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May 20, 2016 - (18) An instant synthesis of brain-derived neurotrophic factor (BDNF) in the hippocampus has been observed quickly after a single dose ...
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Instant and Persistent Antidepressant Response of Gardenia Yellow Pigment Is Associated with Acute Protein Synthesis and Delayed Upregulation of BDNF Expression in the Hippocampus Ruyan Wu,† Weiwei Tao,† Hailou Zhang,† Wenda Xue,† Zhilu Zou,† Haoxin Wu,† Baochang Cai,‡ Ravid Doron,§,∥ and Gang Chen*,† †

Center for Translational Systems Biology and Neuroscience, Key Laboratory of Integrative Biomedicine for Brain Diseases, and School of Pharmacy, Nanjing University of Chinese Medicine, 210023 Nanjing, People’s Republic of China § School of Behavioral Sciences, The Academic College of Tel Aviv-Yaffo, Tel- Aviv 61083, Israel ∥ Department of Education and Psychology, The Open University of Israel, 108 Ravutski St., P.O. BOX 808, Raanana 43107, Israel ‡

S Supporting Information *

ABSTRACT: Gardenia yellow pigment (GYP) is a collection of compounds with shared structure of crocin, which confers antidepressant activity. GYP is remarkably enriched in Gardenia jasminoides Ellis, implicated in rapid antidepressant effects that are exerted through enhanced neuroplasticity. This study aims to investigate the rapid antidepressant-like activity of GYP and its underlying mechanism. After the optimal dose was determined, antidepressant responses in tail suspension test or forced swim test were monitored at 30 min, 1 day, 3 days, and 7 days post a single GYP administration. Rapid antidepressant potential was tested using learned helplessness paradigm. The expression of proteins involved in hippocampal neuroplasticity was determined. The effect of blockade of protein synthesis on GYP’s antidepressant response was examined. Antidepressant response was detected at 30 min, and lasted for at least 3 days post a single administration of GYP. A single administration of GYP also reversed the deficits in learned helplessness test. Thirty minutes post GYP administration, ERK signaling was activated, and its downstream effector phosphorylated eukaryotic elongation factor 2 was inhibited, contributing to increased protein translation. Expression of synaptic proteins GluR1 and synapsin 1 was upregulated. Blockade of protein synthesis with anisomycin blunted the immediate antidepressant response of GYP. CREB signaling and BDNF expression were upregulated at 24 h, but not at 30 min. In conclusion, GYP-induced immediate antidepressant response was dependent on synthesis of proteins, including synaptic proteins. This was followed by enhanced expression of CREB and BDNF, which likely mediated the persistent antidepressant responses. KEYWORDS: Gardenia yellow pigment, Yueju, protein synthesis, BDNF, CREB

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a single administration.9,12 A pilot study also demonstrated the rapid antidepressant role of Yueju on MDD patients.13 Gardenia yellow pigment (GYP) is a collection of compounds, and mainly contains crocin.14 Preclinical and clinical studies indicated that chronic administration of crocin alleviated depression behaviors or symptoms.15,16 GYP, which is remarkably enriched in Gardenia jasminoides Ellis (GJ), is also the constituent component that plays a primary role in rapid antidepressant effects of Yueju.17 These observations raise the possibility that GYP holds an antidepressant potential, and may act in an instant and persistent manner after a single administration. The molecular mechanisms underlying the antidepressant response overlap with those of neuroplasticity to a striking

ajor depression disorder (MDD) is one of the most prevalent and relapsing mental illnesses worldwide, imposing heavy burdens on the family and society.1,2 The current medical needs for treatment of the disease remain unmet, partly due to some disadvantages of mainstream pharmacological treatment using selective serotonin reuptake inhibitors (SSRIs), including a considerable population of nonresponse patients, side effects, and a delayed onset of efficacy.3−5 A recent study demonstrated that a subanesthetic dose of ketamine, one of the N-methyl-D-aspartate (NMDA) antagonists, resulted in robust fast-onset of antidepressant effects within hours and lasted for 1 week in depressive patients.6−8 The rapid and persistent antidepressant effects were also demonstrated in animal models.9,10 However, the wide use of ketamine may be restricted by its neurotoxin effects and abuse potential.11 Yueju, a traditional Chinese medicine formulated to treat mood disorders, can rapidly and persistently attenuated depression-like symptoms in animal models of depression after © 2016 American Chemical Society

