Neurochem Res (2015) 40:36–40 DOI 10.1007/s11064-014-1462-4
ORIGINAL PAPER
Acute Restraint Stress Impairs Induction of Long-Term Potentiation by Activating GSK-3b Yu Jin • Takeshi Kanno • Tomoyuki Nishizaki
Received: 12 August 2014 / Revised: 17 October 2014 / Accepted: 20 October 2014 / Published online: 30 October 2014 Ó Springer Science+Business Media New York 2014
Abstract The present study investigated the effect of acute restraint stress on long-term potentiation (LTP) and the underlying mechanism. Induction of Schaffer collateral-CA1 LTP was suppressed in hippocampal slices from mice with 1-h restraint stress. Cell surface localization of the N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B and the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2 was not affected in the hippocampus from mice with 1-h restraint stress. Phosphorylation of Akt at Ser473, but not at Thr308, and phosphorylation of GSK-3b at Ser9, but not at Tyr216, were significantly inhibited in the hippocampus with 1-h restraint stress. Taken together, the results of the present study show that acute restraint stress impairs induction of LTP by enhancing GSK-3b activity following suppressed Akt activity, without affecting cell surface localization of the NMDA and AMPA receptor subunits. Keywords Restraint stress Long-term potentiation Suppression Akt GSK-3b
Introduction Stress perturbs functioning in the some regions of the brain including the hippocampus, amygdala, and prefrontal cortex
Yu Jin and Takeshi Kanno have contributed equally to this work. Y. Jin T. Kanno T. Nishizaki (&) Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan e-mail: [email protected]
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[1–6]. Restraint is a preferred means of stressing animals [7], and restraint stress induces depression-like behaviors [8, 9]. Lines of evidence have pointed to the relation between 5-HT1A receptor and depression [10]. We have earlier found that restraint stress significantly inhibits translocation of 5-HT1A receptor, but not 5-HT2A receptor, from the cytosol towards the plasma membrane in the mouse hypothalamus as compared with that for non-stress control mice [8]. The reduction in the cell surface localization of 5-HT1A receptor in the hypothalamus highly correlated to prolongation of immobility time in the forced swim test, i.e., the progression of depression-like behavior [8]. Glycogen synthase kinase 3b (GSK-3b), a serine/threonine protein kinase, is an integral regulator in serotonergic transmission-mediated signaling networks relevant to mental illnesses including mood disorder, psychosis, and depression [10–13]. In support of this note, depression-like behavior-inducible restraint stress enhanced GSK-3b activity in the brain [8, 9]. Lithium has the potential to inhibit GSK-3b, and therefore, lithium is clinically used as an antidepressant [13]. GSK-3b is well-recognized to phosphorylate Tau, a microtuble-associated protein, resulting in Tau aggregation to form neurofibrillary tangles (NFTs), responsible for tauopathy including Alzheimer’s disease [14]. Moreover, GSK-3b regulates synaptic plasticity relevant to learning and memory such as long-tem potentiation (LTP) and longterm depression (LTD). Notably, inhibition of GSK-3b facilitates induction of LTP [15], suggesting that activation of GSK-3b impairs LTP, i.e., learning and memory. Overall, depression and dementia appear to be caused at least in part by the common mechanism GSK-3b activation. Then, we postulated that restraint stress to induce depression-like behavior might have some effect on the expression of LTP.
Neurochem Res (2015) 40:36–40
To address this issue, we examined the effect of acute restraint stress on LTP, subcellular distribution of the Nmethyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B and the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, and activities of Akt and GSK-3b in the mouse hippocampus. We show here that acute restraint stress suppresses induction of LTP by enhancing GSK-3b activity in association with reduced Akt activity, without affecting cell surface localization of NR1, NR2B, GluA1, and GluA2.
