Neuroscience Letters 612 (2016) 32–37
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Research paper
Involvement of nucleotide diphosphate kinase 2 in the reopening of the sensitive period of filial imprinting of domestic chicks (Gallus gallus domesticus) Shinji Yamaguchi a , Naoya Aoki a , Akihiko Takehara a , Masaru Mori b , Akio Kanai b , Toshiya Matsushima c , Koichi J. Homma a,∗ a
Department of Life and Health Sciences, Faculty of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan c Department of Biology, Faculty of Science, Hokkaido University, Hokkaido 060-0810, Japan b
h i g h l i g h t s • Phosphoproteome analysis revealed the upregulation of NDPK2 phosphorylation following exogenous T3 injection. • NDPK2 was prerequisite for the opening and reopening of the sensitive period downstream of T3 action. • NDPK2 participated in the memory priming process by T3 signaling which endowed the potential for learning.
a r t i c l e
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Article history: Received 3 August 2015 Received in revised form 19 November 2015 Accepted 4 December 2015 Available online 7 December 2015 Keywords: Sensitive period Thyroid hormone Filial imprinting NDPK2 Learning Non-genomic signaling
a b s t r a c t Filial imprinting is a behavior characterized by the sensitive or critical period restricted to the first few days after hatching. Once the sensitive period is closed, it is widely believed that chicks can never be imprinted under natural conditions. Previously, we showed that the exogenous injection of T3 reopened the sensitive period which was already closed. That study suggested that T3 functioned by way of a rapid non-genomic action; however, the molecular mechanism of how T3 reopens the sensitive period remains unknown. Here, we show that the phosphorylation level of nucleotide diphosphate kinase 2 (NDPK2) was upregulated following T3 injection. Pharmacological deprivation of the kinase activity of NDPK hampered the molecular process prerequisite for the reopening of the sensitive period of filial imprinting. Moreover, it is shown that the kinase activity of NDPK2 participates in the priming process by T3 signaling which endows the potential for learning. Our data indicate that NDPK2 plays a crucial role downstream of T3 action and that its phosphorylation is involved in the non-genomic signaling during imprinting. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Certain types of learning can be acquired during a limited period called the sensitive or critical period [4,12]. A typical example of such learning is filial imprinting of precocial birds [6,15,18]. Newly hatched domestic chicks and ducklings have specific
Abbreviations: T3 , 3,5,3 -triiodothyronine; NDPK, nucleotide diphosphate kinase; TR, thyroid hormone receptor;; PI3K, phosphoinositide 3-kinase; PCR, polymerase chain reaction; HAMMOC, hydroxy acid-modified metal oxide chromatography; LC/MS, Liquid Chromatography/Mass Spectrometry; IMM, intermediate medial mesopallium; DMSO, Dimethyl sulfoxide; MP, memory priming. ∗ Corresponding author. Fax: +81 3 3964 8132. E-mail addresses:
[email protected],
[email protected] (K.J. Homma). http://dx.doi.org/10.1016/j.neulet.2015.12.004 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
predispositions and acquire preferences for particular features (e.g. colors, shape and configuration) of a conspicuous moving object [6,19] during a limited period of time soon after hatching. The object is the biological mother under natural condition, but it can be other objects in laboratory [8,26]. The learning that occurs during this sensitive period is considered to exert a long-lasting influence on the development of the individual’s social and emotional behaviors. In domestic chicks, the sensitive period lasts about 3 days after hatching under experimental conditions [26]. If the chicks are kept in darkness until the sensitive period closes, they can no longer be imprinted. Recently, we found that thyroid hormone (T3 ) is a critical factor in opening and reopening of the sensitive period of imprinting [14]. Imprinting training at day 1 after hatching during the sensitive period causes the acute influx of T3 into the brain from capillaries,
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were placed in dark plastic enclosures in a breeder at 30 ◦ C in order to prevent exposure to light [8]. 2.2. Phosphoproteome analysis
Fig. 1. The phosphorylation level of NDPK was upregulated following T3 injection. (A) Immunoblotting analysis validated that NDPK phosphorylation level in brain increased after T3 injection on 4-day-old chicks. (B) Quantitative analysis of immunoblotting. Data are presented as mean ± SEM. (U-test, *p < 0.05).