Received: January 9, 2016 Accepted: May 20, 2016 Published: May 20, 2016 1068

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Figure 1. UPLC profiles of (A) control solution and (B) GYP. Peak 1 is crocin-1; peak 2 is crocin-2.

prefrontal cortex (PFC), including increased expression of preand post-synaptic proteins such as synapsin 1 and GluR1, has been observed at a later time after ketamine administration.9 This enhanced synaptic plasticity can sustain for a long time and is therefore considered to underlie the rapid and prolonged antidepressant responses in the MDD patients. Reversal of deficits in synaptic proteins expression in the chronically stressed animals has also been observed a few days after a single dose of Yueju or ketamine.9 Additionally, ketamine induces the activation of cAMP-response element binding protein (CREB),

degree, including the promotion of neurotrophic factor, synaptogenesis and neurogenesis.18 An instant synthesis of brain-derived neurotrophic factor (BDNF) in the hippocampus has been observed quickly after a single dose of ketamine or Yueju.12,19 This synthesis of proteins depends on the enhanced protein translation after deactivation of phosphorylated eukaryotic elongation factor2 (eEF2) kinase.19 Protein synthesis of BDNF is required for the acute antidepressant response of ketamine as the response was blunted in the BDNF knockout mice.19,20 Additionally, an increase in synaptic plasticity in the 1069

DOI: 10.1021/acschemneuro.6b00011 ACS Chem. Neurosci. 2016, 7, 1068−1076

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ACS Chemical Neuroscience a transcriptional factor and a neural plasticity player that regulates expression of BDNF.21 A number of other rapid antidepressant-like agents also demonstrated immediate and persistent promotion of neural plasticity. Here we aim to assess the potential of GYP as a rapid and lasting antidepressant agent and to investigate the associated mechanism. We found that an acute administration of GYP induced an instant antidepressant response, which lasted for at least 3 days. We thus tested the contribution of neuroplasticity to the instant and lasting antidepressant response to GYP. We found that GYP instantly deactivated eEF2 and upregulated the expression of synaptic proteins, but not BDNF. On the other hand, CREB expresion was enhanced at the later time, concurrent with the delayed upregulation of BDNF expression.

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RESULTS AND DISCUSSION Using UPLC, the chemical markers in GYP were identified as the main constituent compounds with crocin-1 (42.6%) and crocin-2 (10.1%) (Figure 1), and the chemical structures are illustrated in Supporting Information Figure S1 for reference. To investigate rapid antidepressant potential of GYP, mice were administrated with a low (100 mg/kg), intermediate (200 mg/kg), or high (400 mg/kg) dose of GYP, and tested for tail suspension (TST) at 24 h later, a time when ethanol extract of GJ or Yueju as well as ketamine demonstrated the rapid antidepressant potential.12,17,22 There was a significant dose effect on the immobility time in the TST (F(3, 27) = 4.870, p = 0.008). Only the intermediate dose (200 mg/kg) of GYP significantly reduced the immobility time compared to control (p = 0.002), with the low dose ineffective (p = 0.614) but a trend for the high dose (p = 0.088) (Figure 2a). Similarly, in the forced swimming test (FST) at 1 day after a single GYP administration, only mice treated with the intermediate dose (200 mg/kg) of GYP showed significantly decreased immobility time compared with the control group (p = 0.002) (Figure 2b). Interestingly, like ketamine, ethanol extract of GJ or Yueju, there was an inverse U-shape dose−effect relationship in this test.17,23,24 To further examine the onset and duration of the antidepressant response, we evaluated the performance in the TST at different time points after a single dose of 200 mg/kg GYP, ranging from 30 min to 7 days. GYP reduced the immobility time almost significantly at 30 min (t = 2.091, p = 0.05), and 1 day (t = 5.312, p = 0.000) and 3 days (t = 4.278, p = 0.001) significantly, but not at 7 days (t = 0.691, p = 0.501), indicating that GYP can induce an instant and enduring antidepressant effect (Figure 2c). In the open field test, 200 mg/kg GYP did not change the total distance traveled or the time spent in the central area 30 min after administration (Supporting Information Figure S2). Learned helplessness behavior paradigm has been used for testing the rapid antidepressant efficacy of ketamine or Yueju25,26 after a single administration, and here it was used with a single administration of 200 mg/kg GYP. There was a significant effect of treatment (F(2, 18) = 13.051, p = 0.000). The number of escape failures was significantly increased after learned helplessness training (Figure 3a). Compared to vehicle, GYP significantly reduced the number of escape failures (p < 0.05), an effect that was also observed in mice administrated with Yueju or ketamine.17 Similarly, there was also a significant effect of treatment in latency test (F(2, 19) = 17.944, p = 0.000). The latency to avoid escapable shocks was significantly increased after learned helplessness training (p = 0.000), and GYP significantly reduced the latency (p = 0.005) (Figure 3b).

