GSK-3β activity in the hippocampus is required for memory retrieval

Neurobiology of Learning and Memory 98 (2012) 122–129

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GSK-3b activity in the hippocampus is required for memory retrieval Jin Gyu Hong a,c, Dong Hyun Kim a,c, Chang Hwan Lee a,c, Se Jin Park a,c, Jong Min Kim a,c, Mudan Cai a,c,1, Dae Sik Jang a,c, Jong Hoon Ryu a,b,c,⇑ a

Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea c Kyung Hee East–West Pharmaceutical Research Institute, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 12 January 2012 Revised 27 June 2012 Accepted 5 July 2012 Available online 16 July 2012 Keywords: Memory retrieval GSK-3b Passive avoidance test Hippocampus

a b s t r a c t Several molecules were recently found to be important for the memory retrieval process in the hippocampus; however, the mechanisms underlying the memory retrieval remain poorly understood. GSK3b has been implicated in the control of synaptic plasticity and memory formation. Here, we investigated the relationship between hippocampal GSK-3b activity and memory retrieval using behavioral and Western blotting methods. We found that GSK-3b was activated in the hippocampus after a retention session in the passive avoidance task. An intrahippocampal injection of the GSK-3b inhibitor, SB 216763, before the retention session blocked memory retrieval (but not reconsolidation) without affecting locomotor activity. These results suggest that GSK-3b activation would be essential for memory retrieval in the hippocampus. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Memory retrieval is an essential component of memory processing, and the deterioration of memory retrieval is a major symptom in the primary stage of Alzheimer’s disease. Although most studies have been focused on the roles of the hippocampus in memory consolidation and storage, the retrieval phase is also considered to be dependent on the hippocampus (Anderson et al., 2004; Vanelzakker et al., 2011). For example, the failure of cholinergic receptors in the hippocampus completely inhibits retrieval, and the activation of metabotropic glutamate receptors or protein kinase A in the hippocampus is necessary for retrieval (Barros et al., 2001; Szapiro et al., 2000). Moreover, extracellular signal-regulated kinase 1/2 (ERK 1/2) activation in the hippocampus is critical for the retrieval of spatial or fear memory (Huang, Chiang, Liang, Thompson, & Liu, 2010; Izquierdo et al., 2001). Furthermore, studies have suggested that phosphatidylinositol 3 kinase (PI3K) activation in the hippocampus, which is dependent on the upregulation of ERK 1/2 activation, is also required during memory retrieval (Chen et al., 2005). ⇑ Corresponding author. Address: Department of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, #1 Hoegi-dong, Dongdeamun-gu, Seoul 130-701, Republic of Korea. Fax: +82 2 966 3885. E-mail address: [email protected] (J.H. Ryu). 1 Present address: Department of Medical Research, Korea Institute of Oriental Medicine, 483 Expo-ro, Yuseong-gu, Daejeon 305-811, Republic of Korea. 1074-7427/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nlm.2012.07.003

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase. Four different phosphorylated regions have been described in GSK-3, and phosphorylation of serine 9 correlates with the inhibition of its kinase activity (Hooper, Killick, & Lovestone, 2008; Medina & Wandosell, 2011). It is now widely accepted that GSK-3 plays an important role in various essential physiological processes, such as development, cell cycle, or apoptosis, in the mammalian brain (Medina & Wandosell, 2011). Recent studies have indicated a relationship between GSK-3b, which is one of the isoforms that is rich in the mammalian brain, and memory processing. For example, inactivation of GSK-3b by phosphorylation at serine 9 (Ser 9) facilitates the induction of long-term potentiation (LTP), which is implicated in memory induction and formation, in the hippocampal CA1 and dentate gyrus regions (Hooper et al., 2007). GSK-3b-mediated b-catenin also contributes to a normal consolidation of new memories in the amygdala (Maguschak & Ressler, 2008), and contextual memory reconsolidation requires the activation of GSK-3b in the hippocampus (Kimura et al., 2008). However, it remains unclear whether GSK-3b signaling is associated with memory retrieval. In the present study, we investigated the role of GSK-3b in memory retrieval using intrahippocampal injections of SB 216763, which is a potent GSK-3b inhibitor, and a passive avoidance task and Western blot analysis. Here, we demonstrate evidence that the activation of hippocampal GSK-3b is required for memory retrieval in the passive avoidance task.

