Repeated administration of phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate (NMDA) receptor blocker, produces schizophrenia-like behaviors in humans and rodents. Although impairment of synaptic function has been implicated in the effect of PCP, the molecular mechanisms have not yet been elucidated. Considering that brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity, we examined whether exposure to PCP leads to impaired BDNF function in cultured cortical neurons. We found that PCP caused a transient increase in the level of intracellular BDNF within 3 h. Despite the increased intracellular amount of BDNF, activation of Trk receptors and downstream signaling cascades, including MAPK/ERK1/2 and PI3K/Akt pathways, were decreased. The number of synaptic sites and expression of synaptic proteins were decreased 48 h after PCP application without any impact on cell viability. Both electrophysiological and biochemical analyses revealed that PCP diminished glutamatergic neurotransmission. Furthermore, we found that the secretion of BDNF from cortical neurons was suppressed by PCP. We also confirmed that PCP-caused downregulation of Trk signalings and synaptic proteins were restored by exogenous BDNF application. It is possible that impaired secretion of BDNF and subsequent decreases in Trk signaling are responsible for the loss of synaptic connections caused by PCP.
Phencyclidine (PCP), a noncompetitive and use-dependent blocker of the N-methyl-d-aspartate (NMDA)-type glutamate receptors, induces psychotic symptoms that are similar to schizophrenia in humans (Allen and Young 1978; Javitt and Zukin 1991). Unlike other psychotomimetic drugs, such as amphetamine, PCP induces negative symptoms (e.g., flattening of affect, alogia, anhedonia, and social withdrawal) and cognitive deficits in addition to positive symptoms (e.g., delusions, hallucinations, and formal thought disorder) of schizophrenia (Andreasen 1995; Olney and Farber 1995; Jentsch and Roth 1999). In rodents, PCP causes schizophrenia-related behaviors, such as disruption in prepulse inhibition (Mansbach and Geyer 1989), stereotyped behavior, and social isolation (Sams-Dodd 1996), increased immobility in forced swimming (Noda et al. 1995), and impaired learning and memory in various maze tasks (Kesner et al. 1983; Handelmann et al. 1987; Danysz et al. 1988; Wass et al. 2006). Extensive reduction in the number of spines in the rat prefrontal cortex has been demonstrated with subchronic PCP treatment (Hajszan et al. 2006), indicating a substantial contribution of abnormal synaptic function to the development of schizophrenia-related behaviors (Mirnics et al. 2001; Frankle et al. 2003; McCullumsmith et al. 2004). Moreover, recent findings suggest that altered expression of genes encoding synapse-associated proteins also play a critical role in the development of schizophrenia (Harrison and Weinberger 2005). Human postmortem studies show reduced dendritic spine density of pyramidal cells in the prefrontal cortex of subjects with schizophrenia (Glantz and Lewis 1997, 2000; Knable et al. 2004). However, the molecular mechanisms underlying the effect of PCP on reduced synaptic connection have not yet been elucidated.
BDNF, a member of the neurotrophin family, plays an important role in synaptic plasticity (Stoop and Poo 1996; Lu 2003; Arancio and Chao 2007, Numakawa et al. 2011) through activation of its receptor Tropomyosin-related kinase B (TrkB) and consequent stimulation of downstream signaling pathways, including mitogen-activated protein/extracellular signal-regulated kinase (MAPK/ERK), phosphoinositide 3-kinase/Akt (PI3K/Akt), and phospholipase C-γ (PLC-γ). We have recently reported important regulatory roles of BDNF in synaptic functions of cortical neurons (Kumamaru et al. 2011). BDNF shows broad expression in the developing and adult mammalian brain (especially, the hippocampus, cerebral cortex, cerebellum, and amygdala) (Ernfors et al. 1990; Hofer et al. 1990; Yan et al. 1997; Conner et al. 1997). As expected, impairment of BDNF/TrkB function has been implicated in the pathogenesis of schizophrenia (Durany and Thome 2004; Angelucci et al. 2005; Lewis et al. 2005), as well as other neuropsychiatric diseases, such as depression (Altar 1999), drug addiction (Davis 2008), Huntington's disease (Zuccato et al. 2001; Gauthier et al. 2004), and Rett syndrome (Chen et al. 2003; Nelson et al. 2008). We found that disrupted-in-schizophrenia 1 (DISC1) and dysbindin, both of which confer susceptibility to schizophrenia, are involved in the regulation of ERK1/2 or Akt signaling (Numakawa et al. 2004; Hashimoto et al. 2006). We have also revealed that glucocorticoid, a stress hormone closely linked to depression (e.g., Kunugi et al. 2006), hampers the synaptic function of BDNF in cortical neurons (Numakawa et al. 2009).
Since expression and secretion of BDNF are facilitated by neuronal activity (Lessmann et al. 2003; Kuczewski et al. 2009), it is likely that PCP may decrease the expression and/or secretion of BDNF via blockade of neuronal activity. Importantly, increased expression of BDNF was reported in rat hippocampal tissue after acute (Kalinichev et al. 2008) and chronic (Takahashi et al. 2006; Harte et al. 2007) treatment with PCP, although one study reported conflicting results (Semba et al. 2006). In the present study, we investigated changes in expression and secretion of BDNF, activity of downstream signaling cascades stimulated via Trk receptors and synaptic function after PCP exposure.