Figure 2. Acute and persistent antidepressant effects post a single dose of GYP in KM mice. Antidepressant response was measured with immobility time during the last 4 min of a 6 min tail suspension test and forced swimming test. (a) Antidepressant response at 1 day post GYP in KM mice using tail suspension test. There was a significant dose effect on the immobility time in TST. Only the middle dose (200 mg/kg) of GYP significantly reduced the immobility time compared with the control, with the low dose ineffective but a trend for the high dose. (b) Antidepressant response at 1 day post GYP in KM mice using forced swimming test. Only mice treated with the middle dose (200 mg/kg) of GYP showed significantly decreased immobility time in FST compared with the control group. One way ANOVA, ***p < 0.001, compared to control with Bonferroni post hoc test. Data represent means ± SEM. (c) Onset and duration of the antidepressant response of GYP (200 mg/kg) in KM mice. GYP significantly reduced the immobility time at 30 min, 1 day, and 3 days, but not at 7 days. Student’s t test, *p < 0.05, ***p < 0.001, compared to control. Data represent means ± SEM.

Figure 3. Rapid antidepressant effects of GYP in the learned helplessness paradigm. (a) Learned helplessness was quantified as number of escape failures in an active avoidance test. Mice received inescapable shock training for 2 days prior to a 30-trial active avoidance test. (b) Learned helplessness was also quantified as latency to avoid the escapable shock. ***p < 0.001 compared to control, #p < 0.05; ###p < 0.001 compared to vehicle; Bonferroni post hoc tests.

ERK and eEF2 singaling pathways are implicated in rapid antidepressant effects,12,27 and thus, we examined their involvement in the acute and persistent antidepressant response of GYP. At 30 min after GYP administration, there was a trend to significant increase in ERK phosphorylation (t = −2.352, p = 1070

DOI: 10.1021/acschemneuro.6b00011 ACS Chem. Neurosci. 2016, 7, 1068−1076

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Figure 4. GYP acutely increased p-ERK and decreased eEF2 phosphorylation. (a) ERK phosphorylation was significantly activated at 30 min after GYP administration, but not at 24 h. (b) The expression of total ERK was not significantly changed by the treatment either at 30 min or 24 h. (c) The ratio of p-ERK/ERK was significantly increased at 30 min but not 24 h after administration. (d) There was no significant change in PKA activation either at 30 min, or 24 h after the GYP administration. (e) Phosphorylated eEF2 was significantly decreased at 30 min and 24 h post GYP administration. (f) The expression of total eEF2 was not significantly changed by the treatment either at 30 min or 24 h. (g) The ratio of p-eEF2/eEF2 was significantly decreased at 30 min but not 24 h. Student’s t test, *p < 0.05, ***p < 0.001, compared to control. Data represent means ± SEM.

