Neurochem Res (2014) 39:785–792 DOI 10.1007/s11064-014-1270-x

ORIGINAL PAPER

Impairments in Brain-Derived Neurotrophic Factor-Induced Glutamate Release in Cultured Cortical Neurons Derived from Rats with Intrauterine Growth Retardation: Possible Involvement of Suppression of TrkB/Phospholipase C-c Activation Tadahiro Numakawa • Tomoya Matsumoto • Yoshiko Ooshima • Shuichi Chiba Miyako Furuta • Aiko Izumi • Midori Ninomiya-Baba • Haruki Odaka • Kazuo Hashido • Naoki Adachi • Hiroshi Kunugi

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Received: 10 November 2013 / Revised: 10 February 2014 / Accepted: 25 February 2014 / Published online: 6 March 2014 Ó Springer Science+Business Media New York 2014

Abstract Low birth weight due to intrauterine growth retardation (IUGR) is suggested to be a risk factor for various psychiatric disorders such as schizophrenia. It has been reported that developmental cortical dysfunction and neurocognitive deficits are observed in individuals with IUGR, however, the underlying molecular mechanisms have yet to be elucidated. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are associated with schizophrenia and play a role in cortical development. We previously demonstrated that BDNF induced glutamate release through activation of the TrkB/phospholipase C-c (PLC-c) pathway in developing cultured cortical neurons, and that, using a rat model for IUGR caused by maternal administration of thromboxane A2, cortical levels of TrkB were significantly reduced in IUGR rats at birth. These Electronic supplementary material The online version of this article (doi:10.1007/s11064-014-1270-x) contains supplementary material, which is available to authorized users. T. Numakawa (&)  Y. Ooshima  S. Chiba  M. Furuta  A. Izumi  M. Ninomiya-Baba  H. Odaka  N. Adachi  H. Kunugi Department of Mental Disorder Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan e-mail: [email protected] T. Numakawa  T. Matsumoto  N. Adachi  H. Kunugi Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), 4-1-8, Honmachi, Kawaguchi, Saitama 332-0012, Japan T. Matsumoto Department of Psychiatry and Neurosciences, Institute of Biomedical and Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima, Hiroshima 734-8553, Japan

studies prompted us to hypothesize that TrkB reduction in IUGR cortex led to impairment of BDNF-dependent glutamatergic neurotransmission. In the present study, we found that BDNF-induced glutamate release was strongly impaired in cultured IUGR cortical neurons where TrkB reduction was maintained. Impairment of BDNF-induced glutamate release in IUGR neurons was ameliorated by transfection of human TrkB (hTrkB). Although BDNFstimulated phosphorylation of TrkB and of PLC-c was decreased in IUGR neurons, the hTrkB transfection recovered the deficits in their phosphorylation. These results suggest that TrkB reduction causes impairment of BDNF-stimulated glutamatergic function via suppression of TrkB/PLC-c activation in IUGR cortical neurons. Our findings provide molecular insights into how IUGR links to downregulation of BDNF function in the cortex, which might be involved in the development of IUGR-related diseases such as schizophrenia.

S. Chiba Faculty of Pharmacy, Research Institute of Pharmaceutical Science, Musashino University, 1-1-20, Shinmachi, Nishi-Tokyo, Tokyo 202-8585, Japan M. Furuta Department of Physiology, St Marianna University School of Medicine, 2-16-1, Sugou, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan A. Izumi Division of Structural Cell Biology, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan

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Keywords Schizophrenia  Intrauterine growth retardation  BDNF  TrkB  Glutamatergic transmission

