Skip to content

Advertisement

You're viewing the new version of our site. Please leave us feedback.

Learn more

BMC Neuroscience

Open Access

Effects of escitalopram and paroxetine on mTORC1 signaling in the rat hippocampus under chronic restraint stress

  • Mi Kyoung Seo1,
  • Cheol Min Choi2,
  • Roger S. McIntyre3, 4,
  • Hye Yeon Cho1,
  • Chan Hong Lee1,
  • Rodrigo B. Mansur3, 4,
  • Yena Lee3,
  • Jae-Hon Lee5,
  • Young Hoon Kim6,
  • Sung Woo Park1, 2Email author and
  • Jung Goo Lee1, 2, 3, 7Email author
BMC NeuroscienceBMC series – open, inclusive and trusted201718:39

https://doi.org/10.1186/s12868-017-0357-0

Received: 1 December 2016

Accepted: 20 April 2017

Published: 26 April 2017

Abstract

Background

Recent studies have suggested that the activation of mammalian target of rapamycin (mTOR) signaling may be related to antidepressant action. Therefore, the present study evaluated whether antidepressant drugs would exert differential effects on mTOR signaling in the rat hippocampus under conditions of chronic restraint stress. Male Sprague–Dawley rats were subjected to restraint stress for 6 h/days for 21 days with either escitalopram (10 mg/kg) or paroxetine (10 mg/kg) administered after the chronic stress procedure. Western blot analyses were used to assess changes in the levels of phospho-Ser2448-mTOR, phospho-Thr37/46-4E-BP-1, phospho-Thr389-p70S6 K, phospho-Ser422-eIF4B, phospho-Ser240/244-S6, phospho-Ser473-Akt, and phospho-Thr202/Tyr204-ERK in the hippocampus.

Results

Chronic restraint stress significantly decreased the levels of phospho-mTOR complex 1 (mTORC1), phospho-4E-BP-1, phospho-p70S6 K, phospho-eIF4B, phospho-S6, phospho-Akt, and phospho-ERK (p < 0.05); the administration of escitalopram and paroxetine increased the levels of all these proteins (p < 0.05 or 0.01). Additionally, chronic restraint stress reduced phospho-mTORC1 signaling activities in general, while escitalopram and paroxetine prevented these changes in phospho-mTORC1 signaling activities.

Conclusion

These findings provide further data that contribute to understanding the possible relationships among mTOR activity, stress, and antidepressant drugs.

Keywords

Chronic restraint stressHippocampusmTOR signalingAntidepressantsNeuroplasticity

Background

Depression is a chronic mental illness that involves multiple episodes [1]. The lifetime prevalence of depression in the United States has been estimated at up to 17% [2]. Moreover, this disorder is associated with substantial morbidity, reduced quality of life, and premature mortality [3, 4]. As a result, determining the underlying pathoetiological substrates of mood disorders continues to be a focus of ongoing research [5].

Meaningful advances have been made towards identifying the brain regions and neural circuits that may regulate emotions, mood, and anxiety [6]. Additionally, several neurochemical and molecular changes that underlie depression and stress-related disorders have been observed [7]. For example, stress can induce activation of the hypothalamic–pituitary–adrenal (HPA) axis and increase glucocorticoid hormone production during adaptive responses [8]. Thus, the HPA system has a significant impact on the brain and its major functions, such as mood, cognition, and behavior [9]. A remarkable finding regarding the influence of stress on the brain is that the stress response can result in neurodegeneration [10], including decreased brain volume, neuronal atrophy, and decreases in synaptic proteins [11]. Stress is also related to a decrease in the number of spines on neurons [5]. Taken together, these recent findings suggest that stress and depression may cause changes in neuronal and/or glial size, shape, and density in brain regions that regulate mood and emotion [12].

Of the recently reported findings on the molecular mechanisms associated with synaptogenesis, increases in synaptic protein levels after the activation of mammalian target of rapamycin complex 1 (mTORC1) signaling are of particular importance [13]. mTORC1 is a protein serine/threonine kinase that belongs to the phosphatidylinositol 3-kinase (PI3K)-related kinase family and in involved in a variety of biological processes [14]. Two structurally and functionally distinct mTOR-containing complexes have been identified. The defining components of the first, mTORC1, include the regulatory association protein of mTOR (Raptor) and the proline-rich Akt substrate 40 kDa [PRAS40; 15]; the activity of mTORC1 is specifically sensitive to rapamycin. The second complex, mTORC2, is composed of the rapamycin-insensitive companion of mTOR (Rictor), mammalian stress-activated mitogen-activated protein (MAP) kinase-interacting protein 1 (mSin1), and proteins observed with Rictor 1 and 2 (Protor-1 and Protor-2) [14]. mTORC1 is a regulator of cell growth and metabolism, while mTORC2 may be related to cell survival and cytoskeletal organization [16].

Li et al. [13] reported that a sub-anesthetic dose of ketamine (10 mg/kg) in mice increases mTOR phosphorylation and the levels of synaptic proteins, such as postsynaptic density protein 95 (PSD95), glutamate ionotropic receptor α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) type subunit 1 (GluA1), and synapsin I in the prefrontal cortex. These mice also exhibited decreases in immobility time in the forced swimming test (FST) and increases in synaptic protein levels when the ketamine treatment was blocked by rapamycin [13]. Therefore, the increased levels of synaptic proteins after ketamine treatment may be attributable to mTORC1 signaling activation. Park et al. [17] also observed differential influences of antidepressants on mTORC1 phosphorylation, synaptic protein expression, and neurite outgrowth under toxic conditions in rat primary hippocampal neurons.

