L-type 칼슘 채널을 저해하는 저해제, nifedipine에 의한 쥐 뇌실하 영역 신경줄기세포의 신경세포로의 분화 촉진

Increase in Neurogenesis of Neural Stem Cells Cultured from Postnatal Mouse Subventricular Zone by Nifedipine

Abstract

뇌실하 영역은 뇌에서 신경줄기세포가 분포하는 곳으로 평생에 걸쳐 새로운 신경세포를 생성하는 곳이다. 많은 세포 안팎의 인자들이 신경줄기세포의 세포 증식과 신경세포로의 분화에 영향을 미친다. 최근 들어, L-type 칼슘 채널이 신경계의 발달을 조절하고 뇌실하 영역에 있는 신경줄기세포, 신경세포로 분화 중인 세포, 그리고 성숙한 신경세포에 분포한다고 밝혀졌다. L-type 칼슘 채널의 저해제인 nifedipine은 고혈압의 치료제로 오랜 기간 사용되어 왔다. 신경줄기세포에 nifedipine을 사용하여 L-type 칼슘 채널을 저해하는 연구는 많이 없는 상황이다. 이번 연구에서, 우리는 5일령 쥐의 뇌실하 영역에서 배양한 신경줄기세포에 nifedipine을 처리하여 신경세포로의 분화에 미치는 영향을 관찰하였다. Nifedipine은 Tuj1을 발현하는 신경세포의 수를 증가시킨 반면, Olig2를 발현하는 희소 돌기 아교 세포(oligodendrocytes)의 수에는 큰 영향을 미치지 않았다. Nifedipine은 S기를 표지하는 5-ethynyl-2'-deoxyuridine (EdU)가 들어간 세포의 수를 증가시켰고, 세포 분열시 나타나는 인산화된 히스톤 H3(PH3)를 발현하는 세포의 수를 증가시켰다. Nifedipine은 신경세포로의 분화를 촉진하는 Dlx2 유전자의 전사를 증가시켰고, 초기 신경세포에서 보이는 Mash1의 양도 증가시켰다. Nifedipine 외 또다른 L-type 칼슘 채널의 저해제인 verapamil을 처리하자, 신경세포로의 분화가 소폭 증가하였으나, 통계적 유의미성은 매우 낮았다. T-type 칼슘 채널의 저해제 유전자인 Cav3.1, Cav3.2, Cav3.3가 발현함을 관찰하여, T-type 칼슘 채널의 저해제인 pimozide를 신경줄기세포에 처리하였으나, 신경세포로의 분화에는 변화가 없었다. 이러한 결과를 통해 nifedipine이 신경줄기세포의 초기 분화를 증진함을 알 수 있으며, L-type 칼슘 채널이 신경세포로의 분화에 관여함을 알 수 있다.

The subventricular zone (SVZ) in the brain contains neural stem cells (NSCs) that generate new neurons throughout one's lifetime. Many extracellular and intracellular factors that affect cell proliferation and neuronal differentiation of NSCs are already well-known. Recently, L-type calcium channels have been reported to regulate neural development and are present in NSCs, differentiating neuroblasts, and mature neurons in the SVZ. Nifedipine, a blocker of L-type calcium channels, has been long used as a therapeutic drug for hypertension. However, studies on the use of nifedipine to inhibit L-type calcium channels of NSCs are lacking. Herein, we treated NSCs cultured from mouse postnatal SVZ with nifedipine during neuronal differentiation. Nifedipine increased the number of Tuj1-positive neurons but did not significantly change the number of Olig2-positive oligodendrocytes. Nifedipine increased cell division during early differentiation, which was detected using the 5-ethynyl-2'-deoxyuridine incorporation assay and immunocytochemistry assessment by staining the cells with phosphorylated histone H3, a mitosis marker. Nifedipine increased the transcription of Dlx2, a neurogenic transcription factor, and the level of Mash1, a marker for early neurogenesis. In addition to nifedipine, verapamil, which is also an L-type calcium channel blocker, showed a slight increase in neurogenesis, but its statistical significance was very low. In contrast, pimozide, a T-type calcium channel blocker, did not affect neurogenesis, although T-type calcium channel genes Cav3.1, Cav3.2, and Cav3.3 were expressed. In summary, nifedipine might promote the neuronal fate of NSCs during early differentiation and calcium signaling through L-type calcium channels might be involved in neuronal differentiation, especially during the early stages of differentiation.

