NSF test, consistent with findings from some previous reports33,34. Furthermore, we for the first time demonstrated that a single administration of ketamine or Yueju improved the body weight gain at 6 days post-treatment, with a more pronounced restoration by Yueju. Body weight change is an important clinic symptom of depression and the normalization of body weight may represent part of functional recovery from depression. The present study, using a mouse chronic model of depression validated with multiple measurements of depression-like responses, suggests rapid antidepressant effects after a single dose of ketamine or Yueju. A pilot clinical study supported rapid antidepressant effects of Yueju on MDD patients (Wu and Chen, unpublished observations).

Additionally, the present study characterized the onset and duration of rapid antidepressant effects in chronically stressed mice. This is the first demonstration in CMS mice of a rapid antidepressant response (within 2 hours) for acute ketamine or Yueju, but not fluoxetine, a typical SSRI. Two hours post-ketamine is also a time point when MDD patients started to experience an improvement in mood induced by ketamine treatment35. Garcia et al. assessed the early time points for antidepressant responses in chronically stressed rats, but did not detect a significant effect of ketamine on sucrose intake 1 hour after administration33. It takes about 2 hours for ketamine to promote synaptic protein synthesis in rodents, which is crucial for rapid antidepressant responses to ketamine6. Therefore, a shorter time may not be sufficient for an antidepressant action of ketamine. The antidepressant effects of ketamine or Yueju appeared to last for 5 or 6 days in depression-like mice, in agreement with sustained antidepressant effects in studies using chronically stressed rats or mice10 as well as some clinical findings with ketamine8,36.

Activation of mTOR signaling in PFC is crucial for rapid antidepressant effects6. In the current study, mTOR signaling via 4E-BP1 and p70S6K was suppressed in the PFC of CMS mice, consistent with previous results in CMS rats17,37,38. For the first time, we showed that a single dose of ketamine or Yueju reversed the effects of CMS on the activation of mTOR effectors 4E-BP1 and p70S6K in the PFC of depression-like rodents. Ketamine transiently stimulated mTOR signaling via p-mTOR, 4E-BP1 and p70S6K in PFC of non-stressed rats6. Similar effects were also observed after administration of serotonin 2C receptor antagonists that exhibited fast-onset antidepressant effects. Our findings provide new information that in the depression-like mice, ketamine and Yueju induced a sustained increase in phosphorylated 4E-BP1 and p70S6K to the normal level, which may be required for rapid antidepressant responses by promoting synaptic protein synthesis in the PFC of depression-like animals. Particularly, our study suggested that the sustained depression-like behavior was more closely linked with the prolonged deficits in 4E-BP1, whereas p70S6K level recovered at 6 days post termination of stress exposure.

Although chronic stress led to deficits in the activation of mTOR downstream effectors 4E-BP1 and p70S6K, there was no change in the activation of mTOR in the PFC, consistent with previous findings in chronically stressed rats17. In contrast, deficits in ERK and Akt activation were found in the chronically stressed mice in the present and previous studies39. ERK and Akt both are capable of regulating 4E-BP1 or p70S6K in a manner independent of activation of mTOR40. Inhibitors of either ERK or Akt blunted ketamine’s antidepressant effect6, plausibly by suppressing 4E-BP1 and p70S6K activation. The deficits in ERK and Akt may suppress 4E-BP1 and p70S6K activation in the chronically stressed animals. Additionally, increased ERK and Akt activity was consistent with increased 4E-BP1 and p70S6K activity resulting from ketamine or Yueju treatment. Interestingly, ERK activation returned to normal level at PAD 6, whereas Akt signaling remained deficient. Normalization of Akt paralleled the antidepressant effect of Yueju, indicating that signaling regulated by Akt pathway may be crucially involved in maintaining depression-like status and antidepressant effects. An increasing number of studies indicate the requirement of Akt signaling in antidepressant effects of ketamine and some conventional antidepressants in non-stressed animals6,41,42. It is of particular interest to further investigate mechanisms by which improvement of Akt signaling mediates antidepressant efficacy in mTOR-dependent and –independent manner.

