Pharmacology,BiochemistryandBehavior

The involvement of PI3K/Akt/mTOR/GSK3β signaling pathways in the antidepressant-like effect of AZD6765
Vivian B. Neisa,⁎, Morgana Morettia, Priscila B. Rosaa, Yasmim de Oliveira Dalsentera, Isabel Werlea, Nicolle Platta, Fernanda Neutzling Kaufmanna,b, AXel Fogaça Rosadoa,
Matheus Henrique Besena, Ana Lúcia S. Rodriguesa
a Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, 88040-900 Florianópolis, SC, Brazil
b Department of Psychiatry and Neuroscience, Faculty of Medicine and CERVO Brain Research Center, Université Laval, Quebec City, Canada

A R T I C L E I N F O

Keywords:
AZD6765
Antidepressant Ketamine Stress
Signaling pathways

A B S T R A C T

AZD6765 (lanicemine) is a non-competitive NMDA receptor antagonist that induces a fast-acting antidepressant effect without presenting psychotomimetic effects. However, the mechanisms underlying its effects remain to be established. In this context, we demonstrated that a single administration of AZD6765 (1 mg/kg, i.p.) was able to induce an antidepressant-like effect in mice submitted to tail suspension test (TST), an effect reversed by LY294002 (a reversible PI3K inhibitor, 10 nmol/site, i.c.v.), wortmannin (an irreversible PI3K inhibitor, 0.1 μg/ site, i.c.v.) and rapamycin (a selective mTOR inhibitor, 0.2 nmol/site, i.c.v.). In addition, the administration of sub-effective doses of AZD6765 (0.1 mg/kg, i.p.) in combination with lithium chloride (non-selective GSK-3β inhibitor, 10 mg/kg, p.o.) or AR-A014418 (selective GSK-3β inhibitor, (0.01 μg/site, i.c.v.) caused a synergistic antidepressant-like effect. These results suggest the involvement of PI3K/Akt/mTOR/GSK3β signaling in the AZD6765 antidepressant-like effect. In addition, western blotting analysis showed an increased immunocontent of synapsin in the prefrontal cortex and a tendency to an increased immunocontent of this protein in the hip- pocampus 30 min after AZD6765 administration, but no significant effect of AZD6765 was observed in P70S6K (Thr389) phosphorylation and GluA1 immunocontent. A single dose of AZD6765 (3 mg/kg, i.p.), similarly to
ketamine (1 mg/kg, i.p.), decreased the latency to feed in the novelty suppressed feeding (NSF) test, a behavioral paradigm that evaluates depression/anxiety-related behavior. This effect was reversed by rapamycin adminis- tration, suggesting the activation of mTOR signaling in the effect of AZD in the NSF test. In addition, a single administration of AZD6765 (1 mg/kg, i.p.) or ketamine (1 mg/kg, i.p.) reversed the depressive-like behavior induced by chronic unpredictable stress (CUS). Altogether, the results provide evidence for the fast-acting an- tidepressant profile of AZD6765, by a mechanism likely dependent on PI3K/Akt/mTOR/GSK3β.

1. Introduction
NMDA receptors are a class of ionotropic glutamatergic receptors related to synaptic plasticity and cell survival (Glasgow et al., 2015). These receptors have been extensively studied in encephalic structures such as cerebral cortex and hippocampus and are highly implicated in psychiatric disorders such as major depressive disorder (MDD) (Hansen et al., 2014; Li et al., 2009). Glutamate excitotoXicity due to over-ac- tivation of these receptors has been implicated in the pathophysiology of these disorders (Fan and Raymond, 2007; Wang and Michaelis, 2010).
NMDA receptor antagonists have demonstrated beneficial effects for the treatment of MDD (Dang et al., 2014; Zarate et al., 2010).

Preclinical studies have reported that these antagonists exhibit anti- depressant-like effect in several animal models of depression (Belzung, 2014; Li et al., 2010). An NMDA receptor antagonist that has emerged as a promising therapeutic agent is ketamine, a dissociative anesthetic that has demonstrated fast and long-lasting antidepressant effects when acutely administered at subanesthetic doses in patients suffering from severe MDD symptoms (Berman et al., 2000; Zarate et al., 2006; Zarate et al., 2013). The antidepressant response elicited by ketamine is pro- posed to be mediated by glutamate increase with the consequent sti- mulation of BDNF receptors, which leads to the activation of phos- phatidylinositol 3-kinase (PI3K)/Akt, inhibition of glycogen synthase kinase-3β (GSK3β) and stimulation of mammalian target of rapamycin (mTOR) (Monteggia et al., 2013; Wohleb et al., 2017). The activation of

⁎ Corresponding author at: Department of Biochemistry, Center of Biological Sciences, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil.
E-mail address: [email protected] (V.B. Neis).

https://doi.org/10.1016/j.pbb.2020.173020

Received 27 June 2020; Received in revised form 10 August 2020; Accepted 26 August 2020

mTOR results in the synthesis of synaptic proteins such as synapsin, considered essential for the rapid antidepressant effect of ketamine (Li et al., 2010). However, the psychotomimetic effects of ketamine limit its clinical use (Zarate, 2020) raising the interest in other NMDA re- ceptor antagonists with potential antidepressant effects.

AZD6765 (lanicemine), another non-selective, non-competitive NMDA receptor antagonist with lower trapping than ketamine, has demonstrated antidepressant properties in clinical studies without in- ducing psychotomimetic effects (Sanacora et al., 2014; Zarate et al., 2013). A single infusion of AZD6765 induced rapid but not sustained antidepressant effects compared to placebo (Sanacora et al., 2014). Of note, a single AZD6765 infusion was more effective than placebo when administered to 22 subjects without causing psychotic or dissociative effects (Zarate et al., 2013). However, a study by Sanacora et al. (2017) demonstrated no significant difference between lanicemine and placebo treatment on any outcome measures related to MDD. In preclinical studies, AZD6765 elicited antidepressant-like effect in rodents sub- jected to forced swim test and learned helplessness paradigm (Sanacora et al., 2014). Furthermore, the co-administration of hyperforin, a nat- ural and biologically active compound extracted from Hypericum per- foratum (Cervo et al., 2002) that attenuated symptoms of mild to moderate depression in several clinical trials, displayed long-lasting antidepressant-like effect when administered with AZD6765 in mice (Pochwat et al., 2018). Despite some promising antidepressant effects elicited by AZD6765, the mechanisms and signaling pathways related to its effects have not been investigated. Therefore, the purpose of the present study was to evaluate if the antidepressant effects elicited by AZD6765 are dependent on PI3K/Akt/mTOR and GSK-3β, targets im- plicated in fast antidepressant responses (Li et al., 2010; Pazini et al., 2016).