Introduction

As a second messenger, calcium plays a key role in many cellular signaling pathways. Involvement of calcium in neural development has been extensively studied [24]. Various calcium channels regulate different aspects of neural development including cell proliferation, neuronal differentiation, and migration to the target site. Among those calcium channels, L-type calcium channels seem to be the main calcium influx pathway in proliferation and early neurogenesis of NSCs during the brain development [24]. L-type calcium channels play roles in neural induction and neuronal differentiation during neural development of Xenopus embryo [13], zebrafish spinal cord [2], and postnatal mouse brain cortex [3]. L-type calcium channels also participate in neuronal differentiation of adult NSCs. An agonist of an L-type calcium channel increased neurogenesis of adult hippocampal progenitor cells [4]. Adult mice with a deletion of L-type calcium channel gene Cav1.3 had reduced neurogenesis in the hippocampus and cognitive defects [14]. Agonists and antagonists of L-type calcium channels increased and decreased, respectively, the survival of immature neurons of a NSC cell line derived from adult rat hippocampus [23].

Besides hippocampus, SVZ is also a neurogenic region and harbors NSCs through adulthood [16]. Yet, the roles of calcium signaling and L-type calcium channels in SVZ NSCs have not been reported much. NSCs from rat embryo SVZ required a proper function of L-type calcium channels in the cyclic adenosine monophosphate (cAMP)-dependent neuronal differentiation [13]. Calcium dynamics regulate the fate of adult SVZ NSCs between proliferation and quiescence [6]. Expression of two isoforms of L-type calcium channels, Cav1.2 and Cav1.3, was detected in adult SVZ NSCs in vivo [14] as well as in vitro cultures [9]. Therefore, more research is needed to elucidate the function of L-type calcium channels in SVZ NSCs. Nifedipine, an L-type calcium channel blocker, has long been used as a medicine to treat hypertension [25]. Only recently, nifedipine has been used in the field of NSC biology.

Nifedipine decreased electromagnetic fields-induced neurogenesis of adult hippocampal NSCs [12] and postnatal brain cortex [22]. Furthermore, nifedipine decreased fetabl bovine serum-induced neurogenesis of cultured NSCs from postnatal mouse brain cortex [3]. Nifedipine decreased allopregnanolone-induced cerebellar granule cell neurogenesis [8]. In addition, nifedipine decreased cAMP-induced neuronal differentiation of SVZ NSCs from rat embryo [13]. Taken together, nifedipine seems to inhibit neuronal differentiation of NSCs in the various regions of the brain. However, it is not still clear how nifedipine might affect neuronal differentiation of SVZ NSCs.

In this report, we show that nifedipine increased neurogenesis of differentiating SVZ NSCs, nifedipine increased cell division during the early differentiation, nifedipine increased the transcription level of a neurogenic transcription factor, Dlx2, and nifedipine increased expression level of an early neuroblast marker, Mash1. This is the first that reports increased neurogenesis by nifedipine and the possible mechanism underlying nifedipine action.

Materials and Methods

Mouse SVZ NSC culture

SVZ tissues were obtained from 5-day old mouse pups (CD1 (ICR) from Orient Bio, Sungnam, Korea). All the procedures related to animals were done following national guidelines and the protocol approved by Inje University Animal Care and Use Committee (approval ID number: Inje 2020-006). The mouse pups were euthanized using carbon dioxide and brains were obtained. After slicing into approximately 0.5-cm thick coronal section, SVZ tissues were cut out and dissociated in trypsin (Gibco, ThermoFisher, Waltham, MA, USA) in the incubator at 37℃ with 5% carbon dioxide for 10 min. Then, cells were plated in a density that cells obtained from one pup are plated into a well in a 6-well plate. After one week of cell proliferation in N5 medium, the cells are called as p0 passage and split to a 1:2 or 1:3 ratio to new culture dish for maintenance. Cultured cells were grown in an incubator at 37℃ with 5% CO2. If not mentioned, all cells used for experiments were p5 passage.