Materials and Methods Animal Care All procedures have been approved by the Animal Care and Use Committee at Hyogo College of Medicine and were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Restraint Stressed Mice Male C57BL/6J mice were obtained at 8 week of age from Japan SLC Inc. (Shizuoka. Japan). Mice were restricted in a plastic cylinder 11.5 cm in height and 2.7 cm in diameter for 1 h. Under urethane (1.5 g/kg, intraperitoneal injection) anesthesia, mice were sacrificed 1 h after restraint stress and hippocampal slices (400 lm in thickness) were prepared. LTP Monitoring Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region of hippocampal slices from mice without and with 1-h restraint stress by electrically stimulating the Schaffer collateral (0.033 Hz, 0.1 ms in duration). The parameters for high frequency stimulation (HFS) to induce LTP were four trains with an inter-train interval of 200 ms and each train consisted of ten 30-s 200-Hz pulses. Western Blotting The hippocampus isolated from mice without and with 1-h restraint stress were homogenized by sonication in an icecold mitochondrial buffer (210 mM mannitol, 70 mM sucrose, and 1 mM EDTA, 10 mM HEPES, pH 7.5) containing 1 % (v/v) protease inhibitor cocktail and centrifuged at 800g for 5 min at 4 °C. Some supernatants were used as total lysates for Western blotting using antibodies
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against phospho-Thr308-Akt (pThr308) (Cell Signaling, Beverly, MA, USA), phospho-Ser473-Akt (pSer473) (Cell Signaling), Akt (Cell Signaling), phospho-Ser9-GSK-3b (pSer9) (Cell Signaling), phospho-Tyr216-GSK-3b (pTyr216) (BD Biosciences, San Jose, CA, USA), and GSK-3b (Cell Signaling). The remaining supernatants were further centrifuged at 11,000g for 15 min at 4 °C, and the collected supernatants were further ultracentrifuged at 100,000g for 60 min at 4 °C. The supernatants and pellets were used as the cytosolic and plasma membrane fractions, respectively, followed by Western blotting using antibodies against NR1 (EMD Millipore, Billerica, MA, USA), NR2B (EMD Millipore), GluA1 (EMD Millipore), and GluA2 (EMD Millipore). After washing, membranes were reacted with a horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG antibody. Whether the cytosolic and plasma membrane components were successfully separated was confirmed in the Western blot analysis using antibodies against LDH (Santa Cruz Biotechnology, Dallas, Texas, USA), a cytosolic marker, and cadherin (Sigma, St Louis, MO, USA), a plasma membrane marker. Immunoreactivity was detected with an ECL kit (Invitrogen, Carlsbad, CA, USA) and visualized using a chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). Protein concentrations for the supernatants were determined using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Statistical Analysis Statistical analysis was carried out using Dunnett’s test and unpaired t test.
Results Acute Restraint Stress Impairs LTP Induction We initially monitored fEPSPs in the CA1 region of hippocampal slices from mice without and with 1-h restraint stress. High frequency stimulation (HFS) induced a transient increase in the fEPSP slope to approximately 200 % of basal levels followed by gradual decrease to about 150 % of basal levels, being evident 60 min after HFS for non-stress control mice (Fig. 1). In contrast, an HFSinduced increase in the fEPSP slope for stress mice was significantly suppressed in the initial phase as compared with that for control mice (P \ 0.001 during 5 min after HFS, Dunnett’s test) (Fig. 1). This indicates that acute restraint stress suppresses induction of LTP.
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mice, but there was no significant difference in the Thr308 phosphorylation level between two groups (Fig. 3a). This indicates that acute restraint stress reduces Akt activity. GSK-3b is activated through Ser9 dephosphorylation and Tyr216 phosphorylation. Ser9 phosphorylation of GSK-3b in the hippocampus from mice with acute restraint stress was significantly inhibited as compared with that from non-stress control mice, while there was no significant difference in the Tyr216 phosphorylation level between two groups (Fig. 3b). This indicates that acute restraint stress enhances GSK-3b activity.
