Key words
ziprasidone - haloperidol - beta-III-tubulin-positive neurons - adult neural stem
cells - murine hippocampus -
de-novo generation
Introduction
Adult neurogenesis comprises a complex physiological cascade of events finally leading
to the de novo generation of new neurons including the proliferation of resident neural stem cells,
their differentiation, and functional integration into already existing neural circuitries
[1]. As a consequence, disturbed adult neurogenesis has been linked to the etiopathology
of psychiatric disorders. Specifically, decreased neural stem cell proliferation in
the dentate gyrus of the hippocampus has been demonstrated in post-mortem human brain
of schizophrenic patients [2].
These novel findings range among other meanderings, which have been observed in studies
looking into pathological changes in schizophrenic patients. In this context, abnormalities
in brain morphology have been reported frequently [3]. Although the data are still not unequivocal, the most prominent findings include
total brain volume reductions and enlarged ventricles [4]. Also, there is evidence for alterations on the cellular level. They centre around
the loss of regular cell morphology of large pyramidal neurons in the region of the
dorsolateral prefrontal cortex [5] and a selective decrease in the number of parvalbumin-positive GABA-ergic interneurons
in the hippocampus of the diseased [6]. Own experiments involving rat hippocampus have produced similar results after animal
treatment by the NMDA-receptor antagonist MK-801 [7].
Although it seems far-fetched to interpret schizophrenia heretically as a neurodegenerative
disease, the volume and cellular abnormalities might be indicative of a disturbed
cellular repair mechanism by adult neural stem cells (ANSC). Moreover, Reif and co-workers
reported a reduction in the number of stem cells in post-mortem hippocampi of schizophrenic
patients [2].
Therefore, treatment options to restore full stem cell functioning could possibly
become an option. Studies on antipsychotic drug-induced neurogenesis have already
been conducted, but this survey on antipsychotic drugs looked particularly into the
role of the serotonin 5HT1A receptor subtype in terms of the de-novo generation of new neurons, as a previous study by this group has shown that serotonin
served as maintenance factor in hippocampus stem cells and this function was delivered
by the 5-HT1A receptor [8]: In a set of experiments, this particular receptor strongly influenced ANSC proliferation
and stem cell survival. The group of Kempermann has demonstrated a differential impact
of serotonin receptors on proper ANSC functioning emphasizing the 5-HT1A receptor subtype’s effect [9]. In addition, Santarelli [10] has revealed that this serotonin receptor is necessary for proper action of psychiatric
medication improving anxiety. We have therefore chosen ziprasidone with its particular
5-HT1A receptor binding potential to be tested against the first generation antipsychotic
haloperidol with no decisive 5-HT1A receptor binding.
In order to investigate the mechanisms by which antipsychotic compounds may impact
on neuroplastic features, we applied a standardized in-vitro neurosphere assay [11] of ANSC isolated and expanded from the dentate gyrus of mouse hippocampus.
The results demonstrate that ziprasidone significantly generated more new neurons
compared to haloperidol. Furthermore, when the 5HT1A receptors were blocked by WAY100621, ziprasidone lost the neuroplastic superiority
over haloperidol. The loss of cellular mobility after haloperidol or WAY100621 treatment
may account for the disadvantage in terms of differentiation, since stem cell differentiation
requires cellular mobility during the early steps of this process.
Material and Methods
Establishment of primary ANSC cultures
Animals were treated according to NIH equivalent animal care rules. Study procedures
were approved by the Regierung von Oberbayern, Munich (approval ID regarding the use
of rodents for schizophrenia research 209.1/211.2531-78/03). Adult wild-type C57B6
mice (12 weeks old, Charles River Germany, Sulzfeld) were anesthetized by intraperitoneal
injection of pentobarbital (120 mg/kg) and sacrificed by cervical dislocation. Brains
were removed and placed in chilled PBS. Whole hippocampi were carefully removed and
the dentate gyrus was identified. This structure was then digested in Eagle’s balanced
salt solution (EBSS) containing 0.94 mg/mL papain (purchased from Worthington Biochemicals),
0.2 mg/mL cysteine and EDTA (both Sigma) for 50 min at 37°C under gentle rocking.