The N5 medium is DMEM/F12-GlutaMAXTM supplemented with 5% fetal bovine serum, 10% N2 supplement, 35 μg/ml bovine pituitary extract, 20 ng/ml epidermal growth factor (EGF), 20 ng/ml basic fibroblast growth factor (bFGF), and 10% antibiotic/antimycotic. All components were from Gibco (ThermoFisher, Waltham, MA, USA) except for fetal bovine serum from GenDEPOT (Texas, USA).

Neuronal differentiation

To differentiate NSCs, cells were plated onto 8-well CC2 chamberslide (Nunc, ThermoFisher, Waltham, MA, USA) coated in an incubator at 37℃ with 5% CO2 overnight with 5 μg/ml laminin (Invitrogen, ThermoFisher, Waltham, MA, USA). Before the plating, the chamberslide was rinsed with phosphate-buffered saline (PBS) (GenDEPOT, Texas, USA). After 1 day of plating, N5 medium was removed and N6 medium was added for rinsing. Then, fresh N6 medium was added to the cells. N6 medium is differentiation medium of which components are the same as N5 medium except for lack of serum and growth factors (EGF and bFGF). Nifedipine, verapamil, or pimozide were dissolved in dimethyl sulfoxide (DMSO) to make a stock solution and diluted in N6 medium to obtain working concentarations. All four chemicals including DMSO were purchased from Sigma (St. Louis, MO, USA). Every 4 days of differentiation, medium was changed with fresh N6 medium containing DMSO or freshly diluted nifedipine.

Immunocytochemistry

The protocol is the same as previously reported [18]. Before fixation, cells were briefly rinsed with PBS to remove any floating and dead cells. Cells were fixed in 4% paraformaldehyde (Sigma, St. Louis, MO, USA) for 30 min and then, incubated in a blocking solution, PBS containing 10% normal goat serum (Cell Signaling, Danvers, MA, USA) and 0.1~0.3% triton X-100 (Sigma, St. Louis, MO, USA) for 30 min. Cells were incubated with primary antibodies diluted in blocking solution as follows for 2 hr at room temperature: mouse anti-Tuj1 (BioLegend, San Diego, CA, USA) at 1:500, rabbit anti-Olig2 (Millipore, Billerica, MA, USA) at 1:500, rabbit anti-histone H3 phosphorylated at Ser10 (PH3) (Millipore, Billerica, MA, USA) at 1:100, rabbit anti-Mash1 (Abcam, Cambridge, UK) at 1:50. After rinsing with PBS, cells were incubated with secondary antibodies (1:500 dilution) and 4’,6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA) (1:1,000 dilution) in PBS for 1 hr. Secondary antibodies were Alexa-488-conjugated anti-mouse, Alexa-488-conjugated anti-rabbit and Alexa-594-conjugated anti-rabbit, which were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Finally, cells were mounted using Aqua-Poly/Mount (Polysciences, Warrington, PA, USA).

EdU incorporation and detection assay

The procedure was done following manufacturer’s protocol by using Click-iT Plus EdU Imaging Kit (Invitrogen, ThermoFisher, Waltham, MA, USA). For the 1-hr labeling, cells were incubated in medium containing 5 μM EdU. Then, cells were fixed in 4% paraform aldehyde (Sigma, St. Louis, MO, USA) for 15 min. After rinsing with 3% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA) in PBS twice, the blocking solution, PBS containing 0.5% triton X-100 (Sigma, St. Louis, MO, USA) was added as permeabilization reagent for 20 min. After rinsing with 3% BSA twice, cells were incubated in EdU detection cocktail, a mixture containing Alexa Fluor® 594 picolyl azide, for 30 min in dark. After rinsing with 3% BSA, cells were incubated in DAPI solution (1:1,000 diluted in PBS) to stain nuclei for 1 hr in dark and then, mounted using Aqua-Poly/Mount (Polysciences, Warrington, PA, USA).

