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NERVOUS SYSTEM CELL BIOLOGY
1Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, and Department of Medicine (Cardiology), New Jersey Medical School-University of Medicine and Dentistry of New Jersey, Newark, New Jersey; and 2Cardiovascular Research Institute and 3Department of Medical Science and Cardiorenal Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan
Submitted 5 March 2007 ; accepted in final form 20 August 2007
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ABSTRACT |
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neuronal culture; phospholipase C; striatum; adenosine 3',5'-cyclic monophosphate
Such inconsistent findings may be due, at least in part, to the developmental changes in dopamine signaling after birth. Previous reports demonstrated that expression of D1- and D2-like receptors in the striatum was relatively low at birth and progressively increased to adult levels at 
2–3 wk of age (9, 36, 43). In addition, the number of striatal cells was decreased during the postnatal period (7), suggesting that the developmental changes in dopamine signaling play a role in regulating the number of striatal cells, or apoptosis, after birth. To examine this hypothesis, primary culture of striatal neurons from postnatal (2- to 3-wk-old) rodents is useful. However, such primary culture has been difficult, because, in contrast to neurons from the embryo or neonate, those from young adults are easily damaged and their viability is poor after culture. Therefore, we developed a new method for culture of striatal neurons from young (2- to 3-wk-old) mice. Then we examined the difference in dopamine signaling between neonatal and young striatal neurons.
We demonstrate a difference in the ability of dopamine to induce apoptosis between neonatal and young striatal neurons. Dopamine stimulation increased apoptosis in young, but not neonatal, neurons. cAMP signaling did not play a major role in induction of apoptosis, because isoproterenol (Iso), a β-adrenergic receptor agonist, stimulated even higher cAMP accumulation than dopamine, but Iso did not induce neuronal apoptosis. Furthermore, dopamine-induced apoptosis was inhibited by Ca2+ depletion, and PLC expression was higher in young than in neonatal striatal neurons, suggesting that dopamine induced apoptosis most likely via PLC-Ca2+ signaling in the striatum. Indeed, dopamine increased intracellular Ca2+ in young, but not neonatal, striatal neurons, suggesting developmental changes in dopamine-mediated Ca2+ kinetics. Our results showed a potential mechanism for inducing apoptosis in striatal neurons that changes during the postnatal period, as well as the feasibility of culturing striatal neurons from young adults.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Neuronal primary cultures.
Primary striatal culture from neonatal C57BL/6J mice was performed as previously described with some modifications (24). Briefly, the striatum was dissected with forceps and minced with a razor blade. The minced tissues were incubated with 0.3% trypsin (catalog no. 15090-046, GIBCO) with moderate shaking for 15 min at 37°C. Then the cells were triturated 8–10 times with a 0.5-ml-caliber Pasteur pipette and 8–10 times with a 0.2-ml-caliber Pasteur pipette. The cell suspension was centrifuged at 1,500 rpm for 5 min, and the supernatant was removed. The resultant pellet was dissolved with 300 µl of NBM (1x GlutaMax-I, B-27 supplement, 100 µg/ml penicillin, and 100 µg/ml streptomycin). The number of viable cells was counted using trypan blue staining. Cells (4 x 104) were seeded onto an 18-mm cover glass (Matsunami, Kishiwada, Japan) that had been precoated with 6 mg/ml poly-L-lysine (catalog no. P-5899, Sigma) in 0.15 M sodium borate (pH 8.5) for 1 h. Another group of cells (2.5 x 104) were seeded onto a 96-well plate precoated with 6 mg/ml poly-L-lysine in 0.15 M sodium borate (pH 8.5) for 1 h. The cells were incubated at 37°C in 95% air-5% CO2 at 95% humidity. After 4–14 days, the cells were used for assays.