2. Material and methods

2.1. Animals
The present study was performed using adult female Swiss mice (30–40 g). Animals were maintained at 20–22 °C with free access to water and food, under a 12/12 h light-dark cycle (lights on at 07:00 a.m.). Mice were allowed to acclimatize to the holding room for 24 h before the behavioral procedures. All manipulations were carried out between 9:00 a.m. and 5:00 p.m. (N = 6–8 animals per group). The procedures in this study were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and the experiments were performed after protocol approval by the ethics committee of the institution. All efforts were made to minimize animal suffering and to reduce the number of ani- mals used in the experiments.

2.2. Drugs

The following drugs were used: AZD6765, LY2940029 (reversible PI3K inhibitor), AR-A014418 (selective GSK-3β inhibitor), rapamycin (selective mTOR inhibitor), wortmannin (irreversible PI3K inhibitor), fluoXetine (all from Sigma Chemical Co. St Louis, USA) and lithium chloride (non-selective GSK-3β inhibitor) (MERCK, Darmstadt, Germany). Lithium chloride was dissolved in distilled water and was given orally (p.o.) by gavage in a volume of 10 ml/kg body weight. AZD6765 and ketamine dissolved in NaCl (0.9%) were administered intraperitoneally. LY294002, wortmannin, AR-A014418, and rapa- mycin dissolved in saline at a final concentration of 1% DMSO were administered by i.c.v. route, in a volume of 3 μl per mouse. Vehicle treated groups were also assessed simultaneously. The i.c.v. injections were performed by employing a “free hand” method according to the procedure described previously (Moretti et al., 2014). Mice were lightly anesthetized with isoflurane (2.5%; Abbot Laboratórios do Brasil Ltda., Rio de Janeiro, RJ, Brazil). After that anesthesia mice were gently restrained by hand for i.c.v. injections into the left lateral ventricle, according to the following coordinates from bregma: AP: 0.1 mm, ML: 1 mm, DV: 3.0 mm (Paxinos and Franklin, 2004). The injection was given over 30s, and the needle remained in place for another 30s in order to avoid the refluX of the substances injected. The asepsis of the injection site was carried out using gauze embedded in 70% ethanol. To ascertain that the drugs were administered exactly into the cerebral ventricle, the brains were dissected and examined macroscopically after the test.

2.3. Experimental protocols
EXperiments were performed in four cohorts of animals (protocols 1, 2, 3 and 4 described in Figs. 2, 3, 4 and 5). In the first and second protocols, animals were treated with different pharmacological tools in order to investigate possible signaling pathways involved in the anti- depressant-like effect elicited by AZD6765.
2.3.1. Protocols 1 and 2

To investigate the involvement of PI3K in the antidepressant-like effect of AZD6765, mice were treated with AZD6765 (1 mg/kg, i.p., active dose in the TST) or vehicle 15 min before wortmannin (0.1 μg/ site, intracerebroventricular (i.c.v.) or vehicle. After 15 min of the last treatment mice were submitted to TST and open-field test (OFT). In another set of experiments, to confirm the involvement of PI3K in the behavioral effect of AZD6765 in the TST, mice were treated with AZD6765 (1 mg/kg, i.p.) or vehicle 15 min before LY294002 (10 nmol/ site, i.c.v.) or vehicle. The TST was carried out 15 min after LY294002 administration ) (Cunha et al., 2016). To investigate the possibility that the antidepressant-like effect of AZD6765 depends on mTOR partici- pation, mice were treated with AZD6765 (1 mg/kg, i.p.) an after 15 min an i.c.v. injection of rapamycin was administered (0.2 nmol/site). The TST was carried out 15 min after rapamycin administration (Pazini et al., 2016). This first experimental protocol is shown in Fig. 2A.
In the experimental protocol 2 (Fig. 3A), to test the hypothesis that the antidepressant-like effect of AZD-6765 is dependent on the inhibi- tion of GSK-3β, mice were treated with a sub-effective dose of AZD6765 (0.1 mg/kg, i.p.) or vehicle and immediately after, a sub-effective dose of lithium chloride (a non-selective GSK-3β inhibitor, 10 mg/kg, p.o.) or vehicle was administered.

The TST was carried out 60 min later. In another set of experiments mice were treated with a sub-effective dose of AZD6765 (0.1 mg/kg, i.p.) or distilled water and after 15 min, they were injected with a sub-effective dose of the selective GSK-3β in- hibitor, AR-A014418 (0.01 μg/site, i.c.v.) or vehicle. After 15 min of treatments, mice were submitted to the TST (Moretti et al., 2014; Rosa et al., 2019).

The doses of drugs used in the present study were chosen based on previous studies, namely: wortmannin (Gonçalves et al., 2017; Pazini et al., 2016), LY294002 (Cunha et al., 2016), AR-A014418 (Cunha
et al., 2016; Moretti et al., 2014), lithium chloride (Cunha et al., 2016; Moretti et al., 2014), rapamycin (Bettio et al., 2012; Cunha et al., 2016; Moretti et al., 2014; Pazini et al., 2016).
Mice administered with AZD6765 (1 mg/kg, i.p., active dose in the TST) and subsequently submitted to the behavioral tests (after 30 min) were euthanized and the prefrontal cortex and hippocampus were dis- sected to quantify P70S6K (Thr389) phosphorylation, as well as GluA1 and synapsin immunocontent.

2.3.2. Protocol 3: Novelty suppressed feeding test (NSF)
The NSF was performed as previously described (Camargo et al., 2019; Fraga et al., 2020). This test measures the latency of mice in approaching and eating food in a novel environment following an ex- tended period (up to 24 h) of food deprivation. The latency to begin eating reflects how the animal copes with a behavioral conflict. Because the ability to solve conflicts is inversely related to anxiety and de- pression, and since NSF test assesses anhedonia in a situation where there is a conflict between food reward and novel open space, this test has been used for depression-related assessments (Dulawa and Hen, 2005; Powell et al., 2012). Mice were weighed, and food was removed from their cages, although water continued to be provided with free access. ApproXimately 24 h after the removal of the food, mice were placed in an illuminated and soundproofed wooden boX (40 × 60 cm and 50 cm height).