which enables the chick to acquire imprinting. When the sensitive period for imprinting is closed at day 4, the brain T3 level remains low even after imprinting training is performed. However, chicks exogenously injected with T3 were able to be imprinted after the sensitive period closed, showing that T3 is a critical factor to reopen the sensitive period. The molecular mechanism of the reopening by T3 involved the heterodimer of T3 and a thyroid hormone receptor (TR) which transduced their information to phosphoinositide 3-kinase (PI3 K) in a rapid non-genomic way [26]. However, a more precise mechanism by which T3 reopens the sensitive period remains unexplained. The family of nucleotide diphosphate kinase (NDPK) proteins is known to regulate a vast variety of cellular and physiological events such as proliferation, differentiation, molecular transport, apoptosis, and metastasis formation in tumor progression [17,25]. In addition to the multifunctional role of NDPKs, there is evidence that NDPK2 can modulate neurite outgrowth [2]. It was reported that NDPK1 is the ubiquitous form in most tissues, whereas NDPK2 is highly expressed in the cerebrum and the testes [21] and in the central nervous system during early embryonic development in mammals [14]. Here, we examined the role of NDPK, which acts downstream of T3 , in the opening and reopening of the sensitive period of filial imprinting. We performed phosphoproteome analysis to identify the proteins whose phosphorylation levels were upregulated accompanying the injection of T3 using the brains of 4-day-old chicks. We found that NDPK2 exhibited an increase in phosphorylation level following T3 injection, and we analyzed its roles in the opening and reopening of the sensitive period.
Whole brains, excluding hypothalamus, of T3 -injected chicks (0.5 h after injection, n = 13; 1 h after injection, n = 14; 3 h after injection, n = 14; and control dark-reared chicks n = 13) were dissected and homogenized in a lysis buffer at pH 8.5 (12 mM sodium deoxycholic acid, 12 mM sodium dodecanoyl sarcosinate, 100 mM triethylammnonium bicarbonate) containing protease inhibitors (P2714, Sigma–Aldrich, St. Louis, MO, USA) and protein phosphatase inhibitor cocktails 2 and 3 (P5726 and P0044, Sigma–Aldrich, USA). To distinguish male chicks from female, genomic DNA was extracted from the blood of individual chicks and used in genomic PCR analysis [3] with the following primers: sense, GTTACTGATTCGTCTACGAGA; antisense, ATTGAAATGATCCAGTGCTTG. The homogenates were separated and mixed in eight groups of chicks: 1, male dark-reared (n = 7); 2, female dark-reared (n = 6); 3, male at 0.5 h after injection (n = 6); 4, female at 0.5 h after injection (n = 7); 5, male at 1 h after injection (n = 7); 6, female at 1 h after injection (n = 7); 7, male at 3 h after injection (n = 7); and 8, female at 3 h after injection (n = 7). We performed phosphoproteome analysis to identify the phosphoproteins associated with T3 injection (group 1 versus group 3, group 2 versus group 4, group 1 versus group 5, group 2 versus group 6, and group 2 versus group 8). The proteins in homogenates were treated with the previous described method with some modifications [24]. The samples were digested using trypsin (Promega, Madison, WI, USA) and Lys-C (Wako Pure Chemical Co., Osaka, Japan) at 37 ◦ C for 16 h, and then, hydroxy acid-modified metal oxide chromatography (HAMMOC) method using the stage tip [19] packed with titanium dioxide beads (GL Sciences, Tokyo, Japan) was performed for phosphopeptide enrichment. The concentrated phosphopeptide fraction was analyzed with nanoLC/MS system using the same conditions as the previous report [24] for the separation of phosphopeptides. These peptide sequences were identified by the MS/MS Ion Search using MASCOT Server ver. 2.4.0 (Matrix Science, London. U.K.) with SwissProt database at the significant level of 0.05. The ratios of phosphorylated peptides were calculated as the maximal signal intensity of each peptide ions from T3 -injected chicks (after 0.5, 1, and 3 h) divided by that from dark-reared chicks. Mann–Whitney U-test was performed using the triplicate values from each experimental condition and p value was used to select the candidate phosphoproteins. The level was set at p ≤ 0.121 in the experiment, and the analysis of triplicate values were as follows: all three values in T3 -treated chicks were higher (or lower for downregulated phosphorylation) than those in dark-reared chicks (p = 0.050), two of the three values in T3 -treated chicks were higher (or lower for downregulated phosphorylation) than all three values in dark-reared chicks and one of the values in T3 -treated chicks was not determined (p = 0.083), and the two of the three values in T3 -treated chicks were higher (or lower for downregulated phosphorylation) than those in dark-reared chicks and that one of the three values was not determined in each condition (p = 0.121).