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of the highest grade available and were obtained from normal commercial sources.

2. Materials and methods 2.1. Animals

2.3. Microinfusion of drugs Male ICR mice (27–30 g, 7 weeks) were purchased from the Orient Co. Ltd., which is a branch of Charles River Laboratories (Seoul, Korea), and kept in the University Animal Care Unit for 1 week until the experiments. The mice were housed 5 per cage and were allowed access to water and food ad libitum. The environment was maintained under a constant temperature (23 ± 1 °C) and humidity (60 ± 10%) under a 12-h light/dark cycle (lights on 07:30–19:30 h). The treatment and maintenance of the mice were carried out in accordance with the Principles of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Kyung Hee University, Korea. 2.2. Materials SB 216763 was purchased from Tocris Bioscience (Ellisville, MO), and Zoletil 50Ò was obtained from Virbac (06516 Carros, France). CT-99021 was obtained from Axon Medchem (Groningen, Netherlands). LiCl was purchased from Sigma–Aldrich (St. Louis, MO). Complete protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail were purchased from Roche (Palo Alto, CA). Goat polyclonal anti-GSK-3b, rabbit polyclonal anti-phosphorylated GSK-3b (pGSK-3b at Ser 9) and rabbit monoclonal anti-phosphorylated GSK-3a (pGSK-3a at Ser 21) antibodies were purchased from Cell Signaling Technology (Cell Signaling, Danvers, MA). Goat polyclonal anti-GSK-3a antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). All of the other materials were

The mice were placed on a stereotaxic frame (Stoelting Co., Wood Dale, IL) under Zoletil 50Ò anesthesia (10 mg/kg, i.m.), and guide cannulae (26 G) were aimed at the hippocampus (stereotaxic coordinates: AP, 2.00 mm from the bregma; ML, ±1.50 mm from the midline; DV, 1.00 mm from the bregma) using an atlas of the mouse brain (Paxinos & Franklin, 2001). The guide cannulae were fixed to the skull with dental cement and covered with dummy cannulae. Following surgery, the mice were allowed to recover for 7 days. The GSK-3b inhibitor, SB 216763, was dissolved in 100% DMSO (1 ng/0.5 lL) before the injection as previously described (Xu et al., 2009). Fifteen minutes before the retention session, the mice were carefully restrained by hand and bilaterally infused with SB 216763 (1 ng/0.5 lL/side) or vehicle through the injector cannulae (30 G) that were extended 1.0 mm beyond the tips of the guide cannulae. After 2 min of infusion, the infusion needle was left in the guide cannulae for 1 min to ensure proper delivery of the reagents. At the end of the experiment, the position of the cannulae in the hippocampus was verified by injection of 0.5 lL of 1% Evans Blue solution and Nissl staining (Fig. 1). 2.4. The step-through passive avoidance task To manipulate memory phases, we employed the step-through passive avoidance task (Izquierdo et al., 2001; Izquierdo et al., 2007; Myhrer, 2003; Park et al., 2010). In the passive avoidance

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Fig. 1. The site of intrahippocampal microinjections. (A) Summary of the site of microinjection in the hippocampus (n = 15). (B) Photomicrographs of Nissl staining to confirm the injection sites of the guide cannulae (coordinates: AP, 2.00 mm from the bregma; ML, ±1.50 mm from the midline; and DV, 1.00 mm from the bregma).