A small piece of mouse chow was placed in the center of the boX and each mouse was placed in the corner of the testing arena, and the time until the first feeding episode was recorded within 10 min. Immediately after the mouse began to eat the chow, the tested animal was placed alone in a cage with a weighed piece of chow for 5 min and, at the end of this period, the amount of food consumed was determined by weighing the piece of chow. After all mice from a single cage had been tested, mice returned to the cage with free access to food and water (Blasco-Serra et al., 2017). In this protocol, AZD6765 was administered at doses of 1 and 3 mg/kg. After 30 min of treatment, mice were submitted to NSF test. In another experiment, animals were treated with AZD6765 (3 mg/kg, i.p.) or ketamine (1 mg/kg, i.p.) and after 15 min they received rapamycin (0.2 nmol/site). After 15 min of rapamycin administration, mice were submitted to NSF test in order to verify the involvement of mTOR in the AZD6765 effects. Ketamine was administered as a positive control at a dose previously shown to be effective in this test (Fraga et al., 2020).

After NSF test and evaluation of amount of food consumed, the following OFT parameters were recorded and analyzed: grooming epi- sodes, grooming latency, number of rostral grooming, total time of rearing, number of rearing, crossings and total time in the apparatus center.

2.3.3. Protocol 4: Chronic unpredictable stress (CUS)
The third protocol performed was CUS, that consisted of a variety of stressors randomly applied at different times of day for 21 days to prevent habituation (Kaster et al., 2015; Moretti et al., 2019) (Table 1). Mice were divided into control (non-stressed) and stressed groups; control mice were kept in their home cages according to conditions previously reported. Control and stressed mice were weighed once a week during the twenty-one days of procedure.

Two hours after the last stress exposure (day 21), control and stressed animals received a single i.p. administration of AZD6765 (1 mg/kg), ketamine (1 mg/kg) or fluoXetine (10 mg/kg) and were subjected to behavioral evaluation 24 h after the treatments. The doses of ketamine and fluoXetine were chosen based on a previous study (Neis et al., 2016).

Schedule of stressor agents used in the 21 days of chronic stressful stimuli.
Day Stressor

1 Damp bedding (24 h)
2 Paired housing (1 h)
3 Cold bath (15 °C, 20 min)
4 Restraint stress (2 h)
5 Inescapable shock (0.7 mA, 3–3 s, 5 min)
6 Apparatus exposure, no footshock (1 h)
7 Damp bedding (24 h)
8 Cage tilt (45°, 24 h)
9 Stroboscopic light (3 h)
10 Tail pinch (10 min)
11 Light/dark cycle inversion, removal of bedding (12 h)
12 Paired housing (1 h)
13 Cold bath (15 °C, 20 min)
14 Restraint stress (3 h)
15 Inescapable shock (0.7 mA, 3–3 s, 5 min)
16 Apparatus exposure, no footshock (1 h)
17 Stroboscopic light (3 h)
18 Tail pinch (10 min)
19 Restraint stress (4 h)
20 Light/dark cycle inversion, removal of bedding (12 h)
21 Cage tilt (45°, 24 h)

2.4. Behavioral tests
2.4.1. Tail suspension test (TST)
The total duration of immobility in the TST was measured according to the method described by Steru et al. (1985). Briefly, mice visually isolated were suspended 50 cm above the floor by adhesive tape placed approXimately 1 cm from the tip of the tail. Immobility time was re- corded during a 6 min period by an experienced observer blinded to the experimental conditions. Mice were considered immobile only when they hung passively and completely motionless.

2.4.2. Open-field test (OFT)
In order to rule out the interference of alterations in overall loco- motor activity in the TST, mice were evaluated in the OFT as previously described (Rodrigues et al., 2002). The apparatus consisted of a wooden boX measuring 40 × 60 × 50 cm with floor of the arena divided into 12 equal squares. The number of squares crossed with all paws (crossing) was counted in a 6-min session. The apparatus was cleaned with a so- lution of 10% ethanol between tests to hide animal clues.

2.5. Western blotting analyses
Western blotting analyses were performed as previously described (Neis et al., 2016; Pazini et al., 2016). Mice were euthanized by rapid decapitation and prefrontal cortex and hippocampus were quickly dis- sected and snap-frozen with liquid nitrogen prior to storage at −80 °C until use. Briefly, samples were mechanically homogenized in 300 μl of 50 mM Tris pH 7.0, 1 mM EDTA, 100 mM NaF, 0.1 mM PMSF, 2 mM
Na3VO4, 1% Triton X-100, 10% glycerol, and Amresco Protease In- hibitor Cocktail catalog number M222 (Working concentration: 0.5 mM AEBSF, 0.3 μM aprotinin, 10 μM bestatin, 10 μM E-64, 10 μM leupeptin, 50 μM EDTA). Lysates were centrifuged (10.000g for 10 min, at 4 °C) to eliminate cellular debris. The supernatants were diluted 1/1 (v/v) in 100 mM Tris pH 6.8, 4 mM EDTA and 8% SDS, followed by boiling for 5 min. Thereafter, sample dilution (40% glycerol, 100 mM Tris, bro- mophenol blue, pH 6.8) in the ratio 25:100 (v/v) and β-mercap- toethanol (final concentration 8%) were added to each sample. Protein content was quantified using bovine serum albumin as a standard (Peterson, 1977). The same amount of protein (60 μg per lane) for each sample was electrophoresed in 10% SDS-PAGE minigels and transferred to nitrocellulose membranes using a semi-dry blotting apparatus (1.2 mA/cm2; 1.5 h).