Figure 5. GYP acutely enhanced expression of synaptic proteins GluR1 and synapsin 1 but not BDNF. (a) Expression of GluR1 was significantly enhanced in the hippocampus but not 24 h. (b) Expression of synapsin 1 was significantly enhanced in the hippocampus but not 24 h. (c) BDNF was not changed at 30 min after GYP administration, whereas there was a significantly elevated BDNF protein expression at 24 h. Student’s t test, *p < 0.05, ***p < 0.001, compared to control. Data represent means ± SEM.

1071

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ACS Chemical Neuroscience 0.051) (Figure 4a). The expression of total ERK trended to decrease by the treatment (t = 2.031, p = 0.082) (Figure 4b), and the ratio of p-ERK/ERK was significantly increased (t = −3.171, p = 0.016) (Figure 4c). At 24 h after administration, there was no significant change in the levels of p-ERK (t = −0.382, p = 0.714) (Figure 4a), total ERK (t = 1.538, p = 0.175) (Figure 4b), or the ratio of p-ERK/ERK (t = −0.891, p = 0.402) (Figure 4c). In contrast, PKA, a signaling closely related to ERK, was not activated either at 30 min (t = 1.145, p = 0.290) or 24 h (t = 1.390, p = 0.207) after the GYP administration (Figure 4d). ERK is a regulator of eEF2 activation,28 which controls protein synthesis.19 At 30 min after GYP administration, the level of phosphorylated eEF2 (t = 2.851, p = 0.025) (Figure 4e) as well as the ratio of p-eEF2/eEF2 (t = 3.361, p = 0.012) (Figure 4g) were significantly decreased, with the unaltered expression of the total expression of eEF2 (t = 0.951, p = 0.373) (Figure 4f). At 24 h after the treatment, there was a trend for a reduced eEF2 phosphorylation (t = 2.295, p = 0.055) (Figure 4e), without a change in the level of total eEF2 (t = 0.986, p = 0.357) (Figure 4f) or the ratio of p-eEF2/eEF2 (t = 0.494, p = 0.645) (Figure 4g). As GYP rapidly reduced eEF2 phosphorylation, which has been demonstrated to promote protein synthesis,19 we further examined whether level of synaptic proteins was changed after treatment of GYP. At 30 min after GYP administration, the expression of GluR1 and synapsin 1 was significantly enhanced in the hippocampus (t = −2.568, p = 0.025; t = −3.815, p = 0.002) (Figure 5a and b). The expression level of both proteins was not different from that of the control mice at 24 h after the treatment (t = 0.703, p = 0.498; t = 0.514, p = 0.617) (Figure 5a and b). Conversely, BDNF was not changed at 30 min after GYP administration (t = −0.412, p = 0.693) (Figure 5c), whereas there was a significantly elevated BDNF protein expression at 24 h (t = −3.089, p = 0.018) (Figure 5c). To determine whether the increased synaptic protein translation induced by eEF2 deactivation is crucial for the antidepressant effects of GYP, mice were pretreated with 100 mg/kg anisomycin, followed by the administration of GYP. There was a significant effect of anisomycin and GYP interaction (F(1, 24) = 6.068, p = 0.021). The treatment of anisomycin alone did not have effects on the performance in the TST (p = 1.000), but it significantly reversed the decrease in immobility time induced by GYP (p = 0.027), suggesting that enhanced protein synthesis might play an important role in the acute antidepressant effect of GYP (Figure 6). At 30 min after GYP administration, neither phosphorylated nor total CREB levels were changed (p-CREB: t = −1.004, p = 0.349; CREB: t = −0.983, p = 0.359) (Figure 7a and b), and thus the ratio of p-CREB/CREB was not significantly changed (t = −0.085, p = 0.936) (Figure 7c). However, at 24 h after the administration, levels of phosphorylated form and total CREB were significantly elevated (p-CREB: t = −2.781, p = 0.039; CREB: t = −4.878, p = 0.002) (Figure 7a and b), with a trend for the decreased ratio of p-CREB/CREB (t = 2.462, p = 0.070) (Figure 7c). The temporal activation pattern of CREB signaling was thus consistent with BDNF expression. Gardenia yellow pigment is enriched in GJ that plays a primary role in rapid antidepressant effects of Yueju. Here we show that a certain dose of GYP induced antidepressant response at 30 min after a single administration and this effect lasted at least for 3 days. Moreover, a single dose of GYP showed antidepressant response in learned helplessness paradigm. GYP significantly increased the translation of synaptic proteins such as GluR1 and synapsin 1 at 30 min after the treatment by deactivation of eEF2.