2. Material and methods 2.3. Immunoprecipitation 2.1. Animals The experiments were performed under the guidelines and approval of the Committee on animal experiments at Teikyo University. The guidelines are based on the national regulations for animal welfare in Japan. Newly hatched domestic chicks of the Cobb strain (Gallus gallus domesticus) were used in the experiments. Fertilized eggs were obtained from a local supplier (3-M, Nagoya, Japan) and incubated at 37 ◦ C for 21 days. After hatching, the chicks
Immunoprecipitation and western blotting were performed as previously described by Yamaguchi et al. [27] with minor modifications. Chick brains were lysed at 4 ◦ C in a lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 10 mM NaF, 10 mM EDTA, 1% NP-40, 1 mM sodium orthovanadate, 10 mM sodium diphosphate decahydrate, 0.5 mM DTT, pH 7.4) containing protease inhibitors (P2714, Sigma, USA) and phosphatase inhibitors (P5726, Sigma, USA). The lysate was then centrifuged at 10000 × g for 10 min. Pro-
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Fig. 2. NDPK kinase activity plays a role in the opening and reopening of the sensitive period of imprinting. (A) The scheme to test the effect of inhibitors on the reopening of the sensitive period. Imprinting training and simultaneous choice test were carried out by the method of Izawa et al. [8] After hatching, chicks were kept in dark enclosures to prevent exposure to light until day 4. The chicks were then trained with a yellow LEGO object and the preference for the yellow LEGO object was evaluated 1 h later. Imprinting training was initiated 30 min after the inhibitor injection. (B) Inhibitors injected in the IMM 30 min before the T3 injection hampered the effect of exogenous T3 on chicks whose sensitive period had closed on day 4. Each value represents the mean ± S.E.M. (The Kruskal–Wallis test and subsequent multiple comparisons, *p < 0.05 vs. chicks injected with T3 alone.) (C) The schemes to test the effect of inhibitors on imprinting. After hatching, chicks were kept in dark enclosures to prevent exposure to light until day 1, and then trained and tested. (D) Inhibitors injected in the IMM 30 min before T3 injection hampered the acquisition of imprinting on day 1. Each value represents the mean ± S.E.M. (The Kruskal–Wallis test and subsequent multiple comparisons, *p < 0.05 vs. dark-reared chicks).
tein A-Sepharose (GE Healthcare UK Ltd., Little Chalfont, UK) was incubated with anti-NDPK1/2 antibody (1:200, C-20, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 6 h followed by overnight incubation with each supernatant (0.5 mg of protein). The
immunoprecipitates were subjected to western blotting. Phosphorylated NDPK was detected using biotinylated Phos-tagTM (Wako Pure Chemical Industries, Ltd., Osaka, Japan) [11]. The horseradish peroxidase-conjugated antibody (1:2000, GE Healthcare, UK) was
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Fig. 3. NDPK kinase activity plays a role in the acquiring memory priming (MP). (A) The schemes to test the effect of inhibitors on the acquisition of MP. (B) The inhibitor injected into IMM just before intravenous treatment with T3 on day 1 hampered the MP activity of T3 , but did not hamper when injected just before the training on day 4. Data are presented as mean ± SEM (Kruskal–Wallis test and subsequent multiple comparisons, *p < 0.05 vs. dark-reared chicks).