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task, the consolidation phase is appeared between acquisition session and retention session, and retrieval is considered an event that is appeared during the retention session. Thereafter, retrieved memory re-stabilizes overtime, known as memory reconsolidation phase (Alberini, 2011). In the present study, testing was carried out in identical illuminated and nonilluminated boxes (20  20  20 cm) that were separated by a guillotine door (5  5 cm) as previously described (Kim et al., 2007). The illuminated compartment contained a 50 W bulb, and the floor of the nonilluminated compartment was composed of 2 mm stainless steel rods that were spaced 1 cm apart. An acquisition session was carried out in two trials (Bansal & Parle, 2010). Each mouse was initially placed in the illuminated compartment, and the door between the two compartments was opened 10 s later. When the mouse entered the dark compartment, the door automatically closed and a 3 s electrical foot shock (0.5 mA) was delivered through the stainless steel rods. The second trial was carried out 20 min after the first trial. During the second trial, if the mouse entered the dark compartment within 60 s, electric shocks were delivered for 3 s. If the mouse did not enter the dark compartment within 60 s, the mouse was removed from the shock-free zone and subjected to a retention session. The retention session was composed of one trial and was conducted 24 h after the acquisition session in a similar manner, except for the electric shocks. The time taken for a mouse to enter the dark compartment after the door opened was defined as the latency for the retention session and was recorded for up to 300 s. To investigate whether a GSK-3b inhibitor affects memory retrieval, mice were treated with SB 216763 or vehicle 15 min before the retention session. To investigate the state dependency of SB 216763, mice were treated with SB 216763 15 min before both the acquisition and retention sessions. 2.5. Spontaneous locomotor behavior To investigate the motor effect of the indicated dose of SB 216763, spontaneous locomotor behavior was measured as previously described (Jung et al., 2006). Briefly, mice were placed in the center of a horizontal locomotor activity box (40  40  40 cm), and locomotor activity was measured for 20 min immediately after SB 216763 or vehicle injection. The data were analyzed using the video-based Ethovision System (Noldus, Wageningen, The Netherlands). 2.6. Nissl staining For the preparation of Nissl staining samples, the mice were anesthetized with an intramuscular injection of Zoletil 50Ò (10 mg/kg) immediately after the passive avoidance task. The mice were transcardially perfused with phosphate buffer (100 mM, pH 7.4) followed by ice-cold 4% paraformaldehyde prior to decapitation. The brains were removed and postfixed in phosphate buffer (50 mM, pH 7.4) containing 4% paraformaldehyde overnight. Then, the brains were immersed in a 30% sucrose solution [in 50 mM phosphate-buffered saline (PBS)] and stored at 4 °C until sectioning. Brain sections were obtained by the coronal plane (30 lm) using a cryostat (Leica, Nussloch, Germany) and kept in storage solution at 4 °C. After mounting the sections onto gelatin-coated slides, the sections were stained with 0.5% cresyl violet, dehydrated through graded alcohols (70%, 80%, 90%, and 100%  2), placed in xylene, and coverslipped using Histomount medium. 2.7. Slice preparations and the administration of GSK-3b inhibitors To test the effect of GSK-3b inhibitors on GSK-3b phosphorylation state, mouse was sacrificed by dislocation of the neck and then decapitated. The brain was rapidly removed and placed in ice-cold

artificial CSF (aCSF) composed of following concentrations (in mM): 124 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgSO4, and 10 D-glucose (bubbled with 95% O2/5% CO2). Transverse hippocampal slices (400 lm thick) were prepared using a McIllwain tissue chopper (Mickle Laboratory Engineering Co. Ltd., Gomshall, UK). Hippocampal slices were stored in aCSF (20–25 °C) for 1–2 h before transferring to the incubation chamber. Acute hippocampal slices were incubated in aCSF containing SB216763 (5 lM), LiCl (20 nM), or CT-99021 (1 lM) for 30 min. 2.8. Western blot analysis For the preparation of Western blot samples, the mice were decapitated at designated time points after the retrieval of the passive avoidance task, and their isolated hippocampal tissues were homogenized in an ice-chilled Tris–HCl buffer (20 mM, pH 7.4) containing 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, complete protease inhibitor cocktail (1 tablet/50 ml) and PhosSTOP phosphatase inhibitor cocktail (1 tablet/10 ml). After the administrations of GSK-3b inhibitors for 30 min into the incubation chamber, hippocampal slices were homogenized in the same solution as mentioned above. Samples of the homogenates (20 lg of protein) were then subjected to SDS–PAGE (8%) under reduced conditions. Proteins were transferred onto PVDF membranes in transfer buffer [25 mM Tris–HCl (pH 7.4), containing 192 mM glycine and 20% v/v methanol] for 2 h at 400 mA and 4 °C. Western blots were then incubated for 2 h with a blocking solution (5% skimmed milk) prior to incubation with a rabbit anti-pGSK-3b antibody (1:3000 dilution) overnight at 4 °C. After the antibody incubation, the membranes were washed ten times with Tween 20/Tris-buffered saline (TTBS), incubated with horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) for 1 h at room temperature, washed twelve times with TTBS, and developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL). The blots were then stripped and incubated with a goat antiGSK-3b antibody (1:3000 dilution). The membrane was analyzed with the bio-imaging program of the LAS-4000 mini (Fujifilm Lifescience, Stamford, CT, USA). The pGSK-3b levels were normalized to the GSK-3b levels in the same membranes. In the case of Western blotting for GSK-3a or pGSK-3a levels, all procedures were the same as the GSK-3b or pGSK-3b except for the dilution rate (anti-pGSK-3a antibody, 1:2000 dilution; anti-GSK-3a antibody, 1:2000 dilution). 2.9. Statistical analysis Data from the passive avoidance task and the locomotor activity were analyzed by Student’s t-test. Western blot data were analyzed by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test for multiple comparisons. Significance was accepted for P values <0.05. 3. Results 3.1. GSK-3b inhibitor increases the phosphorylation level of GSK-3b in the hippocampal slices Previously, it was reported that GSK-3b inhibitors increase the phosphorylation levels of GSK-3b at Ser 9 residue, an inhibitory phosphorylation site (Liang & Chuang, 2007; Zhang, Phiel, Spece, Gurvich, & Klein, 2003). To test those findings, we conducted Western blotting using mouse brain slice incubated with GSK-3b inhibitors. We found that GSK-3b inhibitors including LiCl, SB 216763, and CT-99021 significantly increased pGSK-3b (Ser 9) levels compared to control group [F(3, 8) = 9.057, P < 0.05, Fig. 2].