To verify transfer efficiency process, membranes were stained with Ponceau Stain. The membranes were blocked with 5% bovine serum albumin (BSA) in TBS (10 mM Tris, 150 mM NaCl, pH 7.5). P70S6K phosphorylation (Thr389), GluA1, synapsin and β-actin
(loading control) immunocontent were detected after overnight in- cubation with specific antibodies diluted in TBS-T containing 2% BSA. The primary rabbit-antibodies were diluted 1:1000 for P70S6K (Thr389) (Cell Signaling Technology), GluA1 (Santa Cruz Biotechnology), sy- napsin (Cell Signaling Technology) and 1:2000 for mouse anti-β-actin (Santa Cruz Biotechnology). Membranes were incubated for 1 h at room temperature with horseradish peroXidase (HRP)-conjugated anti-rabbit or anti-mouse antibody (1:5000, Millipore) for protein detection.

The reactions were developed by chemiluminescence substrate (LumiGLO). All blocking and incubation steps were followed by washing 3 times (5 min) with TBS-T (10 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5). Optical density of the bands was quantified using Imagelab Software and the phosphorylation of P70S6K (Thr389) was determined based on the ratio between optical density of the phosphorylated band and the optical density of the total P70S6K. The immunocontent of synapsin-I and GluA1 was determined based on the ratio between the optical density of their bands and the optical density of the β-actin band.

Fig. 1. Effect of acute administration of AZD6765 in the immobility time in the TST (panel A) and in the locomotor activity in the OFT (panel B). Each column represents the mean ± S.E.M of 7 animals. *P < 0.05; **P < 0.01 as compared with the vehicle-treated group (one-way ANOVA followed by Duncan’s multiple range post hoc test).

2.6. Statistical analysis
All data are presented as mean ± SEM. Differences among ex- perimental groups were determined by two-way ANOVA followed by Duncan’s post-hoc test. A value of P < 0.05 was considered to be significant.

3. Results
3.1. Behavioral tests
3.1.1. Effect of AZD6765 administration in the TST and OFT
Fig. 1A shows that the administration of AZD6765 (1 mg/kg, i.p.) produced a significant reduction in the immobility time of animals in the TST [F(1,30) = 4.47, P < 0.01]. Regarding OFT, only the dose of
3 mg/kg of AZD6765 increased the locomotor activity of mice, as compared to the control group (Fig. 1B) [F(1,30) = 6.23, P < 0.01].

3.1.2. Involvement of PI3K and mTOR pathway in the antidepressant-like effect induced by AZD6765 in the TST
As illustrated in Fig. 2A, we next investigated the influence of wortmannin, LY294002 or rapamycin on the antidepressant-like effect of AZD6765 in the TST. Fig. 2B shows that wortmannin (0.1 μg/site, i.c.v.) abolished the decrease of immobility elicited by AZD6765 (1 mg/ kg) in the TST. The two-way ANOVA revealed significant differences for AZD6765 treatment [F(1,28) = 17.27, P < 0.01], wortmannin treat- ment [F(1,28) = 10.00, P < 0.01] and AZD6765 treatment wort- mannin interaction [F(1,28) = 6.02, P < 0.05] (Fig. 2B). None of the treatments caused alterations in the locomotor activity in the OFT (Fig. 2C). A two-way ANOVA showed no significant differences for AZD6765 treatment [F(1,28) =0.64, P = 0.43], wortmannin treatment [F(1,28) = 0.58, P = 0.45], and AZD6765 wortmannin interaction [F (1,28) = 0.07, P = 0.79] (Fig. 2C). To confirm the hypothesis that PI3K/Akt signaling is involved in the antidepressant-like effect of AZD6765 in the TST, the influence of the administration of the re- versible PI3K inhibitor LY294002 (10 nmol/site) on the anti-immobility effect of AZD6765 (1 mg/kg) in the TST was also investigated as shown in Fig. 2D. The two-way ANOVA revealed significant differences for AZD6765 treatment [F(1,28) = 15.06, P < 0.01], LY294002 treat- ment [F(1,28) = 14.39, P < 0.01] and AZD6765 X LY294002 treat-
ment interaction [F(1,28) = 9.25, P < 0.01] (Fig. 2D). Post-hoc analysis indicated that treatment with LY294002 also prevented the decrease in the immobility time induced by AZD6765 in the TST. Fig. 2E shows that none of the treatments caused alterations in the locomotor activity in the OFT. A two-way ANOVA showed no sig- nificant differences for AZD6765 treatment [F(1,28) = 0.18, P = 0.67],

LY294002 treatment [F(1,28) = 0.64, P = 0.43], and AZD6765 X
LY294002 treatment interaction [F(1,28) = 0.04, P = 0.85] (Fig. 2E). Fig. 2F shows that treatment of mice with rapamycin (selective mTOR inhibitor, 0.2 nmol/site, i.c.v.) prevented the decrease in im- mobility time induced by AZD6765 (1 mg/kg) in the TST. The two-way ANOVA revealed significant differences for AZD6765 treatment [F (1,28) = 11.18, P < 0.01], rapamycin treatment [F(1,28) = 15.43, P < 0.01] and AZD6765 treatment X rapamycin interaction [F (1,28) = 14.53, P < 0.01] (Fig. 2F). Post-hoc analysis showed that the administration of rapamycin abolished the behavioral response of AZD6765 in the TST.

The number of crossings in OFT was not altered by any treatment (Fig. 2G). A two-way ANOVA showed no significant differences for AZD6765 treatment [F (1,28) = 1.03; P = 0.32], ra- pamycin treatment [F(1,28) = 3.35; P = 0.08], and AZD6765 rapa-
mycin interaction [F(1,28) =0.83; P = 0.37] (Fig. 2G).

3.1.3. Involvement of GS3-β inhibition in the antidepressant-like effect induced by AZD6765 in the TST

In order to verify the involvement of GSK-3β in the antidepressant- like effect of AZD6765 in the TST, mice were treated with sub-effective doses of AZD6765 (0.1 mg/kg) and the nonselective GSK-3β inhibitor, lithium chloride (10 mg/kg) in the TST, as indicated in the experi- mental protocol illustrated in Fig. 3A and depicted in Fig. 3C. The two- way ANOVA revealed significant differences for AZD6765 treatment [F (1,24) = 10.74, P < 0.01], lithium chloride treatment [F (1,24) = 12.99, P < 0.01] and AZD6765 treatment X lithium chloride interaction [F(1,24) = 8.78, P < 0.01] (Fig. 3C). Post-hoc analysis indicated that treatment with a sub-effective dose of AZD6765 pro- duced a synergistic antidepressant-like effect with a sub-effective dose of lithium chloride in the TST. The administration of lithium chloride alone or in combination with AZD6765 did not modify the locomotor activity of mice in the OFT (Fig. 3D). A two-way ANOVA showed no significant differences for AZD6765 treatment [F(1,24) = 2.29, P = 0.14], lithium chloride treatment [F(1,24) = 0.54, P = 0.47], and AZD6765 X lithium chloride treatment interaction [F(1,24) = 0.74, P = 0.39] (Fig. 3D). Fig. 3E shows the effect of the co-administration of sub-effective doses of AZD6765 (0.1 mg/kg) and the selective GSK-3β inhibitor, AR-A014418 (0.01 mg/site, i. c.v.).