used as the secondary antibody. Band intensities were quantified using CS Analyzer ver. 3.0 (ATTO Corporation, Tokyo, Japan), and the ratios of the band intensities were calculated. 2.4. Injection Intravenous injection of T3 and IMM injection of various inhibitors were administered as previously described by Yamaguchi et al. [26,28]. In case of IMM injection, chicks were anesthetized with a 2% isoflurane/air mixture and fixed on a stereotaxic apparatus as isoflurane anesthesia was maintained. The skin over the skull was incised and reflected, and a small piece (0.2 mm × 0.2 mm) of the dura mater was cut to expose the telencephalon in order to insert a micropipette for injection. The stereotaxic coordinates were as follows: intermediate medial mesopallium (IMM): 2.9 mm anterior to the bregma, 1.3 mm lateral to the midline. Ellagic acid (1 mM in DMSO, Sigma–Aldrich, USA), ebselen (1 mM in DMSO, Tocris Bioscience, Bristol, UK) were injected at a rate of 14 nl/min for 35 min through the micropipette using an automated injector (Nanoject II, Drummond). The chicks were then returned to the dark breeder and allowed to recover from anesthesia for 30 min. Soon after the recovery, training and tests for imprinting were performed using the methods described by Izawa et al. [8]. 2.5. Training and test procedures Training was carried out according to the methods presented by Izawa et al. [8] after minor modifications. A custom-made I-shaped maze was equipped with a rubber belt (width: 8 cm, length: 43 cm) controlled by a PIC-based sequencer (Tri State Co., Ltd., Japan). An imprinting object (made of yellow LEGO® blocks, 4.7 cm × 6.2 cm × 5.0 cm) at one end of the maze rotated under illumination, and an infrared sensor was placed near the object. When the chick hit the sensor, the belt moved backward and pulled the chick away from the object. Two 1-h training sessions were conducted, and we counted the number of sensor hits as a behavioral
measure of activity during training. Those chicks that hit more than 500 times during the two training sessions were selected and subsequently tested. In choice tests, a cross maze with a pair of 20cm-long start arms and a pair of 46-cm-long side arms was used. The imprinting object (yellow) was placed at the end of one side of the arm, and a novel control object of the same shape (but of different color, red) at the end of the other side of the arm. The successfully trained chick was placed in one start arm, while the other arm was occluded; the start arms were counter-balanced in each test trial. We measured the total time spent near the objects during a 120-s-long test trial, which was repeated four times and the data were averaged. The preference score was given as (time near the imprinting object) − (time near the control object) in sec. 3. Results In order to understand the signal transduction pathway by T3 in the reopening of the sensitive period, we identified proteins whose phosphorylation levels were upregulated or downregulated by the treatment of T3 in chick brains. We injected T3 intravenously into 4-day-old chicks, kept them in dark enclosures for 30 min, 1 h, or 3 h, and collected the whole brains that were then used for phosphoproteome analysis. In our previous report, the genes which play important roles in imprinting were upregulated regardless of gender [26]. Then, we tried to identify the proteins whose phosphorylation levels were upregulated both in male and in female chicks. We identified 1079 phosphopeptides from 520 proteins. Of the phosphopeptides, nine proteins exhibited more than 1.2fold increase in phosphorylation level in both male and female chicks (Table 1) and six phosphopeptides showed less than 0.8-fold decrease (Table 2) following T3 injection compared to the levels detected in the brain of dark-reared chicks. We focused on NDPK2 because the increase in the phosphorylation level of NDPK2 following the T3 injection showed the most significant difference in p value (male, p = 0.05; female p = 0.05) among the nine identified proteins (Table 1). To confirm the increase in the phosphorylation level, we performed immunoblotting. The results confirmed
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Table 1 List of proteins whose phosphorylation level increased after T3 injection. Time after T3 injection
Name of phophorylated protein
Phosphorylated peptide
Fold ♂
Fold ♀
p♂
p♀
30 min
Muscarinic acetylcholine receptor M4 Cytoplasmic dynein 1 light intermediate chain 1 Nucleoside diphosphate kinase 2
