- Open Access
Null effect of antidepressants on the astrocytes-mediated proliferation of hippocampal progenitor cells in vitro
© Ko et al; licensee BioMed Central Ltd. 2007
- Received: 14 May 2007
- Accepted: 15 June 2007
- Published: 15 June 2007
It is well known that antidepressants increase neurogenesis in the dentate gyrus of the hippocampus. The increase of neurogenesis might contribute to the behavioral effects of antidepressants. However, the mechanism by which antidepressants increase hippocampal neurogenesis is largely unknown. It has been recently reported that astroglia induce the neurogenesis of the hippocampal neural progenitor cells (NPCs). Therefore, we hypothesized that antidepressants may act on astrocytes, and this in turn induces neurogenesis of NPCs.
To examine this hypothesis, we used two co-culture systems, i.e., a contact-independent Banker culture and a contact-dependent overlay co-culture. In both of these systems, in comparison with naïve astrocytes, antidepressant-treated astrocytes did not further increase the proliferation of NPCs.
These results suggest that astrocytes increase the proliferation of hippocampal NPCs, however, this may not be directly involved in the antidepressant-induced proliferation of NPCs.
- Glial Fibrillary Acidic Protein
- Dentate Gyrus
- Neural Progenitor Cell
Depressive disorder is a chronic, life-threatening illness that may be caused by genetic and environmental factors such as stress . Of all the brain regions related to depressive disorders, the hippocampus has received the most attention because many patients suffering from depressive disorders exhibit atrophy in the hippocampus, which regulates the stress response by participating in the hypothalamic-pituitary-adrenal (HPA) axis [2, 3]. Recently, it has been reported that antidepressant treatment increases neurogenesis in the dentate gyrus of the hippocampus, and this increased neurogenesis is required for the behavioral effects of antidepressants [4, 5]. However, the mechanism by which antidepressants induce neurogenesis in the hippocampus is largely unknown.
It has been well known that astroglia support the functions of neurons. However, a number of recent studies have also shown that astroglia actively control many neuronal functions and development; moreover, this is related to many neurological disorders [6–11]. Many antidepressants are serotonin or norepinephrine reuptake inhibitors . Astrocytes express a serotonin transporter and a norepinephrine transporter [13, 14], and this raises the possibility that these cells may be activated and play a role in the action of antidepressants. Moreover, it was recently reported that astroglia induce neurogenesis by increasing the proliferation and differentiation of neural progenitor cells (NPCs) in the dentate gyrus of the hippocampus .
We thus hypothesized that when astrocytes are activated by antidepressants, they may release factors that can induce neurogenesis in the dentate gyrus of the hippocampus. In addition, neurogenesis induced by astrocyte-releasing factors may be involved in the behavioral effects of antidepressants. To examine our hypothesis, we used two types of astrocyte-NPC co-culture systems and measured the proliferation of NPCs by using BrdU immunocytochemical analysis.
In this study, we have examined the possibility that astrocytes are involved in antidepressant-induced proliferation of NPCs. After treated with 10 μM of desipramine or fluoxetine for 48 h, astrocytes were co-cultured with NPCs in a contact-independent or contact-dependent manner. In the former case, although naïve astrocytes induced the proliferation of NPCs in comparison with NPCs that were grown alone, desipramine or fluoxetine treatment of astrocytes did not further increase the proliferation of NPCs (Fig. 2). In addition, treatment with desipramine or fluoxetine did not enhance the proliferation mediated by astrocytes in a contact-dependent manner (Fig. 3). These results, however, cannot exclude the possible involvement of astrocytes in antidepressant-induced neurogenesis. First, the response to antidepressants could be different depending on the mouse strains. For example, C57BL/6 mice are less reactive to stressor and antidepressants than BALB/c mice. These two strains show different 5-HT2C pre-mRNA editing responses in the forebrain . Second, we used the experimental scheme to treat BrdU for 24 h because we hypothesized that antidepressants-activated astrocytes might affect the proliferation of NPCs chronically. However, if antidepressants-activated astrocytes affected the proliferation of NPCs transiently, we might be unable to detect the significant effects. Finally, neurogenesis involves three developmental stages: proliferation, differentiation of NPCs, and survival of immature or mature neurons . Although we did not observe the antidepressant-induced proliferating effect of astrocytes, the differentiation of NPCs or survival of immature or mature neurons might be increased by neighboring astrocytes that had been activated by antidepressants. Moreover, a number of signals provided by the microenvironment around the NPCs in the dentate gyrus of the hippocampus have been known to regulate the maintenance, proliferation and differentiation of NPCs [19, 20]. Thus, we cannot exclude a possibility that such signals might be additionally required for the astrocyte-induced proliferation of NPCs in the intact brain that had been exposed to antidepressants.