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3.2. GSK-3b is activated during memory retrieval

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To investigate the temporal changes of GSK-3b activity during memory retrieval, we measured the levels of pGSK-3b in the hippocampus after the retention session of the passive avoidance task. Naive mice, which did not receive a shock but spent the same amount of time in the chamber during both acquisition and retrieval, and control mice, which were trained with the shock but were not introduced to the retention session, were employed. The immunoreactivity of pGSK-3b (Ser 9) was significantly decreased [F(4, 37) = 5.646, P < 0.05, Fig. 3A] compared with control group, and the minimum level was reached 10 min after the retention session. However, the phosphorylation level of GSK-3b at Tyr 216, which is another regulatory site of GSK-3b activity, was not changed (data not shown). The phosphorylation levels of GSK-3a at Ser 21 were not significantly changed during retrieval session [F(4, 20) = 0.8167, P > 0.05, Fig. 3B]. The present results indicate that GSK-3b is activated by dephosphorylation during memory recalls, and may be critically involved in the retrieval phase. In subsequent studies, pGSK-3b levels were measured at 10 min after retrieval.

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Fig. 2. The effect of GSK-3 inhibitors on phosphorylation of GSK-3b at serine 9. Mouse was sacrificed by dislocation of the neck and then decapitated. The brain was rapidly removed and placed in ice-cold artificial CSF (aCSF) composed of following concentrations (in mM): 124 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgSO4, 10 D-glucose, and 0.1 picrotoxin (bubbled with 95% O2/5% CO2). Transverse hippocampal slices (400 lm thick) were prepared using a McIllwain tissue chopper (Mickle Laboratory Engineering Co. Ltd., Gomshall, UK). Hippocampal slices were stored in aCSF (20–25 °C) for 1–2 h before transferring to the incubation chamber. Acute hippocampal slices were incubated in aCSF containing SB216763 (5 lM), LiCl (20 nM), or CT-99021 (1 lM) for 30 min. After homogenization, each sample was conducted Western blot for detecting pGSK-3b and GSK-3b levels. The values are expressed as the means ± S.E.M (n = 3–4/group). P < 0.05 and P < 0.01 compared with the control.

Time after retrieval (min)

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To confirm whether memory retrieval-induced dephosphorylation of GSK-3b was blocked by SB 216763 injection, mice were injected with SB 216763 or vehicle in the hippocampus 15 min before retrieval. Ten minutes after the retention session, the hippocampus was isolated for Western blot analysis. The memory retrieval-induced dephosphorylation of GSK-3b was blocked by the injection of SB 216763 15 min before retrieval [F(2, 16) = 10.93, P < 0.01, Fig. 4A]. The phosphorylation levels of GSK-3a at Ser 21 were not significantly changed by the injection of SB216763 [F(2, 16) = 0.8658, P > 0.05, Fig. 4A]. To investigate whether memory retrieval is affected by GSK-3b inhibition, mice were administered SB 216763 (1 ng/0.5 lL/side) 15 min before the retention session of the passive avoidance task. The latency time in the SB 216763treated group was decreased compared with the vehicle-treated Time after retrieval (min)