For primary striatal culture from 2- to 3-wk-old mice, we extensively modified the above-described procedures for neonates (see RESULTS). Briefly, the striatum was dissected with forceps and minced with a razor blade. The minced tissues were incubated with 0.3% collagenase type I (catalog no. C-0130, Sigma) in HBSS for 15 min at 37°C. The cell suspension was centrifuged at 1,500 rpm for 3 min, and the supernatant was removed. The resultant pellet was triturated one to three times with 1 ml of HBSS and 1% DNase (10 µl/mouse) (catalog no. D-5025, Sigma) with a 0.5-ml-caliber Pasteur pipette. Then the cell suspension was incubated with 0.3% trypsin with moderate shaking for 15 min at 37°C, triturated 8–10 times with 0.1% DNase (50 µl/mouse) with a 0.5-ml-caliber Pasteur pipette, and divided into two tubes. The cell suspension was triturated 8–10 times with a 0.2-ml-caliber Pasteur pipette to dissociate cells. Tissue debris was removed by centrifugation with a cushion of 17% Percoll in HBSS at 1,500 rpm for 10 min. The supernatant was removed, and the resultant pellet was suspended with 300 µl of NBM and transferred to a new tube. The cell suspension was centrifuged at 1,500 rpm for 5 min, and the supernatant was removed. The resultant pellet was dissolved with 300 µl of NBM, and the number of viable cells was counted using trypan blue staining. Seeding and incubation procedures were similar to those described for the neonate. After 4–14 days, cells were used for each assay. This study was approved by the Animal Care and Use Committee at Yokohama City University School of Medicine and New Jersey Medical School.
Western blot analysis. Western blot analysis of cultured striatal neurons was performed as previously described by us with some modifications (13). Briefly, cultured striatal neurons in a 96-well plate were lysed and collected with 40 µl of lysis buffer [25 mM Tris·HCl (pH 8.0), 10 mM EGTA, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 10 mM Na3VO4, 20 µg/ml Na-tosyl-L-lysine chloromethylketone, 10 µg/ml leupeptin, 1 mM PMSF, 50 U of 5,8,11-eicosatriynoic acid, 2 µg/ml aprotinin, and 1% Nonidet P-40]. Cell lysates were subjected to SDS-PAGE and Western blotting and then to densitometric analysis.
TdT-mediated biotin nick end-labeling assay.
In situ labeling of fragmented DNA in cultured striatal neurons was performed with the TACS2 TdT-Blue Label In Situ Apoptosis Detection kit according to the manufacturer's instructions as previously described by us (13). Briefly, striatal neurons cultured on a poly-L-lysine-coated cover glass were fixed with 3.7% formaldehyde and then incubated in proteinase K (5 µg/ml) at 37°C for 15 min. The cells were incubated with 3% hydrogen peroxide in methanol for 5 min, washed with labeling buffer consisting of 50 mM Tris·HCl (pH 7.5), 5 mM MnCl2, 60 µM 2-mercaptoethanesulfonic acid, and 0.05 mg/ml BSA, and then incubated for 60 min at 37°C in labeling buffer containing 150 µM dATP, 150 µM dGTP, 150 µM dTTP, 5 µM biotinylated dCTP, and 40 µM TdT. The cells were incubated with streptavidin-horseradish peroxidase, and the fragmented DNA was stained blue with TACS2 TdT-Blue Label. All nuclei were stained red with nuclear fast red solution. Nuclear staining was viewed under a light microscope. The percentage of TdT-mediated dUTP nick end label-positive striatal neurons (relative to total cells) was determined in a blinded manner by counting 1,000–3,000 cells in randomly chosen fields per cover glass. Usually, blue-stained apoptotic cells under basal condition were <15% of total cells.
Analysis of DNA fragmentation by ELISA. Histone-associated DNA fragments were quantified by the cell death detection ELISA kit according to the manufacturer's instructions with minor modifications (13). Briefly, striatal neurons in 96-well plates were gently washed with warm PBS, incubated with lysis buffer [PBS containing 10 mM EDTA (pH 7.2) and 0.1% Triton X-100] at 37°C for 1 h, and then shaken vigorously for 30 s. The cell lysates, which contained cytoplasmic histone-associated DNA fragments, were applied to a streptavidin-coated microtiter plate. Subsequently, a mixture of biotin-labeled anti-histone antibody and peroxidase-conjugated anti-DNA antibody was added, and the plates were incubated for 2 h with moderate shaking. We washed the plates to remove unbound antibodies and then quantified nucleosomes by the peroxidase retained in the immune complex. The activity of peroxidase was determined photometrically with 2,2-azino-di-[3-ethylbenzthiazoline sulfonate] as substrate. The values from triplicate absorbance (at 405 nm) measurements were averaged.