DKPTTEILPAGQGQSPAHPR AGSFGSSSASGAANNASAELR NIIHGSDSVESAQK
1.3 1.4 1.5
1.5 1.7 1.5
0.121 0.121 0.050
0.121 0.121 0.050
1h
Creatine kinase B-type Heat shock protein HSP 90-alpha
TDLNADNLQGGDDLDPNYVLSSR LGIHEDSQNR
1.8 2.6
1.6 1.5
0.121 0.083
0.121 0.050
3h
Endophilin-A2 Catenin alpha-2 Creatine kinase B-type Phosphoglycerate mutase 1
SIPHLDQPCCK TPEELEDDSDFEQEDYDVR TDLNADNLQGGDDLDPNYVLSSR HGESAWNLENR
1.2 1.7 1.8 2.2
1.6 1.3 1.6 2.0
0.121 0.121 0.121 0.121
0.121 0.121 0.121 0.121
Table 2 List of proteins whose phosphorylation level decreased after T3 injection. Time after T3 injection
Name of phophorylated protein
Phosphorylated peptide
Fold ♂
Fold ♀
p♂
p♀
3h
Drebrin Heat shock protein HSP 90-alpha Hemoglobin subunit beta 14-3-3 protein epsilon Neuromodulin Zinc finger Ran-binding domain-containing protein 2
TADSGPPSSSSSSSSPPR TEDKPEIEDVGSDEEEEK LHVDPENFR EALQDVEDENQ TDAVEETKPTESAQQEEVK YNLDASEEEDTNK
0.26 0.37 0.47 0.56 0.72 0.79
0.15 0.13 0.58 0.23 0.49 0.63
0.121 0.121 0.121 0.121 0.121 0.121
0.121 0.121 0.121 0.121 0.121 0.121
the upregulation of NDPK2 phosphorylation 30 min after the intravenous injection of T3 (Fig. 1A and B), suggesting that the kinase activity of NDPK2 might play a role in the reopening of the sensitive period. It is interesting to note that there was an increase in the phosphorylation level of one peptide derived from Heat shock protein (HSP) 90-alpha 30 min after T3 treatment and an decrease in the phosphorylation level of the other peptide of HSP 90-alpha after 3 h T3 treatment. Next, we investigated whether the kinase activity of NDPK2 is involved in the reopening of the sensitive period when it closed on day 4. It has been shown that the phosphorylation of NDPKs links their kinase activities to other substrates [9]. Ellagic acid and ebselen are structurally different chemicals, but both are known to be the potent and selective inhibitors of NDPK2 kinase activities [16,20]. We found that these inhibitors hampered the effect of T3 in the reopening of the sensitive period (Fig. 2A and B). During the course of imprinting training in the sensitive period 1 day after hatching, intrinsic T3 flows into the brain rapidly and the increased amount of T3 enables the chicks to be imprinted [26]. We then examined whether the kinase activity of NDPK2 is necessary for the acquisition of imprinting. As shown in Fig. 2, the NDPK2 kinase inhibitors injected in one-day-old chicks hampered the acquisition of imprinting on day 1 (Fig. 2C and D), indicating that the signaling pathway through NDPK2 is crucial for the opening of the sensitive period in addition to its reopening. The chicks treated with T3 on day 1 were able to be imprinted on day 4 and even on day 8 [26]. We named this potential given by the temporal wave of T3 “memory priming” (MP). Once chicks have achieved MP, it primes later reinforcement learning other than imprinting [26]. Since the increase in phosphorylation of NDPK2 is involved in the opening and reopening of the sensitive period, we next investigated whether the NDPK2 kinase activity could affect MP. When we injected ellagic acid into the IMM just before intravenous T3 injection on day 1, the inhibitor hampered the MP activity of T3 . However, the inhibitor injected before imprinting training on day 4 did not hamper the MP activity of T3 (Fig. 3B). These results indicate that the signaling pathway through NDPK2 is a prerequisite for the process of acquiring MP. 4. Discussion NDPK proteins are a family of highly conserved proteins in eukaryotes [13] of which seven genes have been identified in
chickens. Among seven chick NDPK proteins, NDPK1 and NDPK2 especially show high homology over all sequence each other (93.2%). NDPK2 is expressed more abundantly than NDPK1 in the central nervous system of rats [21]. Since we identified significant amount of phosphopeptides for NDPK2 but negligible amount of phosphopeptides for NDPK1 on the phosphoproteome analysis, we assume that the phosphorylated NDPK detected in immunoblotting is NDPK2 We showed that NDPK2 acts downstream of T3 signaling. How does T3 transduce the signal to NDPK2? In a previous study, we found that the heterodimer of T3 and TR transduced their information to PI3 K in a non-genomic way to reopen the sensitive period [26]. There are several reports that NDPK2 functions downstream of PI3 K; for example, in the activation of human CD4 T-cell, PI3 K sends signal to NDPK2 for the opening of K+ channels [23]. PI3 K in chick brain