Effects of antipsychotic drugs on the expression of synaptic proteins and

dendritic outgrowth in hippocampal neuronal cultures

Sung Woo Park 1, Chan Hong Lee 1, Hye Yeon Cho 1, Mi Kyoung Seo 1,2, Jung Goo Lee 1,2,3,

Bong Ju Lee 2, Wongi Seol 4, Baik Seok Kee 5, Young Hoon Kim 1,2,3*

1 Paik Institute for Clinical Research, Inje University, Busan, Republic of Korea.

2 Department of Psychiatry, School of Medicine, 3 Haeundae Paik Hospital, Inje University,

Busan, Republic of Korea.

4 InAm Neuroscience Research Center, Wonkwang University, Sanbon Hospital, Gunpo,

Republic of Korea.

5 Department of Psychiatry, School of Medicine, Chungang University, Seoul, Republic of

Korea.

Running Title: Effects of antipsychotics on synaptic proteins

* Corresponding author

Young Hoon Kim, M.D., Ph.D,

Department of Psychiatry, Inje University Haeundae Paik Hospital, 1435, Jwa-dong,

Haeundae-gu, Busan, Republic of Korea

Zip code: 612-030

Tel.: +82 51 890 6749

Fax: +82 51 894 6709

E-mail address: neuro109@hanmail.net (Y.-H. Kim)

Research Article SynapseDOI 10.1002/syn.21634

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an‘Accepted Article’, doi: 10.1002/syn.21634

© 2013 Wiley Periodicals, Inc.Received: Oct 17, 2012; Revised: Dec 18, 2012; Accepted: Dec 18, 2012

- 2 -

ABSTRACT

Recent evidence has suggested that atypical antipsychotic drugs regulate synaptic plasticity.

We investigated whether some atypical antipsychotic drugs (olanzapine, aripiprazole,

quetiapine, and ziprasidone) altered the expression of synapse-associated proteins in rat

hippocampal neuronal cultures under toxic conditions induced by B27 deprivation. A typical

antipsychotic, haloperidol, was used for comparison. We measured changes in the expression

of various synaptic proteins including postsynaptic density protein-95 (PSD-95), brain-

derived neurotrophic factor (BDNF), and synaptophysin (SYP). Then we examined whether

these drugs affected the dendritic morphology of hippocampal neurons. We found that

olanzapine, aripiprazole, and quetiapine, but not haloperidol, significantly hindered the B27

deprivation-induced decrease in the levels of these synaptic proteins. Ziprasidone did not

affect PSD-95 or BDNF levels, but significantly increased the levels of SYP under B27

deprivation conditions. Moreover, olanzapine and aripiprazole individually significantly

increased the levels of PSD-95 and BDNF, respectively, even under normal conditions,

whereas haloperidol decreased the levels of PSD-95. These drugs increased the total

outgrowth of hippocampal dendrites via PI3K signaling, whereas haloperidol had no effect in

this regard. Together, these results suggest that the up-regulation of synaptic proteins and

dendritic outgrowth may represent key effects of some atypical antipsychotic drugs but that

haloperidol may be associated with distinct actions.

KEY WORDS: atypical antipsychotics, synaptic proteins, hippocampal dendritic outgrowth

- 3 -

INTRODUCTION

Schizophrenia is a severe psychiatric illness characterized by positive, negative, and

cognitive symptoms (Kinon and Lieberman, 1996). Although the underlying causes of the

disorder remain largely unknown, many studies have proposed that schizophrenia may be

attributable to abnormalities in brain circuitry that cause reduced and aberrant connectivity in

various brain areas (Gisabella et al., 2005; Hashimoto et al, 2005; Mendrek et al, 2005). In

particular, several postmortem studies have noted decreased expression of genes responsible

for synaptic proteins in the hippocampus and other regions, suggesting impairment or

reduction in synaptic connectivity in schizophrenia (Eastwood et al, 2001; Eastwood and

Harrison, 2005). Additionally, these neuronal alterations might underpin the cognitive deficits

observed in schizophrenic patients.

Two classes of drugs, typical and atypical antipsychotics, are used in the treatment of

schizophrenia. Atypical antipsychotic drugs have shown greater therapeutic efficacy for

negative symptoms and cognitive deficits and less likelihood of inducing extrapyramidal side

effects (EPS) than typical antipsychotic drugs (Kinon and Lieberman, 1996). However, the

Clinical Antipsychotic Trial of Intervention Effectiveness (CATIE) found no difference in the

cognitive effects of typical and atypical antipsychotic drugs (Keefe et al., 2007). Atypical

antipsychotic drugs also failed to show significant efficacy with respect to neurocognition

when compared with typical antipsychotic drugs (Manschreck and Boshes, 2007). Moreover,

some atypical antipsychotic drugs may increase the risk of developing metabolic syndrome

(Nasrallah, 2006; Newcomer, 2007). Although the underlying mechanisms of these

therapeutic actions are not fully understood, it has recently been suggested that atypical

antipsychotic drugs positively regulate dendritic spine formation and synaptogenesis, whereas

the typical antipsychotic drug haloperidol down-regulates them or has no significant effect

- 4 -

(Critchlow et al., 2006; Delotterie et al., 2010). Therefore, it can be suggested that the

positive effects of atypical antipsychotic drugs on synaptic plasticity reflect a significant

difference between typical and atypical antipsychotic drugs. Thus, investigation of the

regulation of synaptic proteins and dendritic morphology may provide a better understanding

of the molecular mechanism underlying the comparative efficacy and safety of typical and

atypical antipsychotic drugs.

The postsynaptic density protein PSD-95 is located largely in dendritic spines and plays

a critical role in regulating dendritic spine size and shape (Ehrlich et al,. 2007; Han and Kim,

2008) by acting as a scaffolding protein that regulates the clustering of glutamate receptors in

the dendritic spine (Han and Kim, 2008). Appropriate levels of PSD-95 are required for

synaptic maturation, strengthening, plasticity, and activity-driven synapse stabilization

(Ehrlich et al,. 2007). PSD-95 overexpression is associated with increases in the size of

excitatory synapses but reductions in the number of inhibitory synaptic contacts (Prange et al.,

2004). PSD-95 may also be relevant to the pathophysiology of schizophrenia (Toro and

Deakin, 2005).

