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Hippocampal signaling pathways are involved in stress-induced impairment of memory formation in rats
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Hippocampal signaling pathways are involved in stress-induced impairment of memory formation in rats Maryam Sardari, Ameneh Rezayof, Fariba Khodagholi
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S0006-8993(15)00632-0 http://dx.doi.org/10.1016/j.brainres.2015.08.015 BRES44418
To appear in: Brain Research
Accepted date: 16 August 2015 Cite this article as: Maryam Sardari, Ameneh Rezayof, Fariba Khodagholi, Hippocampal signaling pathways are involved in stress-induced impairment of memory formation in rats, Brain Research, http://dx.doi.org/10.1016/j. brainres.2015.08.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hippocampal signaling pathways are involved in stress-induced impairment of memory formation in rats
Maryam Sardaria, Ameneh Rezayofa, Fariba Khodagholib
a
Department of Animal Biology, School of Biology and Center of Excellence in Phylogeny of
Living Organisms, College of Science, University of Tehran, Tehran, Iran b
Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Correspondence to: A. Rezayof, PhD. Professor, Department of Animal Biology, School of Biology, College of Science, University of Tehran, P. O. Box 4155-6455, Tehran, Iran Fax: (+9821)-66405141 Tel: (+9821)-61112483 e-mail: [email protected]
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Abstract Stress is a potent modulator of hippocampal-dependent memory formation. The aim of the present study was to assess the role of hippocampal signaling pathways in stress-induced memory impairment in male Wistar rats. The animals were exposed to acute elevated platform (EP) stress and memory formation was measured by a step-through type passive avoidance task. The results indicated that post-training or pre-test exposure to EP stress impaired memory consolidation or retrieval respectively. Using western blot analysis, it was found that memory retrieval was associated with the increase in the levels of phosphorylated cAMP-responsive element binding protein (P-CREB), peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1α) and its downstream targets in the hippocampus. In contrast, the stress exposure decreased the hippocampal levels of these proteins. In addition, stress-induced impairment of memory consolidation or retrieval was associated with the decrease in the PCREB/CREB ratio and the PGC-1α level in the hippocampus. On the other hand, the hippocampal level of nuclear factor E2-related factor 2 (Nrf2) and gamma-glutamylcysteine synthetase (γ-GCS) which are the master regulators of defense system were decreased by the stress exposure. The increased hippocampal levels of Nrf2 and it’s downstream was observed during memory retrieval, while stress-induced impairment of memory consolidation or retrieval inhibited this hippocampal signaling pathway. Overall, these findings suggest that downregulation of CREB/PGC-1α signaling cascade and Nrf2 antioxidant pathways in the hippocampus may be associated with memory impairment induced by stress.
Keywords: Acute stress; Memory formation; Hippocampal signaling pathway; Rat(s)
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Abbreviations AD, Alzheimer's disease; APP/PS1, Amyloid precursor protein/presenilin 1; ARE, Antioxidant responsive element; CAT, Activity of catalase; CBP, cAMP-responsive element binding protein (CREB)-binding protein; DTNB, dithionitrobenzoic acid; ECL, Electrochemiluminescence; γGCS, Gamma-glutamylcysteine synthetase; GSH, glutathione; HO-1, Heme oxygenase-1; Keap1, Kelch-like ECH-associated protein1; mRNA, messenger ribonucleic acid; NRF-1, neuronal expression of transcription factors including nuclear respiratory factor-1; Nrf2, nuclear factor E2-related factor 2; P-CREB, Phosphorylated cAMP-responsive element binding protein; PGC-1α, Peroxisome proliferator-activated receptor gamma coactivator-1a; PVDF, Poly vinylidene fluoride membrane; ROS,
reactive oxygen species; TFAM, mitochondrial
transcription factor A.
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1. Introduction A large body of evidence suggests that the hippocampus plays a vital role in memory formation via increasing the efficiency of synaptic transmission which is called long-term potentiation (LTP; Pittenger and Kandel). Hippocampal cellular and molecular processes that underlie memory consolidation and retrieval mediate the storage of new information (Whitlock et al., 2006). Hippocampal structure and function have been suggested to be very sensitive to stress (Huang et al., 2005; Han et al., 2013). In addition, while the induction of dendritic branches in the hippocampus is necessary for long-term memory storage (Govindarajan et al., 2011), exposure to stress induces spatial memory deficits through the atrophy of dendrites of hippocampal CA3 neurons (Conrad et al., 1996). It has been shown, using animal learning models, that exposure to acute elevated platform (EP) stress impairs memory consolidation or retrieval (Sardari et al., 2014; Segev et al., 2012). On the other hand, hippocampal LTP induction can be interrupted by different stressors like acute EP (Rocher et al., 2004) or foot shock stress (Xiong et al., 2003). A variety of signaling pathways which are important in LTP induction including cAMP response element binding protein (CREB) are the main targets of stress (Bilang-Bleuel et al., 2002; St-Pierre et al., 2006). CREB is a transcription factor that activates transcription of target genes by binding to a certain sequence of DNA which are called cAMP response elements (Mizuno et al., 2000). It is well known that long-term memory is dependent on CREB function (Izquierdo et al., 2002; Kogan et al., 2000). It should be noted that upstream signaling pathways such as brain-derived neutrophic factor (BDNF) signaling pathway (Alonso et al., 2002) phosphorylate CREB on a critical serine residue which leads to the activation of this factor (Ying et al., 2002; Lisman et al., 2002). CREB activity madulates the maintenance of LTP and induces 4
memory consolidation of passive avoidance learning (Bernabeu et al., 1997; Barco et al., 2002). Izquierdo et al. (2001) also found that exposure to a novel environment can enhance memory retrieval via increasing the hippocampal CREB phosphorylation. Additionally, some studies have supported a neuroprotective role for CREB which regulates the antioxidant gene expression (Bedogni et al., 2003; Krönke et al., 2003). On the other hand, CREB mediates the activity of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) which is a potent stimulator of mitochondrial biogenesis for the formation and maintenance of hippocampal dendritic spines and synapses under environmental challenges (Cheng et al., 2012). PGC-1α stimulates the neuronal expression of transcription factors including nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (TFAM) in response to stress (Miranda et al., 1999). CREB also interacts with nuclear factor E2-related factor 2 (Nrf2) by CREB binding protein (CBP; Shen et al., 2004). Nrf2 is a heterodimer transcription factor that binds to the antioxidant responsive element (ARE) sequence of DNA, and elevates the level of defense enzymes in the cell (Itoh et al., 1997). Under unstressed conditions, Nrf2 can be anchored in the cytoplasm via binding to Kelch-like ECH-associated protein1 (Tong et al., 2006), while stress exposure leads to the release of Nrf2 from Keap-1 (Dinkova-Kostova et al., 2005) which allows Nrf2 entry into the cell nucleus. There is evidence that enhancing Nrf2 antioxidant signaling pathway improves the learning and memory in animal models (Kanninen et al., 2009). Interestingly, Alzheimer disease decreases the expression levels of NRF 1 and TFAM along with nuclear levels of PGC-1α in the hippocampus, indicating the importance of mitochondrial biogenesis in memory formation (Sheng et al., 2012). Considering the above-mentioned points and findings, the present study was designed with the following four aims: (i) to determine the effect of exposure to 30 min acute stress in 5
memory consolidation and retrieval of passive avoidance learning; (ii) to investigate the role of P-CREB and its target genes, PGC-1α, NRF-1 and TFAM in the hippocampus during memory formation and stress-induced memory impairment; (iii) to examine the role of Nrf2 and its downstream gamma-glutamylcysteine synthetase (γ-GCS) and heme oxygenase-1 (HO-1); (iiii) to evaluate whether the activation of hippocampal γ-GCS pathway leads the changes of glutathione (GSH) levels and the activity of catalase (CAT; an antioxidant enzyme).
