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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
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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
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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
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(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
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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-
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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).
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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)
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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
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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
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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
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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 =
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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
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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
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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
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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
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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).
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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).
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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.
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