Postsynaptic brain-derived neurotrophic factor (BDNF), the most abundant neurotrophin

in the brain, contributes to axonal branching, dendritic differentiation, and connectivity

among neurons (Ji et al., 2005; Lessmann et al., 2003; Poo, 2001). Numerous studies have

reported BDNF-induced changes in dendritic spine density and morphology in various

neuronal populations (Shen and Cowan, 2010). BDNF has been implicated in activity-

dependent synaptic regulation (Yoshii and Constantine-Paton, 2010). For example, it has

been reported that BDNF induces PSD-95 trafficking to dendrites via PI3K/Akt signaling

after NMDA activation (Yoshii and Constantine-Paton, 2007). BDNF and its receptor, TrkB,

are also required for NMDA-dependent long-term potentiation (Kovalchuk et al., 2002; Pang

- 5 -

et al., 2004). BDNF is also activated at glutamatergic synapses, increases AMPA receptor

currents and induces the maturation of inhibitory neurons (Huang et al., 1999; Pang et al.,

2004; Poo, 2001). Changes in BDNF levels have been implicated in both the etiology of

schizophrenia and the action of antipsychotic drugs (Bai et al., 2003; Durany and Thome,

2004; Parikh et al., 2004; Park et al., 2006; Park et al., 2009a; Park et al., 2009b).

Synaptophysin (SYP), the major integral membrane protein of presynaptic vesicles, is

required for vesicle formation and exocytosis (Valtorta et al., 2004) and is widely used as a

marker for synapse activity (Chambers et al., 2005). SYP plays a regulatory role in activity-

dependent synapse formation in cultures of hippocampal neurons (Tarsa and Goda.,

2002).Thus, increased SYP expression may reflect increased synaptic activity, density, and

vesicles, indicating the improved functioning of synapses. Loss of SYP in the hippocampus

correlates with the cognitive decline associated with Alzheimer’s disease, reflecting its role in

cognitive function (Sze et al., 1997). Postmortem studies of patients with schizophrenia have

shown decreased expression of SYP in the hippocampus (Vawter et al., 1999) and prefrontal

cortex (Karson et al., 1999).

The synapse-associated proteins described above collectively play crucial roles in

synapse formation and synaptic plasticity. We propose that atypical antipsychotic drugs may

act as key regulators of these proteins. Changes in the expression of synaptic proteins induced

by atypical antipsychotic drugs may be more apparent when measured under reduced

synaptic protein levels. We found previously that serum deprivation in SH-SY5Y cell

cultures decreased the levels of neuroprotective proteins, such as Bcl-2 (Kim et al., 2008).

Thus, we used a model of toxicity, a procedure that omits B27 in primary culture of the

hippocampus, an area particularly relevant to cognitive processes, thereby causing cell death

(Bastianetto et al., 2006). In the present study, we investigated the expression of synapse-

- 6 -

associated proteins under conditions in which rat primary hippocampal cells were and were

not subjected to B27 deprivation to assess whether atypical antipsychotic drugs (olanzapine,

clozapine, quetiapine, aripiprazole, ziprasidone) or the typical antipsychotic drug haloperidol

affected the expression of these proteins. Next, we investigated whether these drugs affected

the dendritic morphology of hippocampal neurons.

MATERIALS AND METHODS

Drugs and reagents

Neurobasal medium, fetal bovine serum (FBS), horse serum (HS), B27 supplement, L-

glutamine, and penicillin–streptomycin were purchased from Invitrogen (Carlsbad, CA,

USA), and trypsin and haloperidol were from Sigma (St. Louis, MO, USA). Olanzapine was

supplied by Lilly Research (Indianapolis, IN, USA), quetiapine by AstraZeneca (London,

UK), aripiprazole by Otsuka Pharmaceutical (Tokushima, Japan), and ziprasidone by Pfizer

(New York, NY, USA). Antibodies used for the Western blotting were purchased from the

following sources: anti-synaptophysin (sc-7568), anti-BDNF (sc-546), and anti-goat and anti-

rabbit IgG-horseradish peroxidase conjugates from Santa Cruz Biotechnology (Santa Cruz,

CA, US), anti-α-tubulin from Sigma; anti-PSD95 (AB9634) from Millipore (Temecula, CA,

US), and ECL anti-mouse IgG-horseradish–peroxidase-linked species-specific whole

antibodies from Amersham Pharmacia (Little Chalfont, Buckinghamshire, UK). Antibodies

used for immunostaining were purchased from the following sources: anti-microtubule-

associated protein 2 (MAP-2) from Millipore and Alexa Fluor 568 goat anti-mouse IgG and

Hoechst 33258 from Invitrogen. The PI3K inhibitor LY294002 was purchased from Cell

Signaling Technology (Beverly, MA, USA).

- 7 -

Primary hippocampal cell cultures

All animal manipulations were performed in accordance with the animal-care guidelines

of the US National Institutes of Health (NIH publication no. 23–85, revised 1996) and the

Korean Academy of Medical Science. This experiment was approved by the Committee for

Animal Experimentation and the Institutional Animal Laboratory Review Board of Inje

Medical College (approval no. 2009-36).

Primary cultures of hippocampal neurons were prepared from fetal brains (embryonic

day 17; E17) obtained from Sprague–Dawley rats (Orient Bio, Gyeonggi-Do, Korea) in a

manner similar to that developed by Kaech and Banker (Kaech and Banker, 2006). Briefly,

brains were exposed, and the hippocampi were carefully removed and dispersed in

neurobasal medium containing 0.03% trypsin for 20 min at 37°C (5% CO2). Cells were

suspended in a neurobasal medium including 1% FBS, 1% HS, 2% serum-free growth

medium B27 (components: biotin, α-tocopherol acetate, α-tocopherol, vitamin A, bovine

serum albumin, catalase, insulin, transferrin, superoxide dismutase, corticosterone, galactose,

ethanolamine, glutathione, carnitine, linoleic acid, linolenic acid, progesterone, putrescine,

selenium, and triodo-L-thyronine), 0.25% L-glutamine, and 50 U/mL penicillin–streptomycin.