The two-way ANOVA revealed significant differences for AZD6765 treatment [F (1,28) = 16.19, P < 0.01], ARA014418 treatment [F(1,28) = 4.52, P < 0.05] and AZD6765 treatment X AR-A014418 treatment interac- tion [F(1,28) = 10.97, P < 0.01] (Fig. 3E). As depicted in Fig. 3E, post-hoc analysis indicated that the combined administration of sub- effective doses of AZD6765 and AR-A014418 produced an anti- depressant-like effect in the TST. The administration of AR-A014418 alone or in combination with AZD6765 did not affect locomotor activity.

Effect of the treatment of mice with wortmannin (0.1 μg/site, i.c.v.), LY294002 (10 nmol/site, i.c.v.) or rapamycin (0.2 nmol/site) on the AZD6765-induced (1 mg/kg, i.p.) antidepressant-like effect in the TST (panels B, D and F, respectively) and on locomotor activity in the OFT (panels C, E and G, respectively). Values are expressed as mean ± S.E.M of 8 mice. **P < 0.01 compared with the vehicle treated control group. ##P < 0.01 compared with AZD6765-treated group (two-way ANOVA followed by Duncan’s multiple range post hoc test). The experimental protocol is shown in panel A.

Effect of the treatment of mice with lithium chloride (10 mg/kg, p.o.) or AR-A014418 (0.01 mg/site, i.c.v.) in combination with a sub-effective dose of AZD6765 (0.1 mg/kg, i.p.) in the TST (panels C and E, respectively) and on the number of crossings in the OFT (panels D and F, respectively). Values are expressed as mean ± S.E.M of 8 mice. **P < 0.01 compared with the vehicle-treated control group (two-way ANOVA followed by Duncan’s multiple range post hoc test).

The experimental protocol is shown in panels A and B of mice in the OFT (Fig. 3F). A two-way ANOVA showed no significant differences for AZD6765 treatment [F(1,28) = 1.07, P = 0.31], AR- A014418 treatment [F(1,28) = 2.12, P = 0.16], and AZD6765 X AR-

A014418 treatment interaction [F(1,28) = 0.05, P = 0.83] (Fig. 3F).

3.1.4. Effect of AZD6765 on P70S6K (Thr389) phosphorylation and GluA1 and synapsin immunocontent

Taking into account that we showed that the antidepressant-like effect of AZD6765 is dependent on molecular targets related to rapid antidepressant response, we next aimed to reinforce the role of these targets in the AZD6765 effects by evaluating P70S6K (Thr389) phos- phorylation and the immunocontent of synapsin and GluA1.
These neurochemical parameters were determined in the prefrontal cortex and hippocampus 30 min after the administration of AZD6765 (1 mg/kg, i.p., dose that produces an antidepressant-like effect in the TST) by Western blotting. As revealed by Student’s t-test, there was no significant alteration in the phosphorylation of P70S6K (Thr389) in the prefrontal cortex ([t(12) = −1.6713, P = 0.12]), or hippocampus ([t(14) = −0.4166, P = 0.68]) of mice treated with AZD6765 (Fig. 4A and D). In addition, there was no significance alteration in GluA1 im- munocontent in the prefrontal cortex ([t(10) = −0.2268, P = 0.82]) or hippocampus ([t(10) = −1.7092, P = 0.12]) of AZD6765-treated mice (Fig. 4B and E). However, as illustrated in Fig. 4C and F, mice treated with AZD6765 presented a tendency to an increase in synapsin immunocontent in the hippocampus ([t(10) = −2.0752, P = 0.06]) and presented a significant higher immunocontent of synapsin in the prefrontal cortex as compared with their vehicle-treated counterparts ([t(10) = −2.8994, P < 0.05]).

3.1.5. Involvement of mTOR in AZD6765 and ketamine effects in the NSF test
In order to determine an effective dose of AZD6765 in the NSF test, two doses of this compound were tested (1 mg/kg and 3 mg/kg). As revealed by one-way ANOVA, AZD6765 significantly reduced the

Fig. 4. Effect of AZD6765 treatment in P70S6K (Thr389) phosphorylation (panels A and D), GluA1 (panels B and E) and synapsin immunocontent (panels C and F) in the hippocampus and prefrontal cortex of mice. The representative images are on top of the graphics. Results are presented as percentual of control (considered 100%) and are expressed as mean + S.E.M of 6–7 mice. *P < 0.05 compared with the vehicle-treated group.

latency to feed at a dose of 3 mg/kg [F(1,18) = 3.65, P < 0.05], without altering food consumption [F(1,18) = 0.0006, P = 0.99] (Fig. 5A and B, respectively).
Therefore, the dose of AZD6765 of 3 mg/kg was chosen for the experiment that investigated the influence of inhibiting mTOR signaling on the effect of AZD6765 in the NSF test as represented in Fig. 6A. In Fig. 6C, the two-way ANOVA revealed no significant differences for administration of AZD6765 (3 mg/kg) [F(1,26) = 3.18, P = 0.08]. Effect of AZD6765-induced (1 mg/kg and 3 mg/kg, i.p.) (panels A and B) reduction on the latency to feed and food consumption in mice submitted to the NSF test. Values are expressed as mean ± S.E.M of 6–8 mice. *P < 0.05 as compared with the vehicle-treated control (one-way ANOVA followed by Duncan’s multiple range post hoc test).

Effect of rapamycin (0.2 nmol/site, i.c.v.) in the AZD6765 (3 mg/kg, i.p.) (panels C and D) or ketamine-induced (1 mg/kg, i.p.) (panels E and F) reduction on the latency to feed and food consumption in mice submitted to the NSF test. Values are expressed as mean ± S.E.M of 7–8 mice. * P < 0.05; **P < 0.01 as compared with the vehicle-treated control. # P < 0.05 compared with AZD6765-treated group. (two-way ANOVA followed by Duncan’s multiple range post hoc test). The experimental protocol is shown in panel A.