Fluorescence imaging and image analysis

After immunocytochemistry or EdU detection, images were taken using a fluorescence microscope (Olympus, Tokyo, Japan). For each condition, 3 wells of cells were used and 5 fields of view in each well were randomly imaged for analysis for the most of experiments. Six fields of view were imaged per well for Olig2 counting and seven fields per well were imaged for PH3 counting as well as for Tuj1 counting in p7, p8, or p9 passaged cultures. For quantitative analysis, the counting of DAPI, Mash1-positive cells, PH3- positive cells, EdU-positive cells, and Olig2-positive cells was performed using a cell count macro in iSolution software (IMT i-Solution Inc., Vancouver, BC, Canada). The number of Tuj1-positive cells was counted manually.

Reverse transcription and quantitative real-time PCR

The procedure is similar as previously reported [17] and total RNA extraction was performed as a manufacturer’s instruction using QIAshredder column and RNeasy mini kit (both from Qiagen, Hilden, Germany). SVZ NSCs plated on 8-well chamberslides were lysed and RNA was extracted. To remove genomic DNA, RNase-free DNase (Qiagen, Hilden, Germany) was added to the RNeasy column for 15 min. Extracted RNA was subject to reverse transcription using SuperScript III (Invitrogen, ThermoFisher, Waltham, MA, USA) or GoScript reverse transcriptase (Promega, Madison, WI, USA) with oligo (dT) primers following each manufacturer’s protocol. The cDNA equivalent to 50 ng of RNA was used for each quantitative real-time PCR (qPCR) reaction, which consists of reaction mixture from Real Helix qPCR kit (Nanohelix, Daejeon, Korea) and primers. Primer sequences are as followings: Dlx2 (forward: 5’-GGCCTCACC CAAACTCAG-3’, reverse: 5’-AGGCACAAGGAGGAGAA GC-3’), Tuj1 (forward: 5’-TGAGGCCTCCTCTCACAAGT-3’, reverse: 5’-GGCCTGAATAGGTGTCCAAA-3’), and GAPDH (forward: 5’-CAAGGCTGTGGGCAAGGT-3’, reverse: 5’-TC ACCACCTTCTTGATGTCATCA-3’). The qPCR was done in AriaMx real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) with a following program: 95℃, 12 min; then 40 cycles of 95℃, 10 s; 58℃, 10 s; 72℃, 30 s. Melting curves were obtained to verify a single amplicon for each primer set with a following program: 95℃, 30 s; 65℃, 30 s; 95℃, 30 s.

Reverse transcription and PCR

RNA (200 ng) was added to a tube of DiaStarTM 2X OneStep RT-PCR Pre-Mix (Solgent, Daejeon, Korea) with primers. Primer sequences for Cav3.1, Cav3.2, and Cav3.3 are from the previous report [15] and primer for GAPDH is the same as mentioned above. The amplicon sizes for Cav3.1, Cav3.2, and Cav3.3 are 437 bp, 351 bp, and 346 bp, respectively. Cav3.1 The PCR was done in MJ MiniTM Gradient Thermocycler (Bio-Rad Laboratories, CA, USA) with the following program: 50℃, 30 min; 95℃, 15 min; then 40 cycles of 95℃, 20 s; 58℃, 40 s; 72℃, 1 min and finally 72℃, 5 min. PCR reaction was subject to gel electrophoresis with a 2% agarose gel.

Statistical analysis

Statistical significance was analyzed using Student’s T-test (two-tailed distribution and two-sample equal variance). When p value was below 0.05 or 0.01, the difference was considered as statistically significant.