Introduction A growing body of evidence suggests that low birth weight caused by intrauterine growth retardation (IUGR) is linked with a risk of psychiatric disorders including schizophrenia [1, 2]. It has been demonstrated that children with low birth weight exhibit deficits in neurocognitive functions, a major phenotype of schizophrenia [3, 4, 5, 6]. The cerebral cortex is a major region involved in various neurocognitive functions. Interestingly, it was reported that premature infants (12-month corrected age) with cognitive impairments already showed abnormal electroencephalogram patterns at 6 weeks of age [7]. Taken together, it is speculated that low birth weight is associated with abnormal cortical activities leading to developmental neurocognitive deficits. However, molecular mechanisms underlying IUGR-related cortical dysfunction remain elusive. Glutamate is the primary excitatory neurotransmitter in the brain, and abnormal glutamatergic transmission in the cortex is involved in neurocognitive dysfunction. It is well known that brain-derived neurotrophic factor (BDNF) plays a key role in the regulation of glutamatergic neurotransmission, and influences cortical development and cognitive function [8]. BDNF activates several signaling pathways via its receptor TrkB, including extracellular signal-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K)/Akt, and phospholipase C-c (PLC-c) pathways. We previously showed that BDNF acutely increased glutamatergic transmission by inducing glutamate release through TrkB/PLC-c activation in cultured cortical neurons [9] [10]. Furthermore, we recently found that levels of TrkB in the cortical tissues obtained from postnatal day-1 rats with IUGR were greatly decreased [11]. Therefore, it is possible that reduced TrkB levels in IUGR cortex at an early developmental stage have a

M. Ninomiya-Baba Department of Pharmacology, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1, Okubo, Shinjuku-ku, Tokyo 169-8555, Japan H. Odaka Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1, Okubo, Shinjuku-ku, Tokyo 169-8555, Japan K. Hashido Administrative Section of Radiation Protection, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawa-Higashi, Kodaira, Tokyo 187-8502, Japan

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negative impact on the BDNF-regulated glutamatergic system via suppressing TrkB-stimulated intracellular signaling. In the present study, we used cultured cortical neurons prepared from IUGR rats to investigate whether BDNF-induced glutamate release was impaired in IUGR neurons by affecting the TrkB/PLC-c system.

Materials and Methods Rat Model for IUGR Pregnant Long-Evans rats (Institute for Animal Reproduction, Ibaraki, Japan) were housed in a 12-h light/dark cycle with free access to water/food. IUGR was induced as shown previously [12]. Briefly, on the thirteenth day of pregnancy, synthetic TXA2 analog (9,11-dideoxy-9a, 11a-methanoepoxy-prosta-5Z, 13E-dien-1-oic acid; Cayman Chemical, MI, USA) was maternally applied at a rate of 125 ng/h until the delivery using an osmotic pump (2ML1, Alzet Corp., CA, USA) containing TXA2 solution (12.5 lg/ml). PBS was applied as a control. The pump was implanted into the lower portion of the peritoneal cavity under anesthesia with sodium pentobarbital (35 mg/kg body weight). After delivery, newborn rats were weighed, followed by removing their brains for dissociated cultures. To obtain cortical tissues of postnatal day-7 rats, twelve male pups from control and IUGR litters (n = 6 each) were moved to Wistar foster mothers (Tokyo Laboratory Animals Science Co., Tokyo, Japan) on postnatal day 1 in order to reduce the effect of the wound caused by the pump implantation on breast feeding. Wistar foster mothers were used because they were easily available and relatively inexpensive compared to Long-Evans rats. The body weight of IUGR pups from TXA-treated mothers was 86.56 % that of control pups at birth (n = 6 each). Pups were allowed to grow until sampling on postnatal day 7 (IUGR weight; 116.75 % of control). These animal experiments were approved by the Ethics Review Committee for Animal Experimentation of the National Institute of Neuroscience, Japan. Cortical Neuron Culture Cultured neurons were prepared from the cerebral cortices of postnatal day-1 rats with or without IUGR as previously reported [11]. Briefly, dissociated cortical neurons (5 9 105 cells/cm2) were plated on culture dishes/plates (Becton–Dickinson, NJ, USA) coated with polyethylenimine (Sigma–Aldrich, MO, USA), and maintained with 1:1 mixture of D-MEM/F-12 medium (Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium) (Invitrogen, CA, USA) supplemented with 5 % fetal bovine serum and