Materials and Methods
Cortical Cultures and PCP Treatment
Cortical cultures were prepared from postnatal day 1 or 2 old rats (Wistar, SLC, Shizuoka, Japan) as described previously (Numakawa et al. 2002). Dissociated cells were plated at a final density of 5 × 105/cm2 on polyethyleneimine-coated culture dishes for immunoprecipitation, immunoblotting, and amino acid measurement. For Ca2+ imaging, cortical neurons were cultured on polyethylenimine-coated cover glasses (Matsunami, Osaka, Japan) with FlexiPERM (Greiner Bio-One GmbH, Germany). For immunostaining or electrophysiological recording, neurons were plated on glass-bottom dishes (Matsunami) with a glial feeder layer. The culture medium (5/5 DF) contained 5% fetal bovine serum, 5% heated–inactivated horse serum, 90% of a 1:1 mixture of Dulbecco's modified Eagle's medium, and Ham's F-12 medium. PCP (Sigma–Aldrich, MO) was added to the neurons by bath application at 10–11 days in vitro (DIVs), followed by incubation of the cultures for 3, 6, or 48 h in the presence of PCP before immunocytochemistry, immunoprecipitation, immunoblotting, amino acid measurement and electrophysiological recording. Pure astroglial cultures were prepared as described previously (Hatanaka et al. 1988). Astroglial cells were obtained from cerebral cortex of postnatal day 1 or 2 old rats. All animals were treated according to the institutional guidelines for the care and use of animals.
Cells were lysed in sodium dodecyl sulfate (SDS) lysis buffer containing 1% SDS, 20 mM Tris–HCl (pH 7.4), 5 mM ethylene-diamine-tetraacetic acid (EDTA) (pH 8.0), 10 mM NaF, 2 mM Na3VO4, 0.5 mM phenylarsine oxide, and 1 mM phenylmethylsulfonyl fluoride. The protein concentration was quantified using a BCA Protein Assay Kit (Pierce Biotechnology Inc., IL), and equivalent amounts of total protein were assayed for each immunoblotting. Primary antibodies were used at the following dilutions: anti-BDNF (1:500, Santa Cruz Biotechnology Inc., CA), anti-TrkB (1:1000, BD Biosciences, NJ), anti-Trk (1:1000, Santa Cruz Biotechnology Inc.), anti-pTyr (1:1000, Upstate, VA), anti-Akt (1:1000, Cell Signaling, MA), anti-pAkt (1:1000, Cell Signaling), anti-ERK (1:1000, Cell Signaling), anti-pERK (1:1000, Cell Signaling), anti-synaptotagmin (1:1000, Calbiochem, Darmstadt, Germany), anti-GluR1 (1:500, Sigma), anti-NR2B (1:500, Sigma), anti-SNAP25 (1:1000, Synaptic Systems, Goettingen, Germany), anti-Bcl-2 (1:1000, BD Biosciences), anti-Bad (1:1000, BD Biosciences), anti-TUJ1 (1:5000, Berkeley Antibody Company, CA), and βactin (1:5000, Sigma) antibodies. The immunoreactivity was quantified by using Lane and Spot Analyzer software (ATTO Corporation, Tokyo, Japan). At least 3 independent series of cultures were used for each set of experiments.
To detect the phosphorylation of Trk receptors, immunoprecipitation was carried out (Numakawa et al. 2002; Numakawa et al. 2009). After cells were lysed with 1% Triton-X buffer (20 mM Tris–HCl (pH 7.4), 5 mM EDTA, 150 mM NaCl, 1% Triton-X100), anti-Trk antibody (Santa Cruz Biotechnology Inc.) prebound Protein G-Sepharose beads (Amersham Pharmacia, NJ) were mixed with the lysates containing 300 μg of total protein and rotated for 3 h at 4 °C. After 3 washes with the lysis buffer, the proteins that bound the affinity beads were separated by SDS-polyacryl- amide gel electrophoresis (SDS-PAGE) and analyzed for immunoblotting with anti-pTyr (1:1000, Upstate) antibody.
Detection of Cell Surface TrkB
After a gentle wash with ice-cold phosphate-buffered saline (PBS), cells were incubated with 1.1 mg/mL of EZ-Link Sulfo-N-hydroxy- succinimidobiotin (NHS-biotin)-LC-Biotin (Pierce Biotechnology Inc.) in PBS at 4 °C for 20 min. Then, excessive NHS Biotin was quenched with 0.1 M of glycine in PBS and removed by extensive wash with ice-cold PBS. The biotinylated cells were lysed in 1% Triton-X buffer (20 mM of Tris–HCl (pH 7.4), 5 mM of EDTA, 150 mM of NaCl, 1% Triton-X100). Lysates containing equal amounts of protein were mixed with 50 μL of immobilized Neutravidin beads (UltraLink Immobilized NeutrAvidin protein, Pierce Biotechnology Inc.) and incubated at 4 °C for 1 h with gentle rotation. Centrifugated beads were washed 3 times with lysis buffer. The biotin-labeled cell surface proteins were separated by SDS-PAGE and immunoblotted with anti-TrkB antibody (1:1000, BD Biosciences).