2. Results 2.1. Effect of post-training or pre-test exposure to acute EP stress on memory retrieval Fig. 1 shows the effect of post-training or pre-test exposure to 30 min stress on step-through latency. One-way ANOVA revealed that both post-training and pre-test exposure to acute EP stress reduced the step-through latency in the passive avoidance task [F (2, 18) = 42.98, P<0.001], indicating stress-induced memory impairment.
2.2. Changes of hippocampal CREB/PGC-1α signaling cascade in stress-induced memory impairment As shown in Fig. 2, the level of P-CREB was measured to assess the possible changes of hippocampal signaling pathways in memory formation under acute EP stress. Since P-CREB directly regulates PGC-1α which is a stimulator for the expression of NRF-1 and TFAM transcription factors (see introduction), the levels of PGC-1α, NRF-1 and TFAM were also measured in the hippocampus. Western blot analysis revealed that successful memory retrieval in the L group was associated with the increase in hippocampal levels of P-CREB (1.51 fold, P<0.001; Fig. 2B), PGC-1α (1.32 fold, P<0.001; Fig. 2C), NRF-1 (1.27, P<0.001; Fig. 2D) and
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TFAM (1.35, P<0.001; Fig. 2E). In contrast, the group that received 30 min exposure to EP stress (S group) had a significantly lower hippocampal levels of P-CREB (48.49%, P<0.001; Fig. 2B), PGC-1α (25.85%, P<0.001; Fig. 2C), NRF-1 (24.76%, P<0.001; Fig. 2D) and TFAM (23.21%, P<0.001; Fig. 2E) in comparison with intact group. The densitometric analysis also showed that post-training stress exposure decreased the hippocampal levels of P-CREB, (54.04%, P<0.001; Fig. 2B), PGC-1α (13.5%, P<0.01; Fig. 2C), NRF-1 (25.71%, P<0.001; Fig. 2D) and TFAM (15.51%, P<0.01; Fig. 2E) in the LS1 group. Moreover, exposure to EP stress before the testing phase decreased the levels of P-CREB (79.88%, P<0.001; Fig. 2B), PGC-1α (57.8%., P<0.001; Fig. 2C), NRF-1 (51.34%, P<0.001; Fig. 2D) and TFAM (36.79%, P<0.001; Fig. 2E) in LS2 group as compared with the L group.
2.3. Changes of hippocampal level of Nrf2 and its downstream target genes in stress-induced memory impairment Fig. 3 shows the effect of post-training or pre-test stress exposure on the hippocampal level of Nrf2 which is a stress-sensing genetic transcription factor and increases the level of defense enzymes such as γ-GCS and HO-1 in the passive avoidance task. Western blot analysis revealed that successful memory retrieval in the L group was associated with the increase in hippocampal levels of Nrf2 (1.5 fold, P<0.001; Fig. 3B), γ-GCS (1.31 fold, P<0.001; Fig. 3C) and HO-1(1.37 fold, P<0.001; Fig. 3D). In contrast, exposure to 30 min EP stress (S group) decreased the hippocampal levels of Nrf2 (26.62%, P<0.001; Fig. 3B) and γ-GCS (54%, P<0.001; Fig. 3C), but not HO-1 (P>0.05; Fig. 3D), in comparison with the I group. Further analysis also showed that post-training stress exposure (30 min) decreased the hippocampal levels of Nrf2 (20.31%,
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P<0.001; Fig. 3B), γ-GCS (42.8%, P<0.001; Fig. 3C) and HO-1 (18.06%, P<0.001; Fig. 3D) in the LS1 group as compared with the L group. Moreover, the hippocampal levels of Nrf2 (46.87%, P<0.001; Fig. 3B), γ-GCS (46.68%, P<0.001; Fig. 3C) and HO-1 (32.4%, P<0.001; Fig. 3D) were decreased in the animals that were exposed to 30 min stress before testing phase in comparison with the L group.
2.4. Changes of catalase activity and GSH level decreased in stress-induced memory impairment To assess whether hippocampal Nrf2 signaling pathway, which changed in stress-induced memory impairment, can affect the activity of catalase (CAT; an antioxidant enzyme), the enzyme activity were measured in the hippocampus. As can be seen in Fig. 4, the level of CAT activity was increased (1.43 fold, P<0.001) in the rats that showed successful memory retrieval (L group), while exposure to 30 min EP stress decreased the enzyme activity in S group (38.74%, P<0.001) as compared with the rats that had no manipulation (I group). The analysis also revealed that post-training (LS1 group; 26.84%, P<0.001) or pre-test exposure to stress (LS2 group; 34.88%, P<0.001) decreased the hippocampal levels of CAT as compared with the rats that showed successful memory retrieval (L group). In order to determine whether the activation of γ-GCS pathway modulates intracellular reactive oxygen species (ROS) in the hippocampus during the stress or not, the GSH levels, in addition to CAT, were measured in different conditions. According to the data presented in Fig. 4, the level of GSH was increased about 1.18 fold (P<0.01) in the L group and was decreased 17.92% (P<0.01) in the S group compared to their control group. Moreover, the level of GSH was decreased in the animals that were exposed to stress before test (13.74%, P<0.01), but not after training (P>0.05), as compared with the L group. 8
3. Discussion The results obtained in experiment 1 showed that post-training or pre-test 30 min exposure to acute elevated platform (EP) stress reduced the step-through latency, indicating stress-induced memory impairment. In agreement with our results, exposure to 30 min predator stress (Park et al., 2008) or EP stress (Segev et al., 2012) impaired the memory consolidation and retrieval in rats. Evidence suggests that acute stress exposure decreased the hippocampal number of newly generated cells (Thomas et al., 2007) and dendritic spine density in the animal models (Chen et al., 2010). On the other hand, exposure to stressful conditions has been suggested to alter the molecular signaling pathways related to memory formation (Smith et al., 1995; Sananbenesi et al., 2003). Impairment of hippocampal long-term potentiation (LTP), which is a putative cellular mechanism underlying learning and memory processes, can be induced by EP stress (Wong et al., 2007; Xu et al., 1997) or restraint stress (Yang et al., 2004). In view of the fact that the hippocampus has a crucial role in memory formation (Kogan et al., 2000) and may be sensitive to stress (Kim and Diamond, 2002), the present study was designed to investigate the effect of acute stress on hippocampal signaling pathways involved in stress and/or memory formation. Our results revealed that the hippocampal levels of phospho-cAMP responsive element binding protein (P-CREB) were increased in the rats that showed successful memory retrieval compared with untrained control rats. In contrast, the levels of P-CREB were decreased in the hippocampus of rats exposed to 30 min stress. Electrophysiological studies have highlighted a critical role for CREB-mediated gene expression in hippocampal LTP induction (Barco et al., 2002; Bourtchuladze et al., 1994). This view has also been supported by Taubenfeld et al (1999) who showed that the disruption of hippocampal CREB impaired the consolidation of inhibitory avoidance memory. Moreover, an immunocytochemistry study has shown that the hippocampal 9
level of the P-CREB was increased following inhibitory avoidance training in rats (Bernabeu et al., 1997). Vianna and colleagues (2000) have also shown that hippocampal-dependent associative learning was associated with the increase in the level of P-CREB. Altogether, these studies indicate that CREB plays an essential role in hippocampal-based memory formation. On the other hand, numerous studies have attempted to evaluate the effect of stress on the level of PCREB in the brain. It has been shown that exposure to acute or chronic foot shock stress decreased the CREB and P-CREB levels in the hippocampus and the amygdala (Lin et al., 1998; Kuipers et al., 2013), while exposure to swimming stress increased P-CREB in these sites (Bilang-Bleuel et al., 2002; Shen et al., 2004). Moreover, exposure to acute immobilization stress as well as increase in the level of stress hormone can reduce the hippocampal level of the brain-derived neutrophic factor (BDNF) in rats (Adlard and Cotman, 2004). Another important finding of the present study was that post-training stress or pre-test stress exposure decreased the hippocampal level of P-CREB in the rats. In line with our finding, Song and coworkers (2006) have shown that exposure to chronic mild stress impaired the spatial memory and also decreased the CREB messenger ribonucleic acid (mRNA) levels in the hippocampus of mice. Moreover, chronic stress-induced performance deficits in the shuttle-box task were associated with decreased hippocampal levels of P-CREB (Xu et al., 2006). In support of the effect of acute stress on hippocampal signaling pathways involved in stress and/or memory formation, we designed the second part of the study to investigate whether post-training or pre-test EP stress exposure can also affect the levels of PGC-1α, NRF-1 and TFAM in the hippocampus. The obtained results showed that the hippocampal levels of PGC-1α, NRF-1 and TFAM were increased in the rats that showed successful memory retrieval compared with untrained control rats. Evidence suggests that the increased levels of P-CREB can lead to