Some of other NMDA receptor antagonists are also capable of producing rapid antidepressant effects experimentally9,43. This suggests a role for NMDA systems in the pathology and therapy of depression. We found a significant increase in NR1 subunit of NMDA receptor but not in other two subunits NR2A and NR2B in the PFC of CMS mice. In fact, ketamine, as a noncompetitive NMDA receptor antagonist, acts at the phencyclidine site in NR1 subunit. As NR1 binds both to NR2A and NR2B, our finding suggests a non-selective up-regulation of NMDA activity in the PFC of depression-like mice. Increased expression of NR1 and/or NR2A/NR2B in depressed subjects has been reported previously44,45. However, other studies indicate down-regulation or no changes46,47. Differences in experimental results may be due to heterogeneity within MDD patients, different disease stages of MDD, differences in chronic stress procedures, animal genetic backgrounds or regional differences in brain NMDAR subunit expression patterns. It has been shown that blocking NMDA but not AMPA receptors attenuates dendrite atrophy, suggesting that the NMDA receptor is particularly responsible for stress-induced synaptic loss48,49. A recent study also supports that activation of NMDA (but not AMPA) receptors is required for abolishment of synaptic plasticity and destruction of dendritic spines through the stress-released neuropeptide corticotropin-releasing hormone50. Therefore, the enhanced NR1 expression by chronic stress may contribute to sustained NMDAR-mediated dentritic atrophy and spine loss underlying depression-like responses.

Additionally, we found the selective decrease in NR1 by ketamine and Yueju, in contrast to the selectively increased NR1 in the chronically stressed mice. The reduction of NR1 expression, together with the inhibition of NMDA transmission, may result in a strong suppressing effect on NMDA neurotransmission by ketamine and/or Yueju. NR1 expression was also reduced by MK801, an NMDA antagonist similar to ketamine that also shows rapid antidepressant effects9. The markedly reduced expression of NR1 has been observed after chronic administration of selective serotonin and norepinephrine uptake inhibitors51. The important role of NR1 in the antidepressant effect was especially evident by the finding that inhibition of NR1 was associated with antidepressant effects at 6 days post Yueju administration, whereas ketamine did not reverse NR1 expression and no longer showed antidepressant effects at this time point. It has yet to be elucidated how chronic stress up-regulates NR1 and the molecular mechanisms underlying down-regulation of NR1 by ketamine and Yueju.

Here we found persistent deficits in GluR1 expression. Consistent with our findings, stress enhanced NMDAR and reduced AMPAR activities52. A concurrent increase in NR1 and decrease in GluR1 after exposure to chronic stress may indicate an imbalance of AMPAR and NMDAR activities by favoring NMDA throughput. The AMPA receptor subunit GluR1 expression was up-regulated shortly after acute ketamine administration and lasted for several days in chronically stressed rats, in an mTOR signaling-dependent manner6. Here we found a similar up-regulation of GluR1 in the chronically stressed mice post ketamine or Yueju, which can last for an extended time period. Although AMPAR activities apparently are not required for mediating stress induced dendritic atrophy, GluR1 containing synapses are resistant to stress50. Increased expression of GluR1 and decreased expression of NR1 at 2 and 6 days post acute administration of Yueju or 2 days post ketamine may increase the ratio of AMPAR/NMDAR activity, in agreement with the findings from chronic ketamine administration25. These results support the idea of the association between increased AMPAR/NMDAR ratios and antidepressant responses23. Increasing the ratio of AMPAR-to-NMDAR activities by favoring AMPAR throughput may represent an enhanced capability of glutamate machinery to counteract adverse consequences of stress.

The current study is the first to demonstrate that changes in mTOR-related activity and glutamate receptors caused by chronic stress can be reversed by two distinct rapid antidepressants, ketamine and Yueju. We confirmed that ketamine and Yueju acted as quickly as 2 hours post-drug administration and result in a long-lasting reduction in depression-like symptoms by using a comprehensive behavioral test battery in a chronic model of depression. Based on our findings, a framework for the pathological changes in the PFC after exposure of chronic stress and molecular therapeutic mechanisms is proposed in Fig. 6. We show that a single dose of ketamine or Yueju reversed deficits in mTOR-related signaling, synaptic protein synthesis as well as the imbalance of AMPA/NMDA receptor ratio in the PFC of CMS mice, contributing to the fast-acting and lasting antidepressant effects. This study sheds the new light on targeting pathological changes in glutamate neurotransmission and mTOR-related signaling in depressed subjects using fast-acting antidepressants.