Figure 6. Acute antidepressant effect of GYP was blocked by the protein synthesis inhibitor anisomycin. There was a significant effect of anisomysin and GYP interaction. The treatment of anisomycin alone did not have effects on the test, but it significantly reversed the decrease in immobility time induced by GYP. Two-way ANOVA, *p < 0.05, compared to control with Bonferroni post hoc test. Data represent means ± SEM.

In contrast, CREB and BDNF were significant elevated at 24 h but not 30 min after the GYP treatment (Figure 8). Pretreatment with the protein synthesis inhibitor blocked GYP’s instant antidepressant response, suggesting the dependence of instant antidepressant response on rapid synthesis of proteins. It is somewhat surprising that the medium but not high dose of GYP was effective to modify the immobility time in the TST and FST tested at 1 day after treatment. This is in agreement with the observations in ketamine,29 and ethanol extract of Yueju12 or GJ.17 For example, studies showed that high dose of ketamine did not elicit antidepressant effect, but a lower dose did. In terms of disinhibition theory of ketamine,30 which hypothesizes that a certain dose of ketamine only inhibits the interneurons, and thus disinhibits the pyramidal neurons and increase extracellular glutamate that promotes the rapid antidepressant responses, whereas a high dose may directly inhibit the pyramidal neurons and thus fails to elicit an antidepressant effect. Similar mechanism may also account for the action of Yueju, GJ and GYP, although further studies are warranted. Previous work has demonstrated that the instant antidepressant effect of ketamine required rapid protein synthesis.19 eEF2 mediates the translocation of the ribosome during the elongation phase of mRNA translation, where most of the energy and amino acids required for protein synthesis is used.31,32 Both ketamine and Yueju instantly deactivated eEF2 in the hippocampus,12 leading to the desuppression of protein synthesis. Similarly, a single dose of GYP also deactivated eEF2 and the protein synthesis is required for GYP’s antidepressant action. Interestingly, GYP rapidly elevated the expression of GluR1 and synapsin 1 at 30 min after the administration. The deactivation of eEF2 likely resulted in the upregulated expression of GluR1 and synapsin 1, proteins that are crucial for synaptic plasticity for antidepressant responses.9 Recently, it is demonstrated that ketamine can instantly enhance surface expression of GluR1 in the hippocampus.19 It is thus feasible that the immediate activation of synaptic proteins is responsible for the instant antidepressant activity of GYP. GYP failed to induce a prolonged upregulation of GluR1 and synapsin 1 in the hippocampus. In contrast, ketamine and Yueju can both induce a lasting GluR1 expression in the PFC, which is associated with the long-lasting antidepressant responses.9 This may be explained by the involvement of different signaling pathways in the hippocampus; the GYPinduced eEF2 deactivation led to enhanced protein synthesis but 1072

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Figure 7. CREB was activated at 24 h post GYP. ( a) CREB phosphorylation was not changed at 30 min, but was significantly elevated at 24 h after GYP administration. (b) Total CREB was not changed at 30 min, but was significantly elevated at 24 h after GYP administration. (c) The ratio of p-CREB/ CREB was not significantly changed at 30 min or 24 h after administration. Student’s t test, *p < 0.05, ***p < 0.001, compared to control. Data represent means ± SEM.