Immunostaining of cultured striatal neurons. Immunostaining of striatal neurons was performed as previously described with some modifications (22). At 4 days after plating, neonatal and young striatal cells were washed and fixed in 4% paraformaldehyde in PBS, washed three times with PBS containing 1% bovine serum, incubated with primary antibody with mild shaking overnight, and then washed and incubated with secondary antibody for 1 h with mild shaking. Then the cells were mounted in 4',6-diamidino-2-phenylindole for visualization of all nuclei. To obtain the image of immunofluorescence, the slides were placed on an inverted microscope (model TE2000-E, Nikon).
cAMP measurement. Intracellular cAMP content was measured with the cAMP enzyme immunoassay kit (GE Healthcare) using the nonacylation protocol (40). Briefly, 7 days after plating was completed, striatal neurons from neonatal or young mice in 96-well plates were washed twice with NBM. Then the cells were stimulated by reagents in the presence of 0.5 mM IBMX, an inhibitor of phosphodiesterase, at 37°C for 5 min. Incubation was terminated by addition of lysis buffer followed by 20 min of incubation at room temperature. Then the cell lysate was transferred to the donkey anti-rabbit immunoglobulin-coated plate. Rabbit anti-cAMP antibody was added, and the plate was incubated for 2 h at 4°C. Subsequently, the cell lysate was incubated with cAMP-peroxidase conjugate at 4°C for 1 h. After incubation with enzyme substrate at room temperature for 1 h, the values from absorbance (at 450 nm) were measured, and cAMP contents were determined.
RT-PCR. RT-PCR was performed as previously described (49). Total RNA was isolated from cultured striatal neurons using TRIzol reagent as recommended by the manufacturer. RT was conducted with 1 µg of total RNA in a final volume of 20 µl at 42°C for 50 min. Aliquots (0.08 µl) of RT product were applied to each PCR and 35 cycles of amplification. The 5' and 3' primers specific for endogenous adenylyl cyclase (AC) type 5 (AC5) were 5'-TTCCACCTGAACCAGAGCAGTCTC-3' and 5'-CTTCTTGTGCCAGGTGGTCTAC-3'. The PCR cycle consisted of predenaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 30 s. Amplification products were analyzed by agarose gel electrophoresis and ethidium bromide staining. A fragment of GAPDH was amplified as internal control.
Measurement of intracellular Ca2+ concentration. Intracellular Ca2+ concentrations of individual cells were examined as previously described with some modifications (8, 21). Striatal neurons on a glass-based culture dish (Asahi Techno, Tokyo, Japan) were incubated with the fluorescent Ca2+ indicator dye fura 2-AM (2 µM; Dojin, Kumamoto, Japan) in NBM at 37°C for 45 min. After they were washed with a basal salt solution [130 mM NaCl, 2.5 mM KCl, 2.0 mM CaCl2, 1.2 mM MgCl2, 10 mM HEPES-KOH, and 10 mM D-glucose (pH 7.3)], the cells were sequentially illuminated with a xenon lamp (model UXL 75XB) at 5-s intervals, first at 340 nm and then at 380 nm. Fluorescence emission at 510 nm was monitored for each excitation wavelength, and images were obtained with a charge-coupled device camera (model ORCA-ER, Hamamatsuhotonics, Hamamatsu, Japan). Fluorescence signals were converted to absolute intracellular Ca2+ values with C imaging software (Compix, Sewickley, PA).
Statistical analysis. Values are means ± SE. Student's t-test was used for comparisons between two groups. For statistical analysis of data from multiple groups, one-way ANOVA with Bonferroni's post hoc test was used. P < 0.05 was taken as a minimal level of significance.