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Fig. 3. GSK-3b or GSK-3a activity during memory retrieval in the hippocampus. Photomicrographs and quantitative analysis of the Western blot data of pGSK-3b (Ser 9) (A) or pGSK-3a (Ser 21) (B) in the hippocampus were presented. The mice were sacrificed at 5, 10 or 15 min after retrieval. N, naïve mice without shock; Con (control), mice received shock but no retrieval task. Values are expressed as the means ± S.E.M (n = 8–9/group, data for pGSK-3b levels; n = 5/group, data for pGSK-3a levels). P < 0.05 compared with the control group.

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Fig. 4. The inhibition of GSK-3b activity and memory retrieval in the passive avoidance task. (A) Photomicrographs and quantitative analysis of the Western blot data of pGSK-3b (Ser 9) or pGSK-3a (Ser 21) in the hippocampus were presented. SB 216763 or vehicle was administered bilaterally into the hippocampus 15 min before the retrieval trial, and the mice were sacrificed 10 min after the retrieval trial for Western blot analysis (n = 7–10/group, data for pGSK-3b levels; n = 5–7/group, data for pGSK-3a levels). Control (horizontal striped bar), mice received shock but no retrieval task; Vehicle (white bar), vehicle-treated group; SB 216763 (hatched bar), SB 216763-treated group. (B) Intrahippocampal injection of SB 216763 (1 ng/0.5 lL/side) 15 min before the retention session decreased the latency time compared with the vehicle-treated group (n = 7–8/ group). (C) Intrahippocampal injection of SB 216763 (1 ng/0.5 lL/side) 15 min before the acquisition and retention sessions decreased the latency time compared with the vehicle-treated group (n = 8/group). The values are expressed as the means ± S.E.M. P < 0.05 and P < 0.01 compared with the control (A) or vehicle-treated groups (B and C). ## P < 0.01 compared with the vehicle-treated group (A).

group (P < 0.05, Fig. 4B). The injection of SB 216763 15 min before both the acquisition and retention sessions also decreased the latency time, which suggested that there was no state-dependency (P < 0.01, Fig. 4C). To assess the effects of the intrahippocampal

injection of SB 216763 on motor activity, we measured spontaneous locomotor behavior for 20 min after the administration of SB 216763. As shown in Fig. 5A and B, there were no significant changes in the distance traveled. Taken together, these results sug-

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Fig. 5. The effects of SB 216763 on spontaneous locomotor behavior in the open field test. SB 216763 or vehicle was administered bilaterally into the hippocampus, and locomotor activity was recorded for 20 min. (A) The distance traveled (5 min time bins) and (B) the total distance traveled for 20 min were shown. There were no differences in the distance traveled between the SB 216763 (hatched bar) and the vehicle groups (white bar). The values are expressed as the means ± S.E.M (n = 7/group).

gest that the impairment of memory retrieval by SB 216763 injection 15 min before the retention session is due to the blockade of GSK-3b dephosphorylation induced by memory retrieval. 3.4. Inhibition of GSK-3b activity does not affect memory reconsolidation To determine whether the injection of SB 216763 also affects reconsolidation, we trained mice on day 1 and introduced a retrieval session 24 h (test 1), 48 h (test 2) and 72 h (test 3) after the SB infusion

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training session. Fifteen minutes before test 2, SB 216763 or vehicle was infused into the hippocampus. Administration of SB 216763 significantly blocked memory retrieval compared with the vehicle-treated group (P < 0.001, Fig. 6A) and restored the decreased latency time to the level of the vehicle-treated group after one round of reconsolidation (test 3). We also employed another paradigm in which we trained mice on day 1 and introduced a retrieval session 24 h (test 1), 48 h (test 2), 72 h (test 3) and 96 h (test 4) after the training. Fifteen minutes before test 1 or test 3, SB 216763 or vehicle was infused into the hippocampus. Injections