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increase in the expression of peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1α; Lonard et al., 2006) which is an important factor for the antioxidant enzyme expression and the mitochondrial biogenesis (see introduction). It has been shown that PGC-1α is the upstream for other mitochondrial biogenesis factors such as nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (TFAM; Miranda et al., 1999). Since the knockdown of PGC-1α inhibited spinogenesis and synaptogenesis in cultured hippocampal neurons (Cheng et al., 2012) and hippocampal neuritogenesis may be associated with the formation of trace memories (Shors, 2004), one may suggest that hippocampal increase of PGC1α is involved in passive avoidance learning. This hypothesis can be supported by the findings which suggested that the levels of the PGC-1α and its down streams were reduced in Alzheimer disease (Ashabi et al., 2012). Moreover, our results also indicated that 30 min exposure to acute stress decreased the hippocampal levels of PGC-1α, NRF-1 and TFAM. Interestingly, posttraining stress or pre-test stress exposure decreased the hippocampal levels of PGC-1α, NRF1 and TFAM in rats. It has been reported that exposure to chronic aluminium treatment, which increases the reactive oxygen species (ROS) formation and induces oxidative stress, decreased the levels of PGC-1α, NRF1 and TFAM in the hippocampus (Sharma et al., 2013). It may suggest that the reduction in the levels of PGC-1α and its downstream proteins is the result of a decrease in CREB activation as the upstream factor of these proteins. The current study also showed that the hippocampal levels of nuclear factor E2-related factor 2 (Nrf2), gamma-glutamylcysteine synthetase (γ-GCS) and heme oxygenase-1 (HO-1) were increased in the rats that showed successful memory retrieval as compared with the rats that had no manipulation (intact group). In contrast, 30 min exposure to acute stress or post-training and pre-test exposure to 30 min stress decreased the hippocampal levels of Nrf2, γ-GCS. The
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present findings seem to be consistent with those of Kanninen et al. (2009) who found that significant reductions in spatial learning deficits can be achieved by modulating levels of Nrf2 in the hippocampus of 9-month-old male amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice (a model for Alzheimer's disease; AD). It has been suggested that enhancing the hippocampal Nrf2/γ-GCS signaling pathways ameliorated ageing-induced cognitive dysfunction of mouse (Li et al., 2012). Additionally, post-training intrahippocampal injection of hemeoxygenase inhibitor, impaired the memory formation in both habituation and avoidance tasks in rats (Fin et al., 1994). Therefore, it seems that hippocampal Nrf2 levels and its downstream signal molecules may be involved in learning and memory processes. On the other hand, Morrison and coworkers (2012) have shown that exposure to high fat diet impaired memory retention, suggesting that exposure to this kind of stress decreased the Nrf2 signaling and induced oxidative damage in animals. Nrf2/ARE signaling pathway modulates intracellular reactive oxygen species (ROS) via the changes of glutathione (GSH) levels and the activity of catalase (CAT; Ha et al., 2006; Yan et al., 2010). Previous studies have shown that exposure to acute or chronic exercise, immobilization or restrain stress induced oxidative stress and changed the antioxidant activities in the brain (Liu et al., 1996; Liu et al., 2000; Zaidi et al., 2003). Our results revealed that the hippocampal levels of GSH and CAT were increased in the rats that showed successful memory retrieval, while these levels were decreased in the hippocampus of rats exposed to 30 min stress compared with the rats that had no manipulation. Catalase mimetic compounds are reported to reverse the age-related learning deficits and brain oxidative stress in mice (Liu et al., 2003). Additionally, enhancing the cortico-hippocampal glutathione levels improved the active avoidance task at AD model of rat’s brain (Hashimoto et al., 2002). Hence, it could conceivably
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be hypothesized that catalase activity and GSH have an important role in memory formation. In another major study, Zaidi et al. (2003) found that exposure to immobilization stress decreased the activity of catalase and glutathione-S-transferase in rats. The induction of the oxidative stress reduced the enzymatic defense activity of the catalase and GSH in mice hippocampus (Pires et al., 2014). On the other hand, our results showed that post-training exposure to 30 min stress decreased the hippocampal levels of CAT, but not GSH, in rats.Moreover, pre-test exposure to stress (30 min) decreased the hippocampal levels of GSH and CAT. In line with our results, Schimidt and colleagues (2014) have shown that exposure to oxidative stress impairs the passive avoidance memory formation and reduces the hippocampal level of the CAT, GSH in rats. Exposure to hyperoxia decreased the memory retrieval of spatial memory and remarkably decreased CAT activity in hippocampal neurons of rats (Fukui et al., 2001). In addition, exposure to noise-stress impaired the working memory and decreased the hippocampal level of GSH (Manikandan et al., 2006). In conclusion, considering the effects of post-training or pre-test stress exposure on hippocampal signaling pathways in the passive avoidance task, it is possible that stress-induced memory impairment may be related to down-regulation of hippocampal CREB/PGC-1α signaling cascade and Nrf2 antioxidant pathways.
4. Experimental procedures 4.1. Animals Male Wistar rats (bred in the animal house of School of Biology, University of Tehran) weighing 220–250 g were housed in groups of four per cage in animal house under normal conditions of 12 hr light/dark cycle (lights from 7:00 am to 7:00 pm) and 22±2ºc temperature. The animals had free access to food and water, but not during the experimental hours. All experiments were
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performed in the light phase of the cycle between 8:00 AM and 14:00 PM. Behavioral tests and animal care were conducted in accordance with the standard ethical guidelines (NIH, publication no. 85-23, revised 1985; European Communities Directive 86/609/EEC) and approved by the local ethical committee. All efforts were made to minimize the number of animals used and their suffering.
4.2. Drugs Antibodies directed against CREB, P-CREB and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies directed against PGC-1α and γ-GCS, HO-1 were purchased from ABCAM (Cambridge, UK). TFAM antibody was obtained from Bio Vision (Palo Alto, CA). Antibodies directed against Nrf2 and NRF-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Electrochemiluminescence (ECL) kit was taken from Amersham Bioscience (Piscataway, NJ, USA). Poly vinylidene fluoride membrane (PVDF) was obtained from Millipore (Billerica, MA, USA). H2O2 and dithionitrobenzoic acid (DTNB) were purchased from Merck (Darmstadt, Germany) and Sigma Aldrich (St. Louis, MO, USA), respectively.