Digested dentates were washed twice in DMEM (Gibco Life), mechanically dissociated
using a fire-polished Pasteur pipette, and finally placed in serum-free DMEM/F12 medium
(1:1 v/v; Gibco Life) containing 20 ng/mL EGF and 10 ng/mL FGF-2 (both human recombinant;
Peprotech), 2 mM L-glutamine, 0.6% glucose, 9.6 µg/mL putrescine, 6.3 µg/mL progesterone,
5.2 ng/mL sodium selenite, 0.025 mg/mL insulin, 0.1 mg/mL transferrin and 0.2 µg/mL
heparin (all Sigma) at a density of 20 000 cells/cm2 onto sterile, non-coated Petri dishes (Corning).
Cell culturing and propagation
Cells were serially subcultured by mechanical dissociation every 4–7 days. Cells were
collected as neurospheres and the total number of viable cells was assessed each passage
by trypan blue exclusion (Sigma). Self-renewal and multipotency were assessed every
3 subculturing passages [11]
[12]. For the experiments, 3 different ANSC cell lines at passages 5, 6, and 8 were used.
ANSC were collected 5 days after the last subculturing passage. The total number of
experimental samples for each experiment was n=20 for differentiation and n=40 for
migration. All samples were analyzed in a blinded way.
Cell culture treatments with different agents
To ensure that the observed effects on proliferation and migration were not due to
cytotoxicity, the toxic threshold levels of the administered drugs were assayed. For
both haloperidol and ziprasidone 500 nM was determined as an appropriate concentration,
while 50 µM was used for the 5-HT1A receptor antagonist WAY100635. All concentrations were below LD50 by at least 2 orders of magnitude.
Mobility assay
For these experiments blind well or Boyden chambers were used [13]. Briefly, PVP-free polycarbonate filters with 8 µm pores (Costar) were coated with
Matrigel. DMEM (negative control), DMEM plus 500 nM haloperidol or 500 nM ziprasidone
or regular growth medium as an internal standard containing EGF and FGF2 were placed
in the lower chambers. Undifferentiated ANSCs with a regular cell diameter of ~16 µm
were used for the assays 24 h following the last subculturing passage. 50 000 cells
were resuspended in 200 µL DMEM and then placed in the upper chambers, where they
were incubated for 6 h. ANSCs remaining on the upper surface of the filters were mechanically
removed, while those that had migrated to the lower surface were fixed with ethanol,
Giemsa stained, and counted at 400-fold magnification in 5 random fields per filter.
Differentiation of stem cell progeny, and immunocytochemistry
Undifferentiated ANSC were plated onto Matrigel-coated glass coverslips (Gibco Life)
in growth medium. 6 h after plating, cultures were shifted to FGF2-containing medium,
grown for 2 days, shifted to mitogen-free medium containing 2% FCS, grown for 5 days
and then fixed (4% paraformaldehyde in phosphate-buffered saline – PBS – pH 7.4, for
20 min) and processed for immunofluorescence. In these cell cultures we evaluated
the proportions of neurons. Primary antibody for the detection of neurons was monoclonal
anti-TUJ1 (1:100, Covance) staining the neuron-specific class III beta-tubulin. Primary
antibodies were incubated for 2 h at room temperature before adding Texas-red conjugated
donkey-anti-sheep (1:500) (Chemicon) for 30 min at room temperature. All cell nuclei
were counterstained with DAPI (1:1000 dilution of stock solution, 10 min at room temperature)
in order to assess the total number of differentiated cells. Samples were examined
and photographed using a Nikon Eclipse 3 000 fluorescence microscope at 640-fold magnification.
Immunoreactive cells were counted in at least 5 non-overlapping fields in each sample
(>500 cells/sample) and expressed as a percentage of the total number of nuclei. Fluorescent
signals from single optical sections were sequentially acquired and analyzed by Photoshop
7.0 (Adobe).
Statistical analysis
Experimental data stem from differentiation and mobility experiments on adult neural
stem cells. Within each group 20 differentiation experiments were performed independently
(n=20). Collected data passed one-way analysis of variance normality testing (p=0.666)
and subsequent equal variance testing (p=0.053). The differences in the mean values
between groups were greater than would be expected by chance (p<0.001). All experimental
groups were then pairwise tested according to the Holm-Sidak method with an overall
level of significance set at p≤0.05). The data from the mobility shift experiments
(n=40) failed in the normality test. Therefore, ANOVA on ranks was performed revealing
a statistically significant difference between the groups (p<0.001). In order to isolate
the group or the groups that differ from the others, we used Dunn’s method as multiple
comparison procedure with p<0.05 as the indicator of significance.