Rapamycin administration [F(1,26) = 0.08, P = 0.78], but significant differences for AZD6765 and rapamycin interaction [F(1,26) = 6.23, P < 0.05] in the NSF test. Post-hoc analysis revealed that the ad- ministration of rapamycin abolished the behavioral response (de- creased the latency to feed) of AZD6765 in the NSF test. A similar protocol was carried out to investigate the influence of rapamycin on the effect of ketamine (1 mg/kg) in the NSF test (Fig. 6 B). In Fig. 6E, the two-way ANOVA revealed significant differences for administration of ketamine (1 mg/kg) [F(1,26) = 4.43, P < 0.05] and ketamine and rapamycin interaction [F(1,26) = 5.24, P < 0.05] but no significant differences for rapamycin administration [F(1,26) = 0.25, P = 0.62].

In order to rule out a possible interference of the treatments on food consumption, the amount of food consumed was determined for 5 min. As illustrated in Fig. 6D and F, the treatments with AZD6765 or keta- mine (with or without rapamycin) did not affect the consumption of food in the NSF test. A two-way ANOVA revealed no significant effects for AZD6765 treatment [F(1, 26) = 0.15, P = 0.70], rapamycin treatment [F(1, 26) = 0.03, P = 0.86] and AZD6765 treatment × rapamycin treatment interaction [F(1, 26) = 0.27, P = 0.61] in food consumption (Fig. 6D). In addition, a two-way ANOVA revealed no significant effects for ketamine treatment [F(1, 28) = 0.13, P = 0.72], rapamycin treatment [F(1, 28) = 0.13, P = 0.72] and ketamine treatment × rapamycin treatment interaction [F(1, 28) = 0.51, P = 0.48] in food consumption (Fig. 6F). These results indicate that the effects elicited by AZD6765 and ketamine were not influenced by the food consumption behavior of mice.

3.1.6. OFT parameters
The OFT parameters evaluated in mice previously subjected to NSF (Fig. 6A) are presented in Table 2 (AZD6765) and Table 3 (ketamine).

Table 2
Effect of rapamycin (0.2 nmol/site, i.c.v.) and AZD6765 (3 mg/kg, i.p.) in the behavioral parameters analyzed in the OFT. Values are expressed as mean ± S.E.M of 7–8 mice (two-way ANOVA followed by Duncan’s multiple range post hoc test).
Vehicle AZD6765 Rapamycin AZD6765 + Rapamycin
Grooming episodes 11.00 ± 1.77 9.43 ± 0.78 8.00 ± 0.82 7.87 ± 0.77
Grooming latency 79.75 ± 14.67 75.86 ± 2.60 78.71 ± 6.24 71.87 ± 11.48
Number of rostral groomings 4.12 ± 0.79 4.57 ± 0.37 4.71 ± 0.56 5.25 ± 0.53
Total time of rearing 36.37 ± 7.29 19.28 ± 2.18 27.28 ± 5.64 34.87 ± 5.10
Number of rearings 29.12 ± 5.90 19.28 ± 1.71 24.86 ± 4.16 28.50 ± 4.00
Crossings 89.12 ± 12.03 66.43 ± 7.41 85.57 ± 13.08 85.50 ± 9.31
Total time in center 10.87 ± 1.94 11.57 ± 2.51 13.00 ± 3.99 11.12 ± 2.07

The only significant alteration observed was in mice treated with ke- tamine, that was able to increase total time in center of the apparatus (P < 0.05), a parameter related to anxiolytic effect induced by this compound. The administration of rapamycin was effective to reverse the ketamine-induced increase in the time in the center of the open field apparatus (P < 0.05). The two-way ANOVA revealed significant dif- ferences for administration of ketamine (1 mg/ kg) [F(1,27) = 4.51, P < 0.05], rapamycin administration [F(4,51) < 0.05, P < 0.05], and AZD6765 X rapamycin interaction [F(1,27) = 6.25, P < 0.05] for the total time in the apparatus center (Fig. 6A).

3.1.7. Effects of AZD6765 in CUS protocol
As shown in Fig. 7A, we next investigated the ability of a single dose of AZD6765, ketamine or fluoXetine to counteract the depressive-like behavior elicited by CUS. Fig. 7B shows the effect of AZD6765, keta- mine or fluoXetine on the immobility time in the TST in control and stressed mice. The two-way ANOVA revealed no significant differences for CUS procedure [F(1,56) = 0.39, P = 0.53], but significant differ- ences for treatment [F(3,56) = 33.49, P < 0.01] and CUS procedure X treatment interaction [F(3,56) = 10.78, P < 0.01] (Fig. 7B). Post hoc analyses indicated that CUS-exposed mice presented a significant in- crease on the immobility time in the TST, as compared to control mice. Acute administration of AZD6765 and ketamine was able to reverse the stress-induced increase in the immobility time (P < 0.01). However, in non-stressed mice, only ketamine caused a reduction in the immobility time in the TST (P < 0.01). The OFT was carried out to rule out the possibility of an interference of CUS and/or treatments in the locomotor activity of mice. The two-way ANOVA revealed differences for CUS