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RESULTS |
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Therefore, we first performed primary culture of young mouse striatum according to the conventional method used for culture of neonatal striatum (24). However, this method was unsuccessful, because the yield of cells was very poor and the cells were not viable. Thus we extensively modified each step in the revised protocol (Fig. 1A). The effect of trypsin on cell dissociation was weaker in young than in neonatal tissues, because connective tissues are much more abundant in young samples. Therefore, we treated the tissues with collagenase and then with trypsin to optimize concentration, duration, and temperature (Table 1). We also added DNase after collagenase treatment, because collagenase caused clustering of minced tissues by DNA, which attenuated the effect of trypsin (Table 2). After many trials and errors, we found that treatment with 0.3% collagenase at 37°C for 15 min combined with DNase (10 µl/mouse) achieved the highest number of viable cells. Under such conditions with collagenase and DNase, we also optimized trypsin treatment (Table 3). Furthermore, we found that the presence of tissue debris was cytotoxic; thus we added a Percoll gradient purification step to delete such debris. Centrifugation of the tissues with a 17% Percoll cushion at 1,500 rpm for 10 min yielded the highest number of viable cells (Table 4). Usually, we obtained 0.5–1.5 x 105 cells from the striatum of a mouse, and the cells remained viable for >2 wk (Fig. 1B). Over 90% of 4',6-diamidino-2-phenylindole-stained cells expressed microtubule-associated protein type 2, a marker for differentiated neurons (Fig. 1C) (27, 45), indicating that most of the cells that survived were neuronal, rather than glial. In addition, calbindin, a marker for striatal medium spiny projection neurons, but not for interneurons (3, 6), was detected in >90% of cultured neurons (Fig. 1D), indicating that most cultured cells were medium spiny projection neurons. Moreover, most cultured cells expressed D1- and D2-like receptors (Fig. 1E), which is in accordance with a previous report demonstrating coexpression of these receptors in cultured striatal neurons (6). We used these neonatal and young striatal neurons in the following experiments.
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AC5 was not involved in dopamine-induced apoptosis in young striatal neurons. Because the increase in cAMP was not proportional to induction of apoptosis, we thought that a particular subtype of AC, a cAMP-generating enzyme, may be involved in dopamine-induced apoptosis. AC5 is a major AC subtype in the striatum and the heart (12, 14), and we previously demonstrated that AC5 plays a major role in Iso-induced apoptosis in cardiac myocytes (13); therefore, we thought that dopamine may selectively stimulate AC5 to induce apoptosis in striatal neurons. Using young striatal neurons from AC5-knockout (AC5KO) mice, we examined whether AC5 plays a role in dopamine-induced apoptosis. However, although AC5KO mice produced significantly less cAMP (Fig. 3A), dopamine-induced apoptosis was similar to that in wild-type mice (Fig. 3, B and C), suggesting that AC5 was not involved in dopamine-induced apoptosis in the striatum. These results were confirmed by stimulation of adenosine A1 receptors. Dopamine signaling is modified by adenosine A1 receptor stimulation, which decreases cAMP production via inhibition of AC5 through activation of Gi protein (13, 42). Therefore, if AC5 is involved in inducing apoptosis in striatal neurons, dopamine-induced apoptosis would be decreased by A1 receptor stimulation. However, similar to the results in AC5KO mice, N6-cyclopentyladenosine, a selective A1 receptor agonist, did not decrease dopamine-induced apoptosis, whereas cAMP was significantly decreased (Fig. 3, E and F). These data support the results shown in Fig. 2, A–C, and suggest that AC-cAMP played a very minor role in dopamine-induced apoptosis in young striatal neurons.