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Fig. 6. The effects of GSK-3b inhibition on memory reconsolidation. SB 216763 was administered 15 min before test 2 (A) or 15 min before test 1 and test 3 (B). The latency time in the SB 216763-treated group was decreased compared with the vehicle-treated group; however, the latency time had recovered 24 h later and was similar to the vehicle-treated control group (n = 7–10/group). (C) Photomicrographs and quantitative analysis of the Western blot data of GSK-3b (Ser 9) in the hippocampus were presented. SB 216763 or vehicle was administered bilaterally into the hippocampus 15 min before the test 1, and the mice were sacrificed 10 min after the test 2 for Western blot analysis (n = 4/group). Control, mice were received shock and introduced test 1 but not test 2; Vehicle, vehicle-treated group. The values are expressed as the means ± S.E.M. P < 0.05 and P < 0.001 compared with the vehicle-treated group in A and B; P < 0.01 compared with the control group in C. Control, horizontal stripped bar; vehicle, white bar; SB 216763, hatched stripped bar.

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of SB 216763 15 min before both test 1 and test 3 impaired memory retrieval (P < 0.05 and P < 0.001, respectively, Fig. 6B), and the impaired memory retrieval was recovered after 24 h (test 2 or 4). We also confirmed the changes of GSK-3b phosphorylation after one round of reconsolidation (Fig. 6C). Mice were injected with SB 216763 or vehicle in the hippocampus 15 min before test 1. Mice were introduced to test 2 at 24 h after the test 1 and sacrificed 10 min after test 2. The memory retrieval significantly decreased GSK-3b phosphorylation [F(2, 9) = 14, P < 0.01, Fig. 6C]. However, there were no any significant changes on the pGSK-3b levels between vehicle- and SB 216763-treated groups. Taken together, these results suggest that injections of SB 216763 impaired memory retrieval but not affect reconsolidation.

4. Discussion In the present study, we found that GSK-3b in the hippocampus was activated by memory retrieval, and the inhibition of GSK-3b before the retention session blocked memory retrieval but not reconsolidation. These results indicate that GSK-3b activity is required for memory retrieval. Memory processing can be experimentally divided into several phases: acquisition, consolidation, retrieval and reconsolidation. Each stage is believed to be essential for memory formation or expression. Researches for the molecular mechanisms involved in memory processing are actively progressing, and studies have focused on the memory retrieval phase because memory retrieval is seriously disturbed in Alzheimer’s disease (Backman et al., 1999). In early Alzheimer’s disease patients, retrieval deficit is typically the earliest and most noticeable clinical symptom (Sperling et al., 2010). Therefore, an understanding of the mechanism(s) involved in the retrieval phase would be helpful to overcome the retrieval deficit in diseases such as Alzheimer’s disease. In the present study, we wanted to investigate whether GSK-3b in the hippocampus affects the retrieval phase using the passive avoidance task. Retrieval phase has been shown to be correlated with LTP or long-term depression (LTD) (Bast, da Silva, & Morris, 2005; Cammarota et al., 2005), and it is well known that LTP or LTD could be regulated by GSK-3b (Hooper et al., 2007; Peineau et al., 2007). We observed temporal changes of GSK-3b activity during memory retrieval, which suggested that retrieval regulates GSK-3b activity. Interestingly, the GSK-3b inhibitor, SB 216763, impaired memory retrieval in the passive avoidance task, and increased phosphorylation form of GSK-3b at serine 9. Several studies have reported the effects of GSK-3b activity on behavioral functions in various mammalian brain regions. For example, GSK-3b activity in the nucleus accumbens core contributes to the development and expression of cocaine-induced locomotor sensitization (Xu et al., 2009). In addition, the Akt and GSK-3 pathway may contribute to dopamine-dependent hyperactivity in the striatum (Beaulieu et al., 2004). Moreover, studies have shown that intraperitoneal injection of SB 216763 decreased locomotor activity (Beaulieu et al., 2004). We also observed similar effects after intraperitoneal administration of SB 216763 (data not shown); however, intrahippocampal injection of SB 216763 did not affect general locomotor behavior, which suggested that hippocampal GSK-3b regulates the memory retrieval phase without affecting motor function. It has been reported that the inhibition of GSK-3b blocks the activation of protein phosphatase-1 (PP-1) by inhibitor-2 (I-2), with consequent increase of phosphorylation levels of GSK-3b at Ser 9 (Zhang et al., 2003). In our in vitro studies, we also found that GSK-3b inhibitors including SB 216763 increased the phosphorylation levels of GSK-3b at Ser 9. However, we did not confirm whether the inhibition of GSK-3b by SB 216763 prevents the activation of PP-1 involved in the activation of GSK-3b. PP-1 activity is