4. Discussion
The present study suggests that the antidepressant-like effect in- duced by AZD6765 in the TST is possibly related to the stimulation of PI3K/Akt/mTOR signaling and inhibition of GSK3β. The anti- depressant-like effect of AZD6765 observed 30 min after its adminis- tration was accompanied by an increase in the immunocontent of sy- napsin in prefrontal cortex of mice. We also suggest that the rapid behavioral effect of AZD6765 in the NSF test is dependent on the mTOR-mediated signaling pathway, since the effect of this NMDA re- ceptor antagonist was reversed by rapamycin treatment. In addition, a single administration of AZD6765 was able to reverse the increase in the immobility time in mice submitted to CUS, a result similar to the one caused by ketamine, reinforcing the fast antidepressant responses elicited by both compounds.
Ketamine, an NMDA receptor antagonist, has been reported to elicit a fast antidepressant effect when administered at sub-anesthetic doses to rodents and humans (Berman et al., 2000; Li et al., 2010; Pazini et al., 2016; Zarate et al., 2006; Zhou et al., 2014). The mechanisms related to its fast response seem to involve the activation of PI3K/Akt (Pazini et al., 2016), culminating in Ser9 phosphorylation of the en- zyme GSK3β, an event that causes the inhibition of its activity (Mai et al., 2002). A downstream target of PI3K/Akt/GSK3β signaling is the mechanistic target of rapamycin protein (mTOR), leading to phos- phorylation p70S6K (Thr389) and the translation of synaptic proteins (Li et al., 2010, 2011; Zhang et al., 2018). Despite being an effective treatment for treatment-resistant depression, there are several limita- tions for the chronic use of ketamine, including potential of drug abuse, psychotomimetic or dissociative side effects, peripheral toXicity and procedure [F(1,56) = 48.18, P < 0.01] but no significant differences neurotoXicity (Behrens et al., 2007; Bokor and Anderson, 2014; for treatment [F(3,56) = 0.54, P = 0.66] and CUS procedure X treat- ment interaction [F(3,56) = 0.35, P = 0.78], indicating that all ani- mals submitted to CUS protocol (treated or not with AZD6765, keta- Muetzelfeldt et al., 2008).

In this context, the present study investigated AZD6765 as a po- tential fast-acting antidepressant agent in female mice, since the pre- mine and fluoXetine) presented increased ambulation in the OFT valence of MDD is about two-fold higher in women than in men . Regarding body weight of animals, stressed mice had a de- creased weight gain, independent on drug administration (data not shown), similarly to previous findings from our group (Moretti et al., 2019).

female rodents are more vulnerable to the deleterious effects of stress protocols (Yoshimura et al., 2003). Previous data evi- denced that most of the effects attributed to AZD6765 appear to be associated with enhanced hippocampal-prefrontal coupling, reflecting the improvement of reward networks connectivity (Becker et al., 2019). In addition, a combination of hyperforin and AZD6765 evoked long Effect of rapamycin (0.2 nmol/site, i.c.v.) and ketamine (1 mg/kg, i.p.) in the behavioral parameters analyzed in the OFT. Values are expressed as mean ± S.E.M of 7–8 mice. *P < 0.05 as compared with the vehicle-treated group; #P < 0.05 as compared with ketamine-treated group (two-way ANOVA followed by Duncan’s multiple range post hoc test).

Vehicle Ketamine Rapamycin Ketamine + rapamycin
Grooming episodes 11.00 ± 1.77 8.00 ± 1.05 8.25 ± 0.70 7.25 ± 0.99
Grooming latency 79.75 ± 14.67 59.57 ± 8.05 77.62 ± 6.66 30.75 ± 9.60
Number of rostral groomings 4.12 ± 0.79 4.43 ± 0.48 4.87 ± 0.55 4.75 ± 0.82
Total time of rearing 36.37 ± 7.29 36.71 ± 6.13 30.00 ± 5.79 36.50 ± 3.44
Number of rearings 29.12 ± 5.90 28.43 ± 4.76 25.62 ± 4.48 28.62 ± 2.91
Crossings 89.12 ± 12.04 97.00 ± 7.42 81.37 ± 12.04 102.50 ± 7.49
Total time in center 10.87 ± 1.94 32.43 ± 9.15 * 12.62 ± 3.58 10.87 ± 1.22 #

Fig. 7. Effect of the treatment of mice with a single administration of AZD6765 (1 mg/kg, i.p.), ketamine (1 mg/kg, i.p.) or fluoXetine (10 mg/kg, p.o.) on immobility time in the TST (panel B) and locomotor activity (panel C) in mice subjected to CUS protocol. Values are expressed as mean + S.E.M of 8 mice. **P < 0.01; compared with the vehicle-treated control group. ## P < 0.01 as compared with CUS + vehicle group. The experimental protocol is illustrated in panel A.

lasting antidepressant-like activity in naïve and corticosterone-treated male mice (Pochwat et al., 2018). Although several studies have re- ported the antidepressant effects of AZD6765, here we evaluated, at our knowledge for the first time, the role of PI3K/Akt/mTOR/GSK3β sig- naling pathways in the antidepressant-like effect of this compound. We suggest, by using pharmacological tools, that the stimulation of PI3K/ Akt/mTOR signaling pathway and inhibition of GSK3β by AZD6765 administration possibly underpins the antidepressant response of this compound, indicating that mTOR-mediated signaling has a key role in the AZD6765 effects and similarities to ketamine, since studies report the crucial role of these signaling pathways to the effects elicited by this drug (Li et al., 2010; Liu et al., 2013). In order to reinforce this notion, we analyzed some neurochemical parameters in the hippocampus and prefrontal cortex related to the activation of the mTOR signaling pathway. We demonstrated that AZD6765 increased synapsin im- munocontent in prefrontal cortex of mice and presented a tendency to increase the level of this protein in the hippocampus, a result similar to that obtained with ketamine administration in a previous study (Li et al., 2010). These data are in line with a previous study that showed elevated levels of synapsin after a combined administration of AZD6765 and hyperforin 1 h after the administration (Pochwat et al., 2018).

However, there was no alteration in the phosphorylation of P70S6K
(Thr389) and GluA1immunocontent in the prefrontal cortex and hip-
pocampus, suggesting that other time points or cerebral regions should be investigated in future studies. Although the similarities between AZD6765 and ketamine effects, previous studies have indicated that AZD6765 causes reduced psychotomimetic or dissociative adverse ef- fects while retaining antidepressant efficacy, since it has much lower level affinity to NMDA receptor when compared to ketamine (Machado- Vieira et al., 2017; Zarate et al., 2013). Besides that, it remains to be established the differences between AZD6765 and ketamine regarding the involvement of other signaling pathways in the rapid effect of both compounds.

In the present study, we also demonstrated that acute administra- tion of AZD6765, similar to ketamine, produced a fast behavioral re- sponse in mice submitted to NSF test. This paradigm detects behaviors related to depression and anxiety in mice presenting a conflict between the anxiogenic environment and hunger-induced behavior (Stedenfeld et al., 2011). NSF test is a paradigm sensitive to chronic but not acute effects of conventional antidepressants (Dulawa and Hen, 2005; Powell et al., 2012).