or chemicals showing antidepressant-like effects.38−41 A recent study showed that neurotoxic amyloid-β down-regulates BDNF expression via inhibition of the CREB expression without change of the relative expression of pCREB.42 Additionally, an activation of a domain distinct from the phosphorylation domain in CREB can still activate gene expression independent of CREB phosphorylation.43,44 Therefore, the increased expression of CREB by GYP may play a role in upregulatation of BDNF expression and antidepressant response. Our findings suggest that the action of GYP also involves in some mechanisms underlying classic antidepressants. There are multiple mechanisms for classical antidepressants, including increase in monoamines, increase in CREB mRNA levels activation of CREB signaling, increase in BDNF or other neurotrophic factors, synaptogenesis and neurogenesis et al.45−47 GYP leads to increase in expression of CREB, BDNF, and some synaptic proteins. The major difference probably lies in the rapid synthesis of protein by GYP, which has been reported from the study of ketamine but not classical antidepressants. Currently, there is no toxicological study of GYP yet. A small number of toxicological studies of crocin may provide some relevant information. Hosseinzadeh et al.48 found that an acute treatment of high dose of crocin (3 g/kg, orally or intraperitoneal for 2 days) did not cause any damage to major organs, while a dose of 180 mg/kg of crocin in subactute study (21 days) caused increased platelets and weight loss. Another study indicated that a daily dose of 50 mg/kg crocin for 8 days did not affect hepatic function but a higher dosage of 100 mg/kg for a duration of 2 weeks induced hepatic damage and black pigmentation.49 Here we used acute GYP to induce fast-onset and persistent antidepressant effects. Based on previous findings, it is plausible that the chronic administration of the 200 mg/kg GYP that contains about 100 mg/kg of crocin-1 and crocin-2 in total may lead to toxic effects whereas the acute or intermittent administration may not. However, this issue needs further study. The present study demonstrated for the first time that GYP has a rapid antidepressant-like potential, and revealed a novel mechanism in which an instant antidepressant effect is dependent on protein synthesis, exclusive of the upregulation of BDNF expression. A delayed upregulation of CREB-BDNF may be responsible for the persistent antidepressant response of the chemicals. As the GYP is much less expensive than crocin, GYP may be translationally more realistic for development of an

only transitory upregulation of GluR1 and synapsin 1, whereas ketamine-induced mTOR signaling activation in the PFC led to long-term upregulation of protein expression.9 This possibility merits further investigation. The present study also observed a significant increase in expression of p-ERK at 30 min after the administration, paralleled the rapid decrease of p-eEF2. Study demonstrated that ERK was activated rapidly in antidepressant response of ketamine and inhibition of ERK blocked this rapid antidepressant effect.27 Activity of eukaryotic elongation factor 2 (eEF2) is controlled by multiple signaling pathways including ERK.28 An inhibition of ERK leads to increase in phosphorylated eEF2 levels, thus suppressing the protein synthesis.19,27 It is feasible that GYP rapidly activated ERK, which in turn suppressed the phosphorylation of eEF2 and enhanced the expression of synaptic protein synthesis, thus contributing to the instant antidepressant response. Surprisingly, BDNF expression was not upregulated immediately after GYP administration, and thus may not be involved in the instant antidepressant response as by ketamine or Yueju administration.12,19 Instead, the present study demonstrated a delayed upregulation of BDNF and CREB. Both CREB and BDNF play a major role in neuroplasticity and are implicated in the pathology of depression as well as in the response to antidepressants.24,33−36 Administration of conventional antidepressants SSRIs increased the expression of total and phorsphorylated CREB24 and its downstream target gene BDNF in the hippocampus.37 Although they were unlikely responsible for the instantly increased expression of protein and the antidepressant response of GYP, the significant increase in the expression of CREB and BDNF at 24 h after GYP administration may have a part in the antidepressant activity in the late time period. This delayed upregulation of CREB and BDNF expression may similarly regulate neuroplasticity which underlie the persistent antidepressant effects of GYP at the transcriptional level regulated by CREB. In the present study, both phosphorylated and total CREB were significantly increased, without signanificant increase in the ratio of p-CREB to total CREB. Increasing evidence indicates that increase of CREB expression itself may mediate gene expression and be involved in antidepressant response. For example, chronic stress reduces the expression of CREB, which is reversed after repeated administration of some antidepressants 1073