The present study sought to assess whether antidepressants would exert varying effects on mTOR signaling in the rat hippocampus under conditions of chronic stress. A 21-day chronic restraint model was employed as the stress condition and the phosphorylation levels of mTORC1 upstream regulators (Akt and extracellular signal regulated protein [ERK]) and downstream effectors (eukaryotic translation initiation factor 4E binding protein 1 [4E-BP-1], p70 ribosomal S6 kinase [p70S6 K], eukaryotic translation initiation factor 4B [eIF4B], and small ribosomal protein 6 [S6]) in the rat hippocampus were assessed with Western blot analyses (Fig. 1).
Fig. 1

Schematic diagram of the experimental schedule. Escitalopram (ESC, 10 mg/kg) or paroxetine (PAR, 10 mg/kg) was administered 1 h prior to restraint stress for a total of 21 days (6 h/days). The rats were sacrificed on the 22nd day

Results

Effects of antidepressants on the expression of phosphorylated mTORC1 following chronic restraint stress

A two-way analysis of variance (ANOVA; Table 1) was performed to evaluate changes in phosphorylated mTORC1 levels and revealed significant individual effects of stress and drug (escitalopram and paroxetine) as well as significant interactions between stress and drug (stress × escitalopram and stress × paroxetine). Chronic restraint stress significantly decreased phospho-Ser2448-mTORC1 expression by 52.78% in the hippocampus compared with the vehicle control group (p = 0.013; Fig. 2). Escitalopram and paroxetine markedly prevented the chronic restraint stress-induced decrease in phospho-Ser2448-mTORC1 (stress + escitalopram = 98.91% of control, p = 0.016; stress + paroxetine = 96.73% of control, p = 0.023; Fig. 2). Neither antidepressant affected phospho-Ser2448-mTORC1 levels under normal conditions.
Table 1

Summary of the two-way analysis of variance on changes in phosphorylated mTORC1, downstream effectors of mTORC1, and upstream activators of mTORC1 related to treatment, stress and on the interaction of treatment and stress

 

Escitalopram

Paroxetine

F

P

F

P

mTORC1

 Drug

7.498

0.013

8.198

0.010

 Stress

8.402

0.009

10.616

0.004

 Drug × stress

10.662

0.004

6.990

0.016

Downstream effectors of mTORC1

 4E-BP-1

  Drug

13.592

0.001

12.403

0.002

  Stress

20.717

<0.001

28.600

<0.001

  Drug × stress

9.579

0.006

11.679

0.003

 p70S6 K

  Drug

8.868

0.007

5.866

0.025

  Stress

17.816

<0.001

17.947

<0.001

  Drug × stress

10.815

0.004

7.900

0.011

 eIF4B

  Drug

8.157

0.010

6.669

0.018

  Stress

19.425

<0.001

16.753

0.001

  Drug × stress

5.919

0.024

8.980

0.007

 S6

  Drug

4.194

0.050

6.878

0.016

  Stress

12.204

0.002

12.117

0.002

  Drug × stress

7.238

0.014

14.918

0.001

Upstream activators of mTORC1

 Akt

  Drug

21.744

<0.001

18.971

<0.001

  Stress

47.192

<0.001

51.327

<0.001

  Drug × stress

17.682

<0.001

13.422

0.002

 ERK

  Drug

17.581

<0.001

21.123

<0.001

  Stress

14.075

0.001

25.637

<0.001

  Drug × stress

21.477

<0.001

14.569

0.001

mTORC1 mammalian target of rapamycin complex 1, 4E-BP-1 eukaryotic initiation factor 4E-binding protein 1, p70S6 K p70 ribosomal protein S6 kinase, eI4FB eukaryotic translation initiation factor 4B, S6 small ribosomal protein 6, ERK extracellular signal-regulated kinase

Fig. 2

Effects of antidepressants on levels of phospho-mTORC1 in the rat hippocampus. Rats (n = 6 animals/group) were given a daily injection of vehicle (Veh; 1 mL/kg), ESC (10 mg/kg), or PAR (10 mg/kg) for 21 days with or without restraint stress (6 h daily for 21 days). Levels of phosphorylated mTORC1 in brain homogenates from the hippocampus were detected by SDS-PAGE and Western blot analyses using anti-phospho-Ser2448-mTORC1 antibodies. A representative image and quantitative analysis normalized to the levels of total mTORC1 are shown. Results are expressed as a percentage of vehicle control and represent the mean ± standard error of the mean (SEM) of 6 animals per group. *p < 0.05 versus vehicle control; p < 0.05 versus stress + vehicle

Effects of antidepressants on the expression of phosphorylated mTORC1 downstream effectors (4E-BP-1, p70S6 K, eIF4B, and S6)

There were significant individual effects of stress and drug (escitalopram and paroxetine) on the phosphorylated levels of 4E-BP-1, p70S6 K, eIF4B, and S6 (Table 1) as well as an interaction between these two factors (stress × escitalopram and stress × paroxetine) that significantly affected these levels (Table 1). Specifically, chronic restraint stress decreased the expression of mTORC1 downstream regulators and produced significant decreases in the levels of phospho-Thr37/46-4E-BP-1 (56.25%, p = 0.001, Fig. 3a), phospho-Thr389-p70S6 K (49.29%, p = 0.002, Fig. 3b), phospho-Ser422-eIF4B (62.88%, p = 0.005, Fig. 3c), and phospho-Ser240/244-S6 (61.24%, p = 0.004, Fig. 3d) compared with the vehicle control group. However, treatment with escitalopram and paroxetine prevented the chronic restraint stress-induced decreases in the phosphorylated levels of these mTORC1 downstream effectors (4E-BP-1: stress + escitalopram = 93.69% of control, p = 0.003; stress + paroxetine = 88.02% of control, p = 0.010, Fig. 3a; p70S6 K: stress + escitalopram = 91.68% of control, p = 0.012; stress + paroxetine = 87.40% of control, p = 0.034, Fig. 3b; eIF4B: stress + escitalopram = 85.55% of control, p = 0.048; stress + paroxetine = 86.56% of control, p = 0.042, Fig. 3c; S6: stress + escitalopram = 86.53% of control, p = 0.046; stress + paroxetine = 92.09% of control, p = 0.014, Fig. 3d). The phosphorylated levels of these mTORC1 downstream effectors were not affected by antidepressants under normal conditions.
Fig. 3