Fig. 1 Effect of acute restraint stress on LTP. fEPSPs were monitored in the CA1 region of hippocampal slices from mice without (Control) and with 1-h restraint stress (Stress), and HFS (arrow) was applied to induce LTP. fEPSPs illustrated were recoded just before (20 min) (1) and 5 min (2) after HFS. In the graph, each point represents the mean (±SEM) percentage of basal fEPSP slope (20 min) (n = 7 independent mice without restraint stress and 6 independent mice with restraint stress)
Acute Restraint Stress does not Affect Subcellular Distribution of the NMDA and AMPA Receptor Subunits We next examined subcellular distribution of the NMDA receptor subunits NR1 and NR2B and the AMPA receptor subunits GluA1 and GluA2, which are the major mediator of excitatory synaptic transmission, in the hippocampus from mice without and with 1-h restraint stress. There was no significant difference in the cell surface localization of NR1 and NR2B in the hippocampus between mice without and with acute restraint stress (Fig. 2a). Likewise, acute restraint stress did not affect cell surface localization of GluA1 and GluA2 in the hippocampus (Fig. 2b). Taken together, these results suggest that acute restraint stressinduced impairment of LTP induction is not ascribed to reduction of cell surface localization of the NMDA receptor subunits NR1 and NR2B or the AMPA receptor subunits GluA1 and GluA2. Acute Restraint Stress Inhibits Akt and Activates GSK3b We finally examined the effect of acute restraint stress on activities of Akt and GSK-3b in the hippocampus from mice without and with 1-h restraint stress. Akt is activated by being phosphorylated at Thr308 and Ser473. Ser473 phosphorylation in the hippocampus from mice with acute restraint stress was significantly inhibited as compared with that from non-stress control
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Discussion Depression causes neuroplastic changes due to suppression of neural stem cell proliferation, decreased survival of neuroblasts and immature neurons, impairment of the cortical–striatal–limbic circuits, reduction of neurotrophins including BDNF, VEGF, NGF and IGF-1, and attenuation of spine density and dendritic retraction [16]. Moreover, emerging evidence has pointed to an intimate cross-talk between neuroimmune factors and neuroplasticity in depression. Neuroimmune factors such as IL-1, IL-6, and TNF-a, which are associated with reduced neurogenesis in the rodent models of depression, regulate cognitive functions by affecting synaptic plasticity like LTP [17, 18]. In the present study, acute restraint stress suppressed induction of LTP. Indeed, chronic restraint stress induced spatial memory disorder as well as depression-like behavior in mice [9]. The results of the present study also suggest that acute stress, but not chronic stress, could still impair learning and memory. Then, the question addressing is how acute restraint stress suppresses induction of LTP. A consistent finding is that Schaffer collaretral-CA1 LTP is dependent upon NMDA receptor. Interestingly, the LTP monitored in hippocampal slices from chronically epileptic rats is abolished by the NR2B-preferring antagonist Ro25-6981 [19], indicating the implication of NR2B in the expression of LTP. In the present study, however, acute restraint stress did not affect cell surface localization of NR2B nor NR1 in the hippocampus. Moreover, cell surface localization of GluA1 and GluA2 in the hippocampus was not also affected by acute restraint stress. Taken together, these results indicate that acute restraint stress-induced impairment of LTP induction is not ascribed to changes in the subcellular distribution of the NMDA and AMPA receptor subunits. Akt, a serine/threonine kinase, is activated by being phosphorylated at Thr308 and Ser473 through receptor
Neurochem Res (2015) 40:36–40
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Fig. 2 Effect of acute restraint stress on subcellular distribution of NMDA and AMPA receptor subunits. The hippocampus isolated from mice without (Control) and with 1-h restraint stress (Stress) were separated into the cytosolic (C) and plasma membrane fractions (M), followed by Western blotting. a Subcellular distribution of NR1 and
NR2B. b Subcellular distribution of GluA1 and GluA2. In the graphs, each column represents the mean (±SEM) signal intensity for each subunit on the plasma membrane relative to that for total cells (n = 8 independent experiments). C control, RS restraint stress, NS not significant; unpaired t test
