The present study reports that the long-term treatment with chlorpromazine, decreased the number of Sox-2 expressing cells in the rat brain SVZ. The results imply cautiously the effect of chlorpromazine on the early stages of adult neurogenesis, although its molecular mechanism is so far unexplained. In spite of the study limitations, such as low animal numbers and the lack of Sox-2/Musashi-1 gene expression, our findings suggest that chronic treatment with chlorpromazine may depress adult neurogenesis in the rat SVZ and possibly impede the rostral migratory stream formation.
The effect of APDs on adult neurogenesis is heterogeneous in animal models, where data suggest that the greatest differences can be observed between typical and atypical APD; yet even within one group, there are some distinct internal differences [1, 5]. As stated previously, data on the impact of chlorpromazine on neurogenesis are limited, with few studies demonstrating contradicting results. Collectively suggesting that chlorpromazine may have various effects at different stages of neurogenesis and that the kinetics of prescribing may also be relevant. By analyzing the neurogenesis markers in the present study, we anticipated to reveal timing influences on the relationship of this antipsychotic drug with CNS stem cell proliferation.
According to some studies, the antagonistic affinity of the D2 receptor leads to an increase in neurogenesis. The fact that chlorpromazine among others blocks this receptor is an argument for the pro-neurogenic properties of this APD. Moreover, chlorpromazine does not block the D5 receptor, and stimulation of this receptor leads to an enzymatic cascade causing an increased tendency for cell proliferation [6, 15, 16]. Another important element of the pharmacodynamics of chlorpromazine in this context is blockade of the 5-HT2 receptor. Its blocking leads to the induction of the same enzymatic pathway as the stimulation of the D5 receptor [1, 17]. For the above reasons, it seemed reasonable to expect that chronic administration of chlorpromazine should support adult neurogenesis. Sox-2, (SRY [sex determining region Y]-box 2 is a transcription factor that is actively involved in the mechanisms of stem cell niche maintenance, their self-renewal and progenitor formation. However, as the cell grows, this factor decreases until silenced, and is therefore only an early stem cell marker. The Mann–Whitney U test revealed that treatment with chlorpromazine significantly decreased the number of Sox-2-immunopositive cells in SVZ compared to untreated controls (Figs. 2, 3; z = 2.119; N1 = 12; N2 = 10; p = 0.034) and the difference was 28%.
Another marker used in the study was the Musashi-1 (MSI1), RNA-binding protein with distinct expression in neural progenitor cell populations. Chlorpromazine administration decreased the number of Musashi1-immunopositive cells in SVZ compared to controls (Figs. 2, 3; z = 0.968; N1 = 0.12; N2 = 10; p = 0.332) and the difference was 19.97%; however, it was not statistically significant. Further research would dispel doubts as to whether the downward trend observed here is a real effect of the administered drug, or rather a coincidence resulting from the internal diversity of the studied animals. Nevertheless, the fact that Sox2-expressing cells are reduced under the influence of chlorpromazine, suggest that a similar phenomenon should be observed using the marker Musashi-1, as the expression of these factors is closely related to progenitor cells at a similar level of development.
The inverse trend was observed during examination of DCX-positive cells—a 7.89% increase in the number of cells was detected in the test group as compared to the control group. The Mann–Whitney U test revealed that treatment with chlorpromazine slightly increased the number of DCX-immunopositive cells in SVZ compared to untreated controls (Figs. 2, 3; z = 0.880; N1 = 12; N2 = 10; p = 0.378). However, this result is not statistically significant, which does not allow for final conclusion. Nonetheless, two possibilities can be assumed hypothetically. Either cells stained for DCX expression do not change their quantity after chronic administration of chlorpromazine, or there is some increase in cell density. In both cases, the same process occurs, but with a different intensity. As previously shown, cells reactive to the Sox2 antibody decreased in number as a result of drug administration, which makes it much less possible for later DCX-expressing cells to develop. Thus, it seems logical to hypothesize that the study group would have a lower density of DCX-immunopositive cells. However, for some reason, their number is equal or greater than the control group. This leads to the conclusion that despite the reduced potential (by reduced number), early progenitors and stem cells in chlorpromazine group had higher survivability compared to the control group (in which the development of nerve cells was undisturbed). Therefore, it can be hypothesized that chlorpromazine has a potential neuroprotective effect on maturing nerve cells (neuroblasts). For instance Kuruba et al.  mentioned the neuroprotective effect of chlorpromazine in CNS. In this study, no increase in the number of DCX-positive neuroblasts in SGZ was observed as a result of drug administration.