4.3. Passive avoidance learning The step-through passive avoidance apparatus (Borj Sanat, Tehran, Iran) was used to evaluate memory retrieval in the animals. This rectangular apparatus included two chambers that were equal in size (20 cm×20 cm×30 cm); one was light (white) and the other one was dark (black). These two chambers were connected via a portable door (7cm×3cm×9 cm) which could be lifted manually. The apparatus was connected to a stimulator and each animal was given a shock in the 14
dark chamber by stainless steel rods which were 2.5mm in diameter and were placed with 1-cm intervals in the floor. The shocking was done (50 Hz, 3s, 1mA intensity) in the trial sections of the training day. The animals were allowed to habituate in the experimental room for 1 h prior to the experiments. Then, each animal was gently placed in the brightly lit compartment of the apparatus; after 5 s the guillotine door was opened and the animal was allowed to enter the dark compartment. The latency with which the animal crossed into the dark compartment was recorded. The animals that waited for more than 100 s to cross to the dark compartment were discarded from the experiments. Once the animal crossed with all four paws to the next compartment, the guillotine door was closed and the rat was taken into its home cage. The trial was repeated after 30 min as in the acquisition trial where after 5 s the guillotine door was opened and as soon as the animal crossed to the dark (shock) compartment the door was closed and a foot shock (50 Hz, 1 mA, 3 s) was immediately delivered to the grid floor of the dark room. After 20 s, the animal was removed from the apparatus and placed temporarily into its home cage. Two min later, the animal was retested in the same way as the previous trials; if the animal did not enter the dark compartment during 120 s, successful acquisition of passive avoidance response was recorded. Otherwise, when the animal entered the dark compartment (before 120 s) for a second time, the door was closed and the animal received the same shock again. After retesting, if the rat acquired the acquisition of passive avoidance, the training was done successfully. Twenty-four hours after training, a retrieval test was performed to measure long-term memory. Each animal was placed in the light compartment for 20 s, the door was opened, and the step-through latency was measured for entering into the dark compartment. The test session
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ended when the animal entered the dark compartment or remained in the light compartment for 300 s (criterion for retrieval). During these sessions, no electric shock was applied. It is important to note that all animals were sacrificed immediately after finishing the test and their hippocampal formation was extracted.
4.4. Stress protocol An elevated platform (EP) apparatus (Borj Sanat, Tehran, Iran) was used to induce stress. The apparatus is based on that used by Xu et al. (1998) and consisted of a plexiglas platform (21×20 cm2 and 100 cm above ground level) in the middle of a brightly lit room. In order to induce an acute inescapable stress, the animals were picked-up and placed on the EP for 30 min (Sardari et al., 2014). In first 10 min,, the animals showed the behavioral signs of stress such as immobility, urination and defecation (Xu et al., 1998).
4.5. Western blot analysis The hippocampi of animal brains were extracted immediately in minor time (Chiu et al., 2007). The tissues were lysed in buffer containing complete protease inhibitor cocktail. Protein concentrations were determined according to Bradford’s method (Bradford et al., 1976). Bovine serum albumin was used for a standard plot. Sixty µg of proteins were electrophoresed on 12% SDS−PAGE gel, transferred to PVDF membrane, and probed with specific antibodies. Immunoreactive band of proteins were detected by ECL reagents and subsequent autoradiography. Quantification of the results was performed by densitometric scan of films. Data analysis was done by Image J, measuring integrated density of bands after background subtraction.
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4.6. Catalase activity assay Catalase activity was measured by the method of Aebi (Aebi et al., 1984). Briefly, a solution of H2O2 (%30) and distilled water in the volumes of 153 µl and 50 ml respectively was prepared. Reaction was started by the addition of 60 µg of tissue lysate to a cuvette containing 500 µl of prepared solution. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm.
4.7. Measurement of glutathione levels The concentration of glutathione (GSH) was determined in hippocampus tissues using DTNB method at 412 nm (Ellman et al., 1959).
4.8. Experimental design In these experiments, the animals were divided into five groups. Group 1 was rats that had no treatment and acted as an intact control group (I group). Group 2 (S group) included rats that were placed on the EP apparatus for 30 min. Group 3 (L group) consisted of rats that were trained in the passive avoidance task and showed successful memory retrieval. Group 4 (LS1 group) included rats that were exposed to 30 min stress immediately after training (post-training stress) and whose memory retrieval was measured on the testing day. Group 5 (LS2 group) included rats that were trained and whose memory retrieval was tested after 30 min exposure to stress on the test day (pre-test stress). Immediately after the end of experiments, each animal was sacrificed for extracting the hippocampi, and hippocampal signaling pathways of memory formation under different treatments were evaluated via western blotting and enzyme activity assays.
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4.9. Statistical analysis All data are expressed as means ± SEM. For evaluating the difference between groups, one-way analysis of variance (ANOVA) followed by a specific post-hoc test, was used. P<0.05 was considered statistically significant.
Acknowledgments The authors have no conflict of interest to declare.
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and
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Legends
Fig. 1. The effects of post-training or pre-test exposure to acute stress on memory formation. Three groups of animals were trained and tested in the passive avoidance task (n=6). One group of animals was placed on the EP for 30 min immediately after training (post-training stress), and after 24 h was tested (LS1 group). The other group was trained in the task, and after 24 h, was exposed to 30 min stress before measuring step-through latency (LS2 group). A control group was also trained and tested without exposure to stress (L group). Each value represents mean ± SEM of six animals per group. *** P<0.001 compared with the control group.
Fig. 2. The effect of acute stress on the hippocampal CREB/PGC-1α signaling cascade. Five groups of animals were used in this experiment (n=4). The first group of animals received no manipulation (I group). The second group was exposed to 30 min stress (S group). The third group was trained and tested in the passive avoidance task (L group). The other two groups were exposed to 30 min stress immediately after training (post-training stress, LS1 group) or before testing (pre-test stress, LS2 group) with their memory retrieval measured then. Panel A is the representative immunoblots of P-CREB, CREB, PGC-1α, NRF1, TFAM proteins in the hippocampus. Graphs B, C, D and E show the mean ± S.E.M of the ratios of P-CREB/CREB, PGC-1α/β-actin, NRF1/β-actin and TFAM/β-actin which were calculated from densitometric quantification of the corresponding bands. *** P<0.001 compared with the I group. ++P< 0.01, +++P<0.001 compared with the L group.
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Fig. 3. The effect of acute stress on the hippocampal level of Nrf2 and its downstream signaling pathways. Five groups of animals were used in this experiment (n=4). The first group of animals received no manipulation (I group). The second group was exposed to 30 min stress (S group). The third group was trained and tested in the passive avoidance task (L group). The other two groups were exposed to 30 min stress immediately after training (post-training stress, LS1 group) or before testing (pre-test stress, LS2 group) with their memory retrieval measured then. Panel A is the representative immunoblots of Nrf2, γ-GCS and HO-1 proteins in the hippocampus. Graphs B, C and D show the mean ± S.E.M of the ratios of Nrf2/β-actin, γ-GCS /β-actin and HO-1 /β-actin which were calculated from densitometric quantification of the corresponding bands. *** P < 0.001 compared with the I group. +++P<0.001 compared with the L group.
Fig. 4. Biochemical assessment of the hippocampal level of GSH and catalase activity in stressinduced memory impairment. Five groups of animals were used in this experiment. The first group of animals received no manipulation (I group). The second group was exposed to 30 min stress (S group). The third group was trained and tested in the passive avoidance task (L group). The other two groups were exposed to 30 min stress immediately after training (post-training stress, LS1 group) or before testing (pre-test stress, LS2 group) with their memory retrieval measured then. Each value represents the mean ± S.E.M (n = 4). ** P <0.01, ***P<0.001 compared with the GSH level and catalase activity of I group respectively. ++ P <0.01, +++P<0.001 compared with the GSH level and catalase activity of the L group respectively.
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Research Highlights ►Post-training or pre-test exposure to acute stress induced memory impairment. ► Stress exposure decreased the levels of P-CREB, PGC-1α and Nrf2 in the hippocampus. ► Posttraining or pre-test exposure to stress decreased levels of P-CREB and PGC-1α. ►Stressinduced memory impairment is associated with the decrease of level of Nrf2.