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5 % heated-inactivated horse serum for 5 days. In some experiments, cultured neurons were transfected with constructs encoding human TrkB (see below) by using Lipofectamine 2000 reagent (Invitrogen) at 4 days in vitro. A vector encoding GFP was used as a control (pAcGFP1-N1, Clontech, CA, USA). Unless otherwise indicated, cultured neurons at 5–6 days in vitro were used for glutamate release assay and Western blotting analysis. In these experiments, BDNF (final 100 ng/ml) dissolved in PBS containing BSA (1 mg/ml) was bath-applied to cultured neurons. SH-SY5Y Cell Culture The human neuroblastoma cell line, SH-SY5Y (ECACC, Public Health England, London, UK), was plated on culture dishes (Becton–Dickinson) in 1:1 D-MEM/F-12 medium (Invitrogen) supplemented with 10 % fetal bovine serum and penicillin (18 units/ml)/streptomycin (18 mg/ ml) (Sigma–Aldrich). The medium was changed with a fresh one every 3 days. Subconfluent cells were used for human TrkB transfection with Lipofectamine 2000 (Invitrogen). Forty-eight hours later, the cell lysates were collected for Western blotting.

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release, and then BDNF (final 100 ng/ml) was applied to KRH buffer freshly added to the culture for 1 min as BDNF-stimulated release. Immunoprecipitation To detect the phosphorylation of PLC-c, immunoprecipitation was performed as previously shown [10]. Briefly, cultured neurons were lysed using 1 % Triton-X buffer (pH7.4) containing 20 mM Tris–HCl, 150 mM NaCl and 5 mM EDTA. The lysates (300 lg of total protein) were incubated with Protein G-Sepharose beads (GE Healthcare, PA, USA) prebound to anti-PLC-c antibody (200 lg/ml, Santa Cruze Biotechnology Inc., CA, USA) for 3 h at 4 °C. After washes with the lysis buffer, the immunoprecipitates were collected for Western blotting. Western Blotting

For cloning of cDNA encoding full-length human TrkB, total RNA (0.3 lg) isolated from SH-SY5Y cells was reverse transcribed using SuperScriptTM III First-Strand Synthesis System for RT-PCR (Invitrogen) and Oligo(dT) primer. The cDNAs were then PCR-amplified using the following primers to amplify the coding sequence of full-length human TrkB: forward primer, 50 -CGGAATTCGCCACCATGTCG TCCTGGATAAGGTG-30 and reverse primer, 50 -CGGGAT CCGCCTAGAATGTCCAGGTAGA-30 . The PCR products were inserted into the multiple cloning site in the pFLAGCMVTM-5a EXPRESSION VECTOR (Sigma–Aldrich) and sequenced using Dye Terminator Cycle Sequencing with Quick Start kit (BECKMAN COULTER, CA, USA) and sequencer CEQ8000 (BECKMAN COULTER).

Cultured cells were lysed in 1 % SDS buffer (pH 7.4) containing 10 mM Tris–HCl, 5 mM EDTA, 10 mM NaF, 2 mM Na2VO4, 0.5 mM phenylarsine oxide, and 1 mM phenylmethylsulfony fluoride. Using the cell lysates or immunoprecipitates indicated in the above, SDS-PAGE and Western blotting were carried out. For Western blotting, the following primary antibodies were used at the indicated dilutions: anti-bactin (1:3,000, Sigma–Aldrich), anti-p75 (1:1,000, Promega, WI, USA), anti-phosphoTrkA (1:200, Cell Signalling, MA, USA), anti-PLC-c (1:1,000, Santa Cruz Biotechnology Inc.), anti-TrkB (1:500, BD Biosciences, NJ, USA), and anti-phosphoTyr (1:1000, Millipore, MA, USA) antibodies. According to the datasheet of the anti-phosphoTrkA antibody, it detects not only phosphorylated TrkA but also phosphorylated TrkB. We previously confirmed that TrkB (but not TrkA) is a major Trk receptor in rat cortical neurons [13]. Therefore, we used the antiphosphoTrkA antibody for detection of phosphorylated TrkB. Densitometry analysis was performed to quantify the intensities of the immunoreactivities by using CS Analyzer software (ATTO Corp., Tokyo, Japan).