Results

Increase of neurogenesis by nifedipine

NSCs cultured from mouse subventricular zone were differentiated in N6 medium containing DMSO as a vehicle control or 50 µM nifedipine for 9 days. Cells were subject to immunocytochemistry to detect Tuj1-positive neurons and Olig2-positive oligodendrocytes. Treatment of NSCs with 50 μM nifedipine significantly increased the number of Tuj1-positive neurons compared to the vehicle control by 2.6 fold (Fig. 1A). The number of Olig2-positive oligodendrocytes was slightly less in nifedipine-treated cells than in the control cells, but the difference was not statistically significant (Fig. 1B). The average number of cells counted with DAPI staining was significantly higher in nifedipine-treated cells than in the control by 1.2 fold, which might be caused by increased cell proliferation during neuronal differentiation (Fig. 1C). Then, NSCs with different passage numbers were subject to the similar process. In the control group, 1.6% cells of passage 7 were Tuj1 positive, but only 0.1% cells of passage 8 or 9 were Tuj1 positive (Fig. 1D). In nifedipine treated group, the neurogenesis was 4.6% in the passage 7 cells, 1.2% in the passage 8 cells, and 0.7% in the passage 9 cells. In all three kinds of passaged NSCs, nifedipine significantly increased the neurogenesis. Nifedipine seems to maintain neurogenic capability of NSCs during cell passaging.

Fig. 1. Increased neurogenesis by an L-type calcium channel blocker nifedipine. A and B. Cultured SVZ NSCs were differentiated for 9 days in N6 medium containing DMSO (control) or 50 μM nifedipine. Immunocytochemistry was performed to detect Tuj1 (A), Olig2 (B), and nuclei using DAPI (both A and B). Scale bar is 100 μm in A and 40 μm in B. The number of Tuj1-positive cell (A) or Olig2-positive cells (B) were counted and divided with the total number of cells (DAPI). Data are mean and standard error from three independent sets of experiments. Student’s T-test was used for statistical analysis (** p<0.01). C. The average numbers of total cells (DAPI) counted per well from the experiments in A and B are plotted. Data are mean and standard error and Student’s T-test was used for statistical analysis (*p<0.05). D. SVZ NSCs with different passage numbers (p7, p8, and p9) were differentiated in the presence of DMSO (control) or 50 μM nifedipine. Images were taken and the numbers of Tuj1-positive neurons were counted. The data are mean and standard deviation from three samples for each condition. Compared to the control, statistical significance was analyzed using Student’s T-test (*p<0.05).

Increased cell proliferation during neurogenic differentiation by nifedipine

In the Fig. 1C, the total number of cells was higher in nifedipine-treated cells than in control cells. This suggests that the increased number of cells might have been caused by higher cell division in the presence of nifedipine. To examine this possibility, EdU, a thymidine analogue, was applied to NSCs during neuronal differentiation. When a cell goes through DNA replication, EdU is incorporated into the nucleus. EdU was applied to cells that are incubated in differentiation medium containing DMSO or 50 μM nifedipine for 1, 2, or 3 days (Fig. 2A). In the vehicle control groups, EdU incorporation ratio increased to approximately 2 fold in 2 days of differentiation, compared to 1-day differentiation (Fig. 2B). When cells were differentiated for 3 days, EdU incorporation drops to 36% of 1-day differentiation group. This indicates that cells are dividing more during the early neurogenesis such as 1 or 2 days of differentiation and the cell division slows down at around 3 days of differentiation. In the case of nifedipine-treated groups, EdU incorporation was significantly higher than the control groups during 1 or 2 days of differentiation (Fig. 2B). At 2 days of differentiation, EdU incorporation ratio was almost doubled in nifedipine-treated cells, compared to the control cells. At 3 days of differentiation, EdU incorporation was higher in nifedipine-treated cells than in the control with no statistical significance.

Fig. 2. Increased EdU incorporation by nifedipine. A. SVZ NSCs were differentiated in N6 medium containing DMSO for vehicle control (Cont) or 50 μM nifedpine (Nif) for 1 (1d),2 (2d), or 3 days (3d). EdU was applied to the N6 medium for 1 hr and cells were fixed for EdU detection. Images were taken for EdU and nuclei (DAPI) (Scale bar=100 μm). B. The number of EdU-positive cells and the total number of cells (DAPI) were counted for each condition. The data are mean and standard error from three independent experiments with Student’s T-test between the control and nifedipine groups at each time point (*p<0.05).