For Western blotting, neurons were plated in 6-well dishes coated with poly-L-lysine at a

density of 2 × 105 per well. For the neurite assay, neurons were plated in 12-well dishes at a

density of 2 × 104 per well. They were grown under the above condition (normal condition)

for 5 days (for neurite assay) or 10 days (for Western blotting). B27 has been demonstrated to

facilitate optimal growth and long-term survival of rat embryonic hippocampal neurons. Our

preliminary experiment showed that hippocampal cells were reduced by about 32% under

B27-free conditions (data not shown). After incubation for 5 or 10 days, the cells were treated

with antipsychotic drugs in the presence or absence of B27 for 4 days (for Western blotting)

- 8 -

or under normal condition for 5 days (for the neurite assay) and harvested for further analysis.

The culture media and drugs were changed every 2 days.

Drug treatment

Antipsychotic drugs (10 mM) were dissolved in dimethyl sulfoxide (DMSO). The

solutions were diluted to various concentrations (final concentration of 1% DMSO) with

neurobasal medium before use. For Western blotting and the neurite assay, cells were

cultured for 4 and 5 days, respectively, with olanzapine (10, 50, and 100 µM), quetiapine (0.1,

1, and 10 µM), aripiprazole (0.1, 1, and 10 µM), ziprasidone (0.1, 1, and 10 µM), or

haloperidol (0.1, 1, and 10 µM) in the presence or absence of B27. Control cells were

cultured without antipsychotics under normal condition. The drug concentrations used in this

study were based on the preliminary studies showing that lower concentrations (<0.1 µM)

had not effects on key proteins (Akt or extracellular signal-regulated kinase (ERK))

responsible for the activation of signaling pathways regulating neurite outgrowth and

neuronal differentiation, and higher concentrations reduced the viability of hippocampal cells.

Additionally, Lu et al (2005) reported a similar narrow concentration range for positive

effects of atypical antipsychotic drugs on neuronal cells.

Western blot analysis

The cells were washed twice with ice-cold phosphate-buffered saline (PBS, pH 7.4).

Lysis buffer [20 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1% sodium

dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 2 mM EDTA, and one tablet complete

protease inhibitor (Roche, Laval, Quebec, Canada), pH 8.0] was added, and the lysates were

centrifuged (1000×g, 15 min, 4°C), and the supernatants were boiled in lysis buffer. Equal

- 9 -

amounts of protein (20 μg) derived from the cell extracts under each treatment condition were

separated on SDS-polyacrylamide gels and transferred electrophoretically onto

polyvinylidene fluoride membranes. The blots were blocked by incubation in 5% (w/v) non-

fat milk in Tris-buffered saline (TBS, pH 8.0) with 0.15% Tween 20 (TBS-T) for 1 h. After

incubation with a primary antibody (anti-PSD-95, 1:1000; anti-BDNF, 1:1000; anti-

synaptophysin, 1:1000; and anti-α-tubulin, 1:2000) in TBS-T at 4°C overnight, the

membranes were washed three times in TBS-T for 10 min. The membranes were

subsequently incubated for 1 h in TBS-T containing the horseradish peroxidase-conjugated

secondary antibody (anti-synaptophysin, 1:5000; goat-anti-rabbit IgG for anti-PSD95, 1:2000

and anti-BDNF, 1:2000; and anti-mouse IgG for anti-α-tubulin, 1:10000). Immunoreactive

bands were visualized and quantified using ECL+ Western blotting reagents, with

chemifluorescence detected using the Las-3000 Image Reader (Fuji Film, Tokyo, Japan)

software. The amount of protein was normalized based on α-tubulin, which was unaffected

by the drug treatments, to adjust for protein-loading variation.

Neurite assay

Neurites were visualized using immunostaining with a MAP-2 antibody, a dendritic

marker, as follows. Cells were fixed for 20 min at room temperature using 4%

paraformaldehyde; they were then permeabilized with 0.1% Triton X-100 and blocked with

4% bovine serum albumin in PBS for 2 h to reduce non-specific binding. Cells were

incubated with the anti-MAP-2 antibody diluted 1/1000 in PBS for 2 h. Alexa Fluor 568 goat

anti-mouse IgG was used as a secondary antibody, and Hoechst 33258 was used for nuclear

staining. Stained cells were mounted on cover glasses and observed using a fluorescent

microscope (Olympus, Tokyo, Japan). For the neurite analysis, two independent experiments

- 10 -

for each sample were performed and five fields were randomly selected from each sample.

The images were captured through an automatic FISH (Fluorescence in situ hybridization)

system with an Olympus BX51 (Olympus, Japan) by a person blinded to their identities.

Photomicropraphs were cropped and adjusted for brightness and contrast with Adobe

photoshop CS3. At least 300-400 cells were analyzed using MetaMorph software, version 6.1,

an automated image-analysis program (Molecular Devices, Downingtown, PA, US)

(Klimaschewski et al., 2002).

Statistical analysis

For Western blotting and the neurite assay, we obtained samples from three experiments

and two independent experiments, respectively. Western blotting was perfromed two or three

times form each sample. The three final values obtained from the three independent smaples

were used for quantification and statistic anlaysis. In the neurite assay, 300-400 cells obtained

from five randomly picked areas were analyzed from each sample. Cells obtained from one

typical sample out of two simalr samples were included for quantification and statistic

anlaysis.

All statistical analysis were conducted using one-way ANOVA followed by Scheffe’s test.

P-values ≤0.05 were deemed to indicate statistical significance.

Results

Effects of antipsychotic drugs on postsynaptic proteins PSD-95 and BDNF and

presynaptic protein synaptophysin

- 11 -

Antipsychotic drug-induced changes in the expression of the postsynaptic proteins PSD-

95 and BDNF and presynaptic protein SYP were measured in rat hippocampal neurons

maintained separately in media with or without B27. Olanzapine and four other antipsychotic

drugs (aripiprazole, quetiapine, ziprasidone, haloperidol) were added at concentrations of 10–

100 µM and 0.1–10 µM, respectively, for 4 days. B27 deprivation reduced the levels of PSD-

95, BDNF, and SYP expression significantly, to approximately 60.9%, 61.3%, and 51.2% of

control levels, respectively, (p < 0.05 or p < 0.01, Fig. 1-3).