For immunocytochemical staining, neurons were fixed with 4% paraformaldehyde (Sigma) and 4% sucrose in Dulbecco's PBS for 20 min at room temperature. The cells were incubated with PBS containing 0.2% Triton-X (Sigma) for 5 min and blocked by 10% horse serum in PBS for 1 h at 37 °C. Then, anti-MAP2 monoclonal antibody (isotype: IgG1, 1:250, Sigma), anti-BDNF polyclonal antibody (2 μg/μL, produced by Dr Ritsuko Katoh-Semba: Katoh-Semba et al. 1997), anti-GAD67 (glutamic acid decarboxylase 67 kD) monoclonal antibody (1:2000, Millipore, CA) and anti-synaptotagmin monoclonal antibody (isotype IgG2a, 1:100, Chemicon, CA) were applied overnight at 4 °C. BDNF and synaptotagmin were visualized by isotype-specific secondary antibody conjugated with Alexa 488 (1:200, Molecular Probes, CA). MAP2 was visualized by anti-mouse secondary antibody conjugated with Alexa 546 (1:2000, Molecular Probe). A fluorescent microscope (Axiovert 200, Carl Zeiss, Oberkochen, Germany) was used to obtain images. When quantification of BDNF immunoreactivity was conducted, we measured mean intensity of randomly selected areas of cell body or primary dendrites by using imaging software Slide Book TM 3.0 (Intelligent Imaging Innovations Inc., CO).
To calculate cell viability, the metabolic activity of mitochondria was estimated by measuring the mitochondrial-dependent conversion of the tetrazolium salt, MTT (Sigma) (Numakawa et al. 2009). In brief, after treatment of PCP for 48 h, cultured cells were incubated with MTT solution. Two hours later, cultures were lysed and the metabolic activity of the mitochondrial reductase was estimated.
Detection of BDNF Secretion
Following washes with neurobasal medium and 0.1 mg/mL BSA, cultured neurons were incubated with or without PCP (1 μM) in neurobasal medium containing anti-BDNF antibody (2 μg/mL, Santa Cruz Biotechnology Inc.) for 6 h. Then, medium was carefully collected and the secreted BDNF captured by the antibody was immunoprecipitated. After 3 washes with the lysis buffer, BDNF in immunoprecipitates was detected by immunoblotting with the same anti-BDNF antibody (Santa Cruz Biotechnology Inc.) or ELISA assay (BDNF-ELISA E-max; Promega, WI) with another anti-BDNF antibody, a component of the ELISA kit. For detection of BDNF secretion from cortical acute slices, 200-μm-thick coronal sections were prepared using a microtome (VT1000S, Leica, Nussloch, Germany) from prefrontal cortex of postnatal 30–40 days old male rats in ice-cold HEPES-buffered solution (containing 120 mM NaCl, 4 mM KCl, 1.2 mM KH2PO4, 1 mM MgSO4, 2 mM CaCl2, 30 mM glucose, and 20 mM HEPES, pH 7.4). Each slice, which was obtained from right and left cortical hemisphere, was assigned to control and PCP treatment. Freely floating slice sections were incubated with HEPES-buffered solution at 37 °C for 4 h before sampling. Then, after washing several times, secreted BDNF was determined in a similar way used in the culture experiments.
Detection of Amino Acid Neurotransmitters
The amount of amino acid released from cultured neurons was measured as described previously (Numakawa et al. 2002). Briefly, high-performance liquid chromatography (HPLC; Shimazu Co., Kyoto, Japan) was used to measure the amino acids released into the modified HEPES-buffered Krebs Ringer solution (KRH; containing 130 mM NaCl, 5 mM KCl, 1.2 mM NaH2PO4, 1.8 mM CaCl2, 10 mM glucose, 1% bovine serum albumin, and 25 mM HEPES, pH 7.4). After the cultures were washed 3 times with KRH buffer, fresh KRH buffer was added to the cultures and collected without stimulation (1 min) as the basal release. Then, potassium (50 mM KCl for 1 min) was added to the cultures in order to induce depolarization.