We hypothesized that astrocytes might be involved in the antidepressant-mediated neurogenesis since it was well known that astrocytes induce the hippocampal neurogenesis and have serotonin or norepinephrine transporter which are targets of the many antidepressants. However, in both contact-dependent and contact-independent co-culture system, astrocytes did not further increase the proliferation of the hippocampal NPCs. Considering that astrocytes are involved in many other neurological functions, more investigations should be carefully performed to elucidate the role of astrocytes on the antidepressant-induced hippocampal neurogenesis.
Primary NPCs from the hippocampus of postnatal day 1 or 2 (P1 or P2) C57BL6/J mice were prepared essentially as described previously [21, 22]. Hippocampal tissue was dissociated and plated on poly-L-lysine/laminin-coated dishes (25 μg/ml poly-L-lysine; Sigma, 10 μg/ml laminin; Invitrogen) in the presence of Dulbecco's modified Eagle's medium/Nutrient mixture (DMEM/F12) (1:1) containing B27 supplement (without vitamin A), 100 U/ml penicillin, 100 μg/ml streptomycin, and 20 ng/ml bFGF (Gibco BRL). Half of the medium was replaced with fresh medium every 3 days. At least after DIV 6, NPCs were used for co-culturing with astrocytes. For the overlay culture, NPCs were infected with retrovirus to express GFP as a marker at DIV 3.
Primary astrocytes from the hippocampus of P1 or P2 C57BL6/J mice were prepared as described previously [21, 22]. Hippocampal tissue was dissociated and plated on a 25 cm2 tissue culture flask (SPL) in DMEM/F12 (1:1), 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS) (Gibco BRL). The medium was replaced every 5 days. When the cells became confluent (DIV 10–11), the flasks were shaken at 200 rpm and 37°C for 3–4 h, and the medium was replaced with fresh medium. Additionally, in case of the overlay co-culture, the cells were treated with cytosine arabinoside (20 μM; Sigma) for 72 h and then washed with phosphate buffered saline (PBS) three times before co-culture.
For the Banker culture, astrocytes were passaged on the poly-L-lysine-coated coverslips (20 μg/ml; Sigma) at a density of 3.7 × 104/cm2. On the following day, the cells were treated with 10 μM of fluoxetine (Sigma) or desipramine (Sigma) dissolved in the culture medium for 48 h; this was followed by washing with PBS three times. The concentration of each antidepressant was determined on the basis of the steady-state brain concentrations estimated from a clinical study . With regard to the length of time used for incubation with the antidepressants, we treated the cells with the drugs for the longest possible time, because only chronic antidepressant treatment, not acute, is known to induce neurogenesis. However, the treatment of cells with antidepressants longer than 48 h induced cell death (data not shown) . After washing out the antidepressants, the astrocytes were co-cultured with NPCs that were plated on the poly-L-lysine/laminin-coated glass-bottom dish (10 μg/ml poly-L-lysine, 5 μg/ml laminin) at a density of 1.5 × 104/cm2 in the N2 medium containing 20 ng/ml bFGF.
For the overlay culture, astrocytes were plated on the poly-L-lysine-coated glass- bottom dish at a density of 3.8 × 104/cm2. After one day, the cells were treated with 10 μM of fluoxetine or desipramine for 48 h; this was followed by three washes with PBS. After washing out the antidepressants, NPCs expressing GFP were overlaid onto astrocytes in the N2 medium containing 20 ng/ml bFGF.