Fig. 3 Effect of acute restraint stress on activities of Akt and GSK3b. The hippocampus isolated from mice without (C) and with 1-h restraint stress (RS) were lysed, followed by Western blotting. a In the graph, each column represents the mean (±SEM) ratio: signal intensity for phospo-Thr-308-Akt (pThr308) and phospo-Ser-473-Akt (pSer473) relative to that for total Akt (n = 8 independent
experiments). P values, unpaired t test. NS not significant. b In the graph, each column represents the mean (±SEM) ratio: signal intensity for phospho-Ser9-GSK-3b (pSer9) and phospho-Tyr216GSK-3b (pTyr216) relative to that for total GSK-3b (n = 8 independent experiments). P values, unpaired t test. NS not significant
tyrosine kinases (RTKs). RTKs include receptors for the neurotrophins BDNF, VEGF, NGF and IGF-1. In the present study, acute restraint stress significantly inhibited Akt phosphorylation at Ser473, but not at Thr308, in the hippocampus. This implies that acute restraint stress suppresses Akt activity. Reduced levels of BDNF, VEGF,
NGF and IGF-1 in depression, therefore, may be a factor responsible for suppression of Akt activity [16]. GSK-3b, a serine/threonine kinase, is implicated in the regulation of diverse cellular functions including metabolism, transcription, and cell survival. GSK-3b is activated by being dephosphorylated at Ser9 and autophosphorylated
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at Tyr216. GSK-3b is a substrate of Akt, and Akt inactivates GSK-3b by phosphorylating at Ser9. In the present study, acute restraint stress significantly inhibited GSK-3b phosphorylation at Ser9, but not at Tyr216, in the hippocampus. This implies that acute restraint stress enhances GSK-3b activity. Taken together, these results indicate that acute restraint stress suppresses Akt activity, to inhibit Ser9 phosphorylation of GSK-3b, resulting in the enhancement of GSK-3b activity. Consequently, these results indicate that acute restraint stress impairs induction of LTP by enhancing GSK-3b activity following suppressed Akt activity. In conclusion, the results of the present study show that acute restraint stress inhibits Akt, thereby activating GSK3b, resulting in the impairment of LTP induction, without affecting cell surface localization of the NMDA receptor subunits NR1 and NR2B or the AMPA receptor subunits GluA1 and GluA2. This may represent the mechanism for cognitive impairment in association with depressive disorders. Conflict of interest of interest.
The authors declare that they have no conflict
References 1. Eiland L, Ramroop J, Hill MN, Manley J, McEwen BS (2012) Chronic juvenile stress produces corticolimbic dendritic architectural remodeling and modulates emotional behavior in male and female rats. Psychoneuroendocrinology 37:39–47 2. Zamora-Gonzales EO, Santerre A, Palomera-Avalos V, MoralesVillagran A (2013) A chronic combinatory stress model that activates the HPA axis and avoids habituation in BALB/C mice. J Neurosci Methods 213:70–75 3. Mika A, Mazur GJ, Hoffman AN, Talboom JS, Bimonte-Nelson HA, Sanabria F, Conrad CD (2012) Chronic stress impairs prefrontal cortex-dependent response inhibition and spatial working memory. Behav Neurosci 126:605–619 4. Mitra R, Jadhav S, McEwen BS, Vyas A, Chattarji S (2005) Stress duration modulates the spatiotemporal patterns of spine formation in the basolateral amygdala. Proc Natl Acad Sci USA 102:9371–9376
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5. Palazidou E (2012) The neurobiology of depression. Br Med Bull 101:127–145 6. Vyas A, Mitra R, Shankaranarayana Rao BS, Chattarji S (2002) Chronic stress induces contrasting patterns of dendritic remodeling in hippocampal and amygdaloid neurons. J Neurosci 22:6810–6818 7. Buynitsky T, Mostofsky DI (2009) Restraint stress in biobehavioral research: recent developments. Neurosci Biobehav Rev 33:1089–1098 8. Kanno T, Tanaka A, Nishizaki T (2014) Linoleic acid derivative DCP-LA ameliorates stress-induced depression-related behavior by promoting cell surface 5-HT1A receptor translocation, stimulating serotonin release, and inactivating GSK-3b. Mol Neurobiol, in press 9. Kanno T, Yu J, Nishizaki T (2014) DL-/PO-phosphatidylcholine restores restraint stress-induced