Results
Neurogenesis
Based on previous work, we hypothesized the serotonin 5-HT1A receptor subtype to play a pivotal role for differentiation of adult neural stem
cells. As proven before, neurogenesis among other factors (EGF, FGF2, e. g.,) also
depends on serotonin in general and on the 5HT1A receptor availability in particular. Since number and availability of neurons in
the hippocampus seem to be crucial, we tested antipsychotic agents on their capacity
to provide neurons.
At the end of the de-novo differentiation after 8 days, the newly generated neurons derived from undifferentiated
murine hippocampus stem cells were identified by immunocytochemistry using the TUJ1
monoclonal antibody. In the untreated control group 10.8% (mean value, SEM±0.1%) of
the de-novo differentiated cells in culture were neurons (i. e., beta-III-tubulin-positive),
which is in due accordance with the general literature in this field ([Fig. 1b, g]).
Fig. 1 Outcome after differentiation. Microscopic images (a–f) showing 400-fold magnification of all cell nuclei stained by DAPI (blue) and by
the neuronal marker beta-III-tubulin (red), white scale bar f measures 35 µm. Bar columns g representing the mean amount of neurons among all DAPI stained nuclei, column ticks
showing the SEM; p≤0.05 for §, #, $, *, and &.
After 500 nM haloperidol treatment 7.5% (SEM±0.1%) of the cells became beta-III-tubulin-positive
neurons ([Fig. 1c]). The difference of this group compared to control was statistically not significant
(p>0.05). The second antipsychotic used was ziprasidone. Here, 16.2% (SEM±0.1%) neurons
surfaced after differentiation ([Fig. 1d]), which was statistically significant in comparison to both controls (p<0.05, [Fig. 1g§]) and haloperidol treatment (p<0.05; [Fig. 1g#]).
Based on previous studies, we hypothesized that these significant effects were due
to the 5HT1A receptor activity of ziprasidone. Therefore, we impeded this receptor by adding 50 µM
of WAY100621 to 500 nM ziprasidone. As a result of this, the proportion of neurons
([Fig. 1e]) decreased to 5.2% (SEM±0.1%) and there was no statistical difference between this
group and the haloperidol group. By contrast, when the ziprasidone/WAY group was tested
against ziprasidone there was a significant difference between the 2 groups (p<0.05,
[Fig. 1g*]).
Finally, WAY100621 alone was assayed. In this scheme, the number of de-novo generated neurons ([Fig. 1f]) was 7.4% (SEM±0.1%) revealing a significant effect compared to ziprasidone (p<0.05,
[Fig. 1g&]), while no statistical effect was observed between this group and control or haloperidol
(p>0.05 each).
Cell mobilization
It has become increasingly evident that stem cell mobilization represents a stem cell
characteristic, which is as important as proliferation or differentiation. Stem cell
mobility brings appropriate numbers of stem cells to their correct locations in the
brain, which is essential for establishing, maintaining and modifying the neural circuitry
[14].
Haloperidol decreased the number of mobile cells in a statistically significant manner.
After 6 h of incubation in the blind-well or Boyden chambers only 1 cell (median value)
migrated ([Fig. 2]), whereas in the untreated control group 2.5 cells (median value) did so. This was
significant (p<0.05, [Fig. 2§]) as well as the comparison of haloperidol and ziprasidone (p<0.05, [Fig. 2#]). In the latter group 4 cells (median value) were chemotactically attracted by ziprasidone.
This effect vanished after 5HT1A blockade by WAY100621 added to ziprasidone (p<0.05, [Fig. 2$]) resulting in one migrating cell during the given incubation period. Compared to
control (2.5 cells) there was a negative chemotactic effect associated with 5HT1A blockade, which was significant (p<0.05, [Fig. 2*]).
Fig. 2 Mobility assays. Box-and-whisker plots of the mobility assays representing the median
value, with 50% of all data falling within the box. The “whiskers” extend to the 5th and 95th percentiles. Mobilized undifferentiated ANSC after no (C), haloperidol (HAL), ziprasidone
(ZIP), ziprasidone + WAY100621 (ZIP+WAY), and WAY100621 (WAY) treatment; p≤0.05 for
§, #, $, and *.