As another method to investigate cell proliferation, immunocytochemistry was performed to detect phosphohistone H3 (PH3), a proliferation marker, since it appears in the nucleus when chromatin is condensed during mitosis. At the beginning of differentiation (0 day differentiation), 5.7% of total cells were positive for PH3 (Fig. 3). After 1 day of differentiation, PH3 positivity decreased to 0.8% in the control and 1.1% in the nifedipine treated cells. After 2 days of differentiation, it was 1.1% in the control and 1.7% in the nifedipine treated cells. Even though the fold difference was small, nifedipine significantly increased the number of PH3-positive proliferating cells at 2 days of differentiation (Fig. 3B). This is consistent with the result of EdU, which showed more cells going through S phase of the cell cycle in the presence of nifedipine. Together, nifedipine seems to cause more cell division during the early neuronal differentiation.

Fig. 3. Increased mitosis by nifedipine. A. Immunocytochemistry was done with antibody against phosphorylated histone H3 (PH3) to detect cells undergoing mitosis. Cultured SVZ NSCs were fixed without differentiation (0d) or differentiated for 1 (1d) or 2 days (2d) followed by fixation. During differentiation, N6 medium containing DMSO (cont) or 50 μM nifedipine (Nif) was used (Scale bar=100 μm). Arrows indicate PH3-positive cells. B. The number of PH3-positive cells and total number of cells (DAPI) were counted and plotted. Data are mean and standard deviation from three wells with statistical analysis using Student’s T-test between control and nifedipine groups (*p<0.05).

Increase of early neurogenic markers by nifedipine

To further investigate the neurogenic effect of nifedipine, we examined the transcription level of Dlx2, a neurogenic transcription factor, using quantitative real-time PCR after reverse transcription. In the vehicle control groups, the level of Dlx2 transcript increased to 4.4, 20.9, and 2.4 fold at 1, 2, and 5 days of differentiation, respectively, compared to the cells without differentiation (0 day differentiation) (Fig. 4A). This trend is consistent with the previous observation [19]. Cells treated with 50 µM nifedipine showed a similar pattern of a transient increase in Dlx2 transcript. Yet, the level of Dlx2 transcript of nifedipine-treated cells was almost 2-fold more than the control at 1 day differentiation. Along with Dlx2, the transcription level of Tuj1, a neuronal specific beta-tubulin, was also measured. In both control and nifedipine groups, Tuj1 transcript was greatly increased at 2 days of differentiation by 23 fold, compared to 0 day differentiation (Fig. 4B). At 5 days of differentiation, the average quantity of Tuj1 transcript was much higher in nifedipine treated cells than in the control, but its difference was not statistically significant.

Fig. 4. Transcriptional level of Dlx2 and Tuj1 during differentiation in the presence of nifedipine. A and B. SVZ NSCs were differentiation for 1 (1d), 2 (2d), or 5 days (5d) in N6 medium containing DMSO (cont) or 50 μM nifedipine (Nif). Then, RNA obtained from each condition was subject to reverse transcription followed by quantitative real-time PCR. RNA from proliferating SVZ NSCs without differentiation (0d) was also obtained. Dlx2 (A) or Tuj1 (B) expression level was normalized with GAPDH transcript level. For both control and nifedipine conditions, the relative quantity of Dlx2 (A) or Tuj1 (B) at 0d was set as 1. Data are mean and standard error from three independent experiments. At each time point, Student’s T-test was performed between the control and the nifedipine groups (*p<0.05).

In addition, the expression of Mash1, a transcription factor required for neuronal commitment, was observed using immunocytochemistry after applying nifedipine during neuronal differentiation. After 1 day of differentiation, both control cells and nifedipine-treated cells had Mash1-positive cells in apporoximately 37% of the total number of cells (Fig. 5). At 2 days of differentiation, the ratio of Mash1-positive cells among total number of cells decreased to 16% in the control cells, but it was 25% in the nifedipin-treated cells (Fig. 5B). The difference between the control and nifedipine-treated cells statistically significant (p<0.01). All these results of Dlx2 transcript and Mash1 protein expression support that nifedipine might cause more NSCs to take the neuronal fate.