Whole-cell voltage clamp recordings were performed on cultured cortical neurons using an AxoClamp 2B amplifier (Molecular Devices). Cells were continuously superfused with external solution containing 150 mM NaCl, 4 mM KCl, 2 mM CaCl, 1 mM MgCl2, 10 mM HEPES, 10 mM d-glucose, 10 μM glycine (pH = 7.4, 310 mOsm). Miniature excitatory postsynaptic currents (mEPSCs) were isolated by adding picrotoxin (100 μM) and tetrodotoxin (0.5 μM) to the bath. Recordings were performed for 5 min, which enabled us to collect more than 1000 events. Patch electrodes (7–12 MΩ) contained 130 mM Cs-methane sulfonate, 10 mM CsCl, 10 mM MgCl2, 10 mM HEPES, 5 mM MgATP, and 1 mM Na2GTP (pH 7.3, 300 mOsm). All experiments were carried out at room temperature (27 °C). Cells with an input resistance >200 MΩ (range: 300–800 MΩ) were used. Each neuron was voltage-clamped at −70 mV. For each recording, series resistance and input resistance were continuously monitored, and if these values changed by >15%, the data were discarded. Signals were filtered at 3 kHz and digitized at 10 kHz (Digidata 1320A: Molecular Devices). Off-line analysis of mEPSCs was carried out using Clampfit v 9.2 (Molecular Devices). Miniature events were detected using Mini Analysis software (Synaptosoft) with an amplitude threshold of −5 pA. The events were further inspected visually to exclude inappropriate data, such as overlapped events or events with a noisy baseline.
Imaging of Intracellular Ca2+
Ca2+ imaging was performed using fluo-3 dye (Molecular Probes) as previously reported (Numakawa et al. 2002). The changes in the fluo-3 intensity through the fluorescent microscope were analyzed and quantified using Slide Book TM 3.0 (Intelligent Imaging Innovations Inc.) from randomly selected cell bodies. Ca2+ imaging experiments were performed at least 3 times for each experimental condition.
Total RNA Extraction and Reverse Transcription
Total RNA was extracted from cultured cells using the RNeasy Plus Kit (QIAGEN, CA) according to the manufacturer's manual. Cells were homogenized by QIAshredder (QIAGEN). To prevent genomic DNA contamination, gDNA Eliminator column (QIAGEN) was used. Total RNA (1 μg) was mixed with SuperScript VILO enzyme and reaction mixes (Invitrogen, CA) in a total volume of 20 μL. After incubation at 25 °C for 10 min, the mixture was heated to 42 °C for 60 min and followed by an inactivation step (85 °C, 5 min). The cDNA solutions were stored at −80 °C until used.
For real-time PCR analyses of mRNA, cDNA was prepared from cultured cortical neurons or pure astrocytes using a TaqMan Cells-to-Ct kit (Applied Biosystems, CA) according to the manufacturer's protocol. Each cDNA was amplified with specific TaqMan Gene Expression Assays (Rn02531967_s1 for rat BDNF; Rn00565046_m1 for rat MAP2; Rn00566603_m1 for rat GFAP: glial fibrillary acidic protein). ABI prism 7000 was used for amplifications. Ct of target mRNA was obtained using Sequence Detection System software (ABI). Serial dilution (1:1, 1:3.3, 1:10, 1:33, and 1:100) of pooled samples was used as a standard. Each gene amplification was normalized with rat GAPDH control (4352338E).
Data are expressed as mean ± standard deviation, and statistical significance was calculated using a one-way ANOVA followed by Scheffe's post hoc test in SPSS ver.18 (SPSS Japan, Tokyo, Japan) if not otherwise specified. The probability values of less than 5% were considered significant.
Transient Increase of Intracellular BDNF Levels by PCP
To determine the acute effects of PCP on neurons, cortical cultures (at DIV 10 and 11) were exposed to PCP, followed by examination of BDNF and its receptor expression. Interestingly, Western blot analyses revealed that 3-h PCP treatment significantly increased levels of intracellular BDNF (14 kD, mature BDNF) (e.g., PCP 1 μM: 152 ± 21.7% of control, P < 0.01) (Fig. 1A,B). BDNF expression was increased 6 h after 0.1 μM PCP application as well, though statistical significance was not reached (Fig. 1A,B). Expression of TrkB and p75 (low affinity receptor for BDNF) were not altered by 3- or 6-h PCP application (Fig. 1A, Supplementary Fig. 1A). Expression of β-actin and TUJ1 (both are controls) were also intact after PCP exposure (Fig. 1A). To check involvement of de novo synthesis in the up regulation of BDNF, we investigated BDNF mRNA after PCP stimulation. Unexpectedly, a significant reduction in BDNF mRNA expression was induced by PCP (1 μM, 3 h) (Supplementary Fig. 1B), suggesting that the PCP-induced increase in intracellular BDNF expression is not due to an increase of BDNF translation. Although the cell body size of neurons was not affected (Fig. 1C), immunostaining with anti-BDNF antibody showed that the intensity of granular signals in MAP2-positive neurons increased after PCP treatment (1 μM, 3 h) (cell body: 123 ± 10% of control, P < 0.001, dendrite: 115 ± 13% of control, P < 0.001, t-test) (Fig. 1D,E). Such granular signals of BDNF were observed in only non–GAD-positive neurons (Supplementary Fig. 1C), suggesting that the selected BDNF-positive neurons used to estimate immunoreactivity were glutamatergic neurons. In this study, we confirmed a very small proportion of glial cells in our cortical cultures (Supplementary Fig. 2A) and much higher expression of BDNF mRNA in the cortical cultures than that in pure astrocyte cultures. Furthermore, PCP decreased rather than increased the BDNF levels in the pure astrocytes (Supplementary Fig. 2B), indicating that the increased expression of BDNF was specific to neurons.