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Hippocampal signaling pathways are involved in stress-induced impairment of memory formation in rats Maryam Sardari, Ameneh Rezayof, Fariba Khodagholi
www.elsevier.com/locate/brainres
PII: DOI: Reference:
S0006-8993(15)00632-0 http://dx.doi.org/10.1016/j.brainres.2015.08.015 BRES44418
To appear in: Brain Research
Accepted date: 16 August 2015 Cite this article as: Maryam Sardari, Ameneh Rezayof, Fariba Khodagholi, Hippocampal signaling pathways are involved in stress-induced impairment of memory formation in rats, Brain Research, http://dx.doi.org/10.1016/j. brainres.2015.08.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hippocampal signaling pathways are involved in stress-induced impairment of memory formation in rats
Maryam Sardaria, Ameneh Rezayofa, Fariba Khodagholib
a
Department of Animal Biology, School of Biology and Center of Excellence in Phylogeny of
Living Organisms, College of Science, University of Tehran, Tehran, Iran b
Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Correspondence to: A. Rezayof, PhD. Professor, Department of Animal Biology, School of Biology, College of Science, University of Tehran, P. O. Box 4155-6455, Tehran, Iran Fax: (+9821)-66405141 Tel: (+9821)-61112483 e-mail: [email protected]
1
Abstract Stress is a potent modulator of hippocampal-dependent memory formation. The aim of the present study was to assess the role of hippocampal signaling pathways in stress-induced memory impairment in male Wistar rats. The animals were exposed to acute elevated platform (EP) stress and memory formation was measured by a step-through type passive avoidance task. The results indicated that post-training or pre-test exposure to EP stress impaired memory consolidation or retrieval respectively. Using western blot analysis, it was found that memory retrieval was associated with the increase in the levels of phosphorylated cAMP-responsive element binding protein (P-CREB), peroxisome proliferator-activated receptor gamma coactivator-1a (PGC-1α) and its downstream targets in the hippocampus. In contrast, the stress exposure decreased the hippocampal levels of these proteins. In addition, stress-induced impairment of memory consolidation or retrieval was associated with the decrease in the PCREB/CREB ratio and the PGC-1α level in the hippocampus. On the other hand, the hippocampal level of nuclear factor E2-related factor 2 (Nrf2) and gamma-glutamylcysteine synthetase (γ-GCS) which are the master regulators of defense system were decreased by the stress exposure. The increased hippocampal levels of Nrf2 and it’s downstream was observed during memory retrieval, while stress-induced impairment of memory consolidation or retrieval inhibited this hippocampal signaling pathway. Overall, these findings suggest that downregulation of CREB/PGC-1α signaling cascade and Nrf2 antioxidant pathways in the hippocampus may be associated with memory impairment induced by stress.
Keywords: Acute stress; Memory formation; Hippocampal signaling pathway; Rat(s)
2
Abbreviations AD, Alzheimer's disease; APP/PS1, Amyloid precursor protein/presenilin 1; ARE, Antioxidant responsive element; CAT, Activity of catalase; CBP, cAMP-responsive element binding protein (CREB)-binding protein; DTNB, dithionitrobenzoic acid; ECL, Electrochemiluminescence; γGCS, Gamma-glutamylcysteine synthetase; GSH, glutathione; HO-1, Heme oxygenase-1; Keap1, Kelch-like ECH-associated protein1; mRNA, messenger ribonucleic acid; NRF-1, neuronal expression of transcription factors including nuclear respiratory factor-1; Nrf2, nuclear factor E2-related factor 2; P-CREB, Phosphorylated cAMP-responsive element binding protein; PGC-1α, Peroxisome proliferator-activated receptor gamma coactivator-1a; PVDF, Poly vinylidene fluoride membrane; ROS,
reactive oxygen species; TFAM, mitochondrial
transcription factor A.
3
1. Introduction A large body of evidence suggests that the hippocampus plays a vital role in memory formation via increasing the efficiency of synaptic transmission which is called long-term potentiation (LTP; Pittenger and Kandel). Hippocampal cellular and molecular processes that underlie memory consolidation and retrieval mediate the storage of new information (Whitlock et al., 2006). Hippocampal structure and function have been suggested to be very sensitive to stress (Huang et al., 2005; Han et al., 2013). In addition, while the induction of dendritic branches in the hippocampus is necessary for long-term memory storage (Govindarajan et al., 2011), exposure to stress induces spatial memory deficits through the atrophy of dendrites of hippocampal CA3 neurons (Conrad et al., 1996). It has been shown, using animal learning models, that exposure to acute elevated platform (EP) stress impairs memory consolidation or retrieval (Sardari et al., 2014; Segev et al., 2012). On the other hand, hippocampal LTP induction can be interrupted by different stressors like acute EP (Rocher et al., 2004) or foot shock stress (Xiong et al., 2003). A variety of signaling pathways which are important in LTP induction including cAMP response element binding protein (CREB) are the main targets of stress (Bilang-Bleuel et al., 2002; St-Pierre et al., 2006). CREB is a transcription factor that activates transcription of target genes by binding to a certain sequence of DNA which are called cAMP response elements (Mizuno et al., 2000). It is well known that long-term memory is dependent on CREB function (Izquierdo et al., 2002; Kogan et al., 2000). It should be noted that upstream signaling pathways such as brain-derived neutrophic factor (BDNF) signaling pathway (Alonso et al., 2002) phosphorylate CREB on a critical serine residue which leads to the activation of this factor (Ying et al., 2002; Lisman et al., 2002). CREB activity madulates the maintenance of LTP and induces 4
memory consolidation of passive avoidance learning (Bernabeu et al., 1997; Barco et al., 2002). Izquierdo et al. (2001) also found that exposure to a novel environment can enhance memory retrieval via increasing the hippocampal CREB phosphorylation. Additionally, some studies have supported a neuroprotective role for CREB which regulates the antioxidant gene expression (Bedogni et al., 2003; Krönke et al., 2003). On the other hand, CREB mediates the activity of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) which is a potent stimulator of mitochondrial biogenesis for the formation and maintenance of hippocampal dendritic spines and synapses under environmental challenges (Cheng et al., 2012). PGC-1α stimulates the neuronal expression of transcription factors including nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (TFAM) in response to stress (Miranda et al., 1999). CREB also interacts with nuclear factor E2-related factor 2 (Nrf2) by CREB binding protein (CBP; Shen et al., 2004). Nrf2 is a heterodimer transcription factor that binds to the antioxidant responsive element (ARE) sequence of DNA, and elevates the level of defense enzymes in the cell (Itoh et al., 1997). Under unstressed conditions, Nrf2 can be anchored in the cytoplasm via binding to Kelch-like ECH-associated protein1 (Tong et al., 2006), while stress exposure leads to the release of Nrf2 from Keap-1 (Dinkova-Kostova et al., 2005) which allows Nrf2 entry into the cell nucleus. There is evidence that enhancing Nrf2 antioxidant signaling pathway improves the learning and memory in animal models (Kanninen et al., 2009). Interestingly, Alzheimer disease decreases the expression levels of NRF 1 and TFAM along with nuclear levels of PGC-1α in the hippocampus, indicating the importance of mitochondrial biogenesis in memory formation (Sheng et al., 2012). Considering the above-mentioned points and findings, the present study was designed with the following four aims: (i) to determine the effect of exposure to 30 min acute stress in 5
memory consolidation and retrieval of passive avoidance learning; (ii) to investigate the role of P-CREB and its target genes, PGC-1α, NRF-1 and TFAM in the hippocampus during memory formation and stress-induced memory impairment; (iii) to examine the role of Nrf2 and its downstream gamma-glutamylcysteine synthetase (γ-GCS) and heme oxygenase-1 (HO-1); (iiii) to evaluate whether the activation of hippocampal γ-GCS pathway leads the changes of glutathione (GSH) levels and the activity of catalase (CAT; an antioxidant enzyme).
2. Results 2.1. Effect of post-training or pre-test exposure to acute EP stress on memory retrieval Fig. 1 shows the effect of post-training or pre-test exposure to 30 min stress on step-through latency. One-way ANOVA revealed that both post-training and pre-test exposure to acute EP stress reduced the step-through latency in the passive avoidance task [F (2, 18) = 42.98, P<0.001], indicating stress-induced memory impairment.