Fig. 5. Increased expression of Mash1 by nifedipine. A. Cultured SVZ NSCs were fixed before differentiation (0d) or after 1 (1d) or 2 days (2d) of differentiation. The differentiation medium contained either DMSO (Cont) or 50 μM nifedipine (Nif). After fixation, immunocytochemistry was done to detect Mash1 and nuclei (DAPI) (Scale bar=50 μm). B. Mash1-positive cells and the total number of cells (DAPI) were counted. Mean and standard deviation from three wells of cells are plotted. Student’s T-test was performed between the control and nifedipine groups at each time point (**p<0.01).

Calcium channel gene expression during differentiation

According to the recent report, Cav1.2 was not detectable in SVZ NSCs, but detectable in NeuN-positive mature neurons as shown in immunohistochemistry [14]. With the transgenic mouse reporter system, Cav1.3 seemed to be expressed in SVZ NSCs as well as mature neurons [14]. To examine whether or not these genes encoding the L-type calcium channel are expressed in our culture system, reverse transcription followed by real-time quantitative PCR was performed. Even though the transcriptional level of Cav1.2 was very low with approximately 38 Ct value before differentiation (0d) (Fig. 6A), transcript was detectable. At 2 days of differentiation, the quantity of Cav1.2 transcript increased to 10 fold, compared to the 0 day differentiation. At 7 days, the level was approximately 200 fold more. Similar pattern was observed when Cav1.3 transcript was measured (Fig. 6B). A relative quantity of Cav1.3 increased approximately 15 fold at 2 days of differentiation and 40 fold at 7 days, compared to 0 day differentiation. These results suggest that both Cav1.2 and Cav1.3 are expressed in both proliferating cultured NSCs from SVZ and differentiated cells with much greater expression.

Fig. 6. Expression of calcium channel genes. A and B. RNA was extracted from cultured SVZ NSCs at right before differentiation (0d) and after 2 days (2d) or 7 days (7d) of differentiation. After reverse transcription, real time quantitative PCR (qPCR) was done to measure Cav1.2 transcript (A) and Cav1.3 transcript (B). Data are mean and standard error from three independent sets of experiments. C. The same RNA from A and B was subject to reverse transcription followed by PCR with primer sets of GAPDH, Cav3.1, Cav3.2, and Cav3.3.

Besides L-type calcium channel genes, T-type calcium channel genes were examined using reverse transcription and PCR. PCR reaction was loaded to agarose gel for electrophoresis. All three Cav3.1, Cav3.2, and Cav3.3 genes were expressed independently of duration of differentiation, even though the expression level of Cav3.3 from the cells differentiated for 2 days was slightly less than the other conditions (0d or 7d differentiation) (Fig. 6C). Therefore, genes for Ttype calcium channels are expressed in cultured SVZ NSCs at least at the transcription level.

Effects of other calcium channel inhibitors on differentiation

Besides nifedipine, another inhibitor of L-type calcium channel, verapamil, was added to differentiating NSC cultures. In our preliminary experiments, 50 μM verapamil was toxic to the cells and many cells died during differentiated. Therefore, we added 0.1 μM or 2 μM verapamil to the cells. Along with verapamil, a T-type calcium channel blocker, pimizode, was also used. Since SVZ NSCs seem to express genes encoding T-type calcium channels (Fig. 6C), the effect of T-type calcium channel blocker on the differentiating NSCs was tested. Among many blockers, we chose pimozide since it has a very low IC50 value for T-type calcium channel (36 nM~54 nM) and does not block L-type calcium channels [10]. Both verapamil and pimozide do not seem to greatly affect neurogenesis (Fig. 7A). Yet, cells treated with 2 μM verapamil showed a tendency of slight increase in neurogenesis (The p value was 0.054 in T-test.). In the case of oligodendrogenesis, the number of Olig2-positive cells was not affected by verapamil, but significantly increased by pimozide (Fig. 7B). There results show that verapamil has a less effect on neurogenesis than nifedipine and T-type calcium channels might not be involved in neurogenesis, but in oligodendrogenesis.