PCP Diminished Activity of BDNF/Trks Signaling
The effect of PCP on activation (phosphorylation) of Trk receptors and downstream signaling pathways was examined. In spite of the increased intracellular BDNF, phosphorylation of Trk receptors was suppressed by 3- and 6-h PCP treatment (PCP 1 μM, 3 h: 49.3 ± 16.3% of control; PCP 1 μM, 6 h: 56.8 ± 10.1% of control, P < 0.001 and P < 0.05, respectively) (Fig. 2A). Three-hour PCP treatment suppressed Trk phosphorylation in all doses tested. Six hours after PCP application, 1–25 μM doses achieved significant inhibition of Trk phosphorylation (Fig. 2A). When alterations in cell surface expression of the TrkB receptor were examined, we confirmed that PCP (10 μM) did not change the amount of surface TrkB, while the ligand BDNF indeed reduced surface TrkB levels (Ji et al. 2010) (Fig. 2B). As shown in Figure 2C,D, activation of Akt (a component of the PI3K pathway) and ERK1/2 (MAPK pathway) were also decreased by PCP (e.g., PCP 1 μM, 3 h: phosphorylated Akt: 62.9 ± 11.7% of control, P < 0.001; phosphorylated ERK1: 61.4 ± 6.7% of control, P < 0.01, phosphorylated ERK2: 51.6 ± 10.3% of control, P < 0.01), whereas expression of total Akt and ERK1/2 were intact. The reduced activation of Akt and ERK1/2 were maintained until 6 h after PCP treatment (Fig. 2C,D). To elucidate which Trk receptor (i.e., TrkA, TrkB, or TrkC) is responsible for the activation of intracellular signaling, cortical neurons were treated with various neurotrophins (Fig. 2E,F). BDNF and neurotrophin 4/5 (NT-4/5), both of which are specific ligands for TrkB, strongly activated Trk receptors, Akt and ERK1/2, whereas nerve growth factor (NGF, a specific ligand for TrkA) did not. NT-3 (for TrkC, and weakly for TrkA and TrkB) also demonstrated low activation ability. These data suggest that TrkB signaling is responsible for the activation of Akt and ERK1/2 pathways in our cultures.
Impaired Secretion of BDNF Caused by PCP
In cortical neurons, intracellular BDNF was increased following PCP exposure, whereas activation of Trk and downstream signaling cascades were decreased. To examine whether BDNF secretion was affected by PCP, conditioned medium of cultures incubated in the presence of anti-BDNF antibody was collected. The immunoprecipitated BDNF using conditioned media showed the same molecular weight as that of recombinant mature BDNF in Western blot analysis, and we found reduced amounts of secreted BDNF after PCP exposure (1 μM, 6 h, Fig. 3A). A significant reduction in the secreted BDNF was observed (60.7 ± 9.9% of control, P < 0.001) in PCP-treated cultures (Fig. 3B). Such reduction in immunoprecipitated BDNF was further confirmed by enzyme-linked immunosorbent assay (ELISA) (Fig. 3C). Furthermore, we examined BDNF secretion in acute cortical slices and found significant suppression in the amount of secreted BDNF caused by PCP (1 μM, 3 h, Supplementary Fig. 3). It is well known that secretion of BDNF depends on neuronal activity (Hartmann et al. 2001; Lessmann et al. 2003). We tested the effect of glutamate (excitatory neurotransmitter) and tetrodotoxin (Na+ channels blocker) on BDNF secretion in cultured neurons. As expected, increased BDNF by glutamate and decreased BDNF by TTX in conditioned media were observed (glutamate: 395 ± 114% of control, P < 0.001; TTX 49.1 ± 21.0% of control, P < 0.001) (Fig. 3D,E). These data suggest that PCP repressed the activity-dependent secretion of BDNF from cortical neurons.
Exposure to PCP for 48 h Did Not Affect Neuronal Viability
How does the suppression of BDNF/Trk signaling induced by PCP influence cortical neurons at the cellular and neuronal network level? To approach this issue, the survival of neurons after exposure to PCP was examined. After 48 h of exposure to 1 μM PCP, cortical neurons were immunostained with anti-MAP2 antibody (Fig. 4A). Quantified data of MAP2-positive living cells revealed no difference in neuronal survival between control and PCP-treated cultures (Fig. 4B). MTT assay also indicated that PCP (0.1–25 μM, 48 h) treatment did not affect cell viability (Fig. 4C). As shown in Fig. 4D, expression of Bcl-2, an antiapoptotic protein, and Bad, a proapoptotic protein, were unchanged by PCP (1 μM, 48 h).