No significance was detected between control and ziprasidone or WAY100621, or between
haloperidol and WAY100621 or ziprasidone/WAY100621.
Discussion
In this survey, we examined the impact of haloperidol and ziprasidone on the de-novo differentiation of adult neural stem cells derived from mouse hippocampus. The outcome
after the differentiation of ANSC into neurons has demonstrated that the second generation
antipsychotic (SGA) ziprasidone generated significantly more new neurons than the
classic first-generation antipsychotic (FGA) haloperidol. As underlying mechanism
for these neuroplastic effects we suggest the particular affinity of ziprasidone to
the serotonin receptor subtype 5-HT1A
[15]. After 5-HT1A was blocked pharmacologically by WAY100621 the pro-neurogenic and chemotactic advantage
of ziprasidone over haloperidol disappeared as this study indicates. In this vein,
we suggest that the mobility of the stem cells represents a necessary condition for
proper differentiation.
This study comparing the classic antipsychotic drug haloperidol with the second-generation
antipsychotic ziprasidone looked particularly into the role of the 5-HT1A receptor. This receptor was also subject of a study with the genetic 5-HT1A receptor knock-out mouse model demonstrating the necessity of this receptor for the
impact on behaviour and neurogenesis of the drug fluoxetine [10]. Here, this survey was conducted in a kind of “pharmacological knock-out model of
the 5-HT1A receptor”. Further elucidating the possible mechanism how 5-HT1A receptors influence adult neurogenesis, in-vivo data suggest that they can modulate
cell proliferation in the hippocampus by a direct post-synaptic effect [16]. Apart from Santarelli’s work it is further known that antidepressant drugs drive
neurogenesis in the hippocampus acting on the serotonergic system through 5-HT1A receptors [17]
[18], while 5-HT1A antagonists decrease cell proliferation in the dentate gyrus [19]. Although there are conflicting results on how serotonin impacts on neuroplastic
effects like stem cell proliferation, survival and differentiation, Klempin et al.
have suggested that the differential display of the several serotonin receptor subtypes
explains some of the results, which might seem contradictory at first view [9]. Also, in-vitro data showed the enhancement of epidermal growth factor (EGF) signalling
and an increased cell-turnover rate in fibroblasts transfected with the 5-HT1A receptor [20]
[21]. Additional 5-HT1A receptor stimulation was not undertaken in our experiments, since previous results
have already elucidated the impact of the serotonergic system on ANSC and the positive
correlation between the serotonergic system, its receptors and the neurogenic process
have been investigated in detail [8]
[22]
[23]
[24].
Among the 2 other monoamines, dopamine has also been tested in relation to neurogenesis
[25]
[26]
[27]. This connection is important, since the dopamine D2 receptor represents the classic target of first generation antipsychotics such as
haloperidol, whereas the SGA ziprasidone has a comparably high affinity for serotonin
receptors. Data from animal studies assessed ziprasidone’s 5-HT2A receptor antagonizing affinity (pKi 9.38). This was about 10 times higher than the affinity to the D2 receptor (pKi 8.32). For the purpose of this study, it is noteworthy to remember that ziprasidone
is an agonist on the 5-HT1A receptor (pKi 8.47), and it antagonizes the 5-HT2A and the dopamine D2 receptor. Contrary to this, haloperidol does not bind to the 5-HT1A receptor [28].
Still, in order to exclude synergistic toxic effects of the WAY100621 compound and
haloperidol apart from receptor antagonism, we included a combined WAY/haloperidol
group in our experimental set-up although knowing there is no haloperidol 5-HT1A receptor interaction.
Although stem cell proliferation was not subject of this report, it should be mentioned
that previous work suggested an influence of the D2 receptor inactivation by haloperidol, which led to an increase in ANSC proliferation
in rodent animal models [29]
[30]. This finding is contrasting with results showing that chronic clozapine, but not
haloperidol, treatment increased the number of proliferating cells in the hippocampus.
Both of these drugs had no effect on cell survival [31].