2.2. Changes of hippocampal CREB/PGC-1α signaling cascade in stress-induced memory impairment As shown in Fig. 2, the level of P-CREB was measured to assess the possible changes of hippocampal signaling pathways in memory formation under acute EP stress. Since P-CREB directly regulates PGC-1α which is a stimulator for the expression of NRF-1 and TFAM transcription factors (see introduction), the levels of PGC-1α, NRF-1 and TFAM were also measured in the hippocampus. Western blot analysis revealed that successful memory retrieval in the L group was associated with the increase in hippocampal levels of P-CREB (1.51 fold, P<0.001; Fig. 2B), PGC-1α (1.32 fold, P<0.001; Fig. 2C), NRF-1 (1.27, P<0.001; Fig. 2D) and
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TFAM (1.35, P<0.001; Fig. 2E). In contrast, the group that received 30 min exposure to EP stress (S group) had a significantly lower hippocampal levels of P-CREB (48.49%, P<0.001; Fig. 2B), PGC-1α (25.85%, P<0.001; Fig. 2C), NRF-1 (24.76%, P<0.001; Fig. 2D) and TFAM (23.21%, P<0.001; Fig. 2E) in comparison with intact group. The densitometric analysis also showed that post-training stress exposure decreased the hippocampal levels of P-CREB, (54.04%, P<0.001; Fig. 2B), PGC-1α (13.5%, P<0.01; Fig. 2C), NRF-1 (25.71%, P<0.001; Fig. 2D) and TFAM (15.51%, P<0.01; Fig. 2E) in the LS1 group. Moreover, exposure to EP stress before the testing phase decreased the levels of P-CREB (79.88%, P<0.001; Fig. 2B), PGC-1α (57.8%., P<0.001; Fig. 2C), NRF-1 (51.34%, P<0.001; Fig. 2D) and TFAM (36.79%, P<0.001; Fig. 2E) in LS2 group as compared with the L group.
2.3. Changes of hippocampal level of Nrf2 and its downstream target genes in stress-induced memory impairment Fig. 3 shows the effect of post-training or pre-test stress exposure on the hippocampal level of Nrf2 which is a stress-sensing genetic transcription factor and increases the level of defense enzymes such as γ-GCS and HO-1 in the passive avoidance task. Western blot analysis revealed that successful memory retrieval in the L group was associated with the increase in hippocampal levels of Nrf2 (1.5 fold, P<0.001; Fig. 3B), γ-GCS (1.31 fold, P<0.001; Fig. 3C) and HO-1(1.37 fold, P<0.001; Fig. 3D). In contrast, exposure to 30 min EP stress (S group) decreased the hippocampal levels of Nrf2 (26.62%, P<0.001; Fig. 3B) and γ-GCS (54%, P<0.001; Fig. 3C), but not HO-1 (P>0.05; Fig. 3D), in comparison with the I group. Further analysis also showed that post-training stress exposure (30 min) decreased the hippocampal levels of Nrf2 (20.31%,
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P<0.001; Fig. 3B), γ-GCS (42.8%, P<0.001; Fig. 3C) and HO-1 (18.06%, P<0.001; Fig. 3D) in the LS1 group as compared with the L group. Moreover, the hippocampal levels of Nrf2 (46.87%, P<0.001; Fig. 3B), γ-GCS (46.68%, P<0.001; Fig. 3C) and HO-1 (32.4%, P<0.001; Fig. 3D) were decreased in the animals that were exposed to 30 min stress before testing phase in comparison with the L group.
2.4. Changes of catalase activity and GSH level decreased in stress-induced memory impairment To assess whether hippocampal Nrf2 signaling pathway, which changed in stress-induced memory impairment, can affect the activity of catalase (CAT; an antioxidant enzyme), the enzyme activity were measured in the hippocampus. As can be seen in Fig. 4, the level of CAT activity was increased (1.43 fold, P<0.001) in the rats that showed successful memory retrieval (L group), while exposure to 30 min EP stress decreased the enzyme activity in S group (38.74%, P<0.001) as compared with the rats that had no manipulation (I group). The analysis also revealed that post-training (LS1 group; 26.84%, P<0.001) or pre-test exposure to stress (LS2 group; 34.88%, P<0.001) decreased the hippocampal levels of CAT as compared with the rats that showed successful memory retrieval (L group). In order to determine whether the activation of γ-GCS pathway modulates intracellular reactive oxygen species (ROS) in the hippocampus during the stress or not, the GSH levels, in addition to CAT, were measured in different conditions. According to the data presented in Fig. 4, the level of GSH was increased about 1.18 fold (P<0.01) in the L group and was decreased 17.92% (P<0.01) in the S group compared to their control group. Moreover, the level of GSH was decreased in the animals that were exposed to stress before test (13.74%, P<0.01), but not after training (P>0.05), as compared with the L group. 8
3. Discussion The results obtained in experiment 1 showed that post-training or pre-test 30 min exposure to acute elevated platform (EP) stress reduced the step-through latency, indicating stress-induced memory impairment. In agreement with our results, exposure to 30 min predator stress (Park et al., 2008) or EP stress (Segev et al., 2012) impaired the memory consolidation and retrieval in rats. Evidence suggests that acute stress exposure decreased the hippocampal number of newly generated cells (Thomas et al., 2007) and dendritic spine density in the animal models (Chen et al., 2010). On the other hand, exposure to stressful conditions has been suggested to alter the molecular signaling pathways related to memory formation (Smith et al., 1995; Sananbenesi et al., 2003). Impairment of hippocampal long-term potentiation (LTP), which is a putative cellular mechanism underlying learning and memory processes, can be induced by EP stress (Wong et al., 2007; Xu et al., 1997) or restraint stress (Yang et al., 2004). In view of the fact that the hippocampus has a crucial role in memory formation (Kogan et al., 2000) and may be sensitive to stress (Kim and Diamond, 2002), the present study was designed to investigate the effect of acute stress on hippocampal signaling pathways involved in stress and/or memory formation. Our results revealed that the hippocampal levels of phospho-cAMP responsive element binding protein (P-CREB) were increased in the rats that showed successful memory retrieval compared with untrained control rats. In contrast, the levels of P-CREB were decreased in the hippocampus of rats exposed to 30 min stress. Electrophysiological studies have highlighted a critical role for CREB-mediated gene expression in hippocampal LTP induction (Barco et al., 2002; Bourtchuladze et al., 1994). This view has also been supported by Taubenfeld et al (1999) who showed that the disruption of hippocampal CREB impaired the consolidation of inhibitory avoidance memory. Moreover, an immunocytochemistry study has shown that the hippocampal 9
level of the P-CREB was increased following inhibitory avoidance training in rats (Bernabeu et al., 1997). Vianna and colleagues (2000) have also shown that hippocampal-dependent associative learning was associated with the increase in the level of P-CREB. Altogether, these studies indicate that CREB plays an essential role in hippocampal-based memory formation. On the other hand, numerous studies have attempted to evaluate the effect of stress on the level of PCREB in the brain. It has been shown that exposure to acute or chronic foot shock stress decreased the CREB and P-CREB levels in the hippocampus and the amygdala (Lin et al., 1998; Kuipers et al., 2013), while exposure to swimming stress increased P-CREB in these sites (Bilang-Bleuel et al., 2002; Shen et al., 2004). Moreover, exposure to acute immobilization stress as well as increase in the level of stress hormone can reduce the hippocampal level of the brain-derived neutrophic factor (BDNF) in rats (Adlard and Cotman, 2004). Another important finding of the present study was that post-training stress or pre-test stress exposure decreased the hippocampal level of P-CREB in the rats. In line with our finding, Song and coworkers (2006) have shown that exposure to chronic mild stress impaired the spatial memory and also decreased the CREB messenger ribonucleic acid (mRNA) levels in the hippocampus of mice. Moreover, chronic stress-induced performance deficits in the shuttle-box task were associated with decreased hippocampal levels of P-CREB (Xu et al., 2006). In support of the effect of acute stress on hippocampal signaling pathways involved in stress and/or memory formation, we designed the second part of the study to investigate whether post-training or pre-test EP stress exposure can also affect the levels of PGC-1α, NRF-1 and TFAM in the hippocampus. The obtained results showed that the hippocampal levels of PGC-1α, NRF-1 and TFAM were increased in the rats that showed successful memory retrieval compared with untrained control rats. Evidence suggests that the increased levels of P-CREB can lead to