depression-related behaviors and spatial memory impairment. Behav Pharmacol 25:575–581 10. Latapy C, Rioux V, Guitton MJ, Beaulieu JM (2012) Selective deletion of forebrain glycogen synthase kinase 3b reveals a central role in serotonin-sensitive anxiety and social behaviour. Philos Trans R Soc Lond B Biol Sci 367:2460–2474 11. Li X, Jope RS (2010) Is glycogen synthase kinase-3 a central modulator in mood regulation? Neuropsychopharmacology 35:2143–2154 12. Polter AM, Yang S, Jope RS, Li X (2012) Functional significance of glycogen synthase kinase-3 regulation by serotonin. Cell Signal 24:265–271 13. Beaulieu JM, Gainetdinov RR, Caron MG (2009) Akt/GSK3 signaling in the action of psychotropic drugs. Annu Rev Pharmacol Toxicol 49:327–347 14. Kolarova M, Garcı´a-Sierra F, Bartos A, Ricny J, Ripova D (2012) Structure and pathology of tau protein in Alzheimer disease. Int J Alzheimers Dis 2012:731526 15. Hooper C, Markevich V, Plattner F, Killick R, Schofield E, Engel T, Hernandez F, Anderton B, Rosenblum K, Bliss T, Cooke SF, Avila J, Lucas JJ, Giese KP, Stephenson J, Lovestone S (2007) Glycogen synthase kinase-3 inhibition is integral to long-term potentiation. Eur J Neurosci 25:81–86 16. Eyre H, Baune BT (2012) Neuroplastic changes in depression: a role for the immune system. Psychoneuroendocrinology 37:1397–1416 17. McAfoose J, Baune BT (2009) Evidence for a cytokine model of cognitive function. Neurosci Biobehav Rev 33:355–366 18. Molina-Holgado E, Molina-Holgado F (2010) Mending the broken brain: neuroimmune interactions in neurogenesis. J Neurochem 114:1277–1290 19. Muller L, Tokay T, Porath K, Ko¨hling R, Kirschstein T (2013) Enhanced NMDA receptor-dependent LTP in the epileptic CA1 area via upregulation of NR2B. Neurobiol Dis 54:183–193
ORIGINAL PAPER
Acute Restraint Stress Impairs Induction of Long-Term Potentiation by Activating GSK-3b Yu Jin • Takeshi Kanno • Tomoyuki Nishizaki
Received: 12 August 2014 / Revised: 17 October 2014 / Accepted: 20 October 2014 / Published online: 30 October 2014 Ó Springer Science+Business Media New York 2014
Abstract The present study investigated the effect of acute restraint stress on long-term potentiation (LTP) and the underlying mechanism. Induction of Schaffer collateral-CA1 LTP was suppressed in hippocampal slices from mice with 1-h restraint stress. Cell surface localization of the N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B and the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2 was not affected in the hippocampus from mice with 1-h restraint stress. Phosphorylation of Akt at Ser473, but not at Thr308, and phosphorylation of GSK-3b at Ser9, but not at Tyr216, were significantly inhibited in the hippocampus with 1-h restraint stress. Taken together, the results of the present study show that acute restraint stress impairs induction of LTP by enhancing GSK-3b activity following suppressed Akt activity, without affecting cell surface localization of the NMDA and AMPA receptor subunits. Keywords Restraint stress Long-term potentiation Suppression Akt GSK-3b
Introduction Stress perturbs functioning in the some regions of the brain including the hippocampus, amygdala, and prefrontal cortex
Yu Jin and Takeshi Kanno have contributed equally to this work. Y. Jin T. Kanno T. Nishizaki (&) Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan e-mail: [email protected]
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[1–6]. Restraint is a preferred means of stressing animals [7], and restraint stress induces depression-like behaviors [8, 9]. Lines of evidence have pointed to the relation between 5-HT1A receptor and depression [10]. We have earlier found that restraint stress significantly inhibits translocation of 5-HT1A receptor, but not 5-HT2A receptor, from the cytosol towards the plasma membrane in the mouse hypothalamus as compared with that for non-stress control mice [8]. The reduction in the cell surface localization of 5-HT1A receptor in the hypothalamus highly correlated to prolongation of immobility time in the forced swim test, i.e., the progression of depression-like behavior [8]. Glycogen synthase kinase 3b (GSK-3b), a serine/threonine protein kinase, is an integral regulator in serotonergic transmission-mediated signaling networks relevant to mental illnesses including mood disorder, psychosis, and depression [10–13]. In support of this note, depression-like