known to be regulated by phosphorylation. For example, phosphorylation of I-2, an inhibitory subunit of PP-1, at Thr 72 by GSK-3b restores PP-1 activity and unphosphorylated I-2 inhibits PP-1 activity (Connor et al., 2000; DePaoli-Roach, 1984). Szatmari et al. (2005) reported that the inhibition of GSK-3b by LiCl reduced I-2 phosphorylation and increased the phosphorylation levels of GSK-3b at Ser 9, known as autoregulatory loop (Zhang et al., 2003). Thus, it is likely that the increased levels of pGSK-3b by SB 216763 are due to the inhibition of PP-1 activity although additional experiments are required to confirm this. GSK-3a is also involved in contextual and cued memory in the fear conditioning (Kaidanovich-Beilin et al., 2009). However, there were no significant changes in the levels of pGSK-3a during memory retrieval with or without SB 216763 in the hippocampal tissues. Additional studies are needed to examine the precise effects of the memory retrieval process on GSK-3a phosphorylation. Memory reconsolidation, which is triggered by memory recall, serves to maintain, strengthen and/or modify acquired reactivated memories (Stickgold & Walker, 2005; Tronson & Taylor, 2007). There is a controversy in the field regarding the effects of GSK-3b on the reconsolidation phase. For example, studies have shown that GSK-3b activity is required for memory reconsolidation in the amygdala (Wu et al., 2011). In addition, other studies have shown that GSK-3b activity in the hippocampus is required for the reconsolidation phase (Kimura et al., 2008). However, Chen et al. (2011) reported that an intrahippocampal injection of SB 216763 does not affect the reconsolidation phase. In the present study, we observed that the administration of SB 216763 after one round of reconsolidation or repeated administration before the retention session produced memory retrieval impairment. Interestingly, the impairment of memory retrieval by the injection of SB 216763 was completely restored within 24 h after the retrieval test. In addition, there were not any significant changes on the levels of pGSK-3b between vehicle- and SB 216763-treated groups after one round of reconsolidation. Although GSK-3b activation is required for the retrieval phase, the present results suggest that GSK-3b activation in the hippocampus may not be essential for memory reconsolidation. In addition, the involvement of GSK-3b activity in other regions (e.g., the amygdala) for the reconsolidation phase should be clarified (Wu et al., 2011). Several studies have reported that the activation of PI3K, ERK, or cAMP response element-binding protein signaling in the hippocampus are required for memory retrieval and its enhancement (Chen et al., 2005; Leon, Bruno, Allard, Nader, & Cuello, 2010; Viosca et al., 2009). Because GSK-3b signaling could be inhibited by PI3K/ Akt or ERK activation (Delcommenne et al., 1998; Ding et al., 2005), the inhibition of GSK-3b activity might result in the enhancement of memory retrieval. However, the present study showed the opposite results. Currently, we do not have any concrete data to explain this discrepancy, and we can only speculate on the differing results. Studies have suggested that NMDA receptor signaling is important for memory retrieval (Cammarota et al., 2005) and that GSK-3b activation is required for NMDA receptor-dependent LTD induction (Peineau et al., 2007). Recently, studies have reported that D-serine enhanced spatial memory retrieval through the induction of LTD in the hippocampus, which suggested a relationship between LTD and spatial memory retrieval (Zhang, Gong, Wang, Xu, & Xu, 2008). Thus, the effect of GSK-3b activity on memory retrieval in the present study is likely to be associated with NMDA receptordependent LTD induction. Akt is known to phosphorylate and inhibit GSK-3b activity. However, there were no significant changes in the Akt phosphorylation levels between vehicle- and SB 216763treated groups after the retrieval of the passive avoidance test (data not shown). Previous report suggested that Akt was not involved in NMDA receptor-dependent LTD (Peineau et al., 2007). Additional experiments are needed to clarify these issues.

J.G. Hong et al. / Neurobiology of Learning and Memory 98 (2012) 122–129

Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science and Technology (2011-0010884).

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