Olanzapine, aripiprazole, and quetiapine attenuated the B27 deprivation-induced

decrease in PSD-95 protein in a concentration-dependent manner (F = 88.949, p = 0.003 for

olanzapine; F = 88.499, p = 0.003 for aripiprazole; F = 72.974, p = 0.001 for quetiapine; Fig.

1A-C). Moreover, high concentrations of olanzapine and aripiprazole increased the PSD-95

level significantly, by 30%, under normal conditions (F = 88.949, p = 0.003 for olanzapine; F

= 88.499, p = 0.003 for aripiprazole). In contrast, ziprasidone and haloperidol had no effect

on the hippocampal levels of PSD-95 under B27-deprivation conditions (Fig. 1D), while

haloperidol itself at 10 µM decreased this protein levels in normal conditions (F = 122.132, p

= 0.044, Fig. 1E).

Olanzapine and aripiprazole increased the BDNF protein level in hippocampal cells with

and without B27 in a concentration-dependent manner (F = 60.668, p = 0.001 for olanzapine;

F = 56.291, p = 0.014 for aripiprazole Fig. 2A, B). At concentrations up to 10 μM, quetiapine

produced this effect only under B27 deprivation conditions (F = 65.429, p < 0.001, Fig. 2C).

Neither ziprasidone nor haloperidol changed the BDNF level in hippocampal neurons (Fig.

2D and E).

Olanzapine, aripiprazole, quetiapine, and ziprasidone increased SYP protein levels

significantly, in a concentration-dependent manner in B27-deprived cultures (F = 16.591, p =

- 12 -

0.004 for olanzapine; F = 8.577, p = 0.037 for aripiprazole; F = 20.358, p = 0.001 for

quetiapine; F = 32.239, p < 0.001 for ziprasidone; Fig. 3A-D) but not in normal cultures. In

contrast, the levels of this presynaptic protein in the hippocampal neurons were unchanged by

haloperidol (Fig. 3E).

Effects of antipsychotic drugs on dendritic outgrowth

We performed a neurite outgrowth assay to investigate whether antipsychotic drugs

regulated dendritic morphology in hippocampal neurons. Primary hippocampal cells were

incubated for 5 days with different antipsychotic drugs at different concentrations (olanzapine

at 100 μM and the other four antipsychotic drugs at 10 μM) that produced maximum effects

on the expression of synapse-associated proteins.

Hippocampal cells treated without antipsychotic drugs produced modest dendritic

differentiation, with an average dendrite length of around 40 µm (Fig. 4B). These results

indicate that all atypical antipsychotic drugs tested increased dendritic outgrowth and

branching in hippocampal cells (all p < 0.001, Fig. 4A-C). This enhancement was most

pronounced in olanzapine-treated cells. In contrast, no effect on dendrite outgrowth and

branching was observed following haloperidol treatment.

BDNF is believed to induce neuronal differentiation and synaptogenesis by binding to

TrkB receptors and activating phosphatidylinositol 3-kinase (PI3K)/Akt and other pathways

(Chambers et al., 2005). We found that atypical antipsychotic drugs increased the levels of

BDNF (Fig. 2). To explore a possible relationship between PI3K and the morphological

changes induced by these drugs, we examined the effects of the PI3K inhibitor LY294002 on

the morphological changes induced in neurons by atypical antipsychotic drugs. LY294002

concentrations ranging from 0.1 to 10 µM had no significant effects on dendritic outgrowth

- 13 -

and branching (data not shown). Representative images and data in Figure 5 show that

LY294002 (10 µM) inhibited the enhancement of dendritic outgrowth and branching induced

by atypical antipsychotic drugs (dendritic outgrowth: F= 82.965, p < 0.001 for olanzapine; F

= 44.113, p < 0.001 for aripiprazole; F = 31.296, p < 0.001 for quetiapine; and F = 32.757, p

< 0.001 for ziprasidone, branching: F = 93.744, p < 0.001 for olanzapine; F = 23.656, p <

0.001 for aripiprazole; F = 19.616, p < 0.001 for quetiapine; and F = 24.978, p < 0.001 for

ziprasidone). These data suggest that PI3K activity is required for atypical antipsychotic

drugs to enhance hippocampal dendritogenesis.

DISCUSSION

The present study is the first report of positive effects of some atypical antipsychotic

drugs (olanzapine, aripiprazole, quetiapine, ziprasidone) on the levels of synapse-associated

proteins and dendritic outgrowth in hippocampal neurons. We found that these atypical drugs,

but not haloperidol, increased the expression of several proteins associated with synapse

structure and activity in B27-deprived hippocampal cultures. We also showed the

contribution of PI3K signaling to increases in hippocampal outgrowth induced by atypical

antipsychotics. However, haloperidol was not associated with these effects.

Olanzapine and aripiprazole increased BDNF and PSD-95 protein levels in hippocampal

cultures with and without B27. Quetiapine inhibited the reduction of these proteins, which

would have otherwise been induced by B27 deprivation. Ziprasidone had no effects, but

haloperidol decreased the levels of PSD-95 under normal conditions. There is considerable

evidence that BDNF and PSD-95 may play a direct role in postsynaptic morphogenesis by

increasing the number and size of dendritic spines in hippocampal neurons (El-Husseini et al.,

2000; Tyler and Pozzo-Miller, 2003). This is supported by other studies demonstrating that

- 14 -

BDNF treatment in cultured neurons promotes the growth of spines by regulating the

formation of PSD-95-TrkB complexes (Yoshii and Constantine-Paton, 2010), and NMDA

receptor-dependent BDNF activation facilitates the rapid transport of PSD-95 to dendritic

spines through PI3K signaling (Yoshii and Constantine-Paton, 2007). Thus, increase in these

proteins at glutamatergic synapses may have a beneficial impact on information flow to other

neurons. Given the roles of BDNF and PSD-95 in spine formation, the results of the present

study could suggest that some atypical antipsychotic drugs may induce the promotion of new

synapse formation in hippocampal neurons. However, further research examining the density

and morphology of synapses is required to confirm this.