Glutamate Release Assay

MTT Assay

The amounts of glutamate released from cultured cortical neurons were measured using high-performance liquid chromatography (Shimadzu Co, Kyoto, Japan) as previously reported [10]. Briefly, cultured neurons at 5 days in vitro were washed with the modified Hepes-buffered Krebs–Ringer (KRH) solution containing 130 mM NaCl, 5 mM KCl, 1.2 mM NaH2PO4, 1.8 mM CaCl2, 10 mM glucose, 1 % BSA, and 25 mM Hepes (pH7.4). The KRH buffer was collected after 1-min incubation as a basal

To check the effect of transfection on cell viability, an MTT assay was conducted as previously reported [11]. Briefly, cultured neurons at 6 days in vitro (48 h after transfection) were incubated with the tetrazolium salt, MTT (Sigma–Aldrich), for 2 h during which the metabolic conversion of MTT was initiated by mitochondrial reductase of surviving cells. The activity of the reductase was then measured with a plate reader (absorbance of 570 nm, Bio-Rad Laboratories Inc., CA, USA).

Generation of Constructs for Expressing Human TrkB

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Statistical Analysis Data are presented as mean ± standard deviation (SD). We performed a student’s t test, or two- or three-way analysis of variance (ANOVA) followed by Bonferroni post hoc test in SPSS ver. 21 (IBM, Japan) to evaluate statistical significance. Probability values less than 5 % were considered statistically significant.

Results TrkB Reduction Not Only in Cortical Tissues But Also in Cultured Cortical Neurons Obtained from Rats with IUGR As reported previously [11], birth weight of rats with IUGR induced by maternal administration of TXA2 was lower by 13–22 % compared with those without TXA2 administration (control). As also reported [11], levels of TrkB were significantly reduced in cortical tissues from postnatal day-1 rats with IUGR (Fig. 1a, b). Importantly, we found that the downregulation of TrkB was still observed in cortical tissues obtained from IUGR pups 7 days after their birth compared with control (Fig. 1c, d). In contrast, cortical levels of p75 (a low affinity receptor for neurotrophin) did not change at postnatal days 1 and 7 (Fig. 1a–d). We then confirmed our previous findings [11] that levels of TrkB, but not p75, were significantly reduced in cultured cortical neurons at 5 days in vitro prepared from postnatal day-1 IUGR rats compared with those from control rats (Fig. 2a, b). Impairment of BDNF-Induced Release of Glutamate due to Reduced TrkB Levels in IUGR Neurons Next, we examined whether IUGR-related reduction in TrkB levels caused an impairment of BDNF-induced release of glutamate. To this end, we used cultured cortical neurons prepared from postnatal day-1 rats, because (1) TrkB reduction observed in IUGR cortical tissues was similarly maintained in cultures (see Figs. 1, 2), (2) IUGR did not affect cell survival under basal conditions [11], and (3) dissociated neurons in culture can develop and are available for DNA transfection [10]. As shown in Fig. 3, acute application of BDNF induced glutamate release in control cultures, while the effect of BDNF on glutamate release was markedly impaired in IUGR neurons. Basal release of glutamate before BDNF stimulation was not affected (Fig. 3). Since we and others have confirmed that 100 ng/ml of BDNF exerts maximal effects on glutamate release [10] [14], this concentration was used in the present study. To