Fig. 7. Effects of verapamil a nd pimozide on neurogenesis and oligodendrogenesis of cultured SVZ NSCs. A and B. Cells were differentiated for 10 days in N6 medium containing 0.1 μM or 2 μM verapamil (Ver) or 50 nM pimozide (Pim). All controls (Cont) contained DMSO at the same concentration as each chemical treatment. Data are mean and standard deviation from three different wells of cells. Student’s T-test was performed between the control and each chemical (*p<0.05).

Discussion

Previous reports have shown that nifedipine decreases neurogenesis of different types of cells in the nervous system. Nifedipine inhibited neurogenesis of NSCs in the cortex [3] and cerebellar granule cells [8]. Neurogenesis of NSCs from embryonic SVZ was also decreased by nifedipine [13], of which neurogenesis was cAMP-mediated. Notably, differentiation of our cultured NSCs was induced by removing growth factors (EGF and bFGF) and fetal bovine serum. Just switching medium from N5 to N6 medium induced neurogenesis of our cultured SVZ NSCs without adding any other chemicals as inducers of neurogenesis. We suspect that blocking L-type calcium channel activity by nifedipine rather increases neurogenesis in this more spontaneous neuronal differentiation. Interestingly, NSCs with a high passage number, with a low rate of cell proliferation and neurogenesis, maintained the neurogenic capability in the presence of nifedipine (Fig. 1D). This hints that nifedipine might increase neurogenesis of aged SVZ, because both proliferation and neurogenesis are decreased in the aged SVZ [1].

To investigate the mechanism underlying enhanced neurogenesis of SVZ NSCs by nifedipine, we observed cell division during differentiation as well as early neurogenic markers. Nifedipine increased (1) the number of cells as counted using DAPI after 9 days of differentiation (Fig. 1C), (2) EdU incorporation that indicates S phase progression (Fig. 2), and (3) PH3 that marks mitotic phase of the cells (Fig. 3). Furthermore, those increases were dependent on the timing of detection. While increased EdU incorporation was observed both after 1 and 2 days of differentiation, PH3 detection increased after 2 days of differentiation in nifedipine, but not at 1 day. This might be due to S phase occurs earlier than mitotic phase in the cell cycle. These also suggest that nifedipine might act on early neurogenesis more than late one, which is consistent with the previous report that proliferation is required for early neurogenesis [20].

To further examine the mechanism of nifedipine action on neurogenesis, we measured transcriptional level of Dlx2, a transcription factor that induces neuronal fate and represses oligodendrocyte fate [21]. Nifedipine greatly increased Dlx2 level at early differentiation, which is consistent with the trend of the higher transcription of Tuj1 at later time point (5 days of differentiation) and also with more protein level of Tuj1 at 9 days of differentiation. Interestingly, the number of Olig2-positive cells was not increased and rather had a trend to be decreased by nifedipine, which suggests that much expression of Dlx2 might have repressed the oligodendrogenesis. Another early neurogenic marker, Mash1 was higher in nifedipine-treated NSCs during 2 days of differentiation. In the images of EdU detection, some cells seemed to gather into a group (Fig. 2A) at 3 days of differentiation in the presence of nifedipine, but not in the control cells. Formation of this type of cell clusters indicates early neurogenesis [17]. All these results suggest that nifedipine increased early neurogenesis of SVZ NSCs.

In this study, we observed that nifedipine enhanced neurogenesis of cultured NSCs from mouse postnatal SVZ. Also, we think that increased cell division followed by neuronal fate decision during early neurogenesis might have caused the enhancement. To our knowledge, this is the first report showing increased neurogenesis of SVZ NSCs by nifedipine and its possible mechanisms. Still, more detailed information how L-type calcium channel plays a role in SVZ NSC proliferation and early neurogenesis is needed.