behavior-inducible restraint stress enhanced GSK-3b activity in the brain [8, 9]. Lithium has the potential to inhibit GSK-3b, and therefore, lithium is clinically used as an antidepressant [13]. GSK-3b is well-recognized to phosphorylate Tau, a microtuble-associated protein, resulting in Tau aggregation to form neurofibrillary tangles (NFTs), responsible for tauopathy including Alzheimer’s disease [14]. Moreover, GSK-3b regulates synaptic plasticity relevant to learning and memory such as long-tem potentiation (LTP) and longterm depression (LTD). Notably, inhibition of GSK-3b facilitates induction of LTP [15], suggesting that activation of GSK-3b impairs LTP, i.e., learning and memory. Overall, depression and dementia appear to be caused at least in part by the common mechanism GSK-3b activation. Then, we postulated that restraint stress to induce depression-like behavior might have some effect on the expression of LTP.
Neurochem Res (2015) 40:36–40
To address this issue, we examined the effect of acute restraint stress on LTP, subcellular distribution of the Nmethyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B and the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, and activities of Akt and GSK-3b in the mouse hippocampus. We show here that acute restraint stress suppresses induction of LTP by enhancing GSK-3b activity in association with reduced Akt activity, without affecting cell surface localization of NR1, NR2B, GluA1, and GluA2.
Materials and Methods Animal Care All procedures have been approved by the Animal Care and Use Committee at Hyogo College of Medicine and were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Restraint Stressed Mice Male C57BL/6J mice were obtained at 8 week of age from Japan SLC Inc. (Shizuoka. Japan). Mice were restricted in a plastic cylinder 11.5 cm in height and 2.7 cm in diameter for 1 h. Under urethane (1.5 g/kg, intraperitoneal injection) anesthesia, mice were sacrificed 1 h after restraint stress and hippocampal slices (400 lm in thickness) were prepared. LTP Monitoring Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region of hippocampal slices from mice without and with 1-h restraint stress by electrically stimulating the Schaffer collateral (0.033 Hz, 0.1 ms in duration). The parameters for high frequency stimulation (HFS) to induce LTP were four trains with an inter-train interval of 200 ms and each train consisted of ten 30-s 200-Hz pulses. Western Blotting The hippocampus isolated from mice without and with 1-h restraint stress were homogenized by sonication in an icecold mitochondrial buffer (210 mM mannitol, 70 mM sucrose, and 1 mM EDTA, 10 mM HEPES, pH 7.5) containing 1 % (v/v) protease inhibitor cocktail and centrifuged at 800g for 5 min at 4 °C. Some supernatants were used as total lysates for Western blotting using antibodies
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against phospho-Thr308-Akt (pThr308) (Cell Signaling, Beverly, MA, USA), phospho-Ser473-Akt (pSer473) (Cell Signaling), Akt (Cell Signaling), phospho-Ser9-GSK-3b (pSer9) (Cell Signaling), phospho-Tyr216-GSK-3b (pTyr216) (BD Biosciences, San Jose, CA, USA), and GSK-3b (Cell Signaling). The remaining supernatants were further centrifuged at 11,000g for 15 min at 4 °C, and the collected supernatants were further ultracentrifuged at 100,000g for 60 min at 4 °C. The supernatants and pellets were used as the cytosolic and plasma membrane fractions, respectively, followed by Western blotting using antibodies against NR1 (EMD Millipore, Billerica, MA, USA), NR2B (EMD Millipore), GluA1 (EMD Millipore), and GluA2 (EMD Millipore). After washing, membranes were reacted with a horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG antibody. Whether the cytosolic and plasma membrane components were successfully separated was confirmed in the Western blot analysis using antibodies against LDH (Santa Cruz Biotechnology, Dallas, Texas, USA), a cytosolic marker, and cadherin (Sigma, St Louis, MO, USA), a plasma membrane marker. Immunoreactivity was detected with an ECL kit (Invitrogen, Carlsbad, CA, USA) and visualized using a chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). Protein concentrations for the supernatants were determined using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Statistical Analysis Statistical analysis was carried out using Dunnett’s test and unpaired t test.