Many in vitro and in vivo studies have found that BDNF mRNA and protein are up-

regulated by atypical antipsychotics, including olanzapine, clozapine, quetiapine, aripiprazole,

and ziprasidone, whereas haloperidol down-regulates or does not affect BDNF levels (Bai et

al., 2003; Park et al., 2006; Park et al., 2009a; Park et al., 2009b). The transcription factor

cAMP response element binding protein (CREB) induces expression of BDNF, which

contributes to the stabilization of synaptic plasticity. CREB activity is also elevated following

treatment with atypical drugs (Hammonds and Shim, 2009; Park et al., 2009a). Thus, atypical

drugs may influence synaptic plasticity via CREB activation.

Several studies have investigated the effects of treatment with antipsychotic drugs on

PSD-95. One study reported that chronic treatment with haloperidol increased PSD-95

mRNA expression significantly in the rat striatum, consistent with its action at nigrostriatal

projections and its propensity to produce motor side effects, but did not affect the

hippocampus significantly (Iasevoli et al., 2010). Another study demonstrated that acute

administration of haloperidol or olanzapine did not modulate PSD-95 expression in rat

forebrains (de Bartolomeis et al., 2002). Little is known about the mechanism of action of

- 15 -

antipsychotic drugs on PSD-95, despite its potential importance for understanding the

pathophysiology of schizophrenia. Only one reported study has focused on the role of PSD-

95 in mediating 5-HT2A receptors, the main site of action of atypical antipsychotic drugs

(Abbas et al., 2009). According to this study, the absence of PSD-95 decreased the targeting

of 5-HT2A receptors, the total expression of 5-HT2A receptors, and 5-HT2A-mediated signaling

(p-ERK1/2 and p-GSK-3β). Moreover, the antipsychotic-like action of clozapine was

dramatically impaired in PSD-95-null mice. Together with our data, this evidence suggests

that up-regulation of PSD-95 expression with atypical antipsychotic drugs may increase

synapse formation by modulating 5-HT2A-receptor function.

All atypical antipsychotic drugs tested in the present study effectively enhanced

dendritogenesis via the PI3K pathway when administered at concentrations that produced

maximum effects on BDNF and PSD-95 levels. Although levels of BDNF and PSD-95 were

not affected by ziprasidone treatment, enhancement of dendritic outgrowth and branching

was observed in ziprasidone-treated hippocampal neurons. This discrepancy may be

attributable to the differential effects of different durations of drug treatment (4 days for

protein expression vs. 5 days for dendritic outgrowth). The PI3K inhibitor LY294002 did not

affect dendritogenesis but did decrease the differentiation induced by atypical antipsychotic

drugs, which suggests that PI3K contributes to responses to these drugs. This finding is

similar to those of a previous study showing the involvement of the PI3K/Akt pathway in the

positive effects of olanzapine, quetiapine, and clozapine on neurite outgrowth in PC12 cells

(Lu and Dwyer, 2005). Recent studies have underscored the importance of the PI3K/Akt

pathway in schizophrenia [for review, see Zheng et al (2012)]; these studies suggest that

reduced activity of this signaling could at least partially explain the cognitive impairment,

synaptic morphological abnormalities, neuronal atrophy, and dysfunction of neurotransmitter

- 16 -

signaling observed in schizophrenia. Moreover, it is thought that reduced levels of Akt may

reduce the response of patients to antipsychotic drugs (Zheng et al., 2012). Thus, this

signaling pathway appears to be essential for the potentiating effects of olanzapine,

aripiprazole, quetiapine, and ziprasidone. On the other hand, haloperidol did not influence

these effects. Moreover, an important role of PI3K/Akt signaling in the determination of

dendritic morphogenesis has been reported. Kumar et al. (2005) showed that PI3K/Akt

signaling in rat hippocampal neurons increased dendrite size and dendritic complexity as well

as density of dendritic spine via activation of mammalian target of rapamycin (mTOR),

which controls new protein synthesis required for synaptic connections that lead to

synaptogenesis (Yang et al., 2008). Thus, we hypothesized that some atypical antipsychotic

drugs might also play a role in increasing not only dendritic outgrowth but also expression of

synaptic proteins via activation of PI3K.

This study revealed that atypical antipsychotic drugs increase SYP expression under B27

deprivation but not under normal conditions. No change was observed in haloperidol-treated

hippocampal cells. It has been reported that haloperidol decreased SYP levels in the

hippocampus of rats (Eastwood et al., 1997). This discrepancy might be attributable to the

difference in experimental paradigms (the dosage, duration of administration, and animals vs.

cells) used in the present and previous studies. Only one study on atypical antipsychotic

drugs showed that clozapine increased SYP levels in rat frontal cortex (Bragina et al., 2006).

SYP plays a central role in the rapid retrieval of synaptic vesicles, which is required for

efficient synaptic transmission (Daly et al., 2000). The increased expression of SYP induced

by atypical antipsychotic drugs may reflect an increase in the complement of synaptic

vesicles, leading to an increased potential for neurotransmitter release (Bragina et al., 2006).

- 17 -

Overall, our results suggest that some atypical antipsychotic drugs, but not typical

haloperidol, may mediate synaptic plasticity by modulating synaptic proteins levels and

dendritic outgrowth. It may also provide evidence that some atypical antipsychotic drugs

induce synaptic reorganization. These effects may contribute to some degree to improvements

in cognitive function in patients with schizophrenia.

ACKNOWLEDGEMENT

This research was supported by Basic Science Research Program (2009-0075340 to YH

Kim and 2010-0005057 to SW Park) and Global Partnership Program (2009-00505 to W

Seol) through the National Research Foundation of Korea (NRF) funded by the Ministry of

Education, Science and Technology (MEST).

REFERENCES

Abbas AI, Yadav PN, Yao WD, Arbuckle MI, Grant SG, Caron MG, Roth BL. 2009. PSD-95

is essential for hallucinogen and atypical antipsychotic drug actions at serotonin

receptors. J Neurosci 29:7124-7136.

Bai O, Chlan-Fourney J, Bowen R, Keegan D, Li XM. 2003. Expression of brain-derived

neurotrophic factor mRNA in rat hippocampus after treatment with antipsychotic drugs.

J Neurosci Res 71:127-131.

Bastianetto S, Danik M, Mennicken F, Williams S, Quirion R. 2006. Prototypical

antipsychotic drugs protect hippocampal neuronal cultures against cell death induced by

growth medium deprivation. BMC Neurosci 7:28.