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Drug Administration. GYP was dissolved in 0.9% sterile saline and intragastriclly administrated at a dose of 0.1 mL/10 g (body weight). A pilot study showed no effect with a single dose of 50 mg/kg or lower when screening for rapid antidepressant potential. Behavior was tested at designated time points after a single administration of GYP or vehicle. Anisomycin (Sigma, St. Louis, MO), dissolved in HCl/saline (final pH7.4), was injected i.p. 30 min before GYP administration at a dosage of 100 mg/kg according to a previous study.19 Learned Helplessness Test. The procedures for learned helplessness were followed as reported previously,17,22,46 with minor modifications. Learned helplessness experiments were performed in sound-proofed two-way shuttle boxes (40 × 10 × 13 cm3), with walls made of clear Plexiglass. The chamber was divided into two identical compartments. For the induction of helplessness, mice received 120 inescapable shocks (18−44 s, average 30 s; 0.45 mA for 15 s) once daily for 2 consecutive training days. Two hours after termination of the helplessness induction, mice were treated with GYP or vehicle. Mice were tested for helplessness 24 h after the drug treatment. For testing of learned helplessness response, animals were subjected to 30 avoidance trials (18−44 s, average 30 s; 0.45 mA for 3 s). Animals were also tested with tail suspension test and forced swim test, with at least 2 h between test times. Tail Suspension Test. In acoustic and visual isolated chambers, a single mouse was suspended in 50 cm above the floor, with a tape placed at about 1 cm of the tail. Activities of the animals were videotaped. The computer calculated the total duration of immobility during the last 4 min in a 6 min testing time. Immobility was defined when the animals hung passively without any struggling movements. Forced Swimming Test. The FST was carried out in mice, individually forced to swim in an open cylindrical container (diameter 10 cm, height 25 cm), containing 15 cm of water at 25 ± 1 °C. The sessions of the animals were videotaped. Data were calculated for the total duration of immobility in the final 4 min of the 6 min test. Each mouse was considered to be immobile when it failed to struggle and remained floating motionless in the water, making only those movements necessary to keep the nose above water. A decrease in the duration of immobility during the FST or TST was taken as a measure of antidepressant activity. Open Field Test. Open field test was used to assess the locomotory as well as the exploratory behavior in an open area. In the test, spontaneous locomotor activity was measured in a square arena (40 × 40 × 15 cm3) for monitoring horizontal activity, namely, total distance traveled. Mice were tested in a well-illuminated (∼300 lx) transparent acrylic cage for 5 min. Activity of mice in the two compartments, near the bulkhead and central regions, was tracked. Distance (cm) and time spent in the central zone were analyzed. The test device was thoroughly cleaned before each animal using 75% ethanol. Western Blotting. The whole hippocampus (ventral and dorsal) is rapidly dissected, frozen, and lysed in buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was determined colorimetrically via BCA assay (Pierce, Rockford, IL). Protein lysates were separated by SDS-PAGE electrophoresis and were transferred onto polyvinyllidene difluoride (PVDF) membranes. Primary antibodies for BDNF (Santa Cruz Biotechnology, 1:200), pERK (Cell Signaling Technology, 1:500), ERK (Cell Signaling Technology, 1:1000), p-CREB (Cell Signaling Technology, 1:500), CREB (Cell Signaling Technology, 1:1000), p-eEF2 (Cell Signaling Technology, 1:500), eEF2 (Cell Signaling Technology,1:500), and βtubulin (Cell Signaling Technology, 1:5,000) were used, and anti-rabbit secondary antibodies were used at 1:2000. The blots were visualized using the Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.). The amount of each protein was normalized to βtubulin bands. All experiments were performed three times. Statistics. Two-sample comparisons were carried out using the twotailed Student’s t test; multiple comparisons were made using one-way ANOVA or two-way ANOVA, followed by the Bonferroni multiple comparison tests. All data are presented as the mean ± SEM, and statistical significance was accepted at the 5% level, unless otherwise indicated.