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increase in the expression of peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1α; Lonard et al., 2006) which is an important factor for the antioxidant enzyme expression and the mitochondrial biogenesis (see introduction). It has been shown that PGC-1α is the upstream for other mitochondrial biogenesis factors such as nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor A (TFAM; Miranda et al., 1999). Since the knockdown of PGC-1α inhibited spinogenesis and synaptogenesis in cultured hippocampal neurons (Cheng et al., 2012) and hippocampal neuritogenesis may be associated with the formation of trace memories (Shors, 2004), one may suggest that hippocampal increase of PGC1α is involved in passive avoidance learning. This hypothesis can be supported by the findings which suggested that the levels of the PGC-1α and its down streams were reduced in Alzheimer disease (Ashabi et al., 2012). Moreover, our results also indicated that 30 min exposure to acute stress decreased the hippocampal levels of PGC-1α, NRF-1 and TFAM. Interestingly, posttraining stress or pre-test stress exposure decreased the hippocampal levels of PGC-1α, NRF1 and TFAM in rats. It has been reported that exposure to chronic aluminium treatment, which increases the reactive oxygen species (ROS) formation and induces oxidative stress, decreased the levels of PGC-1α, NRF1 and TFAM in the hippocampus (Sharma et al., 2013). It may suggest that the reduction in the levels of PGC-1α and its downstream proteins is the result of a decrease in CREB activation as the upstream factor of these proteins. The current study also showed that the hippocampal levels of nuclear factor E2-related factor 2 (Nrf2), gamma-glutamylcysteine synthetase (γ-GCS) and heme oxygenase-1 (HO-1) were increased in the rats that showed successful memory retrieval as compared with the rats that had no manipulation (intact group). In contrast, 30 min exposure to acute stress or post-training and pre-test exposure to 30 min stress decreased the hippocampal levels of Nrf2, γ-GCS. The
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present findings seem to be consistent with those of Kanninen et al. (2009) who found that significant reductions in spatial learning deficits can be achieved by modulating levels of Nrf2 in the hippocampus of 9-month-old male amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice (a model for Alzheimer's disease; AD). It has been suggested that enhancing the hippocampal Nrf2/γ-GCS signaling pathways ameliorated ageing-induced cognitive dysfunction of mouse (Li et al., 2012). Additionally, post-training intrahippocampal injection of hemeoxygenase inhibitor, impaired the memory formation in both habituation and avoidance tasks in rats (Fin et al., 1994). Therefore, it seems that hippocampal Nrf2 levels and its downstream signal molecules may be involved in learning and memory processes. On the other hand, Morrison and coworkers (2012) have shown that exposure to high fat diet impaired memory retention, suggesting that exposure to this kind of stress decreased the Nrf2 signaling and induced oxidative damage in animals. Nrf2/ARE signaling pathway modulates intracellular reactive oxygen species (ROS) via the changes of glutathione (GSH) levels and the activity of catalase (CAT; Ha et al., 2006; Yan et al., 2010). Previous studies have shown that exposure to acute or chronic exercise, immobilization or restrain stress induced oxidative stress and changed the antioxidant activities in the brain (Liu et al., 1996; Liu et al., 2000; Zaidi et al., 2003). Our results revealed that the hippocampal levels of GSH and CAT were increased in the rats that showed successful memory retrieval, while these levels were decreased in the hippocampus of rats exposed to 30 min stress compared with the rats that had no manipulation. Catalase mimetic compounds are reported to reverse the age-related learning deficits and brain oxidative stress in mice (Liu et al., 2003). Additionally, enhancing the cortico-hippocampal glutathione levels improved the active avoidance task at AD model of rat’s brain (Hashimoto et al., 2002). Hence, it could conceivably
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be hypothesized that catalase activity and GSH have an important role in memory formation. In another major study, Zaidi et al. (2003) found that exposure to immobilization stress decreased the activity of catalase and glutathione-S-transferase in rats. The induction of the oxidative stress reduced the enzymatic defense activity of the catalase and GSH in mice hippocampus (Pires et al., 2014). On the other hand, our results showed that post-training exposure to 30 min stress decreased the hippocampal levels of CAT, but not GSH, in rats.Moreover, pre-test exposure to stress (30 min) decreased the hippocampal levels of GSH and CAT. In line with our results, Schimidt and colleagues (2014) have shown that exposure to oxidative stress impairs the passive avoidance memory formation and reduces the hippocampal level of the CAT, GSH in rats. Exposure to hyperoxia decreased the memory retrieval of spatial memory and remarkably decreased CAT activity in hippocampal neurons of rats (Fukui et al., 2001). In addition, exposure to noise-stress impaired the working memory and decreased the hippocampal level of GSH (Manikandan et al., 2006). In conclusion, considering the effects of post-training or pre-test stress exposure on hippocampal signaling pathways in the passive avoidance task, it is possible that stress-induced memory impairment may be related to down-regulation of hippocampal CREB/PGC-1α signaling cascade and Nrf2 antioxidant pathways.
4. Experimental procedures 4.1. Animals Male Wistar rats (bred in the animal house of School of Biology, University of Tehran) weighing 220–250 g were housed in groups of four per cage in animal house under normal conditions of 12 hr light/dark cycle (lights from 7:00 am to 7:00 pm) and 22±2ºc temperature. The animals had free access to food and water, but not during the experimental hours. All experiments were
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performed in the light phase of the cycle between 8:00 AM and 14:00 PM. Behavioral tests and animal care were conducted in accordance with the standard ethical guidelines (NIH, publication no. 85-23, revised 1985; European Communities Directive 86/609/EEC) and approved by the local ethical committee. All efforts were made to minimize the number of animals used and their suffering.
4.2. Drugs Antibodies directed against CREB, P-CREB and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies directed against PGC-1α and γ-GCS, HO-1 were purchased from ABCAM (Cambridge, UK). TFAM antibody was obtained from Bio Vision (Palo Alto, CA). Antibodies directed against Nrf2 and NRF-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Electrochemiluminescence (ECL) kit was taken from Amersham Bioscience (Piscataway, NJ, USA). Poly vinylidene fluoride membrane (PVDF) was obtained from Millipore (Billerica, MA, USA). H2O2 and dithionitrobenzoic acid (DTNB) were purchased from Merck (Darmstadt, Germany) and Sigma Aldrich (St. Louis, MO, USA), respectively.