Bottenstein JE, Sato GH. 1979. Growth of a rat neuroblastoma cell line in serum-free

supplemented medium. Proc Natl Acad Sci USA 76:514-517.

- 18 -

Bragina L, Melone M, Fattorini G, Torres-Ramos M, Vallejo-Illarramendi A, Matute C,

Conti F. 2006. GLT-1 down-regulation induced by clozapine in rat frontal cortex is

associated with synaptophysin up-regulation. J Neurochem 99:134-141.

Castillo-Gómez E, Gómez-Climent MA, Varea E, Guirado R, Blasco-Ibáñez JM, Crespo C,

Martínez-Guijarro FJ, Nácher J. 2008. Dopamine acting through D2 receptors modulates

the expression of PSA-NCAM, a molecule related to neuronal structural plasticity, in the

medial prefrontal cortex of adult rats. Exp Neurol 214:97-111.

Chambers JS, Thomas D, Saland L, Neve RL, Perrone-Bizzozero NI. 2005. Growth-

associated protein 43 (GAP-43) and synaptophysin alterations in the dentate gyrus of

patients with schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 29:283-290.

Cohen-Cory S, Fraser SE. 1995. Effects of brain-derived neurotrophic factor on optic axon

branching and remodelling in vivo. Nature 378:192-196.

Critchlow HM, Maycox PR, Skepper JN, Krylova O. 2006. Clozapine and haloperidol

differentially regulate dendritic spine formation and synaptogenesis in rat hippocampal

neurons. Mol Cell Neurosci 32:356-365.

Daly C, Sugimori M, Moreira JE, Ziff EB, Llinás R. 2000. Synaptophysin regulates clathrin-

independent endocytosis of synaptic vesicles. Proc Natl Acad Sci USA 97:6120-6125.

de Bartolomeis A, Aloj L, Ambesi-Impiombato A, Bravi D, Caracò C, Muscettola G, Barone

P. 2002. Acute administration of antipsychotics modulates Homer striatal gene expression

differentially. Brain Res Mol Brain Res 98:124-129.

Delotterie D, Ruiz G, Brocard J, Schweitzer A, Roucard C, Roche Y, Suaud-Chagny MF,

Bressand K, Andrieux A. 2010. Chronic administration of atypical antipsychotics

improves behavioral and synaptic defects of STOP null mice. Psychopharmacology (Berl)

208:131-141.

- 19 -

Durany N, Thome J. 2004. Neurotrophic factors and the pathophysiology of schizophrenic

psychoses. Eur Psychiatry 19:326-337.

Eastwood SL, Heffernan J, Harrison PJ. 1997. Chronic haloperidol treatment differentially

affects the expression of synaptic and neuronal plasticity-associated genes. Mol

Psychiatry 2:322-329.

Eastwood SL, Cotter D, Harrison PJ. 2001. Cerebellar synaptic protein expression in

schizophrenia. Neuroscience 105:219-229.

Eastwood SL, Harrison PJ. 2005. Decreased expression of vesicular glutamate transporter 1

and complexin II mRNAs in schizophrenia further evidence for a synaptic pathology

affecting glutamate neurons. Schizophr Res 7:159-172.

Ehrlich I, Klein M, Rumpel S, Malinow R. 2007. PSD-95 is required for activity-driven

synapse stabilization. Proc Natl Acad Sci USA 104:4176-4181.

El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS. 2000. PSD-95

involvement in maturation of excitatory synapses. Science 290:1364-1368.

Elmariah SB, Crumling MA, Parsons TD, Balice-Gordon RJ. 2004. Postsynaptic TrkB-

mediated signaling modulates excitatory and inhibitory neurotransmitter receptor

clustering at hippocampal synapses. J Neurosci 24:2380-2393.

Elmariah SB, Hughes EG, Oh EJ, Balice-Gordon RJ. 2005. Neurotrophin signaling among

neurons and glia during formation of tripartite synapses. Neuron Glia Biol 1:1-11.

Gisabella B, Bolshakov VY, Benes FM. 2005. Regulation of synaptic plasticity in a

schizophrenia model. Proc Natl Acad Sci USA 102:13301-13306.

Hammonds MD, Shim SS. 2009. Effects of 4-week treatment with lithium and olanzapine on

levels of brain-derived neurotrophic factor, B-cell CLL/lymphoma 2 and phosphorylated

- 20 -

cyclic adenosine monophosphate response element-binding protein in the sub-regions of

the hippocampus. Basic Clin Pharmacol Toxicol 105:113-119.

Han K, Kim E. 2008. Synaptic adhesion molecules and PSD-95. Prog Neurobiol 84:263-283.

Hashimoto T, Bergen SE, Nguyen QL, Xu B, Monteggia LM, Pierri JN, Sun Z, Sampson AR,

Lewis DA. 2005. Relationship of brain-derived neurotrophic factor and its receptor TrkB

to altered inhibitory prefrontal circuitry in schizophrenia. J Neurosci 25:372-383.

Huang ZJ, Kirkwood A, Pizzorusso T, Porciatti V, Morales B, Bear MF, Maffei L, Tonegawa

S. 1999. BDNF regulates the maturation of inhibition and the critical period of plasticity

in mouse visual cortex. Cell 98:739-755.

Iasevoli F, Tomasetti C, Marmo F, Bravi D, Arnt J, de Bartolomeis A. 2010. Divergent acute

and chronic modulation of glutamatergic postsynaptic density genes expression by the

antipsychotics haloperidol and sertindole. Psychopharmacology (Berl) 212:329-344.

Ji Y, Pang PT, Feng L, Lu B. 2005. Cyclic AMP controls BDNF-induced TrkB

phosphorylation and dendritic spine formation in mature hippocampal neurons. Nat

Neurosci 8:164–172.

Kaech S, Banker G. 2006. Culturing hippocampal neurons. Nat Protoc 1:2406-2415.

Karson CN, Mrak RE, Schluterman KO, Sturner WQ, Sheng JG, Griffin WS. 1999.

Alterations in synaptic proteins and their encoding mRNAs in prefrontal cortex in

schizophrenia: a possible neurochemical basis for 'hypofrontality'. Mol Psychiatry 4:39-

45.