Effects of antidepressants on the levels of mTORC1 downstream effectors (phospho-4E-BP-1, phospho-p70S6 K, phosphor-eIF4B, and phospho-S6) in the rat hippocampus. Rats (n = 6 animals/group) were given a daily injection of Veh (1 mL/kg), ESC (10 mg/kg), or PAR (10 mg/kg) for 21 days with or without restraint stress (6 h daily for 21 days). Levels of phosphorylated 4E-BP-1, p70S6 K, eIF4B, and S6 in brain homogenates from the hippocampus were detected by SDS-PAGE and Western blot analyses using anti-phospho-Thr37/46-4E-BP-1 (a), anti-phospho-Thr389-p70S6 K (b), anti-phospho-Ser422-eIF4B (c), and anti-phospho-Ser240/244-S6 (d) antibodies. A representative image and quantitative analysis normalized to the levels of total 4E-BP-1 (a), p70S6 K (b), eIF4B (c), and S6 (d) are shown. The results are expressed as a percentage of vehicle control and represent the mean ± SEM of 6 animals per group. **p < 0.01 versus vehicle control; p < 0.05 or †† p < 0.01 versus stress + vehicle

Effects of antidepressants on the expression of phosphorylated mTORC1 upstream activators (Akt and ERK)

Stress, drug (i.e., escitalopram and paroxetine), and their interaction (stress × escitalopram and stress × paroxetine) had significant effects on the levels of phosphorylated Akt and ERK (Table 1). Specifically, chronic restraint stress decreased mTORC1 upstream activators and significantly decreased the levels of phospho-Ser473-Akt (47.88% of control, p < 0.001, Fig. 4a) and phospho-Thr202/Tyr204-ERK (51.22% of control, p < 0.001, Fig. 4b) in the hippocampus. However, treatment with escitalopram and paroxetine prevented the chronic restraint stress-induced decreases in the phosphorylated levels of mTORC1 upstream activators (Akt: stress + escitalopram = 89.92% of control, p < 0.001; stress + paroxetine = 86.54% of control, p = 0.001, Fig. 4a; ERK: stress + escitalopram = 102.91% of control, p < 0.001; stress + paroxetine = 97.57% of control, p < 0.001, Fig. 4b). These levels were not affected under normal conditions.
Fig. 4

Effects of antidepressants on the levels of mTORC1 upstream activators (phospho-Akt and phospho-ERK) in the rat hippocampus. Rats (n = 6 animals/group) were given a daily injection of Veh (1 mL/kg), ESC (10 mg/kg), or PAR (10 mg/kg) for 21 days with or without restraint stress (6 h daily for 21 days). Levels of phosphorylated Akt and ERK in brain homogenates from the hippocampus were detected by SDS-PAGE and Western blot analyses using anti-phospho-Ser473-Akt (a) and anti-phospho-Thr202/Tyr204-ERK (b) antibodies. A representative image and quantitative analysis normalized to the levels of total Akt (a) and ERK (b) are shown. The results are expressed as the percentage of vehicle control and represent the mean ± SEM of 6 animals per group. **p < 0.01 versus vehicle controls; p < 0.05 or †† p < 0.01 versus stress + vehicle

Discussion

The main findings of this study were that chronic restraint stress decreased the expression of phospho-mTORC1, phospho-4E-BP-1, phospho-p70S6 K, phospho-eIF4B, phospho-S6, phospho-Akt, and phospho-ERK in the rat hippocampus. Additionally, this study showed that escitalopram and paroxetine prevented changes in the levels of phospho-mTORC1, phospho-4E-BP1, phospho-p70S6 K, phospho-eIF4B, phospho-S6, phospho-Akt, and phospho-ERK that were induced by chronic restraint stress. Therefore, escitalopram and paroxetine activated mTORC1 signaling pathways in the rat hippocampus under chronic restraint conditions (Fig. 5).
Fig. 5

Possible mechanisms underlying antidepressant-induced molecular changes related to antidepressant effects. Antidepressants increase BDNF [please spell out] expression. The release of BDNF and the stimulation of associated signaling cascades (PI3 K/Akt and MEK/ERK) activate mTORC1 signaling and translation which, in turn, increases synaptic protein levels and synaptogenesis. These effects contribute to the sustained antidepressant actions of antidepressants. TrkB tyrosine-related kinase B, PI3 K phosphoinositide 3-kinase, MEK MAP/ERK kinase, ERK extracellular signal-regulated kinases, GSK-3, glycogen synthase kinase-3, mTORC1 mammalian target of rapamycin complex 1, 4E-BP-1 4E-binding protein 1, p70S6 K p70ribosomal protein S6 kinase, eEF2 eukaryotic elongation factor 2, eIF4E eukaryotic translation initiation factor 4E, S6 small ribosomal protein 6, eIF4B eukaryotic translation initiation factor 4B, PSD-95 post-synaptic density 95, GluA1 glutamate ionotropic receptor AMPA type subunit 1, BDNF brain-derived neurotrophic factor. The molecular pathways shown in red illustrate novel observations from the present study while those in black are generally accepted signaling pathways involved in antidepressant action

Stress can facilitate the activity of the HPA axis and the production of glucocorticoids, which are the major stress-reactive hormones [8]. Heightened levels of glucocorticoid hormones may cause neuronal toxicity in certain brain structures and have been associated with mood and emotional dysregulation [18]. However, the underlying cellular mechanisms mediated by stress are not fully understood [19].

Stress can also reduce the expression of growth factors, such as brain-derived neurotrophic factor (BDNF), which may affect neurogenesis in the brain, especially the hippocampus [5, 11, 20]. The hippocampus is a limbic structure implicated in the pathogenesis of mood disorders and related symptoms [9, 11, 20] that establishes circuits with other brain structures, such as the amygdala and prefrontal cortex, and affects learning, memory, and regulation of the HPA axis [18, 21]. The hippocampus also contains considerable quantities of glucocorticoid receptors [22, 23]. Thus, stress can induce neuronal damage and atrophy in the hippocampus as well as cause changes in its structure [2426].