Twenty-four hours after the co-culture, NPCs were treated with BrdU (10 μM, Banker culture and 2.5 μM, overlay co-culture, respectively) (Sigma) for 24 h. In case of the overlay co-culture, NPCs and astrocytes were treated with BrdU. Finally, co-cultured NPCs were subjected to the immunocytochemical analysis. Briefly, the cells were fixed with 4% paraformaldehyde in PBS. For the detection of BrdU, the cells were treated with 1 N HCl at 37°C for 30 min and then neutralized by washing with 0.1 M sodium borate buffer. After the treatment with HCl, cells were incubated with cold PBT (0.1% Triton X-100 and 0.1% BSA in PBS) for 15 min. After blocking with a preblock solution (2% BSA and 0.08% Triton X-100 in PBS) at room temperature for 2 h, the cells were incubated overnight with the following primary antibodies in a preblock solution: BrdU (1:100, mouse; Sigma), GFP (1:500, rabbit; Abcam), nestin (1:400, mouse; BD biosciences), GFAP (1:400, rabbit; DAKO). After washing with PBT, the cells were incubated with the fluorescence-labeled secondary antibodies for 2 h. Cells were counterstained for 15 min with 10 ng/ml 4', 6'-diamidino-2-phenylindole (DAPI) (Molecular Probes) or 50 μg/ml propidium iodide (PI). Immunofluorescence was visualized using a fluorescence microscope (Carl Zeiss). Fluorescence-positive cells were quantified in 20 fields (×10) per dish either manually (overlay culture) or by using Image J software (Banker culture).
Total RNA was collected using the TRIzol Reagent according to the manufacturer's instructions (Invitrogen). Reverse transcription (RT) reactions were performed using SuperScript™ III Reverse Transcriptase (Invitrogen). cDNA was synthesized from total RNA (2 μg) using a random primer (0.1 μg). One microliter of the RT reaction product was utilized as the template in the PCR with each of the following primer sets. GAT ATT GCA GCG GTT CCT GT and TTC GCA CTG TAG CAG GAA TG were used for GDNF, while TGC ACC ACC AAC TGC TTA and GGA TGC AGG GAT GAT GTT C were used for GAPDH. The number of PCR cycles was set such that it was below saturation conditions and was 30 and 23 for GDNF and GAPDH, respectively.
We thank Dr. Sunyoung Kim for retroviral constructs. This work was supported by the Creative Research Initiative Program of the Korean Ministry of Science and Technology (to B.-K.K.) and by the Korea Research Foundation grant (KRF-20005-005-J16001, to B.-K.K.).
- Berton O, Nestler EJ: New approaches to antidepressant drug discovery: beyond monoamines. Nature reviews 2006,7(2):137–151. 10.1038/nrn1846PubMedView ArticleGoogle Scholar
- Duman RS: Depression: a case of neuronal life and death? Biological psychiatry 2004,56(3):140–145. 10.1016/j.biopsych.2004.02.033PubMedView ArticleGoogle Scholar
- Manji HK, Drevets WC, Charney DS: The cellular neurobiology of depression. Nature medicine 2001,7(5):541–547. 10.1038/87865PubMedView ArticleGoogle Scholar
- Malberg JE, Eisch AJ, Nestler EJ, Duman RS: Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci 2000,20(24):9104–9110.PubMedGoogle Scholar
- Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S, Weisstaub N, Lee J, Duman R, Arancio O, Belzung C, Hen R: Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 2003,301(5634):805–809. 10.1126/science.1083328PubMedView ArticleGoogle Scholar
- Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha BK, Von Zastrow M, Beattie MS, Malenka RC: Control of synaptic strength by glial TNFalpha. Science 2002,295(5563):2282–2285. 10.1126/science.1067859PubMedView ArticleGoogle Scholar
- Christopherson KS, Ullian EM, Stokes CC, Mullowney CE, Hell JW, Agah A, Lawler J, Mosher DF, Bornstein P, Barres BA: Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell 2005,120(3):421–433. 10.1016/j.cell.2004.12.020PubMedView ArticleGoogle Scholar
- Panatier A, Theodosis DT, Mothet JP, Touquet B, Pollegioni L, Poulain DA, Oliet SH: Glia-derived D-serine controls NMDA receptor activity and synaptic memory. Cell 2006,125(4):775–784. 10.1016/j.cell.2006.02.051PubMedView ArticleGoogle Scholar