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test whether TrkB reduction is the cause of impairment in BDNF-induced glutamate release from IUGR neurons, we generated a construct encoding human TrkB (hTrkB) and performed a rescue experiment. Transfection of the hTrkB construct into SH-SY5Y cells indeed showed an increase in the TrkB levels without any effect on p75 expression (please see Fig. 4a, b). In cells transfected with hTrkB, immunoblotting with anti-TrkB antibody showed doublet bands, which may be derived from glycosylated and non-glycosylated TrkB [15]. Importantly, impairment of BDNFinduced glutamate release in IUGR neurons was largely recovered after hTrkB transfection (Fig. 3). Induction of hTrkB slightly increased BDNF-induced release in control neurons compared to those without hTrkB transfection, whereas no change in basal release after hTrkB transfection was observed (Fig. 3). No alteration of cell viability after transfection in cultured cortical neurons was observed (Fig. 4c). These results suggest that downregulation of TrkB by IUGR has a negative impact on glutamatergic neurotransmission mediated by BDNF. Suppression of PLC-c Signaling in IUGR Neurons As BDNF-induced glutamate release is dependent on the PLC-c pathway in our cultures [9], we hypothesized that activation of PLC-c by BDNF was impaired in IUGR neurons. As expected, phosphorylation of TrkB was clearly observed within 60 s after BDNF stimulation in control neurons, though the TrkB response to BDNF in IUGR neurons was weak (Fig. 5a, b). Importantly, decreased TrkB phosphorylation in IUGR neurons was recovered by hTrkB induction (Fig. 5a, b). No alteration in levels of actin as a control was confirmed in any conditions tested (Fig. 5a, b). Interestingly, although BDNF-stimulated phosphorylation of PLC-c was hardly detectable in IUGR neurons, the deficit in PLC-c signaling was reversed when hTrkB was transfected (Fig. 5c, d). While total levels of TrkB were reduced, those of PLC-c did not change in IUGR neurons (Fig. 2 and supplementary Fig. 1). These results indicate that activation of TrkB/PLC-c signaling triggered by BDNF is repressed in IUGR neurons, which is likely due to reduced levels of TrkB.

Discussion In this study, we found that BDNF-induced glutamate release was impaired in cultured cortical neurons from IUGR rats. BDNF-stimulated activation of TrkB/PLC-c signaling, which is required for BDNF-induced glutamate release [9], was also suppressed in IUGR neurons. Furthermore, transfection of hTrkB into IUGR neurons

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Fig. 2 Reduction in TrkB, but not p75, levels in cultured IUGR cortical neurons. a Western blotting with anti-TrkB, p75 and actin antibodies were performed using protein lysates of cultured cortical neurons at 5 days in vitro prepared from postnatal day-1 rats with IUGR or those without IUGR (CON). Actin levels were shown as a control. b Quantification of a. TrkB and p75 levels normalized to those in control were shown. Data present mean ± SD (n = 5, n indicates the number of dishes for each experimental condition). ***P \ 0.001 by Student’s t test. ns indicates no significant difference. The reproducibility was confirmed with several independent series of cultures

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Fig. 1 Reduction in TrkB, but not p75, levels in cortical tissues obtained from IUGR rats. a Western blotting with anti-TrkB, p75 and actin antibodies was performed using protein lysates of cortical tissues obtained from postnatal day-1 (P1) rats with IUGR or those without IUGR (CON). b Quantification of a (n = 5, n indicates the number of control and IUGR rats respectively). c Levels of TrkB, p75, and actin in the cortex of postnatal day-7 (P7) rats with IUGR or those without IUGR (CON) were shown (Western blotting). d Quantification of c (n = 6, n indicates the number of control and IUGR rats respectively). Actin levels were shown as a control. TrkB and p75 levels normalized to those in control were shown. Data present mean ± SD. ***P \ 0.001 by Student’s t test. ns indicates no significant difference

ameliorated the impairments of BDNF-stimulated glutamate release and TrkB/PLC-c activation. Low birth weight measures are used as a crude marker of IUGR [16]. In the present study, we used a rat model for IUGR by maternal TXA2 application because lower birth weight can be achieved as we previously reported [11]. Using this model, we previously showed that the levels of TrkB were reduced in the cortex of IUGR rats at birth [11]. In the present study, we found that TrkB reduction in the cortex was maintained at least until postnatal day 7, suggesting that TrkB downregulation at birth continues during the early stage of postnatal cortical development. Decreased TrkB levels were consistently observed after dissociation for neuronal cultures [11]. Therefore, we used this culture system for investigating what occurred in cortical neurons as a result of TrkB reduction due to IUGR. We previously demonstrated that the survival-promoting