PCP Diminished the Number of Presynaptic Sites and Synaptic Transmission
We next investigated the effect of PCP (48 h) on synaptic function. Western blot analysis revealed decreased expression of presynaptic proteins (synaptotagmin and synaptosome-associated protein of 25 kD [SNAP25]) and postsynaptic glutamate receptors (NR2B and GluR1) (Fig. 5A). Immunostaining with anti–synaptotagmin antibody also demonstrated a decrease in the number of presynaptic puncta after PCP (1 μM, 48 h) exposure (13.8 ± 2.9 per 50 μm of dendrite in control and 8.3 ± 3.0 in PCP, P < 0.001) (Fig. 5B,C). Importantly, basal and depolarization-induced release of glutamate were both decreased after 48 h of PCP treatment (Fig. 5D), suggesting that PCP decreases the number of glutamatergic synapses. Electrophysiological recordings on cultured cortical neurons revealed that PCP (1 μM, 48 h) reduced the frequency of mEPSCs in both NMDA receptor and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor components (Fig. 6A,B). Cumulative plots also indicated a shift to longer intervals in both AMPA receptor- and NMDA receptor-mediated mEPSCs after PCP treatment (Fig. 6C), while the amplitude of mEPSCs was not changed (Fig. 6D).
Exogenous BDNF Prevented PCP's Suppression of Intracellular Signaling and Synaptic Protein Expression
We investigated whether coapplication of BDNF with PCP could prevent the reduction in ERK1/2 and Akt activation at 3 h later as well as prevent the decrease in synaptic protein expression at 48 h later. As expected, exogenous BDNF (10 ng/mL) completely prevented the reduction of intracellular signaling (ERK 1/2 and Akt activation, Fig. 7A) and of synaptic proteins (synaptotagmin, SNAP25, NR2B, and GluR1, Fig. 7B). Delayed BDNF application at 3 h after PCP addition also prevented down regulation of synaptic proteins caused by PCP (Supplementary Fig. 4). These data suggest that the PCP-induced loss of synaptic connectivity is due to the impaired secretion of endogenous BDNF.
PCP Blocked Intracellular Ca2+ Mobilization and Other NMDA Receptor Antagonists Induced Intracellular BDNF Increase
Increase in the intracellular Ca2+ concentration is required for the activity-dependent secretion of BDNF (Hartmann et al. 2001; Lessmann et al. 2003). Thus, we assessed the possibility that the PCP-suppressed BDNF secretion is due to an inhibition of Ca2+ influx mediated by glutamate receptors. First, as shown in Fig. 8A, change in levels of glutamate receptors including NR2A, NR2B, and GluR1 during in vitro maturation were determined. We found that these proteins gradually increased during in vitro maturation, while very low expression levels at DIV 4 and 5 occurred (Fig. 8A). Synaptotagmin and SNAP25, presynaptic proteins, also increased during in vitro maturation in our cultures. Previously, we reported that cultured cortical neurons develop spontaneous Ca2+ oscillations during in vitro maturation, and the endogenous phenomenon is mediated via glutamatergic neurotransmission (Numakawa et al. 2002). Therefore, we determined whether PCP affected spontaneous Ca2+ oscillations at DIV 12 and found that PCP treatment (1 μM, 3 h) dramatically reduced the endogenous Ca2+ activity (Fig. 8B,C). In some cases, we observed a resting series of sister cultures, in which all neurons showed no Ca2+ oscillations as previously reported (Numakawa et al. 2002). Then, the glutamate-evoked increase in intracellular Ca2+ concentration was also checked using such resting neurons (Fig. 8D,E and Supplementary Fig. 5A). Importantly, PCP treatment for 3 h repressed the glutamate-stimulated Ca2+ (Fig. 8D,E and Supplementary Fig. 5A). Such suppression of Ca2+ elevation was still observed at 6 h after PCP addition (Supplementary Fig. 5B). In line with the low expression level of glutamate receptors, immature neurons at DIV 5 did not respond to glutamate with or without PCP (Fig. 8E). In the DIV 5 immature neurons, PCP had no effect on intracellular BDNF expression (Supplementary Table 1). Furthermore, we confirmed a positive effect of other noncompetitive and competitive antagonists for NMDA receptors (MK-801 and APV, respectively) on intracellular BDNF levels in DIV 12 neurons (but not in DIV 5 neurons) (Supplementary Table 2), indicating that the effect is not specific to PCP but it holds true for NMDA receptor antagonists in general. These results suggest that inhibition of Ca2+ influx via NMDA receptors was involved in the decrease in BDNF secretion by PCP.
In the present study, we found that intracellular BDNF protein was transiently increased by 3-h PCP exposure. Despite such a BDNF increase, activation of Trks and downstream signaling pathways (ERK1/2 and Akt) were diminished. Importantly, the number of synaptic sites and expression of synaptic proteins were reduced 48 h after PCP application. Furthermore, both basal and depolarization-evoked glutamate release were decreased in PCP-treated neurons, and the electrophysiological studies also revealed a reduced frequency of mEPSCs by PCP. Interestingly, we discovered suppression in BDNF secretion after PCP treatment both in cortical cultures and acute slices. Application of exogenous BDNF prevented the PCP-induced reduction in expression of synaptic proteins in cortical cultures. It is possible that the PCP-induced impairment of BDNF secretion results in the transient accumulation of intracellular BDNF and leads to down regulation of BDNF/TrkB signaling, which is required for maintenance of synaptic protein expression. As PCP treatment significantly inhibited the spontaneous Ca2+ activity and evoked Ca2+ influx by glutamate, the inhibition of Ca2+ mobilization may contribute to the PCP-induced impairment of BDNF secretion.