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Dopamine stimulation decreased phosphorylation of Akt in young striatal neurons. Since the molecular mechanism for dopamine-induced apoptosis in the striatum is not well studied (11, 26, 38), we investigated changes in downstream molecules of dopamine stimulation. We examined two major molecules in apoptosis/survival signals in neuronal cells, i.e., ERK1/2 (48) and Akt (34). In neonatal striatal neurons, dopamine did not change the level of phosphorylation of Akt (Fig. 5A). In contrast, in young striatal neurons, dopamine significantly decreased phosphorylation of Akt (Fig. 5C). On the other hand, ERK1/2 was activated by Iso, but not dopamine, in young neurons (Fig. 5D). Since Iso did not induce apoptosis (Fig. 2, B and C), these data indicate that Akt, but not ERK1/2, played a role in dopamine-induced apoptosis in young striatal neurons. Also, these data are consistent with a previous report demonstrating that inactivation of Akt was involved in neurotoxin-induced apoptosis in a dopaminergic cell line (39).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Primary culture of objective neurons is a useful tool in the study of neuroendocrine mechanisms, electrophysiology, and signal transduction, which are difficult to pursue in vivo (24). Primary neuronal cultures from embryonic and neonatal rodents have been widely used for such studies because of their simplicity; however, there have been few reports demonstrating primary neuronal culture from rodents after the postnatal period. This may be due to the difficulty involved in obtaining viable neurons after the preparation, because neurons in the postnatal period have less resistance to the environmental changes than neurons in the embryonic and neonatal stages. Indeed, when we tried to culture young striatal neurons according to the method conventionally used for neonatal striatal neurons, no cells were viable. Therefore, we extensively modified the neonatal method and, finally, after many trials and errors, established a new method for culturing young striatal neurons. The major and critical modified points are as follows: 1) the striatum was removed only with forceps, without cutting or slicing, to minimize damage to neurons, 2) collagenase was added before trypsin, 3) a Percoll gradient purification step was added before neurons were plated to remove tissue debris, which was cytotoxic in the culture medium. In addition to the major points described above, from our experiences, the tissues and cells should be always treated gently, and the quality of the enzyme should be monitored, especially when the lot number of enzymes, such as collagenase, DNase, or trypsin, is changed. We also found that this method was useful for the culture of young hippocampal neurons as well (data not shown), implying that it can be used to culture neuronal cells from multiple regions. We thus believe that the method for young neurons will be of great value for investigators in this field.
Our results show that dopamine stimulation induced apoptosis in young, but not neonatal, striatal neurons, indicating that dopamine signaling changes during the postnatal period. Several previous reports demonstrated the increasing expression of dopamine receptors in the striatum. In situ hybridization (36) and radioligand binding assay (32, 43) showed that expression of D1- and D2-like receptors was low at birth and progressively increased to reach adult levels at 3 wk of age in the rat. We also demonstrated that expression of Gs and Golf proteins was increased after birth and reached a plateau at postnatal day 14 (12). These data indicate that the dopamine-cAMP pathway in the striatum matures by 2–3 wk after birth and are consistent with our cAMP accumulation assay results, which showed significant increases in young neurons (Fig. 2A). However, we found that cAMP did not play an important role in dopamine-induced apoptosis. Iso did not induce apoptosis (Fig. 2, B and C), whereas a greater increase in cAMP was stimulated by Iso than by dopamine (Fig. 2A). Furthermore, deletion of AC5, a major subtype of AC in the striatum, did not decrease dopamine-induced apoptosis, but it dramatically reduced cAMP production (Fig. 3, A and B). Moreover, stimulation of the Gi protein-coupling A1 receptor did not prevent dopamine-induced apoptosis but inhibited cAMP production in young neurons (Fig. 3, E and F), indicating that cAMP was not a key molecule in dopamine-induced apoptosis. These data suggest that although dopamine-cAMP signaling develops in the postnatal period, cAMP is not critical in dopamine-induced apoptosis.
Intracellular Ca2+ signaling induces apoptosis mainly via activation of caspase-7 and/or caspase-12 (33), and Ca2+ signaling plays a major role in neuronal apoptosis (17, 19, 46). Also, in the striatum, D1-like receptors are known to activate Gq protein and PLC, leading to the elevation of intracellular Ca2+ (31, 44, 46). Interestingly, although stimulation of D1-like receptors did not increase intracellular Ca2+ in the striatum of the neonatal rat (21), such stimulation increases intracellular Ca2+ in adult rat brain slice preparations (41), implying that the change in dopamine-Ca2+, rather than dopamine-cAMP, signaling after birth is a major mechanism involved in induction of apoptosis in young neurons. Indeed, we demonstrated that dopamine increased intracellular Ca2+ in young, but not neonatal, striatal neurons (Fig. 4, A–D). In addition, since, to our knowledge, there have been no reports on the developmental changes of PLC in the striatum, we compared PLC expression in young neurons with that in neonatal neurons. PLC expression was markedly increased in young striatal neurons (Fig. 4G). Therefore, developmental changes in the dopamine-PLC-Ca2+ signaling pathway can make a difference in dopamine-induced apoptosis between neonatal and young striatal neurons. Previous reports showed that the number of striatal cells reduces to the adult level by postnatal day 8 (7, 37), implicating the occurrence of apoptosis, and changes in dopamine-PLC-Ca2+ may play a role in such developmental cell death in the striatum.