However, NSF test is sensitive to a single administration of ketamine, which enables the research of potential fast-acting anti- depressants (Brachman et al., 2016; Fraga et al., 2020; Wu et al., 2017). In the present study we confirm that a single administration of keta- mine is effective in the NSF test. The reduction in the latency to feed observed in this test is probably not related to an increase of appetite since the amount of food consumed was not affected by the treatments used. Of note, the administration of the inhibitor of mTOR rapamycin was effective to abolish the effects of both AZD6765 and ketamine in the NSF test, characterizing that the activation of mTOR plays a crucial role for the behavioral effect of these drugs in this test.
Regarding OFT parameters, ketamine increased total time in the center of the apparatus, reinforcing its anxiolytic effect reported pre- viously (Fraga et al., 2018; Krystal et al., 1994). These results reinforce the notion that ketamine is effective in inducing behavioral responses related to the reduction in anxiety and depression-related behaviors. In addition, the effect elicited by ketamine was reversed by rapamycin treatment. This result is in line with a previous study reporting that ketamine induced an anxiolytic effect in elevated plus maze (increased the time spent in open arms) that was blocked by rapamycin administration (Holubova et al., 2016), reinforcing the notion that the anxiolytic effect of ketamine is dependent on mTOR signaling pathway. In addition, the fact that ketamine modulates GABA synaptic function (Ghosal et al., 2020; Wang et al., 2017) may contribute for its anxiolytic effect. AZD6765 failed to increase the total time in the center of the open field apparatus, indicating that this compound exerts no anxiolytic effect, as opposed to ketamine, at least under the experimental condi- tions used.

This reason by which AZD6765 caused no anxiolytic-like effect in the OFT is not clear and requires future studies. It remains to be determined if this drug exerts anxiolytic effect when administered at higher doses. Interestingly, a recent study reported that AZD6765 modulated the GABAergic neuronal activity in mice subjected to chronic unpredictable mild stress when administered at a higher dose (15 mg/kg) than the one used in the present study (Mishra et al., 2020). Although a previous study reported that AZD6765 was not able to exert antidepressant-like effects in a chronic social defeat stress model of depression in mice (Qu et al., 2017), here we provide evidence that a single administration of either AZD6765 or ketamine abolished the depressive-like behavior induced by CUS, one of the most commonly used, reliable, and effective rodent model of depression (Antoniuk et al., 2019; Katz et al., 1981). This divergent result may be due to differences in the stress protocol, gender and strain of mice and other methodological issues. In line with our results, Mishra et al. (2020) demonstrated that AZD6765 was able to reverse the behavioral effects elicited by chronic stress in C57BL6 male mice.

Interestingly, ketamine, but not AZD6765 (24 h after administra- tion), was able to reduce the immobility time of animals in the TST in mice not submitted to stress, indicating that AZD6765 does not have a persistent effect, differing from ketamine in this aspect. Literature data have also reported differences between ketamine and AZD6765. For instance, AZD6765 was not able to increase prefrontal connectivity in depressed patients, different from ketamine (Abdallah et al., 2018). However, another study showed that AZD6765 and ketamine have si- milar effects in activating the anterior cingulate cortex in adults and that this may affect mood (Downey et al., 2016). The lack of anti- depressant-like effect in non-stressed control mice that received AZD6765 indicates different sensitivity to AZD6765 and ketamine, that was effective to cause an antidepressant-like effect under this condition. Although another study demonstrated the antidepressant-like effect of AZD6765 in the TST 24 h after its administration, a higher AZD6765 dose (10 mg/kg) was used (Pochwat et al., 2018), raising the possibility that a higher dose of AZD6765 is required to elicit this effect at this time point.

Despite the possibility that AZD6765 and ketamine elicit behavioral responses with different profile in unstressed mice, all the experimental protocols employed point to the conclusion that AZD6765 presents fast- acting antidepressant properties that seems to be dependent on mTOR signaling, reinforcing the notion that the activation of this signaling pathway plays a crucial role for rapid antidepressant responses (Li et al., 2010; Neis et al., 2016; Pazini et al., 2016). However, it is im- portant to highlight that a clinical study demonstrated that AZD6765 was not able to reduce depressive symptoms when compared to placebo in patients with MDD and history of inadequate response to anti- depressants (Sanacora et al., 2017), as AR-A014418  opposed to ketamine (Berman et al., 2000; Zarate et al., 2006).

5. Conclusion
The identification of potential fast-acting antidepressants with no psychotomimetic effects is highly important for treatment of depres- sion. Our results suggest that AZD6765 possibly acts by enhancing PI3K/Akt/mTOR signaling pathways inhibiting GSK3β and increasing synapsin level in prefrontal cortex of mice, mechanisms related to strengthened synaptic connections. Although we did not show the correlation of PI3K/Akt, mTOR and GSK3β in the antidepressant-like effects of AZD6765, it has been shown that the stimulation of mTOR signaling may occur as a consequence of PI3K/Akt activation and GSK3β inhibition (Ignácio et al., 2016; Liu et al., 2013). This suggests that mTOR signaling may be a crucial target for fast antidepressant-like effects of AZD6765. We further reinforced this hypothesis by demon- strating that a single administration of AZD6765, similarly to ketamine, is able to induce a fast behavioral response in NSF test that was reversed by rapamycin. Moreover, the fast antidepressant-like effect of AZD6765 was indicated by its ability to reverse the depressive-like behavior in- duced by CUS in the TST. However, considering that ketamine, but not AZD6765, has a persistent antidepressant-like effect in the TST (ob- served 24 h after its administration) in mice not submitted to CUS protocol, and that only ketamine was effective to elicit anxiolytic re- sponse in the OFT, more studies are necessary in order to clarify the differences between both NMDA receptor antagonists.

Role of the funding source

VBN, MM and ALSR designed the study and wrote the protocol. VBN, MM, PBR, YD, IW, AFR and MHB conducted experiments. VBN and FNK analyzed data. VBN, PBR and ALSR wrote the manuscript and managed the literature searches. All authors contributed to the design, acquisition, analysis and interpretation of data. All authors read and approved the manuscript.

Acknowledgements

This study was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) #310113/2017-2 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). ALSR is a CNPq Research Fellow. V.B.N. acknowledges post- doctoral funding from CNPq (158126/2018-1).
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