may transduce the TR-mediated signal to NDPK2 in the reopening of the sensitive period of imprinting. What is the function of NDPK2 in the signaling pathway of T3 ? Accumulating evidence suggest that NDPK2 interacts with various proteins which regulate the actin cytoskeleton [22]. The binding of NDPK2 to RhoGEFs is shown to regulate the dynamics of the actin cytoskeleton in fibroblasts through the mode of action of Rho GTPases [1,7]. In neurons, the structural changes in actin cytoskeleton of dendritic spines are suggested to be the basis for learning and memory [5,10]. NDPK2 in neuronal cells may regulate the actin cytoskeleton remodeling and give molecular basis for imprinting. Why 4-day-old chicks cannot be imprinted by imprinting training without the exogenous T3 ? On day 1, T3 flows into the brain following the imprinting training, which leads to the increase of T3 levels in the brain and enables chicks to be imprinted. In contrast, little amount of T3 flows into the brain by imprinting training on day 4; therefore the T3 level in the brain remains low, which keeps the sensitive period unopened [26]. As we showed in the present report, NDPK2-mediated signaling pathway is necessary for imprinting on either 1-day-old or 4-day-old chicks, suggesting that the mechanisms of the opening and reopening of the sensitive period share the common molecular cascade downstream of T3 in the neuronal network to cause imprinting. Our previous study has also shown that T3 confers MP to chicks. In case of imprinting, MP is endowed simultaneously with the training through the inflow of intrinsic T3 in brains. Under experimental conditions, the chicks can achieve MP by exogenous T3 injection during the sensitive period without imprinting training, indicating
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that the process of acquiring MP conferred by T3 is separable from the learning itself [26]. As shown in Fig. 3B, the NDPK2 inhibitor hampered the MP activity of T3 when injected just before T3 injection on day 1, but not before imprinting training on day 4. The data suggest that NDPK2 is involved in the process of acquiring MP, but not in the process of the execution of imprinting behavior, supporting the idea that T3 is the primer that confers the learning potency. 5. Conclusions We showed that NDPK2 plays a crucial role downstream of T3 action and that its phosphorylation is involved in the non-genomic signaling during imprinting. Our data also indicate that NDPK2 participates in the process of memory priming which endows the learning potency. Thus, chicks will acquire imprinting on the basis of the priming process through the acute action of T3 . Conflict of interest There are no conflicts of interest. Acknowledgements This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (N.A., 24790089; S.Y., 24590096, 15K07945; T.M., 25291071; K.J.H., 26440182); the Grant-in-Aid for Scientific Research on Innovative Areas “Memory dynamism” (26115522) and “Adaptive circuit shift” (15H01449) from the Ministry of Education, Culture, Sports, Science and Technology (K.J.H.); the Naito Foundation (K.J.H.); the Japan Foundation of Applied Enzymology (K.J.H.); NOVARTIS Foundation (Japan) for the Promotion of Science (K.J.H.); the Uehara Memorial Foundation (K.J.H., S.Y.); the Sagawa Foundation for Promotion of Cancer Research (S.Y.); Sumitomo Foundation (S.Y.); Takeda Science Foundation (S.Y.); and the Pharmaceutical Society of Japan Award for Young Scientists (S.Y.). We thank Dr. Kazuhiro Wada for providing the protocol for genomic PCR used for gender identification of the chicks. We thank Takaaki Kitajima for technical assistance. References [1] H.N. Fournier, C. Albigés-Rizo, M.R. Block, New insights into Nm23 control of cell adhesion and migration, J. Bioenerg. Biomembr. 35 (2003) 81–87. [2] F. Gervasi, I. D’Agnano, S. Vossio, G. Zupi, A. Sacchi, D. Lombardi, nm23 influences proliferation and differentiation of PC12 cells in response to nerve growth factor, Cell Growth Differ. 7 (1996) 1689–1695. [3] R. Griffiths, M.C. Double, K. Orr, R.J. Dawson, A DNA test to sex most birds, Mol. Ecol. 7 (1998) 1071–1075. [4] E.H. Hess, Imprinting, Science 130 (1959) 133–141. [5] A. Holtmaat, K. Svoboda, Experience-dependent structural synaptic plasticity in the mammalian brain, Nat. Rev. Neurosci. 10 (2009) 647–658. [6] G. Horn, Pathways of the past: the imprint of memory, Nat. Rev. Neurosci. 5 (2004) 108–120.
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