4.3. Passive avoidance learning The step-through passive avoidance apparatus (Borj Sanat, Tehran, Iran) was used to evaluate memory retrieval in the animals. This rectangular apparatus included two chambers that were equal in size (20 cm×20 cm×30 cm); one was light (white) and the other one was dark (black). These two chambers were connected via a portable door (7cm×3cm×9 cm) which could be lifted manually. The apparatus was connected to a stimulator and each animal was given a shock in the 14
dark chamber by stainless steel rods which were 2.5mm in diameter and were placed with 1-cm intervals in the floor. The shocking was done (50 Hz, 3s, 1mA intensity) in the trial sections of the training day. The animals were allowed to habituate in the experimental room for 1 h prior to the experiments. Then, each animal was gently placed in the brightly lit compartment of the apparatus; after 5 s the guillotine door was opened and the animal was allowed to enter the dark compartment. The latency with which the animal crossed into the dark compartment was recorded. The animals that waited for more than 100 s to cross to the dark compartment were discarded from the experiments. Once the animal crossed with all four paws to the next compartment, the guillotine door was closed and the rat was taken into its home cage. The trial was repeated after 30 min as in the acquisition trial where after 5 s the guillotine door was opened and as soon as the animal crossed to the dark (shock) compartment the door was closed and a foot shock (50 Hz, 1 mA, 3 s) was immediately delivered to the grid floor of the dark room. After 20 s, the animal was removed from the apparatus and placed temporarily into its home cage. Two min later, the animal was retested in the same way as the previous trials; if the animal did not enter the dark compartment during 120 s, successful acquisition of passive avoidance response was recorded. Otherwise, when the animal entered the dark compartment (before 120 s) for a second time, the door was closed and the animal received the same shock again. After retesting, if the rat acquired the acquisition of passive avoidance, the training was done successfully. Twenty-four hours after training, a retrieval test was performed to measure long-term memory. Each animal was placed in the light compartment for 20 s, the door was opened, and the step-through latency was measured for entering into the dark compartment. The test session
15
ended when the animal entered the dark compartment or remained in the light compartment for 300 s (criterion for retrieval). During these sessions, no electric shock was applied. It is important to note that all animals were sacrificed immediately after finishing the test and their hippocampal formation was extracted.
4.4. Stress protocol An elevated platform (EP) apparatus (Borj Sanat, Tehran, Iran) was used to induce stress. The apparatus is based on that used by Xu et al. (1998) and consisted of a plexiglas platform (21×20 cm2 and 100 cm above ground level) in the middle of a brightly lit room. In order to induce an acute inescapable stress, the animals were picked-up and placed on the EP for 30 min (Sardari et al., 2014). In first 10 min,, the animals showed the behavioral signs of stress such as immobility, urination and defecation (Xu et al., 1998).
4.5. Western blot analysis The hippocampi of animal brains were extracted immediately in minor time (Chiu et al., 2007). The tissues were lysed in buffer containing complete protease inhibitor cocktail. Protein concentrations were determined according to Bradford’s method (Bradford et al., 1976). Bovine serum albumin was used for a standard plot. Sixty µg of proteins were electrophoresed on 12% SDS−PAGE gel, transferred to PVDF membrane, and probed with specific antibodies. Immunoreactive band of proteins were detected by ECL reagents and subsequent autoradiography. Quantification of the results was performed by densitometric scan of films. Data analysis was done by Image J, measuring integrated density of bands after background subtraction.
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4.6. Catalase activity assay Catalase activity was measured by the method of Aebi (Aebi et al., 1984). Briefly, a solution of H2O2 (%30) and distilled water in the volumes of 153 µl and 50 ml respectively was prepared. Reaction was started by the addition of 60 µg of tissue lysate to a cuvette containing 500 µl of prepared solution. The rate of decomposition of H2O2 was measured spectrophotometrically at 240 nm.
4.7. Measurement of glutathione levels The concentration of glutathione (GSH) was determined in hippocampus tissues using DTNB method at 412 nm (Ellman et al., 1959).
4.8. Experimental design In these experiments, the animals were divided into five groups. Group 1 was rats that had no treatment and acted as an intact control group (I group). Group 2 (S group) included rats that were placed on the EP apparatus for 30 min. Group 3 (L group) consisted of rats that were trained in the passive avoidance task and showed successful memory retrieval. Group 4 (LS1 group) included rats that were exposed to 30 min stress immediately after training (post-training stress) and whose memory retrieval was measured on the testing day. Group 5 (LS2 group) included rats that were trained and whose memory retrieval was tested after 30 min exposure to stress on the test day (pre-test stress). Immediately after the end of experiments, each animal was sacrificed for extracting the hippocampi, and hippocampal signaling pathways of memory formation under different treatments were evaluated via western blotting and enzyme activity assays.
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4.9. Statistical analysis All data are expressed as means ± SEM. For evaluating the difference between groups, one-way analysis of variance (ANOVA) followed by a specific post-hoc test, was used. P<0.05 was considered statistically significant.
Acknowledgments The authors have no conflict of interest to declare.
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Legends
Fig. 1. The effects of post-training or pre-test exposure to acute stress on memory formation. Three groups of animals were trained and tested in the passive avoidance task (n=6). One group of animals was placed on the EP for 30 min immediately after training (post-training stress), and after 24 h was tested (LS1 group). The other group was trained in the task, and after 24 h, was exposed to 30 min stress before measuring step-through latency (LS2 group). A control group was also trained and tested without exposure to stress (L group). Each value represents mean ± SEM of six animals per group. *** P<0.001 compared with the control group.
Fig. 2. The effect of acute stress on the hippocampal CREB/PGC-1α signaling cascade. Five groups of animals were used in this experiment (n=4). The first group of animals received no manipulation (I group). The second group was exposed to 30 min stress (S group). The third group was trained and tested in the passive avoidance task (L group). The other two groups were exposed to 30 min stress immediately after training (post-training stress, LS1 group) or before testing (pre-test stress, LS2 group) with their memory retrieval measured then. Panel A is the representative immunoblots of P-CREB, CREB, PGC-1α, NRF1, TFAM proteins in the hippocampus. Graphs B, C, D and E show the mean ± S.E.M of the ratios of P-CREB/CREB, PGC-1α/β-actin, NRF1/β-actin and TFAM/β-actin which were calculated from densitometric quantification of the corresponding bands. *** P<0.001 compared with the I group. ++P< 0.01, +++P<0.001 compared with the L group.
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Fig. 3. The effect of acute stress on the hippocampal level of Nrf2 and its downstream signaling pathways. Five groups of animals were used in this experiment (n=4). The first group of animals received no manipulation (I group). The second group was exposed to 30 min stress (S group). The third group was trained and tested in the passive avoidance task (L group). The other two groups were exposed to 30 min stress immediately after training (post-training stress, LS1 group) or before testing (pre-test stress, LS2 group) with their memory retrieval measured then. Panel A is the representative immunoblots of Nrf2, γ-GCS and HO-1 proteins in the hippocampus. Graphs B, C and D show the mean ± S.E.M of the ratios of Nrf2/β-actin, γ-GCS /β-actin and HO-1 /β-actin which were calculated from densitometric quantification of the corresponding bands. *** P < 0.001 compared with the I group. +++P<0.001 compared with the L group.
Fig. 4. Biochemical assessment of the hippocampal level of GSH and catalase activity in stressinduced memory impairment. Five groups of animals were used in this experiment. The first group of animals received no manipulation (I group). The second group was exposed to 30 min stress (S group). The third group was trained and tested in the passive avoidance task (L group). The other two groups were exposed to 30 min stress immediately after training (post-training stress, LS1 group) or before testing (pre-test stress, LS2 group) with their memory retrieval measured then. Each value represents the mean ± S.E.M (n = 4). ** P <0.01, ***P<0.001 compared with the GSH level and catalase activity of I group respectively. ++ P <0.01, +++P<0.001 compared with the GSH level and catalase activity of the L group respectively.
29
Research Highlights ►Post-training or pre-test exposure to acute stress induced memory impairment. ► Stress exposure decreased the levels of P-CREB, PGC-1α and Nrf2 in the hippocampus. ► Posttraining or pre-test exposure to stress decreased levels of P-CREB and PGC-1α. ►Stressinduced memory impairment is associated with the decrease of level of Nrf2.
30
350
Step-Through Latency (S)
300
250
200
150
100
***
***
50
0
L
1 LS
Fig. 1
31
2 LS
A)
P-CREB
43 kDa
CREB
43 kDa
PGC-1α
91 kDa
NRF1
68 kDa
TFAM
27 kDa
β-actin
45 kDa
B)
C) ***
+++
*** +++
D)
E)
Fig. 2 32
B)
A) Nrf2
57 kDa
γ-GCS
73 kDa
HO-1
32 kDa
β-actin
45 kDa
*** +++ +++
C)
D)
Fig. 3
33
A)
B) ++
Fig. 4 34