Magnetic resonance imaging studies have shown that reductions in the hippocampal volume of patients with depression are associated with more frequent episodes [27] and a meta-analysis observed reduced hippocampal volume in patients with unipolar depression [28]. A loss of hippocampal volume has also been observed in patients with first-episode depression [29] and it has been suggested that reduced hippocampal volume might be a biomarker of the progression of depression [29, 30]. Taken together, these findings suggest that the pathophysiology of depression may be associated with the decreased volumes of cortical and limbic brain regions, atrophy of neurons, and decreased numbers of synaptic connections [25, 31, 32].

As mentioned above, stress reduces the expression and function of BDNF in brain structures related to the pathogenesis of depression. Reduced levels of BDNF or growth factors may be related to the structural and neural plastic changes associated with stress and depression [32, 33] because decreases in BDNF may cause neuronal death and atrophy; this factor is necessary for neuronal remodeling. An increased vulnerability to depression-like behaviors was observed in BDNF-heterozygous knockout mice [34, 35], while human studies have reported that the presence of the BDNF Val66Met allele blocks the normal maturation of BDNF and may cause neuronal atrophy in hippocampal neurons [36].

These effects may be due to the modification of intracellular signaling pathways by BDNF. The major intracellular signaling pathways involved in neuronal survival and synaptogenesis are the PI3 K-Akt and mitogen-activated protein kinase (MAPK) signaling pathways [37, 38], which have multiple downstream targets that regulate neuronal survival, neuroprotection, and synaptic plasticity [39, 40]. An important downstream target for the regulation of synaptic plasticity and production of synaptic proteins is mTORC1 [13, 14, 32]. Neurotrophic factors regulate mTORC1 signaling; however, one’s nutritional, energy, endocrine, and metabolic status can also regulate mTORC1 signaling activity [40, 42]. For example, the expression of mTORC1 in primary rat hippocampal neurons decreases under B27-deprivation conditions [17], while treadmill exercise increases the level of mTORC1 and synaptic proteins in the rat hippocampus following 7 days of immobilization stress [41]. Additionally, ketamine increases mTORC1 activity and the production of synaptic proteins in the mouse prefrontal cortex and rat primary hippocampal neurons [5, 13, 17, 32]. Therefore, it is possible that mTORC1 is a convergence pathway for synaptic plasticity and the production of synaptic proteins [5, 32, 43].

Chronic restraint stress is one experimental method that can be used to create stressful conditions in animals [44]. Therefore, the present study adopted a repeated restraint stress paradigm [6 h/days for 21 days; 45, 46]. Previous studies have shown that chronic restraint stress decreased the levels of BDNF, PSD95, and β-catenin in the rat hippocampus [47] and resulted in the retraction of dendrites in hippocampal CA3 neurons and spatial memory deficits in rats [48]. A murine study reported that chronic restraint stress impaired neurogenesis in the hippocampus and produced hippocampus-dependent fear memory [19]. Similarly, the use of a 7-day immobilization stress paradigm decreased levels of synaptic proteins, such as PSD95 and synaptophysin [41].

In a previous study, 8 weeks of chronic unpredictable stress resulted in reduced levels of phosphorylated mTORC1, ERK-1/2, Akt1, and GluA1 in the rat amygdala [49]. However, there were no significant changes in these proteins in the frontal cortex, hippocampus, or dorsal raphe [49]. These discrepant results may be due to the different types of stressors and varying periods of stress used in the experiments. Similarly, 21 days of chronic unpredictable stress decreased the expression levels of PSD95, GluA1, and synapsin I, as well as decreased the number of spines and inhibited excitatory postsynaptic currents in the rat prefrontal cortex [50]. Moreover, 21 days of chronic unpredictable stress decreased mTORC1 expression and increased levels of regulated in development and DNA damage response-1 (REDD1), which is an inhibitor of mTORC1 in the rat prefrontal cortex [51]. Although a different stress paradigm was used in the present study, decreased levels of phospho-mTORC1 and its downstream effectors phospho-4E-BP-1, phospho-p70S6 K, phospho-eIF4B, and phospho-S6 were observed in the rat hippocampus. Furthermore, there were decreased expression levels of phospho-Akt and phospho-ERK, which are upstream activators of mTORC1.

Activated mTORC1 phosphorylates 4E-BP and p70S6 K [52, 53] and activated p70S6 K phosphorylates S6 and eIF4B [5254], which subsequently facilitate protein translation [5052]. Thus, decreased expression levels of mTORC1 may be related to decreased levels of 4E-BP-1, P70S6 K, eIF4B, and S6. Previous studies have reported that 21 days of restraint stress in mice decreased levels of Akt and ERK in the hippocampus and that 7 days of immobilization stress reduced Akt in the rat hippocampus [41, 55]. The decreased levels of Akt and ERK may be related to the lower levels of mTORC1 and the decreased effect on mTORC1 downstream effectors. In the present study, the effects of 21-day chronic restraint stress on the expression of synaptic proteins was not assessed; however, the present and previous studies have shown that 21 days of restraint stress significantly reduces the levels of BDNF, PSD95, and synaptophysin in the rat hippocampus [47, 56]. Therefore, it is possible that chronic restraint stress decreased activation of the mTORC1 signaling pathway in the present study.

The present study also showed that treatment with escitalopram and paroxetine prevented the chronic restraint stress-induced reduction of phospho-mTORC1 expression in the rat hippocampus. Escitalopram and paroxetine also prevented the chronic stress-induced reduction in the levels phospho-4E-BP1, phospho-p70S6 K, phospho-eIF4B, phospho-S6, phospho-Akt, and phospho-ERK. A previous study of rat primary hippocampal neurons showed that escitalopram and paroxetine increased the levels of phospho-mTORC1, phospho-4E-BP-1, phospho-p70S6 K, phospho-eIF4B, and phospho-S6 under B27-deprived toxic conditions [17]. Moreover, escitalopram and paroxetine also increased the levels of phospho-Akt and phospho-ERK [17]. Although different doses of antidepressant drugs were used in the present study, the findings were similar to those of the in vitro study [17]. Therefore, escitalopram and paroxetine could prevent decreases in the levels of mTORC1 as well as its downstream effectors and upstream regulators after chronic restraint stress. In other words, chronic restraint stress could decrease the activation of mTORC1 signaling but this may be prevented by some antidepressant treatments.