Results Acute Restraint Stress Impairs LTP Induction We initially monitored fEPSPs in the CA1 region of hippocampal slices from mice without and with 1-h restraint stress. High frequency stimulation (HFS) induced a transient increase in the fEPSP slope to approximately 200 % of basal levels followed by gradual decrease to about 150 % of basal levels, being evident 60 min after HFS for non-stress control mice (Fig. 1). In contrast, an HFSinduced increase in the fEPSP slope for stress mice was significantly suppressed in the initial phase as compared with that for control mice (P \ 0.001 during 5 min after HFS, Dunnett’s test) (Fig. 1). This indicates that acute restraint stress suppresses induction of LTP.
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mice, but there was no significant difference in the Thr308 phosphorylation level between two groups (Fig. 3a). This indicates that acute restraint stress reduces Akt activity. GSK-3b is activated through Ser9 dephosphorylation and Tyr216 phosphorylation. Ser9 phosphorylation of GSK-3b in the hippocampus from mice with acute restraint stress was significantly inhibited as compared with that from non-stress control mice, while there was no significant difference in the Tyr216 phosphorylation level between two groups (Fig. 3b). This indicates that acute restraint stress enhances GSK-3b activity.
Fig. 1 Effect of acute restraint stress on LTP. fEPSPs were monitored in the CA1 region of hippocampal slices from mice without (Control) and with 1-h restraint stress (Stress), and HFS (arrow) was applied to induce LTP. fEPSPs illustrated were recoded just before (20 min) (1) and 5 min (2) after HFS. In the graph, each point represents the mean (±SEM) percentage of basal fEPSP slope (20 min) (n = 7 independent mice without restraint stress and 6 independent mice with restraint stress)
Acute Restraint Stress does not Affect Subcellular Distribution of the NMDA and AMPA Receptor Subunits We next examined subcellular distribution of the NMDA receptor subunits NR1 and NR2B and the AMPA receptor subunits GluA1 and GluA2, which are the major mediator of excitatory synaptic transmission, in the hippocampus from mice without and with 1-h restraint stress. There was no significant difference in the cell surface localization of NR1 and NR2B in the hippocampus between mice without and with acute restraint stress (Fig. 2a). Likewise, acute restraint stress did not affect cell surface localization of GluA1 and GluA2 in the hippocampus (Fig. 2b). Taken together, these results suggest that acute restraint stressinduced impairment of LTP induction is not ascribed to reduction of cell surface localization of the NMDA receptor subunits NR1 and NR2B or the AMPA receptor subunits GluA1 and GluA2. Acute Restraint Stress Inhibits Akt and Activates GSK3b We finally examined the effect of acute restraint stress on activities of Akt and GSK-3b in the hippocampus from mice without and with 1-h restraint stress. Akt is activated by being phosphorylated at Thr308 and Ser473. Ser473 phosphorylation in the hippocampus from mice with acute restraint stress was significantly inhibited as compared with that from non-stress control
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Discussion Depression causes neuroplastic changes due to suppression of neural stem cell proliferation, decreased survival of neuroblasts and immature neurons, impairment of the cortical–striatal–limbic circuits, reduction of neurotrophins including BDNF, VEGF, NGF and IGF-1, and attenuation of spine density and dendritic retraction [16]. Moreover, emerging evidence has pointed to an intimate cross-talk between neuroimmune factors and neuroplasticity in depression. Neuroimmune factors such as IL-1, IL-6, and TNF-a, which are associated with reduced neurogenesis in the rodent models of depression, regulate cognitive functions by affecting synaptic plasticity like LTP [17, 18]. In the present study, acute restraint stress suppressed induction of LTP. Indeed, chronic restraint stress induced spatial memory disorder as well as depression-like behavior in mice [9]. The results of the present study also suggest that acute stress, but not chronic stress, could still impair learning and memory. Then, the