In hippocampal and cortical neurons, BDNF showed a vesicular expression pattern in dendrites and axons and appeared to be sorted into a regulated pathway in which BDNF is secreted in response to neuronal activity (Goodman et al. 1996; Farhadi et al. 2000; Hartmann et al. 2001,Kojima et al. 2001; Kohara et al. 2001; Gartner and Staiger 2002; Lessmann et al. 2003; Wu et al. 2004; Adachi et al. 2005). The activity-dependent secretion of BDNF is triggered by an increase in intracellular Ca2+ concentration via ionotropic glutamate receptors, voltage-gated Ca2+ channels, and internal Ca2+ stores (Hartmann et al. 2001; Lessmann et al. 2003). In the present study, we examined the possibility that PCP attenuated an increase of intracellular Ca2+ concentration at basal and evoked conditions, contributing to the transient increase of intracellular BDNF and suppression of its secretion. In mature cortical neurons, we found that PCP induced an inhibition of Ca2+ mobilization, and that BDNF protein released into the culture medium was reduced after PCP application compared with control. This reduction in BDNF secretion was demonstrated by both immunoblotting and ELISA methods after immunoprecipitation with anti-BDNF antibody. Interestingly, when we examined the effect of PCP on de novo synthesis of BDNF, we found decreased levels of BDNF mRNA (Supplementary Fig. 1B), suggesting that PCP-dependent BDNF increase is not due to transcriptional activity. Resultant down regulation of BDNF protein levels might appear as a reduction in the amount of intracellular BDNF at 6 h or later. Importantly, other NMDA antagonists, MK-801 (noncompetitive) and APV (competitive), also elevated BDNF levels in neurons, suggesting that the PCP-increased BDNF would be due to the inhibitory effect of PCP on NMDA receptors. Furthermore, such an increase of BDNF by NMDA antagonists occurred only in mature cortical neurons (DIV 12) that express adequate glutamate receptors. PCP-dependent impairment of BDNF secretion and subsequent decrease in synaptic function may only occur in mature fully developed neurons that express adequate NMDA receptors.
The reduction of BDNF secretion is considered to be a neuronal response to PCP, as we confirmed that the majority of cultured cells in the experiment were indeed neurons. Furthermore, PCP did not cause an intracellular increase of BDNF in pure astroglial cultures. In our cortical cultures, vesicular expression of BDNF was observed only in GAD-negative neurons. If this vesicular pattern of BDNF expression reflects the activity-dependent population of BDNF secretion, it is possible that PCP specifically impacts the regulatory release of BDNF from glutamatergic (not GAD-positive) neurons. Importantly, some reports suggest that preferential binding of PCP to NMDA receptors on GABAergic interneurons results in the activation of glutamatergic pyramidal neurons in vivo (Homayoun and Moghaddam 2007; Kargieman et al. 2007). Homayoun and Moghaddam reported that firing rates in ∼69% of GABAergic neurons and 86% of pyramidal neurons were decreased after PCP injection (Homayoun and Moghaddam 2007). Interestingly, Kargieman et al. showed that PCP increases and decreases the activity of 45% and 33% of the pyramidal neurons, respectively (Kargieman et al. 2007). Our results indicated a decreased synaptic activity in cultured cortical neurons after 48 h PCP exposure. Furthermore, PCP-dependent decrease in the secretion of BDNF from acute cortical slices, in which local neuronal circuits remain intact, was confirmed. It is possible that differences in experimental conditions including dose of PCP and neuronal maturity may influence such a different neuronal response to PCP, although future studies will be needed using in vivo and in vitro systems.
Secretion of BDNF to the extracellular space is required to generate its biological effects via activation of TrkB. Indeed, activation of Trk receptors and downstream signaling cascades (ERK1/2 and Akt) were reduced by PCP. In our system, BDNF and NT-4/5 functioned as major contributors for the activation of Trk receptors, suggesting that TrkB signaling is predominant. A subset of the BDNF/TrkB downstream signaling molecules, especially ERK1/2 activity, is known to be regulated by NMDA receptor-mediated Ca2+ influx (Xia et al. 1996; Sutton and Chandler 2002). Therefore, it is possible that decreased activity of ERK1/2 may be attributable, at least in part, to the blockade of the NMDA receptor by PCP directly. However, simultaneous application of exogenous BDNF blocked the PCP-dependent suppression of synaptic protein levels as well as ERK1/2 and Akt signaling pathways even when NMDA receptors were blocked in the presence of PCP. Recently, we reported that ERK1/2 activity is involved in the maintenance of synaptic protein expression (Kumamaru et al. 2011). Furthermore, delayed application of exogenous BDNF reversed the suppression of some synaptic proteins inhibited by PCP. All things considered, impaired BDNF secretion substantially contributed to the down regulation of ERK1/2 activity and synaptic protein expression.