The major part of studies that investigated neurogenesis and antipsychotics used haloperidol
either as a reference or as the primary drug of interest. Concerning atypical or second-generation
antipsychotic drugs most of the reports analyzed olanzapine [32] and risperidone [33]
[34]. As a common denominator, nearly all studies took haloperidol as the comparator
check with the SGA. By contrast, studies on ziprasidone examining this antipsychotic
compound and neurogenesis with particular interest for the role of the 5-HT1A receptor are very rare or not present.
Adult neural stem cells derived from murine hippocampus cultured in serum-free medium
with EGF and FGF differentiated into more new neurons in a statistically significant
manner after ziprasidone was added to the media compared to both control and ANSC
treated with haloperidol as this survey has proven. By contrast to the results reported
here, haloperidol caused an increase of de-novo generated neurons in a different set-up, which used the salamander, a non-mammalian
model [35]. The species difference may explain the divergent results to some extent, since
it has been shown that even within the same species, different mouse strains (Balb/cJ
and 129SvEvTac) could produce different results [36].
Most of the models commonly employed mimic the so-called negative symptomatology (apathy,
anhedonia, lack of emotion) and adjunctive cognitive defects, which are produced by
psychostimulants [e. g., MK-801 or phencyclidine (PCP)] inferring with the regular
balance of the glutamatergic and the GABAergic system. In such a model clozapine,
but not haloperidol, prevented the decrease in neurogenesis after mice were treated
by PCP [37].
We want to emphasize that the target of our cell culture model is not mimicking schizophrenia.
Instead, we want to decipher pharmacological mechanisms of drug action. Therefore,
we used a genetically unaltered, non-viral and non-oncogenic cell culture taking into
account that most of the in-vitro cell culture experiments were done with cell lines
derived from tumor tissue like PC12 cells [38]. The advantage of ANSC lies in the ability to generate permanent cell lines, which
differentiate into neurons under given conditions without possible interference with
– for the purpose of understanding pharmacological pathways – the surrounding tissue.
In doing so, we achieve a reduction in the complexity for the gain of insight into
molecular function of single cell types and the process of neurogenesis. Due to this
experimental paradigm, it was not possible to address potentially cardiotoxic effects
caused by ziprasidone, although a recent study has shown that ziprasidone’s putative
cardiotoxicity is within the regular range of SGA drugs [39].
Furthermore, we did not test the various hypotheses and their underlying mechanism
leading to the onset of schizophrenia like other groups who showed, for example, that
risperidone inhibited the production of interferon gamma induced microglial activation,
which in turn possibly could contribute to schizophrenia [34]
[40]. Ziprasidone produced similar results in analogous experiments, respectively [41]. Haloperidol did not have any effect on neurogenesis in the rat hippocampus in a
kanaic acid hippocampus deletion model [32]. In terms of mobility, another SGA drug, olanzapine, revealed a positive chemotactic
effect [42].
Although the total of the migrated cells appears small, the statistically significant
results from the mobility experiments reflect the main message of this study – that
ziprasidone’s neuroplastic properties are due to its 5-HT1A receptor activation. Impeding this receptor by WAY100621 produces similar results
resembling those of the haloperidol and the WAY/ziprasidone group. Additional studies
going beyond the approach used here would be needed to rule out the notion that other
factors than the direct inhibition of the 5-HT1A receptor by WAY100621 have caused this outcome taking into account that the neurosphere
assay used is considered controversial by some authors [43]. In general, the results presented here are in accordance with previous studies
on the serotonergic system [8] and may be clinically involved in conferring potential SGA benefits of memory function
and cognition. The newly generated neurons become part of existing neural networks
and project to the CA3 layer where they form synapses and receive input from the perforant
path [44].
Taken together, our data indicate a role for ziprasidone in augmenting de-novo neural differentiation and maintaining mobilization of ANSC-derived neuronal progenitors.
It is tempting to speculate that this may partially account for improved cognition
in patients treated with ziprasidone as compared to patients treated with haloperidol
[45], which hampered ANSC migration and differentiation in our experiments. In general,
the neurogenic region of the hippocampus is crucial for learning and memory, all cognitive
functions compromised in schizophrenic patients. To put it cautiously, our observations
might partially explain the often postulated positive effects of SGA on cognition.
We are very well aware that this interpretation is highly speculative, but hope that
this study becomes a pilot to stimulate future translational studies connecting the
laboratory bench to the bedside.