Overall, the effect of chlorpromazine on postnatal neurogenesis remains inconclusive. The present study showed that, in fact, chlorpromazine reduces the number of stem cells and early progenitors (Sox-2). Surprisingly however, this effect does not appear to affect the later stages of neuron formation. However, the upward trend in the number DCX-positive neuroblasts after drug treatment suggests cautiously that chlorpromazine may support the late stage of adult neurogenesis.The suppression of Sox-2+ progenitors origin by chlorpromazine may potentially be caused by the inhibition of dopamine receptors because neural stem cells (NSCs) in the rat SGZ express D2 receptors . In contrast to chlorpromazine, clozapine, a second-generation antipsychotic (atypical) drug, was pro-neurogenic and anti-apoptotic. More than a two-and-a-half-time increase in the number of new cells in SGZ in the rat was demonstrated after clozapine treatment, but it did not affect the total number of new cells after three weeks. For this reason, the authors concluded that clozapine does not have protective properties against newly formed nerve cells . An anti-apoptotic effect of clozapine was also observed . Another atypical antipsychotic olanzapine, supports adult neurogenesis mainly within the SVZ, although there are some reports that also suggest this effect occurs in the SGZ . It should be taken into account that the proliferative rates in the SVZ and in the SGZ are different. Moreover, an increased proliferation of progenitor cells in the rats’ prefrontal cortex was observed as a result of a 3-week treatment of this drug . The results of the studies conducted so far show that haloperidol does not stimulate neurogenesis in the hippocampus  while it is stimulated by olanzapine , and some atypical neuroleptics reverse the inhibition of neurogenesis caused by repeated restraint stress (quetiapine)  and phencyclidine (clozapine, risperidone) [22, 23]. Based on these observations, a hypothesis has been formulated that it is atypical APDs, and not typical ones, that stimulate neurogenesis in the hippocampus. This hypothesis, however, is not confirmed, for example, by the studies of Halim et al.  who showed that clozapine does not stimulate neurogenesis in the hippocampus. The neuroprotective properties of olanzapine have also been proven and it is believed that the higher level of secretion of brain-derived neurotrophic factor (BDNF) and neural growth factor (NGF) is induced after treatment with olanzapine . There is also a study reporting that adult neurogenesis in the hibernating hamsters (in the state of torpor) is more reduced in the SVZ than in the SGZ. Interestingly, this proliferative rate is normalized after 3–4 days of artificial hibernation. Of note, tau3R protein with microtubule-binding subunits turned out to be an appropriate marker for the analysis of canonical adult neurogenesis in this rodent species .
Data about another classic APD, haloperidol diverge, but most sources indicate that haloperidol does not increase the proliferation and vitality of newborn cells [3, 20, 21]. It has been suggested that this neuroleptic may exert pro-apoptotic effects on adult neurogenesis via inhibiting BDNF secretion . However, there are also reports suggesting proneurogenic properties of haloperidol [4, 15]. It is possible that differences in experimental data are caused by use of different markers and their limited selectivity/affinity for specific neurogenic cells or even alterations in microbiota at differing sites of study that can influence APD function .
The mechanism responsible for the neurogenesis promoting effects of APD varies greatly due to the broad spectrum of the drugs themselves. Starting with typical APD that primarily affect dopamine D2 receptors by blocking them. Research shows, although not entirely clearly, that stimulation of the D2 receptor, which is located on the surface of CNS stem cells, inhibits proliferation. Thus, blocking the receptors by some APD may be one of the mechanisms enhancing neurogenesis (studies that have shown a positive effect of haloperidol on neurogenesis usually refer to this D2 receptor antagonistic mechanism of drug action) [6, 15, 16]. Stimulation of the D5 receptor, e.g. by ziprasidone—a second-generation antipsychotic agent, causes phosphorylation of Akt, which reduces the activity of the glycogen synthase kinase 3β (GSK-3β) enzymatic cascade. Inhibition of GSK-3β activates β-catenins and p53 and finally triggers cyclin D1 action, which in turn induces neurogenesis. Despite the research conducted so far, it is still unknown what element of the antipsychotic drug mechanism is responsible for stimulating neurogenesis . Many reports show that the discussion dealing with the stimulatory effects of APD on adult neurogenesis requires detailed further study.