- Pascual O, Casper KB, Kubera C, Zhang J, Revilla-Sanchez R, Sul JY, Takano H, Moss SJ, McCarthy K, Haydon PG: Astrocytic purinergic signaling coordinates synaptic networks. Science 2005,310(5745):113–116. 10.1126/science.1116916PubMedView ArticleGoogle Scholar
- Seifert G, Schilling K, Steinhauser C: Astrocyte dysfunction in neurological disorders: a molecular perspective. Nature reviews 2006,7(3):194–206. 10.1038/nrn1870PubMedView ArticleGoogle Scholar
- Ullian EM, Sapperstein SK, Christopherson KS, Barres BA: Control of synapse number by glia. Science 2001,291(5504):657–661. 10.1126/science.291.5504.657PubMedView ArticleGoogle Scholar
- Hisaoka K, Nishida A, Koda T, Miyata M, Zensho H, Morinobu S, Ohta M, Yamawaki S: Antidepressant drug treatments induce glial cell line-derived neurotrophic factor (GDNF) synthesis and release in rat C6 glioblastoma cells. Journal of neurochemistry 2001,79(1):25–34. 10.1046/j.1471-4159.2001.00531.xPubMedView ArticleGoogle Scholar
- Inazu M, Takeda H, Ikoshi H, Sugisawa M, Uchida Y, Matsumiya T: Pharmacological characterization and visualization of the glial serotonin transporter. Neurochemistry international 2001,39(1):39–49. 10.1016/S0197-0186(01)00010-9PubMedView ArticleGoogle Scholar
- Inazu M, Takeda H, Matsumiya T: Functional expression of the norepinephrine transporter in cultured rat astrocytes. Journal of neurochemistry 2003,84(1):136–144. 10.1046/j.1471-4159.2003.01514.xPubMedView ArticleGoogle Scholar
- Song H, Stevens CF, Gage FH: Astroglia induce neurogenesis from adult neural stem cells. Nature 2002,417(6884):39–44. 10.1038/417039aPubMedView ArticleGoogle Scholar
- Mercier G, Lennon AM, Renouf B, Dessouroux A, Ramauge M, Courtin F, Pierre M: MAP kinase activation by fluoxetine and its relation to gene expression in cultured rat astrocytes. J Mol Neurosci 2004,24(2):207–216. 10.1385/JMN:24:2:207PubMedView ArticleGoogle Scholar
- Banker G K. Goslin (Eds.): Culturing Nerve Cells. Massachusetts Institute of Technology; 1991.Google Scholar
- Englander MT, Dulawa SC, Bhansali P, Schmauss C: How stress and fluoxetine modulate serotonin 2C receptor pre-mRNA editing. J Neurosci 2005,25(3):648–651. 10.1523/JNEUROSCI.3895-04.2005PubMedView ArticleGoogle Scholar
- Abrous DN, Koehl M, Le Moal M: Adult neurogenesis: from precursors to network and physiology. Physiological reviews 2005,85(2):523–569. 10.1152/physrev.00055.2003PubMedView ArticleGoogle Scholar
- Lie DC, Song H, Colamarino SA, Ming GL, Gage FH: Neurogenesis in the adult brain: new strategies for central nervous system diseases. Annual review of pharmacology and toxicology 2004, 44: 399–421. 10.1146/annurev.pharmtox.44.101802.121631PubMedView ArticleGoogle Scholar
- Palmer TD, Takahashi J, Gage FH: The adult rat hippocampus contains primordial neural stem cells. Molecular and cellular neurosciences 1997,8(6):389–404. 10.1006/mcne.1996.0595PubMedView ArticleGoogle Scholar
- Takahashi J, Palmer TD, Gage FH: Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures. Journal of neurobiology 1999,38(1):65–81. 10.1002/(SICI)1097-4695(199901)38:1<65::AID-NEU5>3.0.CO;2-QPubMedView ArticleGoogle Scholar
- Strauss WL, Layton ME, Hayes CE, Dager SR: 19F magnetic resonance spectroscopy investigation in vivo of acute and steady-state brain fluvoxamine levels in obsessive-compulsive disorder. The American journal of psychiatry 1997,154(4):516–522.PubMedView ArticleGoogle Scholar
- Bartholoma P, Erlandsson N, Kaufmann K, Rossler OG, Baumann B, Wirth T, Giehl KM, Thiel G: Neuronal cell death induced by antidepressants: lack of correlation with Egr-1, NF-kappa B and extracellular signal-regulated protein kinase activation. Biochemical pharmacology 2002,63(8):1507–1516. 10.1016/S0006-2952(02)00882-1PubMedView ArticleGoogle Scholar
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