To our knowledge, this is the first report of the effects of antidepressants on the mTORC1 signaling pathway in the rat hippocampus. Notwithstanding, this study has several limitations. First, although previous studies have shown that chronic restraint stress induces depression-like behavior in behavioral tests [5759], the present study did not confirm the behavioral effects of this chronic restraint stress paradigm. Second, the levels of synaptic proteins, such as PSD95, GluA1, and synapsin I, were not assessed in the present study. Third, the effects of mTORC1 inhibitors, such as rapamycin, and other signal pathway inhibitors were not evaluated in the present study. Therefore, additional work that addresses these limitations is needed to strengthen the findings of this study.

Conclusions

In summary, chronic restraint stress reduced mTORC1 signaling activities in the rat hippocampus but these decreases were prevented by treatment with escitalopram and paroxetine. The findings of this study may allow for a better understanding of the possible relationships between mTOR activity and the biology of stress. Furthermore, the present findings highlight that some antidepressants may regulate mTOR signaling activity in chronic stress situations.

Methods

Drugs and reagents

Escitalopram oxalate (Pangbourne, UK), and paroxetine HCl (Holzkirchen, DE) were gifts from Sandoz. The antibodies for the Western blot analyses were obtained from the following sources: anti-phospho-mTOR (Ser2448, #2971), anti-mTOR (#2972), anti-phospho-Akt (Ser473, #9271), anti-Akt (#9272), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204, #9101), anti-p44/42 MAPK (ERK1/2, #4695), anti-phospho-4E-BP-1 (Thr37/46, #2855), anti-4E-BP-1 (#9452), anti-phospho-eIF4B (Ser422, #3591), anti-eIF4B (#3592), anti-phospho-S6 (Ser240/244, #2251), anti-S6 (#2217), anti-phospho-p70S6 K (Thr389, #9205), and anti-p70S6 K (#9202) from Cell Signaling Technology (Beverly, MA, USA); anti-BDNF (sc-546) and goat anti-rabbit IgG-horseradish-peroxide conjugates (sc-2004) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and monoclonal anti-α-tubulin (T9026) and anti-mouse IgG peroxidase conjugates (A4416) from Sigma (St. Louis, MO, USA).

Animals

Male Sprague–Dawley rats (Orient Bio, GyeongGi-Do, Korea) weighing 200–250 g were housed 2 or 3 per cage with ad libitum food and water in an environment maintained at 21 °C on a 12/12-h light/dark cycle.

After 7 days of acclimatization, the rats were randomly divided into 6 groups of 6 rats each. All drugs were dissolved in vehicle (0.7% glacial acetic acid in 0.9% saline) and intraperitoneally (i.p.) injected into the animals. The first group (vehicle) received vehicle (1 mL/kg, i.p.) without immobilization stress; the second (escitalopram) and third (paroxetine) groups received escitalopram (10 mg/kg, i.p.) and paroxetine (10 mg/kg, i.p.), respectively, without restraint stress; and the sixth group (vehicle + stress) received the vehicle at 10:00. Then, 1 h later, the rats were completely restrained for 6 h (from 11:00 to 17:00) in specially designed plastic restraint tubes (dimensions: 20-cm high, 7 cm in diameter). The rats in the fourth (escitalopram + stress) and fifth (paroxetine + stress) groups received escitalopram (10 mg/kg, i.p.) or paroxetine (10 mg/kg, i.p.), respectively, and were then restrained in the same way as the rats in the sixth group. These procedures were repeated once daily for 3 weeks (Fig. 1).

The dose of escitalopram (10 mg/kg) used in the present study was selected based on a report showing that this dose exerted antidepressant-like effects in rats with depression-like behaviors induced by chronic mild stress [60]. In rats receiving chronic treatment with paroxetine (3 weeks, 10 mg/kg), the hippocampus exhibited increases in BDNF expression and synaptic levels of AMPA receptor subunits [GluA1 and GluA2/3; 61]. In particular, these doses of escitalopram and paroxetine significantly prevented chronic restraint stress-induced decreases in BDNF mRNA in the rat hippocampus (Additional file 1: Figure S1).

Protein extraction and Western blotting

The rats were sacrificed by rapid decapitation, 24 h after the final restraint session. Immediately after decapitation and rapid removal of the brain, hippocampus was dissected out. The detailed procedure for western blot analysis was described previously [47].

The membranes were probed with antibodies against anti-phospho-mTORC1 (Ser2448), anti-mTORC1, anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-p44/42 MAPK (ERK1/2) (Thyr202/Tyr204), anti-p44/42 MAPK (ERK1/2), anti-phosho-4E-BP-1 (Thr37/46), anti-4E-BP-1, anti-phospho-p70S6 K (Thr389), anti-p70S6 K, anti-phosho-eIF4B (Ser422), anti-eIF4B, anti-phosho-S6 (Ser240/244), anti-S6, anti-BDNF, 1:1000; and anti-α-tubulin, 1:2000. The membranes were subsequently probed with horseradish peroxidase-conjugated secondary antibody, goat-anti-rabbit IgG for anti-phospho-mTORC1 (Ser2448), anti-mTORC1, anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-p44/42 MAPK (ERK1/2) (Thyr202/Tyr204), anti-p44/42 MAPK (ERK1/2), anti-phosho-4E-BP-1 (Thr37/46), anti-4E-BP-1, anti-phospho-p70S6 K (Thr389), anti-p70S6 K, anti-phosho-eIF4B (Ser422), anti-eIF4B, anti-phosho-S6 (Ser240/244), anti-S6, 1:1000; anti-BDNF, 1:2000; and anti-mouse IgG for anti-α-tubulin 1:10,000. Proteins were detected by Pico EPC Western blot reagents (ELPIS, Daejeon, Korea).