effect of BDNF on cultured cortical neurons from IUGR rats was attenuated [11]. Indeed, MRI studies demonstrated a reduction in gray matter volume in the cerebral cortex in children with low birth weight [17], implying that IUGRrelated reduction in cortical TrkB expression in the early stage of development may affect the survival of cortical neurons. In addition to such an anatomical abnormality, functional impairments of the cortical regions were reported in premature infants [7]. Here, we found that BDNFinduced glutamate release was impaired in developing cortical neurons from IUGR rats, though basal release of glutamate was intact, suggesting that BDNF-mediated glutamatergic neurotransmission is specifically affected by IUGR. In our previous study, marked downregulation of glutamate receptors including GluA1 and GluN2A in IUGR cortical neurons was observed [11]. Including data of the present study, these results indicate that both preand post-synaptic impairments in glutamatergic transmission are caused by IUGR. It is important to confirm whether the functional change of glutamate system in vitro occurs in the cortex by measuring glutamatergic transmission in vivo [18]. In IUGR cortical neurons, TrkB levels were reduced. In the experiment where hTrkB was transfected into cultured cortical neurons, we found that the impairments of BDNFtriggered glutamate release and TrkB/PLC-c activation in IUGR neurons were reversed, suggesting the reduction in TrkB as a cause for the impairments of BDNF function. In contrast, IUGR did not affect the levels of PLC-c during in vitro maturation. Further, we previously observed that

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the levels of ERK1/2 and of Akt, molecules downstream of TrkB in addition to PLC-c, were also unchanged in IUGR neurons [11]. These results suggest that IUGR specifically affects TrkB expression. It has been revealed that a deficiency in the gene encoding methyl CpG binding protein 2, a transcriptional repressor that binds to methylated DNA, increases expression of brain TrkB [19]. Further, in cortical tissue, it has been reported that levels of truncated TrkB mRNA and the degree of cytosine methylation at the promoter region of its gene are negatively correlated in suicide subjects [20]. Thus, it is interesting to approach whether DNA methylation of the promoter region of full-length TrkB in the early development of the cortex is altered by IUGR. The present study may provide new molecular and cellular mechanisms underlying the notion that low birth weight is linked with a risk of schizophrenia [1, 2]. Previously, we suggested that an alteration in expression of dysbindin-1, a protein encoded by DTNBP1 associated with schizophrenia, influences glutamate release in cultured cortical neurons [21], which is supported by recent evidence using mice lacking dysbindin-1 as an animal model for schizophrenia [22]. Here, we showed an impairment of the glutamatergic system and reduced levels of TrkB (but not PLC-c) in IUGR neurons. Interestingly, postmortem studies have shown a decrease in expression of TrkB [23], but not of PLC-c [24], in the cerebral cortex of patients with schizophrenia. Taken together, our study might contribute to understanding a link between IUGR and schizophrenia at the molecular and cellular level, while further studies are needed.

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Fig. 3 Decreased release of glutamate stimulated by BDNF in IUGR neurons. Cultured cortical neurons from control- or IUGR-rats with or without human TrkB (hTrkB) transfection were used. BDNF (100 ng/ ml, 1 min) was added to neuronal cultures (black bar) after collecting the assay buffer solution for measuring 1-min basal release (white bar). Data present mean ± SD (n = 6). ***P \ 0.001 by three-way ANOVA followed by Bonferroni post hoc test. There was a significant interaction between IUGR and hTrkB transfection [F(1,40) = 24.6, P \ 0.001], IUGR and BDNF application [F(1,40) = 56.6, P \ 0.001], hTrkB transfection and BDNF application [F(1,40) = 129, P \ 0.001]

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Fig. 4 Transfection of human TrkB increased the levels of TrkB, but not p75, in SH-SY5Y cells. a SH-SY5Y cells were transfected with human TrkB (hTrkB), 2 days later, cell lysates were collected for Western blotting with anti-TrkB, p75, and actin antibodies. Actin was shown as a control. b Quantification of a. TrkB and p75 levels normalized to those in control were shown. Data present mean ± SD (n = 4, n indicates the number of dishes for each experimental condition). **P \ 0.01 by Student’s t test. ns, no significant difference. The reproducibility was confirmed with independent series of cultures. c Cell viability of cultured cortical neurons after transfection was assessed with MTT assay. No changes in viability were observed in any conditions tested. Data present mean ± SD (n = 8, n indicates the number of wells for each experimental condition). The reproducibility was confirmed with independent series of cultures