Figure 8. Working model of GYP on proteins involved in hippocampal neuroplasticity expressions underlying the fast-acting and persistent antidepressant effects. At 30 min, GYP increases ERK phosphorylation, reduces eEF2 phosphorylation, and thus increases synaptic protein such as GluR1 and synapsin 1, which may leading the rapid antidepressant response in mice. At 24 h, GYP activates CREB and BDNF, which may regulate neuroplasticity underlying the persistent antidepressant effects of GYP at the transcriptional level regulated by CREB. The diagram was drawn by Ruyan Wu according to the present experiment.

antidepressant. Future studies should focus on the molecular substrate linking the instant to delayed antidepressant response, and identify the major compounds responsible for Gardenia yellow pigment’s antidepressant effects.

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METHODS

Animals. Male Kunming mice (20−25 g) were purchased from China Academy of Military Medical Sciences (Beijing). Mice, aged 6−8 weeks old, were habituated to animal facilities for 1 week before experiment. The animals were maintained in standard laboratory conditions (temperature 22 ± 2 °C and room humidity, 50 ± 10%) with a 12/12 h light/dark cycle. The animals were fed with standard diet and filtered water. The experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Nanjing University of Chinese Medicine. Preparation of Gardenia Yellow Pigment. Gardenia yellow pigment was prepared according to the previous study.45 The air-dried fruit of Gardenia jasminoides (1 kg) was refluxed with 50% aqueous ethanol for 2 h at 100−110◦C three times. After solvent removal, the combined residues were separated on a macroporous resin column (600 g, 20−40 mesh) with gradient (EtOH/H2O, 0:100 → EtOH;H2O, 20:80 → EtOH/H2O, 50:50) to yield 3 fractions (Fr.1−Fr.3). Fr.3 (104 g) was further subjected to a Sephadex LH-20 column eluted with EtOH/H2O (50:50) to afford total Gardenia yellow (63g). Identification of Chemical Markers in GYP. The major constituents were analyzed to control quality of GYP. The chemical makers were identified by Ultra Performance Liquid Chromatography (UPLC) with a PDA detector (Waters, Milford, MA). A Thermo Syncronic C18 column (100 mm × 2.1 mm, 1.7 μm) was employed, and the column temperature was maintained at 35 °C. The mobile phase was composed of A (0.1% formic acid) and B (acetonitrile) using a gradient elution of 10−30% B at 0−6 min, 30−90% B at 6−9 min with a flow rate set at 0.40 mL min−1. The maximum absorption wavelength was set at 440 nm. The autosampler was conditioned at 4 °C, and the injection volume was 2 μL. GYP (2.0 mg) dissolved in 10 mL of acetonitrile solution was centrifuged at 10 000 rpm for 5 min, and the supernatant was filtered twice through 0.22 mm filter before UPLC analysis. The yields of the major constituents, crocin-1 and crocin-2, were determined. 1074

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.6b00011. Chemical structures of crocin-1 and crocin-2; the effects of GYP on open field test (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: Nanjing University of Chinese Medicine, 138 Xianlin Ave, Nanjing 210023, People’s Republic of China. Tel: +86 25 8581 1160. Fax: +86 25 8581 1160. E-mail: chengang@ njucm.edu.cn. Author Contributions

Conceived and designed the experiments: R.W and G.C. Performed the experiments: R.W, W.T., H.Z., W.X., and Z.Z.. Analyzed the data: R.W., W.T., B.C., and G.C. Contributed to the writing of the manuscript: R.W., H.W., R.D., and G.C. Funding

The study was supported by the Natural Science Foundation of Jiangsu Province (BK20151568, BK20140961, and BK20140962), the Natural Science Foundation of Nanjing University of Chinese Medicine (13XZR06), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Israel Science Foundation (ISF 738/ 11), the National Institute for Psychobiology in Israel (NIPI-72011-12), and the Open University of Israel foundation. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS MDD, major depression disorder; SSRIs, selective serotonin reuptake inhibitors; NMDA, N-methyl-D-aspartate; GYP, gardenia yellow pigment; BDNF, brain-derived neurotrophic factor; eEF2, eukaryotic elongation factor2; PFC, prefrontal cortex; CREB, cAMP-response element binding protein; TST, tail suspension test; FST, forced swimming test

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