Statistical analysis

To determine the individual and interactive effects of drug administration and restraint stress on the protein levels, a two-way ANOVA was performed with Scheffe’s tests for post hoc comparisons. A p value < 0.05 was considered to indicate statistical significance.

Abbreviations

4E-BP-1: 

eukaryotic translation initiation factor 4E binding protein 1

ANOVA: 

analysis of variance

BNDF: 

brian-derived neurotrophic factor

eIF4B: 

eukaryotic translation initiation factor 4B

ERK: 

extracellular signal regulated protein

FST: 

forced swimming test

GluR1: 

glutamate ionotropic receptor α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) type subunit 1

HPA: 

hypothalamic-pituitary-adrenal

MAP: 

mitogen-activated protein

MAPK: 

mitogen-activated protein kinase

mSin1: 

mitogen-activated protein kinase-interacting protein 1

mTOR: 

mammalian target of rapamycin

mTORC1: 

mammalian target of rapamycin complex 1

mTOR2: 

mammalian target of rapamycin complex 2

p70S6K: 

p70 ribosomal S6 kinase

PI3 K: 

phosphatidylinositol 3-kinase

PRAS40: 

proline-rich Akt substrate 40 kDa

protor-1: 

protein obsereved with Rictor 1

protor-2: 

protein obsereved with Rictor 2

PSD-95: 

postsynaptic density protein 95

Rator: 

regulatory association protein of mTOR

REDD1: 

regulated in development and DNA damage response-1

Rictor: 

rapamycin-insensitive companion of mTOR

S6: 

small ribosomal protein 6

Declarations

Authors’ contributions

JG, SW, and YH designed the study. MK, CM, HY and CH performed the experiment of this study. MK and SW wrote the protocol and undertook the statistical analysis. RS, RB, YN, and JH contributed the methods and analysis tools. JG, SW and MK wrote the first draft of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

None.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

Data and materials are available from the corresponding author upon request.

Ethics approval and consent to participate

The animal experiments in this manuscript approved by the Committee for Animal Experimentation and the Institutional Animal Laboratory Review Board of Inje Medical College (Approval No. 2013-003).

Funding

This work was supported by the 2013 Post-doctoral Research Program of Inje University (Reference number; none).

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Paik Institute for Clinical Research, Inje University
(2)
Department of Health Science and Technology, Graduate School, Inje University
(3)
Mood Disorders Psychopharmacology Unit, University Health Network
(4)
Department of Psychiatry, University of Toronto
(5)
Department of Psychiatry, Korea University Ansan Hospital, Korea University College of Medicine
(6)
Department of Psychiatry, Gongju National Hospital
(7)
Department of Psychiatry, School of Medicine, Haeundae Paik Hospital, Inje University