The number of synaptic sites was decreased when chronic PCP exposure was administered. There are 2 lines of evidence: 1) the reduced expression of pre- and postsynaptic proteins assessed with Western blotting and 2) the decreased number of presynaptic sites estimated with immunostaining. We also obtained evidence for functional changes, showing a marked reduction in glutamate release as well as a decreased frequency of mEPSCs mediated by NMDA and AMPA receptors. Taken together, excitatory neurotransmission is suppressed by 48 h of PCP treatment.
It is well known that BDNF/TrkB signaling plays an important role in synaptic plasticity. BDNF stabilizes and increases dendritic synapse density in the optic tectum (Hu et al. 2005; Sanchez et al. 2006). BDNF increases spine density in hippocampal neurons through ERK1/2 activation (Alonso et al. 2004). We also reported that BDNF increases the expression of pre- and postsynaptic proteins via ERK1/2 signaling in cultured cortical neurons (Kumamaru et al. 2011). Overexpression of TrkB or activation of PI3K/Akt signaling enhances motility of dendritic filopodia and synaptic density (Luikart et al. 2008). These findings, including our current results, suggest that decreased BDNF secretion caused by PCP is one of the major factors for loss of synaptic connections and/or overall neuronal function.
In the present study, 48 h of PCP treatment did not change both the number of MAP2-positive cells and mitochondrial activity in cortical cultures. Mitochondrial activity was not influenced even when a critically high concentration of PCP (25 μM) was applied. Furthermore, expression levels of both an antiapoptotic protein Bcl-2 and a proapoptotic protein Bad were unchanged after PCP application. These data suggest that PCP has no major influence on survival of cultured cortical neurons, although it does inhibit synaptic connectivity and function. Interestingly, Lei et al. (2008) reported that PCP causes apoptosis in cultured cortical neurons through suppression of Akt activity and activation of GSK3β and caspase-3. This discrepancy between Lei et al. (2008) and our study may be attributable to differences in culture conditions, as their neurobasal medium contained B27 while our 5/5 DF medium contained serums.
PCP induces schizophrenia-like behaviors in humans (Allen and Young 1978; Javitt and Zukin 1991) and rodents (Noda et al. 1995; Furuta and Kunugi 2008). In vivo administration of PCP causes extensive reduction in the number of spines in the rat prefrontal cortex (Hajszan et al. 2006) and suppression of glutamate release in the prefrontal cortex of mice (Nabeshima et al. 2006; Murai et al. 2007). Postmortem brain studies from schizophrenia patients demonstrate that the number of neurons in the prefrontal cortex is not decreased (Pakkenberg 1993; Akbarian et al. 1995), although synaptophysin immunoreactivity and dendritic spine density of pyramidal cells are reduced (Glantz and Lewis 1997; Glantz and Lewis 2000; Knable et al. 2004). These findings are consistent with our observation of synaptic loss and decreased glutamatergic transmission without any change in cell viability in PCP-treated cortical neurons. Therefore, impairment in BDNF secretion and downstream signaling may be involved in the pathogenesis of schizophrenia-like behaviors. In fact, altered expression of BDNF and TrkB has been reported in postmortem brains of schizophrenia patients in several studies (e.g., Takahashi et al. 2000; Durany et al. 2001; Weickert et al. 2003; Weickert et al. 2005; Hashimoto et al. 2005; Altar et al. 2009). Recently, biological functions of proneurotrophins through the p75 receptor were revealed (Lee et al. 2001; Dechant and Barde 2002; Pagadala et al. 2006). Considering this, it may be valuable to study not only TrkB-stimulated signaling but also p75-stimulated signaling, during PCP exposure.
In conclusion, our results suggest that impaired secretion of BDNF and the resultant decrease in activation of Trk receptor signaling pathways are responsible, at least in part, for the PCP-dependent reduction in synaptic connectivity and function, which may be involved in PCP's ability to elicit schizophrenia-like behaviors. Our experimental system might be a “cell model” suitable for studies to clarify the molecular mechanisms of schizophrenia.
Core Research for Evolutional Science and Technology Program (CREST) Japan Science and Technology Agency (JST) (to N.A., T.N., E.K., and H.K.); Ichiro Kanehara Foundation (to T.N.); Takeda Science Foundation (to T.N.); Hokuto Foundation for Bioscience (to T.N.); Health and Labor Sciences Research Grants (Comprehensive Research on Disability, Health, and Welfare H21-kokoro-002 to H.K.); and Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant-in-Aid for Scientific Research [B]: grant 20390318 to H.K. and for Young Scientists [A]: grant 21680034 to T.N.).
We thank Regeneron Pharmaceutical Co., Takeda Chemical Industries, Ltd., and Dainippon Sumitomo Pharma Co. Ltd. for donating the BDNF. Conflict of Interest : None declared.