The present study revealed that TrkB/PLC-c pathwaydependent glutamate release was impaired in developing cortical neurons from IUGR rats. This in vitro system would thus provide molecular insights into how IUGR fosters the development of abnormal cortical functions leading to neurocognitive deficits. Since decreased TrkB is a main contributor to the impaired BDNF-stimulated PLCc system for release of glutamate in cortical neurons, drugs that upregulate cortical TrkB during development may be beneficial for treatment of IUGR-related diseases such as schizophrenia. We demonstrate that our in vitro system would be a useful tool to uncover the molecular and cellular mechanisms underlying IUGR-related cortical dysfunction.

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Fig. 5 Decreases in BDNF-stimulated activation of TrkB and of PLC-c in IUGR neurons. Cultured cortical neurons from control- or IUGR-rats with or without human TrkB (hTrkB) transfection were used. BDNF (100 ng/ml) was applied to the cultures for 30 or 60 s, respectively. a Western blotting (WB) with anti-phospho-Trk and actin antibodies. b Phosphorylated Trk stimulated by BDNF (1 min) was quantified. Data present mean ± SD (n = 4, n indicates the number of dishes for each experimental condition). ***P \ 0.001 by two-way ANOVA followed by Bonferroni post hoc test. There was a significant interaction between IUGR and hTrkB transfection

[F(1,12) = 8.03, P \ 0.05]. c Immunoprecipitation (IP) of cell lysates with anti-PLC-c antibodies, followed by Western blotting (WB) with anti-phospho-tyrosine and PLC-c antibodies. d Phosphorylated PLC-c stimulated by BDNF (1 min) was quantified. Data present mean ± SD (n = 4, n indicates the number of dishes for each experimental condition). **P \ 0.01, ***P \ 0.001 by two-way ANOVA followed by Bonferroni post hoc test. There was no significant interaction between IUGR and hTrkB transfection [F(1,12) = 3.81, P = 0.075]

Acknowledgments This study was supported by the following research grants: the Health and Labor Sciences Research Grants (Comprehensive Research on Disability, Health, and Welfare H21-kokoro-002) (H. K.), the Core Research for Evolutional Science and Technology (CREST) Program, Japan Science and Technology Agency (JST) (T.N., N.A. and H.K.), Takeda Science Foundation (T. N.), a grant from Grantin-Aid for Scientific Research (B) (JSPS KAKENHI Grant Number 24300139) (T. N.), and Grant-in-Aid for Challenging Exploratory Research (JSPS KAKENHI Grant Number 25640019) (T. N.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

2. Nielsen PR, Mortensen PB, Dalman C, Henriksen TB, Pedersen MG, Pedersen CB, Agerbo E (2013) Fetal growth and schizophrenia: a nested case–control and case–sibling study. Schizophr Bull 39(6):1337–1342 3. Aarnoudse-Moens CS, Weisglas-Kuperus N, van Goudoever JB, Oosterlaan J (2009) Meta-analysis of neurobehavioral outcomes in very preterm and/or very low birth weight children. Pediatrics 124(2):717–728 4. Freedman D, Bao Y, Kremen WS, Vinogradov S, McKeague IW, Brown AS (2013) Birth weight and neurocognition in schizophrenia spectrum disorders. Schizophr Bull 39(3): 592–600 5. Seidman LJ, Buka SL, Goldstein JM, Horton NJ, Rieder RO, Tsuang MT (2000) The relationship of prenatal and perinatal complications to cognitive functioning at age 7 in the New England Cohorts of the National Collaborative Perinatal Project. Schizophr Bull 26(2):309–321 6. Shenkin SD, Starr JM, Deary IJ (2004) Birth weight and cognitive ability in childhood: a systematic review. Psychol Bull 130(6):989–1013

Conflict of interest

The authors declare no conflict of interest.

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