References

  1. Lasserre AM, Marti-Soler H, Strippoli MP, Vaucher J, Glaus J, Vandeleur CL, et al. Clinical and course characteristics of depression and all-cause mortality: a prospective population-based study. J Affect Disord. 2016;189:17–24.View ArticlePubMedGoogle Scholar
  2. Kessler RC, Berglund P, Demler O, Jin R, Koretz D, Merikangas KR, et al. The epidemiology of major depressive disorder: results from the National Comorbidity Survey Replication (NCS-R). JAMA. 2003;289:3095–105.View ArticlePubMedGoogle Scholar
  3. Hawton K, Casanas I, Comabella C, Haw C, Saunders K. Risk factors for suicide in individuals with depression: a systematic review. J Affect Disord. 2013;147:17–28.View ArticlePubMedGoogle Scholar
  4. Richards D. Prevalence and clinical course of depression: a review. Clin Psychol Rev. 2011;31:1117–25.View ArticlePubMedGoogle Scholar
  5. Duman RS. Pathophysiology of depression and innovative treatments: remodeling glutamatergic synaptic connections. Dialogues Clin Neurosci. 2014;16:11–27.PubMedPubMed CentralGoogle Scholar
  6. Savitz J, Drevets WC. Bipolar and major depressive disorder: neuroimaging the developmental-degenerative divide. Neurosci Biobehav Rev. 2009;33:699–771.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Krishnan V, Nestler EJ. The molecular neurobiology of depression. Nature. 2008;455:894–902.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Swaab DF, Bao AM, Lucassen PJ. The stress system in the human brain in depression and neurodegeneration. Ageing Res Rev. 2005;4:141–94.View ArticlePubMedGoogle Scholar
  9. Kino T. Stress, glucocorticoid hormones, and hippocampal neural progenitor cells: implications to mood disorders. Front Physiol. 2015;6:230.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Bao AM, Meynen G, Swaab DF. The stress system in depression and neurodegeneration: focus on the human hypothalamus. Brain Res Rev. 2008;57:531–53.View ArticlePubMedGoogle Scholar
  11. MacQueen G, Frodl T. The hippocampus in major depression: evidence for the convergence of the bench and bedside in psychiatric research? Mol Psychiatry. 2011;16:252–64.View ArticlePubMedGoogle Scholar
  12. Duman RS, Monteggia LM. A neurotrophic model for stress-related mood disorders. Biol Psychiatry. 2006;59:1116–27.View ArticlePubMedGoogle Scholar
  13. Li N, Lee B, Liu RJ, Banasr M, Dwyer JM, Iwata M, et al. mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science. 2010;329:959–64.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Costa-Mattioli M, Monteggia LM. mTOR complexes in neurodevelopmental and neuropsychiatric disorders. Nat Neurosci. 2013;16:1537–43.View ArticlePubMedGoogle Scholar
  15. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274–93.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Laplante M, Sabatini DM. mTOR signaling at a glance. J Cell Sci. 2009;122:3589–94.View ArticlePubMedPubMed CentralGoogle Scholar
  17. Park SW, Lee JG, Seo MK, Lee CH, Cho HY, Lee BJ, et al. Differential effects of antidepressant drugs on mTOR signalling in rat hippocampal neurons. Int J Neuropsychopharmacol. 2014;17:1831–46.View ArticlePubMedGoogle Scholar
  18. Numakawa T, Adachi N, Richards M, Chiba S, Kunugi H. Brain-derived neurotrophic factor and glucocorticoids: reciprocal influence on the central nervous system. Neuroscience. 2013;239:157–72.View ArticlePubMedGoogle Scholar
  19. Yun J, Koike H, Ibi D, Toth E, Mizoguchi H, Nitta A, et al. Chronic restraint stress impairs neurogenesis and hippocampus-dependent fear memory in mice: possible involvement of a brain-specific transcription factor Npas4. J Neurochem. 2010;114:1840–51.View ArticlePubMedGoogle Scholar
  20. McEwen BS, Nasca C, Gray JD. Stress effects on neuronal structure: hippocampus, amygdala, and prefrontal cortex. Neuropsychopharmacology. 2016;41:3–23.View ArticlePubMedGoogle Scholar
  21. Aggleton JP. Looking beyond the hippocampus: old and new neurological targets for understanding memory disorders. Proc Biol Sci. 2014;281:1–9.View ArticleGoogle Scholar
  22. Fastenrath M, Coynel D, Spalek K, Milnik A, Gschwind L, Roozendaal B, et al. Dynamic modulation of amygdala–hippocampal connectivity by emotional arousal. J Neurosci. 2014;34:13935–47.View ArticlePubMedGoogle Scholar
  23. Sapolsky RM. Depression, antidepressants, and the shrinking hippocampus. Proc Natl Acad Sci USA. 2001;98:12320–2.View ArticlePubMedPubMed CentralGoogle Scholar
  24. Kim EJ, Pellman B, Kim JJ. Stress effects on the hippocampus: a critical review. Learn Mem. 2015;22:411–6.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Malykhin NV, Coupland NJ. Hippocampal neuroplasticity in major depressive disorder. Neuroscience. 2015;309:200–13.View ArticlePubMedGoogle Scholar
  26. Woon FL, Sood S, Hedges DW. Hippocampal volume deficits associated with exposure to psychological trauma and posttraumatic stress disorder in adults: a meta-analysis. Prog Neuropsychopharmacol Biol Psychiatry. 2010;34:1181–8.View ArticlePubMedGoogle Scholar
  27. MacQueen GM, Campbell S, McEwen BS, Macdonald K, Amano S, Joffe RT, et al. Course of illness, hippocampal function, and hippocampal volume in major depression. Proc Natl Acad Sci USA. 2003;100:1387–92.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Videbech P, Ravnkilde B. Hippocampal volume and depression: a meta-analysis of MRI studies. Am J Psychiatry. 2004;161:1957–66.View ArticlePubMedGoogle Scholar
  29. Cole J, Costafreda SG, McGuffin P, Fu CH. Hippocampal atrophy in first episode depression: a meta-analysis of magnetic resonance imaging studies. J Affect Disord. 2011;134:483–7.View ArticlePubMedGoogle Scholar
  30. Chan SW, Harmer CJ, Norbury R, O’Sullivan U, Goodwin GM, Portella MJ. Hippocampal volume in vulnerability and resilience to depression. J Affect Disord. 2016;189:199–202.View ArticlePubMedGoogle Scholar
  31. Abdallah CG, Jackowski A, Sato JR, Mao X, Kang G, Cheema R, et al. Prefrontal cortical GABA abnormalities are associated with reduced hippocampal volume in major depressive disorder. Eur Neuropsychopharmacol. 2015;25:1082–90.View ArticlePubMedPubMed CentralGoogle Scholar
  32. Duman RS, Aghajanian GK, Sanacora G, Krystal JH. Synaptic plasticity and depression: new insights from stress and rapid-acting antidepressants. Nat Med. 2016;22:238–49.View ArticlePubMedGoogle Scholar
  33. Holtmaat A, Svoboda K. Experience-dependent structural synaptic plasticity in the mammalian brain. Nat Rev Neurosci. 2009;10:647–58.View ArticlePubMedGoogle Scholar