Keefe RS, Bilder RM, Davis SM, Harvey PD, Palmer BW, Gold JM, et al. 2007.

Neurocognitive effects of antipsychotic medications in patients with chronic

schizophrenia in the CATIE Trial. Arch of gen psychiatry 64:633-47.

Kim NR, Park SW, Lee JG, Kim YH. 2008. Protective effects of olanzapine and haloperidol

- 21 -

on serum withdrawal-induced apoptosis in SH-SY5Y cells. Prog Neuropsychopharmacol

Biol Psychiatry 32:633-642.

Kinon BJ, Lieberman JA. 1996. Mechanisms of action of atypical antipsychotic drugs: a

critical analysis. Psychopharmacology (Berl) 124: 2-34.

Klimaschewski L, Nindl W, Pimpl M, Waltinger P, Pfaller K. 2002. Biolistictransfection and

morphological analysis of cultured sympathetic neurons. J Neurosci Methods 15:63-71.

Kovalchuk Y, Hanse E, Kafitz KW, Konnerth A. 2002. Postsynaptic induction of

BDNFmediated long-term potentiation. Science 295:1729-1734.

Kumar V, Zhang MX, Swank MW, Kunz J, Wu GY. 2005. Regulation of dendritic

morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways. J Neurosci

25:11288-11299.

Lessmann V, Gottmann K, Malcangio M. 2003. Neurotrophin secretion: current facts and

future prospects. Prog Neurobiol 69:341–374.

Lidow MS, Song ZM, Castner SA, Allen PB, Greengard P, Goldman-Rakic PS. 2001.

Antipsychotic treatment induces alterations in dendrite- and spine-associated proteins in

dopamine-rich areas of the primate cerebral cortex. Biol Psychiatry 49:1-12.

Lonze BE, Ginty DD. 2002. Function and regulation of CREB family transcription factors in

the nervous system. Neuron 35:605-623.

Lu XH, Dwyer DS. 2005. Second-generation antipsychotic drugs, olanzapine,quetiapine, and

clozapine enhance neurite outgrowth in PC12 cells via PI3K/AKT, ERK, and pertussis

toxin-sensitive pathways. J Mol Neurosci 27:43-64.

Manschreck TC, Boshes RA. 2007. The CATIE schizophrenia trial: results, impact,

controversy. Harv Rev Psychiatry 15:245-58.

- 22 -

McAllister AK, Katz LC, Lo DC. 1997. Opposing roles for endogenous BDNF and NT-3 in

regulating cortical dendritic growth. Neuron 18:767-778.

Mendrek A, Kiehl KA, Smith AM, Irwin D, Forster BB, Liddle PF. 2005. Dysfunction of a

distributed neural circuitry in schizophrenia patients during a working-memory

performance. Psychol Med 35:187-196.

Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM, Makhinson M, He Y,

Ramsay MF, Morris RG, Morrison JH, O'Dell TJ, Grant SG. 1998. Enhanced long-term

potentiation and impaired learning in mice with mutant postsynaptic density-95 protein.

Nature 396:433-439.

Nasrallah HA. 2006. Metabolic findings from the CATIE trial and their relation to tolerability.

CNS Spectr 11:32-9.

Newcomer JW. 2007. Metabolic considerations in the use of antipsychotic medications: a

review of recent evidence. J Clin Psychiatry 68:20-7.

Pang PT, Teng HK, Zaitsev E, Woo NT, Sakata K, Zhen S, Teng KK, Yung WH, Hempstead

BL, Lu B. 2004. Cleavage of proBDNF by tPA/plasmin is essential for long-term

hippocampal plasticity. Science 306:487-491.

Parikh V, Khan MM, Mahadik SP. 2004. Olanzapine counteracts reduction of brain derived

neurotrophic factor and TrkB receptors in rat hippocampus produced by haloperidol.

Neurosci Lett 356:135-139.

Park SW, Lee SK, Kim JM, Yoon JS, Kim YH. 2006. Effects of quetiapine on the brain-

derived neurotrophic factor expression in the hippocampus and neocortex of rats.

Neurosci Lett 402:25-29.

- 23 -

Park SW, Lee CH, Lee JG, Lee SJ, Kim NR, Choi SM, Kim YH. 2009a. Differential effects

of ziprasidone and haloperidol on immobilization stress-induced mRNA expression in

the hippocampus and neocortex of rats. J Psychiatr Res 43:274-281.

Park SW, Lee JG, Ha EK, Choi SM, Cho HY, Seo MK, Kim YH. 2009b. Differential effects

of aripiprazole and haloperidol on BDNF-mediated signal changes in SH-SY5Y cells.

Eur Neuropsychopharmacol 19:356-362.

Poo MM. 2001. Neurotrophins as synaptic modulators. Nat. Rev. Neurosci 2:24–32.

Pozzo-Miller LD, Gottschalk W, Zhang L, McDermott K, Du J, Gopalakrishnan R, Oho C,

Sheng ZH, Lu B. 1999. Impairments in high-frequency transmission, synaptic vesicle

docking, and synaptic protein distribution in the hippocampus of BDNF knockout mice. J

Neurosci 19:4972-4983.

Prange O, Wong TP, Gerrow K, Wang YT, El-Husseini A. 2004. A balance between

excitatory and inhibitory synapses is controlled by PSD-95 and neuroligin. Proc Natl

Acad Sci USA 101:13915-13920.

Sala C, Rudolph-Correia S, Sheng M. 2000. Developmentally regulated NMDA receptor-

dependent dephosphorylation of cAMP response element-binding protein (CREB) in

hippocampal neurons. J Neurosci 20:3529-3536.

Shen K, Cowan CW. 2010. Guidance molecules in synapse formation and plasticity. Cold

Spring Harb Perspect Biol 2:1-18.

Sze CI, Troncoso JC, Kawas C, Mouton P, Price DL, Martin LJ. 1997. Loss of the

presynaptic vesicle protein synaptophysin in hippocampus correlates with cognitive

decline in Alzheimer disease. J Neuropathol Exp Neurol 56:933-944.