question addressing is how acute restraint stress suppresses induction of LTP. A consistent finding is that Schaffer collaretral-CA1 LTP is dependent upon NMDA receptor. Interestingly, the LTP monitored in hippocampal slices from chronically epileptic rats is abolished by the NR2B-preferring antagonist Ro25-6981 [19], indicating the implication of NR2B in the expression of LTP. In the present study, however, acute restraint stress did not affect cell surface localization of NR2B nor NR1 in the hippocampus. Moreover, cell surface localization of GluA1 and GluA2 in the hippocampus was not also affected by acute restraint stress. Taken together, these results indicate that acute restraint stress-induced impairment of LTP induction is not ascribed to changes in the subcellular distribution of the NMDA and AMPA receptor subunits. Akt, a serine/threonine kinase, is activated by being phosphorylated at Thr308 and Ser473 through receptor
Neurochem Res (2015) 40:36–40
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Fig. 2 Effect of acute restraint stress on subcellular distribution of NMDA and AMPA receptor subunits. The hippocampus isolated from mice without (Control) and with 1-h restraint stress (Stress) were separated into the cytosolic (C) and plasma membrane fractions (M), followed by Western blotting. a Subcellular distribution of NR1 and
NR2B. b Subcellular distribution of GluA1 and GluA2. In the graphs, each column represents the mean (±SEM) signal intensity for each subunit on the plasma membrane relative to that for total cells (n = 8 independent experiments). C control, RS restraint stress, NS not significant; unpaired t test
Fig. 3 Effect of acute restraint stress on activities of Akt and GSK3b. The hippocampus isolated from mice without (C) and with 1-h restraint stress (RS) were lysed, followed by Western blotting. a In the graph, each column represents the mean (±SEM) ratio: signal intensity for phospo-Thr-308-Akt (pThr308) and phospo-Ser-473-Akt (pSer473) relative to that for total Akt (n = 8 independent
experiments). P values, unpaired t test. NS not significant. b In the graph, each column represents the mean (±SEM) ratio: signal intensity for phospho-Ser9-GSK-3b (pSer9) and phospho-Tyr216GSK-3b (pTyr216) relative to that for total GSK-3b (n = 8 independent experiments). P values, unpaired t test. NS not significant
tyrosine kinases (RTKs). RTKs include receptors for the neurotrophins BDNF, VEGF, NGF and IGF-1. In the present study, acute restraint stress significantly inhibited Akt phosphorylation at Ser473, but not at Thr308, in the hippocampus. This implies that acute restraint stress suppresses Akt activity. Reduced levels of BDNF, VEGF,
NGF and IGF-1 in depression, therefore, may be a factor responsible for suppression of Akt activity [16]. GSK-3b, a serine/threonine kinase, is implicated in the regulation of diverse cellular functions including metabolism, transcription, and cell survival. GSK-3b is activated by being dephosphorylated at Ser9 and autophosphorylated
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at Tyr216. GSK-3b is a substrate of Akt, and Akt inactivates GSK-3b by phosphorylating at Ser9. In the present study, acute restraint stress significantly inhibited GSK-3b phosphorylation at Ser9, but not at Tyr216, in the hippocampus. This implies that acute restraint stress enhances GSK-3b activity. Taken together, these results indicate that acute restraint stress suppresses Akt activity, to inhibit Ser9 phosphorylation of GSK-3b, resulting in the enhancement of GSK-3b activity. Consequently, these results indicate that acute restraint stress impairs induction of LTP by enhancing GSK-3b activity following suppressed Akt activity. In conclusion, the results of the present study show that acute restraint stress inhibits Akt, thereby activating GSK3b, resulting in the impairment of LTP induction, without affecting cell surface localization of the NMDA receptor subunits NR1 and NR2B or the AMPA receptor subunits GluA1 and GluA2. This may represent the mechanism for cognitive impairment in association with depressive disorders. Conflict of interest of interest.
The authors declare that they have no conflict
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