We have demonstrated that dopamine stimulation decreased Akt phosphorylation. Akt mediates apoptosis via the mitochondrial pathway, which induces caspase-9 activation (33), and is mainly activated by phosphatidylinositol 3-kinase and prevents apoptosis by inhibiting Bad, a regulator of mitochondria-related apoptosis (4). In striatal neurons from rat embryo, Akt is activated by dopamine stimulation (1). However, little is known about the mechanism of inhibiting Akt. Only a very recent study showed that phosphorylation of Akt was decreased in 6-hydroxydopamine-induced apoptosis in a dopaminergic cell line (39). Our results showed that phosphorylation of Akt was decreased in young striatal neurons and not altered in neonatal striatal neurons. We do not know the exact mechanism for this change; however, the functional difference in negative regulators for Akt may be involved between the prenatal and the postnatal period, as implied in recent studies describing protein phosphatase type 2A (PP2A) (2). PP2A is expressed in the striatum (10) and activated by PKC-
, a novel (diacylglycerol-sensitive) PKC isoform, in dopaminergic neurons (28, 51). Because PKC is a downstream enzyme of PLC, it is possible that stimulation of D1-like receptors inhibits Akt phosphorylation via the PLC-PKC--PP2A pathway. PLC expression was observed in young, but not neonatal, neurons (Fig. 4G); thus increased PLC expression may cause Akt inactivation in young striatal neurons. Also, these data indicated that, besides Ca2+ signaling, dopamine-induced apoptosis was mediated by the Akt-Bad pathway via mitochondria. However, further investigation is necessary to elucidate the underlying mechanism. Several reports have indicated that the ERK signaling pathway is activated in response to D1- and D2-like receptor stimulation in striatal neurons (48, 50). In addition, a recent study demonstrated that ERK is activated by neurotoxin, 6-hydroxydopamine, in the B65 neuronal cell line (20), indicating the role of ERK in neuronal apoptosis. However, our results showed that Iso, but not dopamine, increased ERK1/2 phosphorylation in young neurons, implying that ERK signaling does not play a critical role in dopamine-induced apoptosis in young striatal neurons.
In summary, we have demonstrated that the effect of dopamine on apoptosis varies at different developmental stages. Dopamine-PLC-Ca2+ signaling, which changed during the postnatal period, most likely regulated the induction of apoptosis via dopamine receptor stimulation. The insight gained from our studies may be helpful for understanding the mechanism underlying dopamine-induced apoptosis in striatal neurons, which is involved in neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and schizophrenia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* K. Iwatsubo and S. Suzuki contributed equally to this work. 
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4 independent experiments. Intracellular Ca2+ elevation was detected as fluorescent red. E: effect of Ca2+ chelators on dopamine-induced apoptosis. Striatal neurons from young mice were incubated with 100 µM dopamine in the presence or absence of 1 mM EGTA or 5 µM BAPTA-AM for 72 h. Apoptosis was evaluated by TUNEL staining. Values are means ± SE (n = 4). F: effect of a PLC inhibitor on dopamine-induced apoptosis. Striatal neurons from young mice were incubated with 100 µM dopamine in the presence or absence of U-73122 for 72 h. Apoptosis was evaluated by TUNEL staining. Values are means ± SE (n = 4). G: expression of PLC was increased in striatal neurons from young mice. Striatal neurons from neonatal and young mice were lysed and subjected to SDS-PAGE and Western blotting for PLC and β-actin. Values are means ± SE (n = 4).