Tarsa L, Goda Y. 2002. Synaptophysin regulates activity-dependent synapse formation in

cultured hippocampal neurons. Proc Natl Acad Sci USA 99:1012-1016.

- 24 -

Toro C, Deakin JF. 2005. NMDA receptor subunit NRI and postsynaptic protein PSD-95 in

hippocampus and orbitofrontal cortex in schizophrenia and mood disorder. Schizophr

Res 80:323-330.

Tyler WJ, Pozzo-Miller L. 2003. Miniature synaptic transmission and BDNF modulate

dendritic spine growth and form in rat CA1 neurones. J Physiol 553:497-509.

Valtorta F, Pennuto M, Bonanomi D, Benfenati F. 2004. Synaptophysin: leading actor or

walk-on role in synaptic vesicle exocytosis? Bioessays 26:445-453.

Vawter MP, Howard AL, Hyde TM, Kleinman JE, Freed WJ. 1999. Alterations of

hippocampal secreted N-CAM in bipolar disorder and synaptophysin in schizophrenia.

Mol Psychiatry 4:467-475.

Yang PC, Yang CH, Huang CC, Hsu KS. 2008. Phosphatidylinositol 3-kinase activation is

required for stress protocol-induced modification of hippocampal synaptic plasticity. J

Biol Chem 283:2631-2643.

Yoshii A, Constantine-Paton M. 2007. BDNF induces transport of PSD-95 to dendrites

through PI3K-AKT signaling after NMDA receptor activation. Nat Neurosci 10:702-711.

Yoshii A, Constantine-Paton M. 2010. Postsynaptic BDNF-TrkB signaling in synapse

maturation, plasticity, and disease. Dev Neurobiol 70:304-322.

Zheng W, Wang H, Zeng Z, Lin J, Little PJ, Srivastava LK, Quirion R. 2012. The possible

role of the Akt signaling pathway in schizophrenia. Brain Res 1470:145-158.

- 25 -

Figure 1. Effects of antipsychotic drugs on the expression of PSD-95 in hippocampal

neurons. Cells were treated with different doses of antipsychotic drugs for 4 days with

(+B27) or without (-B27) B27. (A) olanzapine, (B) aripiprazole, (C) quetiapine, (D)

ziprasidone, and (E) haloperidol. For each condition, three independent experiments were

performed. Cell lysates were analyzed by SDS-PAGE and Western blotting with anti-PSD-95

primary antibodies. A representative image and quantitative analysis normalized to the α-

tubulin band are shown. Values are means ± SEMs expressed as percentage of values of the

control cells (+B27, no drug treatment). *p < 0.05 vs. untreated control, **p < 0.01 vs.

untreated control, #p < 0.05 vs. B27 -deprived only, ##p < 0.01 vs. B27-deprived only.

Figure 2. Effects of antipsychotic drugs on the expression of BDNF in hippocampal neurons.

Cells were treated with different doses of antipsychotic drugs for 4 days with (+B27) or

without (-B27) B27. (A) olanzapine, (B) aripiprazole, (C) quetiapine, (D) ziprasidone, and

(E) haloperidol. For each condition, three independent experiments were performed. Cell

lysates were analyzed by SDS-PAGE and Western blotting with anti-BDNF primary

antibodies. A representative image and quantitative analysis normalized to the α-tubulin band

are shown. Values are means ± SEMs expressed as percentage of values of the control cells

(+B27, no drug treatment). *p < 0.05 vs. untreated control, **p < 0.01 vs. untreated control, #p

< 0.05 vs. B27-deprived only, ##p < 0.01 vs. B27-deprived only.

Figure 3. Effects of antipsychotic drugs on the expression of synaptophysin (SYP) in

hippocampal neurons. Cells were treated with different doses of antipsychotic drugs for 4

days with (+B27) or without (-B27) B27. (A) olanzapine, (B) aripiprazole, (C) quetiapine,

(D) ziprasidone, and (E) haloperidol. For each condition, three independent experiments were

- 26 -

performed. Cell lysates were analyzed by SDS-PAGE and Western blotting with anti-

synaptophysin primary antibodies. A representative image and quantitative analysis

normalized to the α-tubulin band are shown. Values are means ± SEMs expressed as

percentage of values of the control cells (+B27, no drug treatment). *p < 0.05 vs. untreated

control, **p < 0.01 vs. untreated control, #p < 0.05 vs. B27--deprived only, ##p < 0.01 vs. B27-

deprived only.

Figure 4. Effects of antipsychotic drugs on the dendritic outgrowth and branching of

hippocampal neurons. Cells were treated with DMSO control (Con, a), olanzapine (Ola, 100

μM, b), aripiprazole (Ari, 10 μM, c), quetiapine (Que, 10 μM, d), ziprasidone (Zip, 10 μM, e),

or haloperidol (Hal, 10 μM, f) for 5 days. For each condition, two independent experiments

were performed. Cells were photographed (A) and scored (B; dendritic outgrowth, C;

dendritic branching) according to the methods described above. In total, 300-400 cells were

analyzed from one of two independent samples. Data are expressed as means ± SEMs. *p <

0.05 **p < 0.01 vs. control.

Figure 5. Effects of PI3K inhibitor on dendritic outgrowth and branching of hippocampal

neurons induced by atypical antipsychotic drugs. Cells were exposed to LY294002 (LY, 10

μM) for 30 min before the addition of DMSO control (a), olanzapine (Ola, 100 μM, b and c),

aripiprazole (Ari, 10 μM, d and e), quetiapine (Que, 10 μM, f and g), or ziprasidone (Zip, 10

μM, h and i) for 5 days. For each condition, two independent experiments were performed.

Cells were photographed (A) and scored (B, dendritic outgrowth; C, dendritic branching)

according to the methods described above. In total, 300-400 cells were analyzed from one of

two independent samples. Data are expressed as means ± SEMs. **p < 0.01 vs. control, ##p <

- 27 -

0.01 vs. olanzapine-treated cells, ††p < 0.01 vs. aripiprazole-treated cells, $$p < 0.01 vs.

quetiapine-treated cells, §§p < 0.01 vs. ziprasidone-treated cells.

Page 29 of 32Synapse

Page 30 of 32Synapse

Page 31 of 32Synapse

Page 32 of 32Synapse

Page 33 of 32 Synapse