  34. Duman CH, Schlesinger L, Kodama M, Russell DS, Duman RS. A role for MAP kinase signaling in behavioral models of depression and antidepressant treatment. Biol Psychiatry. 2007;61:661–70.View ArticlePubMedGoogle Scholar
  35. Yau SY, Lau BW, Tong JB, Wong R, Ching YP, Qiu G, et al. Hippocampal neurogenesis and dendritic plasticity support running-improved spatial learning and depression-like behaviour in stressed rats. PLoS ONE. 2011;6:e24263.View ArticlePubMedPubMed CentralGoogle Scholar
  36. Frodl T, Schule C, Schmitt G, Born C, Baghai T, Zill P, et al. Association of the brain-derived neurotrophic factor Val66Met polymorphism with reduced hippocampal volumes in major depression. Arch Gen Psychiatry. 2007;64:410–6.View ArticlePubMedGoogle Scholar
  37. Dijkhuizen PA, Ghosh A. BDNF regulates primary dendrite formation in cortical neurons via the PI3-kinase and MAP kinase signaling pathways. J Neurobiol. 2005;62:278–88.View ArticlePubMedGoogle Scholar
  38. Patapoutian A, Reichardt LF. Trk receptors: mediators of neurotrophin action. Curr Opin Neurobiol. 2001;11:272–80.View ArticlePubMedGoogle Scholar
  39. Horwood JM, Dufour F, Laroche S, Davis S. Signalling mechanisms mediated by the phosphoinositide 3-kinase/Akt cascade in synaptic plasticity and memory in the rat. Eur J Neurosci. 2006;23:3375–84.View ArticlePubMedGoogle Scholar
  40. Numakawa T, Suzuki S, Kumamaru E, Adachi N, Richards M, Kunugi H. BDNF function and intracellular signaling in neurons. Histol Histopathol. 2010;25:237–58.PubMedGoogle Scholar
  41. Fang ZH, Lee CH, Seo MK, Cho H, Lee JG, Lee BJ, et al. Effect of treadmill exercise on the BDNF-mediated pathway in the hippocampus of stressed rats. Neurosci Res. 2013;76:187–94.View ArticlePubMedGoogle Scholar
  42. Watson K, Baar K. mTOR and the health benefits of exercise. Semin Cell Dev Biol. 2014;36:130–9.View ArticlePubMedGoogle Scholar
  43. Scheuing L, Chiu CT, Liao HM, Chuang DM. Antidepressant mechanism of ketamine: perspective from preclinical studies. Front Neurosci. 2015;9:249.View ArticlePubMedPubMed CentralGoogle Scholar
  44. Gregus A, Wintink AJ, Davis AC, Kalynchuk LE. Effect of repeated corticosterone injections and restraint stress on anxiety and depression-like behavior in male rats. Behav Brain Res. 2005;156:105–14.View ArticlePubMedGoogle Scholar
  45. Hennebelle M, Balasse L, Latour A, Champeil-Potokar G, Denis S, Lavialle M, et al. Influence of omega-3 fatty acid status on the way rats adapt to chronic restraint stress. PLoS ONE. 2012;7:e42142.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Zhao X, Seese RR, Yun K, Peng T, Wang Z. The role of galanin system in modulating depression, anxiety, and addiction-like behaviors after chronic restraint stress. Neuroscience. 2013;246:82–93.View ArticlePubMedGoogle Scholar
  47. Seo MK, Lee CH, Cho HY, You YS, Lee BJ, Lee JG, et al. Effects of antipsychotic drugs on the expression of synapse-associated proteins in the frontal cortex of rats subjected to immobilization stress. Psychiatry Res. 2015;229:968–74.View ArticlePubMedGoogle Scholar
  48. McLaughlin KJ, Gomez JL, Baran SE, Conrad CD. The effects of chronic stress on hippocampal morphology and function: an evaluation of chronic restraint paradigms. Brain Res. 2007;1161:56–64.View ArticlePubMedPubMed CentralGoogle Scholar
  49. Chandran A, Iyo AH, Jernigan CS, Legutko B, Austin MC, Karolewicz B. Reduced phosphorylation of the mTOR signaling pathway components in the amygdala of rats exposed to chronic stress. Prog Neuropsychopharmacol Biol Psychiatry. 2013;40:240–5.View ArticlePubMedGoogle Scholar
  50. Li N, Liu RJ, Dwyer JM, Banasr M, Lee B, Son H, et al. Glutamate N-methyl-D-aspartate receptor antagonists rapidly reverse behavioral and synaptic deficits caused by chronic stress exposure. Biol Psychiatry. 2011;69:754–61.View ArticlePubMedPubMed CentralGoogle Scholar
  51. Ota KT, Liu RJ, Voleti B, Maldonado-Aviles JG, Duric V, Iwata M, et al. REDD1 is essential for stress-induced synaptic loss and depressive behavior. Nat Med. 2014;20:531–5.View ArticlePubMedPubMed CentralGoogle Scholar
  52. Proud CG. Signalling to translation: how signal transduction pathways control the protein synthetic machinery. Biochem J. 2007;403:217–34.View ArticlePubMedGoogle Scholar
  53. Wang X, Li W, Parra JL, Beugnet A, Proud CG. The C terminus of initiation factor 4E-binding protein 1 contains multiple regulatory features that influence its function and phosphorylation. Mol Cell Biol. 2003;23:1546–57.View ArticlePubMedPubMed CentralGoogle Scholar
  54. Kuang E, Fu B, Liang Q, Myoung J, Zhu F. Phosphorylation of eukaryotic translation initiation factor 4B (EIF4B) by open reading frame 45/p90 ribosomal S6 kinase (ORF45/RSK) signaling axis facilitates protein translation during Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication. J Biol Chem. 2011;286:41171–82.View ArticlePubMedPubMed CentralGoogle Scholar
  55. Huang P, Li C, Fu T, Zhao D, Yi Z, Lu Q, et al. Flupirtine attenuates chronic restraint stress-induced cognitive deficits and hippocampal apoptosis in male mice. Behav Brain Res. 2015;288:1–10.View ArticlePubMedGoogle Scholar
  56. Park SW, Phuong VT, Lee CH, Lee JG, Seo MK, Cho HY, et al. Effects of antipsychotic drugs on BDNF, GSK-3beta, and beta-catenin expression in rats subjected to immobilization stress. Neurosci Res. 2011;71:335–40.View ArticlePubMedGoogle Scholar
  57. Chiba S, Numakawa T, Ninomiya M, Richards MC, Wakabayashi C, Kunugi H. Chronic restraint stress causes anxiety- and depression-like behaviors, downregulates glucocorticoid receptor expression, and attenuates glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex. Prog Neuropsychopharmacol Biol Psychiatry. 2012;39:112–9.View ArticlePubMedGoogle Scholar
  58. Suvrathan A, Tomar A, Chattarji S. Effects of chronic and acute stress on rat behaviour in the forced-swim test. Stress. 2010;13:533–40.View ArticlePubMedGoogle Scholar
  59. Bernal-Morales B, Contreras CM, Cueto-Escobedo J. Acute restraint stress produces behavioral despair in weanling rats in the forced swim test. Behav Process. 2009;82:219–22.View ArticleGoogle Scholar
  60. Eren I, Naziroglu M, Demirdas A. Protective effects of lamotrigine, aripiprazole and escitalopram on depression-induced oxidative stress in rat brain. Neurochem Res. 2007;32:1188–95.View ArticlePubMedGoogle Scholar
  61. Martinez-Turrillas R, Del Rio J, Frechilla D. Sequential changes in BDNF mRNA expression and synaptic levels of AMPA receptor subunits in rat hippocampus after chronic antidepressant treatment. Neuropharmacology. 2005;49:1178–88.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s) 2017

Advertisement